Studies on Subterranean clover mottle virus towards development of a gene silencing vector
This thesis is submitted to Murdoch University for the degree of Doctor of Philosophy
by
John Fosu-Nyarko B.Sc (Hons) [Agric. Sci]
Western Australian State Agricultural Biotechnology Centre Division of Science and Engineering Murdoch University February, 2005
Declaration
Declaration
I declare that this thesis is my own account of my research and contains as its main content work which has not been previously submitted for a degree at any tertiary education institute.
……………………
John Fosu-Nyarko
ii
Abstract
Abstract
Subterranean clover mottle virus (SCMoV) is a positive sense, single-stranded RNA virus that infects subterranean clover (Trifolium subterraneum) and a number of related legume species.
The ultimate aim of this research was to investigate aspects of SCMoV that would support its use as a gene silencing vector for legume species, since RNA (gene) silencing is now a potential tool for studying gene function. The ability of viruses to induce an antiviral defense system is being explored by virus-induced gene silencing (VIGS), in which engineered viral genomes are used as vectors to introduce genes or gene fragments to understand the function of endogenous genes by silencing them. To develop a gene silencing vector, a number of aspects of SCMoV host range and molecular biology needed to be studied.
A requirement for a useful viral vector is a suitably wide host range. Hence the first part of this work involved study of the host range and symptom development of SCMoV in a range of leguminous and non-leguminous plants. The aim of this work was to identify new and most suitable hosts among economically important crop and model legumes for functional genomic studies, and also to study symptom development in these hosts for comparison with host responses to any SCMoV-based viral vectors that might be used in later infection studies. A total of 61 plant genotypes representing 52 species from 25 different genera belonging to 7 families were examined for their response to SCMoV infection, including established and new crop legumes, established pasture, and novel pasture and forage legumes, and 12 host indicator plants belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae,
Cucurbitaceae and Solanaceae. Following mechanical inoculation, plants were examined for symptoms and tested for primary and secondary infection by RT-PCR and/or ELISA after 2-3 weeks and 3-9 weeks, respectively. Thirty-six legume hosts belonging to eight different genera of legumes were identified as suitable hosts of SCMoV, 22 of them systemic hosts and 15 were infected locally. Only two non-legumes were infected with SCMoV-P23, one systemically and one as a local host, so confirming that SCMoV is essentially a legume-infecting virus. This work considerably expanded knowledge of the host range of SCMoV.
iii
Abstract
To provide the information needed to modify the SCMoV genome to develop gene vectors, the virus was characterized in detail. The complete genomes of four isolates, SCMoV-AL,
SCMoV-MB, SCMoV-MJ and SCMoV-pFL, were sequenced using high fidelity RT-PCR and molecular cloning, and compared to the first sequenced isolate (SCMoV-P23) to give a complete picture of the genome organisation of the virus. The 4,258 nucleotide (nt) sequence of SCMoV RNA is not polyadenylated. The 5’ non-coding region (NCR) is 68 nt in length and the 3’ NCR is 174 nt. The coding region contains four overlapping open reading frames
(ORFs). The first, ORF1 (nt 68-608), encodes a putative protein containing 179 amino acids with a calculated molecular mass (Mar) of 20.3 kDa. It overlaps with the next ORF, ORF2a, by four bases. ORF2a (nt 605-2347) encodes a putative protein of 580 amino acids with a Mar of
63.7 kDa and contains a motif characteristic of chymotrypsin-like serine proteases. The ORF2b is probably translated as part of a polyprotein by -1 ribosomal frameshifting in ORF2a. The transframe product (Mar = 107.5 kDa) is made up of 966 amino acids. A GDD motif typical of
RNA virus polymerases is present in ORF2b. The 3’ terminal ORF3 (nt 3323-4084) encodes the 27.3 kDa coat protein (CP).
Nucleotide variation between the complete sequences of the isolates was two to three orders of magnitude larger than base misincoporation rates of the polymerases used in RT-PCR.
Molecular relationship analysis between all five isolates, undertaken with the complete nucleotide sequences, clearly separated them into three groups. These groups reflect similar significantly diverse groupings based on the symptoms and their severity in subterranean clover. Intra-isolate sequence variability is therefore a possible cause of the differences in symptom severity. The analysis also showed that there were more nucleotide substitutions at the 5’ terminal half of SCMoV than at the 3’ end. This observation was confirmed by the higher value of nucleotide diversities at nonsynonymous versus synonymous sites (dN/dS ratio) estimated for the ORF1, compared to the near conservation of sequences of the other ORFs.
These results, together with functional and comparative sequence analysis with other sobemoviruses, implicate the ORF1 gene product in pathogenicity of SCMoV, possibly as a severity determinant or as a viral suppressor of RNA silencing in plants.
iv
Abstract
Because more information on SCMoV genome function was required, the possible involvement of the ORF1 gene product (P1) and the CP in movement of SCMoV was studied in cells of grasspea (Lathyrus clymenum L) and chickpea as C-terminal fusion constructs with jellyfish
(Aequorea victoriae) green fluorescent protein (GFP). A transient expression vector, pTEV, for in planta synthesis of reporter gene constructs was developed. The vector was based on pGEM-T with 35S RNA transcriptional promoter of Cauliflower Mosaic virus (CaMV) and nopaline synthase gene transcription terminator signal (T-Nos) separated by a multiple subcloning site. A custom-made particle inflow gun was used to introduce the constructs into plant cells. The bombardment conditions were first optimised for efficient delivery of DNA- coated particles. Transient gene expression of GFP was monitored 24-96 hours after particle bombardment. Fluorescence from GFP alone, GFP:CP and GFP:P1 constructs was observed in the nucleus of single cells, cytoplasm and cell periphery of neighbouring cells. There was limited spread of these fusion proteins from one cell to another 36-48 hours after transformation. These results indicate that the P1 and CP cannot move independently from cell to cell. Other viral/cellular components might be needed to form a complex with these proteins to transport the viral genome. Putative nuclear export signals in the P1 and CP sequences of
SCMoV were identified by sequence comparison. These could be tested by mutagenesis using full-length infectious clones.
To determine the possibility of gene expression of vectors based on SCMoV, three forms of a full-length cDNA clone of SCMoV-pFL were developed: one with no heterologous transcriptional factors (pFL), a second under the control of only 35S (p35SFL) and a third with
35S and T-Nos (pTEVFL). Fifteen day-old in vitro-cultured chickpea, grasspea and subterranean clover seedlings were inoculated by particle bombardment using gold particles coated with plasmid pTEVFL. In vivo-transcribed RNA transcripts were detected by RT-PCR after two weeks in grasspea but not in subterranean clover and chickpea.
v
Abstract
Experiments were undertaken towards developing the SCMoV genome into a VIGS vector.
Three forms each of five major GFP chimeric constructs of pFL (the full length SCMoV cDNA clone) were generated from which in vitro- and in vivo-transcribed RNA transcripts could be derived. The rationale used in developing these constructs was gene insertion and/or replacement with gfp, and duplication of the putative subgenomic RNA promoter (sgPro) of
SCMoV. The major constructs were as follows:
• pFLCPgfp, pFL with the gfp gene fused to the 3’ end of the CP gene,
• pFLP1gfp, pFL with gfp gene fused to the 3’ end of the ORF1,
• pFLCPsgprogfp, pFL with a putative sgPro sequence and a translatable gfp gene cloned
in tandem between the CP gene and the 3’ NCR of SCMoV,
• pFLCP∇sgprogfp, pFL with a putative sgPro sequence and a translatable gfp gene
cloned in tandem between a truncated CP gene and the 3’ NCR and
• pFLREPsgprogfp, pFL with the ORF2b, a putative sgPro sequence and a translatable
gfp gene cloned in tandem between a truncated CP gene and the 3’ NCR
These constructs were all made, but a detailed assessment of their vector potential could not be done because there was a delay of about one year whilst the Office of the Gene Technology
Regulator processed the application for permission for glasshouse testing. Although additional work needs to be undertaken to complete development of a final RNA silencing vector, this study has contributed to new knowledge in terms of extending understanding of SCMoV host range, symptoms, sequence variation and control of gene expression. The constructs made have also laid the groundwork for development of a legume gene silencing vector based on
SCMoV.
vi
Table of Contents
TABLE OF CONTENTS Title page ...... i Declaration...... ii Abstract...... iii Table of Contents...... vii Acknowledgements ...... xiii Dedication...... xv Presentations and Publications ...... xvi List of abbreviations ...... xvii
CHAPTER ONE: General Introduction and Literature Review...... 1
1.0 General Introduction...... 2 1.1 Subterranean clover mottle Sobemovirus (SCMoV)...... 3 1.1.1 Symptoms ...... 3 1.1.2 Isolates ...... 4 1.1.3 Transmission...... 5 1.1.4 Host range and response to infection ...... 5 1.2 Sobemoviruses...... 6 1.2.1 Definitive species and geographical distribution ...... 6 1.2.2 Biology...... 6 1.2.2.1 Strains and isolates ...... 6 1.2.2.2 Host range...... 8 1.2.2.3 Symptoms and cellular localisation ...... 8 1.2.2.4 Transmission...... 9 1.2.3 Structure...... 9 1.2.3.1 Morphology ...... 9 1.2.3.2 Genome organisation...... 10 1.2.4 Gene expression...... 12 1.2.5 Gene products ...... 12 1.2.6 Sequence comparison...... 13 1.2.6.1 Sequences of non-coding regions ...... 13 1.2.6.2 Sequences of coding regions...... 14 1.2.7 Functions of Sobemovirus proteins...... 15 1.2.7.1 The P1 protein ...... 15 1.2.7.2 The polyprotein...... 16 1.2.7.3 The P3 protein ...... 17 1.2.7.4 The coat protein...... 17 1.3 RNA (Gene) silencing in plants...... 19 1.3.1 History and discovery ...... 19 1.3.2 RNA silencing as a universal system...... 20 1.3.2.1 Genes involved in RNA silencing ...... 21 1.3.3 Stages of RNA silencing...... 22 1.3.3.1 Initiation ...... 22 1.3.3.2 Propagation...... 23 1.3.3.3 Maintenance...... 23 1.3.4 Mechanism of RNA silencing in plants ...... 24 1.4. RNA silencing and RNA virus-host interaction ...... 25 1.4.1 Viruses as targets of RNA silencing ...... 25 1.4.2 Viruses as suppressors of RNA silencing ...... 26 1.4.3 Viruses as inducers of RNA silencing ...... 27
vii
Table of Contents
1.5 Virus-induced gene silencing (VIGS)...... 28 1.5.1 VIGS as a tool for functional analysis of endogenous genes ...... 28 1.5.2 Viral vectors for VIGS...... 29 1.5.3 VIGS insert ...... 30 1.6 Plant viruses as gene vectors...... 31 1.6.1 Development of plant RNA viruses as gene vectors, strategies...... 31 1.6.2 Virus-based vectors as transient gene expression systems...... 33 1.6.3 Introduction of viral vectors into host plants ...... 33 1.7 Reporter genes and viral vectors as research tools...... 34 1.7.1 GFP as a research tool...... 36 1.7.2 GFP as a reporter gene for validating viral vectors...... 37 1.7.3 GFP as a reporter gene for studying viral pathogenesis...... 38
1.8 Limitations in construction and use of plant RNA viruses as gene vectors...... 39 1.8.1 Intrinsic viral factors...... 39 1.8.2 Technical factors...... 40 1.8.3 Environmental and regulatory factors...... 41 1.9 Aims and objectives of this project ...... 42
CHAPTER TWO: General Materials and Methods ...... 43
2.0 Virus isolates and culture, and plant inoculation ...... 44 2.1 Plant materials and growth in the glasshouse ...... 44 2.2 Extraction of total nucleic acids from virus-infected leaf tissues ...... 45 2.3 Modifying enzymes ...... 45 2.4 Reverse transcription (RT) ...... 46 2.4.1 RT with MuLV reverse transcriptase ...... 46 2.4.2 RT with M-MLV reverse transcriptase ...... 46 2.5 Polymerase Chain Reaction (PCR) ...... 47 2.5.1 Routine PCRs...... 47 2.5.2 High Fidelity PCRs ...... 47 2.6 Design and primers for RT, PCR and DNA sequencing ...... 48 2.7 Agarose gel electrophoresis and molecular weight standard markers ...... 49 2.8 Cloning...... 51 2.8.1 Cloning of PCR products ...... 51 2.8.1.1 Ligation of PCR products to cloning vectors ...... 51 2.8.2 Cohesive/sticky-end cloning ...... 52 2.8.2.1 Restriction endonuclease digestion ...... 52 2.8.2.2 Dephosphorylation of plasmid vectors ...... 52 2.8.2.3 Disruption of endonuclease recognition sites in plasmids...... 53 2.8.2.4 Ligation of restriction endonuclease DNA fragments ...... 54 2.8.3 Transformation of competent E. coli cells ...... 54 2.8.3.1 Bacterial strains ...... 54 2.8.3.2 Preparation of chemically-competent cells...... 54 2.8.3.3 Transformation efficiency of chemically-competent cells ...... 55 2.8.3.4 Heat-shock transformation and bacteria growth...... 56 2.8.4 Selection for transformants ...... 57 2.8.5 Analysis of transformants...... 57 2.8.5.1 Analysis of transformants by PCR ...... 57 2.8.5.2 Analysis of transformants by plasmid DNA miniprep/restriction digestion...... 58 2.9 DNA purification ...... 58 2.9.1 DNA purification from agarose gels ...... 58
viii
Table of Contents
2.9.2 DNA purification from solutions ...... 59 2.9.2.1 DNA purification with UltraClean™ PCR Clean-up kit ...... 59 2.9.2.2 DNA precipitation ...... 60 2.9.2.3 Phenol:chloroform DNA extraction ...... 60 2.9.3 Plasmid DNA purification from bacterial cell culture...... 60 2.9.3.1 Alkaline lysis method of plasmid purification...... 60 2.9.3.2 QIAGEN Plasmid Mini purification method ...... 61 2.9.3.3 QIAprep Spin Miniprep plasmid purification...... 61 2.10 DNA quantitation/quantification...... 62 2.11 DNA Sequencing ...... 62 2.11.1 Sequencing reactions...... 63 2.11.2 Purification of extension products for sequencing ...... 63 2.11.3 Data analysis ...... 63
CHAPTER THREE: Host Range and Symptomatology of SCMoV ...... 64
3.0 Introduction ...... 65 3.1 Aim of Chapter 3 ...... 66 3.2 Materials and methods...... 66 3.2.1 Potential host plants and growth conditions...... 66 3.2.2 Virus inoculations ...... 66 3.2.3 Detection of infection by RT-PCR...... 67 3.2.5 Detection of infection by Enzyme-linked Immunosorbent Assay (ELISA)...... 67 3.3 Results...... 68 3.3.1 Symptomatology of SCMoV...... 68 3.3.1.1 Development of primary symptoms in test plants ...... 68 3.3.1.2 Secondary symptom development in crop and alternative crop legumes...... 69 3.3.1.3 Secondary symptom development in Trifolium spp...... 72 3.3.1.4 Secondary symptom development in alternative pastures/forage legumes...... 73 3.3.1.5 Secondary symptom development in host indicator plants...... 75 3.3.2 Detection of primary and secondary infection of SCMoV-P23 by RT-PCR ...... 75 3.3.2.1 Detection of SCMoV-P23 infection by RT-PCR in crop/alternative crop legumes ...... 76 3.3.2.2 Detection of SCMoV-P23 infection by RT-PCR in Trifolium spp...... 77 3.3.2.3 Detection of SCMoV-P23 infection by RT-PCR in alternative pasture/forage legumes...... 77 3.3.2.4 Detection of SCMoV-P23 infection by RT-PCR in host indicator plants ...... 78 3.3.3. Detection of primary and secondary infection in P. sativum and M. truncatula by ELISA...... 79 3.4. Summary of results - Host Status of test plants to SCMoV ...... 80 3.5 Discussion ...... 81 3.5.1 Extension of host range of SCMoV ...... 82 3.5.2 Symptomatology of SCMoV...... 82 3.5.3 Importance of host range study to gene silencing studies with SCMoV-based vectors ...... 84 3.5.4 Susceptibility of new pasture cultivars to SCMoV ...... 84 3.5.5 Susceptibility of crop legumes and farming systems in SCMoV-endemic areas ...... 85 3.6 Conclusions...... 86
CHAPTER FOUR: Genetic Diversity in SCMoV and Functional Constraints of its Genes ...... 88
4.0 Introduction...... 88 4.1 Aim and objectives of Chapter 4 ...... 89 4.2 Materials and methods ...... 89 4.2.1 SCMoV isolates and symptom severity ...... 89 4.2.2 Sequencing strategy and primers for sequencing ...... 90
ix
Table of Contents
4.2.3 Primers for RT-PCR...... 90 4.2.4 Sequence analysis...... 92 4.3 Results...... 92 4.3.1 Complete genomic sequences of four isolates of SCMoV...... 92 4.3.1.1 Genome organisation of SCMoV ...... 92 4.3.1.2 Comparative sequence analysis of isolates of SCMoV ...... 93 4.3.1.3 Genetic variation within SCMoV isolates ...... 94 4.3.1.4 Distribution of nucleotide substitutions in all isolates...... 95 4.3.1.5 Nucleotide differences in isolates and symptom severity...... 95 4.3.1.5a Molecular relationship between isolates of SCMoV ...... 95 4.3.1.5b Test of significance of relationship between nucleotide differences and symptom severity ...... 96 4.3.2 Nucleotide and deduced amino acid sequence comparison and variability...... 96 4.3.2.1 Nucleotide sequences of the 5’ and 3’ NCRs ...... 96 4.3.2.2 Deduced amino acid sequences of the ORF1 ...... 97 4.3.2.3 Deduced amino acid sequences of ORFs 2a and 2b ...... 98 4.3.2.4 Deduced amino acid sequences of the coat protein gene...... 101 4.3.3 Selective constraints (dN/dS) for amino acids of SCMoV...... 101 4.3.3.1 dN/dS for amino acids of the ORF1 region...... 102 4.3.3.2 dN/dS for amino acids of the ORF2a/2b region...... 103 4.3.3.3 dN/dS for amino acids of the ORF3 region...... 103 4.4 Discussion ...... 104 4.4.1 Genetic variation in SCMoV isolates...... 104 4.4.2 Distribution of nucleotide differences in SCMoV isolates and symptom severity...... 104 4.4.2 Implications of genetic variation and/or mutations on the biology of SCMoV...... 105 4.4.3 Functional constraints on genes of SCMoV...... 107 4.4.4 Fidelity of in vitro polymerases and genetic variation in SCMoV isolates ...... 108 4.5 Conclusions and further work...... 109
CHAPTER FIVE: Cell-to-Cell Movement of the P1 and Coat Protein of SCMoV...... 111
5.0 Introduction ...... 112 5.1 Aim and objectives of Chapter 5 ...... 113 5.2 Materials and Methods...... 113 5.2.1 Development of transient expression vector, pTEV...... 113 5.2.1.1 Amplification and cloning of 35S and T-Nos...... 113 5.2.1.2 Sequences and map of pTEV...... 114 5.2.2 Development of reporter gene constructs...... 115 5.2.2.1 Amplification, cloning and subcloning of GFP into pTEV as pTEVGFP...... 115 5.2.2.2 Sequences of m-gfp5 and map of pTEVGFP...... 115 5.2.2.3 Amplification, cloning and fusion of CP and ORF1 genes of SCMoV-pFL to GFP...... 116 5.2.2.4 Sequencing and maps of c-terminal GFP fusion constructs ...... 117 5.2.3 Biolistic transformations ...... 117 5.2.3.1 Biolistic device for plant transformation ...... 118 5.2.3.2 DNA for transformations...... 118 5.2.3.3 Microcarrier preparation...... 119 5.2.3.4 Coating DNA onto microcarriers ...... 119 5.2.3.5 Particle bombardment and plant tissues transformed ...... 120 5.2.3.6 Visualisation of GFP expression ...... 120 5.3 Results...... 121 5.3.1 Effect of amount of DNA and shooting pressure on GFP fluorescence...... 121 5.3.2 Effect of dry and wet shooting of DNA-coated particles on transformation...... 122 5.3.3 Transient expression of free GFP in cells of subclover, Trigonella, chickpea and grasspea...... 122 5.3.4 Transient expression of GFP:CP and GFP:P1 in cells of chickpea and grasspea...... 124
x
Table of Contents
5.4 Discussion ...... 125 5.4.1 Expression and localisation of CP and P1 of SCMoV-pFL in host cells ...... 126 5.4.2 Putative nuclear export signals of the SCMoV CP and P1 proteins...... 127 5.4.3 Optimum conditions for transient transformation of legumes...... 128 5.4.4 Limitations of GFP as a reporter gene...... 129 5.5 Conclusion and further work ...... 130
CHAPTER SIX: Development of an Infectious cDNA Clone of SCMoV and GFP chimera cDNA Constructs for use as VIGS Vectors...... 131
6.0 Introduction...... 132 6.1 Aim and objectives of Chapter 6 ...... 132 6.2 Gene expression of SCMoV ...... 133 6.3 Prediction of sg promoter (sgPro) of SCMoV by sequence analysis ...... 134 6.3.1 Eukaryotic sg RNA synthesis...... 134 6.3.2 Transcription start site of sobemoviruses...... 135 6.3.3 Putative sgPro of SCMoV...... 135 6.4 PART 1: Development of a full-length cDNA clone of SCMoV-pFL...... 137 6.4.1 Isolation of SCMoV-pFL viral RNA...... 137 6.4.1.1 Purification and properties of SCMoV-pFL virions...... 137 6.4.1.2 Staining of virus particles and electron microscopy...... 138 6.4.1.3 Preparation of RNase-free glassware and solutions ...... 139 6.4.1.4 vRNA extraction from purified virions of SCMoV-pFL...... 139 6.4.1.5 Purity and identity of SCMoV vRNA ...... 139 6.4.1.5a Formaldehyde gel electrophoresis of SCMoV vRNA ...... 140 6.4.1.5b RT-PCR of SCMoV vRNA ...... 140 6.4.2. Cloning of a full-length cDNA of SCMoV-pFL ...... 141 6.4.2.1 RT-PCR amplification of SCMoV-pFL RNA genome ...... 141 6.4.2.2 Ligation cloning of overlapping cDNA clones to produce pFL ...... 142 6.4.2.3 Screening of transformants for pFL...... 144 6.4.2.3a Restriction digestion screening...... 144 6.4.2.3b PCR screening ...... 144 6.4.2.3c DNA sequence of pFL...... 145 6.4.3 Development of p35SFL and pTEVFL hybrids of pFL ...... 146 PART 2: Development of SCMoV-based gene vector constructs, strategies and clones...... 146 6.5 Construction of plasmid pFLCPgfp...... 148 6.5.1 Development of plasmid pCPgfp3NCR...... 148 6.5.1.1 Cloning of plasmid p1425 ...... 149 6.5.1.2 Development of a mutated plasmid of p1425 for ligation to gfp gene ...... 149 6.5.1.2a Inverse PCR mutagenesis of p1425 to incorporate EcoRI and SacI sites...... 149 6.5.1.2b Primers for IPCR...... 149 6.5.1.2c Optimisation of IPCR ...... 150 6.5.1.2d Religation of the IPCR mutagenesis product...... 151 6.5.1.3 Ligation and cloning of gfp gene and p1425M ...... 152 6.5.2 Assembly and sequential cloning to produce construct pFLCPgfp...... 152 6.5.3 Development of 35S and pTEV hybrids of FLCPgfp ...... 153 6.6 Construction of plasmid pFLP1gfp...... 153 6.6.1 Clones for constructing plasmid pFLP1gfp...... 153 6.6.2 Assembly and sequential cloning to produce construct pFLP1gfp ...... 154 6.6.2.1 Ligation and cloning of p5'P1 and pGFPE ...... 154 6.6.2.2 Ligation and cloning of p5'P1gfp and pP2for37...... 155 6.6.2.3 Ligation and cloning of p5'P1gfpP2 and pFLrev14...... 155 6.6.3 Development of 35S and pTEV hybrds ofpFLP1gfp ...... 156
xi
Table of Contents
6.7 Construction of plasmid pFLCPsgprogfp ...... 156 6.7.1 Clones for constructing plasmid pFLCPsgprogfp ...... 157 6.7.1.1 Cloning of translatable GFP and the 3’ NCR ...... 157 6.7.1.2 Cloning of the 3’ end of ORF2b and the CP gene...... 157 6.7.1.3 Cloning of the putative sgPro ...... 157 6.7.2 Assembly and sequential cloning to produce construct pFLCPsgprogfp...... 158 6.7.3 Development of 35S and pTEV hybrids of pFLCPsgprogfp...... 158 6.8 Construction of plasmid pFLCP∇sgprogfp...... 158 6.8.1 Assembly and sequential cloning of pFLCP∇sgprogfp and its 35S and T-Nos hybrids...... 159 6.9 Construction of plasmid pFLREPsgprogfp...... 159 6.9.1 Clones for developing construct pFLREPsgprogfp...... 160 6.9.1.1 Cloning of ORF2b of SCMoV-pFL as plasmid pP2b...... 160 6.9.1.2 Cloning of 35S with 5’ NCR of SCMoV...... 160 6.9.2 Assembly and ligation cloning of plasmid pFLREPsgprogfp and 35S and T-Nos hybrids ...... 160 6.10 Overlapping clones for development of ORF1 mutants of pFL ...... 161 6.11 Biological activity of in vivo RNA transcripts of full-length cDNA of SCMoV-pFL ...... 162 6.11.1 Host plants used and plant infection...... 162 6.11.2 Detection of in vivo-transcribed RNA transcripts ...... 162 6.12 Discussion ...... 163 6.12.1 Summary of clones developed in chapter 6...... 163 6.12.2 Particle morphology and biochemical properties of SCMoV-pFL...... 163 6.12.3 Amplification and cloning of full-length cDNA of SCMoV-pFL...... 164 6.12.4 Technical issues concerning the development of SCMoV-based cDNA constructs ...... 165 6.12.5 Stability of clones in E. coli ...... 165 6.12.6 Activity of in vivo-transcribed RNA transcripts from pTEVFL...... 166 6.13 Conclusion and further work...... 167
CHAPTER SEVEN: GENERAL DISCUSSION AND CONCLUSION ...... 169
7.0 Review of aim and objectives of the project ...... 170 7.1 The extent to which the aims and objectives were achieved ...... 170 7.1.1 New information on hosts of SCMoV...... 170 7.1.2 Detailed characterisation of SCMoV ...... 173 7.1.2.1 Genetic diversity and biology of SCMoV ...... 173 7.1.2.2 Genome structure of SCMoV...... 175 7.1.2.3 Limited cell-to-cell movement of the P1 protein and CP of SCMoV...... 176 7.1.2.4 Development of gene vector constructs...... 177 7.1.3 Expression of infectious SCMoV cDNA and GFP chimeric derivatives ...... 179 7.2 Further work...... 180 7.3 SCMoV as a VIGS vector for functional genomics research...... 182 7.4 Conclusion ...... 183
Bibliography...... 184
xii
Acknowlegements
Acknowlegements
Most important on my list of appreciation is the Almighty God for the gift of life. When it seemed like the whole project was a mountain, He gave me strength enough to carry on.
I would like to say “thank you” to all my supervisors who helped me throughout the preparation of this thesis. First, to Prof. Michael G. K. Jones, my principal supervisor, I am highly indebted for the support, encouragement and careful reading and correction of the manuscript. It was certainly an amazing experience working with Dr. Roger A. C. Jones, Head of Plant Pathology Section, Department of Agriculture Western Australia, for his readiness to share his writing skills and vast experience and knowledge of viruses. His excellent supervision and involvement in the work presented in Chapter 3 of this thesis could not produce a more successful result. I am also very grateful to Dr Geoffrey I. Dwyer for his help in making my life in Australia less stressful, introducing me to several techniques in Molecular
Biology, for his close supervision, regular advice and his friendship. There are not enough words to convey my appreciation.
To members of the elite Plant Biotechnology Research Group (PBRG) at the WA State
Agricultural Biotechnology Centre (SABC); Drs. Steve Wylie, Modika Perera, Zhaohui Wang, and Ms. Angela Hollams, Dora Li, John Blinco, Peiling Tan, Hui Phing Loo, Dale Whitney,
Kanokwan Ratanasooban, I say thanks for the helpful scientific and non-scientific discussions, and their readiness to provide any material and help I needed to complete my work. It meant a lot to me. Special mention is due Dr. Steve Wylie for his readiness to share his knowledge and
Dr. Modika Perera for her friendship.
To Shawn Seet, Tobias Schoep, Andrea Pizzirani, Carly Palmer, Sharon Wescott, David Dunn,
Suren Suredurai, Andrew Low, Tom La, I say thanks for being the friends they are, the helpful discussions and social gatherings, and the technical help they gave all in the spirit of comradeship and excellence were appreciated.
xiii
Acknowlegements
The spiritual support given me by Full Gospel Assembly, Perth was enough to take me through the dark times. I cannot be more thankful to my fellow cell members and Carolyn Mardell,
James Soon, Justin Goh and Jimmy Teo for their constant concern and encouragement when it was most needed. Special mention of Jocelyn Low, my cell leader, and Mr. Hui Chau Wong is justified for their special friendship, care and concern throughout the period of my project.
Thanks are also due the Grains Reasearch Development Council (GRDC), Division of Science and Engineering (DSE), Murdoch University, Prof. Mike Jones and Dr. Geoff Dwyer for funds to attend conferences and workshops: the 7th International Congress on Plant Molecular
Biology, Barcelona, Spain, in June, 2003; study tour of the Sainsbury Laboratory and the John
Innes Centre at the Norwich Research Park, UK and the Scottish Crop Research Institute,
Dundee, Scotland, in June/July, 2003; First Medicago truncatula Workshop, Rottnest Island,
Perth, Western Australia, in November 2002; the 12th Australasian Plant Breeding Conference,
Perth, Western Australia, in September 2002, and the 11th Combined Biological Sciences
Meeting, Perth, Western Australia, in August, 2000.
It was indeed a blessing to be granted an International Postgraduate Research Scholarship by
Murdoch University and a Completion Scholarship by DSE and to work with state-of-the-art facilities at the SABC and with the staff, Prof. Mike Jones, David Berryman, Andrea Tongue and Frances Brigg.
Last but certainly not the least, had it not been the support, care, concern, love at home not to mention the actual involvement (typing and proofreading of the manuscript) by Monica, my dear wife, I would not have pulled through to the end of the project. I am forever indebted to her.
xiv
Dedication
Dedicated to
The One I Loved And Wished I Could Know More, My Dear Sister, Abigail Serwaa Nyarko, 7th September, 1985 – 19th March, 2001.
xv
Presentations
Publications and Presentations
Refeered Publications Jones, R. A. C., Fosu-Nyarko, J., Jones, M. G. K., and Dwyer, G. I. (2001). Subterranean clover mottle virus. In “AAB Descriptions of Plant Viruses No. 387. Scottish Crop Research Institute, Invergowrie, Dundee, Scotland” (Eds. T. Jones and D. Robinson)
Fosu-Nyarko, J., Jones, R. A. C., Smith, L. J., Jones, M. G. K., and Dwyer, G. I. (2002a). Host range and symptomatology of subterranean clover mottle sobemovirus in alternative pasture, forage and crop legumes. Australasian Plant Pathology 31, 345-350.
Fosu-Nyarko, J., Jones, R. A. C., Smith, L. J., Jones, M. G. K., and Dwyer, G. I. (2002b). Responses of alternative annual pasture and forage legumes to challenge with infectious subterranean clover mottle virus. In Crop Updates: Farming systems, Department of Agriculture - Western Australia, July, 2002.
Fosu-Nyarko, J., Jones, R. A. C., Smith, L. J., Jones, M. G. K., and Dwyer, G. I. (2002c). Testing for disease vulnerability in alternative pasture and forage legumes being assessed in plant improvement programmes. In “Proceedings of the 12th Australasian Plant Breeding Conference - Plant Breeding for the 11th Millennium” (Ed. J. A. McComb)(Australasian Plant Breeding Assoc. Inc., Perth, W. Australia, 15-20 th September 2002.), pp. 382-384.
Dwyer, G. I., Njeru, R., Williamson, S., Fosu-Nyarko, J., Hopkins, R., Jones, R. A. C., Waterhouse, P. M., and Jones, M. G. K. (2003). The complete nucleotide sequence of subterranean clover mottle virus (SCMoV). Archives of Virology 148: 2237–2247.
Fosu-Nyarko, J., Jones, R. A. C.,Dwyer, G. I., and Jones, M. G. K. (2004). Genetic Diversity and functional constraints of genes of Subterranean clover mottle Sobemovirus. In preparation
Poster Presentations Fosu-Nyarko, J., Dwyer, G. I., and Jones, M. G. K. (2000). Host range and gene expression studies of Subterranean clover mottle virus (SCMoV). In “11th Annual Combined Biological Sciences Meeting”, Perth, Western Australia., pp 128.
Fosu-Nyarko, J., Jones, R. A. C., Jones, M. G. K. and Dwyer, G. I. (2002d). Development of a legume-specific gene silencing vector for functional analysis of plant genes. In “Proceedings of the First Australian Medicago truncatula Workshop” Rottnest Island, Perth, Western Australia, 10-13th. November 2002). pp 130
Fosu-Nyarko, J., Jones, R. A. C., Jones, M. G. K. and Dwyer, G. I. (2003). Potential of Subterranean clover mottle virus as a legume-specific gene silencing vector for functional analysis of plant genes. In “Gene Silencing and Epigenetics, Proceedings of the 7th International Congress of Plant Molecular Biology, Barcelona, Spain, June 23- 28, 2003, pp 398.
Submitted Nucleic Acid Sequences
Fosu-Nyarko, J., Jones, R. A. C., Dwyer, G.I. and Jones, M. G. K. (2003). Complete genomic sequence of four Subterranean clover mottle Sobemovirus isolates: SCMoV-AL (Genbank Accession No. AY376451); SCMoV-MB (Genbank Accession No. AY376452); SCMoV- MJ (Genbank Accession No. AY376453) and SCMoV-pFL (Genbank Accession No. AY376454).
xvi
List of Abbreviations
List of frequently used abbreviations
3’ hydroxyl terminus of DNA molecule 35S 35S RNA transcriptional promoter of CaMV 5’ phosphate terminus of DNA molecule bp base pair cDNA complementary DNA CIP Calf intestinal alkaline phosphatase CP coat protein C-terminus carboxy terminus cv cultivar DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate dsRNA double stranded RNA E. coli Eschericia coli EDTA ethylenediaminetetra-acetate acid disodium salt ELISA Enzyme-Linked Immunisorbent Assay GFP green fluorescent protein gRNA genomic RNA GUS β-glucoronidase gene kb kilobases Mar average calculated molecular mass MCR multiple cloning region MP movement protein NCR non-coding region NES Nuclear export signal N-terminus amino terminus ORF open reading frame PCR Polymerase chain reaction PDS Phytoene desaturase PTGS Post-transcriptional gene silencing RdRp RNA dependent RNA polymerase RNA ribonucleic acid RNAi RNA interference RNase ribonuclease RT reverse transcription SatRNA satellite RNA SEL size exclusion limit sgPro subgenomic promoter sgRNA subgenomic RNA siRNA short interfering RNA TAE Tris-acetate-EDTA TE Tris-EDTA T-Nos Nopaline synthase gene transcription terminator signal U Unit VIGS Virus-induced gene silencing Vol volumes vRNA viral RNA
Viruses and scientific names ACMV African cassava mosaic virus AMV Alfalfa mosaic virus BaMV Bamboo mosiac virus BDMV Bean dwarf mosaic virus BMV Brome mosaic virus
xvii
List of Abbreviations
BNYVV Beet necrotic yellow vein virus BSMV Barley stripe mosaic virus BSSV Blueberry shoestring virus BYDV Barley yellow dwarf virus BYDV-PAV Barley yellow dwarf virus-PAV BYMV Beet yellow mosaic virus BYV Beet yellows virus CaMV Cauliflower mosaic virus CCMV Cowpea chlorotic mottle virus CbLCV Cabbage leaf curl virus virus CfMV Cocksfoot mottle virus CLBV Citrus leaf blotch virus CLCuV Cotton leaf curl virus CMV Cucumber mosaic virus CPMV Cowpea mosaic virus CTV Citrus tristeza virus CymRSV Cymbidium ringspot virus CYVV Clover yellow vein virus LTSV Lucerne transient streak virus MoMLV Moloney murine leukaemia virus MuLV Murine Leukaemia virus ORSV Odontoglossum ringspot virus PLRV Potato leaf roll virus PMMoV Pepper mild mottle virus virus PSbMV Pea seedborne mosaic virus PVX Potato virus X PVY Potato virus Y RCNMV Red clover necrotic mosaic virus RGMoV Ryegrass mottle virus RYMV Rice yellow mottle virus SBMV Southern bean mosaic virus SCMoV Subterranean clover mottle virus SCPMV Southern cowpea mosaic virus SCRLV Subterranean clover red leaf virus SeMV Sesbania mosaic virus SHMV Sunn-hemp mosaic virus SNMV Solanum nodiflorum mottle virus SoMV Sowbane mosaic virus STNV Tobacco necrosis satellite virus TAV Tomato aspermy virus TBSV Tomato bushy stunt virus TCV Turnip crinkle virus virus TEV Tobacco etch virus virus TGMV Tomato golden mosaic virus TLCV Tobacco leaf curl Virus TMGMV Tobacco mild green mosaic virus TMV Tobacco mosaic virus TNV Tobacco necrosis virus ToMV Tomato mosaic virus TRoV Turnip rosette Virus TRV Tobacco rattle virus TYMV Turnip yellow mosaic virus VTMoV Velvet tobacco mottle virus
xviii
Chapter One
General Introduction and Literature review
1. General Introduction and Literature review
1.0 General Introduction
Viruses are classified into a hierarchical taxa according to a number of criteria: whether the viral genome consists of DNA or RNA, and whether it is single- or double-stranded; the existence and nature of RNA intermediates that are required for viral replication; the variety of proteins that house or otherwise associate with the virion genome and whether the protein coat is in turn surrounded by a membrane; virion size and host range. RNA viruses make up nearly
93.9% of plant viruses of which over 75 % are positive sense (Matthews, 1991).
Since the discovery of Tobacco mosaic virus (TMV), viruses have been studied intensively as agents for disease induction in plants. They have contributed to the study of basic concepts in molecular biology; e.g. the mechanism of DNA replication, the genetic code, RNA as a genetic material and mechanisms of protein-protein and protein-nucleic acid interactions. As models for mechanisms of molecular evolution, plant virus populations have also been used to understand pathogen-host interactions, the diversity in these species being critical in their rapid adaptation. Even the current era of genomics is not new to the study of viruses following the first complete sequencing of a virus genome 20 years ago.
Viruses have also been used for the development of essential tools for biotechnology. Presently most vectors for molecular cloning contain viral components, and heterologous genes expression systems are often dependent on viral promoters such as the 35S RNA transcriptional promoter from Cauliflower mosaic virus (CaMV). New and improved viral vectors have been used as preliminary tests for expressing genes of interest in transgenic plants, or as a means of expression in themselves where almost every interaction with their hosts has been studied.
Currently, virologists have moved another step towards using viral vectors as triggers of the hosts’ defense system to study gene function by silencing. This project is designed to contribute to the understanding of gene silencing induced by viruses and the immense potential of this technology to plant biology.
2
1. General Introduction and Literature review
1.1 Subterranean clover mottle virus (SCMoV)
Subterranean clover mottle virus (SCMoV) is the most economically important of all viruses infecting pasture in Australia (Jones, 1996). It was first identified infecting plots of subterranean clover cv Dinninup at Karridale in 1979 in Western Australia (WA) (Francki et al., 1983). Infection is now found in T. subterraneum-based pastures in five Australian states,
WA, Tasmania, South Australia, New South Wales and Victoria (Helms et al., 1993). The virus makes a substantial contribution to reduced productivity of pastures containing subterranean clover (Wayne, 1992). Levels of infection as high as 97% have been reported
(Helms et al., 1993; Ferris and Jones, 1995). Infection is more widespread in old pastures
(70% infection) than in new ones (56%). Infection is higher in grazed (64% and 94%) than in ungrazed (20% and 36%) pastures (Wroth and Jones, 1992b).
1.1.1 Symptoms
In a subterranean clover pasture field, SCMoV-infected plants can be distinguished by their patchy distribution in early stages of an epidemic and often distinctive leaf markings (Wayne,
1992). Symptoms in subterranean clover appear 2-3 weeks post-infection. Initial infection signs include vein clearing or yellowing around minor veins of mostly newly expanded leaves after systemic movement of virus particles. This is followed by leaf mottling and deformation and an eventual dwarfing of plants. Stem thickening is sometimes observed and in winter when plant growth is reduced red pigmentation may appear on leaves of infected plants of some cultivars (Wroth and Jones, 1992a). The virus rarely kills its host, however, very severely infected plants fail to flower (Wayne, 1992; Francki et al., 1983). Systemic symptoms in other
Trifolium species are similar to those in subterranean clover but with varied severity (Wroth and Jones, 1992a). Symptoms in other plants include brown local lesions in inoculated leaves of field pea (Pisum sativum L cv Greenfeast), and appearance of small grey lesions in
Chenopodium murale L (Francki et al., 1983).
3
1. General Introduction and Literature review
1.1.2 Isolates
Since the isolation of the type strain of SCMoV in WA, several isolates have been obtained from all states and territories in Australia except Queensland and the Northern Territory. These induce mild to very severe symptoms typical of SCMoV infection on subterranean clover
(Table 1.1) (Wroth and Jones, 1992b; Francki et al., 1983; Ferris and Jones, 1995). The first survey to study isolate variability of SCMoV in WA was conducted by Wroth and Jones
(1992b) using pastures on five different farms which had had SCMoV infection levels of 55-
97%. They characterised eight isolates by studying severity and forms of symptoms they induced. Four other isolates with known levels of symptom severity were used as references; the type isolate (originally from WA), two from Tasmania and one from New South Wales.
The isolates caused symptoms with different levels of severity in subterranean clover and were classified as mild, moderate, severe or very severe. However, the level of symptom severity did not reflect levels of infection. Based on this classification, the four frequently cultured WA isolates; SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV-P23 fall into three severity groups (Table 1.1). The cause(s) of the different symptom severity induced by the isolates is/are not known. Wayne (1992) however, alluded that the presence or absence of satellite
RNAs could alter the severity of symptoms induced by an isolate in infected plants.
Table 1.1: Isolates of SCMoV, their origin and symptom severity status
Isolate Source Severity AL North Dandalup, WA Very severe F4 Donnybrook, WA Mild F44 Donnybrook, WA Moderate F45 Donnybrook, WA Severe M36 Kirup, WA Severe M38 Kirup, WA Moderate MB Mt. Barker, WA Moderate MJ Manjimup, WA Very severe NSW New South Wales Moderate P23 Manjimup, WA Severe R17 Wilgarup, WA Severe T11 Donnybrook, WA Moderate TAS16 Tasmania Very severe TAS19 Tasmania Mild Type Mayanup, WA Very severe
References: Wroth and Jones (1992b); Francki et al.,(1983); Ferris and Jones, (1995)
4
1. General Introduction and Literature review
1.1.3 Transmission
SCMoV is highly infectious and reaches high titres in its hosts (Wroth and Jones, 1992a). The virus is stable in sap and is readily transmitted mechanically by sap inoculation, by livestock during grazing and also through mowing (Johnstone and McLean, 1987; Ferris et al., 1996).
The virus is also transmitted through seed and by graft-inoculation (Njeru et al., 1995).
Depending on the mode and purpose for testing, seed transmission rates of 0.1 to 3% have been recorded for SCMoV (Njeru et al., 1997; Francki et al., 1988). Tests for non-persistent and persistent transmission by Myzus persicae were unsuccessful (Wroth and Jones, 1992a).
Transmission by lucerne fleas (Sminthanus viridis), aphids (Acyrthosiphon kondoi) and mites
(Halotydeus destructor), though not experimentally tested, has been discounted based on their presence on both test and experimental swards without significant contribution to infection
(Wroth and Jones, 1992a). Sehgal (1999) records beetles as a vector of SCMoV in the
Encyclopedia of Virology, however, no experimental reference supports this.
1.1.4 Host range and response to infection
SCMoV has a narrow host range restricted to the family Fabaceae (Wroth and Jones, 1992a).
The natural host of SCMoV is subterranean clover (Trifolium subterraneum L) on which it induces almost all the symptoms outlined above (Wroth, 1992a, 1992b; Francki et al., 1983).
Genotypes of clovers exhibit a range of responses to SCMoV infection from susceptible to highly resistant (Njeru et al., 1995; 1997; Ferris et al., 1996). Resistance of clovers to SCMoV is not expressed at the single cell level as an inhibition of replication but as a result of restricted cell-to-cell movement of the virus (Njeru et al., 1995). Pastures susceptible to SCMoV include some Medicago spp (Wroth and Jones, 1992a). SCMoV also infects field pea locally and C. murale systemically (Francki et al., 1983). Experimentally determined non-hosts of SCMoV include species of Chenopodium and Nicotiana (Francki et al., 1983). The virus was first isolated and characterised by Francki et al., (1983) who supported the earlier proposal of Hull
(1977) to group viruses with similar characteristics to southern bean mosaic virus into a new taxonomic group, presently the genus Sobemovirus.
5
1. General Introduction and Literature review
1.2 Sobemoviruses
1.2.1 Definitive species and geographical distribution
Sobemoviruses are a group of plant viruses with positive-sense, single-stranded RNA (ssRNA) genomes. Named after the type member Southern bean mosaic virus (SBMV), the genus is not assigned to a family (Hull, 1977; Hull et al., 1999) and presently comprises 12 other definitive species (Table 1.2). These are Southern cowpea mosaic virus (SCPMV), Blueberry shoestring virus (BSSV), Cocksfoot mottle virus (CfMV), Lucerne transient streak virus (LTSV),
Ryegrass mottle virus (RGMoV), Rice yellow mottle virus (RYMV) and Solanum nodiflorum mottle virus (SNMV), Sesbania mosaic virus (SeMV), Sowbane mosaic virus (SoMV),
Subterranean clover mottle virus (SCMoV), Turnip rosette virus (TRoV) and Velvet tobacco mottle virus (VTMoV).
Most sobemoviruses have a limited distribution, but as a group, they are found throughout the world (Table 1.2). SBMV and SCPMV have been identified on all continents except Australia.
CfMV has so far only been found in Asia, Europe and New Zealand (Mohamed, 1980;
Mäkinen et al., 1995a; Campbell and Guy, 2001). Some members have been reported only in either a single country or a continent probably because of limited research in this area, for example, SCMoV in Australia (Francki et al., 1983) and SeMV in India (Lokesh et al., 2001).
RYMV exists and spreads in many western and eastern Africa countries (Bakker, 1974;
Bonneau et al., 1998).
1.2.2 Biology
1.2.2.1 Strains and isolates
Reported strains and isolates of sobemoviruses differ in their host range and symptomatology as well as their distribution (Table 1.2). For example, there are four reported strains of LTSV; the Australian (LTSV-Au), South Australian (LTSV-SA), Canadian (LTSV-Ca) and New
Zealand (LTSV-NZ) strains. These have different symptom development in the common host, lucerne and different host ranges (Blackstock, 1978; Forster and Jones, 1979; Paliwal, 1983,
1984; Dall et al., 1990). In addition, they differ in physical and chemical properties. Resistant-
6
1. General Introduction and Literature review
breaking isolates of SBMV and RYMV have also been reported after successive passage of the
viruses through their hosts (Lee and Anderson, 1998; Fargette et al., 2002). SBMV-S, for
instance, is a resistant breaking mutant of SBMV-Ark after 24 successive passages through the
latter’s permissive and restrictive hosts. The only difference in the Ghana (SBMV-GH) and
Mexican (SBMV-M) strains of SBMV is that of symptom development in the host, common
bean (Othman and Hull, 1995). Two isolates of RYMV; RYMV-CI from Cote d’Ivoire and
RYMV-NG from Nigeria differ in their nucleotide sequences. Also five major strains of
RYMV have been differentiated from a pool of forty isolates collected from nine African
countries (Pinel et al., 2000). Phylogeographic relationship between sequences of the CP gene
and the 3’ NCR of these isolates has been associated with pathogenicity (Zakia et al., 2003).
Isolates of SCMoV producing different levels of symptom severities in subterranean clover has
also been reported (see section 1.1.2). Three reported geographically distinct strains of CfMV
from Japan (CfMV-JP), Norway (CfMV-NO) and Russia (CfMV-RU) are known to differ only
in their genomic sequences.
Table 1.2: Definitive species of sobemoviruses, their natural hosts, geographical distribution and mode of transmission
Virus species Geographical distribution Natural hosts Mode of transmission Mechanical Seed Vector SBMV USA, Central and South America Phaseolus vulgaris Yes Yes Beetles (Mexico), Africa (Ghana), Asia SCPMV USA, Central and South America, Vigna unguiculata Yes Yes Beetles Africa, Asia BSSV USA, Canada Vaccinium corymbosum, V. angustifolium Yes No Aphids CfMV Europe (Norway, Russia), New Dactylis glomerata, Triticum aestivum Yes No Beetles Zealand, Asia LTSV New Zealand, Australia, Canada Medicago sativa Yes No ND RYMV Tropical Africa (Kenya, Nigeria, Oryza sativa, Oryza longistaminata Yes No Beetles Cote d'Ivoire) SNMV Australia Solanum nodiflorum, S. nitidibaccatum, ND No Beetles S. nigrum SoMV USA, Europe, Asia, Chenopodium spp; C. murale, Vitis sp., Yes Yes Controv- Central & South America Prunus domestica, Atriplex suberecta,. ersial SCMoV Australia Trifolium subterraneum Yes Yes ND TRoV United Kingdom Brassica campestris ssp. napus, Brassica Yes ND Beetles campestris ssp. rapa VTMo Australia Nicotiana velutina Yes No Mirids V SeMV India Sesbania grandiflora ND ND ND ND-Not determined
7
1. General Introduction and Literature review
1.2.2.2 Host range
The natural hosts of sobemoviruses are listed in Table 1.2. As a group, sobemoviruses infect diverse plant species. Individual viruses, however, have a restricted host range; infecting only a few species in one or two, rarely more than three, plant families. For example, the host range of SBMV, SCMPV and SCMoV are restricted to a few species in the family Fabaceae. CfMV is restricted to the family Poaceae, whereas VToMV is confined to the family Solanaceae.
SNMV, LTSV and TRoV infect a few species in 3-4 different families. Common susceptible plant families include the Fabaceae (Caesalpinioideae and Papilionoideae), Amaranthaceae,
Chenopodiaceae, Asteraceae and Poaceae. Some of the families include species that are common hosts of most sobemoviruses, e.g. Chenopodiaceae.
1.2.2.3 Symptoms and cellular localisation
Symptoms induced by sobemoviruses vary in form and severity depending on the age and species of the plant host, the season of the year, and the strain or isolate of the virus involved.
Sobemoviruses generally induce mottling and mosaic patterns on infected leaves yellowing/chlorosis, deformation/distortion and streaks and rarely kill the hosts. Symptoms persist in susceptible hosts with the exception of those of LTSV, which disappear soon after infection or as the plant ages, hence the name "transient". In addition, symptoms of BSSV vary seasonally (Ramsdell, 1979). BSSV causes shoestring symptoms in which infected leaves are transformed into curled, strap-like structures. In addition, there is "breaking" of flowers and immature berries show reddish streaking or vein banding patterns in BSSV-infected plants
(Ramsdell, 1979). Unusually, infection by CfMV or TRoV occasionally causes lethal necrosis of infected leaves (Rognli et al., 1995; Brunt et al., 1996). Early infection of susceptible rice varieties by RYMV in some cases results in plant death (Bakker, 1974).
Upon infection, particles of sobemoviruses are present in most plant tissues including the epidermis, mesophyll, meristem and the vascular bundles (Hartmann et al., 1973; Francki et al.,
1983; Brunt et al., 1996; Opalka et al., 1998). They occur in the cytoplasm and vacuoles and in the nuclei (with the exception of BSSV, RYMV and CfMV which have not been reported), but
8
1. General Introduction and Literature review have not been found so far in the mitochondria and chloroplasts. Inclusions found in infected cells are either present as crystalline aggregates, as in SoMV and SBMV, or as discrete membraneous bodies as in VTMoV. Other cellular changes associated with infection include proliferation of tonoplasts with concentric membranes bulging into the vacuole and the presence of rod-like materials in pallisade cells of infected tissues (Hartmann et al., 1973).
1.2.2.4 Transmission
Sobemoviruses are highly infectious and reach high concentrations in their hosts. Their virions are highly stable in sap. Consequently, they are readily transmitted mechanically by sap inoculation and via contact through leaf abrasions (Table 1.2). Guttation fluid exuding from
RYMV infected plants is a source of inoculum for subsequent infection and possibly through irrigation water (Abo et al., 1998). Sap inoculation with LTSV is, however, difficult unless a purified virus preparation is used (Blackstock, 1978; Forster and Jones, 1979). All sobemoviruses, except three, are transmitted by insect vectors of the orders Aphididae,
Chrysomellidae, Coccinellidae or Miridae, either semi-persistently or non-persistently (Table
1.2) (Hollings and Stone, 1973; Greber, 1981; Mohamed et al., 1981; Tremaine and Hamilton,
1983). There are conflicting reports as to whether SoMV is vector-transmitted non-persistently by insects or not at all (Kado, 1967, 1971). Seed transmission does not seem to be a common mode of sobemoviral transmission. SBMV, SCPMV, SCMoV and SoMV are the only sobemoviruses shown to be graft- and seed-transmissible and with the exception of SCMoV, they are transmitted from pollen to seed and to pollinated plants (Brunt et al., 1996).
1.2.3 Structure
1.2.3.1 Morphology
Sobemoviruses have uniform isometric particles with a diameter in the range of 25-30 nm (Hull et al., 1999). The virions contain a single CP of about 30 kDa. The structure of the capsid and subunits of some sobemoviruses (e.g. SBMV, SCPMV, SeMV and RYMV) are well known at several resolutions (Abad-Zapatero et al., 1980; Erickson et al., 1985; Subramanya et al., 1993;
Bhuvaneshwari et al., 1995; Murthy et al., 1997; Opalka et al., 2000; Qu et al., 2000). The
9
1. General Introduction and Literature review capsid shell is composed of 80 subunits arranged in a T = 3 icosahedral symmetry lattice stabilised by divalent cations: Ca2+ and Mg2+ (Qu et al., 2000). This organisation gives it a compact nature that excludes negative stains from penetrating the core. The subunits adopt a variation of the canonical eight-stranded jelly-roll β-sandwich fold which gives it enhanced stability. This has been implicated to enhance systemic movement of RYMV particles in its host (Opalka et al., 2000; Qu et al., 2000).
Virions of all sobemoviruses encapsidate a genomic RNA (gRNA). A subgenomic RNA
(sgRNA) molecule, 3’ co-terminal to the gRNA, has been reported for SBMV, SCPMV,
RYMV and CfMV (Rutgers et al., 1980; Weber and Sehgal, 1982; Mäkinen et al., 1995b;
Ryabov et al., 1996; Bonneau et al., 1998; Tamm et al., 1999). The gRNA is a single-stranded
6 messenger-sense molecule with a molecular mass (Mr) of about 1.4 x 10 whereas the Mr of sgRNAs range from 0.3 x 106 - 0.4 x 106. Both RNA molecules are uncapped, have a genome- linked viral protein (VPg) covalently linked to the 5’ terminal end and have no poly (A) tail.
There are no conserved 3’ terminal structures at the termini of either RNA molecule (Ghosh et al., 1981). CfMV is the only sobemovirus reported to encapsidate defective interfering RNAs
(Makinen et al., 2000a). Infection by sobemoviruses is associated with viroid-like satellite
RNAs (satRNAs). These exhibit differential replication in helper sobemoviruses and host plants (Davies et al., 1990; Gould and Hatta, 1981; Randles et al., 1981; Tien-Po et al., 1981;
Francki et al., 1983; 1986; Jones and Mayo, 1983; 1994; Sehgal et al., 1993; Collins et al.,
1998).
1.2.3.2 Genome organisation
The gRNAs of nine sobemoviruses have been completely sequenced (Table 1.3). They are among the smallest plant RNA viruses with genome lengths of less than 4500 nucleotides (nt). The sobemovirus genome is compact and regularly contains four open reading frames (ORFs). The
ORFs are flanked at the 5’ and 3’ termini by varying lengths of non-coding regions (NCR). There are two major types of genome organisation for sobemoviruses; the SCPMV-like and the CfMV- like (Fig. 1.1). In the SCPMV-like organisation, there is a small 5’ proximal ORF1, a long
10
1. General Introduction and Literature review continuous ORF2, a small internal ORF3 situated in the -1 frame of ORF2 and a 3’ terminal
ORF4. Sobemoviruses with this organisation include SCPMV, SBMV, LTSV, SeMV, RYMV and RGMoV. The ORF1 and ORF2 of RYMV and RGMoV do not overlap (Fig. 1.1). The
CfMV-like genome organisation shared by CfMV, SCMoV-P23 and TRoV is characterised by the presence of the small ORF1, two shorter overlapping ORFs, 2a and 2b, instead of the long continuous ORF2, and a 3’ terminal ORF3. Parts of the CfMV-like organisation is similar to that reported for some poleroviruses and enamoviruses (van der Wilk et al., 1989; Demler and de Zoeten, 1991).
Table 1.3: Sobemoviruses/strains with their nucleotide sequences completely determined
Sobemovirus GenBank Length of References Accession No gRNA(bp) CfMV-JP AB040447 4083 Zhang and Toriyama, unpublished CfMV-RU L40905 4083 Ryabov et al., 1996 CfMV-NO Z48630 4082 Mäkinen et al., 1995b LTSV U31286 4275 Jeffries et al., unpublished RYMV-CI L20893 4450 Yassi et al., 1994 RYMV-NG U23142 4451 Pinto and Baulcombe, unpublished RGMoV AB040446 4210 Zhang and Ma, 2001 SeMV AY004291 4149 Lokesh et al., 2001 SBMV-ARK AF055887 4136 Lee and Anderson, 1998 SBMV-S AF055888 4136 Lee and Anderson, 1998 SBMV-B L34672 4109 Othman and Hull, 1995 SCPMV M23021 4194 Wu et al., 1987 SCMoV-P23 AF208001 4258 Dwyer et al., 2003 TRoV AY177608 4037 Callaway et al., unpublished
SCPMV-like genome organisation (SCPMV, SBMV, LTSV, SeMV, RYMV and RGMoV)
ORF3 ORF4 5’ VPg ORF1 Pro RdRp 3’ P1 ORF2 CP
CfMV-like genome organisation (CfMV, SCMoV, TroV)
ORF2b ORF2a RdRp ORF3 3’ 5’ VPg ORF1 Pro CP P1
Fig. 1.1: Genome organisation of sobemoviruses. ORFs are represented by hollow boxes and the VPg is shown as a small circle at the 5’ terminus of the gRNA. Approximate locations of the putative protease (Pro) and putative RdRp domains are represented as bricks and checkerboards respectively. Sites of -1 ribosomal frameshift consensus signals are indicated by light horizontal lines. P1 is the ORF1-encoded protein; CP, coat protein.
11
1. General Introduction and Literature review
1.2.4 Gene expression
Translation of ORF1 and ORF2 from the gRNA demonstrated for SCPMV (Sivakumaran and
Hacker, 1998) and CfMV-NO (Tamm et al., 1999) show that the gRNA functions as a bicistronic mRNA. The results also indicate translation initiation of the two ORFs occur at their respective first AUG codons, though the initiation codon of ORF1 is surrounded by a poor context sequence for translation by plant ribosomes (Lütcke et al., 1987; Cavener and Ray,
1991). Experimental evidence from SCPMV and SBMV-GH show the ORF2, and possibly
ORF2a of CfMV, SCMoV and TroV, is translated by leaky scanning rather than by internal ribosome binding (Wilson, 1985; Sivakumaran and Hacker, 1998). The ORF2b of CfMV is expressed via -1 frame-shifting of ORF2a (Mäkinen et al., 1995a; Tamm et al., 1999). It is expected that a similar expression strategy is used by TRoV and SCMoV-P23. This is supported by the presence of ribosomal frameshifting signals; a heptanucleotide slippery sequence (TTTAAAC) and a predicted stem loop downstream of the heptamer. The signals are also present upstream of the first AUG of ORF3 of SCPMV, SBMV-Ark, SeMV, RYMV-CI, and LTSV. There are no stop codons present between the slippery sequence and the AUG of
ORF3 in either reading frame further supporting the assumption that ORF3 is also likely to be translated via -1 ribosomal frameshifting mechanism. The CP of sobemoviruses has been shown by direct sequencing to be translated via sgRNA synthesis from the first AUG of the 3’ terminal ORF (Mäkinen et al., 1995a).
1.2.5 Gene products
Sobemoviruses characterised so far encode four major proteins; a P1 protein encoded by ORF1, the CP encoded by the terminal ORFs 3/4, a polyprotein encoded by ORF2 (for SCPMV-like viruses) or by ORF2a and 2b (for CfMV-like viruses), a protein product of ORF2a or a trans- frame fusion product for ORF2-3 (Table 1.4). The P1 proteins have molecular masses between
12 and 28 kDa. The slightly different molecular masses observed in vitro and in vivo for
RYMV was suggested to result from degradation, post-transcriptional modifications or from structure or other characteristics of P1 (Yassi et al., 1994; Bonneau et al., 1998). The
12
1. General Introduction and Literature review sobemoviral polyprotein is larger than the other encoded proteins. For SBMV, SBMV-Ark,
SCPMV, RYMV-CI, and LTSV, the approximately 100 kDa polyprotein is synthesised from the continuous single ORF2. In the case of CfMV, however, it is translated by ORF2a and
ORF2b via -1 ribosomal frameshifting mechanism (Mäkinen et al., 1995a). A 71 kDa translation product is produced from the ORF2a of CfMV whereas in SCPMV a similar product thought to be a proteolytic product of the polyprotein encoded by the ORF2 (Wu et al., 1987), has been suggested by Tamm et al., (1999) to represent an ORF2-ORF3 trans-frame fusion protein. In vitro translational studies with SBMV, SCPMV, RYMV-CI, and CfMV-NO show the approximately 30 kDa protein is the viral CP and is translated from the 3’ terminal ORF
(Table 1. 4).
Table 1.4: Size of in vitro translation products of some sobemoviruses (kDa)
Virus Putative Unknown Coat Poly- References ORF1 protein protein product CfMV 12 71 34 100 Mäkinen et al., 1995b; Tamm et al., 1999 LTSV 18 78 33 105 Morris-Krsinich and Forster, 1983 SBMV 14 75 29 105 Salerno-Rife et al., 1980; Mang et al., 1982 SCPMV 20 70 30 100 Mang et al., 1982 SNMoV 28 67 38 100 Kiberstis and Zimmern, 1984 TRoV 67 30 105 Morris-Krsinich and Hull, 1981 RYMV 18 or 19 Bonneau et al., 1998
1.2.6 Sequence comparison
1.2.6.1 Sequences of non-coding regions
The genes of sobemoviruses are flanked at both termini by variable lengths of NCRs.
Sequences of the 3’ NCR vary from 127 nt in LTSV to 284 nt in RYMV-NG and are generally longer than those of the 5’ NCR which range from 48 nt in SCPMV to 99 nt in RgMV. The 5’
NCRs are more similar to each other than those of the 3’ NCR. Multiple sequence analysis reveals no clear conserved sequences in the NCRs. Viruses with similar translation mechanisms have near identity or conserved sequences at the 5’ and 3’ termini of their gRNA
(Ahlquist et al., 1981; Dams et al., 1988; Danthine et al., 1993) or have functional substitutes
13
1. General Introduction and Literature review to ensure efficient translation of their genes through synergistic interaction (Carrington and
Freed, 1990; Leathers et al., 1993; Sarnow, 1995). However, the absence of specialised structures, the low degree of nucleotide identity and lack of consensus in the NCRs of sobemoviruses suggest the individual viruses may use different mechanisms for gene expression (Gallie, 1990; Tarun and Sachs, 1995). The accumulation of sobemoviruses to remarkably high titres in their hosts suggests they contain elements for efficient gene expression. This was demonstrated by the ability of the 5’ NCR of CfMV to enhance heterologous gene expression in both dicotyledonous (tobacco) and monocotyledonous (barley) plants (Mäkeläinen et al., 2003).
1.2.6.2 Sequences of coding regions
Analysis of nucleotide sequences of ORFs as well as primary sequences of proteins of sobemoviruses show a variation pattern. There is more variation in ORF1 and P1 sequences followed by the CP gene and CP and then the polyprotein. For instance, there is overall amino acid sequence identity of <20% in P1 whilst the polyproteins and the CPs are on the average
>32% identical.
Multiple sequence analysis of ORF1 (nucleotide) and P1 (amino acid) of the different sobemoviruses shows they are unrelated, with an average of only 18.9% identity for the amino acids. There are no obvious conserved regions in the primary sequences of the P1. They are also unrelated to any other protein in sequence databases. Primary sequences of sobemoviral polyproteins are related by universal sequence motifs. Based on sequence similarity with other positive strand RNA viruses, the polyprotein is proposed to encode for a serine protease and an
RNA dependent RNA polymerase (RdRp) (Kamer and Argos, 1984; Gorbalenya et al., 1988).
The consensus GDD motif of plant RdRps characterised by SGSYCTSSTNX19-35GDD is present in the ORF2b of CfMV-like viruses and the C-terminus of the ORF2 gene product of other sobemoviruses. A consensus serine protease motif, H(X26–35)[D/E](X61-62)TXXGXSG, similar to cellular and viral proteases (Gorbalenya et al., 1988; Koonin, 1991), is present in the
14
1. General Introduction and Literature review
N-terminus of the ORF2 product of SCPMV-like and the ORF2a of CfMV-like sobemoviruses
(Fig. 1.1). The primary sequence of the CPs is more related than those of proteins encoded by other ORFs. There is however no defined conserved element in the sequences.
1.2.7 Functions of Sobemovirus proteins
Full-length cDNA clones of only two sobemoviruses (RYMV-CI and SCPMV) have been reported (Brugidou et al., 1995; Bonneau et al., 1998; Sivakumaran et al., 1998). These have been used to characterise the genomes using mutational analysis in transgenic plants and in protoplasts. In addition, the functions of the P1 and CP of some sobemoviruses, e.g. CfMV, have been characterised with in vitro systems that examine properties of proteins that are related to their functions.
1.2.7.1 The P1 protein
The function of P1 in the sobemoviral infection cycle has been the most widely studied.
Mutational analyses of the P1 of RYMV-CI and SCPMV assessed in rice and cowpea plants, respectively, show the protein is dispensable for replication but not for cell-to-cell and long distance movement of the viruses (Bonneau et al., 1998; Sivakumaran et al., 1998). The P1 of
SCPMV has also been shown to be non-essential for viral assembly.
The P1 of RYMV-NG has been demonstrated to function as a suppressor for the maintenance of RNA silencing in Nicotiana benthamiana though the virus does not infect Nicotiana species
(Voinnet et al., 1999). This explains the earlier observation where there was more rapid symptom appearance in transgenic plants over-expressing P1 of RYMV-CI than in non- transformed plants following infection with in vitro-transcribed RNA transcripts (Bonneau et al., 1998). In determining the relationship between the roles of pathogenicity determinants and silencing suppressors, cDNA encoding the P1 of RYMV-NG expressed from an infectious
Potato virus X (PVX) vector was tested with other viral suppressors for induction of symptoms in N. benthamiana, a non-host of RYMV-NG (Voinnet et al., 1999). The results indicated that
15
1. General Introduction and Literature review the P1 of RYMV-NG in addition to the AC2 of African cassava mosaic virus (ACMV) were pathogenicity determinants. The suppressor activity of P1 of CfMV-NO tested for RNA silencing of the ChlH gene reveals the protein suppresses RNA silencing by targeting the maintenance stage (Sarmiento et al., 2003). The ChlH gene encodes the H subunit of magnesium chelatase, an enzyme involved in chlorophyll biosynthesis.
Other functions of the P1, which are similar to those determined for other viral suppressors of
RNA silencing, include its involvement in infection and virus spread (Bonneau et al., 1998).
The ability of P1 of CfMV-CI to bind to ssRNA in a non-sequence-specific manner has been reported (Tamm and Truve, 2000). Although these functions are common properties of viral movement proteins (MPs), there is hesitation designating the P1 of sobemoviruses as the MP.
The limited homology in the primary sequences of P1 makes it difficult and perhaps unreasonable to predict similar functions for sobemoviruses for which the function of P1 has not been directly demonstrated. However, the above results suggest the P1 of sobemoviruses could either be a pathogenicity determinant and/or viral suppressor of RNA silencing for particular hosts. The dissimilarity in the sequences suggests the proteins are under constant evolution, as these functions in viral genomes involve proteins that may evolve rapidly to counteract the host’s defence systems.
1.2.7.2 The polyprotein
The polyprotein of sobemoviruses is known to undergo proteolytic processing to produce three main products; a protease, a VPg and an RdRp. Whilst processing of the VPg from the polyprotein has been demonstrated in CfMV and SBMV by N-terminal sequencing
(Gorbalenya et al., 1988; van der Wilk et al., 1998; Makinen et al., 2000b), its expression and those of the protease and RdRp remain to be experimentally determined for all sobemoviruses.
The protease and RdRp are deduced by sequence analysis. Their presence and functions are implicated in the ability of the viruses to successfully replicate and persist in their hosts.
16
1. General Introduction and Literature review
Amino acid sequences of the VPg of SBMV is 63% identical to corresponding sequences of
SCPMV but shares no similarity to sequence of VPg of CfMV or any other viral VPgs.
However, analyses of the cleavage sites known for processing of viral VPgs show potential sites for cleavage of sobemoviral VPgs and place it between the putative serine protease and the
RdRp motifs in the polyprotein (Gorbalenya et al., 1988; van der Wilk et al., 1998; Makinen et al., 2000b). A similar polyprotein arrangement, protease-VPg-RdRp, has been shown for luteoviruses (van der Wilk et al., 1997; Wobus et al., 1998). The specific function(s) of sobemoviral VPgs have not been experimentally determined but assessment of infectivity of in vivo transcribed genome-transcripts of RYMV-CI has shown that the VPg moiety is not an absolute necessity for infectivity (Brugidou et al., 1995).
1.2.7.3 The P3 protein
Function of the P3 in the infection cycle of sobemovirues encoding this protein is not known.
Its function in SCPMV can only be implied from a mutant expressing truncated P3
(Sivakumaran et al., 1998). In vivo expression in cowpea protoplasts of this mutant had no effect on viral RNA synthesis or SCPMV assembly. However, one clone was not infectious in cowpea, indicating that P3 might be needed for SCPMV infection.
1.2.7.4 The coat protein
Structural information relating to functions of sobemovirus CP has been derived from studies with RYMV, SCPMV and SeMV. The CP of these viruses consists of two functional domains; the N-terminal R (random) and the C-terminal S (shell) domains (Abad-Zapatero et al., 1980;
Bhuvaneshwari et al., 1995; Opalka et al., 2000). The S domain is responsible for subunit- subunit interactions in the virus particles whereas the R-domain has been shown to bind efficiently to RNA (Hermodson et al., 1982; Rossmann et al., 1983). A recombinant R domain of the CP of SCPMV, the native CP and a recombinant form of CfMV-NO expressed in E. coli by independent researchers have shown the N-terminal arginine-rich motif of SCPMV CP is required for RNA binding whilst that of CfMV (the recombinant and native) selectively bind to ssRNA in a non-sequence-specific manner (Tamm and Truve, 2000; Lee and Hacker, 2001).
17
1. General Introduction and Literature review
In determining the functions of the CP in the viral infection cycle, CP mutants of full-length clones of RYMV-CI and SCPMV have been expressed in vivo and in vitro in rice and cowpea respectively. In both cases, the clones replicated efficiently in host cells but in intact plants there was no virus accumulation in inoculated or uninoculated leaves. The results indicated the
CP is not required for RNA synthesis but is needed for cell-to-cell and long distance movement of the viruses (Brugidou et al., 1995; Sivakumaran et al., 1998). Movement property of sobemoviral CPs has also been demonstrated with CfMV CP which could rescue movement functions and complement spread of a mutant rod-shaped virus, PVX, lacking this function
(Fedorkin et al., 2001).
The requirement of encapsidation for long distance transport of sobemovirus comes from mixed or single virus infection studies. In co-infection experiments, Sunn-hemp mosaic virus
(SHMV) was shown to facilitate cell-to-cell spread of SBMV in its non-permissive hosts but not systemic infection of SCPMV in beans, a local host of SCPMV (Fuentes and Hamilton,
1991, 1993). The failure was attributed to improper formation of normal T=3 virions of
SCPMV. However, when SBMV was co-inoculated with SCPMV, the latter replicates and virions recovered from inoculated and systemically infected leaves were encapsidated. This was an indication that heterologous encapsidation may play a role in the movement of SCPMV and probably other sobemoviruses. Direct evidence for requirement of encapsidation for long distance movement of sobemovirues comes from the observation of virions in host tissues away from foci of infection such as virus-like particles found within plasmodesmata connecting mesophyll cells infected with RYMV (Opalka et al., 1998).
The RNA binding property of the R domain of sobemoviral CPs, the protein-RNA interaction needed for proper formation of the virion and its involvement in virus movement most likely indicate sobemoviruses are transported by a mechanism well known for begomoviruses, potexviruses and mastreviruses; that the viral RNA moves in complex with the CP and/or the
MP or probably other viral and/or cellular proteins (Jeffery et al., 1996; Kotlizky et al., 2000;
18
1. General Introduction and Literature review
Lough et al., 2000; Palmer and Rybicki, 2001). This movement hypothesis could be investigated with experiments to determine whether the CP and/or P1 can move independently in host cells. Such experiments are best conducted with viral gene vectors expressing reporter genes such as green fluorescent protein (GFP). However, no gene vector has been developed from sobemoviruses although their properties (e.g. efficient infection and high titres in hosts, less complex and small size of the genome) makes them good candidates as viral gene vectors with high levels of foreign gene expression. It is more likely that gene vectors derived from sobemoviruses will be useful for studying the mechanism of RNA silencing especially when the P1, a viral suppressor of RNA silencing, is expressed from a native rather than an unrelated virus (Voinnet et al., 1999; Sarmiento et al., 2003).
1.3 RNA (Gene) silencing in plants
1.3.1 History and discovery
Past genetic events that triggered the study of RNA (gene) silencing in plants were co- suppression, cross-protection and pathogen-derived resistance. Co-suppression was first observed in plant transformation experiments where transgenes, designed to boost expression of endogenous/target gene products, were repressed in addition to the endogenous plant gene.
For example, in an attempt to over-express chalcone synthase (chs), a gene encoding a flower intermediate pigment biosynthesis enzyme in Petunia, chs transgene failed to increase the purple pigment by repressing the floral pigment biosynthetic pathway (Napoli et al., 1990; van
Der Krol et al., 1990; van Blokland et al., 1994). In cross-protection, infection of a plant with two viruses leads to resistance of the host to one or both viruses whereas in pathogen-derived resistance, introduction of a transgene homologous to a portion of a pathogen into an infected host confers resistance to the pathogen (Lindbo and Dougherty, 1992; Dougherty and Parks,
1995; Culver, 1996; Goregaoker et al., 2000). In each of these systems, high levels of transgene transcription did not translate into either cytoplasmic mRNA or protein accumulation, an indication that transgenes were repressed. These and other events are presently explained using a form of gene silencing.
19
1. General Introduction and Literature review
Gene silencing is a conserved cellular defense system for controlling transposon and foreign gene expression in plants, animals and fungi (Cogoni and Macino, 2000; Voinnet, 2001;
Waterhouse et al., 2001). It is referred to as RNA interference (RNAi) in animals e.g.
Caenorhabditis elegans and quelling in fungi e.g. in Neurospora crassa (Romano and Macino,
1992; Sharp, 2001). It occurs either in the nucleus at the level of transcription (transcriptional gene silencing, TGS) or in the cytoplasm after transcription (post-transcriptional gene silencing,
PTGS). Whilst in TGS, target genes are not expressed, in PTGS, with no apparent effect on transcription of target genes, there is gradual reduction in mRNA levels through degradation.
PTGS is then appropriately referred to as RNA silencing.
1.3.2 RNA silencing as a universal system
RNA silencing as a universal host defense mechanism is evident from the similar characteristics observed in plants (Voinnet, 2001), fungi (Cogoni and Macino, 1999), mammals
(e.g. mice) (Bahramian and Zarbl, 1999; Jensen et al., 1999), protozoans (Ruiz et al., 1998a) and in a number of other organisms, including flies (e.g. Drosophila), hydra and nematodes
(Fire et al., 1998; Fire, 1999). The mechanism is triggered by similar factors in these organisms and involves synthesis of double-stranded RNA (dsRNA) and spread from a localised initiating area, consistent with accumulation of small interfering RNAs (siRNA). In all of these systems, putative factors such as RdRps, RNA helicases and proteins of unknown functions are involved, which are the only presently known differences between RNA silencing in different organisms (Dalmay et al., 2000; Mourrain et al., 2000; Dalmay et al., 2001).
Whilst dsRNAs have been shown to result from aberrant processing, siRNAs are known to be degradation intermediates corresponding to subfragments of mRNA (van Eldik et al., 1998).
Metzlaff et al. (1997) first proposed that the presence of these RNA fragments is a result of processes that involves base pairing of internal complementary regions of RNA fragments to form dsRNA structures, followed by endonucleolytic cleavage at both ends and exonuclease digestion to produce siRNAs. The significant accumulation of sense and antisense siRNAs reported in several PTGS systems in plants (Hamilton and Baulcombe, 1999; Zamore et al.,
20
1. General Introduction and Literature review
2000; Elbashir et al., 2001) suggests dsRNA is produced prior to RNA degradation. However, the production of dsRNA is not fully understood. Isolation of genes of similar functions involved in RNA silencing from different organisms further confirms the universality of the system.
1.3.2.1 Genes involved in RNA silencing
Isolation of mutant Arabidopsis lines in which RNA silencing of transgenes is affected provides insights into PTGS as a natural defence mechanism in plants. Mutants in which silencing is increased (enhancers of gene silencing, egs) (Dehio and Schell, 1994), or is suppressed
(suppressors of gene silencing, sgs) (Elmayan et al., 1998; Mourrain et al., 2000) or mutants in which silencing is inhibited, (silencing-defective, sde) (Dalmay et al., 2000) have been shown to be similar to mutants exhibiting impaired quelling in N. crassa, (quelling-defective, qde)
(Cogoni and Macino, 1997) and inhibited RNAi in C. elegans (RNAi defective, rde) (Tabara et al., 1999). These genes have provided details into, and similarities between, gene silencing in different organisms.
Isolation of genes encoding proteins with similar functions from mutants of different organisms suggests possible cellular processes involved in RNA silencing, including DNA methylation confirmed by the MET1 gene which encodes the major DNA methyltransferase in Arabidopsis
(Vongs et al., 1993; Finnegan et al., 1996). The activity of host encoded RdRp is confirmed by proteins encoded by Arabidopsis SGS2 and SDE1 similar to a tomato RdRp (Dalmay et al.,
2000; Mourrain et al., 2000), the QDE-1, which is required for quelling in Neurospora (Cogoni and Macino, 1999), and EGO-1, required for RNAi of some genes in germline of C. elegans
(Smardon et al., 2000). Furthermore, the isolation of the SDE3 gene that positively controls
PTGS in Arabidopsis, similar to MUT-6 required for PTGS in Chlamydomonas (Wu-Scharf et al., 2000), and SMG-2, required for RNAi in C. elegans (Domeier et al., 2000) confirm the involvement of RNA helicases in PTGS.
As a defense mechanism, it is expected that genes involved in PTGS should control development of organisms that exhibit PTGS. For example, ago1 mutants display strong
21
1. General Introduction and Literature review developmental alterations that affect architecture and fertility of Arabidopsis (Fagard et al.,
2000). AGO1 protein shares similarity with a number of proteins containing PIWI and PAZ
(Piwi/Argonaute/Zwille) domains (Cerruti et al., 2000): STING, required for silencing of the repetitive Stellate locus in Drosophila (Schmidt et al., 1999); RDE-1, required for RNAi in C. elegans (Tabara et al., 1999); QDE-2, required for quelling in Neurospora (Catalanotto et al.,
2000); PIWI, required for germline maintenance in Drosophila (Cox et al., 1998), and eIF2C, presumed to play a role in the control of translation initiation in rabbit (Zou et al., 1998).
However, the presence of some Arabidopsis SGS proteins and absence of the corresponding mutants in C. elegans, Drosophila and Neurospora suggest possible differences in the mechanism of PTGS in these organisms.
1.3.3 Stages of RNA silencing
Studies into RNA silencing in plants distinguish three stages in the process into: initiation, spread from initiation sites and maintenance of silencing.
1.3.3.1 Initiation
Two types of transgene loci trigger RNA silencing in transgenic plants. First, it is initiated by highly transcribed single transgene copies, the efficiency of which is dependent on several factors; production of a particular form of RNA above a threshold level (Threshold model), the level of homozygosity of plants for the transgene locus (de Calvalho et al., 1992), strength of promoters driving transgenes (Que et al., 1997) and high transgene transcription (Vaucheret et al., 1997). The second trigger of RNA silencing is diametrically opposed to the threshold model. This type of transgene locus carries two transgene copies arranged as an inverted repeat and thought to produce dsRNA by read-through transcription at very low levels (van Blokland et al., 1994). This hypothesis has been confirmed in plants by efficient silencing of homologous transgenes carrying the same sequence cloned in sense and antisense orientations
(Hamilton et al., 1998; Waterhouse et al., 1998; Tavernarakis et al., 2000; Lacomme et al.,
2003) and in animals by the ability of dsRNA to initiate RNAi (Fire et al., 1998).
22
1. General Introduction and Literature review
1.3.3.2 Propagation
Transmission of RNA silencing of several transgenes from initiation to other sites in transgenic plants suggests the involvement of a systemic signal in the process (Palauqui et al., 1997;
Voinnet and Baulcombe, 1997; Voinnet et al., 1998). Palauqui and co-workers (Palauqui et al.,
1997) first established the existence and specificity of a systemic signal in RNA silencing by grafting experiments between silent transgenic plants and non-silent transgenic plants.
Silencing of nitrate reductase and nitrite reductase transgenes could be transmitted efficiently from a silenced stock to a non-silenced scion expressing a corresponding transgene but not to scions expressing non-homologous transgenes. Silencing signals were also transmitted across the graft union of silenced plants physically separated by up to 30 cm of stem of a non-target wild-type plant, indicating the existence of a diffusible factor in RNA silencing (Palauqui et al.,
1997). Similarly, Voinnet and co-workers triggered systemic silencing in a non-silenced GFP transgenic N. benthamiana plant after inoculation of a single leaf with a GFP transgene
(Voinnet and Baulcombe, 1997; Voinnet et al., 1998). These experiments and others have shown the systemic signal involved in RNA silencing must be nucleic acid to maintain specificity and mobility; either dsRNA or siRNA (Voinnet and Baulcombe, 1997; Ruiz et al.,
1998b; Voinnet et al., 1998).
1.3.3.3 Maintenance
A third stage of RNA silencing involves maintenance of a silenced state in an organism. This stage has been demonstrated by Palauqui and Vaucheret (1998) using grafting experiments.
Whereas silencing was maintained in transgenic plants from which graft-induced silenced scions were removed, it was not the case on wild type stocks on which silenced scions has been grafted indicating that only transgene loci able to initiate PTGS maintain a silenced state by reproducing the systemic signal (Palauqui and Vaucheret, 1998). Maintenance of RNA silencing has also been shown in many examples of transgene and recombinant virus-induced silencing where in the absence of the inducers, a silenced state persists. This is confirmed by resistance of plants to related viruses after silencing was induced by another virus and shows
23
1. General Introduction and Literature review that the maintenance stage, like the propagation and unlike the initiation stages, is independent of the initiator. Also, recovery of silencing in upper parts of virus-infected plants after introduction of viral cDNA transgenes provides evidence of existence of a “memory” of silencing and as an initiator-independent stage of RNA silencing (Lindbo et al., 1993; Tenllado et al., 1995). Although these experiments show RNA silencing is made up of distinct stages, all three stages may not occur in any one particular silencing event.
1.3.4 Mechanism of RNA silencing in plants
Gene silencing was first proposed as a possible explanation for the ability of untranslatable CP transgene to confer resistance to Tobacco etch virus (TEV) about a decade ago (Lindbo and
Dougherty, 1992; Dougherty and Parks, 1995). Since then unprecedented advances have been made towards the present understanding of this mechanism in plants using viruses and viral transgenes as initiators. Several models have been proposed to explain the mechanism [e.g.
Waterhouse et al., (1999); Fire (1999); Dalmay et al., (2000)]. However, much of the mechanism is still unknown and no single model accommodates all phenomena identified in research. By analogy with RNAi and quelling, the mechanism of RNA silencing in plants is understood to involve the following basic steps:
1. Production of dsRNA, probably from aberrant RNAs synthesised with transgene initiators as
template, which might involve host encoded proteins and enzymes such as the RdRps
encoded by the SGS2/SDE1 gene (Metzlaff et al., 1997; Dalmay et al., 2000; Mourrain et al.,
2000),
2. Cleavage of dsRNA into siRNAs of size ranging from 18 to 25 nt long, consistent with
identification of genes encoding plant RNA helicases involved in PTGS (Zamore et al., 2000;
Dalmay et al., 2001),
3. Base pairing of siRNAs to possibly dsRNases, similar to DICER involved in RNAi in
Drosophila, to form an RNA degradation complex, named RNA induced silencing complex
(RISC) in Drosophila (Bernstein et al., 2001)
4. Subsequent guidance of RISC by siRNAs to mRNAs sharing perfect complementarity to the
24
1. General Introduction and Literature review
siRNA and cleavage by RISC (Tuschl et al., 1999),
5. Involvement of other genes, proteins, cellular factors or processes which may facilitate any
of the stages or stabilise transgenes, dsRNA or siRNA e.gs. the SGS3 and SDE3 proteins of
unknown function (Dalmay et al., 2000; Mourrain et al., 2000; Dalmay et al., 2001) and
DNA methylation confirmed by the isolation of Arabidopsis mutant in which impairment of
DNA-methyltransferase leads to impaired maintenance of PTGS (Wassenegger et al., 1994).
1.4. RNA silencing and RNA virus-host interaction
The use of viruses and viral transcripts in RNA silencing studies has not only revealed the complexity of virus-host interactions but also establishes viruses as targets, inhibitors and inducers of RNA silencing in plants and the system as an anti-viral defense mechanism.
1.4.1 Viruses as targets of RNA silencing
PTGS has been shown to target viral RNA the same way it targets non-virus transgenes and in most cases can eliminate a virus completely leading to recovery of host plants (Waterhouse et al., 1998; Rovere et al., 2001). This is exemplified by silencing of a GFP transgene and complete recovery after initial susceptibility of transgenic N. benthamiana to a recombinant
PVX carrying a GFP gene (Ruiz et al., 1998b). The results demonstrated that PTGS triggered after viral replication targets both the transgene and the virus with silencing later spreading throughout the plant. A similar conclusion was drawn by English et al. (1996) using a PVX vector carrying the β-glucuronidase (GUS) gene and tobacco plants. Ratcliff et al. (1999) also demonstrated that PTGS is a general response to virus infection in experiments where infection of N. benthamiana with Tobacco rattle virus (TRV) carrying GFP (TRV-GFP) resulted in induction of PTGS targeted to TRV-GFP. This lead to a recovery phenotype, parts of which were immune to both TRV-GFP and PVX-GFP, but not to PVX carrying a GUS gene. This shows PTGS as a natural response by plants against virus infection as it was entirely based on viruses and independent of homologous sequences in the plant genome as targets. Additional evidence for viruses as targets of PTGS comes from the observation that Arabidopsis mutants
25
1. General Introduction and Literature review that exhibit impaired PTGS are hypersusceptible to infection by Cucumber mosaic virus
(CMV) (Marathe et al., 2000). Furthermore, the fact that plants can resist infection or undergo a preliminary phase of infection or total recovery indicates they can specifically degrade virus
RNA. This can occur at the initiation stage of RNA silencing or by inhibition of spread of the silencing signal or by a recovery from virus infection with the help of a ‘memory’ of a first virus. These processes have been shown to be similar to PTGS targeted against non-viral transgenes (Covey et al., 1997; Ratcliff et al., 1997; Al-Karf et al., 1998; Ratcliff et al., 1999).
These and other experiments with viral vectors in reverse genetics indicate that RNA silencing acts as an antiviral system in plants.
1.4.2 Viruses as suppressors of RNA silencing
The ability of viruses to succeed in infecting their hosts suggests they have evolved to overcome the plants’ RNA-mediated defense through evasion, escape or by suppression
(Matthews, 1991; Voinnet et al., 1999). Evasion and/or escape used by viruses such as
Tobacco mosaic virus (TMV) and Cowpea mosaic virus (CPMV) have been suggested as secondary strategies for counteracting effects of the plant’s RNA defense system (Voinnet et al., 1999). Some DNA and RNA plant viruses encode proteins that suppress PTGS. These include the helper component protease (HC-Pro) of PVX and TRV (Anandalakshmi et al.,
1998), the 2b protein of CMV and Tomato aspermy virus (TAV) (Brigneti et al., 1998; Li et al.,
1999), the AC2 gene product of ACMV (Voinnet et al., 1999), the 19K protein of Tomato bush stunt virus (TBSV) (Voinnet et al., 1999), the P21 protein of Beet yellows virus (BYV), and the
P1 protein of two sobemoviruses, RYMV and CfMV (Voinnet et al., 1999; Sarmiento et al.,
2003).
Viral suppressors of RNA silencing target different stages of the process and produce characteristic patterns of silencing suppression. For instance, HC-Pro suppresses maintenance of silencing whereas Cmv2b only affects silencing initiation (Beclin et al., 1998; Brigneti et al.,
1998; Voinnet et al., 1999). Interestingly, most of the presently known viral suppressors have
26
1. General Introduction and Literature review also been identified as pathogenicity determinants e.g. HC-Pro, Cmv2b and P19 of TBSV (Ding et al., 1995; Scholthof et al., 1995; Kasschau et al., 1997). In an experiment to confirm the relationship between the roles of pathogenicity determinants and silencing suppressors, cDNA encoding the P1 of RYMV-NG and the AC2 of ACMV were inserted into an infectious PVX vector separately and tested for induction of symptoms in N. benthamiana (Voinnet et al.,
1999). A further experiment to test for suppression of a silenced N. benthamiana plant transgenic for GFP indicated that both proteins are pathogenicity determinants and silencing suppressors in N. benthamiana. It is therefore likely that the activity of these proteins in pathogenicity of the encoding virus, including their role in virus movement, is associated with their suppressor activity (Brigneti et al., 1998; Voinnet et al., 2000). This is exemplified by the ability of a movement protein of PVX, the 25 kDA protein, p25 to prevent systemic silencing in
N. benthamiana. It further confirms the observation that the systemic signal of silencing moves between cells through plasmodesmata and over long distances through the vascular system
(Voinnet et al., 2000). These developments make it tempting to assign silencing suppressor functions to pathogenicity determinants of viruses and vice versa. The diversity in nature
(nucleotide and amino acid sequences) and silencing activity of viral suppressors of silencing shows they are specific for a particular virus and could therefore mean that the suppressor function has evolved independently as anti-PTGS. It may also be because the different proteins are at different stages of dynamic evolution.
1.4.3 Viruses as inducers of RNA silencing
The above discussion shows that viruses are not only targets and suppressors of RNA silencing but they also trigger the process in transgenic and non-transgenic plants. Several examples exist where native viruses as well as engineered viral vectors, with viral and non-viral transgenes in different forms: sense and antisense inserts, hairpins and/or loops or inverted repeat inserts, have been shown to induce PTGS of which the viral ssRNA and/or non-viral transgenes are targets (see section 1.4.1). In these systems, dsRNA is thought to be the replication intermediate that causes RISC to target the viral ssRNA. The complex is said to be
27
1. General Introduction and Literature review effective in blocking accumulation of viral ssRNA at later stages of virus infection when the rate of viral RNA replication has increased enough to allow the abundant accumulation of viral dsRNA and siRNA and the formation of RISC (Voinnet, 2001). Like other transgene-induced silencing, silencing induced by viruses in plants takes place if there is sequence similarity between the recombinant virus and either a transgene or an endogenous nuclear gene. This characteristic feature is being exploited as a technology for studying gene function.
1.5 Virus-induced gene silencing (VIGS)
The ability of viruses to effectively induce RNA silencing in host plants is currently being exploited in a gene technology referred to as virus-induced gene silencing (VIGS). VIGS has been validated by silencing reporter genes (GUS and GFP) in transgenic plants using several recombinant DNA and RNA viral vectors referred to as VIGS vectors and transgenes, called
VIGS inserts (English et al., 1996; Ruiz et al., 1998b; Ratcliff et al., 2001). In a typical VIGS experiment, a cell is infected with a VIGS vector with an insert sharing homology with an endogenous gene. A dsRNA replication intermediate is processed into siRNA (corresponding to the endogenous gene) which then guides RISC to the homologous host mRNA. Symptoms observed in the infected plant then reflect the loss of function in the encoded protein.
1.5.1 VIGS as a tool for functional analysis of endogenous genes
VIGS has potential applications in both reverse and forward genetics. As a reverse genetics tool, VIGS can be exploited for functional analysis where it would be possible to silence a gene and thereby determine the role of the gene product from mutants or resulting phenotypes. This system will be much quicker than the use of antisense or sense non-virus transgene suppression or the use of other systems (fungal) for functional analysis due to the enhanced transient foreign gene expression provided by viral vectors (section 1.6.1) (Schweizer et al., 1999; Escobar et al., 2003). This approach has been demonstrated by the use of many VIGS vectors to successfully suppress expression of genes encoding metabolic enzymes (e.g. phytoene desaturase, PDS), defense-related genes (e.g. EDS1), growth regulatory genes (e.g. Leafy) and essential genes (e.g. cofactor of DNA polymerase) in various plants (Table 1.5).
28
1. General Introduction and Literature review
It is also possible to use cDNA libraries in a forward genetics approach based on VIGS where
genes expressed in many tissues could be targeted for silencing. The main limitation is the
inability to target genes expressed in meristems as viruses do not generally replicate in
meristematic zones (Matthews, 1991; Tanzer et al., 1997). This approach would be beneficial
for complementing and characterising the many cDNA libraries being constructed for several
model plants such as rice, Arabidopsis and Medicago truncatula.
Table 1:5 Common VIGS vectors and some endogenous genes targeted for silencing in suitable hosts Vector Silenced endogenous gene Host plant Reference PVX 1. phytoene desaturase gene Nicotiana. Kumagai et al., 1995; Ruiz et al., 1998b 2. chlorophyll biosynthetic enzyme benthamiana Kjemtrup et al., 1998 3. cellulose synthase Burton et al., 2000; Peart et al., 2002 TMV 1. chlorophyll biosynthetic enzyme N. benthamiana Kjemtrup et al., 1998 2. cellulose synthase Burton et al., 2000 TRV 1. PDS, Tomato Liu et al., 2002 2. CTR1 and CTR2 Arabidopsis Dalmay et al., 2000 3. Leafy N. benthamiana Ratcliff et al., 2001 4. ribulose bisphosphate carboxylase Peart et al., 2002 5. EDS1 gene BSMV PDS barley Holzberg et al., 2002 CbLCVa Chlorata 42 gene N. benthamiana Kjemtrup et al., 1998; Turnage et al., CbLCVb PDS Arabidopsis 2002 TGMV 1. Proliferating cell nuclear antigen N. benthamiana Kjemtrup et al., 1998 2. cofactor of DNA polymerase Peele, 2001
1.5.2 Viral vectors for VIGS
Both DNA and RNA viruses have been used successfully as VIGS vectors (Table 1.5) most
popular of which are the rod- or tubular-shaped RNA viruses TRV, TMV and PVX (a tripartite
RNA virus), Barley stripe mosaic virus (BSMV), and geminiviruses such as Cabbage leaf curl
virus (CbLCV) (Turnage et al., 2002) and Tomato golden mosaic virus (TGMV) (Peele, 2001).
These have either been modified from previously constructed forms for biological studies or
have been developed specifically as VIGS vectors (Ratcliff et al., 2001; Holzberg et al., 2002;
Liu et al., 2002; Turnage et al., 2002). Viruses as inducers and suppressors of RNA silencing
(section 1.2.3) have implications for virus vector design and application. A viral vector
intended for foreign protein expression is useful if it does not induce gene silencing on a chosen
host. More importantly, in using VIGS as a tool for functional genomics, a viral vector will be
more useful if it is a strong inducer of silencing but does not encode for a strong suppressor.
29
1. General Introduction and Literature review
Viral activation and suppression of RNA silencing have mostly been studied in solanaceous plants e.g. Nicotiana benthamiana, N. tabacum and tomato and also in barley (Table 1.5).
1.5.3 VIGS insert
Successful VIGS of target genes depends to a large extent on the size, location and orientation of the VIGS insert. The size of inserts used in VIGS vectors is variable and generally ranges from 150 to 500 nt. The PDS gene silenced with as short as 23 nt of complementary sequence exhibits less extensive and more transient silencing than with larger inserts (Thomas et al.,
2001a). This is a good sign for viral vectors where a larger insert will possibly cause genetic instability. The present proposed mechanism of RNA silencing makes the orientation of insert in VIGS vector irrelevant as both sense and anti-sense transgenes hairpins and inverted repeats could trigger PTGS. However, Lacomme et al., (2003) have used 40–60 nt direct inverted- repeats to generate a stronger and more pervasive VIGS phenotype corresponding to GFP and
PDS than constructs carrying corresponding cDNA fragments in sense or antisense orientation.
They proposed this was a result of increased levels of dsRNA molecule production, the mechanism of which is still unknown.
Complementarity between a VIGS insert and the target gene affects the silencing phenotype.
An insert from a unique region of a target gene would produce a highly specific silencing phenotype whereas one with regions similar to related genes, e.g. in a gene family, may silence more than the intended target (Daisuke et al., 2003). The known mechanism of RNA silencing coupled with the analysis of short inserts for VIGS of PDS makes it likely that any gene with more than about 20 nt perfectly matched to the insert in the VIGS vector may be silenced. It is not known whether VIGS is better with coding or noncoding targets though introns are known not to direct VIGS (Ruiz et al., 1998a). Analysis of several silencing events, however, do not support the suggestion that the 3’ end of transgene mRNAs are the targets of gene silencing
(English et al., 1996; Sijen et al., 1996; Marano and Baulcombe, 1998).
30
1. General Introduction and Literature review
1.6 Plant viruses as gene vectors
Gene transfer by Agrobacterium tumefaciens transformation, agroinoculation, microinjection and biolistics has revolutionalised biology (Songstad et al., 1995). However, the efficient use of a limited amount of genetic material by viruses to direct their amplification and propagation makes them more useful, especially when high-level transient gene expression rather than stable transformation is required. Virus-based vector systems for gene transfer began with the isolation, cloning and characterisation of DNA viruses, especially caulimoviruses, and later cloning of RNA viruses into infectious agents first demonstrated for Brome mosaic virus
(BMV) (Ahlquist et al., 1984; Dawson et al., 1986; Meshi et al., 1986). Since then, in vivo- and/or in vitro-transcribed infectious RNA transcripts derived from full-length cDNA clones of several RNA viruses have become important tools for studying various aspects of virus pathogenicity: symptom development, movement, transmission, interaction with resistance genes, replication, proteolysis, disassembly of viral CP, etc.
1.6.1 Development of plant RNA viruses as gene vectors, strategies
Infectious clones, of lengths ranging from 0.9-11.9 kb, from several families and genera of both rod-shaped and isometric plant RNA viruses have been engineered for use in various aspects of plant virology (Boyer and Haenni, 1994; Scholthof et al., 1996; Scholthof, 1999). These are modified into gene vectors with a number of strategies resulting in retention of all or most of their biological functions. Successful modifications depend largely on molecular knowledge of the virus concerned; its genome structure, gene expression strategies and functions of the proteins it encodes. This provides information on which genes are essential and which are not for expression of foreign genes. Strategies used for construction of viral vectors could involve gene replacement, duplication or insertion and complementation of viral or foreign genes.
Nevertheless, there is no guarantee that a useful vector can be derived from an infectious cDNA clone. Therefore, for validation of vector potential, different strategies for construction of the genome should be explored using a variety of foreign gene insertions at different positions within the genome to study their effects on stability of the modified genomes. A summary of strategies for developing RNA viruses into vectors and successful examples is in Table 1.6.
31
1. General Introduction and Literature review
Table 1.6: Summary of strategies for developing RNA viruses into gene vectors
Strategy Gene replacement Gene insertion Gene Epitope presentation complementation Description Expunging viral Addition of foreign Use of viral helper- Translational fusion of genes sequences or genes dependent systems small foreign peptides to viral proteins Purpose To reduce possible To leave viral genes To circumvent To raise specific effects of increased intact limitations of insertions antibodies against small genome size and replacements peptides
Requirement Requires expunged All viral genes are Requires production of Chosen insertion sites viral gene to be intact; (essential and fusion product plus requires resulting nonessential; not nonessential are native viral protein peptides to project involved in needed for overall without interference outwards on the virus replication or fitness of virus) with normal functions particle movement
Modification Genes replaced are i) duplication of i) complementation of i) C-terminal fusions to i) CP when it is sgRNA promoters in mutants by transgenic CP dispensable for cis and in trans plants ii) surface loop infection ii) inter-cistronic ii) Subviral agents e.g insertions to the CP ii) those encoding for insertions regions a. defective interfering iii) Internal peptide insect transmission iii) insertional fusion RNAs fusions factors b. satellites RNAs
Pioneer virus Replacement of i) Intercistronic inser i) mutants of CaMV for i) TMV expresssion of /e.g. of virus i) CaMV geneII with -tions into MSV (7) complementation (14) a. AICEI (19, 20) dhfr (1) ii) duplicating ii) satRNA of BaMV b. malarial epitopes ii) CP of BMV(2) and sgRNA promoter in (15) (21) TMV (3) with CAT TMV (8, 9,10) and PVX (11). iii) DI RNAs of TBSV c. acid murine epitope iii) CP of TGMV ( 16) and CymRSV (22) (4,5) and ACMV (6) iii) insertion between (17,18) with bacterial genes HC-Pro and ii) HIV related products proteinase of TEV expressed from (12, 13) TBSV(23)
Limitations Gene replacement Increased genome Rapid recombination Detailed knowledge of may affect overall size may impede with the use of mutants virus structure at the viral fitness effective packaging atomic level
Widely used Bromo-, caulimo-, Caulimo-, gemini-, Como-, tobamo-, and Alfamo-, caulimo-, virus group furo-, gemini-, potex potex-, poty- and tombusviruses diantho-, gemini-, hordei-, tobamo-, tobamoviruses potex-, tobamo-, and tobra-, and tombusviruses tombusviruses
1. Brisson et al., 1984, 2. French et al., 1986, 3. Takamatsu et al., 1987b, 4. Hayes and Buck, 1988, 5. Hayes et al., 1989, 6. Ward et al., 1988, 7. Shen and Hohn, 1995, 8. Dawson et al., 1989, 9. Donson et al., 1991, 10. Kumagai et al., 1995, 11. Kavanagh et al., 1992, 12. Dolja et al., 1992, 13. Dolja et al., 1998 14. Hirochika and Hayashi, 1991, 15. Fiers et al., 1986, 16. Scholthof et al., 1993, 17. Burgyan et al., 1994, 18. Burgyan et al., 1992, 19. Hamamoto et al., 1993, 20. Sugiyama et al., 1995, 21. Turpen et al., 1995, 22. Fitchen et al., 1995, 23. Oxelfelt et al., 1995
32
1. General Introduction and Literature review
1.6.2 Virus-based vectors as transient gene expression systems
Autonomously replicating viruses provide good systems for transient expression of foreign genes with increased efficiency, effectiveness and control in experimentation. The nature of virus infections allows high levels of multiplication and maximum levels of foreign gene expression within a short time-period from an engineered viral genome, usually within a week or two after inoculation. This property, in addition to ease of mechanical transmission of many viruses will allow rapid and wide scale testing of expression and also screening of genes that induce measurable phenotypic effects in a variety of different host plants. Viral vectors as transient gene expression systems provide increased speed and flexibility during early phases of experimentation. For instance, expression of any foreign gene could be evaluated in numerous plants of the same or different genotypes within a short period. With this system, introduction and expression of the foreign gene can be postponed until early developmental stages are completed avoiding any negative effects on young developing plants, and permitting flexibility in timing with optimum results. The advantages offered by viral based systems coupled with
VIGS would provide a valuable tool in the hands of researchers in providing a diary of possible functions of genes for future discovery.
1.6.3 Introduction of viral vectors into host plants
Infectivity of plant RNA viral vectors has been assessed using in vitro and/or in vivo- transcribed RNA transcripts. RNA virus vectors were originally introduced in hosts after in vitro transcription of infectious RNA from a linearised plasmid DNA template (Donson et al.,
1991; Chapman et al., 1992). However, the insertion of exogenous transcriptional promoters and terminators such as the 35S RNA transcriptional promoter of Cauliflower Mosaic virus
(CaMV) (commonly referred to as 35S) upstream of full-length cDNA derived from RNA viruses, followed by direct DNA inoculation, is also being increasingly used for infection of plants. For some viruses, both in vitro-and in vivo-transcribed RNA transcripts have been successfully used as infectious agents but for others only one of the RNA forms is effective
(Boyer and Haenni, 1994; Scholthof et al., 1996).
33
1. General Introduction and Literature review
Most plant RNA viruses that are easily transmissible mechanically have been shown to give high infectivity with appropriately constructed cDNA clones (Boyer and Haenni, 1994).
However, for viruses that are not amenable to mechanical inoculation, e.g. phloem limited luteoviruses, this limitation is overcome by use of agroinoculation (Grimsley and Bisaro, 1987;
Leiser et al., 1992) where infectious cloned viral DNA or cDNA is introduced into the host nucleus by Agrobacterium T-DNA mediated uptake. An alternative and simpler technique for inoculation of phloem limited viruses involves introduction via vascular puncture of seeds
(Louie, 1995). Particle bombardment with plasmid DNA derived from cloned RNA viruses has been reported to give increased infectivity in hosts compared to mechanical inoculation
(Fakhfakh et al., 1996; Gal-On et al., 1996).
Direct inoculation of cDNA clones or agroinoculation is more beneficial for several reasons. It avoids the relatively costly and tedious in vitro transcription experiment with or without cap analogues. This is beneficial for RNA viruses which require many transcripts for infection, but is difficult to produce in vitro. In using cDNA clones as infectious agents, the expression of viral genes is largely independent of the viral replication process. For example, in studying the role and localisation of viral proteins expressed by mutant viral RNAs unable to replicate in cells, the in vivo-produced transcripts behave as mRNA produced by a host RNA polymerase able to express native or mutant proteins without being replicated. Also, infectivity is independent or less dependent on cellular RNA degradation processes (Boyer and Haenni,
1994).
1.7 Reporter genes and viral vectors as research tools
Plant virus-based vectors have been developed as reporter gene-tagged viral genomes to validate their stability and effectiveness for foreign gene expression and for studying basic aspects of viral pathology. Validation involves direct or indirect detection and/or quantification by flourimetry or with luminescent substrates of viral proteins and/or reporter proteins inserted into the genome. Earlier reporter genes used this way include the luciferase (LUC),
34
1. General Introduction and Literature review dihydrofolate reductase (DHFR), chloramphenicol acetyl transferase (CAT) genes (Brisson et al., 1984; French et al., 1986; Takamatsu et al., 1987) and later the GUS gene (Table 1.5)
(Jefferson et al., 1987).
The first reported plant viral genomes engineered to express GUS were TEV and PVX
(Chapman et al., 1992; Dolja et al., 1992). Detection of GUS activity in infected tissues indicated both TEV.GUS and PVX.GUS gave systemic infections on Nicotiana tabacum
(Chapman et al., 1992; Dolja et al., 1992). The GUS gene thus provided the first opportunity to accurately follow the progress of local and systemic virus infections in situ. Following this success, and the invention of reverse genetics, viral pathogenesis has been studied for several viruses with GUS as a reporter gene including requirements of the CP of PVX and TEV for cell-to-cell movement (Chapman et al., 1992; Dolja et al., 1992), involvement of the HC-Pro in systemic movement of TEV (Cronin et al., 1995), requirement of the p22 protein of TBSV in local movement (Scholthof et al., 1995) and regulation of the plasmodesmatal pore size, and viral cell-to-cell movement by the 25 kDa protein of PVX (Angell et al., 1996). In experiments to determine both the timing and level of expression, translational fusions between BVY- encoded proteins and GUS have been used to determine and compare temporal regulation of individual BYV sgRNA promoters (Hagiwara et al., 1999).
However, GUS is less attractive than other reporter genes. The limitations are related to size, processing and detection of the end product. The relatively larger size of the UidA gene (1.8kb) could potentially interfere with stability of some vectors (Chapman et al., 1992; Dolja et al.,
1993; Guo et al., 1998). The highly diffusible reaction intermediate produced by the oxidation reaction to produce the coloured end product could interfere with its use as a reporter for localisation and in situ detection of gene expression of viral vectors (Guivarc'h et al., 1996).
The detection methods for GUS are also invasive and involve vacuum infiltration of detached plant organs and use of organic solvents for decolouration. GFP is a better marker because of its smaller size, non-invasive detection methods, and independent heterologous expression
(Roberts et al., 1997; Szecsi et al., 1999).
35
1. General Introduction and Literature review
1.7.1 GFP as a research tool
Following discovery (Shimomura et al., 1962), characterisation (Ward and Cormier, 1979;
Ward et al., 1980; Ward et al., 1982; Ward and Bokman, 1992), and cloning and sequencing
(Prascher et al., 1992), the jellyfish (Aequorea victoriae) GFP has become an important research tool (Tsien, 1998). To date, wild-type GFP is known to be a 27 kDa monomer consisting of 238 amino acids and has unique characteristics of emitting green light when excited with UV (360-400 nm) or blue (440-480 nm) lights. Desirable properties of the protein include its capability for heterologous expression indicating the gene contains all information necessary for post-translational synthesis of its chromophore with no need for jellyfish-specific enzymes (Chalfie et al., 1994; Inuoye and Tsuji, 1994), successful fusion to foreign proteins without necessarily compromising the function of the fusion partner (von Arnim et al., 1998), and high levels of constitutive expression in trans without interfering with transformation, regeneration or growth of transgenic plants (Pang et al., 1996). GFP has since been expressed in a variety of organisms; bacteria (Pogliano et al., 1995; Burlage et al., 1996; Christensen et al., 1996), fungi (Heim and Tsien, 1996), invertebrates (Ormo et al., 1996), vertebrates (Ehrig et al., 1995) and plants (Inuoye and Tsuji, 1994; Yokoe and Meyer, 1996; Haseloff et al., 1997;
Wachter et al., 1997).
As a research tool, GFP has been used for two purposes: as a fusion tag and as an indicator to study several processes and systems and to predict the functions of its fusion partners. Its fluorescence is used to determine the levels of gene expression or (sub)cellular localisations and intracellular trafficking of domains or host of proteins fused at either the N- or C-terminus.
It has been targeted successfully to practically every major organelle of the cell, including endoplasmic reticulum (Haseloff et al., 1997; Presley et al., 1997), golgi apparatus (De Giorgi et al., 1996; Presley et al., 1997), mitochondria (Rizzuto et al., 1996), nucleus (Lim et al.,
1995; Rizzuto et al., 1996), peroxisomes (Wiemer et al., 1997), plasma membrane (Mashall et al., 1995; Yokoe and Meyer, 1996), secretory vesicles (Kaether and Gerges, 1995) and vacuoles (Di Sansebastiano et al., 2001). The protein has been used in whole organism studies
36
1. General Introduction and Literature review in plants and animals, in disease and ecological monitoring, and in genetic transformations
(Haseloff et al., 1997; Elliot et al., 1999; Pang et al.,, 1996).
1.7.2 GFP as a reporter gene for validating viral vectors
Qualitative and quantitative methods developed to successfully detect GFP in vitro and in vivo have made the protein a readily scoreable marker for gene expression studies and an invaluable tool for improving virus-based vectors (Baulcombe et al., 1995; Niwa et al., 1999; Dundr et al.,
2002). The first successful report of a GFP-tagged plant virus was PVX-based gene insertion vector (Baulcombe et al., 1995). Since then, vector potential of several viruses from other genera has been demonstrated, and GFP expressed without interference with viral functions, e.g. CPMV (Verver et al., 1998), TEV (Kasschau and Carrington, 1998), TMV (Casper and
Holt, 1996), CMV (Canto et al., 1997), and Bean dwarf mosaic virus (BDMV) (Sudarshana et al., 1998).
Accumulation of GFP as a percentage of total soluble protein in leaves has also been used to determine which strategy is more appropriate in engineering a viral genome into a vector. A classic example was demonstrated by Shivprasad et al., (1999). They compared the efficiency of a series of TMV-based hybrid vectors and four other tobamoviruses [Odontoglossum ringspot virus (ORSV), tobacco mild green mosaic virus (TMGMV) variants U2 and U5, tomato mosaic virus (ToMV), and SHMV] for transient gene expression of GFP. The vectors were constructed with similar designs but differing in the source of heterologous tobamovirus sequence and their effectiveness assessed using GFP accumulation. The most effective vector in this series contained sequences encoding the CP sgRNA promoter (sgPro), the CP ORF, and
3’ NCR from TMGMV-U5 and accumulated up to 10% of total soluble GFP in leaves. The use of GFP in several of such gene expression experiments [e.g. Gopinath et al., (2000)] based on the intensity of fluorescence will greatly facilitate comparative assessments of virus-based vectors.
37
1. General Introduction and Literature review
1.7.3 GFP as a reporter gene for studying viral pathogenesis
Among the many aspects of viral pathogenesis, plant virus vectors expressing GFP as a free protein has provided insights into virus localisation and movement in virus-infected tissue in vivo (Oparka et al., 1996). Also subcellular targeting and association of several virus-encoded proteins with the intracellular transport apparatus of plants has been well characterised with
GFP-tagged viruses. Viral vectors tagged with GFP have contributed to understanding of subcellular distribution of TMV movement protein (MP) (Padgett et al., 1996; Mas and
Beachy, 1998), cellular responses induced by viral infection [e.g. Szecsi et al., (1999)], plasmodesmatal localisation and molecular determinants of MP localisation (Kahn et al., 1998), association between MPs and plant cytoskeleton and tubulins (Heinlein et al., 1995; McLean et al., 1995).
Expression of biologically active GFP-CP fusions has been one of interest in viral pathology although not much success has been achieved in this area. However, the use of fusions to the
CP for peptide presentation at the surface has been successfully employed for viruses with icosahedral, bacilliform and rod-shaped particle morphologies. Using GFP-CP of PVX, Santa
Cruz et al., (1996) demonstrated the incorporation of the fused protein into virus particles that could be imaged in situ by flourescence microscopy. Effective assembly of virus particles incorporating the GFP-CP fusion, however, required the availability of free CP subunits in the absence of which infection was limited to individual epidermal cells. This result, determined with a CP deletion mutant of PVX.GFP (Baulcombe et al., 1995), reflected a requirement for assembled virus in the intercellular movement process rather than some additional, assembly- independent, role of the CP in transport (Santa Cruz et al., 1998). The above discussions describing the applications of GFP in plant virus pathogenesis studies are some of the many ways viral pathology has progressed using virus-based vectors expressing GFP.
38
1. General Introduction and Literature review
1.8 Limitations in construction and use of plant RNA viruses as gene vectors
The popular viral vectors in use have structural and functional characteristics that make them successful. They allow easy genetic manipulation via a full-length infectious clone with little or no size constraint for genome packaging. They mostly infect a wide range of plants effectively following mechanical infection with either RNA or DNA inocula. Also they are relatively genetically stable with optimised viral functions; exhibiting high levels of replication, efficient foreign gene expression; and rapid local and systemic movement in suitable hosts.
Despite significant successes, potential of RNA viruses as gene vectors is limited by intrinsic viral properties, technical factors in their development and regulations about their creation and use.
1.8.1 Intrinsic viral factors
Inherent properties of RNA viruses may hinder their usefulness as gene vectors. First thought to be a major limiting factor, high error frequency in viral RNA replication is now less of a problem compared to the numerous advantages of RNA replicons reported as gene expression and transfer vehicles (Siegel, 1985; Van Vloten-Doting et al., 1985). Rapid recombination, observed as a natural phenomenon in potyviruses, appears to be the predominant cause of instability and deletion of large foreign gene inserts from RNA virus vectors (Greene and
Allison, 1994).
Packaging or other unidentified structural constraints could limit the sizes of foreign genes/inserts that can be maintained in some viruses (e.g. isometric viruses). Inserts in viral genomes may cause instability of modified genomes and deletion of foreign inserts. This observation may reflect a universal evolutionary strategy viruses use to protect themselves against non-essential incorporation of inserts and subsequent amplification of non- advantageous sequences. In addition, it is likely that essential functions of modified viral genomes could be compromised after insertion of foreign material at any given position, even with smaller inserts (Chapman et al., 1992). For example, insertion of a sgPro-GFP cassette
39
1. General Introduction and Literature review between the CP gene and 3’ NCR of Alfalfa mosaic virus (AMV) interferes with RNA accumulation in tobacco (Sanchez-Navarro et al., 2001). Even in viral vectors with no such limitations, they are likely to be less competitive than their wild-type progenitors and this, with the relative instability can be a disadvantage.
An important biological property determining whether a virus might be useful as a gene vector is its ability to infect a large number of host plants efficiently without restriction to specific cell or tissue types. The efficiency of an infectious clone of a vector driven by the popular 35S promoter however, could restrict their potential to just a few plant species because of the limited host range of CaMV (Shepherd, 1989). Though it is likely mechanically transmissible viruses would be infectious when an engineered genome is well constructed, it has been difficult to get infectious clones from several viruses (Boyer and Haenni, 1994).
1.8.2 Technical factors
Genome-length cDNA clones from which infectious in vivo- or in vitro-transcribed RNA transcripts can be synthesised have been obtained for several viruses using RT-PCR and molecular cloning (Boyer and Haenni, 1994). In addition, infectious transcripts have been produced from incomplete and subviral RNAs (Fiers et al., 1986, Scholthof et al., 1993,
Burgyan et al., 1994). Due to different genomic organisations, sizes and morphologies, several methods for retaining cDNA clones derived from RNA viruses have been reported (Okayama and Berg, 1982; Gubbler and Hoffman, 1983; Frankel and Friedmann, 1987; Schmid et al.,
1987; Boyer and Haenni, 1994). These include the use of different reverse transcriptases and
DNA polymerases in RT-PCR using viral or total RNA from infected hosts, and the use of different forms of native and heterologous promoter and terminator sequences. For viruses where it is difficult to obtain genome-length first strand cDNA, infectious full-length clones have been produced by assembly of overlapping cDNA clones (Johansen, 1996; Sivakumaran et al., 1998; Lin et al., 2002).
40
1. General Introduction and Literature review
Whilst the presence of non-viral sequences between transcriptional start sites of promoters and the viral sequence has prevented infectivity of some cDNA clones, it has not been limiting for other viruses (Dore et al., 1990; Rizzo and Palukaitis, 1990; Commandeur et al., 1991). For cDNA clones of some viruses, enhancement of heterologous promoters is needed to make them infectious. In some viral cDNA clones, the orientation of sequences in cloning vectors, and expression of viral and/or some heterologous sequences make them unstable in some E. coli strains (Fakhfakh et al., 1996; Johansen, 1996). For example, the full-length cDNA of Pea seedborne mosaic virus (PSbMV) pathotype P1 under the control of 35S and T-Nos is unstable in E. coli strain DH5α (Johansen, 1996).
1.8.3 Environmental and regulatory factors
Paradoxically, undesirable properties of recombinant viruses are ecological safety features because of the greatly reduced risk of dissemination from containment facilities, and during commercial or agricultural use. With pressure from environmentalists, gene-ethicists and several other interest groups, national regulatory bodies such as The Office of Gene
Technology Regulator (OGTR) of Australia and the Food and Drug Administration of the
USA, have strict regulations concerning the creation, and use of genetically modified organisms (GMOs), including recombinant viruses. This is because potential risks posed by most GMOs are not known and have not been comprehensively assessed. Unknown risks in the use of recombinant viruses are derived from their extreme change and adaptability, possible recombination between transgene RNA and viral RNA leading to creation of new pathogens. A viral vector with a wide host range is more useful as a research tool but this feature is a result of virus evolution and could result in virus transmission to other crops, posing an environmental risk. Until more effort is put into studying expression and evolution of viral vectors, educating the public about the risks they pose, and more importantly how they are being confined and contained, the highest potential of viral gene vectors cannot be realised.
41
1. General Introduction and Literature review
1.9 Aims and objectives of this project
The overall aim of this research was to investigate the possible use of SCMoV as a gene silencing vector for functional analysis of endogenous genes in legume. The few VIGS vectors available are either DNA or rod-shaped RNA viruses with complex genomes and with a limited legume host range. The relatively small genome size and simple organisation of
Sobemoviruses, their efficient infection leading to high titres in hosts, and ease of mechanical transmission is likely to make them successful gene vectors. To achieve the overall aim, a number of aspects of SCMoV host range and molecular biology needed to be studied. Specific objectives laid down to achieve this aim were:
1. To study the host range and symptom development of SCMoV in a range of
leguminous and non-leguminous plants. This work was undertaken to identify new and
the most suitable hosts among economically important crop and model legumes for
functional genomic studies, and also to study symptom development in these hosts for
comparison with host responses to any SCMoV-based viral vectors that might be used
in later gene expression studies.
2. To characterise the genome of SCMoV in detail; to study genetic diversity and
functional constraints of its genes. This was done to provide the information needed to
modify the SCMoV genome to develop gene vectors.
3. To study cellular localisation of gene products of SCMoV. This was done to examine
the possibility of independent cell-to-cell movement of the P1 and CP of SCMoV in
suitable hosts using GFP as a reporter gene, and to provide more information needed to
modify the genome into a gene vector.
4. To develop a series of SCMoV-based cDNA vector constructs for use as gene vectors
for foreign gene expression and functional genomics studies.
42
Chapter Two
General Materials and Methods
2. General Materials and Methods
2.0 Virus isolates and culture, and plant inoculation
Four WA isolates of SCMoV (SCMoV-AL, -MB, -MJ and -P23) used in the project were kindly provided by Dr. R. A. C. Jones, Plant Pathology Unit, Department of Agriculture
Western Australia (DAWA) (Wroth and Jones, 1992a, b). Virus isolates were maintained in T. subterraneum cv. Woogenellup and either kept at DAWA or Murdoch University under the glasshouse conditions stated in section 2.1. Inoculations to maintain cultures and to challenge different potential hosts species were done by grinding young infected leaves in 0.2 M phosphate buffer (pH 7.2) using approximately 0.1 g of leaf per mL of buffer. The sap was mixed with diatomaceous earth, “celite”, (1 in 20 dilution with the buffer) to act as an abrasive and to aid infection. The mixture was then rubbed gently onto the upper surface of dry soft leaves with the forefinger. After about 3 min, the leaves were carefully rinsed with tap water to remove excess celite. Plants were then covered with moist paper for 24 hr to shade inoculated leaves, to reduce water loss through transpiration and to prevent wilting.
2.1 Plant materials and growth in the glasshouse
Seeds of all plants were kindly provided by Ms. Lisa Smith (DAWA) and Dr. Roger Jones.
Plants used at DAWA were grown from seed in steam-sterilised peat, soil and sand mix in volume ratios of 3:1:1 respectively. Some seeds were grown in jiffy pellets and transplanted into pots at the third leaf stage. Potted plants were kept in an insect-proof air-conditioned glasshouse under natural light maintained at 20°C during the day and 18°C at night. Plants and cultures of viruses at Murdoch University were grown in a soil mix (pH 6.0) consisting of pine bark, coarse river sand and peat in volume ratios of 2:2:1 respectively and kept in a cooled
Physical Containment Level 2 (PC2) glasshouse (20 ± 5°C). Legumes were inoculated with appropriate Rhizobium spp. When necessary, plants were fertilised with Osmocote (Scotts
Europe B. V., Heerlen, Netherlands) granules. Washed river sand was used for plants which did not grow well in the soil mixes.
44
2. General Materials and Methods
2.2 Extraction of total nucleic acids from virus-infected leaf tissues
Total nucleic acids were extracted from 0.1-2.5 g leaf tissue or 2-3 virus-infected leaves.
Tissues were macerated in a 1.5 mL microcentrifuge tube with 500 µL of extraction buffer I
(50 mM Tris-HCl pH 8.5, 10 mM EDTA and 200 mM NaCl) or buffer II (200 mM Tris-HCl pH 8.0, 25 mM EDTA, 250 mM NaCl and 0.5% SDS). Larger tissue samples were ground to powder in liquid nitrogen using a mortar and pestle and a sample mixed with 500 µL of extraction buffer in a 1.5 mL microcentrifuge. An equal volume of TE (10 mM Tris-HCl, 1 mM EDTA pH 8.0) saturated phenol:chloroform:isoamyl alcohol (50:49:1) was added, the mixture vortexed for 1 min and then centrifuged at 20,200 x g for a further minute in an
Eppendorf centrifuge 5417C. The aqueous layer was clarified with an equal volume of chloroform:isoamyl alcohol (24:1) and the supernatant transferred to a fresh tube containing 0.7 volumes (vol) ice-cold isopropanol, mixed by inversion and then frozen at -80oC for at least 30 min. After thawing, the nucleic acids were pelleted at 20,200 x g for 30 min and the supernatant discarded. The pellet was washed with 500 µL of ice-cold 70% ethanol and centrifuged at 20,200 x g for 5 min. The ethanol was aspirated off, the pellet dried in a Savant
Speed-vac concentrator (Selby Scientific and Medical, Perth, Western Australia) and resuspended in 50 µL of TE. The nucleic acid extract was stored at -20°C until required.
2.3 Modifying enzymes
A range of common nucleic acid modifying enzymes was used in most experiments to synthesize, degrade, join or remove portions of nucleic acids in a controlled and generally defined manner. These included mesophilic DNA polymerases (e.g. DNA polymerase I Large
[Klenow] fragment, T4 DNA polymerase), thermostable DNA polymerases (e.g. Taq DNA polymerase, Tth DNA polymerase and rTth DNA Polymerase, XL), reverse transcriptases (e.g.
Murine leukemia virus [MuLV] and Moloney murine leukemia virus [MoMLV] reverse transcriptases), nucleases [e.g. RNA nucleases (Ribonuclease H, RNase A)], phosphatase [Calf
Intestinal Phosphatase (CIP)] and ligases (e.g. T4 DNA ligase). The techniques in which these enzymes were used, details of the protocols and suppliers are specified in sections and chapters where they were used. 45
2. General Materials and Methods
2.4 Reverse transcription (RT)
Reverse transcriptions (RT) were done with either MuLV or MoMLV reverse transcriptase obtained from Perkin Elmer (California, USA) and Lifetech Co. (Melbourne, Australia), respectively.
2.4.1 RT with MuLV reverse transcriptase
MuLV reverse transcriptase was used to transcribe RNAs into cDNAs of length less than 1kb.
In most cases, it could not reverse transcribe longer fragments. The reaction mixture consisted of 5 mM MgCl2, 1X PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 1 mM deoxynucleoside triphosphates (dNTPs), 0.5-1.0 Units (U) of RNase inhibitor, 1.25-2.5 U of
MuLV and 0.5 µM of the downstream primer or random hexamers (Perkin Elmer, California,
USA). After adding 1.5 µL of total nucleic acid extract or viral RNA (vRNA), the mixture was made up to a final volume of 10 µL with RNase-free water. The reaction was incubated at
42ºC for 30 min, followed by 95ºC for 5 min to denature the reverse transcriptase. When random hexamers were used for first strand cDNA synthesis, the reaction mixture was incubated at room temperature (25ºC) for 10 min to allow extension of the hexameric primers by the reverse transcriptase. This helped the extended primers to remain annealed to the RNA template on raising the reaction temperature to 42°C.
2.4.2 RT with MoMLV reverse transcriptase
MoMLV reverse transcriptase (obtained as Superscript™ II or III RNase H0 reverse transcriptase) was mostly used to synthesize cDNAs of more than 1 kb. For first strand cDNA synthesis, up to 5 µg (1-2 µL) of total nucleic acid or vRNA was added to 10-20 pmol downstream gene-specific primer or 50-250 ng of random hexamers and the volume made to 12
µL with sterile distilled water in a nuclease-free microcentrifuge tube. The mixture was incubated at 70°C for 15 min (to destabilise secondary structures) and quickly chilled on ice, the reaction tube was briefly centrifuged, after which 1 mM of dNTP, 0.01 M of DTT and 1X first strand buffer (25 mM Tris-HCl, [pH 8.3 at room temperature], 37.5 mM KCl, 1.5 mM
MgCl2) were added. It was further incubated for 2 min at 42°C and 200 U of reverse
46
2. General Materials and Methods transcriptase was added and pipetted gently up and down to mix. When random hexamers were used for reverse transcription, the reaction was left at room temperature (25°C) for 10 min before incubation at 42°C to allow extension of the hexameric primers. The mixture was then incubated at 42ºC for 50 min (for Superscript II) or 55ºC for 60 min (for Superscript III), followed by 70ºC for 15 min to inactivate the reaction and to degrade the reverse transcriptase.
When cDNA fragments of more than 1 kb were produced, 2 U of RNase H was added to the reaction, incubated at 37oC for 30 min to ensure that nascent RNAs were removed. All of the
RT products from MuLV RT reactions or 10% of those by MoMLV were used in PCR reactions (section 2.5).
2.5 Polymerase Chain Reaction (PCR)
2.5.1 Routine PCRs
Routine PCRs were undertaken in a variety situations, for example to amplify second strand cDNA or double stranded (ds)DNA fragments, to determine the direction of cloned fragments in vectors and for screening colonies of bacterial transformants. Taq DNA polymerase used in
PCRs was obtained from several different companies. PCR reaction volumes ranged from 10-
50 µL and consisted of 1X PCR buffer (composition dependent on manufacturer), 1.5-2.0 mM
MgCl2, 0.2 mM dNTPs, 0.1-2.5 U of Taq DNA polymerase, 0.2-0.5 µM of downstream and upstream primers with 10-200 ng of DNA. The PCR reactions were done in a 0.2 ml microfuge tube in a Perkin Elmer GeneAmp® DNA Thermal Cycler model 2400. A typical PCR thermal cycling profile was: a single initial denaturation step of 94ºC for 3 min, followed by 25-35 cycles of denaturation at 94ºC for 30 sec, primer annealing at 55ºC for 30 sec and extension at
72ºC for 1 min. A final incubation step at 72ºC for 7-10 min was included to ensure complete extension. Modifications of this protocol for specific amplifications are described in detail in subsequent chapters.
2.5.2 High Fidelity PCRs
High fidelity PCRs were done with rTth DNA Polymerase, XL (Applied Biosystems,
California, USA). This enzyme is specially-designed and optimized to produce high yields of
47
2. General Materials and Methods long or eXtra Long (XL) PCR products. It has proofreading ability, promotes efficient DNA synthesis and corrects nucleotide misincorporations that might otherwise prematurely terminate synthesis. Typically, the PCR reactions consisted of a lower reagent mix (LRM), upper reagent mix (URM) and sample DNA mix (SDM) as shown in Table 2.1. Optimal concentrations of salt and dNTPs, purity of DNA, and temperature and time profiles for PCR were determined empirically. Optimum Mg(OAc)2 levels were determined empirically by doing titrations in increments of 0.2 mM. DNTP concentrations were balanced by maintaining Mg2+ concentrations at levels 0.2 to 0.4 mM higher than the total concentrations of dNTPs. To prepare reaction samples, the LRM was mixed thoroughly, a sterile wax bead added and incubated at 80oC for 5 min followed by a hold at 25oC for 2 min. After cooling to room temperature, the URM and SDM were added above the hardened wax layer. A typical thermal cycling regime was a two-temperature profile consisting of an initial denaturation step at 94oC for 1 min, followed by 30-35 cycles of denaturing at 94oC for 1 min and anneal/extend at 65oC for 1 min per kb of DNA template, and a final extension of 10 min at 72oC. All PCRs were done in a Perkin Elmer GeneAmp® DNA Thermal Cycler model 2400.
Table 2.1:Composition of reagents for high fidelity PCR with rTth DNA Polymerase, XL
Components Final concentrations Lower reagent mix (LRM) 3.3X XL buffer II 1 X
25 mM Mg(OAc)2 0.8 - 1.5 mM 10 µM downstream primer 0.2 - 0.4 µM (20 - 40 pmol) 10 µM upstream primer 0.2 - 0.4 µM (20 - 40 pmol) 10 mM each dNTP mix 200 µM of each dNTP Sterile water to 40 µl N/A Upper reagent mix (URM) 3.3X XL buffer II 1 X rTth DNA Polymerase, XL 2 - 4 units Sterile water to 20 µl N/A Sample DNA mix (SDM) DNA template 100 – 500 ng Sterile water to 40 µl N/A
2.6 Design and primers for RT, PCR and DNA sequencing
Oligonucleotide primers used for RT of SCMoV RNA, for amplifying cDNA, and for SCMoV
DNA sequencing were either obtained from Dr. Geoff Dwyer or designed from published RNA
48
2. General Materials and Methods sequences of SCMoV-P23 (Dwyer et al., 2003; Genbank accession number AF208001).
Where necessary, these were modified with restriction endonuclease recognition sites for cloning. Similarly, primers for amplifying and sequencing non-SCMoV DNA were designed using published sequences with or without modification. Primers were designed manually and checked for “primability and stability of match” to target sequences using Amplify Version 1.2.
Primer sequences used throughout the project are given in Table 2.2 with restriction enzyme recognition sites in red font. Primers were synthesised either by Life Technologies
Inc.(Melbourne, Australia) or GeneWorks Pty Ltd (Thebarton, South Australia). Modified sequences of the universal primers T7 and SP6 with binding sites in pGEM-T and pGEM-T
Easy cloning vectors were used for sequencing inserts in recombinant plasmids. These primers were also used in PCRs together or with gene-specific primers to determine the presence and direction of cloned inserts in recombinant plasmids.
2.7 Agarose gel electrophoresis and molecular weight standard markers
All agarose gel electrophoresis procedures were carried out in apparatus supplied by Bio-Rad
Laboratories (Hercules, California, USA): the Mini Sub™ DNA cell or the Wide Mini Sub™
DNA cell apparatus. Electrophoresis buffers were made of 1X TAE buffer (40 mM Tris- acetate, 1mM EDTA) as described by Sambrook et al., (1989). Depending on the purpose and size of DNA fragments to be analysed, gels were either low melting point agarose or DNA grade agarose (Progen Industries, Inc., Quensland, Australia) at concentrations between 0.8-
1.5% made up with 1X TAE buffer. DNA fragments were mixed with loading buffer (40%
{w/v} sucrose, 0.25% bromophenol blue) and stained with ethidium bromide (0.3 µgmL-1) and electrophoresed at a constant voltage (60-100V) for 30 min to 2 hr depending on the desired separation. Gels were visualized with a UV illuminator and images captured with a UVP
Video Imager. Molecular weight standard markers used included 1kb DNA marker, 1kb Plus
DNA ladder (Life Technologies, Inc., Melbourne, Australia), 1kb and 2-log DNA markers
(New England Biolabs, NEB, Beverly, Maddison, USA).
49
2. General Materials and Methods
Table 2.2: Sequences of primers used for RT, PCR and DNA sequencing Primer Primer sequence Complementary nt in SCMoV genome 5utrrev 5'GAATTCATAAAATGATCGTC nt 55-68 CprevEcoRI 5'GAATTCTCACTAGGAGTTGAGTGC nt 4070-4087 FLfor 5'GGATCCACAAAATCGCTCGAAGAAG nt 1-19 FLrev 5'GGTACCACACGTCATGCACCCCACGAC nt 4238-4258 P1forBamHI 5'GGATTCAACAATGCCATCAGTCTC nt 69-82 P1rev 5'GAATTCTTGATCGTCTCCTTCTT nt 588-608 P2bforopt 5'GAATTCAACAATGCCCCTTGCCGAC nt 1954-1968 P2brev 5'GAATTCTCAGAGTGGATTGCAG nt 3489-3504 P2for 5'GAGCTCTGATGAATACCGACA nt 603-618 P2foropt 5'GAATTCAACAATGAATACCGACAC nt 605-618 PB-75 5'GGCGCGCCACAAAATCGCTCGAAGAAGAAAGCTTGAATCCGTTTCGC nt 1-39 PB-76 5'P-GTGTCTGAGATGTTCACTAGGAATTCAGTGCAGAAC nt 4100-4065 PB-77 5'P-CTGACTCCTTAGACTCTTGGTCGTCTCCTTCTTGAAGAGG nt 579-604 PB-78 5'P-GGACGAGCTGTACAAGATGAATACCGACAC nt 605-618 SacCPfor 5'GAGCTCTACAAGACAGGCCGCAGGGTGAGA nt 3326-3343 SacCPrev 5'GAGCTCCTAGGAGTTGAGTGCAGAACT nt 4064-4084 SacP1for 5'GAGCTCTACAAGCCATCAGTCTCAATTGAA nt 71-89 SacP1rev 5'GAGCTCTCATTCTTGATCGTCTCCTTC nt 588-608 scm003 5'TGTCCTGAATGGCCCCGGATATCCCAAGTA nt 3238-3267 scm004 5'GGATCCACACGTCATGCACCCCACGACGTC nt 4234-4258 scm005 5'GAATTCCTAGTTCTTGATCGTCTCCTTCTTGAAGAGGC nt 577-609 scm12 5'GAGCCTTTCCTTGATCCTG nt 843-861 scm14 5'CTGTGGAATGACTTAGAGC nt 2671-2689 scm16 5'AGCAGAACCGACAGAGATC nt 3381-3399 scm25 5'GACGTCGACATATGACACGTCATGCACCCCACGCA nt 4240-4258 scm2b 5'GACCGAATTCCCGCTGTATCTAAGTTCGAGAC nt 1536-1557 scm33 5'GCTCTCAGCTAAATATTGC nt 3867–3885 scm34 5'TGCCTCAGGAGAGAGCAGT nt 2212–2230 scm35 5'ATGCTCGAGCATAATGTC nt 1830-1847 scm36 5'GTACACTAGGTCCCCATG nt 2354-2371 scm37 5'TTCTACCGAGTCGTCGC nt 3002-3018 scm38 5'CCAGTTGACTGCTACACC nt 3618-3635 seq1r 5'GTCGCAGGGACTATATGCAG nt 514–533 seq2f 5'CTACGAAGGTAACCTCGTAC nt 533–552 seq2r 5'CCACAGCCAGATCTTTAGCT nt 1051-1070 seq3f 5'GGCTGTGGGTTTCGGGGCTA nt 1063–1082 seq3r 5'GTCTCCGAGCAAGAATTTCC nt 1653–1672 seq4f 5'CCTATGGGGAGTTAGACGAA nt 1575–1594 seq4r 5'GCTTGATAGAGGAGAGAGCC nt 2101–2120 seq5r 5'GGTTGCGACAGCCCCATTCC nt 2635–2654 sgProfor 5'GAATTCGAAGGATATGTTGAG nt 3016-3030 sgProrev 5'GAATTCTTTGTTTTCCCTCTCC nt 3307-3322 Other primers T7 5'TAATACGACTCACTATAGGG pGEM-T primer SP6 5'GATTTAGGTGACACTATAG pGEM-T primer gfp02 5'GGCAGATTGTGTGGACAGGT GFP primer gfpEcoRI 5'GAATTCAGTAAGGGAGAAGAAC GFP primer gfpfor 5'GGATCCAACAATGAGTAAGGGAGAAGAAC GFP primer gfpforEcoRI 5'GAATTCAACAATGAGTAAGGGAGAAGAAC GFP primer gfprev 5'GAGCTCTTATTTGTATAGTTCATCCATGCC GFP primer PB-79 5'P-GACCAAGAGTCTAAGGGAGAAGAACTCTTCACCGGAG GFP primer PB-80 5'P-CTTGTACAGCTCGTCCATGCCATGTG GFP primer scm30B 5'GTACGAATTCCAGTAAGGGAGAAGAACTCTTCACCGGAGTTGTC GFP primer scm31 5'CTTGTAGAGCTCGTCCATGCC GFP primer 35Sfor 5'TGCGCCAAGCTTGCATGCCTGCAGGTCCC 35S sequence primer 35Srev 5'GGTACCCCCGGGGATCCTCTAGAGTCCC 35S sequence primer 35S1b 5'AAGACCAAAGGGCTATTGAGAC 35S sequence primer 35SAscI 5'GGCGCGCCGGTACCCCCGGGGATCCTCTAGAGTCCC 35S sequence primer Nosfor 5'GGTACCGAGCTCGAATTTCCC T-Nos sequence primer Nosrev 5'ACCGGTGAATTCCCGATCTAGTAA T-Nos sequence primer NosforSalI 5'GTCGACGGTACCGAGCTCGAATTTCCC T-Nos sequence primer NosrevSalI 5'ACCGGTGAATTCCCGATCTAGTAA T-Nos sequence primer
50
2. General Materials and Methods
2.8 Cloning
2.8.1 Cloning of PCR products
To clone DNA, purified PCR products were ligated to pGEM-T® or pGEM®-T Easy (Promega
Corp., Annandale, NSW, Australia) cloning vectors, competent E. coli was transformed with ligated products and recombinant plasmid DNA purified from cell cultures.
2.8.1.1 Ligation of PCR products to cloning vectors
Purified PCR products were ligated to pGEM-T or pGEM-T Easy cloning vectors in a 1:3 or
3:1 insert:vector molar ratio using the following formula (Promega Corp. Technical Manual; pGEM-T and pGEM-T Easy Vector Systems, 2002)
ng of insert = ng of vector x kb of insert x insert : vector molar ratio kb size of vector
When PCR products were amplified with rTth DNA Polymerase, XL, which has 3’⇒5’ exonuclease activity, the purified PCR products were treated with Taq DNA Polymerase to add deoxyadenosine to the 3’ termini before TA-cloning into the vector. The A-tailing procedure was as follows: following purification, 100-500 ng of insert DNA was added to a mixture of 2 mM MgCl2, 0.2 mM dATP (deoxyadenine triphosphate), 5 U of Taq DNA Polymerase and the mixture made to 10 µL with deionized water. The mixture was incubated at 72oC for 30 min.
Three microlitres of the A-tailing reaction mixture (equivalent to 30-150 ng of the Taq DNA
Polymerase-treated DNA insert) was ligated to pGEM-T or pGEM-T Easy with 3 Weiss units
T4 DNA ligase for up to 2 hr at room temperature or 16 hr overnight at 4°C. A typical ligation reaction is shown in Table 2.3.
Table 2.3: Ligation of PCR products to pGEM-T or pGEM-T Easy.
Components Standard Positive Background reaction (µL) control (µL) control (µL) 2x T4 DNA ligase buffer 5.0 5.0 5.0 pGEM-T Vector (54 ng /µL) 1. 0 1. 0 1. 0 DNA insert x ------3 Weiss units T4 DNA ligase 1.0 1.0 1.0 Control DNA (2 ng/µL) ----- 2.0 ----- Distilled water to 10.0 10.0 10.0
51
2. General Materials and Methods
2.8.2 Cohesive/sticky-end cloning
DNA fragments were also ligated using cohesive/sticky ends after restriction endonuclease digestion. To do this, they were digested with common restriction enzymes at both ends, purified from solution or gel and ligated with T4 DNA ligase obtained from either NEB or
Promega Corp. When a single restriction endonuclease site was used for cloning, the 5’ termini of the linearised vector was dephosphorylated to prevent self-ligation. Ligated products were then used to transform competent E. coli and plasmid recombinant DNA purified from bacterial culture.
2.8.2.1 Restriction endonuclease digestion
Restriction endonuclease digestion of DNA was done to confirm the presence of restriction sites, to check the sizes of fragments in cloning vectors and for ligating fragments cut with similar restriction endonucleases. All restriction endonucleases and buffers were obtained from
NEB. Reactions were done with or without a supplement of 100 µgmL-1 BSA. Reactions were done using a single (with one enzyme) or a double digest (with two enzymes) or in a sequential digest with an appropriate buffer. The volumes of restriction digestion, amount of DNA and units of endonuclease used were tailored to the purpose of the digestion and specifics are provided in relevant chapters. The reactions were usually incubated at 37°C and for times optimal for the particular endonuclease either according to manufacturers’ instruction or as determined empirically. Desired restriction enzyme fragments were either purified from low melting point agarose gels or from solution.
2.8.2.2 Dephosphorylation of plasmid vectors
When a single restriction endonuclease site was used to ligate DNA fragments, the termini of the linearised vector were treated with CIP (NEB) to remove the 5’ phosphate groups and to prevent self-ligation. After digestion, the linearised vector was either purified from agarose gel or from solution (section 2.9) and treated with 0.5 U of CIP/µg of DNA or 0.01 units/pmol of
52
2. General Materials and Methods ends of DNA. The general formula used for calculating the picomoles of ends of linear dsDNA was: (µg DNA/kb size of DNA) x 3.04 = pmol of ends (Promega Corp. Technical Bulletin)].
Depending on the overhangs left by a particular restriction endonuclease, the reaction mixture was incubated at 37°C for 60 min or 37°C for 30 min followed by a further incubation at 56°C for 30 min. The reaction was stopped with the addition of 2 µL of 0.5 M EDTA followed by incubation at 60oC for 20 min after which the DNA was purified from solution and quantified using a fluorometer.
2.8.2.3 Disruption of endonuclease recognition sites in plasmids.
Undesired restriction endonuclease sites in plasmids were removed to prevent interference with further digestion analysis and to allow the use of other similar site(s) for ligation cloning. This was done by filling in recessed ends or removing overhangs of digested DNA to create blunt ends. T4 DNA polymerase was used for both removal of 3’overhangs and filling 5’overhangs whilst DNA polymerase I, Large (Klenow) fragment was used only for filling in 3’recessed ends. In reactions involving T4 DNA polymerase, digested linearised plasmid DNA was treated with 1-3 U of enzyme/µg DNA in 1X T4 DNA polymerase buffer {50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT (pH 7.9 @ 25°C)} supplemented with 100-200 µM of each dNTP and 50 µgmL-1 BSA. The mixture was incubated at 12°C for 20 min and heat inactivated by a further incubation at 75°C for 10 min. When digested DNA was treated with
DNA polymerase I, Large (Klenow) fragment, the reaction mixture consisted of 1 U of the enzyme/µg DNA, 1X EcoPol buffer (10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 7.5 mM DTT) supplemented with 33-100 µM each dNTPs. It was incubated for 15 min at 25°C after which the reaction was stopped by adding EDTA to a final concentration of 10 mM and heating at
75°C for 10 min. After either treatment, an aliquot of the mixture (containing the treated linearised plasmid DNA) was then religated with T4 DNA ligase for 1 hr and transformed with
E. coli. The recircularised plasmid was purified by alkaline lysis and analysed for success of the fill-in by restriction enzyme digestion.
53
2. General Materials and Methods
2.8.2.4 Ligation of restriction endonuclease DNA fragments
DNA fragments cut with restriction endonucleases were ligated with the longer fragment as the
‘vector’ and the shorter as an insert. 100-150 ng of ‘vector’ and 200-250 ng of insert DNA were ligated in a 20 µL reaction containing 3 Weiss units of T4 DNA ligase and 1X T4 DNA ligation buffer made up to the volume with water as shown in Table 2.4. Control ligation reactions to test the efficiency of ligase and dephosphorylation were performed, where applicable (Table 2.4). Ligation reactions were either kept at room temperature for 1 hr or overnight at 4°C before transformation.
Table 2.4: Ligation reactions for cloning restriction endonuclease DNA fragments Components of reaction Control reactions (µL) Standard reactions (µL) 1 2 3 4 *5 *6 10 X T4 DNA ligase buffer 2.0 2.0 2.0 2.0 2.0 2.0 T4 DNA ligase --- 1.0 --- 1.0 1.0 1.0 Untreated vector z z ------Treated vector ------x x x x Insert ------y y Water to 20 20 20 20 20 20 * Standard reactions were ligations with different vector to insert molar ratio. x, y, z, are empirical volumes.
2.8.3 Transformation of competent E. coli cells
2.8.3.1 Bacterial strains
The E. coli strain JM 109 (endA1, recA1, gyrA96, thi, hsdR17 (rk– , mk+ ), relA1, supE44,
∆(lac-proAB), [F’, traD36, proAB, laqIqZ∆M15]) was used as competent cells for all transformations.
2.8.3.2 Preparation of chemically-competent cells
Competent cells used were used as, or prepared from, commercial stocks using a modified rubidium chloride method (Promega's Protocols and Applications guide, 3rd ed 1996). Stocks of cells stored at -80°C were streaked on LB medium plates and incubated at 37°C overnight.
Ten mL of LB medium was then inoculated with a single colony from a plate and incubated at
54
2. General Materials and Methods
37°C overnight with vigorous shaking (225 strokes per min). Ten percent of the overnight culture was used to inoculate 100 vol of LB in a 2 L flask. The solution was incubated at 37°C on a shaker at 225 strokes/min and monitored until the OD600 was ≈ 0.45 to 0.60, measured with a spectrophotometer (Pharmacia LKB Ultrospec III). A typical growth curve of competent cells used as a guide is shown in Fig. 2.1. The cells were then chilled on ice for 10 min and collected by centrifugation at 4,500 x g for 5 min in a pre-cooled Beckman Avanti™ J-251
High Perfomance Centrifuge at 4°C. Cells were then resuspended in a volume of ice-cold trituration buffer I (100 mM RbCl, 10 mM CaCl2, 50 mM MnCl2.4H2O, 30 mM KOAc, and
15% glycerol adjusted to pH 5.8 with 0.2 M acetic acid and filter sterilized) equivalent to 40% of culture used. The mixture was incubated on ice for 5 min after which it was centrifuged for another 5 min at 4,500 x g at 4°C. The collected cells were gently resuspended by swirling in 4 mL of ice-cold trituration buffer II (10 mM MOPS, 75 mM CaCl2, 10 mM RbCl, and 15% glycerol adjusted to pH 6.5 with 1 M KOH and filter sterilized) and cooled on ice for a further
10 min. The resuspended cells were aliquoted as 100 µL vol into a 1.5 mL microcentrifuge tubes, snap-frozen in liquid nitrogen and then stored at -80°C.
) D 0.6 O y ( t i m
s 0.4 n e 600n D 0.2 at cal i t 0 p
O 1hour 1.5 2 2.15 2.3 3 3.15 3.3 3.45 Time ( hours)
jm109 xlt blue
Fig. 2.1:Growth curve of JM109 and XL1-blue competent cells
2.8.3.3 Transformation efficiency of chemically-competent cells
Bacterial cells were tested for competence before use in transformations with 0.1-10 ng of supercoiled plasmid DNA. The transformation efficiency (TrE) was determined by carrying out a transformation test on 50 µL aliquots of cells prepared as described in section 2.8.3.4 and
55
2. General Materials and Methods calculated using the following formula (Promega Corp., Protocols and Applications Guide)
Transformation efficiency (cfu/µg) = cfu on control plate x 103 ng x final dilution ng of supercoiled vector plated x 1µg
In a typical test, 100 µL aliquot of competent cells were transformed with 1 ng of supercoiled plasmid DNA. Ten microlitres of the transformation reaction (0.1 ng total DNA) was added to
990 µL of LB medium (1:100 dilution). Of that volume, a 100 µL aliquot was plated (1:1000 dilution) and the number of colonies obtained on the plate counted. For one hundred colonies counted from a plate, the TrE of the cells was calculated as {100cfu x 103 ng x 103 } ÷ {0.1 ng of plasmid x 1 µg}. An acceptable TrE was taken as at least 1 x 107 colonies per µg of plasmid
DNA.
2.8.3.4 Heat-shock transformation and bacteria growth
An aliquot of competent cells was taken from –80°C and thawed on ice. The ligation mixture was added to competent cells in a 1.5 mL microcentrifuge tube and mixed by flicking the tubes a few times and left on ice for 30 min. The cells were heat-shocked in a water bath at 42oC for
45-60 sec after which the transformation culture was left for a further 5 min on ice. 700 - 950
µL of LB broth was then added to the cells and the mixture placed on a shaker at 225 strokes per min for 1-1.5 hr at 37°C. 200-400 µL of the transformation culture was plated onto
LB/antibiotic plates. When a higher number of colonies were needed, the cells were pelleted at
1,000 x g for 10 min and resuspended in 100 µL of the supernatant and plated out. The plates were then incubated overnight at 37°C for 16-24 hr. After transformation of E. coli and incubation on antibiotic selective plates, single isolated colonies, positive after analysis, were inoculated to 5-10 mL of LB or CircleGrow® (Bio 101, California, USA) broth with 100 µgmL-
1ampicillin in 20 mL McCartney bottles and incubated on a shaker at 225 strokes/min for 12-18 hr. Culture of cells in CircleGrow allowed purification of plasmid DNA after 6-8 hr.
56
2. General Materials and Methods
2.8.4 Selection for transformants
The pGEM-T and pGEM-T Easy vectors are capable of α-complementation. They contain T7 and SP6 RNA polymerase promoters flanking a multiple cloning region (MCR) within an α- peptide coding region of the enzyme β-galactosidase. Insertion of DNA at the MCR inactivates the α-peptide and allows recombinant clones to be directly identified by colour screening. The vectors also contain a gene for ampicillin resistance permitting selection with this antibiotic.
Transformants of cells with these vectors were selected in two ways; antibiotic selection and blue/white screening. Antibiotic selection was done by spreading aliquots of transformed cells on LB plates containing 100 µgmL-1 ampicillin. Transformants were selected with 5-bromo-4- chloro-3-indoly-B-D-galactopyranoside [(X-Gal), Progen Industries Ltd, Queensland,
Australia] and isopropyl-B-D-thiogalactoside [(IPTG), Progen Progen Industries Ltd,
Queensland, Australia]. LB/ampicillin plates were spread with 20 µL of 50 mgmL-1 X-Gal and
100 µL of 100 mM IPTG and the plates warmed to 37°C for 30 min before plating transformants. Plates were incubated at 37°C for 16 -24 hr. White colonies generally contained inserts whilst the blue ones did not. White colonies were analysed by PCR or plasmid mini preparation and with an appropriate restriction endonuclease to confirm presence and direction of inserts in recombinant plasmids.
2.8.5 Analysis of transformants
2.8.5.1 Analysis of transformants by PCR
The presence and direction of inserts in selected colonies were determined by PCR using modified T7 and SP6 primers or either of the two with a sequence-specific primer. In transformants containing inserts, the amplicon included insert plus 140 bases of the plasmid vector whereas in those colonies lacking inserts, the 140 bp fragment was amplified. Individual isolated colonies were picked with sterile pipette tips and resuspended in 15-20 µL of sterile water of which 5 µL was used as PCR template. The PCR reactions were done in a final volume of 25 µL comprising 4 mM MgCl2, 200 mM dNTPs, 10 pmol of primers, 1X PCR
57
2. General Materials and Methods buffer, and 0.5-1.0 unit Taq DNA polymerase. PCR cycling conditions were an initial denaturing at 94oC for 3 min followed by 25 cycles of denaturing at 94oC for 30 sec, annealing at 55oC for 30 sec and extension at 72oC for 1 min, with a final extension of 7-10 min at 72oC.
2.8.5.2 Analysis of transformants by plasmid DNA miniprep/restriction digestion
Following antibiotic selection and analysis of transformants by PCR, colonies of desired transformants were grown overnight at 37°C in 5-10 mL of LB medium containing 100 µgmL-1 of ampicillin with shaking (225 rpm). Plasmid DNA was purified from bacterial cell culture using an alkaline lysis method described in section 2.9.3.1. An aliquot of plasmid DNA was digested with appropriate enzyme(s) to confirm the presence, size and direction of inserts in a vector. When PCR products were cloned in pGEM-T Easy, a single digest with EcoRI or NotI released the insert. Alternatively, and also in pGEM-T, a double digest with any two restriction endonucleases with sites flanking either side of the MCR released the insert in a plasmid.
Linear, non-recombinant pGEM-T plasmids were run alongside digested products to act as a
DNA marker and to confirm the presence of a desired insert.
2.9 DNA purification
DNA fragments were purified from agarose gels, from solutions or from bacterial cell cultures.
2.9.1 DNA purification from agarose gels
PCR products, linearised plasmid DNA and other non-plasmid DNA fragments were normally purified from low melting point agarose gels using the BRESAClean (UltraClean)TM DNA
Purification Kit (MO BIO Laboratories, Inc, California, USA). After agarose gel electrophoresis desired DNA fragments were excised with a sterile scalpel blade and placed in a 1.5 mL tube containing 3 vol of BRESA-SALTTM and incubated for 5 min at 55°C with occasional shaking. Five to ten microlitres of BRESA-BINDTM was added and the mixture incubated at room temperature with constant or occasional mixing to keep silica in suspension and ensure efficient binding of DNA. The BRESA-BINDTM/DNA complex was pelleted for
58
2. General Materials and Methods
15-30 sec at 20,000 x g and the supernatant discarded. The pellet was resuspended with
BRESA-WASHTM in an equivalent volume to the salt used, pelleted again for 15-30 sec, supernatant discarded and pellet dried in a Speed-vac. The DNA was recovered by resuspending the white pellet in sterile distilled water (in a volume at least twice that of the matrix used) and incubated for 5 min at 55°C, after which the suspension was centrifuged for 1 min at 20,000 x g. The supernatant containing the DNA was then removed and transferred to a new tube avoiding any trace of the silica matrix.
2.9.2 DNA purification from solutions
DNA purification from solution was done with UltraCleanTM PCR clean-up Purification Kit
(MO BIO Laboratories, Inc., California, USA) or by extraction with phenol:chloroform:isoamyl alcohol and/or salt/ethanol precipitation.
2.9.2.1 DNA purification with UltraClean™ PCR Clean-up kit
The UltraClean™ PCR Clean-up Purification Kit was used to purify PCR products and linearised plasmid DNA from solution. Five volumes of SpinBind (Guanidine
HCl/Isopropanol) was added to the DNA solutions and mixed by pipetting or by vortexing.
The DNA/SpinBind mixture was then transferred to a spin filter unit and centrifuged at 20,000 x g for 30-60 sec. The flow through was discarded by decanting and 300 µL of SpinClean buffer added to the spin filter after which the unit was centrifuged for 10-30 sec at 20,000 x g.
The flow through was discarded and the unit centrifuged to remove residual buffer. Spin filter was transferred to a clean 1.5 mL tube and the DNA eluted with 50 µL of sterile water at
20,000 x g for 60 sec. When concentrated DNA was required, 0.04-0.1 vol of 5 M NaCl and
2.0-2.5 vol of ice-cold 100% ethanol was added to the DNA solution, mixed and kept at -20°C for at least 5 min. The mixture was then centrifuged at 12,500 x g for between 5-30 min after which the ethanol was decanted. Residual liquid was removed by evaporating in a Speed-vac.
Precipitated DNA was then resuspended in a desired volume with sterile water.
59
2. General Materials and Methods
2.9.2.2 DNA precipitation
DNA was precipitated with either salt/ethanol solution or with 0.7-0.8 vol of ice-cold isopropanol. Salt/ethanol solutions were made of 0.1 vol of 3 M NaOAc, or 5 M NaCl, pH 5.2 or 0.5 vol of 7.5 M NH(OAc)4 with 2 or 2.5 vol of 100% ethanol. DNA solution was mixed with salt/ethanol solution or isopropanol and left at -20oC for at least 30 min after which it was centrifuged at 12,000 x g for 30 min at room temperature or at 4oC. The supernatant was discarded and the pellet washed with ice-cold 70% ethanol by centrifugation at 12,000 x g for 5 min after which the DNA pellet was dried in a Speed-vac. It was then resuspended in a desired amount of TE or water.
2.9.2.3 Phenol:chloroform DNA extraction
Extraction of DNA from solution using phenol:chloroform involved the addition of an equal volume of TE saturated phenol:chloroform:isoamyl alcohol (25:24:1) to the DNA solution.
The mixture was vortexed for 1-2 min and centrifuged at 12,000 x g for 5 min. The upper aqueous phase was transferred to a fresh tube and the organic phase back-extracted with a small volume of TE to increase recovery. Depending on the level of purity required, the solution was extracted again with an equal volume of chloroform:isoamyl alcohol (24:1), vortexed for 30 sec, centrifuged at 12,000 x g for 2 min and the upper aqueous phase transferred to a fresh tube.
The DNA was then precipitated as in section 2.9.2.2.
2.9.3 Plasmid DNA purification from bacterial cell culture
Plasmid DNA from cell cultures was purified by alkaline lysis or by using one of two kits from
Qiagen Pty Ltd, Victoria, Australia: the QIAprep Spin Miniprep Kit or Qiagen Plasmid Mini purification Kit.
2.9.3.1 Alkaline lysis method of plasmid purification
Bacterial cell cultures were transferred into 1.5 mL centrifuge tubes and the cells pelleted at
20,000 x g for 1-2 min at room temperature. The supernatant was removed and pelleted cells resuspended in 250 µL of resuspension buffer (50 mM Tris.HCl, pH 8.0; 10 mM EDTA; 100
60
2. General Materials and Methods
µgmL-1 RNase A, kept at 4°C). A further 250 µL of lysis buffer (200 mM NaOH, 1% SDS) was added to the suspension and the contents gently mixed by inverting the tube 4-8 times and allowed to stand for less than 5 min after which 350 µL of neutralization buffer (3.0 M KOAc, pH 5.5 kept at 4°C) was added, mixed by inversion and incubated on ice for 10 min. The tube was centrifuged at 20,000 x g for 10 min and the supernatant containing the DNA transfered into a fresh tube containing 0.8 vol ice-cold isopropanol. The DNA was then precipitated from solution by centrifugation at 20,200 x g for 30 min, the supernatant discarded and the DNA washed with ice-cold 70% ethanol and centrifuged for 5 min at 20,200 x g. The pellet was finally dried in a Speed-vac and resuspended in 50 µL of water.
2.9.3.2 QIAGEN Plasmid Mini purification method
This method of plasmid DNA purification uses the same resuspension, lysis and neutralisation buffers as above. After addition of these buffers to the pelleted cells, the mixture was centrifuged 20,200 x g for 10 min. During this period a QIAGEN tip was equilibrated with 1 mL of equilibration buffer (750 mM NaCl; 50 mM MOPS, pH 7.0; 15% isopropanol;
Triton®X-100) which was allowed to flow through the column by gravity. The supernatant was removed promptly after centrifugation to avoid protein precipitation and applied to the
QIAGEN tip and left to enter the resin by gravity flow. The QIAGEN tip was washed four times, each time with 1 mL of wash buffer (1.0 M NaCl; 50 mM MOPS, pH 7.0; 15% isopropanol). The DNA was eluted with 0.8 mL of water and collected into clean 1.5 ml microfuge tubes. The DNA was then precipitated by mixing with 0.7 vol of isopropanol, centrifuged for 30 min at 20,000 x g, the supernatant decanted and washed with 1 ml of 70% ethanol for 5 min at 20,000 x g. The DNA was then dried in a Speed-vac and resuspended in
50 µL of water.
2.9.3.3 QIAprep Spin Miniprep plasmid purification
This method was mainly used to purify plasmid DNA for sequencing. Bacterial cell cultures were pelleted and treated with resuspension and lysis buffer as in the section above, after which
61
2. General Materials and Methods
350 µL of neutralization buffer N3 specially made for use with the resin in the QIAprep spin column was added and mixed immediately to avoid local precipitation. The suspension was then centrifuged at 20,000 x g for 10 min. A QIAprep spin column was placed in a 2 ml collection tube and the supernatant applied to the resin followed by centrifugation for 30-60 sec at 20,000 x g after which the flow through was discarded. After this, 500 µL of buffer PB was added to the spin column and centrifuged at 20, 200 x g for 30-60 sec. The flow through was discarded followed by a washing of the column with 0.75 mL of buffer PE (wash buffer) at
20,200 x g for another 30-60 sec. A further minute of spinning at 20,200 x g was necessary to remove residual wash buffer. The QIAprep spin column was then placed in a clean 1.5 mL centrifuge tube and the DNA eluted with 50 µL of sterile distilled water. After leaving to stand for 1 min, the column was centrifuged for 1 min to ensure complete elution of DNA.
2.10 DNA quantitation/quantification
The presence of DNA in preparations/solutions was observed visually on agarose gels stained with 0.3 µgmL-1 ethidium bromide under UV light. Accurate measurements were obtained with the Hoefer TKO 100 DNA fluorometer (Pharmacia Biotech, California, USA) which uses fluorescence emitted by Hoechst 33258 solution at 460 nm when mixed with dsDNA.
2.11 DNA Sequencing
Complete DNA sequences of cloned inserts were obtained by sequencing double-stranded templates. Modified T7 and SP6 primers were used to sequence all clones in pGEM-T and pGEM-T Easy vectors. Internal sequences of SCMoV cDNA inserts in the vectors were determined with specific primers designed to amplify both strands (Table 2.2). For non-
SCMoV clones, internal sequences and junctions of ligated DNA fragments were determined with primers designed from sequences extracted from public databases. In most cases, sequences of both strands of dsDNA were determined.
62
2. General Materials and Methods
2.11.1 Sequencing reactions
Sequencing was done with the Taq DyeDeoxy™ Terminator Cycle Sequencing Kit from
Applied Biosystems Industries [(ABI), California, USA] using either Rhodamine or BigDye™
Terminator Cycle Sequencing chemistry. Sequencing reactions were half the volumes recommended by ABI, consisting of 300-800 ng of plasmid DNA added to 4 µL of Terminator
Ready Reaction Mix, 1.6/3.2 pmol of primers, made to a final volume of 10 µL with sterile distilled water in a 0.2 mL microfuge tube. The reactions were incubated in a Perkin Elmer
Cetus Thermal Cycler 2400 at 96°C for 2 min followed by 25 cycles of denaturation at 96°C for 15 sec, annealing at 50°C for 5 sec and and extension at 60°C for 4 min.
2.11.2 Purification of extension products for sequencing
After the PCR sequencing reaction, extension products were cooled, briefly centrifuged and then transferred into a 0.5 mL centrifuge tube containing 2.5 vol of 100% ice cold ethanol and
0.1 vol of 3 M NaOAc, pH 5.2. The mixture was vortexed briefly and kept at -20°C for 20 min after which the DNA was precipitated at 20,200 x g for a further 30 min, washed with 125 µL of 70% ice cold ethanol at 20,200 x g for 5 min and dried in a Speed-vac. Sequences were processed by Ms Frances Brigg, SABC, Murdoch University on either an ABI 373 or 377 DNA
Sequencer.
2.11.3 Data analysis
Raw nucleotide sequence data was examined and edited using Seqed V1.0.3 (ABI, California,
USA). Further analyses of sequences were done using multifunctional programmes available through WebANGIS (Australian National Genomic Information Services, www.angis.su.oz.au), Vector NTI V6.0 or BioEdit Sequence Alignment Editor V5.0.9 (Hall,
1999). Restriction enzyme analysis of sequences were done with NEBcutter V2.0
(http://tools.neb.com/NEBcutter2/index.php) and DNA Strider V1.2. Amplify V1.2 was used for analysing the primability and stability of match of designed primers to target sequences.
Sequence databases used included EMBL and GenBank, curated by the National Centre for
Biotechnology Information (NCBI). 63
Chapter Three
Host Range and Symptomatology of SCMoV
3. Host Range and Symptomatology of SCMoV
3.0 Introduction
Most of world’s population is fed by a few species of plants, including three cereals - rice, wheat and maize; two sugars - beet and cane; three root crops - potato, sweet potato, and cassava; grain legumes - chickpea, lentils, peas, common beans, cowpea and soybean; two oil crops - groundnuts and canola, and also coconut and banana. About 15% of viruses recognised by the International Committee for Taxonomy of Viruses (ICTV) have names derived from these host plants. Not surprisingly, much of plant virology research has been concentrated on these viruses. More recently, advances in biotechnology have made almost all plant viruses, irrespective of hosts, potentially important for research: in using engineered virus genomes to study virus-host interactions and as vectors for studying the function of other genes. Viruses of less economically important plants or those with restricted host range have thus become more relevant to virologists. In widening the research scope with these viruses, new hosts, including indicators and those of economic importance for some plant families, have been found.
Indicator plants (e.g. Chenopodium spp. [Chenopodiaceae]; Brassica juncea [Cruciferae];
Phaseolus vulgaris, Vicia faba [Fabaceae]; Cucumis sativus [Curcubitaceae] and Nicotiana glutinosa [Solanaceae]) which are highly susceptible to infection by a wide range of plant viruses and provide good hosts for the maintenance of virus cultures and for virus multiplication. Selection of a suitable virus isolate for research is important because cultivars of the same species and species of the same genus may respond differently to different strains or isolates of a virus.
Use of virus-based vectors for gene silencing studies requires that the virus replicates in a host of interest with little or no inhibition and more importantly, the host becomes systemically infected. To develop SCMoV-based vectors for functional analysis, the host range needs to be known to determine how widely such a gene silencing vector may be used. Also, a detailed study of symptom development in different hosts will provide information that will be useful for subsequent SCMoV-based vector expression studies.
65
3. Host Range and Symptomatology of SCMoV
3.1 Aim of Chapter 3
This chapter describes an extensive host range study involving 52 species belonging to 25 different genera. The work was undertaken to identify new and suitable hosts among legumes including crop, pastures and non-legumes. Systemic hosts were particularly sought for use as model plants for infection studies using SCMoV gene silencing vectors. The information gained would also be of practical value since the results would help establish the potential threat of SCMoV to newly released cultivars of alternative pasture and forage legumes under development, for example, at the Centre for Legumes and Mediterranean Agriculture, WA, as well as crop and alternative crop legumes that could be used in appropriate farming systems.
3.2 Materials and methods
Between five and ten replicates of potential host plants were manually inoculated with sap from
SCMoV-infected plants, observed for development of virus-induced symptoms, and tested for local and systemic infection by RT-PCR and/or ELISA.
3.2.1 Potential host plants and growth conditions
A total of 61 plant genotypes representing 52 species from 25 different genera and belonging to
7 families were studied for their response to SCMoV infection. These included established and alternative crop legumes (24 genotypes of 16 species) [Table 3.1], pasture, alternative pasture and forage legumes (28 genotypes of 24 species) [Table 3.2 & 3.3], and 12 indicator plant species belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae,
Cucurbitaceae and Solanaceae [Table 3.4]. Plants were grown using conditions in section 2.1 and kept at DAWA. Lupinus spp. (lupin) and Vicia faba (faba bean) were grown in washed river sand.
3.2.2 Virus inoculations
Four WA SCMoV isolates (SCMoV-AL, -MB, -MJ and -P23) were used as source of inoculum for infecting test plants. The isolates were cultured in T. subterraneum cv Woogenellup maintained under glasshouse conditions at DAWA. Eighty percent of leaves on 3 or 5 - week
66
3. Host Range and Symptomatology of SCMoV old seedlings of test plants were mechanically inoculated with SCMoV-P23. Medicago truncatula Gaertn. (barrel medic) cv. Caliph and P. sativum cv. Greenfeast were inoculated with all four isolates to compare the reactions of these two plants to published reports. Test plants, which did not become infected after five weeks of inoculation, were inoculated for the second time. Mechanical inoculations were done as described in section 2.0.
3.2.3 Detection of infection by RT-PCR
Leaf samples taken from all plants challenged with infective sap of SCMoV and from control uninoculated plants were tested by RT-PCR. cDNAs of total RNA from test and control plants
(section 2.2) were made using 1.25 U of MuLV reverse transcriptase and 0.5 U of RNase inhibitor (section 2.4.1). Amplification by PCR was done with 1.25 U AmpliTaq® DNA
Polymerase (Perkin Elmer, California, USA) with 1µL total RNA and 10 pmol of primers
(sections 2.5.1). Using the oligonucleotide primer pair scm37 and scm34 in RT-PCR, an 807 bp fragment was amplified corresponding to nt 2212 to 3019 of the SCMoV-P23 genome
(GenBank acc. no. AF208001). PCR cycling conditions were: a single initial denaturation step of 94ºC for 3 min, followed by 35 cycles of denaturation at 94ºC for 30 sec, primer annealing at
55ºC for 30 sec and extension 72ºC for 1 min. A final extension step at 72ºC for 7 min was included to ensure complete extension.
3.2.4 Detection of infection by Enzyme-linked Immunosorbent Assay (ELISA)
Inoculated and tip leaves from test and control plants (0.1 – 2.0 g) were ground in 20 mL of phosphate buffered saline (PBS) solution [10 mM potassium phosphate, 150 mM sodium chloride (pH 7.4)] containing 5 mLL-1 of Tween-20 and 20 gL-1 of polyvinyl pyrrolidone
(PVP). Sap was extracted using a leaf press (Pollahne, Germany) and collected in labelled plastic tubes and used as the source of antigen. ELISA assays for SCMoV using direct double antibody sandwich method (Clark and Adams, 1977) were done by Ms Lisa Smith (DAWA).
All samples were tested in paired wells in microtitre plates. Plates were coated with a polyclonal antibody raised to SCMoV in a high pH carbonate buffer and incubated at 37oC for
67
3. Host Range and Symptomatology of SCMoV
4 hr. The plates were then washed with wash buffer PBST [PBS + 0.5 mL Tween-20 L-1] three times, leaving it for three minutes after each wash and blot-dried by tapping upside down on tissue paper. Sap samples were then added to wells and left at 4oC overnight. The plates were washed as before and then alkaline phosphatase conjugated with rabbit polyclonal antiserum to SCMoV (supplied by Dr. Roger Jones) was added. After incubation at 37oC for 4 hr, plates were washed three times with PBST and 200 µL of freshly prepared substrate [0.6 mgmL-1 of p-nitrophenyl phosphate in 100 mLL-1 of diethanolamine, pH 9.8] was added to each well and the plates incubated at room temperature for 30 min. Absorbance (A405 nm) was measured in a
Titertek Multiscan plate reader (Flow Laboratories, Finland) and values greater than three times those of healthy leaf sap were considered positive. The polyclonal antiserum to SCMoV was prepared by Dr. J. I. Cooper and Ms Dora Li at the SABC, WA.
3.3 Results
3.3.1 Symptomatology of SCMoV
All plants were observed frequently for virus-induced symptoms in the inoculated or uninoculated leaves and the results recorded. For each plant, symptom development was recorded from 1 week after inoculation and continued for 6-9 weeks depending on the length of time symptoms developed.
3.3.1.1 Development of local symptoms in test plants
Only five plant species showed any symptoms of SCMoV-P23 infection in inoculated leaves.
These were P. sativum cv. Greenfeast, Lathyrus clymenum, Trifolium spumosum 24BP,
Trigonella balansae SA5054 and C. quinoa. Local irregularly shaped necrotic spots developed in inoculated leaves of L. clymenum, T. balansae and P. sativum cv. Greenfeast between 6 days and 2 weeks after inoculation (Fig. 3.1a, 3.1b and 3.1c). In addition, there were local necrotic rings and local veinal necrosis in L. clymenum and T. balansae, respectively. The necrotic spots increased in size and became severe with time. Three other cultivars of P. sativum; cvs
Dundale, Laura and Wirrega were symptomless in inoculated leaves. Similar local symptoms
68
3. Host Range and Symptomatology of SCMoV developed in P. sativum cv. Greenfeast when it was challenged with three other SCMoV isolates (SCMoV-AL, -MB and -MJ).
Fig. 3.1a: Spreading necrotic spots Fig. 3.1b: Spreading necrotic spots and Fig. 3.1c: Necrotic spot lesions caused by and veinal necrosis caused by rings caused by SCMoV infection in SCMoV infection in inoculated leaves of SCMoV infection in inoculated inoculated leaves of T. balansae P. sativum cv Greenfeast leaves of L. clymenum
Of the 15 Trifolium spp. tested only T. spumosum 24BP developed necrotic and chlorotic spots on inoculated leaves. Symptoms were observed in the first week after inoculation. C. quinoa developed obvious chlorotic spot lesions in inoculated leaves 7-14 days after inoculation (Fig.
3.1d). Three to four days after the appearance of symptoms, the spots had markedly increased in number, were of similar size and shape, and covered the whole leaf. No other plant was symptomatically infected in the inoculated leaves.
Fig. 3.1d: Local necrotic and chlorotic spots on inoculated leaves of Chenopodium quinoa
3.3.1.2 Systemic symptom development in crop and alternative crop legumes
Three crop (chickpeas, lentils, and narbon bean) and two alternative crop (L. ochrus and L. clymenum) legume species showed systemic symptoms after infection with SCMoV-P23 (Table
3.1). Seven to 9 days after inoculation, the shoot tips of the two cultivars of chickpea (Cicer arietinum L. cvs. Kaniva and Sona) were observed to bend away from the sun (Fig. 3.2).
69
3. Host Range and Symptomatology of SCMoV
Subsequently, the leaves showed vein clearing, chlorosis (pallour), were smaller, and the plants
became stunted two weeks after symptoms first appeared. Noticeable differences in the
severity and development of symptoms between the two chickpea types observed were:
reddening and tip death of the shoots of cv Sona, and chlorosis in cv Kaniva. Lens culinaris
Med. cv. Matilda (lentil) showed mottling and a very slight reduction in leaf size after two
weeks of infection.
Table 3.1: Reactions of different species of crop and alternative crop legumes to inoculation with SCMoV-P23 Species Common name Genotype Symptoms (Accession/ Inoculated Non-inoculated leaves breeding leaves and shoots line/cultivar) Cicer arietinum L. Chickpea ‘kabuli’ cv. Kaniva SI VC, C, RLS, TB, SSt C. arietinum L. Chickpea ‘desi’ cv. Sona SI VC, R, RLS, TB, TD, SSt Lathyrus cicera L. Dwarf Chickling ATC 80521 SI NI L. clymenum L. None C7022 LNR, LNS C L. ochrus (L.) D.C. Grass Pea ATC 80532 SI C, MM, VB, LD, SNS, L. ochrus (L.) D.C. Grass Pea ATC 80123 SI C, M, LD, SNS, RLS, SSt L. sativus L. Grass Pea ATC 80488 SI SS L. sativus L. Grass Pea ATC 80517 SI NI Lens culinaris Med. Lentil cv. Matilda SI M, RLS Lupinus albus L. White lupin cv. Kiev mutant SI NI L. angustifolius L. Narrow-leafed lupin cv. Gunguru SI NI L. luteus L Yellow lupin cv. Wodjil SI NI Phaseolus vulgaris L Climbing bean cv. Stringless blue NI NI Pisum sativum L. Field pea cv. Greenfeast LNS, LVN NI P. sativum L. Field pea cv. Dundale SI NI P. sativum L. Field pea cv. Laura SI NI P. sativum L. Field pea cv. Wirrega SI NI Vicia benghalensis L Purple vetch cv. Popany SI NI V. faba L. Faba bean cv. Ascot NI NI V. faba L. Faba bean cv. Fiesta NI NI V. narbonensis L. Narbon bean None SI C, MM, RLS, St V. narbonensis L. Narbon bean SA26554 SI VC, C, M, LD, RLS, St V. sativa L. Common vetch cv. Namoi SI NI Vigna unguiculata Cowpea None NI NI (L.) Walp.
Symptom codes: LNS = local necrotic spot; LNR = local necrotic rings; LVN = local veinal necrosis; SI = symptomless infection in inoculated leaves; VC = vein clearing; C = chlorosis; R = Reddening; M = mottle; MM = mild mottle; VB =vein banding; LD = leaf deformation/distortion; SNS = systemic necrotic spotting and/or streaking; RLS = reduced leaf size; SRLS = severe reduced leaf size; St = stunting; SSt = severe stunting; SS = symptomless systemic infection; TB = shoot tip bending; TD = shoot tip death; NI = No infection detected from observation.
70
3. Host Range and Symptomatology of SCMoV
Fig. 3.2: Chlorosis (pallour), vein clearing, and Fig. 3.3: Pallour, mottle and leaf deformation in Shoot tip bending in newly emerged leaves of leaves of V. narbonensis SA26554 caused by C. arietinum infection with SCMoV.
Of four species of Vicia challenged with SCMoV-P23, only one developed observable systemic symptoms. The two cultivars of Vicia narbonensis developed chlorosis, mottling, reduction in leaf size, and stunting. In addition to developing the severe form of these symptoms, the genotype SA26554 had distinct vein clearing and leaf deformation, which was absent in the other genotype (Fig. 3.3). The three other species of Vicia; V. benghalensis cv Popany, V. faba cvs Ascot and Fiesta, and V. sativa cv Namoi did not develop systemic symptoms.
Fig. 3.4a: Systemic necrotic Fig. 3.4b: leaf deformation, reduced leaf sizes and stunting spots in new leaves of L. ochrus in new leaves of L. ochrus (left) compared to a healthy plant (right) Two of the four species of Lathyrus tested showed systemic symptoms. The newly expanded leaves of L. clymenum were chlorotic and that was the only symptom observed. The first sign of symptom development in both genotypes of L. ochrus (ATC 80123 and ATC 80532) was a mild mottling followed by an overall pale colour of the leaves. New leaves that emerged 4 weeks post-inoculation were severely reduced in size. There was also a characteristic vein banding in the leaves of genotype ATC 80532 and severe stunting in genotype ATC 80123 that could not be attributed to damage from the mechanical inoculation procedure.
71
3. Host Range and Symptomatology of SCMoV
In addition, the deformed new leaves of both genotypes developed systemic necrotic spots (Fig.
3.4a and 3.4b). L. cicera L. and both types of L. sativus (ATC 80488 and ATC 80517) did not show any signs of systemic infection. There were no observable systemic symptoms on any other crop or alternative crop legume. Although P. sativum cv. Greenfeast developed local symptoms, there was no observable systemic symptoms when challenged with any of the four isolates of SCMoV.
3.3.1.3 Systemic symptom development in Trifolium spp.
The development and expression of disease symptoms in newly expanded leaves for 10 of the
12 Trifolium species tested was similar to that observed in infected subterranean clover plants, the natural host of SCMoV (Table 3.2). This genus was particularly susceptible to infection, with systemic disease symptoms usually appearing within 10 to 14 days after inoculation. In general, the first observable symptom of infection was vein clearing of varied severity, followed by chlorosis (especially of the interveinal regions), which gave the plants an overall mottled appearance. The newly emerged leaves were often deformed and significantly smaller in size, resulting in the plant becoming stunted as it matured. In addition to these general symptoms, T. isthmocarpum Brot Mar14.10 showed a curling of its shoot tip and T. clypeatum
CFD13 had severe vein clearing that persisted through to plant maturity and developed a characteristic rusty appearance (necrosis) on most leaves (Fig. 3.4 and 3.5). Also the two genotypes of T. dasyrum and T. spumosum 24BP developed systemic necrotic spots as well as leaf curling in the latter.
Fig. 3.4. Persistent severe vein clearing in Fig. 3.5. Obvious mottle and necrotic streaking a new leaf of T. clypeatum infected in a new leaf of T. clypeatum infected systemically with SCMoV. systemically with SCMoV.
72
3. Host Range and Symptomatology of SCMoV
Although T. spumosum 24BP, and not T. spumosum 87144, developed local symptoms, the
development and expression of systemic symptoms did not differ from each other or from the
other Trifolium spp. T. vesiculosum cv Seelu was difficult to infect and only became infected at
the third attempt. Moreover, it took 7 weeks for clearly defined symptoms to develop and these
were only mild in comparison with the symptoms shown by other infected Trifolium species.
T. glanduliferum ATC8718 + ATC87182 did not develop observable systemic symptoms and
T. hirtum GCN39 failed to become infected after several attempts.
Table 3.2: Reactions of different species of Trifolium spp to inoculation with SCMoV-P23 Species Common name Genotype Symptoms (Accession/ Inoculated Non-inoculated leaves and breeding leaves shoots line/cultivar) Trifolium cherleri L Cupped clover cv. Lisare SI VC, RLS, St T. clypeatum L. Helmet clover CFD13 SI SVC, C, SM, SNS, RLS, St T. dasyurum C. Eastern Star clover 24GCN39 SI VC, C, SM, LD, SNS, RLS, SSt T. dasyurum C. Eastern Star clover 42BT SI VC, C, SM, VB, LD, SNS, RLS, T. glanduliferum Boiss Gland clover ATC87181+ SI NI ATC87182 T. hirtum L. Rose clover GCN39 NI NI T. incarnatum L. Crimson clover cv. Caprera SI VC, MM, LD, RLS, St T. isthmocarpum Brot. Moroccan clover MAR14.10.1 SI VC, SM, LC, LD, St T. michelianum Savi. Balansa clover Paradana SI VC, C, M, LD, RLS, St T. purpureum Lois Purple clover 136780 SI SVC, M, LD, RLS, St T. resupinatum L. Persian clover Persian Prolific SI VC, C, MM, MLD, RLS, St T. spumosum L. Bladder clover 87144 SI VC, MM, RLS, St T. spumosum L. Bladder clover 24BP LNS, LCS VC, MM, LC, SNS, RLS, MSt T. subterraneum L Sub clover Woogenellup SI VC, C, M, SLD, RLS, St T. vesiculosum Savi Arrowleaf clover cv. Seelu SI VC, C, MM, St
Symptom codes: LCS = local chlorotic spots; LNS = local necrotic spot; SI = symptomless infection in inoculated leaves; VC = vein clearing; SVC = severe vein clearing; C = chlorosis; MM = mild mottle; M = mottle; SM= severe mottle; VB =vein banding; LC = leaf curling; LD = leaf deformation/distortion; MLD = mild leaf deformation/distortion; SLD = severe leaf deformation/distortion; SNS = systemic necrotic spotting and/or streaking; RLS = reduced leaf size; St = stunting; MSt = mild stunting; SSt = severe stunting; NI = No infection detected from observation.
3.3.1.4 Systemic symptom development in alternative pasture/forage legumes
In addition to developing local symptoms, T. balansae was also symptomatic in its newly
expanded leaves (Fig. 3.6). Development and expression of systemic symptoms were similar to
those described for the Trifolium species above. No other alternative annual pasture and forage
legume showed any symptoms in newly expanded leaves (Table 3.3. These included Biserrula
73
3. Host Range and Symptomatology of SCMoV
pelecinus and M. polymorpha cv Circle valley which were symptomlessly infected in
inoculated leaves, and also five species of Medicago: Medicago; M. littoralis cv. Harbinger, M.
murex cv. Zodiac, M. polymorpha cvs. Circle valley and Serena, M. sativa cv. Aquarius, and M.
truncatula cv Caliph.
Fig. 3.6: Severe mottle and leaf deformation in leaves of T. balansae systemically infected with SCMoV-P23
Table 3.3: Reactions of different species of pasture/forage to inoculation with SCMoV P23. Species Common name Genotype Symptoms (Accession/ Inoculated Non-inoculated cultivar /breeding leaves leaves and line) shoots Biserrula pelecinus L Biserrula Casbah SI NI Dorycnium hirsutum Ser. Hairy canary clover SA1111 NI NI Hedysarum coronarium.L Sulla Grimaldi NI NI Medicago littoralis Rohde ex Strand medic Harbinger NI NI Loisel. M. murex L. Murex medic Zodiac NI NI M. polymorpha L. Burr medic Circle Valley SI NI M. polymorpha L. Burr medic Serena NI NI M. sativa L. Lucerne, Alfalfa Aquarius NI NI M. truncatula Gaertn. Barrel medic Caliph NI NI Melilotus alba Medic. White sweet clover 35635 NI NI Ornithopus compressus.L Yellow serradella Santorini NI NI O. sativus Broth. Pink serradella Cadiz NI NI Trigonella balansae Boiss & Trigonella SA5054 LNS VC, SM, SLD, Reuter NL, RLS, SSt Symptom codes: LNS = local necrotic spot lesions; SI = symptomless infection in inoculated leaves; VC = vein clearing; SM = severe mottle; SLD = severe leaf deformation/distortion; RLS = reduced leaf size; SSt = severe stunting; NI = No infection detected from observation.
74
3. Host Range and Symptomatology of SCMoV
3.3.1.5 Systemic symptom development in indicator plants
Newly emerged leaves of C. quinoa showed systemic mottling 3 weeks after inoculation. C. amaranticolor, however, was infected symptomlessly in inoculated leaves without any signs of systemic infection. None of the other 10 indicator plant species from five families challenged with SCMoV-P23 developed any systemic symptoms (Table 3.4).
Table 3.4: Reactions of indicator plants to inoculation with SCMoV P23 Species Common Genotype Symptoms name (Breeding line/ Inoculated Non- Accession/ leaves inoculated cultivar) leaves Brassica juncea L. Mustard cv. Tendergreen NI NI B. napus L. Canola cv. Pinnacle NI NI Capsicum annuum L. Bell pepper cv. Rialto NI NI Chenopodium quinoa Willd. None - LCS M C. amaranticolor Coste & Reyn None - SI NI Cucumis sativus L. Cucumber - NI NI Daucus carota L Carrot cv. Stephano NI NI Gomphrena globosa L. None - NI NI Lycopersicon esculentum Mill. Tomato cv. Grosse Lisse NI NI Nicotiana benthamiana Domin. None - NI NI N. glutinosa L. None - NI NI Physalis floridana Rydb. None - NI NI
Symptom codes: LCS = local chlorotic spots; SI = symptomless infection in inoculated leaves; M = mottle; NI = No infection detected from observation.
3.3.2 Detection of local and systemic infection of SCMoV-P23 by RT-PCR
Samples of inoculated leaves of all potential hosts were tested by RT-PCR 2-3 weeks after inoculation. The tip leaves were tested from the third week through to the ninth (in some cases), depending on the length of time it took for any symptoms to develop. In most cases the assays were done on individual leaf samples, but for M. sativa and M. truncatula, either the inoculated or uninoculated leaf samples of each plant were pooled. This was because more replicates than usual were tested from the first week of infection through to the ninth. Negative and positive PCR controls were done with total RNA from healthy and infected T. subterranean plants respectively as templates.
75
3. Host Range and Symptomatology of SCMoV
3.3.2.1 Detection of infection by RT-PCR in crop/ alternative crop legumes
Ten established and six alternative legume crop species represented by 24 genotypes were tested for infection of SCMoV-P23 in inoculated as well as in newly expanded leaves. Seven of the 10 crop species tested positive for infection in inoculated leaves (Fig. 3.7a). P. vulgaris,
V. faba cv Ascot and Fiesta and V. unguiculata tested negative in inoculated and in tip leaves, indicating that they are not susceptible to infection by SCMoV-P23. The only crop species to test positive in newly expanded leaves were C. arietinum cvs Kaniva and Sona, L. culinaris and
V. narbonensis (Fig. 3.7b).
All the alternative crop legumes tested positive for presence of the virus in inoculated leaves
(Fig. 3.7a) though systemic infection was confirmed in only three of the six, all of which were species of Lathyrus (L. clymenum, L. ochrus ATC 80532 and ATC 80123, and L. sativus ATC
80488 but not genotype ATC 80517). RT-PCR did not confirm systemic infection in Lathyrus cicera, Vicia benghalensis and V. sativus (Fig. 3.7b).
Fig 3.7a
807 bp
M 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 171819 202122 23 2425 2627M Fig. 3.7b
807bp
Fig. 3.7a and 3.7b: Results of RT-PCR detection of SCMoV in inoculated (Fig. 3.7a) and in newly emerged (Fig. 3.7b) leaves of crop and alternative legumes.. Lanes M. Kb plus DNA Ladder 1. Cicer arietinum cv Kaniva 2. C. arietinum cv Sona 3. Lathyrus cicera 4. L. clymenum 5. L. ochrus ATC 80532 6. L. ochrus ATC 80123 7. L. sativus ATC 80488 8. L. sativus ATC 80517 9. Lens culinaris 10. Lupinus albus 11. L. angustifolius 12. L. luteus 13. L. angustifolius 14. Phaseolus vulgaris 15. Pisum sativum cv Dundale 16. P. sativum cv Greenfeast 17. P. sativum cv Laura 18. P. sativum cv Wirrega 19. Vicia benghalensis 20. V. faba cv Ascot 21. V. faba cv Fiesta 22. V. narbonensis SA26554 23. V. narbonensis 24. V. sativa 25. Vigna unguiculata 26. Negative control 27. Positive control NB: Lane 13 is a duplicate of L. angustifolius and is only in Fig. 3.7a
76
3. Host Range and Symptomatology of SCMoV
3.3.2.2 Detection of infection by RT-PCR in Trifolium spp
With the exception of T. hirtum, all Trifolium species tested positive for the presence of
SCMoV-P23 in inoculated leaves (Fig. 3.8a). Similarly, RT-PCR of tip leaves from all
Trifolium species were positive (Fig. 3.8b) except for T. glanduliferum and T. hirtum. T. hirtum was inoculated and tested three times by RT-PCR with negative results in both inoculated and tip leaves. The results of RT-PCR assays confirmed the local symptom development in T. spumosum and T. glanduliferum as symptomlessly infected in inoculated leaves.
Fig. 3.8a
807 bp
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M Fig. 3.8b
807 bp
Fig. 3. 8a and 8b: Results of RT-PCR detection of SCMoV in inoculated (Fig. 3. 8a) and in newly emerged (Fig. 3. 8b) leaves of Trifolium spp Lane M.Kb plus DNA ladder 1. Trifolium cherleri 2. T. clypeatum 3. T. dasyurum 4. T. glanduliferum 5. T. hirtum 6. T. incarnatum 7. T. isthmocarpum 8. T. michelianum 9. T. purpureum 10. T. resupinatum 11. T. spumosum 12. T. spumosum 13. T. vesiculosum S. 14. Negative control 15. Positive control
3.3.2.3 Detection of infection by RT-PCR in alternative pasture/forage legumes
Three alternative pasture and forage legumes tested positive for SCMoV-P23 in their inoculated leaves (Fig. 3.9a). These were B. pelecimus, T. balansae and M. polymorpha cv.
Circle valley, confirming local symptoms observed on T. balansae and indicating symptomless infection for the other two. Of the six Medicago species examined, the presence of SCMoV-
P23 was only detected in inoculated leaves of M. polymorpha cv. Circle Valley. M. sativa and
M. truncatula were planted on two occasions and each time the inoculated leaves were tested until the fourth week after inoculation with negative results each time. The tip leaves were
77
3. Host Range and Symptomatology of SCMoV tested until the ninth week after inoculation and were also negative each time. All other plants tested negative for SCMoV-P23 in the newly expanded leaves (Fig. 3.9b).
Fig. 3.9a
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M 807 bp
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M
Fig. 3.9b
807 bp
Fig. 3.9a and 3.9b: Results of RT-PCR detection of SCMoV in inoculated (Fig. 3.9a) and in newly emerged (Fig. 3.9b) leaves of pasture/forage legumes other than Trifolium spp.Fig. 3.9: Lanes M. Kb plus DNA ladder 1. Biserrula pelecinus 2. Dorycnium hirsutum 3. Hedysarum coronarium 4. Medicago littoralis 5. M. murex 6. M. polymorpha 7. M. polymorpha 8. M. sativa 9. M. truncatula 10. Melilotus alba 11. Ornithopus compressus 12. O. sativus 13. Trigonella balansae 14. Negative control 15. Positive control
3.3.2.4 Detection of infection by RT-PCR in indicator plants
Of the 12 indicator plant species, only C. amaranticolor and C. quinoa tested positive for
SCMoV-P23 in inoculated leaves (Fig. 3.10a). These results confirm the local necrotic lesions observed in C. quinoa and show that C. amaranticolor was infected symptomlessly in its inoculated leaves. The test results indicate C. quinoa was also infected systemically but C. amaranticolor was not. No other indicator plant was positive for SCMoV-P23 in inoculated or tip leaves (Fig. 3.10a&b).
78
3. Host Range and Symptomatology of SCMoV
Fig.3.10a
807 bp
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Fig.3.10b
807 bp
Fig. 3.10a and 3.10b: Results of RT-PCR detection of SCMoV in inoculated (Fig. 3.10a) and in newly emerged (Fig. 3.10b) leaves of indicator plants. Lane M. Kb plus DNA ladder 1. Brassica juncea 2.B. napus 3.Capsicum annuum 4. Chenopodium quinoa 5. C. amaranticolor 6. Cucumis sativus 7. Daucus carota 8. Gomphrena globosa 9. Lycopersicon esculentum 10. Nicotiana benthamiana 11. N. glutinosa 12. Physalis floridana 13 Negative control 14. Positive control
3.3.3 Detection of local and systemic infection in P. sativum and M. truncatula by ELISA
T. subterranean , M. truncatula and P. sativum L. cv Greenfeast were have been designated as diagnostic host species, and T. subterranean and M. truncatula as suitable propagation hosts for
SCMoV (Francki et al., (1998). The SCMoV isolate(s) and plant cultivars used in the study were not mentioned. However, the only other host range study to use M. truncatula concluded that it was not a systemic host (Wroth and Jones, 1992a). To clarify this issue, P. sativum cv.
Greenfeast and M. truncatula cv. Caliph (the only cv available at the time) were each inoculated with four WA isolates of SCMoV each of which induces a different level of symptom severity upon infection. Local and systemic infections were tested by ELISA on the third and fifth week after inoculation because the exact nucleotide sequences of SCMoV-AL, -
MB and -MJ had not been determined. Inoculated leaves of P. sativum cv Greenfeast tested positive for the presence of all four isolates of SCMoV but negative in M. truncatula cv Caliph.
The virus was not detected in the newly expanded leaves of either species (Table 3.5a, 3.5b and
3.5c).
79
3. Host Range and Symptomatology of SCMoV
Table 3.5a: ELISA test readings for inoculated leaves of Medicago truncatula cv Caliph challenged with four different isolates of SCMoV Replicates SCMoV-AL SCMoV-MB SCMoV-MJ SCMoV-P23 Controls Plant 1 0.013, 0.038 0.021, 0.019 0.043, 0.024 0.009, 0.008 Positive control Plant 2 0.010, 0.013 0.010, 0.009 0.033, 0.032 0.008, 0.008 = 0.981, 0.902 Plant 3 0.009, 0.010 0.017, 0.034 0.014, 0.016 0.009, 0.011 Plant 4 0.015, 0.010 0.016, 0.036 0.023, 0.023 0.015, 0.013 Negative control Plant 5 0.017, 0.013 0.017, 0.022 0.014, 0.015 0.027, 0.015 = 0.043, 0.024
Table 3.5b: ELISA test readings for newly expanded leaves of Medicago truncatula cv Caliph challenged with four different isolates of SCMoV Replicates SCMoV-AL SCMoV-MB SCMoV-MJ SCMoV-P23 Controls Plant 1 0.028, 0.014 0.019, 0.028 0.015, 0.023 0.013, 0.014 Positive control Plant 2 0.015, 0.013 0.028, 0.030 0.040, 0.041 0.009, 0.016 = 0.981, 0.902 Plant 3 0.030, 0.027 0.035, 0.038 0.017, 0.058 0.015, 0.010 Plant 4 0.023, 0.037 0.027, 0.027 0.024, 0.031 0.011, 0.019 Negative control Plant 5 0.026, 0.021 0.033, 0.017 0.027, 0.026 0.010, 0.021 = 0.043, 0.024
Table 3.5c: ELISA test readings for inoculated and tip leaves of P. sativum cv Greenfeast challenged with four different isolates of SCMoV SCMoV-AL SCMoV-MB SCMoV-MJ SCMoV-P23 Controls Inoculated leaves 0.483, 0.511 1.836, 1.891 0.785, 0.775 1.000, 1.174 Positive control Tip leaves 0.062, 0.074 0.047, 0.043 0.031, 0.034 0.082, 0.101 = 1.860, 1.520 Negative control = 0.034, 0.030
3.4. Summary of results - Host Status of test plants to SCMoV infection
The plant species tested showed a wide range of responses to SCMoV infection and were either systemic, local or non-host. A summary of the results is provided in Table 3.6 and 3.7. Twenty three genotypes representing 18 species were systemic hosts, 15 genotypes of 12 species were local hosts and 24 other genotypes were non-hosts of SCMoV-P23 (Table 3.7).
Table 3.6: Classification of plants using results of symptom expression studies and RT- PCR. Reactions of plants to SCMoV P23 infection
Symptom expression RT-PCR assay Host status of test Total number of Inoculated New Inoculated New plants species leaves leaves leaves leaves Systemic host 3 Yes Yes +ve +ve 14 No Yes +ve +ve 1 No No +ve +ve Local host 1 Yes No +ve -ve 11 No No +ve -ve Non-host 24 No No -ve -ve
80
3. Host Range and Symptomatology of SCMoV
Table 3.7: Host status of plants test based on results of symptomatology and detection of SCMoV
Systemic hosts Local hosts Non-hosts Chenopodium quinoa Biserulla pelecinus* Brassica juncea Cicer arietinum cv Sona* Chenopodium amaranticolor B. napus C. arietinum cv Kaniva* Lathyrus cicera* Capsicum annum Lathyrus clymenum* L. sativus ATC 80517* Cucumis sativus L. ochrus ATC 80123* Lupinus albus* Daucus carota L. ochrus ATC 80532* L. angustifolius* Dorycnium hirsutum L. sativus ATC 80488* L. luteus * Gomphrena globosa Lens culinaris * Medicago polymorpha cv Circle Valley Hedysarum coronarium Trifolium cherleri* Pisum sativum cv Greenfeast Lycopersicon esculentum T. clypeatum* P. sativum cv Laura* Medicago littoralis T. dasyurum 24GCN39& P. sativum cv Dundale* M. murex T. dasyurum 42BT* P. sativum cv Wirrega* M. polymorpha cv Serena T. incarnatum Trifolium glanduliferum* M. sativa T. isthmocarpum Vicia benghalensis* M. truncatula T. purpureum V. sativa* Melilotus albus T. michelianum Nicotiana benthamiana T. resupinatum N. glutinosa T. spumosum 24BP* Ornithopus compressus T. spumosum 87144* O. sativus T. vesiculosum* Phaseolus vulgaris Trigonella balansae* Physalis floridana V. narbonensis SA26554* Trifolium hirtum Vicia faba cv Ascot V. faba cv Fiesta V. narbonensis Vigna unguiculata * : Genotype is a newly-identified host
3.5 Discussion
The results of this study provide information on the host status of 52 plant species (61 individual genotypes) to SCMoV infection. It extends the known host range of SCMoV in the
Fabaceae and identifies two species of the Chenopodiaceae as susceptible to SCMoV. The importance of several economically important legumes for use as hosts in future analysis of an engineered SCMoV as a legume-specific gene silencing vector is discussed. In addition, the study provides information for plant breeders on the susceptibility of new pasture varieties to
SCMoV and the use of some economically important crop legumes in farming systems in
SCMoV-endemic areas.
81
3. Host Range and Symptomatology of SCMoV
3.5.1 Extension of host range of SCMoV
Results of the study extend the known host range of SCMoV by identifying 18 leguminous plant species as systemic hosts, 12 local hosts and 24 non-host plants species. In addition to the
25 host species reported previously (Francki et al., 1983; Wroth and Jones, 1992a), a further 21 within the Fabaceae were identified, belonging to eight different genera. Nine of these were alternative annual pasture or forage species and 12 were crop legumes. However, its host range outside this family was narrow, as only two other species became infected, both belonging to the Chenopodiaceae.
Of the 12 indicator plants only C. quinoa and C. amaranticolor were infected with SCMoV-
P23. Test plants from Amaranthaceae, Apiaceae, Cruciferae, Cucurbitaceae and Solanaceae were not susceptible to SCMoV. With one of the two new Chenopodium hosts, infection was restricted to inoculated leaves. The first host range study of SCMoV was undertaken by
Francki et al., (1983). Potential hosts in their study included three indicator plants of the family
Chenopodiaceae. They recorded C. quinoa and C. amaranticolor as not susceptible, but not C. murale in which the virus induced very small grey lesions in its inoculated leaves. However,
Brunt (1996) and Franck1 et al.(1998) record only P. sativum as a local lesion assay host. The former also records C. amaranticolor and C. quinoa as non susceptible hosts with nothing said about C. murale, and consider the Chenopodiaceae family not to contain susceptible hosts to
SCMoV although in both cases the work of Francki et al., (1983) was the major reference. The results of this study taken in context with the work of Francki et al., (1983) confirms another susceptible plant family, Chenopodiaceae, to the only plant family previously known to contain susceptible hosts of SCMoV.
3.5.2 Symptomatology of SCMoV
SCMoV-induced leaf symptoms were similar to those reported by Francki et al., (1983) and
Wroth and Jones (1992a, b) with little variation in form and severity. In addition to local necrotic spots typical of local infection of SCMoV in field peas, local necrotic rings and spots,
82
3. Host Range and Symptomatology of SCMoV and local veinal necrosis were observed in some hosts. Typically, systemic symptoms first appeared 10-14 days after inoculation and developed with initial vein clearing in young leaves, followed by mottling, leaf deformation, decreased leaf size and stunting in some plants.
Distinct systemic leafy symptoms developed by some hosts included persistent severe vein clearing, necrotic streaking alongside veins, epinasty and vein banding. Observable systemic symptoms on shoots of host plants not previously reported included tip leaf curling, shoot pigmentation and shoot tip bending and death.
Differences in response to viruses by plant genotypes of the same species, and species of the same genera have long been recorded for several viruses including SCMoV (Drijfhout et al.,
1978; Tomlinson and Ward, 1978; Wroth and Jones, 1992a). Possible stages where a virus might be blocked from infecting a plant and causing systemic disease include: (i) during the initial uncoating of the virus, (ii) during replication in initially infected cells, (iii) during cell-to- cell movement and (iv) by stimulation of the host’s cellular defenses in the region of initial infection and/or away from infection sites to avoid further movement of the virus. Within T. subterraneum, response to sap inoculation of SCMoV is genotype dependent (Wroth and Jones,
1992a; b; Ferris et al., 1996) and is due to impaired cell-to-cell movement of the virus rather than to diminished virus multiplication (Njeru et al., 1995). Differences in response to SCMoV infection were also observed for some cultivars of the same species. Whilst M. polymorpha cv
Circle Valley showed latent but not systemic infection, the cv Serena was not susceptible. In a footnote to the results of their host range study, Wroth and Jones (1992a) noted that among the medics they tested, these two cultivars showed early systemic movement which was detected transiently in tip leaves but not at later times. Also, only a proportion of these cultivars inoculated became systemically infected. Similarly, on infection of P. sativum, the cv
Greenfeast developed local symptoms whereas the three other cvs Dundale, Laura and Wirrega did not. A difference in reaction to SCMoV was observed for genotypes of L. sativus. Whilst genotype ATC 80488 showed subliminal infection genotype ATC 80517 was not systemically infected. Also the different genotypes of T. spumosum and V. narbonensis showed varied reactions to infection.
83
3. Host Range and Symptomatology of SCMoV
3.5.3 Importance of host range study to gene silencing studies with SCMoV-based vectors
Host plants identified included several economically important crop legumes and potential model legumes for biological studies e.g. chickpeas, lentils, narbon beans and burr medic (cv
Circle Valley). P. sativum cv Greenfeast, T. balansae, T. spumosum, L. clymenum and C. quinoa could be used as diagnostic hosts of SCMoV. Also, the severe characteristic systemic symptoms developed by crops like L. ochrus, C. arietinum cv kabuli and cv "desi" Sona, and V. narbonensis make them useful as diagnostic and experimental hosts of SCMoV.
Amongst other factors, infectivity of cloned RNA viruses requires a host plant that produces little or no resistance to infection to the native virus and which produces primary or secondary scorable responses to infection, although there is no guarantee that the response of host plant to field isolates would be the same as for cloned genomes. The systemic and local hosts of
SCMoV identified in this study provide a range of hosts suitable for testing for infectivity and expression of engineered SCMoV gene vectors. In addition, SCMoV vectors when developed could be used as a legume-specific vector for studying gene function by silencing in economically important legumes like chickpeas, lentils, lupins and field peas.
3.5.4 Susceptibility of new pasture cultivars to SCMoV
The potential host plants challenged with SCMoV included new cultivars of alternative pasture and forage legume species recently released by the WA pasture and forage programme, DAWA for sowing in pastures. These made up 19 of the 52 species tested of which 13 were susceptible to SCMoV. The responses of these plants to challenge with SCMoV provide an indication of the risk that SCMoV-induced losses may occur where they are released commercially into pastures in different regions of Australia. Previous extensive surveys of pastures in southern
Australia revealed that those at greatest risk of damage from SCMoV are ones within the highest rainfall zones (Wroth and Jones, 1992b; Helms et al., 1993; Jones, 1996). Whether it is appropriate to deploy SCMoV-susceptible pasture species in which all infected plants become infected systemically and develop severe symptoms, e.g. T. clypeatum, T. dasyurum C. (eastern
84
3. Host Range and Symptomatology of SCMoV star clover), T. isthmocarpum and Trigonella balansae, should be considered carefully before sowing them in such zones.
Instead, deploying species that do not become infected systemically, like B. pelecinus and T. glanduliferum, or non-hosts, like H. coronarium, O. compressus and O. sativus, may be more suitable. It would be advisable to test all new selections of alternative pasture and forage legumes routinely by inoculation with SCMoV before release so that vulnerable species and genotypes can be identified. Depending on the relative importance of any beneficial traits they possess versus their vulnerability to damage caused by SCMoV, it may then be appropriate for such species or genotypes to be targeted only for sowing in lower virus risk regions.
3.5.5 Susceptibility of crop legumes and farming systems in SCMoV-endemic areas
Of the 13 crop legume species that were hosts of SCMoV only six became systemically infected, namely C. arietinum, Lens culinaris, L. clymenum, L. ochrus, L. sativus and V. narbonensis. No surveys have been made to determine whether SCMoV infects crops of these six species naturally and whether they can transmit the virus through seed. However, even though the virus is readily contact transmissible and could sometimes be introduced on contaminated farm machinery, it seems likely that incidences of infection would rarely become high enough to cause economic losses in grain yield, especially since there is no known insect vector. Therefore, in contrast to the situation for alternative pasture and forage legumes grown in pastures, SCMoV does not seem to pose a significant threat to the productivity of crop legumes. These results, however, do provide valuable information to guide the use of such crop legumes as components of crop rotation and ley farming systems in SCMoV-infected areas because even rare infection could be severe enough to cause reduction in yield and also to perpetuate the incidence of the virus in following seasons.
Twelve of the local hosts, most of which were crop legumes, showed symptomless infection in the inoculated leaves. However, other studies have shown that leaf tissue could be strikingly
85
3. Host Range and Symptomatology of SCMoV affected, reflected in different patterns of starch distribution and chlorophyll damage (Holmes,
1964). Such changes could lead to low productivity. These plants could also be reservoirs and/or sources of SCMoV infection in the field. The non-susceptible crop species could be valuable for cropping in appropriate farming systems in SCMoV-endemic areas with subterranean clover and other susceptible plants, without the risk of a yield loss. They could also be used to break the seasonal infection cycle of SCMoV in a crop rotation farming system.
3.6 Conclusions
The work extends the known host range of SCMoV to include new pasture and crop legumes, and hosts suitable for diagnostics and propagation. The study has provided useful hosts for analysing infectivity of SCMoV-based gene silencing vectors and for studying functions of legume genes by silencing.
86
Chapter Four
Genetic Diversity in SCMoV Isolates and Functional Constraints of its
Genes
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
4.0 Introduction
RNA viruses exist as a collection of strains with similar properties. They frequently occur in nature, either in specific host species, or varieties, or in particular locations. Isolates of the same species or of different viruses are distinguished by their biological properties (e.g. host symptoms and range, mode of transmission) or serological properties (e.g. serological reactions and relationships). In addition, molecular properties, including nucleotide sequences, structural and non-structural proteins, can be used to differentiate different viruses or strains of the same virus.
Nucleotide diversity in RNA virus populations results mainly from the accumulation of mutations due to frequent errors in RNA synthesis (Drake, 1993; Domingo and Holland, 1994;
Roossinck, 1997). Genetic variation is further increased by random genetic events such as genome reassortment and recombination (Simon and Bujarski, 1994; Lai, 1995; Aranda et al.,
1997; Bruyere et al., 2000; Garcia-Arenal et al., 2001). This generates diversity in viral genomes and provides variants on which natural selection can act. Evidence for genetic variation of plant viruses was reported as early as 1926, and was based on isolating mutants of the same virus with different symptoms from tissues that showed atypical symptoms in systemically infected plants (McKinney, 1935; Kunkel, 1947). Molecular relationships between isolates or strains of several plant viruses have been studied widely to determine their rate of mutation, selection and fitness, origins, and the nature and biological significance of variation (e.g. Bousalem et al., 2000). Mutations that resulted from selection were later associated with every facet in the life cycle of viruses: transmissibility, strain specialisation, symptom development and severity of strains as evidenced in SBMV, RYMV and BYMV
(Atreya et al., 1992; Lee and Anderson, 1998; Pinel et al., 2000; Wylie et al., 2002; Zakia et al., 2003).
Field isolates of SCMoV in WA have been characterised by the severity of symptoms they induce in subterranean clover (Wroth and Jones, 1992b). Although differences in host symptom are important in recognition of strains, the extent of differences in disease symptoms
88
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
may not be a reliable measure of the degree of relatedness between strains. The relationship between different strains of a virus can be studied effectively either by comparison of full- length nucleotide sequences of several isolates or by comparing the sequences of several different clones covering the same region of the genome. Four WA isolates used in the host range and symptomatology study (Chapter 3) were chosen as good candidates to characterise
SCMoV by studying genetic diversity in the isolates and the functional constraints of SCMoV genes. Differences in the isolates could be a result of intrinsic mutations in SCMoV or natural selection; either of which could be used to explain the differences in the level of symptom severity they induce or conservation of portions of the genome.
4.1 Aim and objectives of Chapter 4
In this chapter the genetic diversity of SCMoV is assessed and the potential impact of accumulation of mutations is evaluated in terms of the function of the genes in the viral infection cycle. This has been achieved by
(i) sequencing the entire genomes of four WA isolates of SCMoV
(ii) estimating variability between isolates, including the first sequenced isolate, SCMoV-P23
(iii) establishing a relationship between genetic variation in the sequences with symptom severity and
(iv) assessing selective constraints on amino acids of the different genes of SCMoV.
4.2 Materials and methods
4.2.1 SCMoV isolates and symptom severity
The four WA isolates of SCMoV used in this study were SCMoV-AL, -MB, -MJ and -pFL.
SCMoV-MJ and SCMoV-MB were originally isolated from T. subterraneum plants within pastures at Manjimup and Mt. Barker respectively (Wroth and Jones, 1992a, b; Ferris and Jones
1995). SCMoV-pFL was derived from SCMoV-P23 which has been maintained in T. subterraneum cv Woogenellup for more than 5 years. SCMoV-P23 was originally isolated from T. subterraneum also in Manjimup. SCMoV-AL was, however, isolated from T. vesiculosum in pastures at North Dandalup. The isolates fall into three severity groups; both
89
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
SCMoV-AL and SCMoV-MJ are very severe isolates, SCMoV-P23 and SCMoV-pFL are severe isolates and SCMoV-MB induces moderately severe symptoms on subterranean clover
(Wroth and Jones, 1992a, b).
4.2.2 Sequencing strategy and primers for sequencing
Nucleotide sequences of SCMoV-AL, SCMoV-MB and SCMoV-MJ were determined from a total of 18 overlapping cDNA clones generated by RT-PCR and molecular cloning from total
RNA extracted from 2-3 leaves of plants infected with each isolate. First strand cDNA was synthesised with gene-specific primers or random hexamers using 200 U of MoMLV reverse transcriptase. PCR of cDNA was done with 2 U of rTth DNA polymerase XL on 20% of RT products. Amplified DNA fragments were cloned into pGEM-T Easy vectors and sequences of both strands of the cDNA insert determined as described below. The nucleotide sequence of
SCMoV-pFL was determined from a full-length cDNA clone produced from vRNA purified from virions and sequenced with primers T7, SP6 and 14 SCMoV-specific primers.
4.2.3 Primers for RT-PCR
The 5’ and 3’ terminal sequences of each of the isolates were determined with primers designed from the published sequences of SCMoV-P23 (Dwyer et al., 2003). Other primers used to amplify overlapping fragments covering the genomes were conserved in all isolates and amplified overlapping 1 kb fragments. These are designated in Fig. 4.1 and the sequences shown in Table 2.2. Both strands of cDNA inserts in selected clones were sequenced with modified T7 and SP6 primers. In addition, cDNA clones were sequenced with internal synthetic oligonucleotide primers to clarify ambiguous sequences. Sequences were determined as described in section 2.11.
1 4258 bp SCMoV RNA FLfor scm2b scm34 scm38 seq2r scm36 cDNA fragments scm12 scm14 scm16
scm35 scm37 scm004
Fig. 4.1: cDNA clones generated from fragments amplified by RT-PCR from SCMoV isolates using the primers indicated and shown in Table 2.2.
90
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
4.2.4 Sequence analysis
Percent identities of nucleotide and amino acid sequences were determined using GAP
(ANGIS, http://www.angis.org.au/). A molecular relationship tree was constructed using
MEGA V2.1 (Kumar et al., 2001). Genetic variation between the isolates, estimated as average nucleotide diversity, was calculated as the average number of nucleotide substitutions per site between all pairs of sequences. Translation of nucleotide sequences and multiple sequence alignments were done with BioEdit Sequence editor (Hall, 1999). Genome organisation was predicted with ORF Finder, an online graphical analysis tool available through NCBI
(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Accumulated mutations in any region of the genome over time were calculated as the ratio of the average number of synonymous substitutions (dS) and non-synonymous substitutions (dN) per site for that region with the formula K= -(3/4) ln(1-4p/3) (Jukes and Cantor, 1969), where p is either pS or pN, proportion of synonymous (pS) and nonsynonymous differences (pN) respectively. The functions, pS and pN, between any pair of sequences, were estimated as pS = Sd/S and pN = Nd/N where S and N are respectively the average number of synonymous and nonsynonymous sites, Sd and Nd are the synonymous and nonsynonymous differences respectively for any two sequences compared
(Nei and Gojobori, 1986). The number of synonymous and nonsynonymous differences between any pair of sequences was calculated by comparing the two sequences codon by codon and counting the number of synonymous and nonsynonymous nucleotide differences for each pair of codons compared for the entire region, considering all the different possible pathways and mutational steps between any two codons (Nei and Gojobori, 1986).
91
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
4.3 Results
4.3.1 Complete genomic sequences of four isolates of SCMoV
The complete nucleotide sequences of SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV- pFL were successfully obtained and have been deposited in the GenBank nucleotide sequence database (Table 4.1). The genome organisation of SCMoV-P23 has recently been described
(Dwyer et al., 2003). The relationships of the four sequenced isolates to SCMoV-P23 were determined from the genome organisation predicted by ORF Finder, and by comparative sequence analysis of the different regions of the genome. Global sequence analyses in
SCMoV, done with all five isolates including SCMoV-P23, were by: assessment of genetic variation within the isolate and distribution of nucleotide substitutions within the genomes and their correlation with symptom severity using a molecular relationship ‘tree construction’ and statistical analysis. Sequences of SCMoV-pFL, a derivative of SCMoV-P23, were included in analyses because there was a possibility that mutations has occurred as a result of successive passage of isolate P23 in different genotypes of subterranean clover over 5 years; such mutation has been reported for SBMV and BYMV (Lee and Anderson, 1998; Wylie et al., 2002).
4.3.1.1 Genome organisation of SCMoV
The organisation of the genome of the sequenced isolates was predicted using ORF Finder and compared to that of SCMoV-P23 for two reasons: to establish their relationship to SCMoV-P23 and to give a complete picture of the genome organisation of SCMoV. Five blocks representing five possible ORFs were predicted for each of the five isolates including SCMoV-
P23 (Fig. 4.2).
Frame from to length
+1 +1 1954...3504 1551 +2a 605…234 7 1743 +2 +2b 3323..4084 762 +3 69….. 608 540 +3 - 2 451…987 537
-2
Fig 4.2: Genome structure of SCMoV isolates as predicted by ORF Finder.
92
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
From the 5’ end, were frame +3 from nt 69 to 608 of the genome followed by frame -2 from base 451 to 987, frame +2a (605-2347 bases), frame +1 (1954-3504 bases) and frame +2b
(3323-4084) in that order. Flanking the terminal ORFs are untranslated regions of 68 and 177 bases respectively at the 5’ and 3’ ends. Among sobemoviruses, CfMV and TRoV are predicted by ORF Finder to have a similar structure.
The genome structure of the isolates is therefore as described earlier for SCMoV-P23 (Dwyer et al., 2003). Based on experimental evidence obtained for CfMV and the prediction by ORF
Finder, SCMoV contains four possible ORFs flanked by 5’ and 3’ NCRs of 68 and 177 nt respectively. There is a 5’ proximal ORF1 (69-608 nt), corresponding to frame +3, a 3’ terminal ORF3 (3323-4084 nt) corresponding to frame +2b, and two middle overlapping ORFs,
ORF2a (605-2347 nt) and ORF2b (1954-3504 nt) corresponding to frames +2a and +1 respectively. The genome is therefore compact with no intergenic regions. Although an additional ORF in frame -2a is predicted for some sobemoviruses, no protein product has been found experimentally for this predicted ORF in any of these viruses.
4.3.1.2 Comparative sequence analysis of isolates of SCMoV
The genomic length of each of the four sequenced isolates, SCMoV-AL, SCMoV-MB,
SCMoV-MJ and SCMoV-pFL, was 4,258 nt which was the same as that published for SCMoV-
P23 (Dwyer et al., 2003). The relationship between SCMoV-P23 and these isolates was assessed using percentage of nucleotide and amino acid sequence identity across entire genomes and between the genes and NCRs (Table 4.1). The isolates share more than 98% nucleotide identity to SCMoV-P23 with SCMoV-pFL being most closely related identical
(99.69%). Nucleotide and amino acid sequences of ORF1 shared least identity with that of
SCMoV-P23, whilst the CP sequences of all isolates were identical.
93
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
Table 4.1: Percentage of nucleotide and amino acid sequence identity between SCMoV-P23 and isolates SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV-pFL
Isolate Complete Percent identity of nucleotide (amino acid) sequences (GenBank acc. nucleotide 5’NCR ORF1 ORF2a/2b CP 3’NCR no.) sequence SCMoV-AL 99.11 98.5 97.1 (96.11) 99.3 (99.17) 100(100) 99.4 (AY376451) SCMoV-MB 98.80 97.0 96.7 (96.67) 98.9 (99.17) 100(100) 100 (AY376452) SCMoV-MJ 98.94 97.0 97.0 (96.11) 99.1 (99.07) 100(100) 100 (AY376453) SCMoV-pFL 99.69 98.5 99.8 (99.44) 99.6 (99.38) 100(100) 100 (AY376454)
4.3.1.3 Genetic variation within SCMoV isolates
Genetic variation within the isolates of SCMoV was assessed to examine how each isolate differed from others by estimating the average nucleotide diversity (Table 4.2) and to compare the average value to nucleotide misincorporation rate of in vitro enzymes used in RT-PCR.
The average nucleotide diversity between the five isolates was 0.0088301 ± 0.0014794 (mean ± s.e.d.)(Table 4.2). Nucleotide substitutions between pairs of complete nucleotide sequences of isolates ranged from 11 between SCMoV-AL and SCMoV-MJ to as much as 52 substitutions between SCMoV-AL and SCMoV-MB.
Table 4.2: Nucleotide diversity D, between pairs of complete genomic sequences of SCMoV isolates SCMoV isolate sequence pairs N D SCMoV P23/pFL 13 0.003053 SCMoV P23/AL 38 0.008924 SCMoV P23/MB 51 0.011977 SCMoV P23/MJ 45 0.010568 SCMoV pFL/AL 33 0.007750 SCMoV pFL/MB 50 0.011742 SCMoV pFL/MJ 38 0.008924 SCMoV AL/MB 52 0.012212 SCMoV AL/MJ 11 0.002583 SCMoV MB/MJ 45 0.010568 Mean 37.6 0.0088301 s.e.d 0.0014794 N = number of nucleotide substitutions, D = number of nucleotide substitutions per site, N/ 4258
94
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
4.3.1.4 Distribution of nucleotide substitutions in all isolates
Deviation of sequences of the five isolates from a consensus sequence was used to investigate the distribution of nucleotide substitutions in the RNA genome. There were 79 nucleotide differences in the first half of the genome (nt 1-2129) and only 35 in the last half (nt 2130-
4258) indicating more substitutions (p≤ 0.05) in the 5’ terminal half of the genome than the 3’ terminal half. Further investigation into this variation was done by comparing nucleotide sequences of the NCRs and to determine how nucleotide substitutions affected the coding regions of the genome (Section 4.3.3).
4.3.1.5 Nucleotide differences in isolates and symptom severity
4.3.1.5a Molecular relationship between isolates of SCMoV
A molecular relationship tree, constructed with the complete genomic nucleotide sequences, clearly distinguishes the five isolates into three groups. The phylogram (Fig. 4.3) shows closer relationships between SCMoV-AL and SCMoV-MJ, and between SCMoV-P23 and SCMoV- pFL, SCMoV-pFL being a derivative of SCMoV-P23 (Fig. 4.3). The moderately severe isolate, SCMoV-MB, is on a separate branch of the tree. This relationship is consistent with grouping based on the level of symptom severity they induce in subterranean clover.
SCMoVP23 SCMoVpFL SCMoVAL SCMoVMJ SCMoVMB
0.005 0.004 0.003 0.002 0.001 0.000
Fig 4.3: Molecular phylogram of SCMoV isolates constructed with complete nucleotide sequences.
95
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
4.3.1.5b Test of significance of relationship between nucleotide differences and symptom severity
A goodness-of-fit test was used to confirm the relationship predicted by MEGA (section
4.3.1.5a). It was used to determine whether the observed nucleotide differences in the isolates is in agreement with grouping based on the level of symptom severity they cause in subterranean clover. A chi squared test of the nucleotide substitutions in the complete sequences of the isolates versus the severity groups identified by the relationship tree and by
2 field characterisation revealed statistically significant deviation (χ = 6.21, degrees of freedom
(df) = 2, P = 0.05) from the null hypothesis of independent relationship between the two distributions. These results suggest that the nucleotide differences in the complete sequences could be responsible for the field behaviour of the isolates.
4.3.2 Nucleotide and deduced amino acid sequence comparison and variability
4.3.2.1 Nucleotide sequences of the 5’ and 3’ NCRs
Protein coding regions of each of the isolates are flanked by 68 nt and 177 nt respectively at the
5’ and 3’ termini (Fig 4.4 and 4.5). Three differences noted in the 5’ NCR occur at positions
19, 21 and 66. The adenine (A) at position 19 in SCMoV-P23 (GenBank acc. no. AF208001) was later detected as error. The base at this position, for all isolates, is guanine (G). Other differences in the 5’ NCR were a guanine (G) in SCMoV-MB at position 21 and cytosine (C) at position 66 in SCMoV-AL instead of C and T respectively for the others.
10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus ACAAAATCGCTCGAAGAAGAAAGCTTGAATCCGTTTCGCTTTATTCAATTGATAGACGATCATTTTAT SCMoV-P23 ·····················A······················································· SCMoV-pFL ·············································································· SCMoV-AL ·············································································· SCMoV-MB ·······················G······················································ SCMoV-MJ ···········································································C··
Fig 4.4: Sequences of the 5’ NCRs of isolates of SCMoV
In all isolates except for SCMoV-AL, the CP gene is terminated with two stop codons, TAG and TGA, with the 3’ NCR beginning with an A. The CP gene for SCMoV-AL however has
96
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
only the first stop codon. Its 5’ NCR then begins with a cytosine, C. Assuming this is not a
polymerase-induced error, this C is the only difference in the 3’NCRs of the isolates.
4090 4100 4110 4120 4130 4140 4150 4160 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus CTAGTGAACATCTCAGACACTATAACCGGAGACCTGGTGGTAAGGTAACCCACCCTCCTGGCAGATGAGGATTCTGCTAC SCMoV-P23 * *·········································································· SCMoV-pFL * *·········································································· SCMoV-AL *·C··········································································· SCMoV-MB * *·········································································· SCMoV-MJ * *··········································································
4170 4180 4190 4200 4210 4220 4230 4240 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus TCGCAACCGTGTTCATATTCGCGGTTTCGCTGAGATCATCATTCAAATATGTGGGGAGATCAATCTCCCCCCCTGACGTC SCMoV-P23 ················································································ SCMoV-pFL ················································································ SCMoV-AL ················································································ SCMoV-MB ················································································ SCMoV-MJ ················································································ 4250 ....|....|....|... Consensus GTGGGGTGCATGACGTGT SCMoV-P23 ·················· SCMoV-pFL ·················· SCMoV-AL ·················· SCMoV-MB ·················· SCMoV-MJ ··················
Fig 4.5: Sequences of the 3’ NCRs of isolates of SCMoV with * representing the stop codons of the coat protein.
4.3.2.2 Deduced amino acid sequences of the ORF1
The ORF1 of all isolates had a single initiation codon at position 1-3 of the gene. Each of the
deduced amino acid sequences of ORF1 consisted of 179 amino acids with an average
calculated molecular mass (Mar) of 20.3 kDa similar to the in vitro product of ORF1 observed for SCPMV (20 kDa)(Morris-Krsinich and Forster, 1983; Mang et al., 1982). There were polymorphisms at ten amino acid positions: 11, 41, 43, 53, 61, 62, 72, 146, 173 and 175 (Fig.
4.6). A prominent difference in the sequences is the absence of a Lysine (K) residue in
SCMoV-P23, SCMoV-pFL and SCMoV-MB whereas SCMoV-MJ and SCMoV-AL have one
97
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
at position 11 (K11). Amino acid residue at position 43 is different for the different severity
groups; Tyrosine (Y) for SCMoV-P23 and SCMoV-pFL, Histidine (H) for SCMoV-AL and
SCMoV-MJ and Cytosine (C) for SCMoV-MB. The amino acids at position 53 were different
for all isolates but the same (H) for SCMoV-AL and SCMoV-MJ. It is Leucine (L) for
SCMoV-P23, Isoleucine (I) for SCMoV-pFL and Glutamine (Q) for SCMoV-MB. In addition
to other polymorphisms, SCMoV-MB is distinguished from the others by the presence of P72
(instead of S72) and R173, 175 rather than Q173 and G175. Sequences of the two very severe isolates, SCMoV-AL and SCMoV-MJ, are the same and differ from the others by having K11,
I41, S61 and I62.
10 20 30 40 50 60 70 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus MPSVSIEVYSRERPTLLLLTHRFWPSSETIPRDYEYDQTVTL·GRFNNEEET·ILCISRCNTCDFWCVLG SCMoV-P23 ORF1 ··········································Y·········L················· SCMoV-pFL ORF1 ··········································Y·········I················· SCMoV-AL ORF1 ··········K·····························I·H·········H·······SI········ SCMoV-MB ORF1 ··········································C·········Q················· SCMoV-MJ ORF1 ··········K·····························I·H·········H·······SI········
80 90 100 110 120 130 140 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus DSNLPGIEIWDSALSCFANAEDCTLSGQCSACRNPPVEEDADSDSSEEYQSLGNRTVIRDSGPEGYWSES SCMoV-P23 ORF1 ······································································ SCMoV-pFL ORF1 ······································································ SCMoV-AL ORF1 ······································································ SCMoV-MB ORF1 ·P···································································· SCMoV-MJ ORF1 ······································································
150 160 170 ....|....|....|....|....|....|....|.... Consensus SDDEDYTRAAYSPCDYEGNLVRLFQLQSIQPLQEGDDQE SCMoV-P23 ORF1 ·····H·································· SCMoV-pFL ORF1 ·····H·································· SCMoV-AL ORF1 ······································· SCMoV-MB ORF1 ································R·R···· SCMoV-MJ ORF1 ······································· Fig 4.6: Deduced amino acid sequences of P1 of isolates of SCMoV
4.3.2.3 Deduced amino acid sequences of ORFs 2a and 2b
Sequences of the ORFs 2a and 2b, which are thought to encode a polyprotein via -1
frameshifting were considered together in all analyses and referred to as ORF2a/2b. Sequences
of the two ORFs were joined by inserting an adenine (A) into the slippery sequence
(TTTAAAC), immediately upstream of the cytosine. With this, the amino acid sequence of the
polyprotein from the slippery sequence site begins with (KLPA) at amino acid position 416 and
makes the ORF2b sequence translatable in-frame with the ORF2a. The deduced amino acid
sequence of ORF2a/2b for each of the isolates was 966 amino acids long with a Mar of 107.5
98
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
kDa similar to the calculated and in vitro products of the polyprotein of LTSV, SBMV and
TroV (Morris-Krsinich and Hull, 1981; Morris-Krsinich and Forster, 1983; Mang et al., 1982).
There were only seventeen polymorphic positions in the sequences (Fig. 4.7). None of these
mutations however affected the conserved amino acids characteristic for serine proteases
(H[X29]D[X63]TTKGWSG between positions 180 and 278) or that of viral RdRps
(SGSYCTSSTNX19GDD present at positions 772 – 802) (Kamer and Argos, 1984).
10 20 30 40 50 60 70 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus MNTDTIRAIFLYWISLALSMAAVVQLETKVDLSNHQSLVMTTAILLPALSVLFGLRVWRRWKVVIVKEVN SCMoV-P23 ORF2a/2b ...... M...... SCMoV-pFL ORF2a/2b ...... M...... SCMoV-AL ORF2a/2b ...... MI...... SCMoV-MB ORF2a/2b ...... V...... A. SCMoV-MJ ORF2a/2b ...... MI......
80 90 100 110 120 130 140 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus PREVINLVGEPFLDPVRGVLMNGISTSGGTFEVLIEPKWWHLFPRSAISKDGEKECAIFNAGYSSVLPGT SCMoV-P23 ORF2a/2b ...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... SCMoV-MJ ORF2a/2b ......
150 160 170 180 190 200 210 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus EPTSLVMLKAKDLAVGFGARVRFNGCSDYLLTAYHVIKPHEKLNLCKGGYMVEDVDLAVTCGSDHDAVDF SCMoV-P23 ORF2a/2b ...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... SCMoV-MJ ORF2a/2b ......
220 230 240 250 260 270 280 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus ALIKVPPAVWSKLKVGVGKLEPMTKKTHITVYGGSDSTRLLSSSGPAYKGKAGYAIIHEASTTKGWSGTP SCMoV-P23 ORF2a/2b ...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... SCMoV-MJ ORF2a/2b ......
290 300 310 320 330 340 350 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus LYSGNTIVGVHTGSGQVGYSNRAVNVKLLLTAVSKFETIFSEISYGELDEDNYLLRNRDDFVEVEILGKG SCMoV-P23 ORF2a/2b ...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... SCMoV-MJ ORF2a/2b ......
Slippery sequence site
360 370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus KFLLGDSSFVDITGKPLGWEKEKRARGEALWHDASDDDFYEDANFLADFYKDSKETVDDIMLEHLKLPAG SCMoV-P23 ORF2a/2b ...... S...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... S...... S.. SCMoV-MJ ORF2a/2b ......
99
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
430 440 450 460 470 480 490 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus GHNFKSCVATLIELASYEWKNLSESISSRGMPLADVGRSSCKFRETVRKELGAAVTAASFEFPELQGYSW SCMoV-P23 ORF2a/2b ...... A...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b .....N...... SCMoV-MB ORF2a/2b ...... AT...... SCMoV-MJ ORF2a/2b ...... D......
500 510 520 530 540 550 560 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus PDRGSKAERGSLLYQAGRFKPTPPPAKLYEAVVKLAPEYPETVPRACLRREQWDKEEIAKEAREIGQKKV- SCMoV-P23 ORF2a/2b ...... G...... K...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... K...... SCMoV-MB ORF2a/2b ...... G...... SCMoV-MJ ORF2a/2b ......
570 580 590 600 610 620 630 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus NPKASPGVPLSILGKTNKEVLDRHGDLVYIAVAERICMLAEADLADAPDPVDLVKAGYCDPIRLFVKQEP SCMoV-P23 ORF2a/2b ...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... SCMoV-MJ ORF2a/2b ......
640 650 660 670 680 690 700 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus HPLKKVVEGRFRLISSVSLVDQLVERLLFGPQNETEIDLWQSVPSKPGMGLSQPWQVSALWNDLEHKHKL SCMoV-P23 ORF2a/2b ...... P...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... SCMoV-MJ ORF2a/2b ......
710 720 730 740 750 760 770 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus SPAAEADISGFDWSVQSWEILADVCIRIDRGGFKGNLRKAALNRFKCFSNAVFQLSDGTLISQGLPGLMK SCMoV-P23 ORF2a/2b ...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... SCMoV-MJ ORF2a/2b ......
780 790 800 810 820 830 840 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus SGSYCTSSTNSRIRCLMAKIIGAPWCIAMGDDSVEGYVEGAREMYDSLGHTCKDYIPCKADLDKLEEVNF SCMoV-P23 ORF2a/2b ...... D...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... V...... SCMoV-MJ ORF2a/2b ......
850 860 870 880 890 900 910 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Consensus CSHTIRKDSYYLQSWAKTLFRFLSQPEDVNELAVELKGCPEWPRISKYLRRIGRISDKTSEGEGKQNDRP SCMoV-P23 ORF2a/2b ...... K...... SCMoV-pFL ORF2a/2b ...... K...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... SCMoV-MJ ORF2a/2b ......
920 930 940 950 960 ....|....|....|....|....|....|....|....|....|....|....|. Consensus QGEKIKEEIRSRDHHPAEPTEIWWETIPEPNDSGQEREDPYGFNSSHREIFPCNPL SCMoV-P23 ORF2a/2b ...... SCMoV-pFL ORF2a/2b ...... SCMoV-AL ORF2a/2b ...... SCMoV-MB ORF2a/2b ...... SCMoV-MJ ORF2a/2b ......
Fig 4.7: Deduced amino acid sequences of ORF2a/2b of isolates of SCMoV aligned with consensus to show differences within isolates.
100
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
4.3.2.4 Deduced amino acid sequences of the coat protein gene
The amino acid sequence of the CP was identical for all five isolates (Fig. 4.8). The Mar of the
CP is 27.3 kDa.
10 20 30 40 50 60 70 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| SCMoV-P23 CP MTGRRVRRSKKKSGQETTTQQNRQRSGGKQSQNQMIQVKRERTPMASTVVIGRSFPAIHSEGTRTRVCHT SCMoV-pFL CP ...... SCMoV-AL CP ...... SCMoV-MB CP ...... SCMoV-MJ CP ......
80 90 100 110 120 130 140 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| SCMoV-P23 CP ELIRAVDINTIFTLNFTWCIPSAFPWLSGVAVNWSKWRWVSLRFTYMPAAATTTQGTVALGYLYDALDAV- SCMoV-pFL CP ...... SCMoV-AL CP ...... SCMoV-MB CP ...... SCMoV-MJ CP ......
150 160 170 180 190 200 210 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| SCMoV-P23 CP PAALQQMSSLAGFSTGAVWSGSEGSVLLRAANKNGNVPGAVSSQLNIADPSKYYRYENLTNFAAIAEGTK SCMoV-pFL CP ...... SCMoV-AL CP ...... SCMoV-MB CP ...... SCMoV-MJ CP ......
220 230 240 250 ....|....|....|....|....|....|....|....|... SCMoV-P23 CP NLYAPARLAVAAANGAADVGGVGTVYATYVVELIDPISSALNS SCMoV-pFL CP ...... SCMoV-AL CP ...... SCMoV-MB CP ...... SCMoV-MJ CP ......
Fig 4.8: Amino acid sequence of the coat protein of SCMoV
4.3.3 Selective constraints (dN/dS) for amino acids of SCMoV
In a homologous set of sequences where “mutational saturation” has not been reached,
synonymous substitution frequencies can provide a linear measure of genetic variation under
conditions of minimal selection pressure. Nonsynonymous substitutions (amino acid
replacements) though a designation of negative and positive Darwinian selection, under certain
circumstances, also serve as a metric. The degree of negative selection in genes, or the degree
of functional constraint for the maintenance of the encoded protein sequence, can be estimated
from the ratio between the nucleotide diversities at nonsynonymous versus synonymous sites
(dN/dS). As nucleotide diversity is a measure of the probability that the base at a certain position differs between two randomly chosen individuals from the population, this ratio indicates the amount of variation in the nucleic acid that results in variation in the encoded
101
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
protein. The selective constraint for amino acids on the ORF1, ORF2a/2b, and the ORF3 genomic regions were estimated using the method described in section 4.2.3 to assess accumulation of mutations over time considering all the different possible pathways and mutational steps between any two codons.
4.3.3.1 dN/dS for amino acids of the ORF1 region
In the ORF1 region, 94 synonymous substitutions from 78 sites across the 4 isolates were scored, compared to 107 nonsynonymous substitutions from 59 sites (Table 4.3). Consequently the average proportion of nonsynonymous substitutions per site (pN =1.59 ± 0.408) [mean + s.e.d] in the set of sequences was more than the proportion of synonymous substitutions per site
(pS= 1.08 ± 0.18). The average ratio of dN/dS for ORF1 was 1.68. This value indicates that more nonsynonymous substitutions have accumulated per site over time in the ORF1 sequence suggesting a positive selection pressure on the ORF1.
Table 4.3: Proportion and number of nucleotide substitutions in ORF1 between pairs of sequences of SCMoV isolates
Sequence pS pN Pairs S Si (S/Si) dS N Ni (N/Ni) dN dN /dS P23/pFL 1 1 1 0.90297 1 1 1 0.90297 1 P23/AL 9 8 1.125 0.51986 8 7 1.14285 0.48497 0.93288 P23/MB 12 12 1 0.90297 8 6 1.3333 0.18848 0.20873 P23/MJ 9 8 1.125 0.51986 8 7 1.14285 0.48497 0.93288 pFL/AL 13 8 1.625 0.11561 18 7 2.5714 0.66547 5.7561 pFL/MB 14 12 1.167 0.44083 20 6 3.3333 0.92757 2.10414 pFL/MJ 13 8 1.625 0.11561 18 7 2.5714 0.66547 5.7561 AL/MB 12 11 1.09 0.59134 13 9 1.4444 0.05772 0.097608 AL/MJ 0 0 0 0 0 0 0 0 0 MB/MJ 12 11 1.09 0.59134 13 9 1.444 0.05772 0.097608 Total 95 79 10.847 107 59 15.9835 16.88605 Mean 1.0847 1.59835 1.6886 s.e.d 0.188 0.408 0.948 pN, average proportion of nonsynonymous substitutions per nonsynonymous site; pS, average proportion of synonymous substitutions per synonymous site; dN, average number of nonsynonymous substitutions per nonsynonymous site; dS, average number of synonymous substitutions per synonymous site; dN/dS, average ratio between nonsynonymous and synonymous substitutions for each pair of comparisons
102
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
4.3.3.2 dN/dS for amino acids of the ORF2a/2b region
In sequences of the ORF2a/b, 118 synonymous substitutions from 117 synonymous sites were recorded whilst there were only 77 nonsynonymous substitutions scored from 76 nonsynonymous sites with consequent closer values for pN (1.0 ± 0.006) and pS (1.01 ± 0.01) for the region (Table 4.4). Though pS for this region is not significantly different from that of
ORF1, pN for ORF2a/2b was less than in the ORF1 region. The value of dN/dS (0.96 ± 0.04) for this region indicate approximately equal numbers of nonsynonymous and synonymous substitutions have accumulated in the ORF2a/2b region of SCMoV isolates over time unlike in the ORF1 region where dN > dS.
Table 4.4: Proportion and number of nucleotide substitutions in ORF2a/2b between pairs of SCMoV isolates
Sequence pS pN Pairs S Si (S/Si) dS N Ni (N/Ni) dN dN /dS P23/pFL 4 4 1 0.90297 5 5 1 0.90297 1 P23/AL 10 10 1 0.90297 7 7 1 0.90297 1 P23/MB 22 21 1.0476 0.69319 8 7 1.1428 0.48497 0.69962 P23/MJ 16 16 1 0.90297 9 9 1 0.90297 1 pFL/AL 6 6 1 0.90297 7 7 1 0.90297 1 pFL/MB 16 16 1 0.90297 11 11 1 0.90297 1 pFL/MJ 12 12 1 0.90297 6 6 1 0.90297 1 AL/MB 16 16 1 0.90297 11 11 1 0.90297 1 AL/MJ 6 6 1 0.90297 3 3 1 0.90297 1 MB/MJ 10 10 1 0.90297 10 10 1 0.90297 1 Total 118 117 10.0476 77 76 10.1428 9.69962 Mean 1.00476 1.01428 0.969962 s.e.d 0.0063 0.0191 0.0402
pN, average proportion of nonsynonymous substitutions per nonsynonymous site; pS, average proportion of synonymous substitutions per synonymous site; dN, average number of nonsynonymous substitutions per nonsynonymous site; dS, average number of synonymous substitutions per synonymous site; dN/dS, average ratio between nonsynonymous and synonymous substitutions for each pair of comparisons.
4.3.3.3 dN/dS for amino acids of the ORF3 region
The CP of SCMoV is produced via subgenomic RNA synthesis from the ORF3. Deduced amino acid sequence of this region of the genome for all isolates is identical; pN and pS is each zero. The coat protein gene could be said to have reached its mutation saturation.
103
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
4.4 Discussion
This study reports on the complete nucleotide sequences of four WA isolates of SCMoV with differences in symptom severity in subterranean clover. Genetic diversity in the genomic sequences and the potential impact of mutations in regions of the genome on the biology of the isolates are discussed. Nucleotide sequence diversity in the isolates with respect to the influence of in vitro polymerases is also discussed.
4.4.1 Genetic variation in SCMoV isolates
The RNA genome length of each of the SCMoV isolates (SCMoV-AL, -MB, -MJ and -pFL) was 4,258 nt which was the same as that published for SCMoV-P23. For isolates of CfMV,
RYMV, and SBMV the genome length varies by up to 27 bases (section 1.2.4.2). Different genome lengths of strains have long been observed for other viruses, e.g. TRV (Cooper and
Mayo, 1972; Angenent et al., 1989). The average genetic variation in SCMoV isolates was
0.00883 ± 0.00147. This estimate is lower than nucleotide diversity in isolates of RYMV
(0.0722) and CfMV (average of 0.03893). However, the intra-isolate variation is higher than those reported for some unrelated viruses; California Citrus tristeza virus (CTV) isolates (Kong et al., 2000), two criniviruses (Rubio et al., 1999; Rubio et al., 2001), two tobamoviruses
(Rodriguez-Cerezo et al., 1989) and Citrus leaf blotch virus (Vives et al., 2002). These results and several others [e.g Lin et al., (2003)] confirm the existence of RNA plant viruses as a distribution of sequence variants around a consensus sequence and not as a single nucleotide sequence.
4.4.2 Distribution of nucleotide differences in SCMoV isolates and symptom severity
The distribution of nucleotide substitutions in isolates of SCMoV is not uniform, but is concentrated in the 5’ half of the genome. This distribution suggests that such differences between isolates may have developed in SCMoV over time by increasing survival of the species or increasing proliferation in hosts. To support this interpretation, there were more mutations in amino acid sequences of the ORF1 gene product than in other gene products. This indicates more positive selection pressure in this region than in other regions of the genome. A
104
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
similar pattern of variation in sequences exists for gene products of other sobemoviruses: there is very little similarity in the P1 protein, but considerable homology in the sequences of the polyprotein and the CP.
Sequences of the CP gene of SCMoV isolates were identical, and those of ORF2a/2b were nearly identical, with no changes in the characteristic serine protease and RdRp motifs.
Variations in amino acid sequences of the ORF1 were associated with the level of severity of symptoms of SCMoV isolates, as shown by the molecular relationship tree and statistical analysis. These results, and the differences in field behaviour of the isolates, imply that genetic variation within each of the RNA genomes may play a role in pathogenicity of the isolates.
Deduced amino acid sequences of the ORF1 for the different severity groups of the isolates reveal characteristic substitutions. Sequences of the very severe isolates (SCMoV-AL and
SCMoV-MJ) are identical. Similarly, primary sequences of P1 of the severe isolates (SCMoV-
P23 and SCMoV-pFL) are the same except at position 53. Sequences of the different isolates differ at several positions, prominent amongst which are the differences at positions 43 and 53
(Fig. 4.6). At position 43, the amino acid residue for the severity groups differ as follows: H for the very severe isolates, Y for the severe isolates and C for the moderately severe isolate,
SCMoV-MB. Except for the difference in SCMoV-pFL and –P23, the residues at position 53 are also correlated with severity. These sequence differences probably underlie the differences in field behaviour of the isolates, and make the P1 the most likely severity determinant of
SCMoV. Examples of how nucleotide and amino acid substitutions influence the biology of sobemoviruses and other viruses are discussed in section 4.4.2.
4.4.3 Implications of genetic variation and/or mutations on the biology of SCMoV
Negative selection is implicated in the accumulation of mutations in passage experiments under different conditions resulting in changes in properties of strains of some viruses (Garcia-Arenal et al., 1984; Atreya et al., 1995; Lee and Anderson, 1998; Wylie et al., 2002). When different hosts are involved it leads to host adaptation (Yarwood, 1979; Kearney et al., 1999; Schneider
105
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
and Roossinck, 2000). For example, the ability of a resistant breaking mutant, SBMV-S, to systemically infect the restrictive host of SBMV-BArk from which it was derived was attributed to a single amino acid mutation in the C-terminus of the RdRp and three in the N-terminus of the CP (Lee and Anderson, 1998). Also, a single mutation within the NAG motif in the CP of aphid transmissible isolate BYMVMI caused a loss of aphid transmissibility in isolate BYMVMI-
NAT after 72 cycles of subculture (Wylie et al., 2002). Mutational studies have confirmed the importance of the DAG motif and its context in the infection cycle of potyviruses, controlling accumulation, systemic movements and aphid transmissibility (Atreya et al., 1990, 1991, 1992,
1995). This is further proof that the sequence variation in isolates of SCMoV, in particular the
ORF1 sequences, could be responsible for the differences in their field behaviour.
It has been observed that the P1 of CfMV and RYMV both suppress RNA silencing by targeting the maintenance stage (Voinnet et al., 1999; Sarmiento et al., 2003). Like other viral suppressors, the P1 of RYMV-NG is a pathogenicity determinant (Ding et al., 1995; Scholthof et al., 1995; Brigneti et al., 1998; Kasschau and Carrington, 1998; Voinnet et al., 1999). The protein also shares striking functional similarities with viral suppressors in that they are all required for efficient accumulation of viral RNAs in protoplasts and long distance movement in their respective hosts (Kasschau, 1997; Bonneau et al., 1998). Studies into the mechanism of
RNA silencing has shown that viral suppressors of RNA silencing are diverse even in similar viruses, with no common structural features, have similar pathogenicity functions making them targets of similar components of the silencing machinery (Brigneti et al., 1998; Voinnet et al.,
1999; 2000). They are also under constant change indicating that this function has probably evolved independently over a period as a strategy to counter the effects of the hosts PTGS. It is becoming increasingly clear that RNA silencing plays a key role in plant-virus interactions and it is conceivable that there will be a dynamic evolution of plant components required for the mechanism and, accordingly, of the virus-encoded components necessary to overcome it.
Based on the effects of different viral suppressors on the different stages of RNA silencing, different suppressors of different viruses probably represent different stages in this dynamic evolution.
106
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
Based on the pattern of sequence variation between ORFs of SCMoV isolates and that between
ORFs of sobemoviruses, it is likely that the P1 of SCMoV shares similar functions with those of other sobemoviruses, in which it is a symptom severity/pathogenicity determinant, is involved in virus movement and is probably a suppressor of RNA silencing in hosts of
SCMoV.
4.4.4 Functional constraints on genes of SCMoV
The values of dN/dS calculated for the ORFs indicate nucleotide substitutions in ORF1 most often resulted in changes in amino acid. It is possible the CP gene of SCMoV has reached saturation mutation hence the perfect homology in the sequences of all isolates. The ORF1 region is therefore more variable, with more accumulated mutations per site than in other coding regions, indicating a more positive selection pressure on this region. The mutations may be advantageous for survival as nucleotide differences in the gene product, reflected as differences in the levels of symptom severity induced by the isolates, contributes to the efficiency of infection. Selection against amino acid changing substitutions in the polyprotein may be detrimental to survival as any changes may affect the roles of the RdRp and VPg in replication and protection of the RNA of SCMoV.
In cellular organisms, certain classes of proteins are always more conserved than others (Nei,
1987). Characterised virus proteins are however multifunctional and are under different selective constraints corresponding to various functions (Callaway et al., 2001). An example is viral CPs, which are never optimised for just one function (Callaway et al., 2001). The HC-Pro of potyviruses is known for its role in transmission, proteolytic processing of the genome translation product and in systemic movement. Its function in aphid transmission is easily lost upon mechanical passage suggesting a balance in optimisation of its different functions (Atreya et al., 1991, 1992, 1993). An extreme case of multiple functional constraints occurs in genomic regions with overlapping ORFs, which is common in tightly packaged viral genomes like
SCMoV. In these genomes one encoded product could constrain the evolution of the other, as
107
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
shown for the overlapping ORFs for the CP and V2 protein of Cotton leaf curl virus (CLCuV) and for the AC1 and AC4 proteins of CLCuV and Tobacco leaf curl virus (TLCV) (Ooi et al.,
1997; Sanz et al., 1999). Environmental conditions also put genes under different selection pressures. For the two groups of BYDV-PAV described, infecting winter cereals and ryegrass
(Mastari et al., 1998), dN/dS for the overlapping CP and MP for one are 0.351 and 0.877, respectively, whereas the values for the other are 1.325 and 0.25 indicating the CP is the most conserved in the first group whereas in the second it is the MP.
Selection pressures have been associated with the maintenance of structural features of amino acids that play a role in assembly and stabilisation of nucleocapsids, e.g. the CP of tobamoviruses (Altschuh et al., 1987) and also in primary, secondary and higher-order structures involved in replication in viral genomes, such as the 3’NCRs of ssRNA genomes
(Kurath et al., 1993; Bacher et al., 1994). However, unlike in SCMoV, where these structures are conserved, those of RYMV are variable in geographically distant isolates (N'Guessan et al.,
2001; Zakia et al., 2003).
4.4.5 Fidelity of in vitro polymerases and genetic variation in SCMoV isolates
Nucleotide sequences of SCMoV isolates were obtained using RT-PCR, molecular cloning and cDNA sequencing. This method has been shown to be simple and valuable for characterising mutant spectra of virus quasispecies (Arias et. al., 2000). However, reverse transcriptases and
DNA polymerases are known to misincorporate bases in templates during in vitro transcription and DNA synthesis. The error frequency of MoMLV reverse transcriptase on RNA template in vitro is less than one error per 28,000 nucleotides (< 3.57 X 10-5 )(Ji and Loeb, 1992). This figure is the same order of magnitude or one order of magnitude smaller than the error rates estimated for viral RNA polymerases (Ji and Loeb, 1992). The estimated substitution error rate of Taq DNA polymerases is 0.2-2.0 X 10-4 errors per bp per cycle for Taq or 9.3 times less for proofreading polymerases (Bracho et al., 1998; Martell et al., 1992). Eckert and Kunkel
(1990), however, have determined conditions necessary to minimise Taq errors. In producing
108
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
cDNAs for cloning sequencing, precautions taken to minimise polymerase-induced errors were
(i) the use of MoMLV and the proofreading enzyme, rTth DNA polymerase, XL, (ii) a balance of amount of MgCl2 in excess of total dNTPs (Eckert and Kunkel, 1990) and (iii) the use of large amounts of cDNA to reduce the probability of cloning several PCR products derived from the same original virus cDNA template (Liu et al., 1996). Thus, genetic analysis of virus variability is unreliable if nucleotide substitution rates is similar to or lower than misincorporation rate of reverse transcriptase and/or DNA polymerase used in RT-PCR
(Bracho et al., 1998). The average genetic variation in SCMoV isolates was 0.00883 ±
0.00147, and even the lowest heterogeneity between SCMoV-AL and SCMoV-MJ, is greater than the error frequency of polymerases plus reverse transcriptases. The high nucleotide diversity and PCR precautions taken indicate that the variation is unlikely to be due to artifacts induced in vitro. However, it cannot be excluded that some mutations may have been introduced by the in vitro polymerases. It is also likely that the variation could have been different if more clones of each insert cDNA were sequenced.
4.5 Conclusions and further work
It has been demonstrated by comparative sequence analysis that the intra-isolate variation in genomic sequences of isolates of SCMoV, is most probably responsible for the different levels of symptom severity they induce in subterranean clover. The ORF1 gene product, whose sequence is more variable and under more positive selection than other genes is the likely severity determinant. These results, together with functional and comparative sequence analysis with other sobemoviruses and proteins of other viruses, implicate the ORF1 gene product in pathogenesis of SCMoV, possibly as a severity/pathogenicity determinant or as a viral suppressor of RNA silencing in plants. It is also likely to be involved in related functions such as genome amplification and virus movement.
Notwithstanding, it is known that the degree of selection on a virus genome is characteristic of the virus itself, as suggested by differences in the diversity of the populations of different viruses (e.g. TMV, CMV, and CCMV) (Schneider and Roossinck, 2000). Secondary mutations
109
4. Genetic Diversity in SCMoV Isolates and Functional Constraints of its Genes
at other sites have also been shown to suppress the effect of deleterious mutations as demonstrated for AMV (Roosien and Van Vloten-Doting, 1982; Van Vloten-Doting and Bol,
1988). This phenomenon might be common in other viruses. Future work could concentrate on determining which specific mutations are responsible for the differences in field biology of the isolates, using mutational analysis and expression of the different ORF1s from an infectious
SCMoV gene vector. Such a vector could be used to assess the possible RNA silencing suppressor activity of the P1 of SCMoV, the foreknowledge of which has been provided by work in this chapter.
110
Chapter Five
Independent cell-to-cell movement of the P1 and CP of SCMoV-pFL
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
5.0 Introduction
To develop viruses into gene vectors for expression of foreign proteins, they should be well characterised: knowledge of their genome structure, control of gene expression and functions of their proteins, is necessary for effective modification. Viral proteins have been characterised in vitro to try to relate their structural features to function (Abad-Zapatero et al., 1980;
Bhuvaneshwari et al., 1995; Opalka et al., 2000; Tamm and Truve, 2000; Lee and Hacker,
2001). However, in vitro systems do not take into account possible interactions with components of a host cell. Recombinant DNA technology that allows engineering of RNA viral genomes into infectious agents, and the discovery of GFP, have contributed immensely to the current knowledge of virus-host interactions. This includes expression of virus proteins, their localisation and movement in host cells. Engineered viral genomes with viral genes fused to GFP have been successfully used to study dynamics of viral movement including association of fused products to the host cellular network, their localisation and trafficking via plasmodesmata (PD) during infection of the virus (Epel et al., 1996; Oparka et al., 1996;
Padgett et al., 1996). To make full use of this approach, a full-length infectious clone is needed and an ability to modify the viral genome without losing infectivity. With a full-length infectious clone, it is possible to study all aspects of localisation and movement of fusion proteins including involvement of hosts and other viral components.
An alternative strategy, which also establishes beyond doubt the ability of a viral protein to move independently between cells, involves in planta production of the protein as a GFP translational fusion product and monitoring of its localisation and direct cell-to-cell trafficking.
This approach has been used as a preliminary investigation to study localisation of viral proteins, to confirm movement functions of virus-encoded proteins and to study the mode of virus movement (Epel et al., 1996; Padgett et al., 1996; Solovyev et al., 2000). The P1 and CP of sobemoviruses are both involved in cell-to-cell and long distance movement in hosts
(Brugidou et al., 1995; Bonneau et al., 1998; Sivakumaran et al., 1998) but their involvement in cell-to-cell movement in SCMoV has not been determined experimentally. It is also not
112
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL known if the P1 or CP of SCMoV is capable of movement independent of other viral components.
5.1 Aim and objectives of Chapter 5
In the absence of a SCMoV gene vector, in planta production of GFP fusions to the ORF1 and
CP gene were monitored to study the possibility of independent cell-to-cell movement in host tissues. This aim was achieved by i) constructing a plant expression vector ii) producing translational fusion constructs of GFP with ORF1 and CP gene of SCMoV-pFL iii) optimising conditions for effective biolistic transformation of legumes for transient gene expression analysis and iv) monitoring in planta production and possible cell-to-cell movement of these proteins by observing GFP fluorescence.
5.2 Materials and Methods
5.2.1 Development of transient expression vector, pTEV
All reporter gene constructs were under the control of 35S and T-Nos. To make cloning easier, sequences of the 35S and T-Nos were cloned and ligated into a single plasmid and used as a transient expression vector, pTEV. The sequences were amplified from the plasmid pPT1 developed by Ms. Peiling Tan (PBRG, SABC).
5.2.1.1 Amplification and cloning of 35S and T-Nos
Sequences of 35S and T-Nos were amplified separately with primers 35Sfor and 35Srev, and
Nosfor and Nosrev (Table 2.2). The PCR products were cloned respectively into pGEMT-Easy as pCaMV35S and pNos. Restriction sites incorporated into the primers created a multiple cloning region (MCR) at the junction of the two sequences, including a KpnI site used for subcloning T-Nos into pCaMV35S. Before subcloning, the SacI site in pCaMV35S was disrupted with T4 DNA polymerase so the SacI in the sequence of T-Nos could be used as one of the sites in the MCR. Recombinant plasmids pCaMV35S and pNos were digested separately, in a double digestion reaction with KpnI and NdeI resulting in a linearised
113
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL pCaMV35S with 83 bases of the MCR of pGEM-T Easy removed whilst the digestion released the T-Nos sequences plus 82 bases from the pGEMT-Easy (Fig. 5.1). Eighty nanograms of linearised pCaMV35S and 55 ng of digested T-Nos were ligated with T4 DNA Ligase as
KpnI/NdeI fragments to produce a recombinant plasmid, pTEV. Ligation was confirmed by
PCR with primers T7 and SP6.
3.894kb
329 bp
M 1 2 3 4 5 6 7
Fig. 5.1: Double restriction digest of pNos (lanes 1-5) and pCaMV35S (lanes 6-7) with KpnI/NdeI
5.2.1.2 Sequences and map of pTEV
The 35S and T-Nos inserts in pTEV, shown in Fig. 5.2, were sequenced with primers T7, 35S1b and SP6. Primer 35S1b anneals to the transcriptional start site of 35S and was kindly provided by Mr. John Blinco and Dr. Steve Wylie (SABC). No PCR or cloning-induced mutations were identified.
SphI KpnI
HindIII PstI EcoRI AgeI BamHI SacI pGEM-T backbone
35S T-Nos
894 bases TGCGCCAAGCTTGCATGCCTGCAGGTCCCCAGATTAGCCTTTTCAATTTCAGAAAG AATGCTAACCCACAGATGGTTAGAGAGGCTTACGCAGCAGGTCTCATCAAGACGAT 289 bases CTACCCGAGCAATAATCTCCAGGAAATCAAATACCTTCCCAAGAAGGTTAAAGATG GGTACCGAGCTCGAATTTCCCCGATCGTTCAAACATTTG CAGTCAAAAGATTCAGGACTAACTGCATCAAGAACACAGAGAAAGATATATTTCTC GCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCT AAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCACAAACCAAG TGCGATGATTATCATATAATTTCTGTTGAATTACGTTAA GCAAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCCACTGAATCAAAGGCCA GCATGTAATAATTAACATGTAATGCATGACGTTATTTAT TGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAA GAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACAT CAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGCCAACAT TTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTA GGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAG GGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGA TCGGGAATTCACCGGT ACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGA TTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGG CTCCTACAAATGCCATCATTGCGATAAAGGAAGGGCCATCGTTGAAGATGCCTCTG CCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAA GACGTTCCAACCACGTCTTCCAAAGCAAGTGGATTGATGTGATATCTCCACTGACG TAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGA AGTTCATTTCATTTGGAGAGAACACGGGGGACTCTAGAGGATCCCCGGGGGTAC Fig. 5.2: A schematic map of inserts in pTEV (not to scale) showing sequences of 35S and T- Nos and restrictions sites at the junction between them and also between the backbone cloning vector, pGEM-T Easy.
114
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
5.2.2 Development of reporter gene constructs
5.2.2.1 Amplification, cloning and subcloning of GFP into pTEV as pTEVGFP
A modified gene of the jellyfish GFP, m-gfp5, without the C-terminal HDEL endoplasmic reticulum (ER) retention signal, provided by Dr. Geoff Dwyer was used as the reporter gene in all experiments and referred to as gfp (Haseloff et al., 1997). Other modifications in this gene included altered codons to eliminate cryptic splicing in Arabidopsis and probably in other plants. The mutation V163A and S175G were made to aid folding of the apoprotein and/or cyclisation of the chromophore to improve fluorescence (Siemering et al., 1996) which in combination with mutation I167T gives the modified gene dual excitation peaks of approximately equal intensity at 400 nm and 475 nm (Siemering et al., 1996). This property allows visualisation of fluorescence with either long wavelength UV or blue light.
To compare expression and localization of free GFP to fusion products, the gfp gene was cloned into pTEV as pTEVGFP. The gene was amplified with primers gfpfor and gfprev.
Primer gfpfor incorporated a BamHI site and a context Kozak sequence for efficient translation at the 5’ end whilst primer gfprev introduced a SacI site and a stop codon at the 3’ end of the gene. The PCR product was cloned into pGEM-T to give pGFPopt and later subcloned into pTEV as a BamHI/SacI fragment to give pTEVGFP (Fig. 5.3). Ligation of GFP into pTEV was confirmed by restriction digests and PCR with primer pairs T7/35Sfor with gfprev, and gfpfor with SP6/nosrev.
5.2.2.2 Sequences of m-gfp5 and map of pTEVGFP
Sequences of GFP, determined with primers T7 and SP6, in pGFPopt confirmed the presence of modifications introduced into m-gfp5 for improved fluorescence (5.2.2.1) but no other PCR or cloning-induced mutations were identified. After subcloning GFP into pTEV, ligated junctions of the 35S and GFP, and GFP and T-Nos were sequenced with primers 35S1b and
SP6 to confirm efficient ligation. A schematic map of pTEVGFP is shown in Fig. 5.3.
115
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
SphI
HindIII PstI EcoRI AgeI BamHI SacI pTEVGFP 35S GFP T-Nos
aacaatg agt aag gga gaa gaa ctc ttc acc gga gtt gtc cca att ctt gtt gaa tta gat ggt M S K G E E L F T G V V P I L V E L D G gat gtt aat ggg cac aaa ttt tct gtc agt gga gag ggt gaa ggt gat gca aca tac gga D V N G H K F S V S G E G E G D A T Y G aaa ctt acc ctt aaa ttt att tgc act act gga aaa cta cct gtt cca tgg cca aca ctt K L T L K F I C T T G K L P V P W P T L gtc act act ttc tct tat ggt gtt caa tgc ttt tca aga tac cca gat cat atg aag cgg V T T F S Y G V Q C F S R Y P D H M K R cac gac ttc ttc aag agc gcc atg cct gag gga tac gtg cag gag agg acc atc ttc ttc H D F F K S A M P E G Y V Q E R T I F F aag gac gac ggg aac tac aag aca cgt gct gaa gtc aag ttt gag gga gac acc ctc gtc K D D G N Y K T R A E V K F E G D T L V aac agg atc gag ctt aag gga atc gat ttc aag gag gac gga aac atc ctc ggc cac aag N R I E L K G I D F K E D G N I L G H K ttg gaa tac aac tac aac tcc cac aac gta tac atc atg gcc gac aag caa aag aac ggc L E Y N Y N S H N V Y I M A D K Q K N G atc aaa gcc aac ttc aag acc cgc cac aac atc gaa gac ggc ggc gtg caa ctc gct gat I K A N F K T R H N I E D G G V Q L A D cat tat caa caa aat act cca att ggc gat ggc cct gtc ctt tta cca gac aac cat tac H Y Q Q N T P I G D G P V L L P D N H Y ctg tcc aca caa tct gcc ctt tcg aaa gat ccc aac gaa aag aga gac cac atg gtc ctt L S T Q S A L S K D P N E K R D H M V L ctt gag ttt gta aca gct gct ggg att aca cat ggc atg gat gaa cta tac aaa taa L E F V T A A G I T H G M D E L Y K *
Fig. 5.3: A schematic map of pTEVGFP (not to scale) showing nucleotide and amino acid sequences of gfp, restriction sites for cloning GFP into pTEV, with an ACAA sequences upstream of the start codon for efficient translation in plants.
5.2.2.3 Amplification, cloning and fusion of CP and ORF1 genes of SCMoV-pFL to GFP
A similar plasmid to pTEVGFP, used for cloning the ORF1 and CP gene of SCMoV-pFL as c- terminal fusion partners to GFP, was pTEVGFPB. In this plasmid, the cloned gfp gene was amplified without a stop codon with primers gfpfor and scm31. The ORF1 and CP genes were each amplified from a full-length cDNA clone of SCMoV-pFL (section 6.2.2) as SacI fragments with primer pairs SacP1for and SacP1rev for ORF1, and SacCPfor and SacCPrev for the CP respectively and cloned into pGEM-T as pSacICP and pSacIP1. They were later subcloned as SacI fragments into SacI linearised, dephosphorylated plasmid pTEVGFPB to give pTEVGFPCP and pTEVGFPP1 (Fig. 5.4). Directional cloning was confirmed with PCR using primer pairs T7/35Sfor and gfprev, and gfpfor and Nosrev/SP6.
116
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
5.2.2.4 Sequencing and maps of c-terminal GFP fusion constructs
Sequences of the ORF1 and CP gene in plasmids pTEVGFPP1 and pTEVGFPCP are the same for those presented for SCMoV-pFL in 4.3.2.2 and 4.3.2.4 respectively. Ligated junctions of the genes and GFP, and T-Nos were determined with primers 35S1b, SP6 and gfpro2. A schematic map of the constructs is shown in Fig. 5.4.
gat gaa cta tac aaa taa gag ctc Bam HI
(A). pTEVGFP 35S GFP T-Nos
gat gaa cta tac aaa gag ctc Bam HI
(B). pTEVGFPB 35S GFP T-Nos
CP sequence
gac gag ctc tac aaa acc ggc...tag gag ctc Bam HI
(C). pTEVGFPCP 35S GFP CP T-Nos
ORF1 sequence
gat gag ctc tac aaa cca tca... tga gag ctc Bam HI (D). pTEVGFPP1 35S GFP ORF1 T-Nos
Fig. 5.4: Schematic map of reporter gene constructs used for cellular studies showing nucleotide modifications in GFP and CP and ORF1 (A) Map of pTEVGFP, (B) Map of pTEVGFPB, (C and D) Map of c-terminal translational fusion of GFP with CP gene and ORF1 is shown as pTEVGFPCP and pTEVGFPP1 respectively.
5.2.3 Biolistic transformations
Introduction of DNA into tissues by biolistics has gained much popularity in the last two decades. The principle behind this method is the high-speed propulsion of DNA-coated microparticles, gold or tungsten, into a wide range of biological samples (Sandford et al.,
1993). Various documented systems have used a burst of a gas, such as a dry steam generated by an electric discharge on a drop of water (Christou, 1995; Shigeru and Kimura, 1997), a shock wave created by capacitive electric discharge (Shigeru and Kimura, 1997), a gun powder explosion (Sandford et al., 1987; Klein et al., 1992), or a burst of compressed helium (He)
117
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
(Sandford et al., 1993). The Bio-Rad Biolistic PDS-1000/He and other He particle delivery systems have been widely used for biolistic transformation experiments because of their recognised efficiency.
5.2.3.1 Biolistic device for plant transformation
A custom-made biolistic device manufactured by the workshop at the Commonwealth Science and Industry Research Organisation (CSIRO), Canberra, Australia, and used for plant transformations at the SABC is shown in Fig. 5.5. In this system, microprojectiles are propelled from a grid by gas shock derived from discharge (high speed solenoid) of compressed
He towards tissues placed in a vacuum chamber with negative pressure of 95-100 kPa provided by a Rotary Vane vacuum pump RZ2 (Vacuubrand GMBH +Co KG, Germany). With the use of the Bio-Rad Biolistic PDS-1000/He System and other modifications, pressures of between
4,000-14,000 kPa have been used for transformation (Sandford et al., 1993) but for safety reasons, the allowable pressure for this equipment was 2,000 kPa.
1 Labels of the biolisitic device 1. Helium pressure gauge 2. Cylinder containing compressed Helium 8 9 gas 3. Vacuum gauge 2 4. Fuse box 5. Regulated power supply 3 6. Vacuum chamber 7. Vacuum pump
8. Oil waste can 9. High speed solenoid 4 7 6 5 Fig. 5.5: The custom-made biolistic device used in the SABC for plant transformation
5.2.3.2 DNA for transformations
Plasmid DNA used for transformations were pTEVGFP and pTEVGFPB (both expressed as free GFP), and two C-terminal GFP fusion constructs, pTEVGFPCP and pTEVGFPP1, expressed as GFP:CP and GFP:P1 respectively. In addition, the vector pUbiGFP containing the maize polyubiquitin promoter usually used for monocot transformations and fused to the synthetic gene sgfpS65T (Chiu et al., 1996; Christensen and Quail, 1996) was used as a standard for optimizing biolistic conditions for efficient transformation. This plasmid was
118
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL obtained from Grain Biotech Australia (GBA), a Western Australian biotechnology company based at the SABC. All plasmid DNA were purified with the QIAprep Spin Minprepi kit and with phenol-chloroform and isopropanol extraction after resuspension, lysis and neutralization of bacterial cells (section 2.9) and dissolved at high concentrations of between 800 ng and 1
µg/µL.
5.2.3.3 Microcarrier preparation
Microcarrier preparation and coating of DNA onto microcarriers were similar to the method described by Sandford et al., (1993). Sixty milligrammes of 0.8-1.0 µm gold microcarrier particles (kindly provided by Ms. Kanokwan Ratanasooban (PBRG, SABC) were washed with
1 mL of freshly prepared 70% ethanol in a 1.5 mL microfuge tube by vortexing for 5 min.
After incubation at room temperature for 15 min, the resulting pellet was centrifuged for 5 sec and the supernatant discarded. The pellet was washed three times as follows: 1 mL of sterile water was added to the pellet, vortexed for 1 min, allowed to settle for a further minute, pelleted again for 2 sec, and the supernatant discarded. The washed microparticles were finally resuspended in 1 mL of sterile 50% (v/v) glycerol solution to make an approximate concentration of 60 mg/mL. This stock was kept at room temperature or 4°C.
5.2.3.4 Coating DNA onto microcarriers
For each control or test construct, a mixture for 6-8 bombardments were prepared as follows: the 50% glycerol microcarrier stock was vortexed vigorously to resuspend and disrupt agglomerated particles and 50 µL (≈3 mg) removed to a fresh tube. The aliquot was continuously vortexed during which 5-10 µg of plasmid DNA, 50 µL of 2.5 M CaCl2 solution and 20 µL of 0.1 M Spermidine solution (kept at -20°C) were added in quick succession. The mixture was continuously vortexed for an additional 3 min, left to settle for 5-10 min or centrifuged for 2 sec after which most of the supernatant was discarded leaving the pellet in a minimum volume necessary for deposition of the particles on the microcarrier grid. This mixture was used for shooting or further treated with ethanol as follows: Excess supernatant was removed and 140 µL of 70% ethanol added without disturbing the pellet and repeated with
119
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
140 µL of absolute ethanol. The supernatant was discarded and the pellet finally resuspended in 50 µL of ethanol, made to settle by gently tapping on the side of the tube or vortexing at a low speed for 2 sec. For particle bombardment, 6-10 µL of the prepared shooting mixture was removed during vortexing, and placed in the centre of a 10 mm plastic grid from which microcarriers were propelled into tissues.
5.2.3.5 Particle bombardment and plant tissues transformed
Leaf tissues of plants used for transient analysis were subterranean clover, Trigonella, grasspea, kabuli chickpea and wheat. The upper surface of fresh leaves excised from 3-5 week-old potted plants or in vitro cultured seedlings were placed on a Petri dish and covered with a 5 cm diameter fine netting with about 120 µm mesh to reduce shock to cells upon bombardment.
Tissues were bombarded by dry or wet shooting at a target distance of 17 cm using a Helium- driven pressure of between 800-2200 kPa and a negative pressure of 95-100 kPa in the vacuum chamber. Wet shooting involved bombarding samples with DNA-coated particles taken from the shooting mixture after a considerable amount of supernatant was discarded without further treatment with ethanol. With dry shooting, DNA-coated particles were treated with ethanol and an aliquot for shooting air-dried for up to 2 min on the grid before bombardment. Following bombardment, leaf tissues were placed in a Petri dish with Murashige and Skoog (MS) medium solidified with 0.6 % (w/v) phytagel sealed with parafilm to keep them moist and placed in a growth room (22°C, 16 h of illumination) until ready for examination.
5.2.3.6 Visualisation of GFP expression
Bombarded leaves were not sectioned but examined as whole and intact leaves. They were mounted on glass slides with cover slips, and pressed before examination 20-96 hr post- bombardment (hpb) with an Olympus BH-2 microscope with a BH-RFL-W Reflected light fluorescence attachment and 100W high pressure mercury burner. GFP expression in host cells was visualised using a blue-green exciter filter and dichromic mirror unit with a Y495 and
Y510 barrier filters. Images were captured with the Olympus DP12 microscope digital camera system attached to the microscope. 120
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
5.3 Results
5.3.1 Effect of amount of DNA and shooting pressure on GFP fluorescence
The biolistic device described in section 5.2.3.1 is regularised for transient and stable transformation of wheat embryos, but not leaf tissues, at an optimum pressure of 1200-1400 kPa. Several attempts to transform legume leaves for transient expression analysis of GFP at these pressures were unsuccessful. After initial bombardment showed no fluorescence in any tissues, some conditions that may influence the efficiency of transformation were optimised: shooting pressure, amount and purity of DNA, and treatment of DNA-coated microcarrier particles before bombardment.
Initially, shooting pressure and amount of DNA optimal for GFP fluorescence was tested with the pUbigfp on wheat tissues. Freshly cut leaves were divided into pieces with surface areas of approximately 4 cm2 and bombarded with varying plasmid DNA concentrations (500, 700 and
1000 ng/≈600 µg of DNA-coated particles per shot) at shooting pressures of 800, 1000, 1400,
1800 and 2000 kPa. Results are shown in Table 5.1. Initially when coated DNA particles were shot in aqueous solution, no fluorescence was observed in tissues shot at 800 and 1000 kPa whilst at a pressure of 1400 kPa, 2-5 fluorescent spots were observed when leaf tissues were bombarded with 500-1000 ng of plasmid DNA. The number of fluorescing cells increased with increasing amount of DNA and shooting pressure. The number of fluorescent spots on wheat leaf tissues shot with pressures of 1600 and 1800 kPa were not different. There was no significant difference in expression when plasmid DNA was purified with either of the methods in section 5.2.3.2.
Table 5.1: Number of fluorescent spots/cells in ≈ 4cm2 wheat leaves bombarded with varying amount of pUbigfp plasmid DNA shot dry and wet at different pressures.
Amount of plasmid DNA per shot (ng of DNA/ ≈ 600 µg of particles) Wet shooting Dry shooting Pressure(kPa) 500 700 1000 500 700 1000 800 ------1000 - - - 2 5 9 1400 2 2 5 6 7 11 1600 10 9 12 11 13 15 1800 10 11 14 13 13 16
121
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
5.3.2 Effect of dry and wet shooting of DNA-coated particles on transformation
Duplication of conditions for biolistic transformation for wheat leaves (section 5.3.1) produced no GFP expression with any of the plasmid constructs in subclover, grasspea, Trigonella and chickpea. Simultaneous increase in shooting pressure to as much as 2200 kPa, a change from wet to dry shooting with increased amount of plasmid DNA finally gave some GFP expression in the legumes. The possible cause for the increased transformation efficiency was investigated with pUbigfp on wheat tissues. Efficiency of transformation of wheat cells increased with dry shooting resulting in expression of GFP even at a shooting pressure of 1000 kPa and as much as
100% fold increase in spots of fluorescence over wet shooting at a pressure of 1400 kPa (Table
5.1). Ethanol treatment of coated particles is thought to allow separation of microcarriers
(eliminates agglomeration) leading to subsequent wider distribution of the microprojectiles. As a result, fewer more uniformly distributed particles, rather than blobs of particles, were expected to enter into single cells to reduce the impact of microprojectiles resulting in single fluorescent cells that allowed easy examination and analysis.
However, fluorescent spots in the legume host tissues were always limited to less than ten. A further increase of shooting pressure from 2200kPa may have increased the efficiency of transformation, however, all analyses were done on tissues bombarded with up to 1 µg of plasmid DNA per ≈ 600 µg of particles shot dry at a pressure of 2200kPa.
5.3.3 Transient expression of free GFP in cells of subclover, trigonella, chickpea and grasspea
Bombarded leaves of subterranean clover and Trigonella showed characteristic greenish-yellow auto-fluorescence that could be attributed to impact of microprojectiles on the tissues.
Comparison of tissues shot with GFP plasmids and control tissues shot with non-GFP plasmids shows there was some GFP fluorescence in the former but was masked by the observed auto- fluorescence (Fig. 5.6a-c). Auto-fluorescence caused by the damage on these tissues was not eliminated by using lower pressures though the number of auto-fluorescent sites was reduced.
This phenomenon has been attributed to activation of unknown (or phenolic) compounds in 122
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL some legumes after particle bombardment. The wavelengths emitted by auto-fluorescence compounds could not be distinguished from GFP fluorescence by the Olympus camera hence appeared similar on photographic film. If this fluorescence were a result of damage it was assumed that transgenic cells of the leaves would not be intact enough to allow accurate expression and analysis of GFP expression. GFP transient analyses were, therefore, done in transgenic cells of chickpea and grasspea.
A B C D
E F G
Fig. 5.6: Epiflourescent micrographs of epidermal cells of host cells used in transient analysis after particle bombardment. (A). Cells of subterranean clover not bombarded with any DNA construct, compared with (B/C) Greenish yellow autofluorescence and/or green fluorescence in epidermal cells of subterranean clover and grasspea 48 hpb with plasmid pTEVGFP. (D). Green fluorescence in single epidermal cell of wheat expressing GFP from pUbiGFP. (E/F). Green fluorescence in epidermal cells of chickpea and grasspea 48 hpb respectively expressed from plasmid pTEVGFP construct. (G). Single epidermal cell of chickpea expressing GFP from pTEVGFPB.
When free GFP was expressed in chickpea and grasspea cells with pTEVGFP and pTEVGFPB,
5 to 8 sites of GFP-specific fluorescence per leaf were detected 20 to 96 hpb. At 20 hpb, a small fluorescence spot in a cell could be maintained at a high level up to 96 hpb. As in the wheat cells (Fig. 5.6d), GFP fluorescence was detected in both the cytoplasm and the nucleus of chickpea and grasspea cells (Fig. 5.6e-g) as reported (Sheen et al., 1995; Chiu et al., 1996).
Fluorescence was generally limited to one cell, but on the few occasions tissues were kept after
72 hpb, the fluorescence began to fade and could be visible in the periphery of two or three
123
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL contiguous cells (Fig. 5.6e). This was more prominent when GFP was near full bleaching after longer exposure to violet light.
5.3.4 Transient expression of GFP:CP and GFP:P1 in cells of chickpea and grasspea
Monitoring of GFP:CP and GFP:P1 expression in cells of chickpea and grasspea after bombardment with pTEVGFPCP and pTEVGFPP1 were always done after 24 hpb. Expression patterns for both GFP:CP and GFP:P1 were similar to that of free GFP i.e. fluorescent signal was observed in the cytoplasm and nucleus of single cells in both chickpea and grasspea, observed as individual spots of green fluorescence. In addition, fluorescence was observed in the cell periphery of neighbouring cells 48 to 60 hpb (Fig. 5.7a-b). However, unlike expressions from pTEVGFP where spread to neighbouring cells was a result of passive diffusion observed after 72 hpb, spread of fluorescence resulting from expression of GFP:CP or
GFP:P1 was more prominent and observed earlier after particle bombardment. Spread was evident when only cell peripheries and not nuclei or cytoplasm of neighbouring cells showed fluorescence. When chickpea leaf tissues were co-bombarded with 1 µg each of plasmids pTEVGFPCP and pTEVGFPP1, the number of fluorescing cells increased significantly compared to fluorescence expressed from either of the plasmids alone. In addition, fluorescence was more prominent and filled the cells such that nuclei of fluorescing cells were not visible. An attempt to monitor this expression over a longer period resulted in photobleaching (Fig. 5.7c).
An interesting observation was the fluorescence from guard cells (Fig. 5.7d-g). Monitored over a period of 72 hpb, fluorescence in guard cells did not show any increase in size or apparent spread to neighbouring cells. Also, there was no spread of fluorescence from fluorescing cells to guard cells present within this area. This is exemplified by the spread of fluorescence from
GFP:CP in veinal cells of grasspea to peripheries of neighbouring cells without spreading into guard cells within the vicinity (Fig. 5.7d).
124
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
A N B C
D E F G
GC GC GC
Fig. 5.7: Fluorescent images obtained using an epifluorescence microscope and blue excitation of cells photographed in the absence (A, B, C and D) or in the presence (E, F and G) of background transmitted light. (A and B). Expression of GFP:P1 in chickpea and grasspea respectively showing the nucleus (N) in both cases and apparent spread of fluorescence into surrounding cells (SC) after 48 hpb. Spread is indicated by the presence of fluorescence in cell peripheries of surrounding cells and the absence of any in their nuclei. (C). Expression in chickpea cells of GFP:CP and GFP:P1 72 hpb showing photobleaching after an attempt to monitor fluorescence over a period. (D). GFP fluorescence in veinal epidermal cells of grasspea showing spread of fluorescence expressed from GFP:CP into periphery of surrounding cells but without spread into guard cells (GC) taken 60 hpb. (E and F). Image of guard cells of chickpea with and without fluorescence 48 hpb with GFP:CP. (G). Fluorescing guard cell of grasspea, image taken to show the position of stomata
5.4 Discussion
In this study, transient expressions of GFP and fusions of GFP with the ORF1 and CP gene of
SCMoV-pFL under the control of 35S promoter and T-Nos terminator sequences were studied in cells of chickpea and grasspea by observing GFP signals. Results of localisation of the P1 and CP and implications for modification of the SCMoV genome into gene vectors are discussed. Optimum conditions for biolistic transformation of legume leaf tissues, difficulties in obtaining intact transformed cells of subclover and Trigonella for transient analysis and limitations of GFP in such studies are also discussed.
125
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
5.4.1 Expression and localisation of CP and P1 of SCMoV-pFL in host cells
Using the transient expression system, the results indicated that GFP:P1 and GFP:CP fusion proteins produced in planta move between cells of chickpea and grasspea but do not accumulate much in the cytoplasm of neighbouring cells. In contrast, the limited ability of GFP to spread between cells (Oparka et al., 1999; Wymer et al., 2001) suggests that it only moves by passive diffusion through the PD. This indicates a limited ability of the CP and P1 of
SCMoV to move independent of viral components. The limited ability of the proteins to move independently, their involvement in movement of sobemoviruses and their ability to bind to ssRNA suggests sobemoviruses may be transported between cells via a complex which may involve ssRNA, viral proteins and/or cellular components similar to movement by some viruses
(Jeffery et al., 1996; Kotlizky et al., 2000; Lough et al., 2000; Palmer and Rybicki, 2001). This could be supported by the over-expression of GFP:CP and GFP:P1 when chickpea leaf tissues were co-bombarded with plasmids encoding the fusion products.
It is known that viral proteins move from cell to cell and over long distances by exploiting and modifying preexisting pathways for macromolecular movement between cells such as through the PD (Ding et al., 1995; Lucas, 1995; Carrington et al., 1996). Interactions of CP and P1 with the PD was not studied and it is therefore not possible to say whether movement of the proteins involves temporary or permanent changes to the PD’s size exclusion limit as observed for some viral proteins (Fujiwara et al., 1993; Ding et al., 1995). However, cell-to-cell movement of the CP and P1 is expected to be via the PD in host cells. Indirect evidence was provided by observed fluorescence patterns in guard cells. That the movement is via PD is supported by the observation that the fused proteins do not spread to guard cells present within the fluorescent patches. Likewise the proteins do not move out of guard cells that have been bombarded with a construct from which the fused proteins are expressed.
The results have implications for the design and modification of SCMoV into a gene vector. If transport of these proteins requires complementation and/or involvement of other viral and cellular factors, a vector derived from sobemoviruses and SCMoV will express and move
126
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL systemically in suitable hosts only when all the necessary viral genes are present in the vector construct.
5.4.2 Putative nuclear export signals of the SCMoV CP and P1 proteins
Mutational and sequence analysis has shown that some viral encoded proteins have nuclear shuttling properties and hence possess nuclear localisation signals (NLS) and/or nuclear export signals (NES), e.g. CPs of the monopartite and bipartite geminiviruses (Kunik et al., 1998; Qin et al., 1998; Kotlizky et al., 2000). For karyophilic proteins, nuclear import is generally mediated by NLS sequences belonging to one of two groups: (i) the monopartite SV40 large T antigen NLS (PKKKRKV) motif (Kalderon et al., 1984; Whittaker and Helenius, 1998) or (ii) the bipartite motif, consisting of two basic regions separated by a variable number of spacer amino acids, typified by the nucleoplasmin NLS (KRPAATKKAGQAKKK) (Robbins et al.,
1991). NESs mediating nuclear export of proteins is characterized by leucine rich motif, exemplified by the HIV-1 Rev molecule (LPPLERLTL) (Bogerd et al., 1995; Fischer et al.,
1995), in the catalytic domain of cAMP dependent protein kinase inhibitor, PKI,
(LALKLAGLDI) and also present in a variety of exported proteins (Fritz and Green, 1996).
Some viral proteins have both NLSs and NESs at different positions e.g. ACMV CP (Unseld et al., 2001).
Interestingly, there is no Lysine (K) residue in the deduced amino acid sequences of the ORF1 gene product of SCMoV-pFL (section 4.3.2.2) and though there are K residues in the deduced amino acid sequences of the CP gene the closest to any of the identified nuclear import signals was at the N-terminus; the three consecutive Ks at positions 10-13 surrounded by a few arginine residues; RRSKKKSG. However, there are leucine-rich sections in both the ORF1 gene product and the CP. Scanning of deduced amino acid sequences of ORF1 revealed two potential NLS sequences at both the N- and C-termini. There are repeats of four Ls at position
16-19 and a series of four Ls at the C-terminus from position 160-172; (LVRLFQLQSIQPL) the latter bearing close resemblance to the typical NES of the HIV-1 Rev molecule. In the CP sequences, a leucine rich section occurs at position 130-150; LGYLYDALDAVPAALQQMSSL,
127
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL with 5 among 21 residues. Proteins with NESs may export RNA molecules and several of these, demonstrated to be involved in virus movement, are also capable of binding to ssRNA molecules like the P1 and CP of CfMV (Citovsky et al., 1990; Osman et al., 1993; Tamm and
Truve, 2000). This comparative sequence analysis and the transient expression studies indicate the P1 and CP may not be karyophilic proteins but may be involved in nuclear and cell-to-cell movement of the viral genome in susceptible hosts.
5.4.3 Optimum conditions for transient transformation of legumes
The ability to visualise green fluorescence in any of the legume tissues after bombardment of reporter gene plasmids with the biolistic device depended on the intrinsic properties of the plant and biolistic conditions including amount of DNA, shooting pressure and treatment of DNA coated particles before shooting. Strong green fluorescence was observed in bombarded wheat leaf tissues even using low pressures and low DNA amounts with the polyubiquitin promoter, whereas similar conditions could not be used to transform legume tissues with plasmids under the control of 35S promoter. There are reports that the 35S is a weaker promoter hence the efficient and widespread use of the polyubiquitin promoter for stable and transient expression in monocots (Sandford et al., 1993; Schenk et al., 1998).
With the device used, Grain Biotechnology Australia, Perth, has obtained both stable and transient transformation of wheat and maize after bombarding immature embryos of these plants. They used plasmids under the control of the polyubiquitin promoter with microcarriers shot into samples in a minimal amount of aqueous shooting mixture without any ethanol treatment. In contrast, transformation of chickpea and grasspea tissues depended on the advantage provided by ethanol treatment of DNA coated particles; effective separation and subsequent increased dispersion and penetration ability. The original biolistic protocol of
Sandford et al., (1993) on which almost all others are based, recommends ethanol treatment and dry shooting of microcarrier particles. In transforming fragile insect tissues, Thomas et al.,
(2001b) compared a wet shooting method developed by Miahle and Miller (1994) with that of
Sandford et al. (1993) and observed 26% better transformation efficiency with the latter
128
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL protocol. The low number of fluorescent cells observed for the legume hosts used in the study and the inability to transform subterranean clover and Trigonella could be attributed to various factors among which is the strength of the 35S promoter. It is possible a further increase in shooting pressure and adjustment of target distance could have increased penetration of microcarriers and efficiency of transformation. However, the optimised conditions for transformation of chickpea and grasspea were not successful for transient expression of subterranean clover and Trigonella. This is an intrinsic property of cells of these plants. In addition, GFP may not be stable in these cells. This is discussed further in section 5.4.4.
5.4.4 Limitations of GFP as a reporter gene
Limitations to the use of GFP are either technical or host-related or both. The technical limitations have to do with the limited ability of GFP to spread from cell to cell on its own
(Wymer et al., 2001). Like some viral MPs, the size of GFP is bigger than the SEL of PDs of most cells and is not expected to move actively between cells. But on rare occasions and in some cells GFP fluorescence may spread passively to neighbouring cells (Wymer et al., 2001).
This does not make GFP less attractive for use in cellular studies, but caution is needed in interpreting results that use GFP fluorescence as a signal to study localisation or movement of proteins in specific cellular compartments. Furthermore, GFP fusions to some proteins have been reported to prevent proper processing of the GFP chimera and causes improper folding, instability or a conformational change in either GFP or the encoded GFP chimera resulting in little or no fluorescence in some plants (Waldo et al., 1999). Although there is no evidence this is the case for subterranean clover and Trigonella, the possibility cannot be ruled out.
Among the host-related factors that may limit the full potential of GFP is the high level of background autofluorescence emitted by tissues of some plants, e.g. subterranean clover, in the natural state or caused by damage e.g. after particle bombardment. This could obscure weak green fluorescence signals leading to low estimation where this property is used as a measure or levels of protein expression. In addition, autofluorescence can be mistaken for a true GFP signal thereby interfering with localisation studies (Mantis and Tague, 2000).
129
5. Independent cell-to-cell movement of the P1 and coat protein of SCMoV-pFL
The ability to visualise GFP may also depend on the condition of, and the cellular compartment within which it is expressed in particular plant species. Insights into effects of pH on GFP expression in cellular compartments came from the study of biogenesis and regeneration of vacuoles (Di Sansebastiano et al., 2001). They attributed failure to visualise GFP in stably transformed tobacco lines to pH of the vacuoles. When GFP markers specific for labelling neutral and acidic vacuoles showed much lower or no GFP fluorescence in acidic vacuoles, it was suggested that the GFP marker was rapidly degraded in its final target compartment as the vacuoles may have accumulated proteases (Hörtensteiner et al., 1992). However, both GFP markers could successfully label different cell types of transgenic Arabidopsis tissues, and there are species differences in the level of GFP expression. The presence of proteases in different cell types and their effect on the level of GFP expression has been reported (Haseloff et al.,
1997; Feilmeier et al., 2000). It is thus possible in subterranean clover and Trigonella that GFP was expressed in an unfavourable compartment or some form of toxicity may have decreased
GFP expression in these cells.
5.5 Conclusion and further work
Limited independent movement of the CP and P1 of SCMoV-pFL between cells and their possible involvement in SCMoV movement and implications for gene vector design and construction has been established in the study. It is not known, however, if any viral component is required for movement of the native SCMoV encoding it and if the putative NESs identified have any role in transport of these proteins and/or the RNA viral genome. Also the interaction of these proteins with the PD is not known. Mutants of the genes expressed from an engineered infectious clone (Chapter 6) as fusion partners to reporter genes will most probably produce, in planta, high concentrations of the proteins in host tissues for further investigation of the role of the proteins in movement, their association with the PD and the role of the putative NESs identified in this study. Such work could not be done at the time because application to OGTR for permission to create and use recombinant SCMoV gene vectors had not been approved.
130
Chapter Six
Development of an Infectious cDNA Clone of SCMoV and GFP
derivatives for use as VIGS Vectors
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.0 Introduction
Infectious full-length cDNA clones of only two sobemoviruses have been reported. Both of these, SCPMV and RYMV-CI, have mainly been used to characterise the genomes (Brugidou et al., 1995; Bonneau et al., 1998; Sivakumaran et al., 1998; Sivakumaran and Hacker, 1998).
In each case, the biological activity was assessed using in vitro-transcribed RNA transcripts.
Reported infectivity of these transcripts were comparable to viruses of other morphologies, indicating successful infection could be gained from engineered genomes of icosahedral RNA viruses. Engineered viral genomes of no other sobemovirus has been reported despite the advantages they have over some other viruses: the relatively small size of the genomes and their efficient infection leading to high titres of the virus in hosts. Moreover, the use of a sobemovirus as a gene vector has not been demonstrated.
Viral gene vectors, including VIGS vectors, are developed by engineering the native genomes using one of more strategies involving gene replacements, insertions, duplications and gene complementation. Usually, they are modified with gene markers and depending on the intended method of introduction into hosts, they are cloned with heterologous sequences such as the CaMV 35S promoter and T-Nos terminator sequences. In GFP chimeric vectors, the protein is used for measuring the level of expression, movement functions and to determine how robust the vectors would be in carrying and expressing a foreign gene. It has also been used for testing gene silencing activity of virus vectors where, as a VIGS insert, it is used for studying GFP silencing in GFP transgenic plants.
6.1 Aim and objectives of Chapter 6
The aim of this chapter was to develop a series of GFP chimeric constructs based on SCMoV.
The best of these, after assessment of their vector potential, will be chosen for routine use as a
VIGS vector. The constructs could also be used in gene expression experiments to characterise
SCMoV. This aim was achieved by i) description of properties of SCMoV necessary for modifying the genome into a gene vector, including
132
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
a) possible gene expression strategies based on the genome organisation
b) prediction of a putative sgPro sequence for synthesis of the CP using comparative
sequence analysis of sobemoviruses and other viruses. The aim was to duplicate the
sgPro sequence in some of the constructs as a promoter for GFP, ii) cloning and preliminary assessment of infectivity of a full-length cDNA of SCMoV-pFL, iii) construction of a series of GFP chimeric derivatives of the full-length clone of SCMoV from which both in vivo-and in vitro-transcribed RNA transcripts could be obtained to assess their vector potential.
6.2 Gene expression of SCMoV
The gene expression strategy of SCMoV is described based on its genome organisation and information from related viruses. Like other sobemoviruses and poleroviruses (D’Arcy et al.,
2000), SCMoV is expected to express its protein products from ORFs using different types of modulation mechanisms (frameshift, initiation bypass, production of sgRNA, proteolysis of primary translation products) (Fig. 6.1). Sequence comparison with sobemoviruses, and the compact nature of SCMoV genome suggests the first AUG of each ORF, except ORF2b, is the initiation codon for translation irrespective of the context sequences of the codons. Both the
ORF1 and ORF2a are probably translated from the gRNA whereas the ORF3 is expressed via a sgRNA. The ORF2b is expressed via -1 frameshifting of ORF2a, confirmed by the presence of the slippery sequence (UUUAAAC) and a predicted stem loop downstream of this heptamer.
All modifications of the genome to develop gene vector constructs assumed that the first AUG was the translation initiation codon of the ORFs.
Translation of the ORF1 is predicted to produce a protein product of about 20 kDa whereas initiation at the first codon of the ORF2a is likely to produce two peptides; a single product from the translation of the ORF2a or a polyprotein from translation of ORFs 2a and 2b. Like all sobemoviruses, the polyprotein is thought to cleave into a protease, RdRP and a VPg
(Gorbalenya et al., 1988; van der Wilk et al., 1998). The CP is produced by the ORF3 via sgRNA synthesis. 133
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
5’ 3’ RNA genome 5’NCR 2b 3’NCR 2a 3 ORFs 1
Expression P1 coat protein P2a
P2a + P2b (frameshift) proteolysis Subgenomic RNA synthesis protease VPg replicase
Fig. 6.1: The genome of SCMoV showing predicted expression strategies: production of subgenomic RNA, initiation at the first AUG (P1), initiation as for P2a and frameshift (P2a+P2b) and proteolysis of the translation product of P2a+P2b. Black lines represent RNA molecules, black boxes are open reading frames (ORFs), grey boxes represent translation products and open boxes represent translation cleavage products.
6.3 Prediction of sg promoter (sgPro) of SCMoV by sequence analysis
6.3.1 Eukaryotic sg RNA synthesis
Eukaryotic RNA viruses employ the production of sgRNAs for expression and regulation of genes from multicistronic RNAs. Several mechanisms of sgRNA synthesis have been proposed for various viruses (Miller et al., 1985; Sawicki and Sawicki, 1998; Sit et al., 1998). Only internal initiation on the negative strand template has been experimentally demonstrated for plant RNA viruses (Miller et al., 1985; Gargouri et al., 1989). The borders of sgPros have been defined for several plant viruses. Generally, sgPros are located upstream of transcription start sites (Miller and Koev, 2000) except in BNYVV and BYDV sgRNAs 2 and 3 (Balmori et al.,
1993; Koev and Miller, 2000). The complete sgPro in many viruses, comprising a core promoter and other elements downstream or upstream of the transcription start site (Marsh et al., 1988; Smirnyagina et al., 1994), are conserved in some genera and families of viruses
(Siegel et al., 1997; Adkins and Kao, 1998). In addition, recent studies have shown that secondary structures contribute to promoter activity. These include stem-loop structures within the sgPros of BYDV (Koev et al., 1999; Koev and Miller, 2000) and Red clover necrotic mosaic virus (RCNMV) (Zavriev et al.,1996), and hairpins for SBMV, SCPMV (Hacker and
Sivakumaran, 1997) and Turnip crinkle virus (TCV)(Wang et al., 1999).
134
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.3.2 Transcription start site of sobemoviruses
Transcription start site of sobemoviruses can be predicted from the mapping studies of SCPMV and SBMV (Hacker and Sivakumaran, 1997). The 5’ ends of the gRNA and sgRNA of
SCPMV were mapped to nts 2 and 3241 respectively whereas the sgRNA of SBMV was mapped to the adenine (A) residue at nt 3163 (Hacker and Sivakumaran, 1997). The terminal nucleotide for all three RNAs was an adenine residue. In addition, there is the sequence
ACAAAA at the end of the RNAs though the 5’ end of the genomic sequence of SBMV is
CACAAAA (Othman and Hull, 1995). Also conserved is a polypurine tract with AG(G)AAA immediately downstream of the ACAAAA sequences of the RNAs. Secondary structures of
RNA near the proposed sgRNA start sites of both viruses are predicted to fold into a hairpin loop, the sequences of which are highly conserved (Hacker and Sivakumaran, 1997). In the loop, there are six conserved base pairs, five GC pairs and one AU pair with the A residue corresponding to the transcription start site. Whilst the significance of the conserved hairpin is not known, it is predicted that it or its complement in minus-strand RNA may function in sgRNA synthesis (Hacker and Sivakumaran, 1997).
6.3.3 Putative sgPro of SCMoV
It has previously been observed that the ACAAAA element is conserved in some positive-sense
RNA viruses, e.g. 5’ ends of gRNAs and sgRNAs of poleroviruses (Veidt et al., 1988; van der
Wilk et al., 1989; Miller and Mayo, 1991; Reutenauer et al., 1993); sgRNA of RCNMV
(Lommel et al., 1988; Xiong and Lommel, 1989; Zavriev et al., 1996) implying that it may be directly or indirectly involved in viral sgRNA synthesis. Based on the results obtained for
SBMV and SCPMV and information from other viruses, sequences of sobemoviruses were compared with the aim of finding the subgenomic transcription start site, leader sequences of the sgRNA and putative sgPro for SCMoV. Multiple sequence alignment of complete genomic sequences show sobemoviruses, except for CfMV, have an AC(A)n motif where n = 2 – 4, at the beginning of the genome and also upstream of the CP gene with a form of a polypurine tract conserved downstream of this motif (Table 6.1).
135
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
Table 6.1: 5’ leader sequences of sobemovirus gRNAs and sgRNAs showing a conserved
AC(A)n polypurine sequences downstream RNA species Sequence (5’ to 3’)
SCMoV gRNA ACAAAAUCGCUCGAAGAAGAAAGCUUGAAUCCGUUUCGCUUU...26nt..AUG SCMoV sgRNA ACAAAACUUCAGAAGGAGAGGGAAAACAAAAUG
LTSV gRNA ACAAAUAAUGCGAAGAAAGAACUGAAGUUUAAUCAAUUUGUG..35nt..AUG LTSV sgRNA ACAAAAUUCCCGAACCAAGAGAAGAAAAUG
SCPMV gRNA ACAAAAUAUAAGAAGGAAAAGUGCUGAUUUUCCUACCUUUGUGUUUCAUG SCPMV sgRNA ACAAAACCGCGCGAGAUAAGAGCAUUUGUAAUG
SBMV gRNA ACAAAAUAUAAGAAGGAAAGCUGGAUUUCCUACCUUUGUGUUUCC..46nt.AUG SBMV sgRNA ACAAAAUUAAUUUAGAAAAUCAAAGUCCGCGCUAUG
RYMV gRNA ACAAUUGAAGCUAGGAAAGGAGCAUAUUGCGAAAGC...43nt..AUG RYMV-CIsgRNA ACAAAGAUGGCCAGGAAGGGCAAGAAAACC RYMV-NGsgRNA ACAAAGAUAGCCAGGAAGGGCAAGAAAACC...156nt...AUG
SeMV gRNA ACAAAAUAUAAGAAGGAAAGCUGGAUUUCCUACCUUUGUGUUUCCAU..28nt..AUG SeMV sgRNA ACAAAAUUGGAAAAUCAAGUCCUCCCUAUG
RgMV gRNA ACAAAUAGAG..40nt..AGGA..45nt..AUG RgMV sgRNA ACAAAGAUGGCAAGGAAGAAGG
TRov gRNA CAAAAUAAAUACAAGAAAGAAAGAUUUUCU...38nt...AUG TRov sgRNA (no particular AC(A)n motif upstream but several downstream of AUG
From results of the mapping studies of SCPMV and SBMV, the transcription start site for sobemovirus sgRNA is expected to be the first A residue of the conserved AC(A)n motif. If sgRNA synthesis starts at this site, then the 5’ leader sequences of these viruses could be as short as five or six and as long as 185 nt in length (Table 6.2). Short leader sequences may not be favourable for efficient translation of mRNA (Kozak, 1991), however, for some eukaryotic mRNAs, e.g. the rat β-subunit of the luteinizing hormone, they are reported to be short
(Jameson et al., 1984). Secondary structures of RNA near the proposed sgRNA start sites of sobemoviruses were predicted with MFOLD (results not shown). Each of these fold into a hairpin loop similar in structure to those predicted for SCPMV and SBMV. The loops appear to be more closely related in sequence to each other than to those of SBMV and SCPMV. In
SCMoV, the transcription start site could either be at nt 3292 or 3319 and the 5’ leader sequence as short as 5 or as long as 30 nt in length. Hence to include the sgPro region of
SCMoV in SCMoV-based cDNA vector constructs, 321 nt upstream of the AUG of the CP gene was amplified and inserted into some vectors.
136
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
Table 6.2: Position of the putative transcription start site and length of the 5’ leader sequence of sgRNAs of sobemoviruses Sobemovirus Putative trancription start Length of 5’ leader site for sgRNA synthesis sequence of sgRNA LTSV 3285 27 RgMV 3328 6 RYMV-CI 3442 6 RYMV-NG 3443 185 SBMV 3163 33 SBMV-Ark 3187 or 3222 20 or 132 SBMV-S 3222 132 SCPMV 3241 30 SCMoV 3292 or 3319 5 or 30 SeMV 3192 27
6.4 PART 1: Development of a full-length cDNA clone of SCMoV-pFL
A full-length cDNA clone of SCMoV-pFL was constructed by ligation of two overlapping cDNA clones. The aim was to use the clone as a template for developing GFP chimeric cDNA constructs in section 6.4 - 6.8. The clone was modified with 35S and T-Nos so that its biological activity could be assessed by mechanical inoculation of plasmid DNA onto leaves of suitable hosts. The results could serve as an indicator for possible expression of any gene vector derived from SCMoV.
6.4.1 Isolation of SCMoV-pFL viral RNA
6.4.1.1 Purification and properties of SCMoV-pFL virions SCMoV-pFL virions were purified from 32.5 g of freshly harvested young leaves of infected T. subterraneum cv Woogenellup by several procedures involving sap extraction in phosphate buffers, clarification with organic solvents, and differential centrifugation followed by density gradient centrifugation, fractionation and sedimentation (Gould, 1981; Walkey, 1991). Sap was extracted from the leaf material by blending in a high performance blender (Vita-mix Corps, model VM0104) in 135 mL of 0.1 M K2HPO4, pH 7.4 containing 0.1% thioglycolic acid
(Mercaptoacetic acid) (Sigma). The sap was filtered through cheesecloth and the filtrate emulsified with an equal volume of chloroform and butan-2-ol by stirring at 4ºC for 20 min.
The slurry was then centrifuged in a Beckman Avanti™ J-251 High Perfomance Centrifuge at
15,344 x g for 10 min. The virus was pelleted from the aqueous supernatant phase by cold
137
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
(4ºC) centrifugation at 229,777 x g for 2 hr in a Beckman L-90 Ultracentrifuge with rotor Type
45 Ti. The resulting pellet was resuspended in 135 mL of 20 mM K2HPO4, pH 7.4 and centrifuged again at 229,777 x g for 1.5 hr. The differential centrifugation was repeated once more at the same speed for 1.5 hr with a 5 mL cushion of 45% sucrose before the pellet was finally resuspended in 1 mL of 20 mM K2HPO4, pH 7.4.
6.4.1.2 Staining of virus particles and electron microscopy
To confirm success of the purification, presence of SCMoV-pFL virions and the absence of any other virus particle, an aliquot of the preparation was stained with 1% phosphotungstic acid
(PTA) in 0.01M Tris (pH 7.3 stored at 4°C) and observed under an electron microscope. A drop of purified virus preparation (1 in 5 dilution of original purification) was added to a two hundred mesh copper grid coated with 0.35% formvar and incubated for 3 min at room temperature. After this period, the film was blotted onto filter paper to remove excess moisture. A drop of PTA was added to the film and left for another 2 min after which excess fluid was drained off by tilting the grid onto an absorbent filter paper. Preparations were examined using a Philips 301 transmission electron microscope. Mr. Peter Fallon of the
Division of Veterinary and Biomedical Science, Murdoch University provided technical assistance. The size of virions of the preparations (Fig. 6.2) was 25 nm in diameter. As expected, virions of SCMoV pFL were isometric in shape and angular in profile.
Fig. 6.2: Electron micrograph of purified virions of SCMoV-pFL
138
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.4.1.3 Preparation of RNase-free glassware and solutions
Glassware, plasticware and solutions used in isolating vRNA were treated with diethylpyrocarbonate (DEPC) (Sigma Aldrich, Sydney, Australia) to destroy enzymatic activity of RNases. RNase free water was prepared by adding DEPC to water at a final concentration of 0.1% (v/v), left at 37°C for 1 hr and then autoclaved for 20 min at 15 psi (1.05 kgcm-2) to break down any remaining DEPC. Glassware and plasticware were filled with 0.1% DEPC in water, allowed to stand for 1 hr at 37°C, rinsed several times with DEPC treated water and then autoclaved for 20 min at 1.05 kgcm-2 (Sambrook et al., 1989). Before any equipment was used, it was again washed with RNAse-Away (Ambion Co., Australia).
6.4.1.4 vRNA extraction from purified virions of SCMoV-pFL
Two lots of 100 µL of the purified virus was each made up to 500 µL with 20 mM K2HPO4 (pH
7.4). One tube was treated with 1% SDS (Sodium dodecyl sulphate) and left at 37ºC for 2 hr, the other left untreated. An equal volume of TE-saturated phenol:chloroform:isoamyl alcohol
(25:24:1) was added to both tubes. The mixture was vortexed for 2 min and then centrifuged at
20,000 x g for a further 2 min. The supernatant was then mixed with an equal volume of chloroform:isoamyl alcohol (24:1), vortexed for 1-2 min and centrifuged at 20,000 x g for 2 min. vRNA in the aqueous layer was precipitated with 0.1 volumes of 3 M NaOAc, pH 5.2 and
0.7 volumes ice-cold isopropanol after freezing at -80oC for 30 min. After thawing, vRNA was pelleted at 20,000 x g for 30 min, the supernatant discarded and the pellet washed with 500 µL of ice-cold ethanol (70%) at 20,000 x g for 5 min. The ethanol was aspirated off, the pellet dried in a Speed-vac and resuspended in 10 µL of DEPC treated water and stored at -20°C.
6.4.1.5 Purity and identity of SCMoV vRNA
Purity of the extracted vRNA was determined on a formaldehyde agarose gel and then amplified by RT-PCR to confirm the presence of SCMoV vRNA.
139
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.4.1.5a Formaldehyde gel electrophoresis of SCMoV vRNA
SCMoV vRNA extracted from purified virions was checked for purity on a 1.2% formaldehyde agarose gel prepared and the electrophoresis run as outlined in the RNeasy® Midi/Maxi
Handbook (QIAGEN Co, 1st Edition, October, 1999). The purity of vRNA was compared to total nucleic acid extracted from SCMoV P23-infected T. subterraneum and Mouse RNA (Fig.
6.4).
M 1 2 3 4 Fig. 6.4: vRNA of SCMoV-pFL electrophoresed on a formaldehyde agarose gel to check for purity. Lanes M- RNA marker (not visible); 1- SDS treated vRNA; 2- Non-SDS treated vRNA; 3- Total nucleic acid; 4- Mouse RNA.
6.4.1.5b RT-PCR of SCMoV vRNA
Three genome-specific primer pairs were used to amplify the 5’ and 3’ ends as well as a mid section of SCMoV genome to confirm the identity of the vRNA. The RT-PCR was done with
MuLV reverse transcriptase and half the volume in Section 2.4.1. Six hundred and five base pairs of the 5’ end was amplified with primers PB-75 and scm005, the 3’ end (750 bp) with primers scm004 and scm003 whilst an 807 bp fragment was amplified from the mid section with primer pair scm34 and scm37.
807 bp 750 bp 605 bp
M 1 2 3 4 5 6 7 Fig. 6.4:Electrophoresis of RT-PCR products to determine the identity of SCMoV vRNA Lanes M: 1kb DNA ladder; 1-3: RT-PCR products of primer pairs PB-75 and scm005, scm004 and scm003, and scm 34 and scm 37 respectively amplified from vRNA treated with SDS; 4-6: RT-PCR products of primer pairs PB-75 and scm005, scm004 and scm003, and, scm34 and scm37 amplified from vRNA not treated with SDS; 7: RT-PCR negative control without any RNA.
140
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.4.2. Cloning of a full-length cDNA of SCMoV-pFL
Several attempts to obtain a genome-length first strand cDNA of SCMoV-pFL from either vRNA or total RNA from infected leaf tissue using either gene-specific primers or random hexamers with several reverse transcriptases were unsuccessful. The full-length cDNA clone, pFL, was then produced by amplifying the genome into two fragments from vRNA by RT-
PCR, cloning each fragment separately into pGEM-T and ligating the overlapping cDNA fragments (Fig. 6.5).
5’NCR 3’NCR ORF2b ORF2a ORF3 ORF1 High fidelity RT-PCR PB75 Pst I
3019 nt scm37 Asc I Pst I Bam H I scm14
1590 nt scm004 Cloning of PCR products and ligation
pGEM-T
pFL
Sph I Bam HI Bam HI PstIX ba I Asc I PCR, cloning and ligation of 35S and T-Nos Hind III Pst I Sal Sal
35S T-Nos
pTEVFL
Fig. 6.5: Schematic diagram for the construction of a full-length cDNA clone of SCMoV- pFL from overlapping clones pPB7537 and pscm14004.
6.4.2.1 RT-PCR amplification of SCMoV-pFL RNA genome
The full-length SCMoV genome was amplified in two pieces, a 3019 bp fragment from the 5’ end and an overlapping 1590 bp fragment extending to the 3’ end of the genome. First strand cDNA of each fragment was synthesised from 250 ng of vRNA with 200 U M-MLV reverse transcriptase, with 10 pmol of downstream gene-specific primers scm37 and scm004 for the 5’ and 3’ fragments respectively. After the RT, nascent RNA was removed by further incubating the reaction mixture at 37°C for 20 min in the presence of 2 U of RNase H. 20% of the first strand reaction mixture was then used in PCR. MuLV reverse transcriptase could not produce
> 2.0 kb first strand cDNA from the vRNA of SCMoV (Fig. 6.6, lane 1a).
141
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
Second strand cDNA was synthesised in a high fidelity PCR using 4U rTth DNA Polymerase,
XL (section 2.5.2) and 40 pmol upstream primers, PB75 and scm14 respectively for the 5’ and
3’ fragments. The PCR was done in a two-temperature cycling profile consisting of a single initial denaturation step at 94°C for 3 min, 30 cycles of denaturation at 94°C for 1 min, anneal/extend at 68°C for 4 min and 2 min respectively for the 5’ and 3’ fragments, followed by a final extension time of 10 min at 72°C. Fig. 6.6 shows the amplified products from the
RT-PCR, a 3019 and 1.590 bp products respectively for primer pairs PB75 and scm37, and scm14 and scm004, on a 1% gel. Synthesis of a second strand cDNA from the > 3.0 kb fragment with Taq Polymerase was not successful (Fig. 6.6, lane 1b).
Fig. 6.6a Fig. 6.6b
3.019 kb 1.590 kb
M 1a 2a 3a 4 M 1b 2b 3b Fig. 6.6a and b: Agarose gel electrographs of RT-PCR products amplified with primer pairs PB75 and scm37, and scm14 and scm004 Lanes: M-1kb Ladder; 1a -Product of primers PB75 and scm37, amplified with rTth DNA polymerase, XL, with first strand cDNA synthesised with MuLV; 2a -Product of primers PB75 and scm37, amplified with Taq DNA polymerase, with first strand cDNA synthesised with M-MLV; 3a, 1b, 2b --Product of primers PB75 and scm37, amplified with rTth DNA polymerase, XL, with first strand cDNA synthesised with M-MLV, 1b and 2b smaller amount of PCR product on lower percentage gel with better separation of marker; 3b- PCR product of primers scm14 and scm004 amplified with rTth DNA polymerase, XL; 4- Negative PCR control (with vRNA) as template amplified with primers PB75 and scm37.
6.4.2.2 Ligation cloning of overlapping cDNA clones to produce pFL
The PCR products in section 6.4.2.1 were ligated separately into pGEM-T to give plasmids pPB7537 and pSCM14004 for the 5’ and 3’ fragments respectively. 500 ng each of the purified
PCR products were A-tailed before ligating to pGEM-T in a 2:1 and 4:1 insert:vector molar ratio respectively for the 3’ and 5’ PCR products. After transformation into E. coli, single colonies were screened by PCR using primer pairs T7/scm35 and SP6/scm2b for plasmid pPB7537, and primer pairs T7 and scm004 for plasmid pSCM14004. Recombinant plasmids with insert in the sense orientation were purified from overnight bacteria cultures.
142
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
A full-length cDNA clone of SCMoV was produced by ligating PstI fragments of the overlapping clones pPB7535 and pSCM14004, using the former as a vector and the latter, an insert. This strategy took advantage of the unique PstI restriction site in the sequence of
SCMoV present in each of the overlapping clones and in that of the pGEM-T vector. To do this, 3 µg each of the plasmids was digested with 10 U of PstI. Digestion of plasmid pPB7537 gave two fragment sizes, 5.672 kb and 0.328 kb whereas digestion of pSCM14004 produced fragments 2.993 kb and 1.586 kb (Fig. 6.7). The DNA fragments 5.672 kb and 1.586 kb were gel purified and used in ligation.
5.672 kb
2.993 kb
1.586 kb
0.328 kb
M 1 1P 2 2P 3 3P 4 4P 5 5P 6 6P 7 7P 8 8P Fig. 6.7: Restriction digests of pPB7537 and pScm14004 with PstI Lanes M: 1kb Plus DNA ladder; 1,2,3&4: four recombinant plasmids of PB7537; 1P,2P,3P&4P: plasmids 1-4 cut with PstI; 5,6,7&8 four recombinant plasmids of pSCM1400; 5P, 6P, 7P&8P: plasmids 5-8 cut with PstI.
After restriction with PstI, and gel purification, 5.385 pmole of ends of the 5.672 kb PstI fragment were dephosphorylated with 5 U of CIP after which the DNA was extracted from solution with phenol:chloroform:isoamyl-alcohol. The 1.586 kb insert was was then ligated to
CIP treated vector with T4 DNA ligase including controls to check for the efficiency of ligase and dephosphorylation (Table 6.4). After transformation, E. coli transformants were screened for inserts containing the full-length cDNA of SCMoV.
Table 6.4: Ligation reactions for subcloning of pPB7537 and pSCM14004
Components of reaction mixture Volumes of reaction mixture (µL) Control Standard 1 2 3 4 *5 *6 10X T4 DNA ligase buffer 2.0 2.0 2.0 2.0 2.0 2.0 T4 DNA ligase --- 1.0 --- 1.0 1.0 1.0 Untreated vector (168.5 ng) 0.5 0.5 ------Treated vector (156.5 ng) ------0.5 0.5 0.5 0.5 Insert (55ng/ul) ------2.5 1.6 Water to 20 20 20 20 20 20 *Reaction 5 and 6 have vector to insert molar ratio of 3:1 and 2:1 respectively.
143
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.4.2.3 Screening of transformants for pFL
Transformants for a full-length cDNA clone were screened in three ways: a) by PstI digestion after plasmid purification to check for ligation of fragments, b) by PCR to confirm the orientation of insert in the vector in a positive transformant, and c) by DNA sequencing to determine the complete sequence of a selected clone.
6.4.2.3a Restriction digestion screening
As many positive transformants as possible were selected and analysed by restriction enzyme digestion. After purification by alkaline lysis, recombinant plasmids were digested with PstI.
Positive plasmids showed two fragments, one the size of the receiving vector and the other the size of the insert (Fig. 6.8).
6000 bp
1650 bp
M 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16
Fig. 6.8: Restriction digest of bacterial clones of pFL with PstI Lanes M: 1kb plus DNA marker; 1-5: clones from ligation reaction 6 in Table 6.4; 6-10: clones from ligation reaction 5 from Table 6.4; 11-13: clones from ligation reaction 2; 14-15: clones from ligation reaction 4; 16: a mix of vector and insert in the same well.
6.4.2.3b PCR screening
PCR was used to confirm presence and orientation of insert in positive transformants such as in lanes 2, 4, 6, 7, 8, 9 of Fig. 6.8. Primers scm34 and scm38 used in the PCR anneal to the viral sequences of the vector and insert respectively and was expected to amplify a fragment of
1.425 kb across the junction of the viral sequences of insert and vector used in ligation in section 6.4.2.2 (Fig. 6.9).
144
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
1.425 kb
M 1 2 3 4 5 6 7 8 9 10 11
Fig. 6.9: PCR screening of pFL to confirm ligation in the correct orientation Lanes M: Kb DNA ladder; 1-6, 10-11: Positive clones with viral sequence ends of insert and vector ligated together; 7-9: Clones of vector and insert ligated in the undesired orientation.
6.4.2.3c DNA sequence of pFL
Three clones were first chosen for sequencing to confirm integrity of the clones. The 5’ and 3’ terminal sequence of SCMoV-pFL cDNA in the clones as well as sequences across junctions of the vector and insert used in creating the recombinant plasmids were determined with primers
T7, SP6, scm34 and scm38. A positive transformant confirmed to contain the full-length cDNA of SCMoV-pFL was sequenced with T7 and SP6 and 14 fourteen other primers (Fig.
6.10) designed to amplify both strands of the cDNA clone to enable systematic sequencing along the whole genome of SCMoV. The nucleotide sequence of pFL has been deposited in
GenBank (accession number AY376454). The sequence was 99.69% similar to SCMoV-P23 with a total of 13 nucleotide differences including an adenine (A) in SCMoV-P23 at position 16
(later recognised as an error). Sequencing finally confirmed successful cloning of a full length cDNA clone of SCMoV-pFL.
T7 seq2f seq3f seq4f scm34 scm14 scm16 scm33
seq1r seq2r seq3r seq4r seq5r scm38 scm37 SP6
SCMoV genome (4.258 kb)
Fig. 6.10: Strategy used for sequencing the full-length SCMoV cDNA clone and the primers used
145
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.4.3 Development of p35SFL and pTEVFL hybrids of pFL
Plasmids p35SFL and pTEVFL were hybrids of pFL modified with the 35S promoter and T-
Nos terminator signal. Plasmid p35SFL was a hybrid with the 35S sequence ligated upstream of the 5’ NCR of the SCMoV sequence in plasmid pFL whereas pTEVFL had, in addition to the 35S sequence, the T-Nos sequence ligated downstream of the 3’ NCR of SCMoV. The 35S promoter and T-Nos terminator sequences were respectively amplified from pTEV (Section
5.2.1) with primer pairs 35Sfor and 35SAscI, and NosforSalI and NosrevSalI. With these primers, PCR product of 35S, with an AscI site at the 3’ end and T-Nos amplified as a SalI fragment, were cloned into pGEM-T Easy as p35AscI and pNosSalI respectively in the sense orientation.
In separate double digest reactions using 10 U each of AscI and SphI, the 35S sequence was digested out of 3 µg of p35SAscI and 5 µg of pFL was linearised. After gel purification, 100 ng of linearised pFL and 150 ng of 35S were ligated to give p35SFL. The T-Nos was digested from 5 µg of pNosSalI as a SalI fragment with 10 U of enyzme at 37°C for 18 hr, gel purified and ligated to SalI linearised, dephosphorylated p35SFL to give a final construct, pTEVFL
(Fig. 6.5).
PART 2: Development of SCMoV-based gene vector constructs, strategies and clones
All pFL hybrid vector constructs were developed by directional cloning of inserts derived from pFL and/or from cDNA clones with heterologous sequences from 35S, T-Nos and the gfp gene
(Fig. 6.10). These inserts overlapped each other by a few nucleotides or by only restriction sites present in SCMoV genomic sequences or incorporated by PCR. All SCMoV-derived inserts were amplified from 38.5 ng pFL with 2U rTth DNA Polymerase, XL (section 2.5.2) and 0.2 µM each of primers, A-tailed and cloned into pGEM-T or pGEM-T Easy. All clones used and the final vector constructs developed are shown in Fig. 6.11.
146
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
ORF2b 3’NCR 5’NCR ORF2a ORF3 ORF1 BamHI SacI PstI KpnI
1 p5’P1 2 pFLrev14
Bam HI 3 AscI PstI EcoRI SacI XbaI AscI
p7537 5 pGFP 4 p35S
Bam HI PstI Xba I Asc I EcoRI 6 p1425 8 p35S5NCR
PstI EcoRI SacI
SacI 7 pCPgfp3NCR 16 p35S5NCRGFP SacI PstI SacI 15 pP2foropt37 EcoRI KpnI 10 pGFP3NCR EcoRI PstI 9 pP2for37 PstI EcoRI SacI Kpn I EcoRI AgeI 13 p14CPrev 12 T-Nos EcoRI EcoRI
11 pP2brev14 EcoRI EcoRI
17 pP2b
EcoRI EcoRI 14 psgpro
Construct Graphical representation Assembled from clones
GFP
1. pFLCPgfp (3), (5) and (6)
2. pFLP1gfp (1), (2), (5) and (9)
sgpro 3. pFLCPsgprogfp (3), (10), (13) and (14)
4. pFLCP∇sgprogfp (3), (10), (11) and (14)
5.pFLREPsgprogfp (8), (10), (14) and (17)
Fig. 6. 11: Clones and strategies used in developing GFP hybrid constructs of pFL, including clones of 35S and T-Nos used in modifying them.
147
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.5 Construction of plasmid pFLCPgfp
Plasmid pFLCPgfp was constructed as a full-length SCMoV-pFL cDNA clone with gfp gene fused to the CP gene. It was constructed by ligation cloning of two overlapping plasmids, pCPgfp3NCR and pPB7537 (section 6.2.2.2) using the unique PstI site in SCMoV genome.
The plasmid, pCPgfp3NCR, was developed by a combination of Inverse PCR (IPCR, section
6.4.1.2) and cloning as in Fig. 6.12.
ORF2a ORF3 3’NCR 5’NCR ORF1 PCR, cloning and disruption of SacI in pGEM-T
p1425 SacI IPCR p7537
PB76 scm29 IPCR primers
p1425
GAATTC GAGCTC IPCR product
Purification, religation and transformation
EcoRI SacI
p1425M pGFPE Ligation of GFP to p1425M
EcoRI SacI 3’NCR
pCPgfp3NCR
Fig. 6.12 Schematic diagram of cloning strategy for constructing pFLCPgfp (not to scale)
6.5.1 Development of plasmid pCPgfp3NCR
Plasmid pCPgfp3NCR was constructed as a partial clone of SCMoV-pFL with the gfp gene fused to the CP gene. This was done in three basic steps: PCR cloning of nt 2671-4258 as p1425, IPCR mutagenesis to incorporate EcoRI and SacI sites in plasmid p1425 to give p1425M and subcloning of gfp gene as an EcoRI/SacI fragment into p1425M.
148
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.5.1.1 Cloning of plasmid p1425
Plasmid p1425 was a clone containing nt sequences 2671-4258 of SCMoV corresponding to the
3’ end of the ORF2b, the CP gene and 3’ NCR. It was developed by Williamson (2000). The sequences were amplified with primers scm14 and scm25 and cloned into pGEM-T. The SacI restriction site in pGEM-T was removed by filling in the 3’ recessed ends left by SacI digestion using DNA polymerase I, Large Klenow fragment. This was done so EcoRI and SacI sites incorporated in p1425 later by IPCR could be used to clone the gfp gene at the 3’ end of the CP gene.
6.5.1.2 Development of a mutated plasmid of p1425 for ligation to gfp gene
Plasmid p1425M was as a mutated form of plasmid p1425 developed to allow the subcloning of the gfp gene. It was generated by IPCR to incorporate EcoRI and SacI sites at the 3’ end of the
CP gene followed by restriction digestion with DpnI and religation of the IPCR mutated product.
6.5.1.2a Inverse PCR mutagenesis of p1425 to incorporate EcoRI and SacI sites
Incorporation of EcoRI and SacI sites at the 3’ end of the CP gene in p1425 to create p1425M was done by IPCR mutagenesis. This PCR technology has been used to disrupt genes lacking convenient restriction enzyme sites to delete regions of targeting gene/sequences and to introduce restriction sites compatible with that of a desired (marker) gene (Wu et al., 1995;
Francois et al., 2002). A schematic diagram of the strategy used in developing pCPgfp3NCR is shown in Fig. 6.12.
6.5.1.2b Primers for IPCR
Two synthetic phosphorylated oligonucleotide primers, scm29b and PB76 were designed to prime within p1425 extending into the rightward and leftward sequences respectively to amplify the whole of the plasmid and to incorporate EcoRI and SacI sites between the 3’ end of the CP gene and the proximal end of the 3’NCR. The two sites were incorporated into the
149
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors primers with a modification of the nucleotide sequences of the last three codons of the CP without a change in encoded amino acids.
6.5.1.2c Optimisation of IPCR
Amplification of plasmid p1425 (5.7 kb) required optimisation of the salt and dNTP concentrations after PCR conditions in section 2.5.2 yielded no results. Optimum Mg(OAc)2 levels were determined empirically by doing titrations in increments of 0.2 mM and the dNTP concentrations balanced by maintaining the Mg2+ concentrations at levels 0.2 to 0.4 mM higher than the total concentrations of dNTP (Table 6.4). The PCR was done with 4U rTth DNA
Polymerase, XL, 10 pmol primers and 500 ng plasmid DNA. PCR was done as follows: an initial denaturing step at 94°C for 1 min, 30 cycles of denaturing at 94°C for 1 min and anneal/extend at 65°C for 10 min, followed by a final extension time of 10 min at 72°C and the results shown in Fig. 6.13.
Table 6.4: Concentrations of Mg2+ and dNTPs for optimising IPCR of p1425
Reagents Reactions 1a 2a 3a 4a 1b 2b 3b 4b 10mM Conc (µM) 400 400 400 400 600 600 600 600 dNTPS Vol (µL) 2.0 2.0 2.0 2.0 3.0 3.0 3.0 3.0 25mM Conc (mM) 0.8 1.0 1.2 1.4 0.8 1.0 1.2 1.4 Mg(OAc)2 Vol (µL) 1.6 2.0 2.4 2.8 1.6 2.0 2.4 2.8 Reactions Reagents 1c 2c 3c 4c 1d 2d 3d 4d 10mM Conc (µM) 800 800 800 800 1000 1000 1000 1000 dNTPS Vol (µL) 4.0 4.0 4.0 4.0 5.0 5.0 5.0 5.0 25mM Conc (mM) 0.8 1.0 1.2 1.4 0.8 1.0 1.2 1.4 Mg(OAc)2 Vol (µL) 1.6 2.0 2.4 2.8 1.6 2.0 2.4 2.8
5.7 kb
M D 1a 2a. 3a 4a 1b 2b 3b 4b 1c 2c 3c 4c 1d 2d 3d 4d Fig. 6.13: Results of PCR mutagenesis of p1425 following optimisation of the concentrations of Mg2+ and dNTPs. Designations of lanes correspond to the different combinations of concentrations of Mg2+ and dNTPs as in Tables 6.4. Lane D shows the amount of plasmid DNA used in each PCR and M is 1kb Ladder. 150
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.5.1.2d Religation of the IPCR mutagenesis product
Following successful amplification of plasmid p1425, a circular plasmid was generated from the blunt-ended IPCR product following digestion with DpnI (a restriction enzyme that cleaves only when its recognition site is methylated), gel purification, and religation. To do this, 200
µL of the IPCR product was digested with 10 U of DpnI at 37°C overnight to remove methylated plasmid DNA (Fig. 6.14). Lanes 1 and 2 showing the untreated and DpnI digested plasmid p1425 respectively indicate the enzyme digests methylated DNA. Lanes 3 and 4 show the IPCR product treated and untreated respectively with DpnI whilst lanes 5 and 6 show that
DpnI digests the cloning vector pGEM-T. The IPCR product digested with DpnI was gel purified, religated and transformed to produce a circular plasmid, p1425M.
5.7 kb
M 1 2 3 4 5 6 Fig. 6.14: Restriction digestion of IPCR mutagenesis product of p1425 with DpnI Lanes: M- Kb Plus DNA marker; 1- Untreated p1425; 2- p1425 treated with DpnI; 3- IPCR product; 4- PCR products treated with DpnI; 5- pGEM-T 6- pGEM-T cut with DpnI
Conditions for re-ligating the IPCR product were optimised for future experimentation. Three sets of re-ligation reactions with different amounts of DNA (25, 50 and 75 ng) were set up.
Each ligation was done in both a short (30 min at room temperature) and longer (overnight) incubation periods. Half of each reaction was transformed with both commercial JM109 competent cells and similar cell lines prepared using the rubidium chloride method to compare efficiency of transformation. There was no significant difference in the amount of DNA used in transformation judging from the number of colonies on plates. The success of ligation was independent of the length of incubation. The efficiency of freshly obtained commercial JM109 competent cells was not significantly different from those made from similar cells. Selected transformants were purified and digested with EcoRI and SacI to confirm the success of the procedure.
151
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.5.1.3 Ligation and cloning of gfp gene and p1425M
The gfp gene used as a fusion partner to CP gene was amplified with primers scm30B and scm31 (5' CTTGTAGAGCTCGTCCATGCC; the red font bases being silent mutations to create a SacI site for cloning the gfp gene). The PCR product was cloned into pGEM-T as an
EcoRI/SacI fragment with no start codon to produce pGFP. Plasmid p1425M was cloned with
GFP as an EcoRI/SacI fragment to produce a plasmid with GFP fused in-frame between the 3’ end of the CP gene of SCMoV-pFL and the 3’ NCR. Sequential digest of p1425M, and pGFP with EcoRI and SacI resulted in a linearised p1425M (as recognition sites for both enzymes were only a few bases apart) and the gfp gene digested out of the plasmid. The ligation of the gfp gene to linearised p1425M to produce pCPgfp3NCR was highly efficient resulting in all transformants screened by PCR being positive. Production of this fusion was confirmed by restriction digests of pCPgfp3NCR with EcoRI, PstI and SacI as shown in Fig. 6.15. The pCPgfp3NCR construct was sequenced with primers T7, SP6, scm16 and scm33 to confirm in- frame fusion of GFP to the CP gene. There was no IPCR or cloning-induced mutation.
5.3 kb 2.3 kb
M 1 2 3 4 5 6 7 8 Fig. 6.15: Restriction digest of two pCPgfp3NCR plasmids to confirm the presence of EcoRI, SacI and PstI sites, and size of the CP:GFP fusion plasmid after IPCR. Lanes M: Kb DNA ladder; 1: pCPgfp3NCR plasmid 1; 2: pCPgfp3NCR plasmid 2; 3&4: plasmids 1&2 respectively digested with EcoRI; 5&6: plasmids 1&2 respectively digested with PstI; 7&8: plasmids 1&2 respectively digested with SacI.
6.5.2 Assembly and sequential cloning to produce construct pFLCPgfp
The last step in the construction of pFLCPgfp was similar to the development of pFL in section
6.2.2.2 which involved the ligation and cloning of pPB7537 to a 3’ half plasmid, pSCM14004 except in this case the 3’ half plasmid is pCPgfp3NCR. PstI digested fragment, the linear dephosphorylated 5.672 kb of pPB7535 (section 6.2.2.2) and the ≈2.3 kb from plasmid pCPgfp3NCR (shown in Fig. 6.13 lanes 5 and 6) were ligated in molar ratio of 3:1 to produce plasmid pFLCPgfp, a gfp chimeric full-length cDNA clone with GFP fused to the 3’ terminus of the CP gene.
152
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.5.3 Development of 35S and pTEV hybrids of FLCPgfp
The 35S and T-Nos were cloned upstream and downstream of the 5’ and 3’ NCRs of SCMoV as in pFLCPgfp (section 6.4) the same way it was done for pFL, i.e. the 35S sequences were digested from p35SAscI as SphI/AscI and ligated to pFLCPgfp to create p35SFLCPgfp.
Plasmid pTEVFLP1gfp was generated by ligating T-Nos (digested from pNosSalI) as a SalI fragment to p35SFLP1gfp.
6.6 Construction of plasmid pFLP1gfp
The construct, pFLP1gfp, as in Fig. 6.11 was developed as a full-length cDNA clone of
SCMoV-pFL with GFP fused to the 3’ end of the ORF1. Initially, pFL was intended to be amplified by IPCR to incorporate restriction sites at the 3’ end of ORF1 sequence for cloning
GFP as a N-terminal fusion partner to the ORF1. Phosphorylated primers, PB-77 and PB-78, were designed to introduce silent mutations corresponding to the BsrGI and AhdI restrictions sites in the ORF1. Similar sites were introduced into primers PB-79 and PB-80 to amplify the gfp gene. However, it was not possible to amplify the 7.258 kb plasmid pFL. The failure was attributed to the size of the plasmid. The 5’ NCR with ORF1 was then amplified, cloned into pGEM-T as pP1 and used as a starting material for IPCR. When it appeared the 3.604 kb plasmid was amplified, the products were all digested by DpnI. The construct was then developed by assembly and ligation cloning of three SCMoV-based cDNA clones (p5'P1, pP2for37 and pFlrev14) and pGFPE as in clones (1), (2), (5) and (9) in Fig. 6.11.
6.6.1 Clones for constructing plasmid pFLP1gfp
Plasmid p5'P1 contained as insert nt sequences 1-605 of SCMoV-pFL. The insert was amplified with primers FLfor and P1rev incorporating BamHI and EcoRI sites at the 5' and 3' ends of the ORF1 respectively after PCR. Plasmid pP2for37 had nt 605-3019 as an insert amplified with primers P2for and scm37 with a SacI site at the 5’ end of the PCR product used with the EcoRI site at the 3’ end of ORF1 to clone the gfp gene. In addition, primer P2for added a few extra bases at the 5’ end of the PCR product to give a stop codon to the gfp gene
153
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors when the latter was cloned in frame as a N-terminal fusion partner to the ORF1. Nucleotides
2671-4258, amplified with primers scm14 and FLrev were cloned as pFlrev14 with a KpnI site at the 3’ end of the sequence. In plasmid pGFPE, the gfp gene was amplified with primer pairs gfpEcoRI and scm31 designed to incorporate EcoRI and SacI at the 5’ and 3’ ends respectively for cloning the gene between the ORF1 and ORF2. The BamHI and the KpnI sites present in
FLfor and FLrev could then be used to ligate 35S and T-Nos to the 5’ and 3’ ends respectively of SCMoV-pFL.
6.6.2 Assembly and sequential cloning to produce construct pFLP1gfp
Plasmid pFLPIgfp was developed by sequential cloning; first the gfp gene (from pGFPE) was ligated to the 3’ terminus of SCMoV sequences in p5’P1 to produce p5’P1gfp followed by ligation of sequences from pP2for37 to the 3’ terminus of the gfp gene to produce pP1gfpP2.
Sequences in pFlrev14 were then ligated to pP1gfpP2 using the unique PstI site at nt 2707 in the SCMoV genome to produce pFLP1gfp.
6.6.2.1 Ligation and cloning of p5'P1 and pGFPE
Ligation and cloning of p5’P1 and pGFPE resulted in a plasmid with 5’ NCR and ORF1 of
SCMoV-P23 fused to the 5’-terminus of GFP. Before subcloning, the SacI restriction site in pGEM-T was deleted by filling in the 3’ recessed ends after digestion with the enzyme. This allowed the use of a similar site in plasmid pP2for37 for further ligation. The gfp gene in pGFPE was excised and ligated to plasmids p5’P1 (without a SacI site) with restriction sites
EcoRI at the 5’ end of the gene and SpeI in pGEM-T. Each of the plasmids was digested in a single reaction with 10 U of EcoRI and SpeI. After gel purification, 50 ng of the resulting linearised p5’P1 was ligated to 100 ng of the EcoRI/SpeI digested gfp gene. Following screening by PCR with T7 and scm31 and sequencing, the plasmid was confirmed to have an additional amino acid, Phenylalanine (F) between the ORF1 product and the gfp gene (Fig.
6.16).
154
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
Native sequences ORF1 GFP
580 590 600 10 20 30 ....|...... |....|....|....|....|....|....|...... |....|....|....|....|....| ATGCCATCA…….ATTCAGCCTCTTCAAGAAGGAGACGATCAAGAATGA ATGAGTAAGGGAGAAGAACTCTTCACCGGA……TACAAATAA M P S I Q P L Q E G D D Q E * M S K G E E L F T G Y
K *
Primer sequences 3’AAGAAGGAGACGATCAAGAATTC 5'GAATTCAGTAAGGGAGAAGAAC
After PCR and ligation cloning
Mutated sequences of P1:GFP
580 590 600 610 620 630 ....|...... |....|....|....|....|....|....|....|....|....|....|....|....|....|... ATGCCATCA……….ATTCAGCCTCTTCAAGAAGGAGACGATCAAGAATTCAGTAAGGGAGAAGAACTCTTCACCGGA………TACAAATAA M P S I Q P L Q E G D D Q E F S K G E E L F T G Y K * Fig. 6.16: Mutated sequences of the ORF1:GFP fusion in plasmid p5’P1gfp
6.6.2.2 Ligation and cloning of p5'P1gfp and pP2for37
By ligating and subcloning inserts in p5’P1gfp and pP2for37 directionally with the SacI site at the 3’ end of gfp gene in p5’P1gfp and the SalI site in pGEM-T sequences, the ORF2a and truncated form of the ORF2b was ligated to the ORF1:GFP. Each of the plasmids was sequentially digested with 10 U SacI (for 3 hr) and SalI (for 18 hr) resulting in the release of
2,414 bp SCMoV DNA insert out of pP2for37 in addition to 89 bases of the pGEM-T whereas
94 bases between the SacI site at the 3’ end of the gfp gene and 84 of the pGEM-T were digested off p5’P1gfp. Fifty five nanograms of the linearised p5’P1gfp and 120 ng of 2,414 bp
SCMoV DNA digested product from pP2for37 were ligated, after which transformants were screened by PCR with primers SP6 and scm34 and by restriction digestion. The resulting plasmid, p5’P1gfpP2, was used for subcloning sequences of the CP gene and the 3’ NCR to give pFLP1gfp.
6.6.2.3 Ligation and cloning of p5'P1gfpP2 and pFLrev14
Plasmid p5’P1gfpP2 had nt 1-3019 of SCMoV-P23 cloned with gfp gene as a fusion partner to
ORF1. The CP gene and 3’ NCR sequences in pFLrev14 was ligated to sequences in p5’P1gfpP2 as PstI fragments to produce pFLP1gfp. PstI digestion of pP2forP2 linearised the
155
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors plasmid whereas two fragments of ≈ 3 kb and 1.58 kb were released from pFLrev14, the latter containing the CP gene and 3’ NCR together with 82 bp of pGEM-T. The termini of the linearised p5’P1gfpP2 were dephosphorylated and 58 ng ligated to 115 ng of the ≈1.58 kb PstI fragment of pFLrev14 and transformed to E. coli. Recombinant plasmid DNA was purified by alkaline lysis and analysed as in section 6.2.2.3. The final construct, pFLP1gfp, was a hybrid of pFL with gfp gene fused to the 3’ terminus of ORF1. Junctions of all ligated fragments in the subclones were sequenced to check for PCR and/or cloning mutations, of which there was none.
6.6.3 Development of 35S and pTEV hybrids of pFLP1gfp
The 35S and T-Nos sequences were ligated to the 5’ and 3’ NCRs of SCMoV in pFLP1gfp as
SphI/BamHI and KpnI/SalI fragments respectively. After digestion of pTEV with 10 U each of
SphI (site in pGEM-T) and BamHI in a single reaction at 37°C for 3 hr, 95 ng of purified linearised pFLP1gfp and 105 ng of purified 35S digested from pTEV were ligated with T4
DNA ligase at room temperature for 2 hr. Transformants were analysed by PCR with T7 and
35Srev. The resulting recombinant plasmid, p35FLP1gfp, was used to create pTEVFLP1gfp by cloning T-Nos behind the 3’ NCR. Plasmids p35FLP1gfp and pTEV were digested separately in a sequential digest with 10 U of KpnI followed by SalI (site in pGEM-T) digestion each time for 18 hr at 37°C and gel purified. 100 ng of linearised p35SFLP1gfp was purified from solution and ligated to 120 ng of T-Nos sequence with T4 DNA ligase and transformed into E. coli. PCR screening of transformants with primer pairs SP6 and NosforSalI, and T7 and
35Srev confirmed the resulting plasmid, pTEVFLP1gfp, as having 35S and T-Nos cloned to the
5’ and 3’ ends of the insert in pFLP1gfp.
6.7 Construction of plasmid pFLCPsgprogfp
The construct, pFLCPsgprogfp, was a SCMoV-based insertion vector containing the full-length genome of SCMoV-pFL with a duplication of a putative sgPro behind the CP gene and a gfp gene inserted in front of the 3’NCR. The construct was assembled from several overlapping clones; (3), (10), (13) and (14) in Fig. 6.11 and generally involved five steps:
156
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
(i) cloning of a translatable GFP and the 3’ NCR to give pGFP3NCR
(ii) cloning of the 3’ sequence of ORF2b and the CP gene as p14CPrev
(iii) subcloning of plasmids in pGFP3NCR and p14CPrev to produce p14CPgfp3NCR
(iv) cloning of the putative sgPro as psgPro, and subsequent subcloning to p14CPgfp3NCR to produce p14CPsgprogfp3NCR and finally,
(v) subcloning of the resulting plasmid in p14CPsgprogfp3NCR to pPB7537 to finally create pFLCPsgprogfp as in Fig. 6.11.
6.7.1 Clones for constructing plasmid pFLCPsgprogfp 6.7.1.1 Cloning of translatable GFP and the 3’ NCR
Unlike in the constructs above, the reporter gene was inserted in the following constructs as a translatable GFP. The gene and the 3’ NCR were amplified from plasmid pCPgfp3NCR
(section 6.4.1) with primers gfpforEcoRI and FLrev incorporating an EcoRI site at the 5’ end of the gene, a start codon with a Kozak context sequence for efficient translation, and a KpnI site at the 3’ end of the PCR product. The product was cloned into pGEM-T in the sense orientation as plasmid pGFP3NCR.
6.7.1.2 Cloning of the 3’ end of ORF2b and the CP gene
With primers scm14 and CPrevEcoRI, the 3’ end of ORF2b and the CP gene of SCMoV-pFL i.e. nt 2671-4082, were amplified with an EcoRI site at the 3’ end of the CP gene. When the
PCR product was cloned into pGEM-T, the recombinant plasmid, p14CPrev, with inserts in the sense orientation were purified for subcloning with pGFP3NCR.
6.7.1.3 Cloning of the putative sgPro
To duplicate and insert the putative sgPro infront of the gfp gene in the final construct, pFLCPsgprogfp, a 321 bp fragment comprising nt 3030-3307 was amplified from pFL with primers sgprofor and sgprorev and cloned as an EcoRI fragment into pGEM-T, referred to subsequently as pSgpro. Direction of inserts in the sense orientation was confirmed by PCR and sequencing. 157
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.7.2 Assembly and sequential cloning to produce construct pFLCPsgprogfp
This construct was developed by subcloning plasmids pGFP3NCR and p14CPrev followed by an insertion of the putative sgPro and finally ligation of the resulting plasmid to p7537 to produce pFLCPsgprogfp. By subcloning plasmids pGFP3NCR and p14CPrev using the EcoRI sites at the 5’ of gfp gene in pGFP3NCR and at the 3’end of the CP gene in p14CPrev respectively, and also SpeI (site in pGEM-T), a translatable gfp gene was cloned behind an intact CP gene. This was done by digesting both plasmids with EcoRI and SpeI in a double digestion reaction at 37°C for 4 hr. This resulted in the release of the insert, GFP with 3’ NCR, from pGFP3NCR and a linear p14CPrev (the two sites are adjacent). The insert was ligated to the linearised p14CPrev to produce plasmid p14CPgfp3NCR which contained a portion of the
3’ end of ORF2b, and a translatable GFP between the CP gene and the 3’ NCR. This was followed by cloning of the putative sgPro, digested from pSgpro as an EcoRI fragment, between the GFP and 3’ NCR in plasmid p14CPgfp3NCR to give p14CPsgprogfp3NCR, similar to p14CPgfp3NCR but with a duplicate of nt 3030-3307 between the CP gene and a translatable gfp gene. Direction of the putative sgPro sequence was determined by PCR in p14CPsgprogfp3NCR. In the final step, p14CPsgprogfp3NCR and pPB7537 were ligated as
PstI fragments making the final construct an insertional SCMoV-pFL full-length cDNA clone with a putative sgPro and a gfp gene between the CP gene and the 3’ NCR.
6.7.4 Development of 35S and pTEV hybrids of pFLCPsgprogfp
Recombinant plasmids p35SFLCPsgprogfp and pTEVFLCPsgprogfp were hybrids of pFLCPsgprogfp the former with 35S cloned infront of 5’ NCR and the latter derived from the former with the T-Nos additionally cloned behind 3’ NCR as in section 6.5.3.
6.8 Construction of plasmid pFLCP∇sgprogfp
This construct, pFLCP∇sgprogfp, was similar to pFLCPsgprogfp except for a 3’ truncation of the CP gene in the former. IPCR mutagenesis of the ≈ 8.3 kb of pFLCPsgprogfp to delete a portion of the CP gene failed and the construct was developed by assembly of clones used in
158
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors developing pFLCPsgprogfp, except pP2brev14 instead of p14CPrev was used. Unlike the insert in p14CPrev, the PCR product cloned into pGEM-T to give plasmid pP2brev14 was amplified with primers P2brev and scm14 resulting in truncation of the CP gene without the portion of the gene that does not overlap with ORF2b. The plasmid, therefore, contained as insert, the last 833 nt of the ORF2b with an EcoRI site at the 3’ end of sequence. The insert thus contained the first 181 nt of the CP gene.
6.8.1 Assembly and sequential cloning of pFLCP∇sgprogfp and its 35S and T-Nos hybrids
Similarly, clones were ligated in the order as in section 6.6.2 for pFLCPsgprogfp; ligation of pGFP3NCR to pP2brev14 using EcoRI sites in both sequences and with SphI site in pGEM-T, followed by insertion of the putative sgPro sequence between the ORF2b and gfp genes and finally ligation and cloning of pPB7537 to give the resulting plasmid pP2bsgprogfp3NCR. The
35S was cloned ahead of the 5’ NCR in pFLCP∇sgprogfp to give p35SFLCP∇sgprogfp, also with T-Nos sequences behind the 5’ and 3’ NCRs to give pTEVFLCP∇sgprogfp as in section
6.6.4.
6.9 Construction of plasmid pFLREPsgprogfp
The difference between constructs pFLCP∇sgprogfp and pFLREPsgprogfp is the absence of
ORFs 1 and 2a in the latter. pFLREPsgprogfp is a construct with a translable ORF 2b, containing the replicase, a truncated CP gene as in pFLCP∇sgprogfp, and with a translatable gfp gene and sgPro sequence cloned in tandem between the ORF2b and 3’ NCR. The construct was developed by first subcloning plasmids pGFP3NCR and pP2b to produce a plasmid with a translatable gfp gene adjacent to the ORF2b followed by insertion of the putative sgPro sequence between the genes and T-Nos behind the 3’NCR. The final cloning steps invloved subcloning of plasmid p35S5NCR to ligate sequences of the 5’ NCR of
SCMoV-pFL and the 35S upstream of the ORF2b.
159
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.9.1 Clones for developing construct pFLREPsgprogfp
Overlapping clones used for developing construct pFLREPsgprogfp were plasmids pGFP3NCR as in section 6.5.1.1, pSgpro as in section 6.5.1.3,. pTEV as in section 5.2.1, pP2b and p35S5NCR.
6.9.1.1 Cloning of ORF2b of SCMoV-pFL as plasmid pP2b
Plasmid pP2b was a clone containing as insert, the ORF2b amplified with primers P2bforopt and P2brev. The PCR product, cloned into pGEM-T as pP2b, had a start codon, a Kozak context sequence at the 5’ of the ORF2b and EcoRI sites at both ends.
6.9.1.2 Cloning of 35S with 5’ NCR of SCMoV
Plasmid p35S5NCR had the 5’ NCR of SCMoV-pFL with 35S ligated upstream to it in pGEM-
T. Both sequences were amplified in a single PCR with primers 35Sfor and 5utrrev from plasmid pTEVFLP1gfp, maintaining all restriction sites at the 5’ end as it was used to clone the promoter into pFL and an EcoRI site at the 3’ end. To prevent disruption of further cloning, the
PstI site in p35S5NCR 35S was disrupted.
6.9.2 Assembly and ligation cloning of plasmid pFLREPsgprogfp and 35S and T-Nos hybrids
This construct was developed by subcloning two major intermediate clones. The first was developed by subcloning ORF2b from plasmid pP2b at the 5’ end of the gfp gene in plasmid pGFP3NCR followed by insertion of the putative sgPro sequence between the genes. The second was developed by ligating 35S with 5’ NCR in p35S5NCR upstream of ORF2b. The resulting plasmids were then subcloned with the PstI site in the SCMoV sequence to produce pFLREPsgprogfp. To subclone ORF2b from pP2b into pGFP3NCR, both plasmids were digested with 10 U of EcoRI at 37°C for 4 hr. The EcoRI digested fragment of the ORF2b was cloned into a linearised dephosphorylated pGFP3NCR resulting in a plasmid, pP2bGFP3NCR, with gfp cloned between ORF2b and the 3’ NCR of SCMoV-pFL. Directional ligation of
160
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
ORF2b to gfp was confirmed by PCR. This was followed by cloning of the putative sgPro sequence between ORF2b and gfp gene, as an EcoRI fragment from pSgpro, into EcoRI linearised, dephosphorylated plasmid pP2bGFP3NCR. After ligation, the putative sgPro sequence was confirmed to be in the 5’ to 3’ direction by PCR.
It was not possible to ligate the 5’ NCR to sequences in pP2bGFP3NCR for lack of restrictions sites for common enzymes in this region. The very short 5’NCR was then amplified with 35S with an EcoRI site at the 3’ end of the product as in p35S5NCR for further ligation to the 2b gene. For the presence of other EcoRI sites, the 5’ NCR was not ligated directly to the 2b gene in pP2bGFP3NCR with the enzyme. The strategy used involved ligation and cloning of the
35S with 5’ NCR to the 5’ end of ORF2b in pP2b using the EcoRI in the sequences and SphI in pGEM-T to give p35S5NCRP2b which was then ligated to pP2bGFP3NCR with the PstI sites in both of the ORF2b sequences and also in pGEM-T making the final construct, p35SFLREPsgprogfp, an SCMoV-based deletion construct containing ORF2b which encodes a putative replicase with a truncation of the CP gene, an insertion of gfp gene behind the 2b gene, a duplication of the putative sgPro sequence infront of the gfp gene and with 35S. One hybrid of p35SFLREPsgprogfp was further developed with T-Nos cloned downstream of the 3’ NCR in pFLREPsgprogfp as a KpnI/SalI fragment to give pTEVFLREPsgprogfp. Another hybrid without heterologous transcriptional factors could be derived by dropping the 35S and T-Nos.
6.10 Overlapping clones for development of ORF1 mutants of pFL
In addition to the constructs above, a few overlapping clones intended for developing full- length clones with various modifications involving ORF1 were developed. These were clones
15 and 16 in Fig. 6:11, i.e. plasmids p35S5NCRGFP and pP2foropt37 which could be assembled with other clones in the the various sections to create at least three constructs: the first with a replacement of ORF1 with GFP, and two without ORF1 but with a 3’ terminal section modified with and without a truncated CP containing a translatable GFP and a duplication of the putative sgPro sequence. Plasmid p35S5NCRGFP was developed by sub-
161
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
-cloning the gfp gene as in pGFPopt (section 5.2.2.1) as an EcoRI and SacI fragment into p35S5NCR (section 6.7.1.2) to produce a plasmid with 35S and 5’ NCR upstream of a translatable gfp gene. Plasmid pP2foropt37 contained as insert in pGEM-T, nt 605-3019 amplified with primers P2foropt and scm37.
6.11 Biological activity of in vivo RNA transcripts of full-length cDNA of SCMoV-pFL
Assessment of infectivity of viral gene vectors via in vivo-transcribed RNA transcripts, though not effective for all cloned RNA viruses, is attractive because it bypasses the need for in vitro transcription and also saves time. It was therefore intended for use as a first approach to testing the infectivity of constructs placed under the control of 35S promoter and T-Nos. However, current regulations concerning the development and use of GMOs placed restrictions on tests of infectivity of the constructs developed until approval was given by OGTR. Strict regulations required that none of the constructs could be used in any form outside a PC2 facility in intact plants. Infectivity of pTEVFL could be tested, but within the confines of a PC2 facility. Any test could only be done in a PC2 laboratory on a small scale because of space limitations.
6.11.1 Host plants used and plant infection
Subterranean clover, chickpea and grasspea, each of which develops conspicuous symptoms on infection with SCMoV-P23 (Chapter 3), were used as host plants for assessing the activity of in vivo-transcribed RNA transcripts from the full-length clone of SCMoV-pFL placed under the control of 35S and T-Nos, pTEVFL. Seeds of each plant were germinated and grown on MS meduim with 0.6% (w/v) phytagel and kept in a growth room at 22°C with 16 hr illumination.
Six weeks after germination, the in vitro cultured plants were inoculated by particle bombardment. Two replicates each of 3 plants grown together in a petri dish or in vitro culture containers were dry shot at a pressure of 2200 kPa with 842, 421 and 200 ng of pTEVFL.
6.11.2 Detection of in vivo-transcribed RNA transcripts
In vitro-transcribed RNA transcripts derived from plasmid pTEVFL were detected by RT-PCR from total RNA extracted (Section 2.2) from bulks of the inoculated plants 15 days after 162
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors particle bombardment. RT-PCR was done with primers (scm14 and scm38) that amplify and detect RNA transcripts corresponding to the C-terminus of the putative replicase and N- terminus of the CP. First strand cDNA was synthesised with M-MLV reverse transcriptase and
10 pmol of primer scm38. PCRs were done with 2 U of Platinum Taq polymerase. There was no amplification in any of the chickpea and subterranean clover plants, but the expected size of
960 bp was amplified from grasspea hosts inoculated with 842, 421 and 200 ng of pTEVFL.
Nucleic acids used in RT-PCR were not treated with DNase but were used as templates in
PCRs to serve as controls.
6.12 Discussion
6.12.1 Summary of clones developed in chapter 6
This chapter describes the cloning of a full-length cDNA clone of SCMoV-pFL and five major
GFP chimeric constructs developed for use as gene vectors. Three forms each of the constructs were generated for assessment of vector potential via in vitro- or in vivo-transcribed RNA transcripts. The major constructs were as follows:
• pFLCPgfp – a pFL with the gfp gene fused to the 3’ end of the CP gene,
• pFLP1gfp – a pFL with gfp gene fused to the 3’ end of the ORF1,
• pFLCPsgprogfp – a pFL with a putative sgPro sequence and a translatable gfp gene
cloned in tandem between the CP gene and the 3’ NCR of SCMoV,
• pFLCP∇sgprogfp – a pFL with a putative sgPro sequence and a translatable gfp gene
cloned in tandem between a truncated CP gene and the 3’ NCR and
• pFLREPsgprogfp – a pFL with the ORF2b, a putative sgPro sequence and a
translatable gfp gene cloned in tandem between a truncated CP gene and the 3’ NCR
6.12.2 Particle morphology and biochemical properties of SCMoV-pFL
Purified preparations of SCMoV-pFL stained with phosphotungstic acid revealed homogenous isometric particles. The virions were 25 nm in diameter, 5 nm smaller than that first published for the type virus (Francki et al., 1983). A variation in size of virions formed by RNA virus
163
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors isolates in hosts during infection has been reported (Nurkiyanova et al., 2000). The reasons for such a difference in size are not known. However, inconsistencies in calibrations or size standards or different types of stains could be a source of variation (Dr. John E Thomas, personal communication). Formaldehyde gel electrophoresis of vRNA of SCMoV-pFL showed the presence of a major band corresponding to the gRNA and a minor band. Njeru
(1996) observed an RNA species of about 700 nt in purified preparations of SCMoV-P23. This is larger than the two viroid-like satRNAs known to be encapsidated by SCMoV (Davies et al.,
1990; Francki et al., 1983). This RNA species has not been thoroughly investigated. It could be a different satRNA or a defective interfering RNA, as reported for CfMV (Makinen et al.,
2000a), or a sgRNA molecule known to be encapsidated by virions of some sobemoviruses
(Rutgers et al., 1980; Weber and Sehgal, 1982; Mäkinen et al., 1995b; Ryabov et al., 1996;
Bonneau et al., 1998; Tamm et al., 1999).
6.12.3 Amplification and cloning of full-length cDNA of SCMoV-pFL
The full-length cDNA clone of SCMoV-pFL was produced by ligation cloning of two overlapping cDNA clones. The entire genome (4,258 kb) could not be amplified in RT-PCR from vRNA extracted from virions. Inability to obtain a full-length first strand cDNA by RT-
PCR for some viruses has been attributed to the presence of secondary RNA structures or higher order RNA conformations though none has been predicted at terminal ends of sobemoviruses (Ghosh et al., 1981; Boyer and Haenni, 1994). The use of M-MLV reverse transcriptase for first strand cDNA synthesis, amplification of the second strand from as much as 20% of the cDNA, and amplification of cDNAs with rTth DNA polymerase, XL, with a balance of amount of MgCl2 in excess of total dNTPs were done to reduce PCR-induced errors
(Eckert and Kunkel, 1990; Liu et al., 1996). As a result, no mutations were observed in amplicons obtained from pFL for further cloning, confirming results of section 4.3.1.5 that the variation of 3 nucleotide substitutions for every 1000 bases between SCMoV-pFL and
SCMoV-P23 was as a result of intrinsic properties of these genomes and unlikely to be due to
PCR or cloning.
164
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
6.12.4 Technical issues concerning the development of SCMoV-based cDNA constructs
Among the advantages of IPCR mutagenesis is the reduction of manipulations in cloning steps allowing many mutants and recombinant plasmids to be obtained within a short time.
Difficulties not only in amplifying bigger fragments by IPCR but also in blunt-end cloning of the IPCR products made it impossible to develop some vector constructs which were then developed by ligation cloning of overlapping clones with cDNAs amplified from plasmid pFL.
Generally the genome of SCMoV lacks restriction sites for commonly used enzymes especially in the 5’ terminal half. This meant longer PCR fragments had to be amplified and generally the common restriction site for PstI at position 2707 in the genome was used for most subcloning.
However on a few instances, sites were introduced into amplified fragments mostly as silent mutations or were incorporated at positions that only facilitated cloning without disrupting the native sequence of the genome. When the T-Nos signal was cloned as a SalI fragment for some of the constructs, it was ligated with the SalI site in pGEM-T, 82 bases away from the last base of the SCMoV genomic sequence. This is not expected to affect activity of in vivo transcripts of any of these constructs as no significant differences in infectivity have been observed for several viruses with and without the terminator signal (Yamaya et al., 1988; Gal-On et al.,
1996).
6.12.5 Stability of clones in E. coli
A full-length cDNA clone of SCMoV-P23, earlier constructed by Williamson (2000), was meant to be the starting material for construction and modification of SCMoV-based constructs for use as gene vectors. The clone was transformed with E. coli strain DH5α. When glycerol stocks of bacterial clones kept at -80°C for about a year were first streaked on plates, the clone produced very small colonies but were stable in E. coli long enough to allow extraction of a minimal quantity of recombinant DNA for restriction digestion analysis to confirm its integrity.
Later when inoculum was taken from plates which had been stored at 4°C or from glycerol stocks kept at -80°C, no plasmid was recovered from overnight cultures: no detectable plasmid band was observed on ethidium bromide-stained gels and the Fluorometer gave no reading.
165
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
The instability of the plasmid in E. coli strain DH5α could be attributed to the production of a particular SCMoV protein, in high concentrations allowed by high copy number of the pGEM-
T backbone, which might be toxic to the cells. Toxicity and instability of virus cDNA sequences has been observed in some viral clones and suggested to be caused by expression of proteins by some viruses in some E. coli strains (Fakhfakh et al., 1996; Johansen, 1996). For example, the full-length cDNA of Pea seedborne mosaic virus (PSbMV) pathotype P1 under the control of 35S and T-Nos is unstable in E. coli strain DH5α (Johansen, 1996).
All SCMoV cDNA clones, including those with heterologous sequences, developed in this project had the high copy number pGEM-T vector as plasmid backbone with ampicillin resistance gene, and were transformed with E. coli strain JM109. From the smallest plasmid in pNos (3,294 kb) to the full-length cDNA of SCMoV-pFL and modified forms with or without foreign inserts (e.g. 35S, T-NOS, gfp) of sizes up to 9.5 kb, all clones were stable and apparently less or non-toxic to E. coli strain JM109 indicated by the large numbers and sizes of bacterial colonies on agar plates, and high plasmid yields of up to 2.5 µg or more per ml of overnight E. coli culture.
6.12.6 Activity of in vivo-transcribed RNA transcripts from pTEVFL
In vivo-transcribed RNA transcripts derived from cloned cDNA of several viruses give successful infection after mechanical inoculation, by rubbing or particle bombardment, using as small an amount of plasmid DNA as 50 ng, and as much as 25 µg per infection (Fakhfakh et al., 1996; Gal-On et al., 1996; Johansen, 1996; Dagless et al., 1997). RT-PCR test of plants mechanically inoculated with a full-length clone under the control of the 35S promoter of
CaMV, pTEVFL, showed positive results for grasspea but not for subterranean clover and chickpea. Why transcripts could not be detected in subterranean clover and chickpea is not clear, but differences in susceptibility of host plants to viral RNAs and the corresponding infectious cDNA constructs have been reported (Weber et al., 1992; Dagless et al., 1997).
166
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors
An explanation of the results obtained for susceptibility of the plants to infection by the full- length cDNA clone includes unknown host-dependent factors, biolistic conditions used for infection, the amount of plasmid DNA used, the strength and position of the heterologous 35S promoter and the sample size used. While the mechanism by which infectious RNAs are synthesised is not entirely clear, it is suggested that specific infectivity may result from the presence of eukaryotic replication signals (Boyer and Haenni, 1994). The presence of non-viral nucleotides between the transcriptional start site of the 35S promoter and the viral sequence reduces or abolishes infection altogether in some viruses (Dore et al., 1990; Rizzo and
Palukaitis, 1990), but not in others (Commandeur et al., 1991).
The amount of plasmid DNA used for infection should have been enough for successful infection, although in some publications, higher levels of DNA have been used. It may also be that the level of transcripts were too low to be detected by RT-PCR. The apparent lack of infection of subterranean clover and chickpea by pTEVFL could be attributed to the method of infection - the biolistic conditions might not have been optimised for viral infection. This is supported by the lower percentage of transformed cells obtained even after optimisation of the biolistic conditions in Chapter 5. The percentage of infection of suitable hosts by cloned RNA viruses is in fact usually lower than that found for native viruses (e.g. Gal-On et al., 1995;
Johansen, 1996).
6.13 Conclusion and further work
A full-length cDNA clone of SCMoV modified with 35S promoter of CaMV and T-Nos was successfully developed. Also, GFP derivative constructs of the full-length cDNA with different modifications such as deletion of the ORF1, insertion of the sgPro and truncation of the CP gene were constructed for use as VIGS vectors (6.11.1).
Infectivity of the full-length clone of SCMoV driven by the 35S was a quick experiment done at the end of the project to provide an indication of whether the gene vector constructs would express in vivo in plant hosts. More comprehensive testing would have to be done to confirm
167
6. Development of a full-length SCMoV cDNA clone & GFP derivatives for use as VIGS vectors the infectivity of the pTEVFL construct and possible expressions of the others developed in this chapter. It will require approval for large scale testing on a range of host plants such as identified in Chapter 3 with several means of infecting plants such as mechanical inoculation by rubbing, and agroinoculation or in vitro testing in protoplasts. This detailed work could not be done because during the period of my candidature, permission had not been given by OGTR for studying in vivo expression of any of the constructs except that of pTEVFL in the confines of a PC2 laboratory. However, the small scale expression studies in these plants is an important step towards obtaining viral and VIGS vectors based on SCMoV for future functional analysis of legume genes.
168
Chapter Seven
General Discussion and Conclusion
7. General discussion and conclusion
7.0 Review of aim and objectives of the project
SCMoV is a positive sense single-stranded RNA virus with a relatively small genome (Francki et al., 1983; Dwyer et al., 2003). The virus mainly infects legume pastures, is readily transmitted mechanically to other hosts and does so efficiently leading to high titres in its host
(Wroth and Jones, 1992a, b). The main aim of the project was to study aspects of the biology of SCMoV important for its development and use as a legume-specific VIGS vector for functional analysis of endogenous genes. A virus gene vector modified or developed for VIGS technology is more useful if it can successfully infect a wide range of hosts. Viral gene vectors have been developed by modifying their structure for efficient expression of foreign genes using one or more strategies. These usually involve gene replacement, insertions, duplication and sometimes complementation. It is therefore important that before cloning, a viral genome should be well characterised. The properties of SCMoV makes cloning and manipulation of the genome relatively straightforward and may enable strong expression levels of a foreign gene cloned into an engineered genome. In addition, the lack of any insect vector potentially reduces environmental risks posed by an engineered genome of SCMoV.
The aims at the start of the project were: a study of the host range of SCMoV and symptom development in its hosts; further characterisation of the genome of this sobemovirus by comparative sequence analysis and cellular studies, development of a full-length infectious cDNA clone; and construction of a range of chimeric GFP cDNA derivatives from which infectious in vivo- and in vitro- transcribed RNA transcripts could be obtained. The last step was to test the constructs for infectivity and their potential use as VIGS vectors. The achievements, progress, limitations and future prospects for the work are discussed below.
7.1 The extent to which the aims and objectives were achieved
7.1.1 New information on hosts of SCMoV
The first aim of the project was to study the host range of SCMoV in detail and to study symptom development in its hosts. This study was undertaken for two main reasons – first to
look for possible new and most suitable hosts amongst legumes and non-legumes that could be
170
7. General discussion and conclusion
used in future functional genomics studies based on SCMoV viral vectors. Second, a record of symptom development in a range of hosts provides new information in characterising the biology of any SCMoV-based viral vectors adapted for gene expression studies. Amongst the
64 plant genotypes, representing 51 species from 25 different genera belonging to 7 families, studied for their response to SCMoV infection, the virus replicated in 37 legumes belonging to
8 different genera. Twenty two of the hosts permitted cell-to-cell and long distance movement, whereas in 15 of them infection was restricted to a group of cells at sites of infection. Twenty five of these species had not been identified as hosts before; they include crop legumes of economic importance and some model legume plants e.g. chickpea, lentil, field peas, common beans, narbon beans and lupins. These results confirm that SCMoV is a virus adapted to infecting legumes, since it replicated in few of the non-legumes that were challenged.
Suitable hosts of viruses have contributed in many ways to the development of plant virology; they have been used to effectively study virus-host interactions, to propagate viruses, to develop diagnostic and infectivity assays to detect and measure virus content and to study molecular biology of viruses (Matthews, 1991). Viruses exploit the translational machinery of susceptible hosts to express their proteins (Matthews, 1991). Indicator plants, susceptible to a wide range of viruses, include Chenopodium spp., Nicotiana spp. and Phaseolus spp. (Walkey,
1991). N. benthamiana is a model host plant for most applications of VIGS (Kjemtrup et al.,
1998; Voinnet et al., 1999; Burton et al., 2000; Voinnet et al., 2000; Peele, 2001; Ratcliff et al.,
2001). This Australian native plant is susceptible to a wide range of viruses and exhibits VIGS symptoms that are generally much more pronounced and more persistent than in other plants, including other Nicotiana spp. the reasons of which has been studied by Waigmann et al.,
(2000). VIGS has also been applied in other species. For example, BSMV, has been used to demonstrate VIGS of PDS in a monocot, barley (Holzberg et al., 2002) whereas TRV vectors support VIGS in Arabidopsis (Dalmay et al., 2000; Dalmay et al., 2001) and tomato (Liu et al.,
2002). However, there is no report demonstrating the application of such a potential biotechnology in any legume species. A trial at the International Centre for Tropical
171
7. General discussion and conclusion
Agriculture (CIAT) using TRV to demonstrate VIGS of PDS and isolation of angular leaf spot resistance genes by silencing yielded results only in solanaceous plants (Romero Catalina,
CIAT, personal communication). This is an indication that even the few legume-infecting virus vectors like TRV are genus -and/or species-specific.
The expanded host range also extends the opportunity to widen the scope of research in legumes using SCMoV viral vectors. For example, chickpea is now being targeted as a model plant for genomics studies by a worldwide consortium, the International Chickpea Genomics
Consortium (ICGC). One objective of this programme is to exploit comparative genomics, and make use of genome information from the model legume species M. truncatula, to speed understanding of the chickpea genome and chickpea gene discovery. The complete genomic sequence of M. truncatula should be available by 2007. This will provide significant new opportunities to undertake functional analysis of chickpea genes, and a legume-specific VIGS vector will be needed for such work. Also the identification of diagnostic plants e.g. Trigonella and grasspea, will provide suitable hosts for rapid assay of infectivity of SCMoV-based viral vectors. With the expansion of cDNA libraries of several model plants, the need to identify functions of their genes will become imperative. Virus based systems are more reliable than other systems because of the increased speed, reliability and accuracy of the exercise.
Considering the broader host-range of SCMoV among legumes, capable of infecting plants from 8 genera, any vector derived from the virus should help to fill the gap in legume research aimed at rapidly screening both genes that exert discernible effects in localised and systemic infections of different legume species and endogenous genes involved in other metabolic pathways by silencing.
An important contribution of this work is that it has provided new insight into the susceptibility to SCMoV infection of some newly released cultivars of pasture and crops. This information has been used by the Western Australia farming community (Fosu-Nyarko et al., 2002a; 2002b) in deciding which new varieties should or should not be planted in SCMoV-endemic areas in farming systems.
172
7. General discussion and conclusion
7.1.2 Detailed characterisation of SCMoV
The second aim of this project was to characterise the genome of SCMoV in more detail to provide the information that was needed to decide how to modify its genome to develop gene vectors. This aspect was achieved by cloning and sequencing four new SCMoV isolates, and comparative sequence analysis of five isolates, combined with cellular studies on the effects of modifying the terminal ORFs of SCMoV.
7.1.2.1 Genetic diversity and biology of SCMoV
The complete genomic sequences of four isolates, SCMoV-AL, SCMoV-MB, SCMoV-MJ and
SCMoV-pFL, were determined in this project using high fidelity RT-PCR, molecular cloning and cDNA sequencing. These isolates induce different levels of symptom severity in subterranean clover. Genetic diversity between genomic sequences of the isolates together with that of SCMoV-P23, and the potential impact of nucleotide differences on the biology of the isolates was studied. The genome length of all five isolates was the same, 4,258 nt in length, but with an average genetic variation of 0.00883 ± 0.0014 (mean ± s.e.d) between them. This estimate is two to three orders of magnitude larger than the error rates of the polymerases used in RT-PCR. A molecular relationship tree clearly separates the five isolates into three groups, similar to the grouping based on the level of symptom severity they induce in subterranean
2 clover. A significant chi squared test (χ = 6.21, df = 2, P = 0.05) of the nucleotide substitutions in the complete sequences of the isolates versus the severity groups indicated that the nucleotide differences in the complete sequences could be responsible for the field behaviour of the isolates. A survey of the distribution of nucleotide differences showed more nucleotide substitutions (p≤ 0.05) at the 5’ terminal half of SCMoV than at the 3’ end. In addition, nucleotide diversities at nonsynonymous versus synonymous sites (dN/dS ratio) was higher for the ORF1 gene compared to the near conservation of sequences of the other genes.
These results, together with pattern of sequence variation and functions of proteins of sobemoviruses and other viruses, implicate the ORF1 gene product in pathogenesis of SCMoV,
173
7. General discussion and conclusion
possibly as a severity/pathogenicity determinant or as a viral suppressor of RNA silencing in plants. It is also likely to be involved in related functions such as genome amplification and virus movement. Plant virus vectors can be used to express high levels of a beneficial foreign gene product, to over-express a specific endogenous gene or to silence detrimental ones in order to confer a particular phenotype. However, high-level expression of transgenes can be reduced by induction of PTGS. Suppressors of silencing have an enormous potential in biotechnological applications. Their ability to directly counter PTGS (Anandalakshmi et al.,
1998; Kasschau and Carrington, 1998) could help improve protein yields of foreign and endogenous genes when plants are transformed together with these protein products.
Suppressors of PTGS also offer an easy assay for identifying silenced lines with potential for high-level transgene expression. Another useful application of suppressors of PTGS is in the area of functional genomics. When silencing of genes is not successful because they are not optimally expressed under normal conditions, their function may be difficult to predict. In such cases, over-expression of genes by transformation may prove to be a superior approach to understanding the function of the encoded protein.
The above applications have implications for virus vector design. For use as a foreign gene expression system, a virus vector is more useful if is not an inducer of gene silencing on a chosen host. Conversely, in VIGS application of endogenous genes it is advantageous if the vector is a strong inducer of silencing and encodes a weak suppressor of PTGS or none at all.
Most studies of viral activation and suppression of RNA silencing have relied on members of the genus Nicotiana, particularly N. benthamiana, and the strength of suppressors is hardly known. For practical applications of plant virus vectors in VIGS and particularly the development of vectors for new hosts, it is important to understand the effects of PTGS and the strength of encoded suppressors in any given virus/host combination. The prediction of the P1 of SCMoV as a suppressor of silencing thus provides an opportunity for the use of SCMoV viral vectors in gene expression systems and functional genomics.
174
7. General discussion and conclusion
7.1.2.2 Genome structure of SCMoV
The genome organisation and proposed mechanisms that control gene expression of one isolate,
SCMoV-P23, based on comparative sequence analysis with related viruses, was published during this work (Dwyer et al., 2003). The complete genomic sequences of the four isolates sequenced in this project, SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV-pFL, together with SCMoV-P23 were used to study the genome structure of SCMoV. The aim was to provide more information and a complete picture of the organisation of the genome and control of gene expression for effective development and modification of the genome as gene vectors.
This was achieved by using genetic analysis tools and comparative sequence analysis with sobemoviruses.
The genome structure of SCMoV was shown to be the same as that published for SCMoV-P23 containing four overlapping ORFs (Dwyer et al., 2003). This structure is similar to those of
CfMV and TRoV and the viruses are likely to share similar gene expression strategies
(Mäkinen et al., 1995a; Mäkinen et al., 1995b; Makinen et al., 2000a; Makinen et al., 2000b;
Callaway et al., 2002). They are likely to encode four proteins, the ORF1 gene product designated P1, a polyprotein produced by ribosomal frameshifting of ORF2a and 2b, a product from ORF2a and the CP produced from the ORF3 via sgRNA synthesis. Comparative sequence analysis was used to predict sequence elements that might influence virus gene expression and hence those of foreign genes. Using information from experiments which studied transcription start sites and subgenomic promoters and multiple sequence alignment of sobemoviruses and other viruses (Balmori et al., 1993; Zavriev et al., 1996; Hacker and
Sivakumaran, 1997), a sgPro sequence for expression of the CP was predicted for SCMoV for duplication in some constructs.
Information from the study of the genome organisation of SCMoV was vital in the development of gene vector constructs, which involved insertion of sequences such as the 35S promoter of CaMV, the T-Nos terminator signal and a putative sgPro, complementation of the genome with gfp gene, deletion of the ORF1 and truncation of the CP gene.
175
7. General discussion and conclusion
7.1.2.3 Limited cell-to-cell movement of the P1 protein and CP of SCMoV
Transient gene expression of the terminal ORFs of SCMoV was studied in cells of grasspea and chickpea to observe mainly the ability of the CP and P1 protein to move independently from cell to cell. The aim was to further characterise SCMoV and to study movement functions of the gene products that will be relevant in the successful modification of the genome into a gene vector. Plasmid DNA of translational fusion constructs of the ORF1 and CP gene each with
GFP driven by the 35S promoter were delivered into leaf tissues by particle bombardment with a custom-made particle inflow gun. The biolistic conditions were first optimised for efficient delivery of DNA-coated particles into tissues for transient gene expression analysis and to provide a rapid method for inoculating host plants with infectious vector constructs. In these experiments, the natural host of SCMoV, subterranean clover, and Trigonella, could not be transformed effectively for transient gene expression analysis; the reason for this result is not known at this stage. The fluorescence from GFP alone, GFP:CP and GFP:P1 constructs was observed in the nucleus of single cells, cytoplasm and the cell periphery of neighbouring cells.
There was thus a limited spread of these fusion proteins from one cell to another. These results indicate that the P1 and CP demonstrated very limited independent cell-to-cell movement.
Viral MPs have been demonstrated to move independently of other viral components in several viruses (Epel et al., 1996; Padgett et al., 1996; Solovyev et al., 2000). For some viruses with no specific MPs, such as begomoviruses, potexviruses and mastreviruses, it is known they traverse from cell to cell and over long distances by forming movement complexes that may involve viral proteins, RNA molecules and other cellular components (Jeffery et al., 1996;
Kotlizky et al., 2000; Lough et al., 2000; Palmer and Rybicki, 2001). The results indicate that neither the P1 protein nor the CP of SCMoV is solely responsible for virus movement although in other sobemoviruses they are both indispensable for cell-to-cell and long distance movement.
This suggests that their involvement in movement of sobemoviruses, and SCMoV, may be similar to viruses where movement is via a complex formation with viral/cellular components.
This hypothesis is supported by the ability of both the P1 and CP of some sobemoviruses to
176
7. General discussion and conclusion
bind to ssRNA (Abad-Zapatero et al., 1980; Bhuvaneshwari et al., 1995; Opalka et al., 2000;
Tamm and Truve, 2000; Lee and Hacker, 2001). The preliminary result contributed to the design and development of the gene vector constructs in this work. For there to be expression and systemic movement in host tissues of any of the viral vectors, it was taken that the P1 and the CP or the presence of the genes encoding these proteins would have to be present.
7.1.2.4 Development of gene vector constructs
The ability to manipulate RNA virus genomes by means of a cloned cDNA intermediate opened the possibility of using RNA viruses as gene vectors (Ahlquist et al., 1984; Dawson et al., 1986; Meshi et al., 1986). Successful viral gene vectors have been developed for optimal expression by modifying the genomes using approaches that include shuffling of viral and foreign genes (Toth et al., 2001), replacement of “non-essential viral genes” with foreign genes
(French et al., 1986; Takamatsu et al., 1987), duplication of viral sequences such as sgPros and addition of foreign genes which also serve as markers e.g. GFP (Dawson et al., 1989; Donson et al., 1991; Kavanagh et al., 1992; Kumagai et al., 1995). Successful modifications depend largely on molecular knowledge of the virus concerned; its genome structure, gene expression strategies and functions of the proteins it encodes. This provides information as to which genes are essential and which are not for expression of foreign genes. For instance the insertion of an sgPro-GFP cassette between the CP gene and 3’ NCR of AMV interferes with RNA accumulation (Sanchez-Navarro et al., 2001), but similar modifications has been shown to increase transient gene expression of GFP in a number of tobamoviruses (Shivprasad et al.,
1999).
Information on the genomic structure, possible gene expression strategies and the predicted sgPro sequence was used to modify the SCMoV genome to develop diverse cDNA constructs for use as gene vectors to speed up research in legume functional genomics. Although the compact nature of the SCMoV genome suggests that all genes might be necessary for the overall fitness of the virus, strategies used in developing the gene vector constructs included
177
7. General discussion and conclusion
gene truncation, fusion or insertion with the gfp gene, and duplication of a putative sgPro, predicted by sequence comparison with other sobemoviruses.
Similar strategies have been used successfully to modify genomes of both DNA and RNA viruses into gene vectors for expression of other viral and heterologous proteins (Sanchez-
Navarro et al., 2001; Shivprasad et al., 1999). Development of the vector constructs generally involved modification of the ORF1, the CP gene, in inserting the gfp gene and duplicating the sgPro in the genome of SCMoV to develop five major GFP chimeric constructs (section
6.11.1). For each of these constructs, three forms were made from which in vitro-and in vivo- transcribed RNA transcripts could be obtained.
It has been predicted that the P1 of SCMoV, like that of CfMV and RYMV, could be a suppressor of RNA silencing in hosts of SCMoV. Gene vector constructs containing the ORF1 could be used to investigate this hypothesis and the strength of its suppression of RNA silencing. A confirmation of this assertion would mean gene vector constructs with intact
ORF1 would not be efficient for use as VIGS vectors especially if they block initiation of RNA silencing. VIGS vectors derived from SCMoV should have no suppressor function and mutation of the P1 in constructs with intact ORF1 would therefore be necessary. Alternatively, gene vector constructs without the ORF1, such as the truncated construct, pFLREPsgprogfp
(section 6.8), would be more efficient as a VIGS vector. Indeed, RNA silencing does not necessarily require the whole genome of a virus, truncated genomes such as a functional RdRp of SCMoV should provide ideal VIGS.
Viruses with similar translation mechanisms have identical or conserved motifs at their NCRs
(Ahlquist et al., 1981; Dams et al., 1988; Danthine et al., 1993) or have functional substitutes which enhance translation (Carrington and Freed, 1990; Leathers et al., 1993; Sarnow, 1995).
Multiple sequence analysis indicates that this is not the case for sobemoviruses. This implies that they may not use common replication strategies found in other viruses. The accumulation
178
7. General discussion and conclusion
of sobemoviruses to remarkably high titres in their hosts does suggest that they contain elements that promote efficient gene expression, but that these are specific for each virus. This is confirmed by the ability of the 5’ NCR of CfMV to enhance foreign gene expression in both dicotyledonous and monocotyledonous plants to levels similar to known translational enhancers of other viruses (Mäkeläinen et al., 2003). Based on this information, the 5’ and 3’ NCRs of
SCMoV were maintained in all the gene vector constructs developed.
7.1.3 Expression of infectious SCMoV cDNA and GFP chimeric derivatives
Following development of a full-length cDNA clone of SCMoV-pFL (pTEVFL) and three forms each of five major cDNA gene vector constructs, the ultimate aim of this project was to study expression of these constructs in different host plants.
The biological activity of the full-length cDNA clone with the 35S and T-Nos was assessed to determine whether a clone of the virus would be infectious, and if any of the GFP chimeric vector constructs would express in legume hosts. When fifteen day-old in vitro-cultured chickpea, grasspea and subterranean clover seedlings were inoculated by particle bombardment with gold particles coated with plasmid DNA, in vivo-transcribed RNA transcripts were detected by RT-PCR after two weeks in grasspea, but not in subterranean clover and chickpea.
Because the results are preliminary, they only provide an indication that gene vectors with GFP chimeric constructs derived from the full-length clone of SCMoV should be active. Biological activity of in vivo-transcribed RNA transcripts of most cloned RNA viruses usually results in low percentages of infection in hosts (Fakhfakh et al., 1996; Gal-On et al., 1996; Johansen,
1996). Testing is best done on a large scale in a range of hosts of different genotypes with different methods of inoculation. This could not be done because of limitations placed on testing of recombinant plasmids in vivo by OGTR. The testing that could be done was on a small scale in a limited space in a growth chamber within a PC2 laboratory using particle bombardment of plasmid DNA as the only method of inoculation. These results need to be extended with more rigorous testing by other forms of inoculation.
179
7. General discussion and conclusion
However, they are an indication that the GFP hybrid plasmid constructs will probably be expressed in one or more of the legume hosts identified in Chapter 3.
A total of fifteen GFP chimeric gene vector constructs of SCMoV intended for use as VIGS vectors were developed. From these it was intended that the best would be selected for use as a routine vector for gene expression studies.
It was indeed unfortunate and frustrating that detailed assessment of the vector potential of these constructs could not be undertaken because there was a delay of one year whilst the
OGTR processed an application for permission to test the vector potential of the constructs on intact plants. The work undertaken nevertheless presents a significant progress towards the development of SCMoV as a legume vector for functional genomics.
7.2 Further work
The characterisation of SCMoV and the constructs developed in this work provide ready-made materials that will facilitate the process of obtaining a VIGS vector based on SCMoV. The additional work will be to assess the vector potential of these constructs using in vitro and/or in vivo transcribed RNA transcripts in several legume hosts. The vector constructs, generated by assembly of overlapping clones, were developed so that their potential could be assessed by mechanical inoculation of plasmid DNA or RNA transcripts (by rubbing or particle bombardment) onto leaves of a range of legume hosts. They were modified with 35S and T-
Nos to make three forms of each. One cloned with both the 35S and T-Nos, another with only the 35S sequence and a “naked” form with no transcriptional factors. The first two forms can be used to assess biological activity of in vivo-transcribed RNA transcripts and to determine the necessity and possible deletion of the T-Nos sequence to reduce the size of constructs and possible negative effects of genome packaging, whereas in vitro-transcribed RNA transcripts can be obtained from the constructs without the transcriptional factors for further testing of infectivity of these constructs.
180
7. General discussion and conclusion
Successful expression and systemic movement of any of the vector constructs will also provide the basic material for use of SCMoV as a gene expression and gene silencing system and to investigate the following with the aim of further characterising the virus and improving the efficiency of the vector: i) possible function and strength of the P1 as a virus suppressor for RNA silencing: This needs to be investigated as such a role will have implications on the biological activity of any
SCMoV viral vector. Virus-host interaction suggests suppressors of RNA silencing influence the final steady-state level of virus accumulation: strong suppressors would allow prolonged virus accumulation at a high level whereas when a virus accumulates at a low level it could be due to weak suppressor activity. Also, the strength of a viral suppressor encoded by SCMoV, if any, will determine the pattern and/or effectiveness of activation or suppression of silencing induced by any of the vectors.
ii) enhancement of movement functions of the P1 and CP by the putative NESs identified:
Proteins with NESs are known to be shuttled in and out of the nucleus and between cells by the help of complexes with RNAs and other cellular components. The putative NESs in the deduced amino acid sequences of the ORF1 and CP gene of SCMoV may have a role in the transport of the viral genome. This could further be investigated by mutational analysis of the
ORF1 and CP gene in a vector containing these. A confirmation of the involvement of such signals in movement functions of the P1 or CP and the viral genome could be exploited to enhance expression and systemic movement of any of the vectors.
In the host range study, M. truncatula, a model plant for genomics studies, was not infected by any of the four isolates of SCMoV used. Moreover, M. polymorpha cv Circle Valley but not cv
Serena supported replication of SCMoV-P23. Also different genotypes of grasspea responded differently to infection with SCMoV-P23. Whilst a significant number of host plants were found for SCMoV in this work, the observed selective susceptibility of species of the same genus and cultivars of the same species to SCMoV (see Chapter 3), and also results from other
181
7. General discussion and conclusion
virus-host combinations (Horvath, 1983; Johnstone et al., 1984), indicate more hosts could have been identified had more genotypes or even new plants been screened with all four or more isolates of SCMoV. A recent screening of about 200 genotypes of M. truncatula has identified genotypes which are susceptible and sensitive to infection with four viruses including
SCMoV-P23 (Saqib, M., Fosu-Nyarko, J., Jones, R.A.C, and Jones M.G.K., unpublished).
Identification of more legume hosts will expand legume research in this field.
7.3 SCMoV as a VIGS vector for functional genomics research
The ability of viruses to activate and suppress the RNA silencing mechanism of their host is being exploited to study gene function by silencing. A VIGS vector based on SCMoV has potential in this field: its ability to efficiently infect legumes will provide a legume-specific virus vector, the lack of which has restricted studies of gene silencing to plants of other families. Also the relatively small genome makes such a vector attractive as it will make manipulation of the vector via infectious clones relatively straightforward.
The potential of viral vectors has not been fully realised because of the threat GMOs pose to human health and the general environment. Other factors such as genetics and gene-ethics, personal, corporate, political, religious, ethical and intellectual property held by individuals and corporations, have delayed progress in the application to agriculture of molecular techniques involving viral vectors. SCMoV and other plant viral vectors, however, pose less risk than other pathogens: they are non-pathogenic to man and animals considering the large quantities of plant virus proteins regularly consumed in food products. Potential risks posed by viral vectors to the environment are mostly associated with virus transmission to other hosts; crops or weeds. For any SCMoV gene vector, the risk is reduced by their non- transmissibility by insect vectors and its somewhat limited host range.
In addition, the risks of plant virus-based vectors to the environment are probably very small as the ideal or reliable and robust vector sought by virologists may never be obtained because of
182
7. General discussion and conclusion
intrinsic properties of RNA viruses, that is, their genetic instability through recombination.
Additional sequences inserted in a viral genome, unless conferring a selective advantage, place genetic load on a virus and makes it less competitive than the wild-type progenitor virus. Thus the more a viral vector is modified, the greater is the chance that recombination may occur to eliminate the changes. This makes accidental escape of more virulent forms of viruses to the environment less of a threat. Loss of relative fitness for a vector may in fact also be an advantage in terms of environmental risk, since the recombinant virus is likely to be outcompeted rapidly by wild-type viruses.
7.4 Conclusion
A significant part of the original aims of this project were achieved despite OGTR delays, contributing to new knowledge of SCMoV. Although additional work needs to be undertaken to complete development of a final RNA silencing vector, this study has provided new information in terms of extending understanding of SCMoV host range, symptoms, genome organisation, control of gene expression and sequence variation. The expression of a full- length clone in a legume host and the GFP chimeric vector constructs made have also laid the groundwork for development of a legume gene silencing vector based on SCMoV. The number of SCMoV-based plasmid constructs ready to be validated for their vector potential would contribute to the study of the mechanism of RNA silencing in legumes and its use in the prediction of gene function.
183
Bibliography
Abad-Zapatero, C., Abdel-Meguid, S. S., Johnson, J. E., Leslie, A. G. W., Rayment, I., Rossmann, M. G., Suck, D. and Tsukihara, T. (1980) Structure of southern bean mosaic virus at 2.8 Å resolution. Nature Biotechnology 286, 33-39
Abo, M. E., Sg, A. A. and Alegbejo, M. N. (1998) Rice yellow mottle virus (RYMV) in Africa: evolution, distribution, economic significance in sustainable rice production and development strategies. Journal of Sustainable Agriculture 11, 85-111
Adkins, S. and Kao, C. C. (1998) Subgenomic RNA promoters dictate the mode of recognition by bromoviral RAN-dependent RNA polymerases. Virology 252, 1-8
Ahlquist, P., Dasgupta, R. and Kaesberg, P. (1981) Near identity of 3' RNA secondary structures in Bromoviruses and cucumber mosaic virus. Cell 23, 183-189
Ahlquist, P., French, R., Janda, M. and Loesh-Fries, L. S. (1984) Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proceedings of the National Academy of Sciences, USA 81, 7066-7070
Al-Karf, N. S., Covey, S. N., Kreike, M. M., Page, A. M., Pinder, R. and Dale, P. J. (1998) Transcriptional and post-transcriptional plant gene silencing in response to a pathogen. Science 279, 2113-2115
Altschuh, D., Lesk, A. M., Bloomer, A. C. and Klug, A. (1987) Correlation of co-ordinated amino acid substitutions with function in viruses related to tobacco mosaic virus. Journal of Molecular Biology 193, 693–707
Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H. and Vance, V. B. (1998) A viral suppressor of gene silencing in plants. Proceedings of the National Academy of Sciences, USA 95, 13079-13084
Angell, S. M., Davies, C. and Baulcombe, D. C. (1996) Cell-cell movement of potato virus X is associated with a change in the size exclusion limit of plasmodesmata in trichome cells of Nicotiana clevelandii. Virology 215, 197-201
Angenent, G. C., Posthumus, E., Brederode, F. T. and Bol, J. F. (1989) Genome structure of tobacco rattle virus strain PLB: Further evidence on the occurrence of RNA recombination among tobraviruses. Virology 171, 271-274
Aranda, M. A., Fraile, A., Dopazo, J., Malpica, J. M. and Garcia-Arenal, F. (1997) Contribution of mutation and RNA recombination to the evolution of a plant pathogenic RNA. Journal of Molecular Evolution 44, 81-88
Arias, A., Lazaro, E., Escarmis, C. and Domingo, E. (2000) Molecular intermediates of fitness gain of an RNA virus: Characterization of a mutant spectrum by biological and molecular cloning. Journal of General Virology 82, 1049-1060
Atreya, C. D., Raccah, B. and Pirone, T. P. (1990) A point mutation in the coat protein abolishes aphid transmissibility of a potyvirus. Virology 178, 161-5.
Atreya, P. L., Atreya, C. D. and Pirone, T. P. (1991) Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus. Proceedings of the National Academy of Sciences, USA 88, 8402–8406
Atreya, C. D., Atreya, P. L., Thornbury, D. W. and Pirone, T. P. (1992) Site-directed mutations in the potyvirus HC-Pro gene affect helper component activity, virus accumulation, and symptom expression in infected tobacco plants. Virology 191, 106–111
Atreya, P. L., Lopezmoya, J. J., Chu, M. H., Atreya, C. D. and Pirone, T. P. (1995) Mutational analysis of the coat protein N-terminal amino acids involved in potyvirus transmission by aphids. Journal of General Virology 76, 265–270
184
Bibliography
Bacher, J. W., Warkentin, D., Ramsdell, D. and Hancock, F. (1994) Selection versus recombination: What is maintaining identity in the 3' termini of blueberry leaf mottle nepovirus RNA1 and RNA2? Journal of General Virology 75, 2133–37
Bahramian, M. B. and Zarbl, H. (1999) Transcriptional and posttranscriptional silencing of rodent alpha1(I) collagen by a homologous transcriptionally self-silenced transgene. Molecular Cell Biology 19, 274-283
Bakker, W. (1974) Characterisation and ecological aspects of rice yellow mottle virus in Kenya. Agricultural Research Report, Cent. Agric. Publ. Doc, Wageningen 829, 152
Balmori, E., Glimer, D., Richards, K., Guilley, H. and Jonard, G. (1993) Mapping the promoter for subgenomic RNA synthesis on beet necrotic yellow vein virus RNA 3. Biochimie 75, 517-521
Baulcombe, D. C., Chapman, S. and Cruz, S. S. (1995) Jellyfish green fluorescent protein as a reporter for virus infections. The Plant Journal 7, 1045-1053
Beclin, C., Berthome, R., Palauqui, J. C., Tepfer, M. and Vaucheret, H. (1998) Infection of tobacco or Arabidopsis plants by CMV counteracts systemic post-transcriptional silencing of nonviral (trans)genes. Virology 252, 313-317
Bernstein, E., Caudy, A., Hammond, S. and Hannon, G. (2001) Role of bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 295-296
Bhuvaneshwari, M., Subramanya, H. S., Gopinath, K., Savithri, H. S., Nayudu, M. V. and Murthy, M. R. N. (1995) Structure of Sesbania mosaic virus at 3 Angstrom resolution. Structure 3, 1021-1030
Blackstock, J. M. (1978) Lucerne transient streak and lucerne latent, two new viruses of lucerne. Australian Journal of Agricultural Research 29, 291-304
Bogerd, H. P., Fridell, R. A., Madore, S. and Cullen, B. R. (1995) Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell 82, 485–494
Bonneau, C., Brugidou, C., Chen, L., Beachy, R. N. and Fauquet, C. (1998) Expression of the Rice yellow mottle virus P1 protein in vitro and in vivo and its involvement in virus spread. Virology 244, 79- 86
Bousalem, M., Douzery, E. J. P. and Fargette, D. (2000) High genetic diversity, distant phylogenetic relationships and intraspecies recombination events among natural populations of Yam mosaic virus : a contribution to understanding potyvirus evolution. Journal of General Virology 81, 243–255
Boyer, J.-C. and Haenni, A.-L. (1994) Infectious transcripts and cDNA clones of RNA viruses. Virology 198, 415-426
Bracho, M. A., Moya, A. and Barrio, E. (1998) Contribution of Taq polymerase-induced errors to the estimation of RNA virus diversity. Journal of General Virology 79, 2921-2928
Brigneti, G., Voinnet, O., Li, W.-X., Ji, L.-H., Ding, S.-W. and Baulcombe, D. C. (1998) Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. The EMBO Journal 17, 6739–6746
Brisson, N., Paszkowski, J., Penswick, J. R., Gronengorg, B., Potrykus, I. and Hohn, T. (1984) Expression of a bacteria gene in plants by using a viral vector. Nature 310, 511-514
Brugidou, C., Holt, C., Yassi, M. N., Zhang, S., Beachy, R. and Fauquet, C. (1995) Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus and mutagenesis of the coat protein. Virology 206, 108-115
Brunt, A. A., Crabtree, K., Dallwitz, M. J., Gibbs, A. J., Watson, L. and Zurcher, E. J. (1996) `Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 20th August 1996.' URL http://biology.anu.edu.au/Groups/MES/vide/.
185
Bibliography
Bruyere, A., Wantroba, M., Flasinski, S., Dzianott, A. and Bujarski, J. J. (2000) Frequent homologous recombination events between molecules of one RNA component in a multipartite RNA virus. Journal of Virology 74, 4214–4219
Burgyan, J., Dalmay, T., Rubino, L. and Russo, M. (1992) The replication of cymbidium ringspot tombusvirus defective interfering-satellite RNA hybrid molecules. Virology 190, 579-586
Burgyan, J., Salanki, K., Dalmay, T. and Russo, M. (1994) Expression of homologous and heterologous viral coat protein-encoding genes using recombinant DI RNAfrom cymbidium ringspot tombusvirus. Gene 138, 159-163
Burlage, R. S., Yang, Z. K. and Mehlhon, T. (1996) A transposon for green fluorescent protein transcriptional fusions: application for bacterial transport experiments. Gene 173, 53-58
Burton, R. A., Gibeaut, D. M., Bacic, A., Findlay, K., Roberts, K., Hamilton, A., Baulcombe, D. C. and Fincher, G. B. (2000) Virus-induced silencing of a plant cellulose synthase gene. The Plant Cell 12, 691–705
Callaway, A., Giesman-Cookmeyer, D., Gillock, E. T., Sit, T. L. and Lommel, S. A. (2001) The multifunctional capsid proteins of plant RNA viruses. Annual Review of Phytopathology 39, 419–460
Campbell, A. W. and Guy, P. L. (2001) Cocksfoot mottle virus spreads to the South Island of New Zealand. Australasian Plant Pathology 30, 217–220
Canto, T., Prior, D. A. M., Hellwald, K. H., Oparka, K. J. and Palukaitis, P. (1997) Characterization of cucumber mosaic virus. 4. Movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus. Virology 237, 237-248
Carrington, J. C. and Freed, D. D. (1990) Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region. Journal of Virology 64, 1590-1597
Carrington, J. C., Kasschau, K. D., Mahajan, S. K. and Schaad, M. C. (1996) Cell-to-cell and long- distance transport of viruses in plants. Cell 8, 1669-1681
Casper, S. J. and Holt, C. A. (1996) Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vector. Gene 173, 69-73.
Catalanotto, C., Azzallin, G., Macino, G. and Cogoni, C. (2000) Gene silencing in worms and fungi. Nature 404, 245
Cavener, D. R. and Ray, S. C. (1991) Eukaryotic start and stop translation sites. Nucleic Acids Research 19, 3185–3192
Cerruti, L., Mian, N. and Bateman, A. (2000) Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain. Trends in Biochemical Science 25, 481-482
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994) Green flourescent protein as a marker for gene expression. Science 263, 802-805
Chapman, S., Kavanagh, T. and Baulcombe, D. C. (1992) Potato virus X as a vector for gene expression in plants. The Plant Journal 2, 549-557
Chiu, W., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H. and Sheen, J. (1996) Engineered GFP as a vital reporter in plants. Current Biology 6, 325–330
Christensen, A. H. and Quail, P. H. (1996) Ubiquitin promoter-based vectors for high-level expres-sion of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Research 5, 213– 218
Christensen, B. B., Sternberg, C. and Molin, S. (1996) Bacterial plasmid conjugation on semi-solid surfaces monitored with green fluorescent protein (GFP) from Aequorea victoria as a marker. Gene 173, 59-65 186
Bibliography
Christou, P. (1995) Particle bombardment. Methods in Cell Biology 50, 375-382
Citovsky, V., Knorr, D., Schuster, G. and Zambryski, P. (1990) The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein. Cell 60, 637-647
Clark, M. F. and Adams, A. N. (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. General Virology 34, 475-483
Cogoni, C. and Macino, G. (1997) Isolation of quelling-defective (qde) mutants impaired in post- transcriptional transgene-induced gene silencing in Neurospora crassa. Proceedings of the National Academy of Sciences, USA 94, 10233-10238
Cogoni, C. and Macino, G. (1999) Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399, 166-169
Cogoni, C. and Macino, G. (2000) Post-transcriptional gene silencing across kingdoms. Current Opinion in Genetic Development 10, 638-643
Collins, R. F., Gellatly, D. L., Sehgal, O. P. and Abouhaidar, M. G. (1998) Self-cleaving circular RNA associated with rice yellow mottle virus is the smallest viroid-like RNA. Virology 241, 269-275
Commandeur, U., Jarausch, W., Li, Y., Koenig, R. and Burgermeister, W. (1991) cDNAs of beet necrotic yellow vein virus RNAs 3 and 4 are rendered biologically active in a plasmid containing the cauliflower mosaic virus 35S promoter. Virology 185, 493-495
Cooper, J. I. and Mayo, M. A. (1972) Some properties of the particles of three tobravirus isolates. Journal General Virology 16, 285-297
Covey, S. N., Al-Karf, N. S., Langara, A. and Turner, D. S. (1997) Plants combat infection by gene silencing. Nature 387, 781-782
Cox, D., Chao, A., Baker, J., Chang, L., Qiao, D. and Lin, H. (1998) A novel class of evolutionary conserved genes defined by piwi are essential for stem cell-renewal. Genes and Development 12, 3715- 3727
Cronin, S., Verchot, J., Heldeman-Cahill, R., Schaad, M. C. and Carrington, J. C. (1995) Long- distance movement factor: a transport function of the potyvirus helper component-protease. The Plant Cell 7, 549-559
Culver, J. N. (1996) Tobamovirus cross protection using a potexvirus vector. Virology 226, 228-235
D’Arcy, C. J., Domier, L. and Mayo, M. A. (2000) Family Luteoviridae. In "Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses". (Eds. Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Carstens, E., Estes, M., Lemon, S., Maniloff, J., Mayo, M.A., McGeoch, D., Pringle, C.R. and Wickner, R.B), New York, San Diego:Academic Press. pp. 775–784
Dagless, E. M., Shintaku, M. H., Nelson, R. S. and Foster, G. D. (1997) A CaMV 35S promoter driven cDNA clone of tobacco mosaic virus can infect host plant tissue despite being uninfectious when manually inoculted onto leaves. Virology 142, 183-191
Daisuke, M., Satoru, M., Rika, I. and Ko, S. (2003) Gene-specific suppression of a multigene family in plants by RNA interference. In "Gene Silencing and Epigenetics". 7th International Congress of Plant Molecular Biology, Barcelona, Spain, June 23-28, 2003. pp. 362
Dall, D. J., Graddon, D. J., Randles, J. W. and Francki, R. I. B. (1990) Isolation of a subterranean clover mottle virus-like satellite RNA from lucerne infected with lucerne transient streak virus. Journal of General Virology 71, 1873-1875
Dalmay, T., Horsefield, R., Braunstein, T. H. and Baulcombe, D. (2001) SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis. The EMBO Journal 20, 2069- 2077
187
Bibliography
Dalmay, T., Hamilton, A., Rudd, S., Angell, S. and Baulcombe, D. C. (2000) An RNA-dependent RNA polymerase gene in Arabidopsis is required for post-transcriptional gene silencing mediated by a transgene but not a virus. Cell 101, 543-553
Dams, E., Hendricks, L., Van de Peer, Y., Neefs, J., Smits, G., Vandenbempt, I. and De Watcher, R. (1988) Compilation of small ribosomal subunit RNA sequences. Nucleic Acid Research 16, 87-173
Danthine, X., Seurink, J., Meulewaeter, F., Van Montagu, M. and Cornelissen, M. (1993) The 3' untranslated region of satelite tobacco necrosis virus RNA stimulates translation in vitro. Molecular Biology Cell 13, 3340-3349
Davies, C., Haseloff, J. and Symons, R. H. (1990) Structure, self-cleavage, and replication of two viroid-like satellite RNAs (virusoids) of subterranean clover mottle virus. Virology 177, 216-224
Dawson, W. O., Becker, D. L., Knorr, D. A. and Grantham, G. L. (1986) cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts. Proceedings of the National Academy of Sciences, USA 83, 1832-1836
Dawson, W. O., Lewandowski, D. J., Hilf, M. E., Bubrick, P., Raffo, A. J., Shaw, J. J., Grantham, G. L. and Desjardins, P. R. (1989) A tobacco mosaic virus-hybrid expresses and loses an added gene. Virology 172, 285-292 de Calvalho, F., Gheysen, G., Kushnir, S., Van Montagu, M., Inze, D. and Castresano, C. (1992) Suppression of beta-1,3-glucanase transgene expression in homozygous plants. The EMBO Journal 11, 2595-2602
De Giorgi, F., Brini, M., Bastianutto, C., Marsault, R., Montero, M., Pizzo, P., Rossi, R. and Rizzuto, R. (1996) Dual-color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant. Gene 173, 19-23
Dehio, C. and Schell, J. (1994) Identification of plant genetic loci involved in a post-transcriptional mechanism for meiotically reversible transgene silencing. Proceedings of the National Academy of Sciences, USA 91, 5538-5542
Demler, S. A. and de Zoeten, G. A. (1991) The nucleotide sequence and luteovirus-like nature of RNA 1 of an aphid non-transmissible strain of pea enation mosaic virus. Journal of General Virology 72, 1819–1834
Di Sansebastiano, G. P., Paris, N., Marc-Martin, S. and Neuhaus, J.-M. (2001) Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles. Plant Physiology 126, 78-86
Ding, S. W., Li, W. X. and Symons, R. H. (1995) A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement. The EMBO Journal 14, 5762-5772
Dolja, V. V., McBride, H. J. and Carrington, J. C. (1992) Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein. Proceedings of the National Academy of Sciences, USA 89, 10208-10212
Dolja, V. V., Herndon, K. L., Pirone, T. P. and Carrington, J. C. (1993) Spontaneus mutagenesis of a plant potyvirus genome after insertion of a foreign gene. Virology 67, 5968-5975
Dolja, V. V., Permyslov, V. V., Keller, K. E., Martin, R. R. and Hong, J. (1998) Isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors. Virology 252, 269- 274
Domeier, M., Morse, D., Knight, S., Portereiko, M., Bass, B. and Mango, S. (2000) A link between RNA interference and nonsence-mediated decay in Caenorhabditis elegans. Science 289, 1929-1931
Domingo, E. and Holland, J. J. (1994) Mutation rates and rapid evolution of RNA viruses. In The Evolutionary Biology of Viruses Edited by S. S. Morse. New York: Raven Press pp 161-184
188
Bibliography
Donson, J., Kearney, C. M., Hilf, M. E. and Dawson, W. O. (1991) Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector. Proceedings of the National Academy of Sciences, USA 88, 7204-7208
Dore, J.-M., Erny, C. and Pinck, L. (1990) Biologically active transcripts of alfalfa mosaic virus RNA3. FEBS Letter 264, 183-186
Dougherty, W. G. and Parks, T. D. (1995) Transgenes and gene suppression: telling us something new? Current Opinion in Cell Biology 7, 399 - 405
Drake, J. W. (1993) Rates of spontaneous mutation among RNA viruses. Proceedings of the National Academy of Sciences, USA 90, 4171-4175
Drijfhout, E., Silbernagel, M. J. and Burke, D. W. (1978) Differentiation of strains of bean common mosaic virus. Netherlands Journal of Plant Pathology 84, 13-26
Dundr, M., McNally, J. G., Cohen, J. and Misteli, T. (2002) Quantitation of GFP-fusion proteins in single living cells. Journal of Structural Biology 140, 92-99
Dwyer, G. I., Njeru, R., Williamson, S., Fosu-Nyarko, J., Hopkins, R., Jones, R. A. C., Waterhouse, P. M. and Jones, M. G. K. (2003) The complete nucleotide sequence of subterranean clover mottle virus (SCMoV) Archives of Virology 148, 2237–2247
Eckert, K. A. and Kunkel, T. A. (1990) High fidelity by the Thermus aquaticus DNA Polymerase. Nucleic Acids Research 18, 3737-3744
Ehrig, T., O'Kane, D. J. and Prendergast, F. G. (1995) Green fluorescent protein mutants with altered fluorescent spectra. FEBS Letter 367, 163-166
Elbashir, S., Lendeckel, W. and Tuschl, T. (2001) RNA interference is mediated by 21-and 22- nucleotide RNAs. Genes and Development 15, 188-200
Elliot, A. R., Campbell, J. A., Dugdale, B., Brettell, R. I. S. and Grof, G. P. L. (1999) Green- fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells. Plant Cell Reports 18, 707-714
Elmayan, T., Balzergue, S., Beon, F., Bourdon, V., Daubremet, J., Guenet, Y., Mourrain, P., Palauqui, J. C., Vernhettes, S., Vialle, T., Wostrikoff, K. and Vaucheret, H. (1998) Arabidopsis mutants impaired in co-suppression. The Plant Cell 10, 1747-1758
English, J. J., Mueller, E. and Baulcombe, D. C. (1996) Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. The Plant Cell 8, 179-188
Epel, B. L., Padgett, H. S., Heinlein, M. and Beachy, R. N. (1996) Plant virus movement protein dynamics probede with a GFP-protein fusion. Gene 173, 75-79
Erickson, J. W., Silva, A. M., Murthy, M. R., Fita, I. and Rossmann, M. G. (1985) The structure of a T = 1 icosahedral empty particle from southern bean mosaic virus. Science 229, 625-629
Escobar, M. N., Haupt, S., Graham, T., Boevink, P., Chapman, S. and Oparka, K. (2003) High- throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata. Cell 15, 1507-1523
Fagard, M., Boutet, S., Bellini, C. and Vaucheret, H. (2000) AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proceedings of the National Acadamy of Sciences, USA 97, 11650-11654
Fakhfakh, H., Vilaine, F., Makni, M. and Robaglia, C. (1996) Cell-free cloning and biolistic inoculation of an infectious cDNA of potato virus Y. Journal of General Virology 77, 519-523
Fargette, D., Pinel, A., Traore, O., Ghesquiere, A. and Konate, G. (2002) Emergence of resistance- breaking isolates of Rice yellow mottle virus during serial inoculations. European Journal of Plant Pathology 108, 585-591 189
Bibliography
Fedorkin, O. N., Solovyev, A. G., Yelina, N. E., Zamyatnin, A. A., Jr, Zinovkin, R. A., Makinen, K., Schiemann, J. and Morozov, S. Y. (2001) Cell-to-cell movement of potato virus X involves distinct functions of the coat protein. Journal of General Virology 82, 449-458
Feilmeier, B. J., Iseminger, G., Schroeder, D., Webber, H. and Phillips, G. (2000) Green fluorescent protein functions as reporter for protein localisation in Escherichia coli. Journal of Bacteriology 182, 4068-4076
Ferris, D. G. and Jones, R. A. C. (1995) Losses in herbage and seed yields caused by Subterranean clover mottle virus in subterranean clover swards. Australian Journal of Agricultural Research 46, 1-17
Ferris, D. G., Jones, R. A. C. and Wroth, J. M. (1996) Determining the effectiveness of resistance to subterranean clover mottle sobemovirus in different genotypes of subterranean clover in the field using the grazing animal as virus vector. Annals of Applied Biology 128, 303-315
Fiers, W., P, A. and J, v. E. (1986) Satellite tobacco necrosis virus: a new vector in plant genetic engineering. Symbiosis 2, 35-41
Finnegan, E. J., Peacock, W. J. and Dennis, E. S. (1996) Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proceedings of the National Academy of Sciences, USA 93, 8449-8454
Fire, A. (1999) RNA-triggered gene silencing. Trends in Genetics 15, 358 - 363
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811
Fischer, U., Huber, J., Boelens, W. C., Mattai, I. W. and Luehrmann, R. (1995) The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475-483
Fitchen, J., Beachy, R. N. and Hein, M. B. (1995) Plant virus expressing hybrid coat protein with added murine epitope elicits autoantibody response. Vaccines 13, 1051-1057
Forster, R. S. L. and Jones, A. T. (1979) Properties of lucerne transient streak virus, and evidence of its affinity to southern bean mosaic virus. Annals of Applied Biology 93, 181-189
Fosu-Nyarko, J., Jones, R. A. C., Smith, L. J., Jones, M. G. K. and Dwyer, G. I. (2002) Testing for disease vulnerability in alternative pasture and forage legumes being assessed in plant improvement programmes. In 'Proceedings of the 12th Australasian Plant Breeding Conference - Plant Breeding for the 11th Millennium' (Ed. JA McComb) pp. 382-384
Fosu-Nyarko, J., Jones, R. A. C., Smith, L. J., Jones, M. G. K. and Dwyer, G. I. (2002) Responses of alternative annual pasture and forage legumes to challenge with infectious subterranean clover mottle virus. In Crop Updates: Farming systems Department of Agriculture - Western Australia, July, 2002
Francki, R. I. B., Grivell, C. J. and Gibb, K. S. (1986) Isolation of velvet mottle virus capable of replication with and without a viroid-like RNA. Virology 148:, 381-384
Francki, R. I. B., Randles, J. W. and Graddon, D. J. (1988) Subterranean clover mottle virus. CMI/AAB Description of Plant Viruses, No. 329. Commonwealth Mycological Institute, Kew, Surrey, and Association of Applied Biologists, Wellesbourne, Warwick, England.
Francki, R. I. B., Randles, J. W., Hatta, T., Davies, C., Chu, P. W. G. and McLean, G. D. (1983) Subterranean clover mottle virus: another virus from Australia with encapsidated viroid-like RNA. Plant Pathology 32, 47-59.
Francois, I. E. J. A., Dwyer, G. I., De Bolle, M. F. C., Goderis, J. W. M., Van Hemelrijck, W., Proost, P., Wouters, P., Broekaert, F. and Cammue, B. P. A. (2002) Processing in transgenic Arabidopsis thaliana plants of polyproteins with linker peptide variants derived from the Impatiens balsamina antimicrobial polyprotein precursor. Plant Physiology and Biochemistry 40, 871-879
190
Bibliography
Frankel, G. and Friedmann, A. (1987) Synthesis of long viral complementary DNA from 7.5 kb poly A+ RNA templates. Journal of Virological Methods 18, 1-12
French, R., Janda, M. and Ahlquist, P. (1986) Bacterial gene inserted in an engineered RNA virus: efficient expression in monocotyledonous plant cells. Science 231, 1294-1297
Fritz, C. C. and Green, M. R. (1996) HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs. Current Biology 6, 848–854
Fuentes, A. L. and Hamilton, R. I. (1991) Sunn-hemp mosaic virus facilitates cell-to-cell spread of southern bean mosaic virus in a nonpermissive host. Phytopathology 81, 1302-1305
Fuentes, A. L. and Hamilton, R. I. (1993) Failure of long-distance movement of southern bean mosaic virus in a resistant host is correlated with lack of normal virion formation. Journal of General Virology 74, 1903-1910
Fujiwara, T., Geiesman-Cookmeyer, D., Ding, B., Lommel, S. A. and Lucas, W. J. (1993) Cell-cell trafficking of macromolecules through plasmodesmata potentiated by the red clover necrotic mosaiac virus movement protein. Plant Cell 5, 1783-1794
Gallie, D. R. (1990) The cap and poly (A) tail function synergistically to regulate mRNA translational efficiency. Genes and Development 5, 2108-2116
Gal-On, A., Meiri, E., Huet, H., Hua, W. J., Raccah, B. and Gaba, V. (1996) Particle bombardment drastically increases the infectivity of cloned DNA of zucchini yellow mosaic potyvirus. Journal of General Virology 76, 3223-3227
Garcia-Arenal, F., Palukaitis, P. and Zaitlin, M. (1984) Strains and mutants of tobacco mosaic virus are both found in virus derived from single-lesion-passaged inoculum. Virology 132, 131-137
Garcia-Arenal, F., Fraile, A. and Malpica, J. M. (2001) Variability and genetic structure of plant virus populations. Annual Review of Phytopathology 39, 157-186
Gargouri, R., Joshi, R. L., Bol, J. F., Astier-Manifacier, S. and Haenni, A. L. (1989) Mechanism of synthesis of turnip yellow mosaic virus coat protein subgenomic RNA in vivo. Virology 171, 386-393
Ghosh, A., Rutgers, T., Ke-Qiang, M. and Kaesberg, P. (1981) Characterization of the coat protein mRNA of southern bean mosaic virus and its relationship to the genomic RNA. Journal of Virology 39, 87-92
Gopinath, K., Wellink, J., Porta, C., Taylor, K. M., Lomonossoff, G. P. and van Kammen, A. (2000) Engineering Cowpea mosaic virus RNA-2 into a vector to express heterologous proteins in plants. Virology 267, 159-173
Gorbalenya, A. E., Koonin, E. V., Blinov, V. M. and Donchenko, A. P. (1988) Sobemovirus genome appears to encode a serine protease related to cysteine proteases of picornaviruses. FEBS Letter 236, 287-290
Goregaoker, S. P., Eckhardt, L. G. and Culver, J. N. (2000) Tobacco mosaic virus replicase-mediated cross-protection: contributions of RNA and protein-derived mechanisms. Virology 273, 267-275
Gould, A. R. (1981) Studies on encapsidated viroid-like RNA. II. Purification and characterisation of a viroid-like RNA associated with velvet tobacco mottle virus. Virology 108, 123-133
Gould, A. R. and Hatta, T. (1981) Studies on encapsidated viroid-like RNA. III. Comparative studies on RNAs isolated from velvet tobacco mottle virus and Solanum nodiflorum mottle virus. Virology 109, 137-147
Greber, R. S. (1981) Some characteristics of solanum nodiflorum virus-a beetle-transmitted isometric virus from Australia. Australia Journal of Biological Sciences 34, 369–378
Greene, A. E. and Allison, R. F. (1994) Recombination between viral RNA and transgenic plant transcripts. Science 263, 1423-1425 191
Bibliography
Grimsley, N. and Bisaro, D. (1987) Agroinfection. In "Plant DNA Infectious Agents", (Eds. T. Hohn, J.Schell), Berlin: Springer-Verlag pp. 87-107.
Gubbler, U. and Hoffman, B. J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269
Guivarc'h, A., Caissard, J. C., Azmi, A., Elmayan, T., Chriqui, D. and Tepfer, M. (1996) In situ detection of the gus reporter gene in transgenic plants: ten years of blue genes. Transgenic Research 5, 281-288
Guo, H. S., Lopez-Moya, J. J. and Garcia, J. A. (1998) Susceptibility to recombination rearrangements of a chimeric plum pox potyvirus genome after insertion of a foreign gene. Virus Research 57, 183-195
Hacker, D. L. and Sivakumaran, K. (1997) Mapping and expression of southern bean mosaic virus genomic and subgenomic RNAs. Virology 234, 317-327
Hagiwara, Y., Peremyslov, V. V. and Dolja, V. V. (1999) Regulation of closterovirus gene expression examined by insertion of a self processing reporter and by northern hybridization. Virology 73, 7988- 7993
Hall, T. A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp. Serv 41, 95-98
Hamamoto, H., Sugiyama, Y., Nakagawa, N., Hashida, E., Matsunaga, Y., Takemoto, S., Watanabe, Y. and Okada, Y. (1993) A new tobacco mosaic virus vector and its use for the systemic production of angiotensin-I-converting enzyme inhibitor in transgenic tobacco and tomato. Biotechnology (N Y) 11, 930-932
Hamilton, A. J. and Baulcombe, D. C. (1999) A species of small antisense RNA in post-transcriptional gene silencing in plants. Science 286, 950-952
Hamilton, A. J., Brown, S., Yuanhai, H., Ishizuka, M., Lowe, A., Solis, A. G. A. and Grierson, D. (1998) A transgene with repeated DNA causes high frequency, post-transcriptional suppression of ACC- oxidase gene expression in tomato. The Plant Journal 15, 737-746
Hartmann, J. X., Bath, J. E. and Hooper, G. R. (1973) Electron microscopy of virus like particles from shoestring-diseased high bush blueberry, Vaccinium corymbosum L. Phytopathology 63, 432–436
Haseloff, J., Siemering, K., Prasher, D. and Hodge, S. (1997) Removal of a cryptic intron and subcellular localisation of green fluorescent protein are required to mark transgenic plants brightly. Proceedings of the National Academy of Sciences, USA 94, 2122-2127
Hayes, R. J. and Buck, K. W. (1988) Replication of tomato golden mosaic virus DNA B in transgenic plants expressing open reading frames (ORFs) of DNA A: requirement of ORF AL2 for production of single-stranded DNA. Nucleic Acids Research 17, 10213-10222
Hayes, R. J., Coutts, R. H. and Buck, K. W. (1989) Stability and expression of bacterial genes in replicating geminivirus vectors in plants. Nucleic Acids Research 17, 2391-2403
Heim, R. and Tsien, R. Y. (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curruent Biology 6, 178-182
Heinlein, M., Epel, B. L., Padgett, H. S. and Beachy, R. N. (1995) Interaction of the tobacco mosaic virus movement protein with the plant cytoskeleton. Science 270, 1983-1985
Helms, K., Muller, W. and Waterhouse, P. (1993) National survey of viruses in pastures of subterranean clover. I. Incidence of four viruses assessed by ELISA. Australian Journal of Agricultural Research 44, 1837-1862
Hermodson, M. A. C., Abad-Zapatero, C., Abdel-Meguid, S. S., Pundak, S., Rossmann, M. G. and Tremaine, J. H. (1982) Amino acid sequence of southern bean mosiac virus coat protein and its relation to the three-dimensional structure of the virus. Virology 119, 133-149
192
Bibliography
Hirochika, H. and Hayashi, K. I. (1991) A new strategy to improve a Cauliflower mosaic virus vector. Gene 105, 293-241
Hollings, M. and Stone, O. M. (1973) Turnip rosette virus. CMI/AAB Description of Plant Viruses, No.125. Commonwealth Mycological Institute, Kew, Surrey, and Association of Applied Biologists, Wellesbourne, Warwick, England.
Holmes, F. O. (1964) Symptomology of viral diseases in plants. In "Plant Virology", (M.K.Corbett and H.D. Sissler, eds), University of Florida Press, Gainesville pp17-38
Holzberg, S., Brosio, P., Gross, C. and Pogue, G. P. (2002) Barley stripe mosaic virus-induced gene silencing in a monocot plant. The Plant Journal 30, 315-327
Hörtensteiner, S., Martinoia, E. and Amrhein, N. (1992) Reappearance of hydrolytic activities and tonoplast proteins in the regenerated vacuole of evacuolated protoplasts. Planta 187, 113-121
Horvath, J. (1983) New artificial hosts and non-hosts of plant viruses and their role in the identification and separation of viruses. Acta Phytopatholgy, Academy of Science, Hungary 18, 121-161
Hull, R. (1977) The grouping of small spherical plant viruses with single RNA components. Journal of General Virology 36, 289-295
Hull, R., Fauquet, C., Gergerich, R. C., Lommel, S. A. and Thottapilly, G. (1999) Sobemovirus. In "Virus Taxonomy", Seventh Report of the International Committee on Taxonomy of Viruses. pp. 1014
Inuoye, S. and Tsuji, F. I. (1994) Aequorea green fluorescent protein: Expression of the gene and fluorescent characteristics of the recombinant protein. FEBS Letter 351, 277-280
Jameson, L., Chin, W. W., Hollenberg, A. N., Chang, A. S. and Habener, J. F. (1984) The gene encoding the rat beta-luteinzing hormone. Journal of Biological Chemistry 259, 15474-15480
Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W. (1987) Gus fusions: beta-glucuronidase as a sensitive and versatile gene marker in higher plants. The EMBO Journal 6, 3901-3907
Jeffery, J. L., Pooma, W. and Petty, I. T. (1996) Genetic requirements for local and systemic movement of tomato golden mosaic virus in infected plants. Virology 223, 208-218
Jensen, S., Gassama, M. P. and Heidmann, T. (1999) Taming of transposable elements by homology- dependent gene silencing. Nature Genetics 21, 209-212
Ji, J. P. and Loeb, L. A. (1992) Fidelity of HIV-1 reverse transcriptase copying RNA in vitro. Biochemistry 31, 954-958
Johansen, I. E. (1996) Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is re-established after transcription in vivo. Proceedings of the National Academy of Sciences, USA 93, 12400–12405
Johnstone, G. R. and McLean, G. D. (1987) Virus diseases of subterranean clover. Annals of Applied Biology 110, 421-440
Johnstone, G. R., Ashby, J. W., Gibbs, A. J., Duffus, J. E., Thottapilly, G. and Fletcher, J. D. (1984) The host ranges, classification and identification of eight persistent aphid-transmitted viruses causing diseases in legumes. Netherlands Journal of Plant Pathology 90, 225-245
Jones, R. A. C. (1996) Virus diseases of Australian pastures. In "Pasture and forage crop pathology". (Eds. S. Chakraborty, K. T. Leath, R. A. Skipp, G. A. Pederson, R. A. Bray, G. C. M. Latch, F. W. Nutter) American Society of Agronomy: Madison, WI. pp. 303-332
Jones, A. T. and Mayo, M. A. (1983) Interaction of lucerne transient streak virus and the viroid-like RNA-2 of Solanum nodiflorum mottle virus. Journal of General Virology 64, 1771-1774
193
Bibliography
Jones, A. T. and Mayo, M. A. (1984) Satellite nature of the viroid-like RNA-2 of Solanum nodiflorum mottle virus and the ability of other plant viruses to support the replication of viroid-like RNA molecules. Journal of General Virology 65, 1713-1721
Jukes, T. H. and Cantor, C. R. (1969) Evolution of protein molecules. In: Munro H.H. (ed.), Mammalian protein metabolism Academic Press, New York, pp. 21-132
Kado, C. I. (1967) Biological and biochemical characterisation of sowbane mosaic virus. Virology 31, 217–229
Kado, C. I. (1971) Sowbane mosaic virus. CMI/AAB Description of Plant Viruses, No. 64. Commonwealth Mycological Institute, Kew, Surrey, and Association of Applied Biologists, Wellesbourne, Warwick, England.
Kaether, C. and Gerges, H. H. (1995) Visualisation of protein transport along the secretory pathway using green fluorescent protein. FEBS Letter 369, 267-271
Kahn, T. W., Lapidot, M., Heinlan, M., Reichel, C., Cooper, B., Gafny, R. and Beachy, R. (1998) Domains of the TMV movement protein involved in subcellular localization. The Plant Journal 15, 15- 25
Kalderon, D., Richardson, W. D., Markham, A. F. and Smith, A. E. (1984) Sequence requirements for nuclear localisation of SV40 large T antigen. Nature 311, 33–38
Kamer, G. and Argos, P. (1984) Primary structural comparison of RNA-dependent polymerases from plant, animal and bacteria viruses. Nucleic Acids Research 12, 7269-7282
Kasschau, K. D., Cronin, S. and Carrington, J. C. (1997) Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component- proteinase. Virology 228, 251-262
Kasschau, K. D. and Carrington, J. C. (1998) A counterdefensive strategy of plant viruses: suppression of postranscriptional gene silencing. Cell 95, 461-470
Kavanagh, T., Goulden, M., Santa Cruz, S., Chapman, S., Barker, I. and Baulcombe, D. (1992) Molecular analysis of a resistance-breaking strain of potato virus X. Virology 189, 609-617
Kearney, C. M., Thomson, M. J. and Roland, K. E. (1999) Genome evolution of tobacco mosaic virus populations during long-term passaging in a diverse range of hosts. Archives of Virology 144, 1513-1526
Kiberstis, P. A. and Zimmern, D. (1984) Translational strategy of Solanum nodiflorum mottle virus RNA: synthesis of a coat protein precursor in vitro and in vivo. Nucleic Acids Research 12, 933-943
Kjemtrup, S., Sampson, K. S., Peele, C. G., Nguyen, L. V., Conkling, M. A., Thompsosn, W. F. and Robertson, D. (1998) Gene silencing from plant DNA carried by a Geminivirus. The Plant Journal 14, 91-100
Klein, T. M., Arentzen, R., Lewis, P. A. and Fitzpatrick-McElligott, S. (1992) Transformation of microbes, plants and animals by particle bombardment. Bio-Technology 10, 286-291
Koev, G. and Miller, W. A. (2000) A positive strand RNA virus with three very different subgenomic RNA promoters. Journal of Virology 74, in Press
Koev, G., Mohan, B. R. and Miller, W. A. (1999) Primary and secondary structural elements required for synthesis of barley yellow dwarf virus subgenomic RNA1. Virology 73, 2876-2885
Kong, P., Rubio, L., Polek, M. and Falk, B. W. (2000) Population Structure and Genetic Diversity within California Citrus tristeza virus (CTV) Isolates. Virus Genes 3, 139 -145
Koonin, E. V. (1991) The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. Journal of General Virology 72, 2197–2206
194
Bibliography
Kotlizky, G., Boulton, M. I., Pitaksutheepong, C., Davies, J. W. and Epel, B. L. (2000) Intracellular and intercellular movement of Maize Streak Geminivirus V1 and V2 proteins transiently expressed as green fluorescent protein fusions. Virology 274, 32-38
Kozak, M. (1991) A short leader sequence impairs the fidelity of initiation by eukayotic ribosomes. Gene 1, 111-115
Kumagai, M. H., Donson, J., della-Cioppa, G., Harvey, D., Hanley, K. and Grill, L. K. (1995) Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proceedings of the National Academy of Sciences, USA 92, 1679-1683
Kumar, S., Tamura, K., Jakobsen, I. B. and Nei, M. (2001) MEGA2: Molecular Evolutionary Genetics Analysis software, Arizona State University, Tempe, Arizona, USA.
Kunik, T., Palanichelvam, K., Czosnek, H., Citovsky, V. and Gafni, Y. (1998) Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells. Plant Journal 31, 393±399
Kunkel, L. O. (1947) Variation in phytopathogenic viruses. Annual Review of Microbiology 1, 85–100
Kurath, G., Heick, J. A. and Dodds, J. A. (1993) RNase protection analyses show high genetic diversity among field isolates of satellite tobacco mosaic virus. Virology 194, 414–418
Lacomme, C., Hrubikova, K. and Hein, I. (2003) Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats. The Plant Journal 34, 543–553
Lai, M. M. C. (1995) Recombination and its evolutionary effect on viruses with RNA genomes. In Molecular Basis of Virus Evolution, Edited by A. Gibbs, C. H. Calisher & F. Garcia-Arenal pp. 119-132
Leathers, V., Tanguay, R., Kobayashi, M. and Gallie, D. R. (1993) A phylogenetically conserved sequence within viral 3' untranslated RNA pseudoknots regulates translation. Molecular Cell Biology 13, 5331-5347
Lee, L. and Anderson, E. J. (1998) Nucleotide sequence of a resistance breaking mutant of southern bean mosaic virus. Archives of Virology 143, 2189-2201
Lee, S. K. and Hacker, D. (2001) In vitro analysis of an RNA binding site within the N-terminal 30 amino acids of the Southern cowpea mosaic virus coat protein. Virology 286, 317-327
Leiser, R. M., Ziegler-Graff, V., Reutenauer, A., Herrbach, E., Lemaire, O., Guilley, H., Richards, K. and Jonard, G. (1992) Agroinfection as an alternative to insects for infecting plants with beet western yellows luteovirus. Proceedings of the National Academy of Sciences, USA 89, 9136-9140
Li, H. W., Lucy, A. P., Guo, H. S., Li, W. X., Ji, L. H., Wong, S. M. and Ding, S. W. (1999) Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism. The EMBO Journal 18, 2683–2691
Lim, C. R., Kimata, K., Oka, M., Nomaguchi, K. and Kohno, K. (1995) Thermosensitivity of a green fluorescent protein utilized to reveal novel nuclear-like compartments. Journal of Biochemistry (Tokyo) 118, 13-17
Lin, S.-S., Hou, R. F. and Yeh, S.-D. (2002) Construction of in vitro and in vivo infectious transcripts of a Taiwan strain of Zucchini yellow mosaic virus. Botanical Bulletin of the Academy of Singapore 43, 261-269
Lin, H.-X., Rubio, L., Smythe, A., Jiminez, M. and Falk, B. (2003) Genetic diversity and biological variation among California isolates of Cucumber mosaic virus. Journal of General Virology 84, 249-258
Lindbo, J. A. and Dougherty, W. G. (1992) Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts. Virology 189, 725-733
195
Bibliography
Lindbo, J. A., Silva-Rosales, L., Proebsting, W. M. and Dougherty, W. G. (1993) Induction of highly specific antiviral state in transgenic plants:implications for regulation of gene expression and virus resistance. The Plant Cell 5, 1749-1759
Liu, Y. L., Schiff, M. and Dinesh-Kumar, S. P. (2002) Virus-induced gene silencing in tomato. The Plant Journal 31, 777-786
Liu, S. L., Rodrigo, A. G., Shankarappa, R., Learn, G. H., Hsu, L., Davidov, O., Zhao, L. P. and Mullins, J. I. (1996) HIV quasispecies and resampling. Science 273, 415-416
Lokesh, G. L., Gopinath, K., Satheshkumar, P. S. and Savithri, H. S. (2001) Complete nucleotide sequence of Sesbania mosaic virus: a new virus species of the genus Sobemovirus. Archives of Virology 146, 209-223
Lommel, S. A., Weston-Fina, M., Xiong, Z. G. and Lomonossoff, G. P. (1988) The nucleotide sequence and gene organisation of red clover necrotic mosaic virus RNA-2. Nucleic Acids Research 16, 8587-8602
Lough, T. J., Netzler, N. E., Emerson, S. J., Sutherland, P., Carr, F., Beck, D. L., Lucas, W. J. and Forster, R. L. S. (2000) Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and the first tripple gene block protein. Molecular Plant Microbe Interaction 13, 962-974
Louie, R. (1995) Vascular puncture of maize kernels for the mechanical transmission of maize white line mosaic virus and other viruses of maize. Phytopathology 85, 139-143
Lucas, W. J. (1995) Plasmodesmata: intercellular channels for macromolecular transport in plants. Current Opinion in Cell Biology 7, 673-680
Lütcke, H. A., Chow, K. C., Mickel, F. S., Moss, K. A., Kern, H. F. and Scheele, G. A. (1987) Selection of AUG initiation codons differs in plants and animals. The EMBO Journal 6, 43–48
Mäkeläinen, K., Wahlström, E., Aspegren, K., Teeri, T. and Mäkinen, K. (2003) Role of untranslated region of cocksfoot mottle virus in translation. In "Plant viruses and viroids: interaction with the host", 7th International Congress of Plant Molecular Biology", Barcelona, Spain, June 23-28, 2003. pp 360
Makinen, K., Generozov, E., Arshava, N., Kaloshin, A., Morozov, S. and Zavriev, S. (2000) Detection and characterization of defective interfering RNAs associated with the cocksfoot mottle sobemovirus. Molecular Biology 34, 291-296
Makinen, K., Makelainen, K., Arshava, N., Tamm, T., Merits, A., Truve, E., Zavriev, S. and Saarma, M. (2000) Characterisation of VPg and the polyprotein processing of Cocksfoot mottle virus (genus Sobemovirus) Journal of General Virology 81, 2783-2788
Mäkinen, K., Næss, V., Tamm, T., Truve, E., Aaspöllu, A. and Saarma, M. (1995a) The putative replicase of the Cocksfoot mottle sobemovirus is translated as a part of the polyprotein by -1 ribosomal frameshift. Virology 207, 566-571
Mäkinen, K., Tamm, T., Naess, V., Truve, E., Puurand, U., Munthe, T. and Saarma, M. (1995) Characterization of cocksfoot mottle sobemovirus genomic RNA and sequence comparison with related viruses. Journal of General Virology 76, 2817-2825
Mäkinen, K., Tamm, T., Naess, V., Truve, E., Puurand, U., Munthe, T. and Saarma, M. (1995b) Characterization of cocksfoot mottle sobemovirus genomic RNA and sequence comparison with related viruses. Journal of General Virology 76, 2817-2825
Mang, K.-Q., Ghosh, A. and Kaesberg, P. (1982) A comparative study of the cowpea and bean strains of southern bean mosaic virus. Virology 116, 264-274
Mantis, J. and Tague, B. W. (2000) Comparing the utility of â-glucuronidase and green fluorescent protein for detection of weak promoter activity in Arabidopsis thaliana. Plant Molecular Biology Reporter 18, 319-330 196
Bibliography
Marano, M. R. and Baulcombe, D. (1998) Pathogen-drived resistance targeted against the negative- strand RNA of tobacco mosaic virus: RNA strand-specific gene silencing? The Plant Journal 13, 537– 546
Marathe, R., Anandalakshmi, R., Smith, T. H., Pruss, G. J. and Vance, V. B. (2000) RNA viruses as inducers, suppressors and targets of post- transcriptional gene silencing. Plant Molecular Biology 43, 295-306
Marsh, L. E., Dreher, T. W. and Hall, T. C. (1988) Mutational analysis of the core and modulator sequences of the BMW RNA3 subgenomic promoter. Nucleic Acids Research 16, 981-995
Martell, M., Esteban, J. I., Quer, J., Genesca, J., Weiner, A., Esteban, R., Guardia, J. and Gomez, J. (1992) Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. Journal of Virology 66, 3225-3229
Mas, P. and Beachy, R. (1998) Distribution of TMV movement protein in single living protoplasts immobilized in agarose. The Plant Journal 15, 835-842
Mashall, J. R., Molloy, R., Moss, G. W. J., Hughes, T. E. and Howe, J. R. (1995) The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function. Neuron 14, 211-215
Mastari, J., Lapierre, H. and Dessens, J. T. (1998) Asymmetrical distribution of barley yellow dwarf virus PAV variants between host plant species. Phytopathology 88, 818-821
Matthews, R. E. F. (1991) Plant Virology. Academic Press, San Diego, CA.
McKinney, H. H. (1935) Evidence of virus mutation in the common mosaic of tobacco. Journal of Agricultural Research 51, 951–981
McLean, B. G., Zupan, J. and Zambryski, P. (1995) Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells. The Plant Cell 7, 2101-2114
Meshi, T., Ishikawa, M., Motoyoshi, F., Semba, K. and Okada, Y. (1986) In vitro transciption of infectious RNAs from full-length cDNA of tobacco mosaic virus. Proceedings of the National Academy of Sciences, USA 83, 5043-5047
Metzlaff, M., O'Dell, M., Cluster, P. D. and Flavell, R. B. (1997) RNA-mediated RNA degradation and chalcone synthase A silencing in petunia. Cell 88, 845-854
Miahle, E. and Miller, L. H. (1994) Biolistic techniques for transfection of mosquito embryos (Anopheles gambiae) Bio. Thechniques 16, 924-931
Miller, J. S. and Mayo, M. A. (1991) The location of the 5' end of the potato leafroll luteovirus subgenomic coat protein mRNA. Journal of Virology 72, 2633-2638
Miller, A. W. and Koev, G. (2000) Synthesis of subgenomic RNAs by positive-strand RNA viruses. Virology 273, 1-8
Miller, W. A., Dreher, T. W. and Hall, T. C. (1985) Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-) sense genomic RNA. Nature 313, 68-70
Mohamed, N. A. (1980) Cocksfoot mottle virus in New Zealand. New Zealand Journal of Agricultural Research 23, 273–275
Mohamed, N. A., Mossop, D. W. and Bath, J. E. (1981) Cynosurus and cocksfoot mottle viruses: a comparison. Journal of General Virology 74, 55-63
Morris-Krsinich, B. A. M. and Hull, R. (1981) Translation of turnip rosette virus RNA in rabbit reticulocyte lysates. Virology 114, 98-112
Morris-Krsinich, B. A. M. and Forster, R. L. S. (1983) Lucerne transient streak virus RNA and its translation in rabbit reticulocyte lysate and wheat germ extract. Virology 128, 176-185
197
Bibliography
Mourrain, A., Beclin, C., Elmayan, T., Feuerbach, F., Gordon, C., Morel, J., Jouette, D., Lacombe, A., Nickic, S., Picault, N., Remoue, K., Sanial, M., Vo, T. and Vaucheret, H. (2000) Arabidoposis SGS2 and SGS3 genes are required for post-transcriptional gene silencing and natural virus resistance. Cell 101, 533-542
Murthy, M. R., Bhuvaneswari, M., Subramanya, H. S., Gopinath, K. and Savithri, H. S. (1997) Structure of Sesbania mosaic virus at 3 A resolution. Biophysical Chemistry 68, 33-42
Napoli, C., Lemieux, C. and Jorgensen, R. (1990) Introduction of a chimeric chalcone synthase gene into a petunia results in reversible co-suppression of a homologous gene in trans. The Plant Cell 2, 279- 289
Nei, M. (1987) Molecular Evolutionary Genetics. New York: Columbia University Press 512 pp
Nei, M. and Gojobori, T. (1986) Simple Methods for Estimating the Numbers of Synonymous and Nonsynonymous Nucleotide Substitutions. Molecular Biology and Evolution 3, 418-426
N'Guessan, P., Pinel, A., Sy, A. A., Ghesquiere, A. and Fargette, D. (2001) Distribution, pathogenicity, and interactions of two strains of Rice yellow mottle virus in forested and savanna zones of West Africa. Plant Disease 85, 59-64
Niwa, Y., Hirano, T., Yoshimoto, K., Shimizu, M. and Kobayashi, H. (1999) Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants. The Plant Journal 18, 455-463
Njeru, R. (1996) Subterranean clover mottle virus. PhD Thesis. Murdoch University, Perth,Western Australia.
Njeru, R., Jones, R. A. C., Sivasithamparam, K. and Jones, M. G. K. (1995) Resistance to subterranean clover motle virus in subterranean clover results from restricted cell to cell movement. Australian Journal of Agricultural Research 46, 633-643
Njeru, R., Ferris, D. G., Jones, R. A. C. and Jones, M. G. K. (1997) Studies on seed transmission of subterranean clover mottle virus and its detection in clover seed by ELISA and RT-PCR. Australian Journal of Agricultural Research 48, 343-350
Nurkiyanova, K. M., Ryabov, E. V., Commandeur, U., Duncan, G. H., Canto, T., Gray, S. M., Mayo, M. A. and Taliansky, M. E. (2000) Tagging Potato leafroll virus with the jellyfish green fluorescent protein gene. Journal of General Virology 81, 617-626
Okayama, H. and Berg, P. (1982) High-efficiency cloning of full-length cDNA. Molecular Cell Biology 2, 161-170
Ooi, K., Ohshita, S., Ishii, I. and Yahara, T. (1997) Molecular phyologeny of geminivirus infecting wild plants in Japan. Journal of Plant Research 110, 247–257
Opalka, N., Brugidou, C., Bonneau, C., Nicole, M., Beachy, R. N., Yeager, M. and Fauquet, C. (1998) Movement of rice yellow mottle virus between xylem cells through pit membranes. Proceedings of the National Academy of Sciences, USA 95, 3323-3328
Opalka, N., Tihova, M., Brugidou, C., Kumar, A., Beachy, R. N., Fauquet, C. M. and Yeager, M. (2000) Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction. Journal of Molecular Biology 303, 197-211
Oparka, K. J., Boevink, P. and Santa Cruz, S. (1996) Studying the movement of plant viruses using green flourescent protein. Trends in Biochemichal Science 1, 412-418
Oparka, K. J., Roberts, A. G., Boevink, P., Santa Cruz, S., Roberts, I., Pradel, K. S., Imlau, A., Kotlizky, G., Sauer, N. and Epel, B. (1999) Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves. Cell 97, 743-754
Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y. and Remington, S. J. (1996) Crystal structure of the Aequora victoria green fluorescent protein. Science 273, 1392-1395 198
Bibliography
Osman, T. A. M., Thommes, P. and Buck, K. W. (1993) Localization of a single-stranded RNA- binding domain in the movement protein of red clover mosaic dianthovirus. Virology 74, 2453-2457
Othman, Y. and Hull, R. (1995) Nucleotide sequence of the bean strain of southern bean mosaic virus. Virology 206, 287-297
Oxelfelt, P., Joelson, T., Akerblom, L., Strandberg, B. and Morris, T. J. (1995) Expression of foreign peptides on the surface of tomato bushy stunt virus. Presented at International symposium. Positive Strands RNA Viruses, 4th, Utrecht, the Netherlands
Padgett, H. S., Epel, B., Khan, T. W., Heinlan, M., Watanabe, Y. and Beachy, R. N. (1996) Distribution of tobamovirus movement protein in infected cells and implications for cell-to-cell spread of infection. The Plant Journal 10, 1079-1088
Palauqui, J.-C. and Vaucheret, H. (1998) Transgenes are dispensable for the RNA degradation step of cosuppression. Proceedings of the National Academy of Sciences, USA 95, 9675-9680
Palauqui, J.-C., Elmayan, T., Pollien, J.-M. and Vaucheret, H. (1997) Systemic acquired silencing: transgene specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non- silenced scions. The EMBO Journal 16, 4738-4745
Paliwal, Y. C. (1983) Identification and distribution in eastern Canada of lucerne transient streak virus, a virus newly recognised in North America. Canadian Journal of Plant Pathology 6, 93-97
Paliwal, Y. C. (1984) Interaction of the viroid-like RNA2 of lucerne transient streak virus with southern bean mosaic virus. Canadian Journal of Plant Pathology 6, 93–97
Palmer, K. E. and Rybicki, E. P. (2001) Investigation of potential of Maize streak virus to act as an infectious gene vector in maize plants. Archives of Virology 146, 1089-1104
Pang, S. Z., DeBoer, D. L., Wan, Y., Ye, G., Layton, J. G., Neher, M. K., Armstrong, C. L., Fry, J. E., Hinchee, M. A. W. and Fromm, M. E. (1996) An Improved Green Fluorescent Protein Gene as a Vital Marker in Plants. Plant Physiology 112, 893-900
Peart, J. R., Cook, G., Feys, B. J., Parker, J. E. and Baulcombe, D. C. (2002) An EDS1 orthologue is required for N-mediated resistance against tobacco mosaic virus. Plant Journal 29, 569–579
Peele, C., Jordan, C.V., Muangsan, N., Turnage, M., Egelkrout, E., Eagle, P., Hanley-Bowdoin, L. and Robertson, D. (2001) Silencing of a meristematic gene, Proliferating Cell Nuclear Antigen (PCNA), using geminivirus-derived vectors. The Plant Journal 27, 357-366
Pinel, A., N'Guessan, P., Bousalem, M. and Fargette, D. (2000) Molecular variability of geographically distinct isolates of Rice yellow mottle virus in Africa. Archives of Virology 145, 1621- 1638
Pogliano, K., Harry, E. J. and Losick, R. (1995) Visualizing the subcellular localisation of sporulation proteins in Bacillus subtilis using immunofluorescence microscopy. Molecular Microbiology 18, 459- 470
Prascher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. and Cormier, M. J. (1992) Primary structure of the Aequorea victoria green flourescent protein. Gene 111, 229-233
Presley, J., Zaal, K., Schrorer, T., Cole, N. B. and Lippincott-Schwartz, J. (1997) ER to Golgi transport visualised in living cells. Nature 389, 81-85
Qin, S., Ward, B. M. and Lazarowitz, S. G. (1998) The bipartite gemi-nivirus coat protein aids BR1 function in viral movement by affecting the accumulation of viral single-stranded DNA. .J. Virol. 72, 9247-9256.
Qu, C. X., Liljas, L., Opalka, N., Brugidou, C., Yeager, M., Beachy, R. N., Fauquet, C. M., Johnson, J. E. and Lin, T. W. (2000) 3D domain swapping modulates the stability of members of an icosahedral virus group. Structure 8, 1095-1103
199
Bibliography
Que, Q., Wang, H.-Y., English, J. J. and Jorgensen, R. A. (1997) The frequency and degree of co- suppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence. The Plant Cell 9, 1357- 1368
Ramsdell, D. (1979) Blueberry shoestring virus. CMI/AAB Description of Plant Viruses. No.204. Commonwealth Mycological Institute, Kew, Surrey, and Association of Applied Biologists, Wellesbourne, Warwick, England.
Randles, J. W., Davies, C., Hatta, T., Gould, A. R. and Franck, R. I. B. (1981) Studies on encapsidated viroid-like RNA. I. Characterisation of velvet tobacco mottle virus. Virology 108, 111–122
Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997) A similarity between viral defense and gene silencing in plants. Science 276, 1558-1560
Ratcliff, F. G., MacFarlane, S. A. and Baulcombe, D. C. (1999) Gene silencing without DNA: RNA- mediated cross-protection between viruses. The Plant Cell 11, 1207-1215
Ratcliff, F., Martin-Hernandez, A. M. and Baulcombe, D. C. (2001) Tobacco rattle virus as a vector for analysis of gene function by silencing. The Plant Journal 25, 237-45.
Reutenauer, A., Ziegler-Graff, V., Lot, H., Scheidecker, D., Guilley, H., Richards, K. and Jonard, G. (1993) Identification of beet western yellows luteovirus genes implicated in viral replication and particle morphogenesis. Virology 195, 692-699
Rizzo, T. M. and Palukaitis, P. (1990) Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2 and 3: Generation of infectious transcripts. Molecular General Genetics 222, 249-256
Rizzuto, R., Brini, M., Rossi, R., Heim, R., Tsien, R. Y. and Possan, T. (1996) Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo. Current Biology 6, 183-188
Robbins, J., Dilworth, S. M., Laskey, R. A. and Dingwell, C. (1991) Two independent basic domains in nucleoplasmin nuclear targeting se-quence: Identification of a class of bipartite nuclear t argeting se- quence. . Cell 64, 615±623
Roberts, A. G., Santa Cruz, S., Roberts, I. M., Prior, D. A. M., Turgeon, R. and Oparka, K. J. (1997) Phloem unloading in sink leaves of Nicotiana benthamiana: Comparison of a flourescent solute with a flourescent virus. The Plant Cell 9, 1381-1396
Rodriguez-Cerezo, E., Moya, A. and Garcia -Arenal, F. (1989) Variability and evolution of the plant RNA virus Pepper mild mottle virus. Virology 63, 2198-2203
Rognli, O. A., Aastveit, K. and Munthe, T. (1995) Genetic variation in cocksfoot (Dactylis glomerata L.) populations for mottle virus resistance. Euphytica 83, 109–116
Romano, N. and Macino, G. (1992) Quelling: Transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Molecular Microbiology 6, 3343-3353
Roosien, J. and Van Vloten-Doting, L. (1982) Complementation and interference of ultraviolet-induced Mts mutants of alfalfa mosaic virus. Journal of General Virology 63, 189–98
Roossinck, M. J. (1997) Mechanisms of plant virus evolution. Annual Review of Phytopathology 35, 191-209.
Rossmann, M. G., Abad-Zapatero, C., Hermodson, M. A. and Erickson, J. W. (1983) Subunit interaction in southern bean mosaic virus. Journal of Molecular Biology 166, 37-73
Rovere, C. V., Asurmendi, S. and Hopp, H. E. (2001) Transgenic resistance in potato plants expressing potato leaf roll virus (PLRV) replicase gene sequences is RNA-mediated and suggests the involvement of post-transcriptional gene silencing. Archives of Virology 146, 1337-1353
200
Bibliography
Rubio, L., Soong, J., Kao, J. and Falk, B. W. (1999) Geographic distribution and molecular variation of isolates of three whitefly-borne closteroviruses of cucurbits : Lettuce infectious yellows virus, Cucurbit yellow stunting disorder virus, and Beet pseudo-yellows virus. Phytopathology 89, 707-711
Rubio, L., Abou-Jawdah, Y., Lin, H.-X. and Falk, B. W. (2001) Geographically distant isolates of the crinivirus Cucurbit yellow stunting disorder virus show very low genetic diversity in the coat protein gene. Journal of General Virology 82, 929-933
Ruiz, M. T., Voinnet, O. and Baulcombe, D. C. (1998b) Initiation and maintenance of virus-induced gene silencing. The Plant Cell 10, 937-946.
Ruiz, F., Vayssie, L., Klotz, C., Sperling, L. and Madeddu, L. (1998a) Homology dependent gene silencing in Paramecium. Mol Biol Cell 9, 931-943
Rutgers, T., Salerno-Rife, T. and Kaesberg, P. (1980) Messenger RNA for the coat protein of southern bean mosaic virus. Virology 104, 506-509
Ryabov, E. V., Krutov, A. A., Novikov, V. K., Zheleznikova, O. V., Morozov, S. Y. and Zavriev, S. K. (1996) Nucleotide sequence of RNA from the sobemovirus found in infected cocksfoot shows a luteovirus-like arrangement of the putative replicase and protease genes. Phytopathology 86, 391-397
Salerno-Rife, T., Rutgers, T. and Kaesberg, P. (1980) Translation of southern bean mosaic virus RNA in wheat embryo and rabbit reticulocyte extracts. Journal of Virology 34, 51-58
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) 'Molecular Cloning, A Laboratory Manual (2nd Edition)'. Cold Spring Harbor Laboratory Press, New York
Sanchez-Navarro, J., Miglino, R., Ragozzino, A. and Bol, J. F. (2001) Engineering of alfalfa mosaic virus RNA3 into an expression vector. Archives of Virology 146, 923-939
Sandford, J., Smith, F. and Russell, J. A. (1993) Optimizing the biolistic process for different biological applications. Methods in Enzymology 217, 483-509
Sandford, J., Klein, T. M., Wolf, E. and Allen, N. (1987) Delivery of substances into cells and tissues using a particle bombardment process. Particulate Science and Technology 5, 27-37
Santa Cruz, S., Roberts, A. G., Prior, D. A. M., Chapman, S. and Oparka, K. J. (1998) Cell-to-cell and phloem-mediated transport of potato virus X: The role of virions. The Plant Cell 10, 495-510
Santa Cruz, S., Chapman, S., Roberts, A. G., Roberts, I. M., Prior, D. A. M. and Oparka, K. (1996) Assembly and movement of a plant virus carrying a green fluorescent protein overcoat. Proceedings of National Academy of Sciences, USA 93, 6286-6290
Sanz, A. I., Fraile, A., Gallego, J. M., Malpica, J. M. and Garcia-Arenal, F. (1999) Genetic variability of natural populations of cotton leaf curl geminivirus, a single-stranded DNA virus. Journal of Molecular Evolution 49, 671–681
Sarmiento, C., Hiriart, J.-B., Meier, M., Lehto, K. and Truve, E. (2003) Gene silencing of chlh gene is differently suppressed by CfMV P1 and PVA P1/Hc-Pro. In: Gene Silencing and Epigenetics. 7th International Congress of Plant Molecular Biology, Barcelona, Spain, June 23-28, 2003. pp 403
Sarnow, P. (1995) Cap-independent translation. In "Current Topics in Microbiology and Immunology". Vol. 203, Springer, Berlin.
Sawicki, S. G. and Sawicki, D. L. (1998) A new model for coronavirus transcription. Advances in Experimental Medical Biology 440, 215-219
Schenk, P. M., Elliot, A. R. and Manners, J. M. (1998) Assessment of Transient Gene Expression in Plant Tissues Using the Green Fluorescent Protein as a. Plant Molecular Biology Reporter 16:, 313–322
Schmid, A., Cattaned, R. and Billeter, M. A. (1987) A procedure for selective full-length cDNA cloning of specific RNA species. Nucleic Acids Research 15, 3987-3996
201
Bibliography
Schmidt, A., Palumbo, G., Bozzetti, M., Tritto, P., Pimpinelli, S. and Schafer, U. (1999) Genetic and molecular characterisation of sting, a gene involved in crystal formation and meiotic drive in the male germline of Drosophila melanogaster. Genetics 151, 749-760
Schneider, W. L. and Roossinck, M. J. (2000) Evolutionarily related Sindbis-like plant viruses maintain different levels of population diversity in a common host. Journal of Virology 74, 3130–3134
Scholthof, H. B. (1999) Rapid delivery of foreign genes into plants by direct rub-inoculation with intact plasmid DNA of a tomato bushy stunt virus gene vector. Journal of Virology 73, 7823-7829
Scholthof, H. B., Morris, T. J. and Jackson, A. O. (1993) The capsid protein gene of tomato bushy stunt virus is dispensable for systemic movement and can be replaced for localized expression of foreign genes. Mol.Plant-Microbe Interact 6, 309-322
Scholthof, H. B., Scholthof, K. B. and Jackson, A. O. (1995) Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector. The Plant Cell 7, 1157-1172
Scholthof, H. B., Scholthof, K. B. and Jackson, A. O. (1996) Plant virus gene vectors for transient expression of foreign proteins in plants. Annual Review of Phytopathology 34, 299-323
Schweizer, P., Pokorny, J., Abderhalden, O. and Dudler, R. (1999) A transient assay system for the functional assessment of defense-related genes in wheat. Molecular Plant Microbe Interaction 12, 647- 654
Sehgal, O. P. (1999) Sobemoviruses. In "Encyclopedia of Virology, 2nd ed", (Eds. A. Granoff and R. G. Webster) pp. 1674-1680
Sehgal, O. P., Sinha, R. C., Gellatly, D. L., Ivanov, I. and AbouHaidar, M. G. (1993) Replication and encapsidation of the viroid-like satellite RNA of lucerne transient streak virus are supported in divergent hosts by cocksfoot mottle virus and turnip rosette virus. Journal of General Virology 74, 785–788
Sharp, P. A. (2001) RNA interference. Genes and Development 15, 485-490
Sheen, J., Hwang, S. B., Y, N., Kobayashi, H. and Galbraith, D. W. (1995) Green fluorescent protein as a new vital marker in plant-cells. Plant Journal 8, 777-784
Shen, W.-H. and Hohn, B. (1995) Vectors based on maize streak virus can replicate to high copy numbers in maize plants. Journal of General Virology 76, 965-969
Shepherd, R. J. (1989) Biochemistry of DNA plant viruses. In "The Biochemistry of Plants", (Ed. A. Marcus), New York: Academic. 15, 563-616
Shigeru, T. and Kimura, M. (1997) Low-voltage electric discharge biolistic device. Bio/Techniques 23, 650-652
Shimomura, O., Johnson, F. H. and Saiga, Y. (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. Journal of Cellular and Comparative Physiology 59, 223-239
Shivprasad, S., Pogue, G. P., Lewandowski, D. J., Hidalgo, J., Donson, J., Grill, L. K. and Dawson, W. O. (1999) Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus- based vectors. Virology 255, 312-323
Siegel, A. (1985) Plant-virus-based vectors for gene transfer may be of considerable use despite a presumed high error frequency during RNA synthesis. Plant Molecular Biology 4, 327-329
Siegel, R. W., Adkins, S. and Cao, C. C. (1997) Sequence specific recognition of a subgenomic RNA promoter by a viral RNA polymerase. Proc Natl Acad Sci U S A 94, 11238-11243
Siemering, K. R., Golbik, R., Server, R. and Haseloff, J. (1996) Mutations that suppress the thermosensitivity of green fluorescent protein. Current Biology 6, 1653-1663
202
Bibliography
Sijen, T., Wellink, J., Hiriart, J.-B. and Van Kammen, A. (1996) RNA-mediated virus resistance: Role of repeated transgenes and delineation of targeted regions. The Plant Cell 8, 2277–2294
Simon, A. E. and Bujarski, J. J. (1994) RNA-RNA recombination and evolution in virus-infected plants. Annual Review of Phytopathology 32, 337-362
Sit, T. L., Vaewhongs, A. A. and Lommel, S. A. (1998) RNA-mediated transactivation of transcription from a viral RNA. Science 281, 829-832
Sivakumaran, K. and Hacker, D. L. (1998) The 105-KDa polyprotein of Southern bean mosaic virus is translated by scanning ribosomes. Virology 246, 34-44
Sivakumaran, K., Fowler, B. C. and Hacker, D. L. (1998) Identification of viral genes required for cell-to-cell movement of southern bean mosaic virus. Virology 252, 376-386
Smardon, A., Spoerke, J. M., Stacey, S. C., Klein, M. E., Mackin, N. and Maine, E. M. (2000) EGO- 1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interferece in C. elegans. Current Biology 10, 169-178
Smirnyagina, E., Hsu, Y. H., Chua, N. and Ahlquist, P. (1994) Second-site mutations in the brome mosaic virus RNA3 intercistronic region partially suppress a defect in coat protein mRNA transcription. Virology 198, 427-436
Solovyev, A. G., Stroganova, T. A., Zamyatnin, A. A., Jr., Fedorkin, O. N., schiemann, J. and Morozov, S. Y. (2000) Subcellular sorting of small membrane-associated tripple gene blocck proteins: TGBp3-assisted targeting of TGBp2. Virology 269, 113-127
Songstad, D. D., Somers, D. A. and Griesbach, R. J. (1995) Advances in alternative DNA delivery techniques. Plant Cell, Tissue and Organ Culture 40, 1-15
Subramanya, H., Gopinath, K., Nayudu, M., Savithri, H. and Murthy, M. (1993) Structure of Sesbania mosaic virus at 4.7 A resolution and partial sequence of the coat protein. Journal of Molecular Biology 229, 20-25
Sudarshana, M. R., Wang, H. L., Lucas, W. J. and Gilbertson, R. L. (1998) Dynamics of bean dwarf mosaic geminivirus cell-to-cell and long-distance movement in phaseolus vulgaris revealed using the green flourescent protein. Molecular Plant-Microbe Interaction 11, 277-291
Sugiyama, Y., Hamamoto, H., Takemoto, S., Watanabe, Y. and Okada, Y. (1995) Systemic production of foreign peptides on the particle surface of tobacco mosaic virus. FEBS Lett 359, 247-250
Szecsi, J., Ding, X., Lim, C. O., Bendahmane, M., Cho, M. J., Nelson, R. S. and Beachy, R. N. (1999) Development of tobacco mosaic virus infection sites in Nicotiana benthamiana. Molecular Plant- Microbe Interaction 12, 143-152
Tabara, H., Sarkissian, M., Kelley, W. G., Fleenor, J., Grishok, A., Tammons, L., Fire, A. and Mello, C. C. (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123-132
Takamatsu, N., M, I., T, M. and Okada, Y. (1987) Expression of bacterial chlormphenicol acetyltransferase gene in tobacco plant mediated by TMV-RNA. EMBO J 6, 307-311
Tamm, T. and Truve, E. (2000) RNA-binding activities of Cocksfoot mottle sobemovirus proteins. Virus Research 66, 197-207
Tamm, T., Makinen, K. and Truve, E. (1999) Identification of genes encoding for the Cocksfoot mottle virus proteins. Archives of Virology 144, 1557-1567
Tanzer, M. M., Thompson, W. F., Law, M. D., Wernsman, E. A. and Uknes, S. (1997) Characterization of post-transcriptionally suppressed transgene expression that confers resistance to tobacco etch virus infection in tobacco. The Plant Cell 9, 1411–1423
203
Bibliography
Tarun, S. Z. and Sachs, A. B. (1995) A common function for mRNA 5' cap and 3' ends in translation initiation in yeast. Genes and Development 9, 2997-3007
Tavernarakis, N., Wang, S. L., Dorovkov, M., Ryazanov, A. and Driscoll, M. (2000) Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genetics 24, 180- 183
Tenllado, F., Garcia-Luque, I., Serra, M. T. and Diaz-Ruiz, J. R. (1995) Nicotiana benthamiana plants transformed with the 54 kDa region of the pepper mild mottle tobamovirus replicase gene exhibit two types of resistance responses against viral infection. Virology 211, 170-83
Thomas, C. L., Jones, L., Baulcombe, D. C. and Maule, A. J. (2001b) Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector. The Plant Journal 25, 417-25.
Thomas, J.-L., Bardou, J., L'hoste, S., Mauchamp, B. and Chavancy, G. (2001a) A helium burst biolistic device adapted to penetrate fragile insect tissues. Journal of Insect Science 19, 1-10
Tien-Po, Davies, C., Hatta, T. and Francki, R. I. B. (1981) Viroid-like RNA encapsidated in Lucerne transient streak virus. FEBS Letter 132, 353–356
Tomlinson, J. A. and Ward, C. M. (1978) The reactions of swede (Brassica napus) to infection by Turnip mosaic virus. Annals of Applied Biology 89, 61-69
Toth, R. L., Chapman, S., Carr, F. and Santa Cruz, S. (2001) A novel strategy for the expression of foreign genes from plant virus vectors. FEBS Letter 489, 215-9.
Tremaine, J. G. and Hamilton, R. I. (1983) Southern bean mosaic virus. CMI/AAB Descriptions of Plant Viruses. No. 274. Commonwealth Mycological Institute, Kew, Surrey, and Association of Applied Biologists, Wellesbourne, Warwick, England.
Tsien, R. Y. (1998) The green flourescent protein. Annual Review of Biochemistry 67, 509-544
Turnage, M. A., Muangsan, N., Peele, C. G. and Robertson, D. (2002) Geminivirus-based vectors for gene silencing in Arabidopsis. The Plant Journal 30, 107-114
Turpen, T. H., Reinl, S. J., Charoenvit, T., Fallarme, V. and Grill, L. K. (1995) Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Bio-Technology 13, 366-374
Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P. and Sharp, P. A. (1999) Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13, 3191-3197
Unseld, S., Hohnle, M., Ringel, M. and Frischmuth, T. (2001) Subcellular Targeting of the Coat Protein of African Cassava Mosaic Geminivirus. Virology 286, 373-383 van Blokland, R., Van der Geest, N., Mol, J. N. M. and Kooter, J. M. (1994) Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover. The Plant Journal 6, 861-877 van Der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. M. and Stuitje, A. R. (1990) Flavinoid genes in petunia:addition of a limited number of genes copies may lead to a suppression of gene expression. The Plant Cell 2, 291-299 van der Wilk, F., Verbeek, M., Dullemans, A. M. and van den Heuvel, J. F. J. M. (1997) The genome-linked protein of potato leafroll virus is located downstream of the putative protease domain of the ORF1 product. Virology 234, 300-303 van der Wilk, F., Verbeek, M., Dullemans, A. and van den Heuvel, J. F. J. M. (1998) The genome- linked protein (VPg) of southern bean mosaic virus is encoded by the ORF2. Virus Genes 17, 21-24 van der Wilk, F., Huisman, M. J., Cornelissen, B. J. C., Huttinga, H. and Goldbach, R. (1989) Nucleotide sequence and organisation of potato leafroll virus genomic RNA. FEBS Lett 245, 51-56
204
Bibliography van Eldik, G. J., Litiere, K., Jacobs, J. J., Van Montagu, M. and Cornellissen, M. (1998) Silencing of beta-1,3-glucanase genes in tobacco correlates with an increased abundance of RNA degradation intermediates. Nucleic Acids Research 26, 5176-5181
Van Vloten-Doting, L. and Bol, J. F. (1988) Variability, mutant selection, and mutant stability in plant RNA viruses In: RNA Genetics, 3:37–52 Boca Raton, FL: CRC Press. 260 pp. ed. E. Domingo, J. J. Holland, P, Ahlquist.
Van Vloten-Doting, L., Bol, J. and Cornelissen, B. (1985) Plant virus-based vectors for gene transfer will be of limited use because of the high error frequency during viral RNA synthesis. Plant Molecular Biology 4, 323-326
Vaucheret, H., Nussaume, L., Palauqui, J.-C., Quilleré, I. and Elmayan, T. (1997) A transcriptionally active state is required for post-transcriptional silencing (co-suppression) of nitrate reductase host genes and transgenes. The Plant Cell 9, 1495-1504
Veidt, I., Lot, H., Leiser, M., Scheidecker, D., Guilley, H., Richards, K. and Jonard, G. (1988) Nucleotide sequence of beet western yellows virus RNA. Nucleic Acids Research 16, 9917-9932
Verver, J., Van Lent, J., Gopinath, K. and Van Kammen, A. (1998) Studies on the movement of cowpea mosaic virus using jellyfish green fluorescent protein. Virology 242, 22-27
Vives, M. C., Rubio, L., Galipienso, L., Navarro, L., Moreno, P. and Guerri, J. (2002) Low genetict variation between isolates of Citrus leaf blotch virus from different host species and of different geographical origins. Journal of General Virology 83, 2587-2591
Voinnet, O. (2001) RNA silencing as a plant immune system against viruses. Trends in Genetics 17, 449-459
Voinnet, O. and Baulcombe, D. C. (1997) Systemic signaling in gene silencing. Nature 389, 553
Voinnet, O., Pinto, Y. M. and Baulcombe, D. C. (1999) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proceedings of the National Academy of Sciences, USA 96, 14147–14152
Voinnet, O., Lederer, C. and Baulcombe, D. C. (2000) A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157–167
Voinnet, O., Vain, P., Angell, S. M. and Baulcombe, D. C. (1998) Systemic spread of sequence- specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA. Cell 95, 177-187 von Arnim, A. G., Deng, X.-W. and Stacey, M. G. (1998) Cloning vectors for the expression of green flouorescent protein fusion proteins in transgenic plants. Gene 221, 35-43
Vongs, A., Kakutani, T., Martienssen, R. and Richards, E. J. (1993) Arabidopsis thaliana DNA methylation mutants. Science 260, 1926-1928
Wachter, R. M., King, B. A., Heim, R., Kallio, K., Tsien, R. Y., Boxer, S. G. and Remington, S. J. (1997) Crystal structure and photodynamic behaviour of the blue-emission variant Y66H/YI45F of green fluorescent protein. Biochemistry 36, 9759-9765
Waigmann, E., Chen, M. H., Bachmaier, R., Ghoshroy, S. and Citovsky, V. (2000) Regulation of plasmodesmatal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein. The EMBO Journal 19, 4875 –4884
Waldo, G. S., Standish, B.M., Berendezen, J. and Terwilliger, T.C. (1999) Rapid protein-folding assay using green fluorescent protein. Nature Biotechnology 17, 691-695
Wang, J., Carpenter, C. D. and Simon, A. E. (1999) Minimal sequence and structural requirements of a subgenomic RNA promoter for turnip crinkle virus. Virology 253, 327-336
205
Bibliography
Ward, W. W. and Cormier, M. J. (1979) An energy transfer protein in coelenterate bioluminescence: Characterisation of the Renilla green-fluorescent protein. Journal of Biological Chemistry 254, 781-788
Ward, W. W. and Bokman, S. H. (1992) Reversible denaturation of Aequorea green-fluorescent protein:physical separation and characterisation of the renatured protein. Biochemistry 21, 4535-4540
Ward, A., Etessami, P. and Stanley, J. (1988) Expression of a bacterial gene in plants mediated by infectious geminivirus DNA. The EMBO Journal 7, 1583-1587
Ward, W. W., Cody, C. W., Hart, R. C. and Cormier, M. J. (1980) Spectrophotomeric identity of the energy transfer chromophores in Renilla and Aequorea green fluorescent proteins. Photochemistry and Photobiology 31, 611-615
Ward, W. W., Prentice, H. J., Roth, A. F., Cody, C. W. and Reeves, S. C. (1982) Spectral perturbations of the Aequorea green-fluorescent protein. Photochemistry and Photobiology 35, 803-808
Wassenegger, M., Heimes, S., Riedel, L. and Sänger, H. L. (1994) RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567-576
Waterhouse, P. M., Graham, M. W. and Wang, M.-B. (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proceedings of the National Academy of Sciences, USA 95, 13959 -13964
Waterhouse, P. M., Smith, N. A. and Wang, M.-B. (1999) Virus resistance and gene silencing: killing the messenger. Trends in Plant Science 4, 452-457
Waterhouse, P. M., Wang, M. B. and Lough, T. (2001) Gene silencing as an adaptive defence against viruses. Nature 411, 834-842
Wayne, R. (1992) Viruses contribute to clover decline. Rural Research 155, 4-10
Weber, K. A. and Sehgal, O. P. (1982) Subgenomic RNAs in virions of southern bean mosaic virus. Phytopathology 72, 909–913
Weber, H., Haeckel, P. and Pfitzner, A., J, P. (1992) A cDNA clone of tomato mosaic virus is infectious in plants. Journal of Virology 66, 3909-3912
Whittaker, G. R. and Helenius, A. (1998) Nuclear Import and Export of Viruses and Virus Genomes. Virology 246, 1-23
Wiemer, E. A., Wenzel, T., Deerinck, T. J., Ellisman, M. H. and Subramani, S. (1997) Visualisation of the peroxisomal compartment in living mammalian cells: dynamic behavavior and association with microtubules. Journal of Cell Biology 136, 71-80
Williamson, S. (2000) Molecular characterisation of Subterranean clover mottle virus. Honours Thesis, Murdoch University, Perth, Western Australia.
Wilson, T. M. (1985) Nucleocapsid disassembly and early gene expression by positive-strand RNA viruses. Journal of General Virology 66, 1201–1207
Wobus, C. E., Skaf, J. S., Schultz, M. H. and de Zoeten, G. A. (1998) Sequencing, genomic localization and initial characterization of the VPg of pea enation mosaic enamovirus. Journal of General Virology 79, 2023-2025
Wroth, J. M. and Jones, R. A. C. (1992a) Subterranean clover mottle sobemovirus: its host range, resistance in subterranean clover and transmission through seed and by grazing animals. Annals of Applied Biology 121, 329-343
Wroth, J. M. and Jones, R. A. C. (1992b) Subterranean clover mottle virus infection of subterranean clover: widespread occurrence in pastures and effects on productivity. Australian Journal of Agricultural Research 43, 1597-1608
206
Bibliography
Wu, S., Rinehart, C. A. and Kaesberg, P. (1987) Sequence and organization of southern bean mosaic virus genomic RNA. Virology 161, 73-80
Wu, M., Shang, H.-S., Yeong, C.-H., Tan, P. H.-N., Lu, Z.-H. and Wong, S.-K. (1995) A novel method employing polymerase chain reaction to disrupt genes lacking convenient restriction enzyme sites in yeast. Molecular Biotechnology 3, 72-74
Wu-Scharf, D., Jeong, B., Zhang, C. and Cerruti, H. (2000) Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-Box RNA Helicase. Science 290, 1159-1162
Wylie, S. J., Kueh, J., Welsh, B., Smith, L. J., Jones, M. G. K. and Jones, R. A. C. (2002) A non- aphid-transmissible isolate of bean yellow mosaic potyvirus has an altered NAG motif in its coat protein. Archives of Virology 147, 1813–1820
Wymer, C. L., Fernandes-Abalos, J. M. and Doonan, J. H. (2001) Microinjection reveals cells-to-cell movement of green fluorescent protein in cells of maize coleoptiles. Planta 212, 692-695
Xiong, Z. G. and Lommel, S. A. (1989) The complete nucleotide sequence and genome organisation of red clover necrotic mosaic virus RNA-1. Virology 171, 543-554
Yamaya, Y., Yoshioka, M., Meshi, T., Okada, Y. and Ohno, T. (1988) Expression of tobacco mosaic virus RNA in transgenic plants. Molecular General Genetics 211, 520-525
Yarwood, C. E. and . (1979) Host passage effects with plant viruses. Advances Virus Research 24, 69– 90
Yassi, M. N., Ritzenthaler, C., Brugidou, C., Fauquet, C. and Beachy, R. N. (1994) Nucleotide sequence and genome characterization of rice yellow mottle virus RNA. Journal of General Virology 75, 249-57
Yokoe, H. and Meyer, T. (1996) Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement. Nature Biotechnology 14, 1252-1256
Zakia, A., Fadhila, A., Agnes, P., Oumar, T., Placide, N. G., Jean-Loup, N., Frances, K., Konate´, G. and Denis, F. (2003) Phylogeography of Rice yellow mottle virus in Africa. Journal of General Virology 84, 733–743
Zamore, P., Tuschl, T., Sharp, P. and Bartel, D. (2000) RNAi:double-stranded RNA directs the ATP- dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33
Zavriev, S. K., Hickey, C. M. and Lommel, S. A. (1996) Mapping of the red clover necrotic mosaic virus subgenomic RNA. Virology 216, 407-410
Zhang, J. and Ma, Y. (2001) Evidence for retroviral intramolecular recombinations. Journal of Virology 75, 6348-58.
Zou, C., Zhang, Z., Wu, S. and Osterman, J. (1998) Molecular cloning and characterisation of a rabbit eIF2C protein. Gene 211, 187-194
207