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Improving the Effectiveness and Delivery of Gene Silencing Triggers to Control Plant Parasitic Nematodes

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Improving the Effectiveness and Delivery of Gene Silencing Triggers to Control Plant Parasitic Nematodes Improving the Effectiveness and Delivery of Gene Silencing Triggers to Control Plant Parasitic Nematodes This thesis is presented to Murdoch University for the degree of Doctor of Philosophy Fareeha Naz (M. Phil) Western Australian State Agricultural Biotechnology Centre (SABC), School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia. 2017 Declaration I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution. I Abstract Plant parasitic nematodes (PPNs) are a major group of crop pests that decrease yields by 7-15%, and locally up to 50% or more. The most economically important groups are the sedentary endoparasites which invade roots and form distinct feeding sites, and the migratory endoparasites which feed from individual cells. Gene silencing through RNA interference (RNAi) could be applied to control a range of plant pests. The overall aim of this study was to try to enhance the effectiveness of RNAi to control PPNs. The research included: targeting multiple genes simultaneously in the sedentary endoparasitic cyst-nematode H. schachtii, investigating the role of splice-leaders as RNAi targets for PPNs, and seeking alternative modes of delivery of double-stranded RNA (dsRNA) (e.g. by spraying) to bypass the need to generate transgenic plants. This could be applied when transgenic technology is not acceptable or financially viable. After H. schachtii had been soaked in dsRNA of different combinations of two different genes (vha-3-vha-8, vha-3-pat-10, and vha3-prp-21), there was around 9-31% greater reduction in their growth and development compared to soaking in mixtures of single genes (vha-3 & vha- 8; vha-3 & pat-10; vha-3 & prp-21). Similarly, in ‘Host-Induced Gene Silencing ‘(HIGS), transgenic A. thaliana plants expressing dsRNA of H. schachtii combinations of vha-3-vha-8, exhibited up to >80% reduction in the development of nematodes compared to transgenic plants expressing genes singly (vha-3: up to ~70% and vha-8: up to ~50%). Splice-leader (SL) sequences were identified for the PPNs P. thornei and H. schachtii. Soaking of mixed stages of P. thornei in SL siRNA significantly reduced penetration of nematodes into wheat roots compared to untreated nematodes. In contrast, J2s of H. schachtii soaked in SL siRNA did not significantly affect their development on A. thaliana roots. II Ectopic delivery of fluorescent oligonucleotides (with a phosphorothioate morpholino oligomer (PMO) backbone) to A. thaliana demonstrated systemic movement, but delivery of naked dsRNA did not appear to initiate endogenous gene silencing. The topically applied nematode dsRNA (dsvha-3) on A. thaliana leaves did not reduce the nematode development on the roots as compared to the controls (dsgfp and no dsRNA). III List of Conferences and Publications John Fosu-Nyarko, Paul Nicol, Fareeha Naz, Reetinder Gill, Michael G. K. Jones. (2016) Analysis of the Transcriptome of the Infective Stage of the Beet Cyst Nematode, H. schachtii. PLosOne 11(1):e0147511 Fareeha Naz, John Fosu-Nyarko and Michael G. K. Jones. (2016) Improving the effectiveness and delivery of gene silencing triggers to control plant nematode pests. Page no. 69. 32nd International Symposium of the European society of nematologists. Braga, Portugal. Fareeha Naz, John Fosu-Nyarko and Michael G. K. Jones. (2015) Application of functional nucleic acids to control plant nematode pests. Functional Nucleic Acids Symposium. Perth, Western Australia. Fareeha Naz, John Fosu-Nyarko and Michael G. K. Jones. (2015) Can we improve the effectiveness and delivery of gene silencing triggers to control plant nematode pests? Royal Society of Western Australia. Perth, Western Australia. IV Table of Contents Declaration ............................................................................................................................................... I Abstract .................................................................................................................................................. II List of Conferences and Publications ....................................................................................................... IV Table of Contents .................................................................................................................................... V Abbreviations .......................................................................................................................................... X Acknowledgement................................................................................................................................. XII 1. Introduction and Literature Review ...................................................................................................... I 1.0 General Introduction.................................................................................................................... 2 1.1 Nematode Biology ............................................................................................................................. 6 1.2 Free-living nematodes ....................................................................................................................... 7 1.3 Parasitic nematodes .......................................................................................................................... 7 1.3.1 Plant parasitic nematodes: the underground enemies of plants .................................................. 8 1.3.1.1 Root-knot nematodes ....................................................................................................... 8 1.3.1.2 Cyst nematodes.............................................................................................................. 10 1.3.1.3 Root lesion nematodes .................................................................................................. 12 1.4 Methods to control PPNs ................................................................................................................. 13 1.5 RNA interference ............................................................................................................................. 14 1.5.1 Mechanism of RNAi in nematodes ............................................................................................ 16 1.5.1.1 DICER mediated cleavage of dsRNA ........................................................................... 17 1.5.1.2 Targeted mRNA degradation through RISC ................................................................ 18 1.5.1.3 Amplification and systemic spread of RNAi effect ....................................................... 18 1.6 In vitro RNAi in PPNs (endoparasitic nematodes) ............................................................................. 21 1.7 Host-induced RNAi in PPNs (endoparasitic nematodes) ................................................................... 25 1.8 Multiple genes silencing .................................................................................................................. 30 1.8.1 Multiple genes silencing in C. elegans ....................................................................................... 30 V 1.8.2 Multiple genes silencing in PPNs ............................................................................................... 33 1.9 Splice leader: a possible target for effective RNAi in PPNs ................................................................ 35 1.9.1 Mechanism of trans-splicing...................................................................................................... 36 1.9.2 SL-trans-splicing in nematodes .................................................................................................. 37 1.10 A novel strategy for plant-mediated RNAi ...................................................................................... 39 1.10.1 Use of carrier peptides and nanoparticles for ectopic delivery of dsRNA to plants ................... 41 1.10.2 Use of Chitosan nanoparticles ................................................................................................. 41 1.10.3 Use of cationic fluorescent nanoparticles ................................................................................ 42 1.10.4 Use of Bioclay nanoparticles ................................................................................................... 43 1.11 Aims and objectives of the research ............................................................................................... 44 2. Materials and Methods...................................................................................................................... 45 2.0 Nematode cultures, plant materials and plant growth ..................................................................... 46 2.1 Nematode extraction ....................................................................................................................... 46 2.2 Extraction of total RNA from nematodes and cDNA synthesis .......................................................... 47 2.3 Quick extraction of genomic DNA from Arabidopsis ......................................................................... 48 2.4 Primer designing .............................................................................................................................. 49 2.5 Polymerase Chain Reactions (PCRs) ................................................................................................
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