Evaluation of Approaches for Enhanced Resistance in Against Phloem Limited Pests

Shaista Javaid

2017

Department of Biotechnology Pakistan Institute of Engineering and Applied Sciences Nilore, Islamabad, Pakistan

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Student’s Name: Shaista Javaid Department: Biotechnology Registration Number: 10-7-1-044-2009 Date of Registration: 20-03-2009 Thesis Title:

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Thesis Submission Approval

This is to certify that the work contained in this thesis entitled Evaluation of Approaches for Enhanced Resistance in Plants Against Phloem Limited Pests was carried out by Shaista Javaid and in my opinion, it is fully adequate, in scope and quality for the degree of PhD. Furthermore, it is hereby approved for submission for review and thesis defense.

Supervisor: ______

Name: Dr. Shahid Mansoor (SI) Date: June 30, 2017 Place: NIBGE, Faisalabad.

Head, Department of Biotechnology: ______

Name: Dr. Shahid Mansoor (SI) Date: June 30, 2017 Place: NIBGE, Faisalabad.

Evaluation of Approaches for Enhanced Resistance in Plants Against Phloem Limited Pests

Shaista Javaid

Submitted in partial fulfillment of the requirements for the degree of PhD.

2017

Department of Biotechnology Pakistan Institute of Engineering and Applied Sciences Nilore, Islamabad, Pakistan

Dedications

I dedicate all of my work to my beloved Mother and lovely Father. They gave me the confidence to do this job and their prayers make me able to complete the task.

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Declaration of Originality

I hereby declare that the work contained in this thesis and the intellectual content of this thesis are the product of my own work. This thesis has not been previously published in any form nor does it contain any verbatim of the published resources which could be treated as infringement of the international copyright law. I also declare that I do understand the terms ‘copyright’ and ‘plagiarism,’ and that in case of any copyright violation or plagiarism found in this work, I will be held fully responsible of the consequences of any such violation.

______(Shaista Javaid)

June 30, 2017 NIBGE, Faisalabad.

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Copyrights Statement

The entire contents of this thesis entitled Evaluation of Approaches for Enhanced Resistance in Plants Against Phloem Limited Pests by Shaista Javaid are an intellectual property of Pakistan Institute of Engineering and Applied Sciences (PIEAS). No portion of the thesis should be reproduced without obtaining explicit permission from PIEAS.

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Table of Contents

Declaration of Originality ...... ii Copyrights Statement ...... iv Table of Contents ...... v List of Figures ...... viii List of Tables ...... x Acknowledgement ...... xi Abstract ...... xiii List of Publications and Patents ...... xiv List of Abbreviations and Symbols...... xv 1 General Introduction ...... 1 1.1 Insecticidal proteins from ...... 1 1.1.1 Limitations of Bt proteins ...... 2 1.2 Other insecticidal proteins and their activity ...... 4 1.2.1 Hvt ...... 4 1.2.2 Lectins ...... 6 1.3 Fusion proteins ...... 14 1.4 Tissue specific expression ...... 15 1.5 Expression of insecticidal proteins in plants ...... 17 1.5.1 Transient expression ...... 17 1.5.2 Stable expression of insecticidal proteins in transgenic plants ...... 19 1.6 Need for the project and objectives ...... 20 2 General Methodology ...... 21 2.1 Selection of promoter ...... 21 2.2 Isolation of insecticidal ...... 21 2.3 Polymerase chain reaction (PCR) ...... 21 2.4 Cloning of PCR-amplified product ...... 21 2.5 Ligation reaction ...... 22 2.6 Preparation of competent cells ...... 22 2.6.1 Preparation of heat shock E. coli competent cells ...... 22 2.6.2 Preparation of electro-competent Agrobacterium tumefaciens cells ...... 22 2.7 Transformation in bacterium ...... 23

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2.7.1 Transformation in E. coli ...... 23 2.7.2 Transformation in Agrobacterium cells ...... 23 2.8 Plasmid isolation ...... 23 2.9 Restriction analysis ...... 24 2.10 DNA analysis techniques ...... 24 2.10.1 Agarose gel electrophoresis ...... 24 2.10.2 Phenol-chloroform precipitation ...... 25 2.11 transformation ...... 25 2.12.1 Shifting in soil ...... 28 2.13 Confirmation of transgenic plants ...... 28 2.13.1 DNA extraction...... 28 2.13.2 RNA extraction ...... 29 2.13.3 cDNA Synthesis ...... 29 2.14 Insect rearing ...... 30 2.14.1 Insect bioassays ...... 30 3 Promoter Characterization ...... 31 3.1 Introduction ...... 31 3.2 Methodology ...... 33 3.2.1 Isolation and cloning of promoters ...... 33 3.2.2 Plant infiltration ...... 35 3.2.3 Histochemical staining assay ...... 35 3.4 Results ...... 35 3.4.1 Promoters analysis ...... 36 3.4.2 Promoter cloning and GUS-assay...... 36 3.5 Discussion ...... 41 4 Expression of Insecticidal Genes under Phloem specific Promoter ...... 43 4.1 Introduction ...... 43 4.2 Methodology ...... 46 4.2.1 Insecticidal gene constructs ...... 46 4.3 Plant transformation ...... 46 4.4 Transgene Analysis ...... 47 4.4.1 DNA extraction...... 47 4.4.2 RNA extraction ...... 47 4.4.3 cDNA synthesis ...... 47 4.4.4 Semi-quantitative PCR ...... 47 4.4.5 Real-time PCR for transgene analysis ...... 50 4.5 Insect rearing ...... 50

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4.5.1 Insect bioassays ...... 50 4.6 Data Analysis ...... 51 4.7 Results ...... 51 4.7.1 Cloning of Hvt and plant transformation ...... 51 4.7.2 Double gene construct: ...... 54 4.7.3 Transgene analysis ...... 55 4.8 Insect Bioassay ...... 58 4.8.1 Mealybugs ...... 58 4.8.2 Aphids (Myzus persicae) ...... 61 4.8.3 Whiteflies (Bemisia tabaci) ...... 64 4.9 Real-time PCR...... 67 4.10 Discussion ...... 69 5 Production of Hybrid Proteins to Control Sucking Pests...... 73 5.1 Introduction ...... 73 5.1.2 PVX ...... 74 5.2 Methodology ...... 76 5.3.1 Cloning of fusion gene constructs ...... 77 5.3.2 Agrobacterium Transformation ...... 77 5.3.3 Plant Infiltration ...... 77 5.3.4 Insect rearing and bioassay ...... 77 5.4 Data Analysis ...... 78 5.5 Results ...... 78 5.6 Discussion ...... 82 6 General Discussion ...... 84 6.1 Can we grow cotton and other crops without pesticides? ...... 84 6.2 Biosafety status of Hvt and lectins ...... 85 6.3 RNA based strategies versus protein based strategies ...... 85 6.4 Double gene versus fusion genes? ...... 87 References ...... 88

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List of Figures

Figure 1-1 Various types of sucking pests (a) Mealybugs (b) Aphids (c) Bugs (d) Whiteflies (e) Thrips (f and g) Mirids (h) Jassids ...... 3 Figure1-2 Mechanism of action of (Auer et al [229]) ...... 5 Figure 1-3 Modulators for ion channels from spider (www.arachnoserver.org) ...... 6 Figure 1-4 Transmission Electron Micrograph for PM of cotton leaf worm S. littoralis (a) normal PM of larvae feeding on control diet (b) PM of larvae feeding on Glehada containing diet...... 13 Figure 1-5 BBTV Genome Organization ...... 17 Figure 1-6 PVX Genome Organization ...... 18 Figure 2-1 Leaf discs on MS-O medium ...... 26 Figure 2-2 Leaf discs on selection medium ...... 26 Figure 2-3 Leaf discs on shooting medium ...... 26 Figure 2-4 Leaf discs on rooting medium...... 27 Figure 2-5 Plants shifted to the sand...... 27 Figure 3-1 Promoter constructs in pJIT 166 ...... 34 Figure 3-2 Constructs JG1 (B) and JG2 (C) with pGreen0029 backbone ...... 34 Figure 3-3 NSP promoter analysis by PlantCARE ...... 37 Figure 3-4 CP promoter analysis by PlantCARE ...... 38 Figure 3-5 CP (a) and NSP (b) promoters cloned in pGreen 0029 ...... 39 Figure 3-6 GUS-Histochemical staining assay for BBTV promoter expression; microscopic view (a) positive control 2X 35S promoter (b) negative control pGreen0029 (c) NSP promoter (d) CP promoter ...... 40 Figure 4-1 Hvt construct in pGreen0029 backbone ...... 48 Figure 4-2 Cassette 1 carrying promoter, Hvt and terminator ...... 48 Figure 4-3 Cassette 2 carrying promoter, lectin and terminator ...... 48 Figure 4-4 Graph representing % mean mortality of mealybugs with time by the effect of Hvt ...... 53 Figure 4-5 Bioassay performed with mealybugs (a) multiplying on healthy N. tabacum (b) plant expressing Hvt (c) some mealybugs multiplied on transgenic plants but nymphs died ...... 53 Figure 4-6 Final Double Gene Construct (DGC) used for Plant Transformation . 54 Figure 4-7 Transgene Analysis (a) DNA extraction from transgenic plants (b) PCR using nptII primers (c) PCR using gene specific primers ...... 55

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Figure 4-8 Transgene Analysis (a) RNA extraction from transgenic plants (b) PCR using gene specific primers from cDNA ...... 56 Figure 4-9 Transgene Analysis (a) semi-quantitative PCR for Hvt (b) semi- quantitative PCR for lectin...... 57 Figure 4-10 Graph representing % mean mortality of mealybugs with time by the effect of Hvt and lectin ...... 60 Figure 4-11 Bioassay performed with mealybugs (a) plant expressing single protein Hvt (b) plant expressing two toxin proteins (c) multiplying on healthy N. tabacum ...... 60 Figure 4-12 Graph representing % mean mortality of aphids with time by the effect of Hvt and lectin toxins ...... 62 Figure 4-13 Bioassay performed with aphids (a) plant expressing single protein Hvt (b) plant expressing two toxin proteins (c) alive and multiplying on healthy N. tabacum (d) exoskeletons of aphids on healthy plants ...... 63 Figure 4-14 Graph representing % mean mortality of whiteflies with time by the effect of Hvt and lectin toxins ...... 65 Figure 4-15 Bioassay performed with whitefly fed on toxin proteins (a) plant expressing single protein Hvt (b) plant expressing two toxin proteins (c) whitefly feeding on healthy N. tabacum (d) whiteflies laid eggs on healthy N. tabacum ...... 66 Figure 4-16 Melt Curve Analysis for Hvt...... 68 Figure 4-17 Normalized gene expression ratio for Hvt as analyzed by real-time PCR ...... 68 Figure 4-18 Melt Curve Analysis for lectin ...... 69 Figure 4-19 Normalized gene expression ratio for lectin as analyzed by real-time PCR ...... 69 Figure 5-1 PVX Genome Organization ...... 75 Figure 5-2 Hvt at N-terminal and lectin at C-terminal ...... 76 Figure 5-3 Lectin at N-terminal and Hvt at C-terminal ...... 76 Figure 5-4 Mortality rate of mealybugs feeding on transiently expressed fusion proteins ...... 80 Figure 5-5 Mealybug Bioassays for fusion protein (a) Hvt (b) lectin (c) Hvt-lectin (d) lectin-Hvt (e) health control (f) PVX control ...... 81 Figure 5-6 Predicted models of proteins using I-TASSER (a) Hvt (b) lectin (c) Hvt-lectin (d) lectin-Hvt ...... 82

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List of Tables

Table3.1 Sequence specific primers used for promoter amplification…………… 33 Table 4.1 Specific primers used for insecticidal genes amplification…………….. 49 Table 4.2 Primers sequence for qPCR analysis………………………………….... 49

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Acknowledgements

A major research project like this is never the work of anyone alone. The contributions of many different people in their different ways, have made this possible. I would like to extend my appreciation especially to the following.

Foremost, I would like to express my sincere gratitude to my advisor Dr. Shahid Mansoor (SI) for the continuous support during my PhD study and research, for his patience, motivation, enthusiasm, and providing me with immense knowledge. I could not have imagined having a better advisor and mentor for my PhD study.

I wish to express my deep sense of gratitude for my foreign supervisor Professor Dr. Georg Jander made a great contribution for the successful completion of my research during my stay at Boyce Thompson Institute for Plant Research (BTI), USA. His skilful advice and learned guidance enabled me to complete this work. I learned a lot from her as she proved a very expert and capable teacher for me.

I offer my special thanks to Dr. Shahid Mansoor (SI), Director NIBGE, and Ex-directors Dr. Zafar Mehmood Khalid and Dr. Sohail Hameed for providing the best possible working environment at NIBGE. I also gratefully acknowledge Dr. Zahid Mukhtar, Head Agricultural Biotechnology Division, NIBGE his moral support throughout my research work. I am profoundly indebted to Dr. Imran Amin (PS), Agricultural Biotechnology Division, NIBGE, for his positive, sincere and valuable guidelines and support. I am also thankful to Dr. Rob W. Briddon Agricultural Biotechnology Division, NIBGE, for his support and guidance.

I am highly obliged to all my lab colleagues at NIBGE especially; Huma Mumtaz, Dr. Ghulam Rasool, Dr. Amir Raza, Dr. Muhammad Nouman Tahir and Mr. Atiq-ur-Rehman for their cooperation and help in experimental work.

Special gratitude is due to Higher Education Commission of Pakistan for providing financial support under Indigenous Scholarship program and International Research Support Initiative program for my studies and making my dreams come true. I offer my special appreciation to National Institute for Biotechnology and Genetic

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Engineering (NIBGE) and Pakistan Institute of Engineering and Applied Sciences (PIEAS) for good academic procedures and research facilities.

Last but not the least special thanks from the core of my heart to my brothers Arsalan Javaid, Armaghan Javaid & Ibtehaj Javaid and sister Warda Javaid for their undying love, support, precious affections, prayers and their valuable encouragement enabled me to complete this task successfully.

Shaista Javaid

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Abstract

To control the phloem feeders’ two insecticidal genes (Hvt and lectin) were expressed under phloem specific promoters. Promoters were isolated from Banana Bunchy Top Virus components Coat Protein and Nuclear Shuttle Protein. Promoters were checked for their tissue specific expression through GUS Histochemical assay. Confirmed promoters were further used for the cloning of toxin genes.

Both genes were expressed transgenically under BBTV promoters Nicotiana tabacum through Agrobacterium transformation. Transgenic plants were confirmed by PCR and semi-quantitative PCR and subjected to the Bioassays with sap sucking pests viz; mealybugs, aphids and whiteflies. The toxin protein controlled the sucking pests effectively and mortality data were recorded on the basis of 24 h. Two best lines, were screened out, able to control the sucking pests up to 100% in minimum time. Transgenic lines proved effective against the sucking pests were also analyzed by real-time PCR in a relative manner to check the gene expression. The lines showing high mortality rate of pests also showed high level of gene expression.

The other approach was used to produce and express the fusion proteins to control sucking pests. For this purpose, Hvt and lectin were translationally fused to produce a single protein. Two constructs were designed to evaluate which orientation of gene proves more effective for the control of sucking pests. The constructs were designed keeping Hvt at N-terminal and lectin at C-terminal and vice versa. Proteins were expressed through PVX expression system and transiently expressed in the Nicotiana tabacum plants. Insects were allowed to feed on plants 14 dpi and mortality data were recorded on the basis of 24 h. The protein having Hvt at N-terminal and lectin at C-terminal proved to be more effective against mealybugs with 85% mortalities followed by the 65% mortalities by the other construct.

These results reveal that both approaches were proved to be effective to control the sap sucking pests.

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List of Publications and Patents

Journal Publication

 Shaista Javaid, Imran Amin, Georg Jander, Zahid Mukhtar, Nasir A. Saeed and Shahid Mansoor. A transgenic approach to control hemipteran insects by expressing insecticidal genes under phloem-specific promoters. Scientific Reports 6, 34706; doi: 10.1038/srep34706 (2016).

US Patent Submitted

 Shahid Mansoor, Shaista Javaid, Imran Amin, Zahid Mukhtar, Nasir A. Saeed. Control of sap-sucking insect pests using transgenic approach. 2016.0210; US Patent. 15/132,986. (2016).

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List of Abbreviations and Symbols

µg microgram µL microliter µM micromolar AGO Argonaute BAP 6-benzylaminopurine BFA Burkina Faso BLAST Basic Local Alignment Search Tool bp Base pair Bt Bacillus thuringiensis

CaCl2 Calcium chloride CCRI Central Cotton Research Institute CLCuD Cotton leaf curl disease CP Coat protein CR Common region CTAB Cetyltrimethylammonium Bromide DDT Dichlorodiphenyltrichloroethane DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate dpi Days post inoculation ds Double-stranded DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid GUS beta-glucuronidase HCl Hydrochloric acid

HgCl2 Mercuric chloride HGT Horizontal gene transfer HL Hvt-lectin ICTV International Committee on Taxonomy of Viruses IPTG Isopropyl-beta-D-1-thiogalactopyranoside IR Intergenic region kDa kilo Dalton KOH Potassium hydroxide

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kV kilo Volt LB Lauri broth LH lectin-Hvt M Molar mg milligram min minute miRNA microRNA mL milliliter mM millimolar MP Movement protein mRNAs messenger RNAs MS Murashige and Skoog medium MS0 MS-zero N Normal NAA Naphthylacetic acid NaCl Sodium chloride NaOH Sodium hydroxide ng nanogram NIG NSP-interacting GTPase NPDR Non-Pathogen derived Resistance NSP Nuclear shuttle protein nt. nucleotide NW New World OD Optical density ORF Open reading frame OW Old World PCNA Proliferating cell nuclear antigen PCR Polymerase chain reaction PD Plasmodesmata PDR Pathogen derived resistance PERK Proline-rich extensin-like receptor protein kinase pH power of hydrogen ion PKC Protein kinase C PM Peritrophic matrix PTGS Post transcriptional gene silencing PVP Polyvinylpyrrolidone PVX Potato virus X

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RCA Rolling circle amplification RCR Rolling circle replication Rep Replication associated protein RIPs Ribosome-inactivating proteins RISC RNA-induced silencing complex RNA ribonucleic acid RNAi RNA interference rpm revolutions per minute SCR Satellite conserved region SDS Sodium dodecyl sulphate SDW Sterile distilled water SEL Size exclusion limit siRNA small interfering RNA ss single stranded SSC Standard saline citrate TAE tris-acetate EDTA Taq Thermus aquaticus TGS Transcriptional gene silencing TrAP Transcriptional activator protein UV Ultra violet VIGS Virus induced gene silencing X-Gal 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside X-Gluc 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid

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1. General Introduction

For food security and agriculture productivity it is very important to control weeds and pathogens, as they cause significant losses to the country’s economy [1]. DDT and organophosphate (synthetic chemical ) have been used, since 1940s, to control insect pests [2]. These chemicals were proved to improve the crop yield but they exert adverse effects on non-target insects as well as human being [3]. To overcome this issue, some other synthetic chemicals with specificity to target pests as pyrethroids, neonicotinoids and growth regulators are being used around the globe [4]. Neonicotinoids, are the general agonists of insect nicotinic acetylcholine receptors but they can bind only to the receptors homolog in higher animals [5]. They make almost 24% of the world market because of their high efficacy and low toxicity [6]. There are also several reports that neonicotinoid pesticides have negative effects on pollinating insects [7, 8] and have recently been banned by European Commission. Insect pollination is an important ecosystem service. For approximately 70% of crops pollination is very essential for fruit setting and it contribute to 35% of the global fruit production [9]. Due to the sub-lethal exposure of neonicotinoid pesticides on pollinating insects, the nectar relevant doses of neonicotinoids impair the Kenyon cells function in the mushroom bodies of honey bees and also reduces their olfactory learning, memory and homing ability [7, 10, 11]. Sub-lethal doses of these pesticides cause foraging failure and bee colonies in bumblebees [12]. While many of the chemical pesticides have high negative effects on bee pollination and other insects [12, 13], but it is hard to ban chemical pesticides without any alternative and can cause great losses to agricultural production. 1.1 Insecticidal Proteins from Bacillus thuringiensis Many of the insecticides not only pollute the environment but also affect the other non- target insects and including humans along with insect pests [14]. One of the most important advancement in biotechnology is the production of transgenic plants capable of expressing toxin proteins [15]. A variety of strategies are being used to

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1. General Introduction avoid the development of resistance in insects against BT toxins. One option is pyramiding the expression of various Cry genes in plants [16, 17] or combining the use of a variety of molecules to express hybrid Cry proteins that manifest greater toxic effect on insects [18, 19].

Here it becomes necessary to design and develop some potential alternatives of pesticides to cope with the insect pests. It may include development of biopesticides that often have high specificity to target pest species [20]. Bacillus thuringiensis is the most common organism, used as a bioinsecticide because of Cry proteins production, to date [21]. δ- Endotoxins crystals are proteolized and dissolved in the insect gut, bind to the cellular membranes of the epithelium, induce cell osmotic lyses and cause death to the insect [22]. The target insects developed resistance against Bt toxins because of the extensive use of Bt gene used in the field [21]. Bt technology has already exceeded the predicted time span before the emergence of insect resistance [21, 23]. The experience of constitutively expressed Bt genes had been very successful. But, tissue specific expression is a better choice e.g. in epidermal cells, which first come under attack from insects or in the phloem for sap sucking insects [24]. It has also been reported that expression can be regulated using transcription factors or chemical inducers. By using this technique, it is also made possible to produce non-refuges within plant refuges where it is observed that some parts of the plants are unable to express the genes and act as non-GM refuge [25].

1.1.1 Limitations of Bt Proteins Although Bt toxin has been proven very effective against insect pests but there are some limitations for these toxins.

Activity Against Chewing Pests But not Sucking Pests Bt toxins have been proven effective against chewing insects. They directly attack the insect’s gut and break down the gut lining, when ingested by the insect pests. But, a big threat to agriculture is “phloem sap sucking pests”. They directly feed on phloem sap and cause damage to the crop by taking up the nutrients. On the other hand, these pests are also important vectors for the transmission of viruses e.g. whitefly is a potent vector for more than 100 begomoviruses species. There are also other insect pests as mealybugs, aphids, jassids, thrips etc. Therefore, it is necessary to find some effective strategy to control sucking pests.

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1. General Introduction

Figure 1-1 Various types of sucking pests (a) Mealybugs (b) Aphids (c) Bugs (d) Whiteflies (e) Thrips (f and g) Mirids (h) Jassids

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1. General Introduction

1.2 Other Insecticidal Proteins and Their Activity Bt is serving for more than two decades but now due to the resistance production in insects against Bt toxin, it is needed to use some other toxins to control insect. Two of the toxins were studied and described here in detail

1.2.1 Hvt Scorpions and are the most important representatives of arthropods. Due to large number of species they are generally called arachnids. To date, more than 42,700 species of spiders have been discussed [26]. This great no. makes them the largest group of terrestrial predators but there are still many remaining to be characterized [27]. The ability of spiders to produce complex venom is the major contribution toward their evolutionary phenomenon [28].

There are several millions of promising components present in the spider venom [29] and numerous peptide toxins present in the scorpion and spider venom affect cellular communication. These toxins have the ability to distinguish the ion channels and can cause modification in their opening and closing mechanism or block them, which leads to the anomalous depolarization of cells [30].

Spiders are active predators of insects and most of them produce venom to detain their prey. But all spiders are not poisonous. Scientists are attracted by the diversity of spiders to find out insecticidal toxins [31, 32, 33, 34] and [35].

How Hvt Affects Insects? Spider are a complex mixture of a variety of compounds including proteins, peptides and low molecular mass compounds (LMM). LMM, often consist of free acids, glucose, free amino acids, neurotransmitters and other biogenic compounds. These LMM have the ability to attack directly the insect nervous system and can cause a wide range of pharmacological effects on synaptic transmission [36]. Among LMMs, several compounds are neurotransmitters while others can block the ion channel at neuronal level, effectively. To explore the nervous system, these neurotoxin LMM could be a great tool and also can contribute in the agrochemical and pharmaceutical industries for the purpose of drug screening [37] (Fig.1-2).

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1. General Introduction

Figure1-2 Mechanism of action of spider neurotoxin (Auer et al [229])

The complex chemical cocktail of spider venom consists of as many as 1000 peptides. It has been estimated that a single venom can contain as many as >10 million bio-agents many of them consisting of disulfide rich peptides, with only 0.01% diversity having been characterized [38]. To date, all the spider-venom peptides and proteinaceous toxins have been described in a manually curated database named ArachnoServer [39, 40].

In excitable cells, the voltage gated sodium channels play a significant role in rising the action potential during rising phase [41]. Insects have only one Nav channel subtype as compared to vertebrates [42]. For the fact, insects are very sensitive for Nav channel modulators. A variety of successful chemicals (DDT, di-hydropyrazoles, indoxacarb, pyrethenoids and N-alkylamides) have been designed as Nav channel modulators [43-45]. Spider venom also contains peptides known as “gating modifier proteins”. These gating modifiers usually modify K+ channel. A 35aa peptide found in spider venom has the ability to bind with K+ channel and is known as hanatoxin. Hanatoxin has three di-sulfide bonds in its structure and binds with the S3b helix from voltage sensor of paddle motif [46]. There are some other toxins also present in spider venom mainly target K+ channel. Some of the K+ channel targeting toxins are guangxitoxin (GxTx1E) which bind with Kv2.1, while stromatoxin (ScTx1) and

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1. General Introduction heteroscodratoxin (HmTx1,2) have tendency to target Kv2 and Kv4, heteropodatoxin (HpTx1-3) and phrixotoxins (PaTx1,2) inhibit preferably Kv4.

There are 186 ion channels listed in ArachnoServer. One third of these ion channels target Nav channels which is not surprising as the presence of these toxins allow spiders to detain their prey (Fig. 1-3).

Figure 1-3 Modulators for ion channels from spider venom (www.arachnoserver.org)

It has been estimated that Hadronyche versuta contains at least 100 polypeptide components [47] whilst cDNA profile shows a high number of polypeptides are expressed by Hadronyche versuta and other Australian funnel web spiders.

1.2.2 Lectins Lectins belong to a super family of proteins expressing the capacity of binding to specific carbohydrates without altering their structure. Usually cereal grains, legumes and fruits have relatively high contents of lectins. Especially beans and grains have significant quantities of lectins [48, 49].

Lectins play a significant role in recognition of biological phenomenon which involves cells and proteins to protect plants against external pathogens e.g. fungi and

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1. General Introduction other organisms. Because of the binding property and agglutination of blood cells lectins are commonly known as hemagglutinins [50]. Due to the binding property of lectins, they are commonly used in the laboratories to study essential biological processes which includes cell proliferation, apoptosis, cell arrest, neoplasm cell metastasis, leukocyte homing and trafficking and most importantly microbial infection. They are also widely used in glycoconjugates (in solution and on cells) as reagents and for cell characterization and separation [51].

Lectins are also being used to as a tool for novel techniques as in microarray for a high throughout analysis of glycans and glycoproteins [52] and in new data storing techniques where carbohydrates are used as hard ware for information coding [53].

Discovery and Classification of Lectins Lectins were first discovered by the end of the nineteenth century. The term “lectins” was derived from the protein named discovered in castor beans (Ricinus communis) [54]. The term hemagglutinin was introduced by finding the capability of ricin protein to agglutinate the red blood cells. Later on, it was discovered that some hemagglutinins have the ability to agglutinate human erythrocytes selectively on the basis of their ABO blood group type. This led to the birth of the word “lectin” derived from a Latin verb “legere” means “to select” [54]. After this discovery, it was proved that the presence of carbohydrate moieties on the erythrocyte’s cell membrane lead to the selection of lectins to bind with the moieties present on erythrocytes.

On the basis of domain architecture, lectins have been categorized in four major categories named hololectins, chimerolectins, merolectins and superlectins [55]. Merolectins are the proteins containing only one carbohydrate-binding domain. This group of lectins cannot agglutinate due to the monovalent nature they have. Hololectins, is a group of lectins, comprise of multiple homologous or identical carbohydrate binding domains which cause the agglutination of cells or precipitation of glyco- conjugates. Most of the plant lectins, isolated and characterized, are hololectins. As compared to hololectins, superlectins carry two domains for the binding of carbohydrates. These domains perform the function of recognition of structurally unrelated carbohydrates. Chimerolectins comprise all the plant lectins carrying single/multiple domains for the binding of carbohydrates. These domains are fused in such a way to make a single domain to perform the biological function. The function of

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1. General Introduction fused domain is solely independent of the parent domains. Sequence analysis of plants have proved that chimerolectins are present abundantly in the kingdom planta [54]. Moreover, recent analysis of plant transcriptome/genome have provided the evidence for the occurrence of many proteins containing one or more lectin domain(s) embedded in one or more multi-domain architecture [54].

Role of Lectins as Defensins in Plants Outside the kingdom planta, many of the lectins show carbohydrate specificity for the glycol-conjugates absent or least abundant in plants (e.g., galactose, sialic acid). A variety of carbohydrate moieties are found to be present in phytophagous insects, nematodes and microorganisms. These carbohydrates are also known to interact with lectins [56-58]. Storage organs and seeds of plants are reservoirs of lectins and predominately on the risk to pathogen attack [59]. Most of the plant lectins are inactive when synthesized and activated when sequestered in specialized organelles. Lectins are also known to play an important role in the defense mechanism of plants to cope with a diverse range of plant pathogens as phyto-pathogenic microorganisms, nematodes or insect pests. Lectins are also used as a source for storage of different proteins especially those needed for the development and growth of plant.

Lectins have been studied extensively for their activity against insects [59-61]. Some of the lectins are known for their ability to affect the growth and development of fungi due to their property of binding with chitin. But this antifungal activity has been proven relatively week as compared to the other antimicrobial or antifungal plants proteins [58, 62]. Many of the lectins have been proved to be highly toxic to many of the phytophagous pests. There are already numerous reports showing negative effects of lectins on pests belonging to different orders as Coleoptera, Hemiptera, Diptera and Lepidoptera when they are allowed to feed on purified toxin or genetically engineered plants expressing toxin genes. Carbohydrate moieties of lectins come into contact with insect midgut when released after ingestion by insect pests.

Other Roles of Plant Lectins Other than the promising role of defense against pathogens and herbivores, plant lectins are also well known to take part in important biological processes. Hevein, another type of lectin, found abundantly in the latex of rubber tree. Hevein is considered to perform the mechanism of latex coagulation using rubber particles [63]. Some other important

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1. General Introduction lectins calnexin and calreticulin are primarily responsible for the proper folding of newly synthesized glycoproteins inside ER or may be possibly involved in the transport of folded glycoproteins to the Golgi [64, 65]. As already mentioned several times, several lectins have been expressed at very low concentration in response to the abiotic and biotic stress inside nucleus and/or cytosol. This study leads to the hypothesis, lectins may also have the capability to act as regulator for various kind of intracellular signaling mechanisms solely involved in the stress physiology of plants [66]. It has been suggested, some extracellular root lectins may play a vital role for establishing a symbiotic relation between microorganism (nitrogen fixing rhizobia and/or mycorrhizal fungi) and the host plant [67].

Lectins in of Crops Due to the plantation of genetically engineered highly resistant crops (GMO’s), the use and production of agricultural pesticides has been decreased [68]. Bt endotoxins from Bacillus thuringiensis have most commonly been used in plants against insect pests but unfortunately they have not been proven effective for the control of bugs, thrips, aphids, mirids and hoppers [69]. Therefore, it is necessary to find out new ways to control sucking pests. Plant lectins belong to the most promising group of plant proteins exhibiting insecticidal properties. Many of the plant lectins have been proven to show entomotoxic properties against various insect pests belonging to the important insect orders Diptera, Coleoptera, Hemiptera and Lepidoptera. To analyze the lectins properties to control the insect pests in a natural system, transgenic plants have been generated. Lectin proteins may affect insect pests in multiple ways depending on the insect species being effected. They may cause severe delay in development to the high mortality rate.

Major Types of Plant Lectins GNA–related Lectins Many lectins with strong entomotoxic properties have been purified from Amaryllidaceous species bulbs. They include Galanthus (snowdrop or Narcissus) daffodils. These lectins majorly contribute to protect plant from the attack of phytophagous insects. The best which lectin has been studied so far for its insecticidal activity is GNA. It binds specifically to the terminal residues of high mannose glycans and known to present frequently on the insect glycoproteins. GNA was found to be

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1. General Introduction toxic against a wide range of insects while insects belonging to the order Hemiptera were proved to be sensitive to GNA toxin. It is also reported that some of the GNA- related lectins also accumulate in the phloem cells of the plant under natural conditions.

To produce a high level of resistance against insect pests, GNA has been expressed in many crops including rice, wheat, sugarcane, tobacco and potato by means of genetic engineering. Severe entomotoxic effects of GNA has been studied on the development and grain survival while expressed in transgenic wheat either expressed under phloem specific promoter or in a constitutive manner [70]. Leaf lectin from garlic ASAL (Allium sativum agglutinin) expressed in transgenic rice cause a high rate of nymph mortalities in the insects belonging to the order Hemiptera brown leafhopper (Nilaparvata lugens), green leafhopper (Nephotettix virescens) and white backed planthopper (Sogatella furcifera) [71]. As a result of ASAL expression in transgenic rice, green leafhopper (N. virescens) was unable to cause tungro disease [72]. It exhibited, pest control also protects plants from being infected by disease transmitted by insect vectors. Genetically engineered GNA-related lectins have also been proved effective against the insects belonging to the order Lepidoptera as tomato moth (Lacanobia oleracea), cotton leaf worm (S. littoralis) and Mexican rice borer (Eoreuma loftini) [73-76].

Legume Lectins This is the kind of lectins usually purified from seeds. There are several legumes which have the ability to bind with the carbohydrate moieties usually not present in plants e.g. complex N-glycan structures having terminal sialic acid and galactose residues or Thomsen-nouveau antigen. The survival and growth of Meligethes aeneus was found to be negatively affected when allowed to feed on PSA diet. A reduction of mass was observed when pollen beetle larvae were allowed to feed on plants expressing PSA in a tissue specific manner [77] but it did not show any negative effect on adult feeding [78]. Glehada, a legume lectin, proved to be toxic to the Colorado potato beetle (Leptinotarsa decemlineata) larvae [79]. In fact, high mortality rate of larvae was recorded while feeding on Glehada solution. Concanavalin A (Con A) from jack bean and GSII lectin from Griffonia simplicifolia were proved to be effective against Acyrthosiphon pisum (a pea aphid) and cowpea weevil (Callosobruchus maculates), respectively [80-82]. High mortality rates of taro planthopper (Durophagous

10

1. General Introduction proserpina) were recorded when ingested Con A but aphid survival was not affected by PSA ingestion [83]. This clearly shows the insecticidal activity of plant legume lectins against variety of insect pests.

Hevein-related Lectins Most of the members of this class of lectins have binding capacity with chitin. Chitin is a hetero-polysaccharide and synthesized by arthropods, nematodes and fungi being important constituent of their exoskeleton. This kind of lectins assume not to have any target interaction in mammals and therefore considered to be a safe tool to use in edible crops [84]. Negative effects were observed in the development of cowpea weevil (Callosobruchus maculatus) when fed on artificial diet mixed with WGA and UDA [85, 86]. WGA is also known to be effective against Lepidopteron and Coleopteran insects [56, 87]. While talking about Hemipteran insects, this class of lectins show very low toxicity towards them. The difference in ultra-structure organization of midgut could be a reason for insect behavior towards different lectin toxins. As the insects belonging to order Hemiptera lack functional peritrophic matrix in the midgut as compared to those belonging to orders Coleoptera or Lepidoptera.

How Lectins Effect Beneficial Insects? Plant lectins play an important role to control the insect pests. However, introducing lectins in genetically modified crops can also produce the unwanted effects especially for beneficial and/or non-target insects. Beneficial insects can be affected directly by feeding on GM plants expressing lectins (honeybees), indirectly by feeding or parasitizing the target insects. To evaluate the effects of lectins upon beneficial insects, the pollen from B. napus transgenic lines expressing PSA mixed with artificial diet and fed on by A. mellifera. Any negative effect on larval survival, development or mass gain was not observed in the larvae fed on pollen as compared to those feeding on control diet [88]. The larval survival and production of offspring of bumblebees were effected severely by feeding on GNA diet. High mortality rates of drones and worker bees were recorded along with significant reduction in the ability to produce offspring [89]. The effect of GNA diet were also studied on solitary bee (Osmia bicornis) larvae [90]. While wasp’s ability to parasitize L. oleracea larvae and its fecundity were not affected by feeding on diet containing GNA. But few sub-lethal effects were observed on Aphelinus abdominalis (a wasp) after parasitizing aphid fed on GNA diet or GNA

11

1. General Introduction expressing plant [91]. After parasitoid uptake GNA was also found to be excreted. A. abdominals is an aphid predator but females are not to be affected by GNA ingested aphids [92]. Honeydew, secreted by phloem feeders, is a great source of carbohydrates and some insects love to feed on. But this could also be a source of lectin if secreted by insects feeding on lectin expressing transgenic plants and can harm beneficial insects. Aphidius colemani, Trichogramma brassicae and Cotesia glomerata are some of the parasitic wasps love to feed on honeydew [93] and showed decrease in survival rate after feeding on lectin containing sucrose syrup.

How Lectins Affect Mammals Lectins are present naturally in a variety of crops while many of them are of daily use including vegetables and fruits (garlic, onion, tomato, bean, pea, rice, lentils, soybean, corn, wheat, banana, mulberry, breadfruit). Most of the lectins present in vegetables and crops are considered as non-toxic and ingested as raw. But there are also some toxic lectins also present as Con A and PHA. In case of kidney beans, if they are not cooked properly and consumed, considered as toxic due to high concentration of PHA. PHA is most likely to have a binding affinity with epithelial cells of digestive tract and cause morphological and metabolic changes. The typical symptoms of PHA toxicity are diarrhea or vomiting and nausea. The lectins cause local and systemic reactions as a result of interactions with the glycoproteins of digestive tract [60]. Due to toxic effects of some of the lectins upon mammals, they are used as anticancer drugs [94] e.g. ricin and are the lectins toxic to mammals but they don’t have toxicity at the same level [95-97]. GNA related lectins have been isolated from edible crops of daily use (onion, garlic, leek etc.), expressed in plants to study against pest control and considered to be safe for mammals. It reduces the threat of negative effects of lectins on consumers upon consumption [98, 99].

Mode of Action for Lectins The epithelium of insect midgut is protected by a physical barrier on the luminal side called peritrophic matrix (PM). PM is secreted by a certain type of epithelial cells [100]. The PM layer consists of a chitinous grid like network held together by chitin binding glycoproteins as peritrophins. PM associated glycoproteins create a molecular sieve and fill the interstitial spaces due to the presence of diverse kind of glycan moieties [100]. Presence of different glycans make insect midgut a hotspot for lectins attack which

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1. General Introduction leads to the malformation of functional PM and disrupted microvilli structures. It can be studied in an Electron microscope electrograph Fig 1-4.

Figure 1-4 Transmission Electron Micrograph for PM of cotton leaf worm S. littoralis (a) normal PM of larvae feeding on control diet (b) PM of larvae feeding on Glehada containing diet

Lectins also caused hyper-secretion of many disorganized layers of PM in European corn borer (O. nubilalis) midgut when allowed to feed on WGA containing diet and the presence of many disintegrated microvilli was also observed [56, 101]. In case of Drosophila, clear changes were observed in morphology of microvilli after feeding on WGA containing diet [102].

In case of some plant lectins, it has been reported that they absorb in the midgut lining through trans cytosis, after ingestion in the insect body, across the gut epithelium. Con A were found to be present in the hemolymph, malpighian tubules and fat tissues of L. oleracea [103]. Similarly, GNA was found in fat tissues, ovaries and hemolymph of rice brown hopper (N. lugens) after ingestion [104]. All of these studies conclude that it is needed to study more potential target sites for lectins other than midgut.

Lectins Target Insect Glycoproteins Glycan arrays were used to study the binding of glycans with lectins, showing plant lectins have high affinity towards binding with insect glycans present frequently in the gut. For plant lectins, glycoproteins are potential target as they are secreted abundantly in the gut lumen as enzymes and transport proteins. A ferritin subunit, an important

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1. General Introduction protein known for iron transport, reported to be targeted by GNA effectively [105, 106]. Membrane bound amino-peptidase in the pea aphid (A. pisum) midgut found to interact with GNA and Con A [107] while some of the released α-amylase in the digestive tract of insect also bind with Con A and GNA [106, 108]. Many of the plant lectins have multiple target cites inside the Drosophila body [109]. It has also been studied in different insects (beetles, caterpillars, aphids, honeybees etc.) that glycoproteins secreted inside the gut have tendency to cluster and make macromolecular complexes that cannot pass through the PM. It leads to the recycling and leakage of digestive enzymes in the digestive tract.

1.3 Fusion Proteins As the biotechnology advancing, it is necessary to find new approaches to control pests along with gene pyramiding or multiple gene resistance. One of the possibility being used worldwide is to construct fusion proteins to control a wider range of insect pests. Fusion of two or more different/ similar toxic proteins lead to the evolution of a new protein with multiple active sites against insect pests. It is important to ensure that toxins to be used should not affect the non-target organisms.

A recent example in this case is, GNA and spider toxin (Hv1a) based fusion protein where GNA act as a carrier for spider venom to move into the insect gut where after proteolysis it becomes easy for spider toxin to attack on insect’s central nervous system. It has been proved to be very effective against insect pests and being synthesized successfully [110]. Pl1a is an insect specific gene and does not harbor the other insects. A Pl1a/GNA fusion protein has been proved very effective against the insects from different orders including aphids [111]. Similarly, other fusion proteins have been produced mostly using Bt toxin genes. As Cry1Ac/ASAL, Cry1c/Cry1E, Cry1Ac/ricin-B chain etc. have been synthesized and proved to be more toxic on a broader range of insects than native/single gene [19, 112].

Here, fusion proteins consisting of one Australian funnel web spider venom toxin (ω-atracotoxin) and other onion leaf lectin were designed to express for pest control. Two different sequences were fused together in such a way to get a single translationally fused protein.

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1. General Introduction

1.4 Tissue Specific Gene Expression Commonly constitutive promoters are used to express a gene throughout the plant for plant transformation studies such as from viruses; cauliflower mosaic virus (CaMV) 35S promoter [113], figwort mosaic virus (FMV) promoter [114]; bacteria (Agrobacterium tumefaciens) Ti plasmid mannopine synthetase (mas) promoter [115] or nopaline synthase (nos) promoter [116]; or plants (the Arabidopsis thaliana Act 2 promoter [117] or Medicago truncatula MtHP promoter [118].

35S promoter derived from Cauliflower mosaic virus is one of the best characterized constitutive promoters. It regulates transgene expression at high levels in dicots [119], including Carrizo citrange [120] and is commonly used as a tandem repeat (2X35S). But now, with advancements in biotechnology, it may not be necessary to express a gene throughout the plant especially in the case when tissue specific gene expression is required. To achieve the goal, some other promoters have also been derived from plant DNA viruses e.g. wheat dwarf geminivirus (WDV) but unlike CaMV several of them are tissue specific promoters showing activity specific to vascular cells [121]. The transgene expression of WDV promoter in tobacco (N. tabacum) and N. benthamiana was found in the leaves, stems, and roots, along with the reproductive organs. Another promoter that has been reported to target vascular tissue is AtSUC2 of Arabidopsis thaliana [122]. It encodes a plasma membrane sucrose H+ symporter essential for transporting sucrose long distances [123] and it is expressed specifically in the phloem companion cells of photosynthetic leaves [122]. AtSUC2 gene promoter has shown its activity in discrete areas in the phloem tissue including that of leaves and stems whilst expressed in N. tabacum [121]. Another phloem specific promoter, now commonly being used, is sucrose synthase gene promoter.

The expression and specificity of a tissue promoter differs in individual plant species, so it’s important to evaluate different promoters within target species to assess affectivity of different promoters in one genetic background. For easy assessment of promoter activity, there are several reporter systems. One of the most commonly employed is the GUS reporter system, based on the Escherichia coli beta-glucuronidase (GUS) gene. When incubated with specific substrates, the enzyme cleaves the substrate to form colored or fluorescent products allowing for visual and quantitative assays. The CaMV 35S promoter drives higher GUS expression in tobacco leaves than in leaves of

15

1. General Introduction alfalfa, Arabidopsis, or canola [124]. The latter two, however; expressed the transgene similarly in all organs whereas tobacco showed high expression in leaves and stems but low expression in flowers and seeds [124].

Targeting transgenes in vascular organs for example is sufficient to express defense related proteins and/or peptides which could potentially confer resistance to pathogens that attack the vascular tissues [125]. Several promoters that target phloem- specific gene expression have been described. These promoter elements are generally associated with genes that express specifically in phloem cells or from organisms that are phloem limited. Over the past few years, several phloem-specific promoters have been utilized to develop genetically superior plants [126]. Two such promoters are RSs1 (isolated from rice) and rolC (isolated from Agrobacterium rhizogenes A4 strain). The RSs1 has already been used to regulate the expression of β-glucuronidase (GUS) gene [127], Galanthus nivalis agglutinin (GNA) gene [128, 129], Allium sativum leaf agglutinin (ASAL) gene [130] and spider toxin [131] in a phloem-specific manner. The rolC has been used for the phloem-specific expression of GUS gene in transgenic tobacco [132, 133] transgenic rice [134] and in transgenic N. tabacum [131].

BBTV is a monocot infecting phloem limited Nanovirus and all of its components have been isolated from Pakistan [135]. BBTV is the type member of genus Babuvirus belonging to the family Nanoviridae. It is transmitted by peach pea aphid (Myzus persicae) and consists of multipartite genome. BBTV carries six circular ssDNAs (≈ 1.1 kb each) and encapsulated individually in icosahedral virions (≈ 18-20 nm in diameter each). The genus Babuvirus distinguished as having six components as compared to the eight components of genus Nanovirus of family Nanoviridae. Each component encodes a single protein in virion sense means BBTV encodes six different proteins. These proteins include a rolling circle replication initiator protein (Rep; DNA- R), capsid protein (CP; DNA-S), movement protein (MP; DNA-M), cell cycle link protein (Clink; DNA-C) and a nuclear shuttle protein (NSP; DNA-N [136-139] (Fig. 1- 5). The intergenic region of coding gene is considered as promoter and can be used for expression in plant phloem cells [140]. The goal was to express different genes under phloem specific promoter to control phloem feeders.

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1. General Introduction

Figure 1-5 BBTV Genome Organization

Different promoters from BBTV were characterized to get optimum expression in phloem cells.

1.5 Expression of Insecticidal Proteins in Plants It is important to see how the genes from different origins behave In Planta. For this purpose, the proteins are expressed in different ways.

1.5.1 Transient Expression Potato Virus X (PVX) There are a no. of RNA and DNA viruses have been modified and being used as vectors for expression and/or silencing of genes in planta. There are some virus vectors being used for the production of heterologous proteins or peptides of commercial importance including vaccine antigens and antibodies in plant cell [141-143, 225]. It is easy and cost effective to overexpress the genes in plants through virus based vectors rather than stable transformation [144-149]. Potato virus X (PVX) is among the top 10 most

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1. General Introduction important plant viruses based on its economical and scientific importance [150]. PVX is used as a model to study the plant-virus interactions and to study the mechanisms of viral propagation as viral replication cell to cell movement and suppression of post- transcriptional gene silencing (PTGS). PVX has a single stranded RNA genome of about 6430 nt. PVX genome consists of five open reading frames (ORFs). ORF1 encodes 167kDa RNA-dependent RNA polymerase (RdRp). Three partially overlapped ORFs encode movement proteins of 25kDa TGBp1, 12kDa TGBp2 and 8kDa TGBp3 (Triple Gene Block). The last ORF5 encodes coat protein (CP) of 25kDa. These gene required for viral replication, encapsidation and cell to cell movement [151].

Figure 1-6 PVX Genome Organization

TGB protein is the largest protein and is multifunctional. TGBp1 (a 25 kDa protein), is essential to build PVX replication complexes [152], initiation of PVX virion translation and for PVX movement [153]. TGBp1 was first discovered RNA gene silencing viral suppressor [154]. It has been reported recently that TGBp1 can interact with AGO proteins and lead to the AGO1 degradation through proteasome pathway [155]. It is also capable to modify the plasmodesmata (PD) aperture to establish the transportation of PVX ribonucleoprotein complexes to the neighbor cells [156]. TGBp1 “gating” activity, i.e. its propensity to expand the plasmodesmata size to the exclusion limit (SEL), is presumably precede or concomitant with its accumulation at pit field PD. TGBp1 functional characterization reveals that its PTGS suppressor activity can be impaired by mutation showing PVX movement is dependent on its PTGS activity while silencing suppression is not sufficient to allow the virus movement between the cells [157].

To serve as expression vector PVX genome was cloned behind a T7 RNA polymerase promoter or 35S promoter derived from cauliflower mosaic virus (CaMV)

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1. General Introduction

[158]. This vector has been successfully used to express the foreign genes in planta [158-161]. A PVX based vector was used to express fusion gene construct.

1.5.2 Stable Expression of Insecticidal Proteins in Transgenic Plants Human being are suffering from different environmental stresses, food shortage and population explosion around the globe. Population of the world is increasing alarmingly and will reach to 8.5 billion by 2025. It is becoming hard to fulfill the food demand of every human being to survive under the natural resources. This food deficiency resulting the malnutrition and leading to the serious health problems. Therefore, it is necessary to improve both, the food quality and quantity, either by following conventional methods of breeding or by genetic engineering [162]. The biotechnology application led to the novel possibilities and opportunities to enhance the qualitative as well as quantitative traits of organisms [163]. Biotechnology combats the food deficiencies by enhancing their carbohydrate, protein, vitamins, lipids and micronutrient composition [164]. It is being emphasized to improve the crop quality for different traits viz; nutritional value, insect and herbicide resistance and biofuel production etc. since 1990. All the traits involve multiple genes to express it completely, so it’s not easy to improve crops while using genetic engineering tools. A number of social, political and legal issues are involved in the production of transgenic plants [162]. WHO has three main concerns with genetically modified crops, particularly GM food crops, including production of allergenic foods, incorporation of modified genes into human body and crossing of GM crops with wild species. All of these factors are a great threat to food security. Regardless of all these threats many countries including USA, China, Canada, and Argentina are growing GM crops [165]. The yield of many crops including tobacco, rice, wheat, soybean and brassica has enhanced using modern techniques of genetic engineering [166]. Biotechnology has the capacity to enhance the capability of biomass-based fuels [167]. Many of the genes have been isolated to combat with the problems regarding biotic and abiotic stresses etc. but it is important to produce transgenic plants expressing such genes [168-171]. The development of such genetically modified crops is too slow because of limited knowledge about mechanisms involved in molecular biology and resistance at cellular and plant level [169]. It’s not easy to pinpoint a single mechanism to enhance the plant stress tolerance whether producing transgenic plants is an ultimate goal to overcome such problems [166]. Tobacco expressing a phyto-hormone (foreign biosynthetic gene)

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1. General Introduction was the first developed transgenic plant [172]. Since then, more than 100 transgenic plant species have been produced showing enhanced resistance against biotic and abiotic stresses including the transgenic plants overexpressing other traits as seed yield, seed size, photosynthesis, leaf size etc. [173-179]. Globally, the production of GM crops is increasing day by day. By 2008, the area under cultivation of GM crops was increased to 125 Mha from 44.2 Mha in 2000 and 1.7 Mha in 1996. USA is the major contributor to produce genetically transformed crops with cultivation area of 62.5 Mha along with other 25 countries. Tomato, rice, alfalfa, soybean, wheat, tobacco, cotton, canola, maize, sugar beet, petunia squash, sweet pepper and carnation are the major biotech crops being cultivated.

1.6 Need for The Project and Objectives

 Characterization of phloem-specific promoter from Banana bunchy top virus.  Production of translationally fused proteins with insecticidal activity.  Expression of fusion gene and separate genes under phloem-specific promoter from BBTV or 35S promoter.

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2. General Methodology

2.1 Selection of Promoter Phloem specific promoters were used to express the insecticidal genes in a tissue specific manner. The promoters were isolated from Nuclear Shuttle Protein (NSP) and Capsid Protein (CP) components of Banana Bunchy Top Virus (BBTV) Pakistan isolates. Intergenic region of BBTV components was considered as proposed promoter and amplified by PCR using sequence specific primers. Clones for BBTV components AM418566 and AM418568 were used to amplify the proposed promoter region [135].

2.2 Isolation of Insecticidal Genes Australian funnel web spider’s venom toxin ω-atracotoxin (Hvt) and onion leaf lectin were decided to express under phloem specific promoter. Hvt (AJ938032) was amplified by using gene specific primers from pSAK II [180] while lectin was amplified from DQ255944 using sequence specific primers.

2.3 Polymerase Chain Reaction (PCR) PCR reaction mixture was prepared using Thermo Scientific (Fermentas) Taq polymerase Kit, containing 1X reaction buffer, MgCl2, 2 mM dNTPs, 10 pm of each primer (forward and reverse), 10 ng of template and 0.5 U of Taq polymerase. PCR reaction was subjected to the following profile, initial denaturation temp. 94°C for 5 min, 1 cycle; followed by 35 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 45 s; and final extension at 72°C for 10 min. SacI and HindIII, restriction sites were used for cloning in vector pJIT166 (expression vector). In the next step, complete expression cassette from pJIT166 was picked and cloned in pGreen0029 (a binary vector), at restriction sites SacI and XhoI. This vector is ready for further cloning of genes under promoter.

2.4 Cloning of PCR-Amplified Product PCR products were initially cloned in pTZR57R/T PCR cloning vector using InsTAclone PCR cloning kit (Fermentas).

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2. General Methodology

2.5 Ligation Reaction For the ligation of vector and gene of interest, T4 DNA ligase (Fermentas kit) was used. Vector and insert were mixed in a tube with 1:3 ratio, respectively along with 1X T4 DNA ligase buffer and 10 U of T4 DNA ligase. Volume was made upto 20 µL using d3H2O and kept in ligation chamber at 16°C for 16-18 h.

2.6 Preparation of Competent Cells 2.6.1 Preparation of Heat Shock E. coli Competent Cells A single colony from a freshly streaked plate of E. coli strain Top10 was picked and cultured in 50 mL LB at 37°C and 180 rpm for 16 h. This culture was called primary culture and used as an inoculum source for the secondary/post culture. Two hundred mL of sterilized LB, in a conical flask, was inoculated by 2-3 mL of primary culture and incubated at 37°C and 180 rpm for 2-3 h until it reaches to OD600 1. Flask was removed from 37°C, kept in ice for 30 min and culture was transferred to cold 50 mL falcon tubes and centrifuged for 10 min at 4000 rpm and 4°C. Supernatant was discarded and pellet was re-suspended in 15mL of 100 mM MgCl2 and again centrifuged for 5 min at 4000 rpm and 4°C. This step was repeated with 10 mL of 100 mM MgCl2. After washing with MgCl2 100mM of CaCl2 was added to the tube and pellet was re-suspended. At this step, re-suspended cells were kept in ice for 30 min. After 30 min, tubes were again centrifuged at 4000 rpm and 4°C. Finally, pellet was re- suspended in 3-4 mL of 10% glycerol in CaCl2 and cells were aliquoted in 1.5 mL cold eppendorf and stored at -80°C for a long time.

2.6.2 Preparation of Electro-competent Agrobacterium tumefaciens Cells A single colony was picked from a freshly streaked plate and cultured in 50 mL LB containing appropriate antibiotic at 28°C and 180 rpm for 36-48 h, called primary culture of Agrobacterium. Two hundred mL of LB was inoculated by 4-5 mL of primary culture and incubated at 28°C and 180 rpm for 4-5 h in a conical flask until it reaches to OD600 1. After incubation, flask was kept in ice for 10 min, culture was poured in 50 mL cold falcon tubes and centrifuged at 4000 rpm and 4°C for 10 min. Supernatant was discarded and pellet re-suspended in 50 mL of sterile cold ddH2O and centrifuged at 4000 rpm and 4°C for 10 min. This step was repeated with 25 mL of sterile cold water

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2. General Methodology and pellet was re-suspended in 10 mL of 10% sterile cold glycerol and again centrifuged at 4000 rpm and 4°C for 5 min. This step was repeated and finally pellet was re- suspended in 3-4 mL of sterile cold 10% glycerol. Aliquots were made in 1.5 mL sterile cold eppendorf and stored at -80°C.

2.7 Transformation in Bacterium 2.7.1 Transformation in E. coli Cell aliquot was kept in ice for few min and allowed to thaw. Five µL of ligation mixture was added in cells and kept in ice for 30 min. After 30 min aliquot was kept in Dri-bath at 42°C for 2 min for heat shock and then again placed in ice for 2 min. After shock 1 mL of LB added to the tube, inverted several times to mix it thoroughly and incubated at 37°C for 1 h. After incubation, it was centrifuged and pellet re-suspended in 200 µL of LB. This re-suspended culture was allowed to spread on LB- agar plate containing desired antibiotic with the help of a glass/metal spreader and then incubated at 37°C for 16 h.

2.7.2 Transformation in Agrobacterium Cell aliquot was kept in ice for few min and allowed to thaw. Two µL of recombinant plasmid was mixed gently with the competent cells. Then cells along with plasmid shifted to the sterile electroporation cuvette (1mm gap) on ice. Electroporator was set at 1400V and cuvette was inserted in the chamber for electric shock and pressed the start button. After shock 1 mL of LB was added to the cuvette and mixed thoroughly. It was taken out in a sterile eppendorf and incubated at 28°C for 2 h. After incubation, the tube was centrifuged and re-suspended the pellet in 200 µL of LB. This suspension was allowed to spread on LB-agar plate containing appropriate antibiotics and incubated at 28°C for 36-48 h.

2.8 Plasmid Isolation Single colonies were picked using autoclaved toothpicks and subjected to grow in 5 mL LB media in culture tubes. Media also contained the desired antibiotic for selection. Cultures were grown at 37°C and 180 rpm for 16 h until late log phase. After 16 h cultures were transferred to 1.5 mL eppendorf tubes and centrifuged at 13,000 rpm for 2 min. Supernatant was discarded and pellet was dried by inverting eppendorf on tissue

23

2. General Methodology paper. After drying 100 µL of re-suspension solution was added to pellet and vortexed for few seconds until the whole pellet was re-suspended. After re-suspension, 150 µL of lyses solution was added and inverted the tube several times to mix it well. It causes lyses of bacterial cell wall and allows plasmid to come out in the solution, tubes were kept at room temperature for 1-2 min to allow bacterial cell wall digestion. Later on, 200 µL of neutralization solution was added which inhibits the lyses reaction. Neutralization of the reaction mixture causes the precipitation of proteins, to remove these proteins, the cultures were again centrifuged at 13,000 rpm for 10 min. After centrifugation, supernatant was separated in a new eppendorf tube. 2.5 volume of 100% ethanol was added to the tube and inverted few times to mix well and kept at -20°C for 30 min. After 30 min, tubes were centrifuged at 13,000 rpm for 15 min. Plasmid was pelleted in the tube bottom and washed with 100 µL of 70% ethanol and allowed to dry at 37°C.

2.9 Restriction Analysis Plasmid isolated from E. coli was analyzed by restriction using appropriate enzymes to get final recombinant plasmid. For restriction 2-3 µL of plasmid was mixed appropriate buffer system for the working of enzymes, enzymes and RNase (to remove RNA).

Volume was made upto 20 µL using d3H2O and incubated at 37°C (or any other suitable temperature) for 1-3 h and finally run on the agarose gel to examine.

2.10 DNA Analysis Techniques 2.10.1 Agarose Gel Electrophoresis One gram of agarose was mixed in 0.5X TAE buffer by heating in microwave oven. Volume of the buffer re-maintained by distilled water and allowed to cool upto 40°C and ethidium bromide was added in appropriate concentration Ethidium bromide was used to stain the gel so that DNA fragments can be seen under UV light. Buffer was poured in the gel tray having combs and allowed to solidify. After solidification, gel tray was kept in tank containing 0.5X TAE buffer and comb removed. Samples were loaded in gel wells and run the gel at 100 V. After running 2/3 part of gel, samples were examined under UV light according to their fragment size.

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2. General Methodology

2.10.2 Phenol-chloroform Precipitation To remove proteins, salts and other impurities from plasmid, phenol-chloroform precipitation was done. Twenty µL of plasmid/ restriction reaction mixture was diluted to 200 µL using d3H2O. An equal volume of phenol-chloroform was added to the tube and mixed thoroughly by inverting several times. Tubes were centrifuged at 13000 rpm for 4 min. After centrifugation, upper layer (containing DNA) was taken in a new sterile eppendorf while lower layer (containing phenol-chloroform and impurities) was discarded. 1/10th volume of DNA, potassium acetate (3M, pH 8) was added and vortexed for at least 30 s to mix it well. Then 2.5 volume of 100% ethanol was added, kept at -20°C for 20-30 min and centrifuged at 13000 rpm for 15 min, supernatant was discarded and pellet was washed by 50 µL of 70% ethanol. After washing pellet was dried at 37°C and dissolved in appropriate volume of d3H2O.

2.11 Plant Transformation N. tabacum was used for stable plant transformation because of its short regeneration time. Ex-plants were grown upto 2-3 leaf stage. Leaves were detached, washed with autoclaved dH2O and sterilized with 10% bleach. Leaf discs were again washed 2-3 times with sterile dH2O to remove bleach residues completely and leaves were cut in small discs. Discs were dried on sterile filter paper and kept on MS-O plates in inverted position.

MS-O plates were kept in growth room under controlled conditions of 28 ± 3°C and 16 L/8 D. After 2 days, plants discs were co-cultured with Agrobacterium strain GV3101 culture transformed with the construct carrying gene of interest for 5 min, dried on sterile filter paper and again kept on MS-O under controlled conditions. After 48 h, there was Agrobacterium growth along the margins of discs. Plant discs were washed with cefotaxime (250 µg/mL) to remove the bacterial growth, dried on sterile filter papers and kept on MS shooting medium (BAP 1 mg/L, NAA 25 µg/L, kanamycin 50 mg/L and cefotaxime 250 mg/L) plates under controlled conditions.

25

2. General Methodology

Figure 2-1 Leaf discs on MS-O medium

Figure 2-2 Leaf discs on selection medium

Figure 2-3 Leaf discs on shooting medium

26

2. General Methodology

Figure 2-4 Leaf discs on rooting medium

Figure 2-5 Plants shifted to the sand

27

2. General Methodology

Plants were checked on/off and shifted to fresh medium plates. After 4 weeks plants started to regenerate. When plantlets (newly emerging plants from ex-plant) grown upto such a level that they cannot grow anymore in plates, plantlets were separated as individual lines and shifted to jars containing shooting medium, one plantlet/one jar and kept under controlled conditions. When plants were grown upto appropriate length of 4-5 inches after 8-10 weeks they were shifted to the rooting medium (BAP 25 µg/L, NAA 1 mg/L, kanamycin 50 mg/L and cefotaxime 250 mg/L) and kept under controlled conditions.

2.12.1 Shifting in Soil Sand was autoclaved and allowed to cool down upto room temperature. Small pots were filled with autoclaved sand and labeled accordingly. Plants were taken out very carefully from jars and their roots washed first by tap water and then sterile water. At the end roots were washed with 0.01% fungicide (Dithane M45 WP80, Dow Agro sciences) and put them in sand. Plants were covered by polythene bags, tied by a rubber band and kept under controlled conditions. Plants were allowed to stabilize in the pots. After 7-8 weeks plants were shifted to glass house and grown upto seed collection.

From To plants T1 seeds were collected and again grown in soil to get T1 plants.

2.13 Confirmation of Transgenic Plants Transgenic plants were confirmed by doing PCR using gene specific primers. PCR positive lines were further used for bioassays.

2.13.1 DNA Extraction DNA of tobacco plants was extracted by using C-TAB buffer protocol. Fresh leaves of tobacco were used for DNA extraction. Leaves were grinded by using pestle and mortar. Initially leaves were hardened by adding liquid nitrogen in mortar with leaves. It helps in grinding and good quality DNA extraction. After grinding 700 µL of C-TAB was added in mortar and mixed well with grinded leaves and then take it in an eppendorf. Eppendorf tubes were kept at 65°C for 30 min and inverted the tubes after intervals. Then samples were allowed to cool at room temperature and added 700 µL of chloroform: isoamyl-alcohol (24:1). Samples were mixed well by inverting the tubes several times. Then tubes were centrifuged at 13000 rpm for 10 min Supernatant was taken in a new eppendorf and 500 µL of isopropanol was added. Samples were kept at

28

2. General Methodology

-20°C for few h or overnight at room temperature. Samples were centrifuged at 13000 rpm for 15 min and supernatant was discarded. The pellet was washed by 70% isopropanol. Then pellet was allowed to dry at 37°C and run on gel to check its quality.

2.13.2 RNA Extraction RNA extraction was done from transgenic tobacco plants using Plant RNA Purification Reagent (Invitrogen Cat no. 12322-012). First of all, leaves were washed with DEPC treated water, detached carefully from plant and kept in liquid nitrogen till further processing. Leaf tissue was homogenized in 1 mL reagent at room temp using pestle mortar. Homogenized tissue was taken out in 1.5 mL eppendorf and centrifuged at 12,000 rpm for 10 min at 4°C. Supernatant was transferred in a new eppendorf tube using 1 mL autoclaved microtip without disturbing the pellet. Two hundred µL of chloroform was added to 1 mL of the extractant and shaken vigorously. The mixture was incubated for 2-3 min at room temperature and centrifuged at 12,000 rpm for 15 min at 4°C. Top layer was taken up into a new tube by tilting the tube at 45° carefully without taking any interface or organic layer. Five hundred µL of isopropanol per 1 mL of reagent was added and incubated at room temperature for 10 min. the mixture was centrifuged at 12,000 rpm for 10 min at 4°C and supernatant was discarded. Pellet was washed with 75% ethanol, vortexed the sample briefly and centrifuged for 5 min at 4°C. Pellet was air dried for 5-10 min and dissolve in nuclease free water.

2.13.3 cDNA Synthesis To synthesize cDNA from mRNA the reaction mixture was prepared using cDNA synthesis Kit (Fermentas). Reaction mixture consisted of template RNA 2 µg, oligo dT primer (0.5 µg/µL), DEPC-treated water, mixed gently and centrifuged briefly. Reaction mixture was incubated at 70°C for 5 min, chilled on ice and centrifuged briefly. Tubes were placed on ice and reaction buffer was added along with RibolockTM ribonuclease inhibitor 20 U and 20 mM dNTPs. Reaction mixture was mixed gently, centrifuged briefly and incubated at 37°C for 5 min. After incubation RevertAid H Minus M-MuLV RT 200 U was added and incubated the mixture at 42°C for 60 min Reaction was stopped by heating at 70°C for 10 min and then chilled on ice.

29

2. General Methodology

2.14 Insect Rearing Multiple sucking pests namely mealybugs, aphids and whiteflies were collected from field and multiplied in glass house under control conditions. Mealybugs were collected from cotton plants and fed on N. tabacum plants to multiply in the glass room at 30 ± 2°C and 16 L/8 D. Mealybugs established on N. tabacum plants and multiplied enormously were used for bioassay. Aphids were collected from tomato field, multiplied under control conditions on tomato plants. Whiteflies were collected from cotton field and multiplied on cotton plants in green house at 37 ± 2°C.

2.14.1 Insect Bioassays

All the bioassays were performed in plant room under controlled conditions of 26 ± 2°C and 16 L/8 D. Transgenic plants were established in plant growth room. After confirmation by PCR, 8 lines of double gene construct and 5 lines of single gene construct, were grown under controlled conditions at T1 stage. Five plants of each line at 3-4 leaves stage were used for bioassay. Different phloem feeding insects as mealybugs, aphids and whiteflies were allowed to feed on plants to check the effects of toxin proteins. Insects were exposed to the plants after starvation of 3-4 h. Equal no. of control plants (wild type N. tabacum) were also used to get comparable mortality data.

30

3. Promoter Characterization

3.1 Introduction Constitutively expressed promoters have commonly being used to express genes for plant transformation studies. Promoters have been isolated from different sources as from viruses; cauliflower mosaic virus (CaMV) 35S promoter [113], figwort mosaic virus (FMV) promoter [114]; bacteria (the Agrobacterium tumefaciens Ti plasmid mannopine synthetase (mas) promoter [115] or nopaline synthase (nos) promoter [116]; or plants (the Arabidopsis thaliana Act 2 promoter [117] or Medicago truncatula MtHP promoter [118]. CaMV 35S promoter is known to be one of the best characterized constitutive promoters. It regulates transgene expression at high levels in dicots [119], including Carrizo citrange [120] and is commonly used as a tandem repeat (2X35S).

But now, as the biotechnology advancing, it may not be necessary to express a gene throughout the plant especially in the case when tissue specific gene expression is required. To achieve the goal, some other promoters have also been derived from plant DNA viruses e.g. wheat dwarf virus (WDV) [121]. The transgene expression of WDV promoter in N. tabacum and N. benthamiana was found in the leaves, stem, root and also along the reproductive organs. Another promoter that has been reported to target vascular tissue is AtSUC2 of Arabidopsis thaliana [122]. It encodes a plasma membrane sucrose H+ symporter essential for long distance transportation of sucrose [123] and expressed specifically in the phloem companion cells of photosynthetic leaves [122]. AtSUC2 gene promoter has shown its activity in discrete areas in the phloem tissue including that of leaves and stems whilst expressed in N. tabacum [121]. Another phloem specific promoter is now commonly being used is sucrose synthase gene promoter.

The expression and specificity of a tissue specific promoter differs in individual plant species, so it’s important to evaluate different promoters within target species to assess affectivity of different promoters in one genetic background. For easy assessment of promoter activity, there are several reporter systems. One of the most

31

3. Promoter Characterization

commonly employed is the GUS reporter system, based on the Escherichia coli beta- glucuronidase (GUS) gene. When incubated with specific substrates, the enzyme cleaves the substrate to form colored or fluorescent products allowing for visual and quantitative assays. The CaMV 35S promoter drives higher GUS expression in tobacco leaves than in leaves of alfalfa, Arabidopsis, or canola [124]. The latter two, however; expressed the transgene similarly in all organs whereas tobacco showed high expression in leaves and stems but low expression in flowers and seeds [124].

It is sufficient to express the proteins/peptides, of defense purpose, in the vascular tissue, can confer resistance effectively to the pathogens attacking vascular tissues [125]. Several promoters, targeting phloem specific gene expression have been discussed earlier. Generally, these promoter elements are associated with the genes being expressed in the phloem cells or belong to phloem limited organisms. Over the past few years, several phloem-specific promoters have been utilized to develop genetically superior plants [126]. Two such promoters are RSs1 (isolated from rice) and rolC (isolated from Agrobacterium rhizogenes A4 strain). The RSs1 has already been used to regulate the expression of β-glucuronidase (GUS) gene [127], Galanthus nivalis agglutinin (GNA) gene [128, 129], Allium sativum leaf agglutinin (ASAL) gene [130] and spider toxin [131] in a phloem-specific manner. The rolC has been used for the phloem-specific expression of GUS gene in transgenic tobacco [132, 133] in transgenic rice [134] and in transgenic N. tabacum [131].

BBTV is a monocot infecting virus belonging to the genus Babuvirus of family Nanoviridae. It is monocot infecting phloem limited virus and all components of BBTV have been isolated from Pakistan [135]. It is transmitted by Myzus persicae (a potato peach aphid) and consists of multipartite genome of six circular ssDNAs (≈ 1.1 kb each) [136-139]. The intergenic region of coding gene is considered as promoter and can be used for expression in plant phloem cells [140]. The goal is to express different genes under phloem specific promoter to control phloem feeders. Different promoters from BBTV components were analyzed to get optimum expression in phloem cells.

32

3. Promoter Characterization

3.2 Methodology 3.2.1 Isolation and Cloning of Promoters To isolate the proposed promoter region from BBTV Pakistan isolates [135] sequences were retrieved from NCBI to get intergenic region of BBTV components NSP and CP. The intergenic region of NSP was supposed to be of 625 bp and for CP it is 547 bp. The promoters NSP promoter efficiency examined by restricting the IR with AccI endonucleases. The deleted region of NSP (352bp) was used to check promoter efficiency [140]. Proposed promoter region was also analyzed by PlantCARE. Sequence specific primers were designed (Table 3.1) and PCR was carried out by the using the protocol described in Chapter 2, SacI and HindIII restriction sites were used for cloning in vector pJIT166 (expression vector) replacing 2X35S promoter upstream of the GUS reporter gene, at forward and reverse end respectively. In the next step, complete expression cassette from pJIT166 was excised and cloned in pGreen0029 (a binary vector), at restriction sites SacI and XhoI which makes it ready for further cloning of genes under phloem specific promoter.

Table3.1 Sequence specific primers used for promoter amplification

Primer Name Primer Sequence

NSP Forward 5’-ATGAGCTCGTATACTAATCTCTGATTGG-‘3

NSP Reverse 5’-ACAAGCTTCATCGCTTCTGCTTTGCTTT-‘3

CP Forward 5’-CAGAGCTCTATGTTTATGTAAACATAAA-‘3

CP Reverse 5’-ACAAGCTTCTAACTCGACACTGGTATTT-‘3

33

3. Promoter Characterization

Figure 3-1 Promoter constructs in pJIT 166

Figure 3-2 Constructs JG1 (B) and JG2 (C) with pGreen0029 backbone

34

3. Promoter Characterization

In the next step, complete expression cassette from pJIT166 was excised by using endonucleases and cloned in pGreen0029 (a binary vector), at restriction sites SacI and XhoI which makes it ready for further cloning of genes under phloem specific promoter. The modified pGreen0029 vector was named JG1 (carrying NSP promoter) and JG2 (carrying CP promoter) (Figure 3.2). 2X35S promoter was used to compare the results of the phloem specific promoter.

After cloning, JG1 and JG2 constructs were confirmed by restriction endonucleases and transformed into Agrobacterium by electroporation following the protocol mentioned in Chapter 2.

3.2.2 Plant Infiltration Single colony culture was established and confirmed by PCR using sequence specific primers (Table 3.1). Culture was centrifuged in a 50 mL falcon tube. Pellet was re-

° suspended in 10 mM MgCl2, added 20 µM acetosyriyngone and kept overnight at 4 C. N. tabacum plants were infiltrated with activated culture using 5cc BD syringe and kept under controlled conditions of 28 ± 2°C and 16 L/8 D for 4-5 days.

3.2.3 Histochemical Staining Assay GUS (β-glucuronidase) was used as a reporter gene to check promoter efficiency. 5-dpi leaves were detached and vacuum infiltrated with sodium phosphate buffer (50 mM and pH 7.0) containing Triton-X100, EDTA and X-Gluc (a substrate for β- glucuronidase) [181]. Leaves were incubated in buffer overnight at 37°C. After incubation buffer was discarded and leaves were bleached with 100% ethanol. Bleaching process was repeated until the removal of all chlorophyll and blue GUS staining became clearly visible.

3.4 Results The IR for NSP and CP promoters was successfully amplified, cloned and sequenced. Homology of the sequences was checked by performing BLAST search analysis. Sequences showed 100% homology with DNA-N (AM418568) and DNA-S (AM418566) of BBTV Pakistan isolates [135].

35

3. Promoter Characterization

3.4.1 Promoters Analysis Sequences of both highly expressed promoters were analyzed by PlantCARE to check its cis-regulatory elements. All the important motifs e.g. A-Box, G-Box, G-box, GC- motif, CAAT-Box and TATA-Box etc. were found in the proposed promoter region. All these elements are necessary for a sequence to act as a promoter (Figure 3.3 and 3.4).

3.4.2 Promoter Cloning and GUS-Assay After cloning, JG1 and JG2 were confirmed by using restriction endonucleases (Figure 3.5). Constructs were transformed into GV3101 followed by the infiltration in N. tabacum. GUS assay was performed showing interesting results. 2X35S promoter was used as positive control and pGreen0029 as negative control, to compare the BBTV promoters result.

As 35S promoter is a constitutive promoter, it showed GUS expression in whole inoculated patch constitutively. BBTV promoters showed GUS expression only in vascular cells but expression by NSP promoter was quite strong as compared to that of CP promoter (Figure 3.6). pGreen0029 (-ve control) did not show GUS gene expression, which proves that there is no role of vector sequence in GUS expression. Detached leaves were studied in detail under light microscope to see the expression at cellular level. Both of these promoters can further be used in future studies or for expression of genes in vascular tissue.

36

3. Promoter Characterization

Figure 3-3 NSP promoter analysis by PlantCARE

37

3. Promoter Characterization

Figure 3-4 CP promoter analysis by PlantCARE

38

3. Promoter Characterization

Figure 3-5 CP (a) and NSP (b) promoters cloned in pGreen 0029

39

3. Promoter Characterization

Figure 3-6 GUS-Histochemical staining assay for BBTV promoter expression; microscopic view (a) positive control 2X 35S promoter (b) negative control pGreen0029 (c) NSP promoter (d) CP promoter

40

3. Promoter Characterization

3.5 Discussion In the present study, two BBTV components proposed promoter region have been checked to get the phloem specific expression of reporter gene (GUS). Promoter activity was checked by GUS reporter gene and studied by using light microscope (Figure 3.6). Promoter isolated from NSP was quite strong almost 2-fold high as compared to the CP promoter. Both promoters showed expression in a phloem specific manner as compared to the control promoter (2X35S).

This approach was specifically used to find some new promoters to express toxin proteins in the phloem cells to control sap sucking insects. So that, insects feed on phloem sap they also engulf toxin proteins. In the case of a constitutive promoter, like CaMV 35S promoter, it expresses almost in all cells of the plant. It’s a drawback for such kind of promoters, they cannot efficiently control the phloem feeding insect pests. Another major drawback of such promoters is the continuous and over expression of toxins under such promoters produces resistance in insects against toxins as many of the insects have been developed resistance against Bt toxins [21]. So, it is very important to express the toxins in the target tissue for target insects to avoid resistance in insects against toxins and to secure non-target insects. It is obvious, from promoter sequence analysis by PlantCARE, promoters do not express in the seedlings. So, seeds of the crops are free from toxins and edible for other organisms. Using such important promoters may facilitate us for the expression of further important genes.

Three phloem specific promoters WDV, CsSUS and AtSuc2 were isolated and used to generate transgenic citrus. GUS assay performed with transgenic citrus leaves showed expression highly confined to the phloem [182]. To get phloem specific expression, three promoters AtSuc2 (Arabidopsis thaliana sucrose transporter 2), AtPP2 (Arabidopsis thaliana phloem protein) and CsPP2 (Citrus phloem protein 2) were expressed in sweet citrus by plant transformation. Phloem specific expression of promoters was experienced by GUS Histochemical staining assay [183]. The expression derived from AtSUC2, RolC, RSsI and RTBV promoters was found to be phloem specific in the GUS staining assay performed with transgenic lines of Mexican lime [184]. A putative promoter region isolated from Tomato yellow leaf curl China virus (TYLCCV) DNAβ was found to be phloem specific when expressed transiently and transgenically in N. benthamiana [185]. Pumpkin PP2 gene promoter showed

41

3. Promoter Characterization

phloem specific expression by GUS and LUC assay, when characterized by deletion at 5’ end [186].

Different phloem specific promoters have already been isolated and used to express the insecticidal genes to control pests. RSsI and RolC promoters used to express Hvt toxin and transgenic plants found resistant against H. armigera [187]. AsusI promoter was used to express ASAII and ASAL genes in N. tabacum. Transgenes were found to be effective to control phloem feeder, Myzus N. e [139].

In this study, the isolated promoters were used further to express insecticidal genes to control phloem feeders. New promoters can also be studied to express different genes in a phloem specific manner.

42

4. Expression of Insecticidal Genes under Phloem Specific Promoter

4.1 Introduction Chemical pesticides have commonly been used for the control of insect pests over the past many decades. Many of insecticides are recalcitrant, they not only pollute the environment but also show negative effects towards vertebrates including humans and non-target insects on ingestion [14]. It needs to develop new strategies to cope with highly resistant insect species as well as to avoid environmental damage caused by these agro-chemicals. One approach is to produce transgenic crops expressing insecticidal toxins as genetically engineered potato, corn, and cotton expressing δ-endotoxins from Bacillus thuringiensis have been developed [189].

Bacillus thuringiensis is the most common organism used as a bioinsecticide for Cry proteins production, to date [21]. δ-endotoxins crystals are proteolized and dissolved in the insect gut, bind to the cells of epithelial membrane in order to form ion channels that induce cell osmotic lyses and cause death of the insect [22]. Resistance have been developed against Bt toxins because of extensive use of Bt gene in many crops [21]. This technology has already exceeded the predicted time span that typically passes in the field before resistance to most conventional neurotoxin pesticides emerges [21, 23].

Constitutively expressed Bt genes have been very successful, but in some cases tissue specific expression is a better option, for example in epidermal cells, which first come under attack from insects or in the phloem for sap sucking insects [24]. In this regard Hemipteran insects cause great damage to crops by taking up the phloem sap and can cause blockage of phloem vessels. These insects also serve as vectors for more than 200 plant viruses [139]. Consequently, resistance should be developed in phloem cells by expressing genes under phloem specific promoters, for phloem feeding insects, to directly affect the insects. Using the approach to express insecticidal genes in tissue specific manner also reduces the metabolic load on plant.

43

4. Expression of Insecticidal Genes under Phloem Specific Promoter

This necessitates the identification and characterization of efficient promoters for tissue specific expression.

Over the past few years, several phloem-specific promoters have been utilized to develop genetically superior plants [126]. Two such promoters are RSs1 and rolC. The RSs1 has already been used to regulate the expression of β-glucuronidase (GUS) gene [127], GNA gene [128, 129] and ASAL gene [130] in a phloem-specific manner. The rolC has been used for the phloem-specific expression of GUS gene in transgenic tobacco [132, 133] and in transgenic rice [134].

The transgenic plants expressing Cry proteins represent one of the most important advances in agricultural biotechnology [15]. A variety of long term strategies are being developed in order to avoid the insect resistance. One option is pyramiding the expression of various Cry genes in plants [16, 17] or combining the use of a variety of molecules to express hybrid Cry proteins that manifest greater toxic effect on insects [18, 19]

A potentially helpful toxin in this perspective is ω-atracotoxin [191]. ω- atracotoxin is a specific antagonist of insect calcium channels that was recently isolated from Australian funnel web spiders by screening their venom for activity against cotton bollworms [191, 192]. Spiders are active predators of insects and most of them produce venom to detain their prey. But all spiders are not poisonous. Scientists are attracted by the diversity of spiders to find out insecticidal proteins and neurotoxins [35]. Insect resistant tobacco, expressing a poisonous toxin from the Hadronyche versuta spider, has also been reported [180].

The venom from insectivorous spiders contains a complex mixture of molecules and peptides with a wide range of mechanisms for activity at a biochemical level, thus having a great biotechnological potential for improving resistance to insects. An important constituent of the spider venom consists of 4–10 kDa strong ligand peptides, that are tightly folded by means of various intramolecular disulfide bridges and which include a great diversity of antagonists, acting on the ion channels of excitable membranes [193, 194].

Spider venoms are complex mixtures of proteins, peptides and low molecular mass organic molecules. LMM compounds often found in the spider venom are free

44

4. Expression of Insecticidal Genes under Phloem Specific Promoter acids, free amino acids, glucose, biogenic amines and neurotransmitters [36]. Several of the LMM are neurotransmitters while others have the ability to block ion channel at neuronal level. These low molecular mass neurotoxins have great potential to explore nervous system as neurochemical tool. They may constitute new models in the drug screening field for pharmaceutical and agrochemical industries, as well [37].

Plant lectins belong to a very heterogeneous group of proteins but they all share an important biological property to recognize and bind to specific carbohydrate moieties. Different analysis of lectins at their genome and transcriptome level reveal that plant lectins are present ubiquitously in the kingdom Planta. To date, hundreds of plant lectins have been purified from different plant species [54]. Lectins can be found in all kingdoms of life ranging from prokaryotes to eukaryotes. Carbohydrate recognition occurs in a large number of different biological contexts [195]. Lectins often are found in the storage plant tissues (seeds, bulbs etc.) at high concentration. The precise function of lectins is still unknown. Due to their high concentration and the tissue source, these proteins are considered as storage proteins. Additionally, they also serve as defense molecules against pathogens [54].

GNA is the first plant lectin known to be active against hemipteran insects. It is one of the most studied lectins and considered to be purified easily from plant [127, 196]. The first candidate lectin receptors are located in the digestive tract of insect. In this case the lectin is able to pass through the gut epithelium barrier [195].

It has been reported that expression can be regulated using transcription factors or chemical induction and with this technique, it is possible to create within plant refuges where parts of the plants do not express the genes and act as non-GM refuge [25]. There are other options e.g. esculentin from , chicken avidin and protease inhibitors from both animals and plants to make insect resistant transgenic plants [197-200].

The goal is to build some effective strategy to control phloem sap feeding insects by gene pyramiding as well as tissue specific expression of insecticidal genes. Thus, for this purpose, Hvt and lectin toxins were used to express under phloem specific promoters isolated from monocot infecting BBTV, in two individual cassettes.

45

4. Expression of Insecticidal Genes under Phloem Specific Promoter

4.2 Methodology Whole of the work, to make transgenic plants and evaluate them against sucking, was planned in two parts.

In first part, transgenic plants expressing Hvt toxin gene under phloem specific promoter were produced. Transgenic plants were challenged by sucking pest and processed for further analysis. In the second part, two insecticidal genes Hvt and lectin were expressed under phloem specific promoters in individual cassettes and cloned in a single vector, allowing to express both genes. Transgenic plants were challenged by multiple sucking pests and other analysis were done for genes presence and expression confirmation.

4.2.1 Insecticidal Gene Constructs Hvt (Accession no. AJ938032) amplified by PCR using gene specific primers (Table # 4.1) and cloned under NSP promoter of BBTV in pGreen0029 vector, using restriction sites HindIII and XbaI. For this purpose, the modified vector built in Chapter 3 containing the phloem specific promoter NSP from BBTV was used for further cloning.

For double gene constructs, two individual cassettes were synthesized by BIO BASIC INC. One cassette containing NSP promoter, Hvt and nos terminator, while other consists of CP promoter, lectin (Accession no. DQ255944) and nos terminator. Both cassettes were cloned in pGreen0029 at restriction sites SacI and XhoI.

4.3 Plant Transformation The final construct in binary vector pGreen0029 was transformed in A. tumefaciens strain GV3101 by the method of electroporation. Single colony culture, after confirmation by PCR, was prepared in 50 mL LB containing antibiotics (50 µg/mL kanamycin, 10 µg/mL tetracycline and 25 µg/mL rifampicin) at 28°C and 180 rpm for 48 h. N. tabacum was transformed by leaf disc method using the modified protocol [172]. MS medium was supplemented by 50 mg/L kanamycin, 250 mg/L cefotaxime, 250 µg/L NAA and 1 mg/L BAP. The putative transformants were shifted to soil for further analysis.

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4. Expression of Insecticidal Genes under Phloem Specific Promoter

4.4 Transgene Analysis 4.4.1 DNA Extraction A good quality DNA with concentration 2-3 µg was extracted from transgenic plants by using C-TAB buffer as mentioned in Chapter 2. DNA was quantified using Nanodrop and 50 ng DNA was used as template to carry out PCR. PCR positive lines were selected and proceeded for further analysis.

4.4.2 RNA Extraction The lines with PCR positive results were selected for total RNA extraction to check either genes are being transcribed or not. Plant RNA Purification Reagent (Invitrogen Cat no. 12322-012) was used to extract high quality RNA from transgenic plants following the protocol mentioned in Chapter 2. Total RNA was run on 2% gel to check its quality and quantified using Nanodrop. A quantified amount of RNA was used to synthesize cDNA, afterwards.

4.4.3 cDNA Synthesis cDNA was synthesized using total RNA extracted from transgenic and control plants. After quantification of samples 2 µg of total RNA was used to synthesize cDNA. cDNA was synthesized by using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific Cat. # K1632). Protocol was followed as mentioned in Chapter 2 to synthesize cDNA. cDNA synthesis was examined by doing PCR using gene specific primers.

4.4.4 Semi-quantitative PCR PCR positive lines were selected for the extraction of total RNA. Total RNA was extracted using Invitrogen Plant reagent and cDNA was synthesized. cDNA was tested by carrying out PCR using gene specific primer (Table 4.1) and further used for semi- quantitative PCR and qPCR. In order to verify the transcription of the genes, semi- quantitative PCR was done using cDNA as template. The reaction was prepared using 500 ng of cDNA and gene specific primers. A set of 5 reaction cycles was set and reaction was subjected to run in Thermal cycler for 15, 20, 25, 30 and 35 cycles.

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4. Expression of Insecticidal Genes under Phloem Specific Promoter

NSP promoter Hvt terminator

Figure 4-1 Hvt construct in pGreen0029 backbone

NSP promoter Hvt terminator

Figure 4-2 Cassette 1 carrying promoter, Hvt and terminator

CP promoter lectin terminator

Figure 4-3 Cassette 2 carrying promoter, lectin and terminator

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4. Expression of Insecticidal Genes under Phloem Specific Promoter

Table 4.1 Specific primers used for insecticidal genes amplification

Primer Name Primer Sequence

Hvt Forward 5’-CG AAGCTTATGTCACCAACTTGCATACC-‘3

Hvt Reverse 5’-ACTCTAGATTAATCGCATCTTTTTACGG-‘3

Lectin Forward 5’-CCAAGCTTATGGCCAGGAACCTACTGAC-‘3

Lectin Reverse 5’-ACTCTAGATTAGTAGGTCCAGTAGAACC-‘3

Table 4.2 Primers sequence for qPCR analysis

Primer Name Primer Sequence

Hvt qPCR Forward 5’-CG AAGCTTATGTCACCAACTTGCATACC-‘3

Hvt qPCR Reverse 5’-ACTCTAGATTAATCGCATCTTTTTACGG-‘3

Lectin qPCR Forward 5’-AGAAACGTATTGGTGAACAA-‘3

Lectin qPCR Reverse 5’-TTTCCTGTACGTACCAGTAG -‘3

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4. Expression of Insecticidal Genes under Phloem Specific Promoter

4.4.5 Real-time PCR for Transgene Analysis Real-time PCR or quantitative PCR (qPCR) was performed to check the expression of transgenes in a relative manner. Here, SYBER Green technology was used to study the gene expression from transgenic plants. All the optimization and experiments were carried out using iQTM5 iCycler BIO-RAD equipment that automatically calculates the

CT values of the samples and sets other parameters for experiment. 18S ribosomal RNA gene was used as a reference and its expression was compared with the expression of transgenes. Gene specific primers as mentioned in the Table 4.2 were used for the real- time PCR studies.

Reaction mixture (25 µL) was consisted of 12.5 µL of 1X iQ SYBER Green Supermix, 50 ng of sample cDNA, and 5 pm of gene specific primers, each (mentioned in the Table 4.2). PCR reactions were carried out into a 96-well optical plate in an iQTM5 iCycler BIO-RAD and all the samples were run in triplicate. PCR was carried out using the profile 1 cycle at 95°C for 5 min followed by 40 cycles, each consisting of 95°C for 30 s (denaturation), 55°C for 30 s (annealing) and 72°C for 45 s (extension) followed by a melt curve analysis starting from 95°C with a decrease in temperature 0.5°C/10 s (85 cycles).

4.5 Insect Rearing Multiple sucking pests e.g. mealybugs, aphids and whiteflies were used to check the effect of toxic proteins. Insects were reared in glass house under controlled conditions of 37 ± 2°C, 16 L/8 D and approximately 65% humidity. Insects were collected from field and allowed to feed and multiply on cotton plants in glass house. Insects settled very well on cotton and multiplied enormously.

4.5.1 Insect Bioassays

Transgenic plants were grown in growth room under controlled conditions of 26 ± 2°C and 16 L/8 D. After confirmation by PCR, 8 lines of double gene construct and 5 lines of single gene construct, were grown in controlled conditions at T1 stage. 5 plants of each line at 3-4 leaves stage were used for bioassay. Different phloem feeding insects as mealybugs, aphids and whiteflies were allowed to feed on plants to check the effects of toxin proteins. Equal no. of control plants (wild type N. tabacum) was also used to get comparable mortality data.

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4.6 Data Analysis The data was analyzed statistically using ANOVA and Tukey’s test.

4.7 Results 4.7.1 Cloning of Hvt and Plant Transformation Initially, Hvt gene was successfully amplified using gene specific primers and sequenced. Sequence was analyzed by BLAST search analysis NCBI. It showed 100% homology to Australian funnel web spider Hadronyche versuta ω-atracotoxin. Gene was further cloned in the vector pGreen0029 under BBTV NSP promoter using the restriction sites Hind III and Xba I.

The construct was confirmed by using restriction endonucleases as explained in Chapter 2 and transformed into GV3101 (A. tumifaciens) by electroporation following the protocol mention in Chapter 2. After Agrobacterium transformation, single colony culture was established, experienced through PCR using gene specific primers and used for tissue culture purpose.

Tissue culture was done by using N. tabacum leaf explants and the whole procedure carried out as elaborated in Chapter 2, when plants get stabilization in the pots, they were exposed to the insect pests (mealybugs).

Bioassay Results Bioassays were performed in order to assess the effectiveness of the toxin protein against sucking insect pests. For this purpose, a model sucking pest mealybug was used.

Randomly, 5 plants of each transgenic line at T0 stage were chosen to perform bioassays and kept in glass house.

Fifteen individuals of mealybugs (mix population) were allowed to feed on each plant in glasshouse under controlled conditions. Mortality data of insects was collected after every 24 h. Insects take some time to adopt the host and during initial 24 h insects were adapted to the host. Bioassays were done in triplicate and the data is being presented in comparative manner.

Mealybug feeding on transgenic plants did not show any significant change up to 48 h but found quite lazy (with slow movements) on SGC 21 and SGC 22 plants. After 3 days, approximately 18% of mealybugs (nymphs) were found dead on SGC 21

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4. Expression of Insecticidal Genes under Phloem Specific Promoter following 11% and 10% mortalities on SGC 22 and SGC 3, respectively. Only 6.7% mealybugs were dead on SGC 17 while no dead individual found on SGC 13. After 4 days, 35% of mealybugs were dead on SGC 21 and approximately 29% on SGC 22. While SGC 17, SGC 3 and SGC 13 showed 20%, 17% and 8% deaths, respectively. After 5 days, 71% mortalities were recorded on SGC 21 and 66.67% on SGC 22. Other lines showed slow mortality rates as 33.34%, 26.67% and 20% on SGC 3, SGC 17 and SGC 13 respectively. After 6 days, 100% mealybugs (15/15) were dead on transgenic tobacco lines SGC 21 and SGC 22. While on other lines, max. 61% mortalities were recorded even after 7-days. The adult individuals survived upto 10-days and also multiplied on plants. But the new ones (nymphs) could not grow to the adult stage and found dead.

Physical characters like body mass lose, browning of bodies with time, stretchening of legs outwards was observed. Adult insects spent more time on plants multiplied, they multiplied in a regular manner but new ones did not grow well and found dead due to toxin feeding. Dead nymphs were also turned to brown.

Insects feeding on healthy plants show normal behavior in all aspects. Body mass of the insects was increased and they multiplied enormously in a healthy way. Adults produced a normal ovisac filled with eggs during multiplication. In case of healthy control, mealybugs were kept on multiplying as much as time they spent on plant in a regular manner. While on transgenic plants adults were also found dead and their multiplication rate was lower than those on healthy controls. In a long run toxin may also effect the fecundity of mealybugs but they were not allowed to survive too long after up taking toxin protein.

ANOVA and Tukey’s analysis reveal the significance of data with p-value < 0.01.

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Figure 4-4 Graph representing % mean mortality of mealybugs with time by the effect of Hvt

Figure 4-5 Bioassay performed with mealybugs (a) multiplying on healthy N. tabacum (b) plant expressing Hvt toxin (c) some mealybugs multiplied on transgenic plants but nymphs died

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4.7.2 Double Gene Construct: In the second step, two cassettes with Hvt and lectin genes were successfully synthesized by Bio Basic INC.

Both cassettes were cloned in the binary vector pGreen0029 by means of restriction endonucleases. For confirmation, appropriate endonucleases were used and construct was also checked before transformation. Homology of all the sequences was checked by performing BLAST search analysis. Hvt gene sequence showed 100% identity with Hadronyche versuta spider neurotoxin [180] and lectin gene sequence showed 100% identity with Allium cepa mannose-binding insecticidal leaf lectin [72]. While promoters were identical to BBTV component IR.

Final construct was further processed for stable transformation in N. tabacum.

Figure 4-6 Final Double Gene Construct (DGC) used for Plant Transformation

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4.7.3 Transgene Analysis Transgenic plants were tested for the confirmation (presence and expression) of the transgenes. Different tests were performed for this purpose.

DNA Extraction and PCR DNA of good quality was extracted using C-TAB buffer method and run on gel to check its quality. DNA was confirmed for the presence of gene by PCR using gene specific primers and nptII gene (coding kanamycin gene) primers.

Figure 4-7 Transgene Analysis (a) DNA extraction from transgenic plants (b) PCR using nptII primers (c) PCR using gene specific primers

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4. Expression of Insecticidal Genes under Phloem Specific Promoter cDNA Synthesis and semi-quantitative PCR cDNA was synthesized using Revert Aid-H-minus strand synthesis kit. cDNA was confirmed by doing PCR using gene specific primers. PCR positive samples were also confirmed by semi-quantitative PCR. After PCR, 5 µL of reaction was loaded in the 1% agarose gel and got the results in ascending order as the no. of cycle increases. Samples with positive PCR were subjected to analyze by qPCR.

Figure 4-8 Transgene Analysis (a) RNA extraction from transgenic plants (b) PCR using gene specific primers from cDNA

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4. Expression of Insecticidal Genes under Phloem Specific Promoter

Figure 4-9 Transgene Analysis (a) semi-quantitative PCR for Hvt (b) semi- quantitative PCR for lectin

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4. Expression of Insecticidal Genes under Phloem Specific Promoter

4.8 Insect Bioassay Bioassays were performed in order to assess the effectiveness of the toxin proteins against a number of insect pests. For this purpose, three different sucking pests mealybugs, aphid and whiteflies (major threat to cotton) were used. Randomly, 5 plants of each transgenic line were selected to perform bioassays and kept in glass house. An equal no. of healthy controls was also utilized. Two best lines from Hvt transgenic plants (the first step towards this study) were chosen to study and compared with

Double Gene Construct plants for effectiveness of toxin at T1 stage.

Bioassays were performed under controlled conditions of 28 ± 3°C, 16 L/8 D and approximately 65% humidity. Insects were allowed to multiply on cotton plants under controlled conditions and then exposed to transgenic plants. Mixed population of mealybugs, aphids and whitefly were collected from greenhouse.

All the experiments were performed in glass house under controlled conditions. The plants expressing both genes (DGC) revealed more interesting results than the plants expressing single toxin (Hvt/SGC).

4.8.1 Mealybugs (Phenacoccus solenopsis) Fifteen individuals of mealybugs (mix population) were exposed to the transgenic plants to feed on. Mortality data of insects was collected after every 24 h. Insects take some time to adopt the host and during initial 24 h insects were adapted to the host. All the experiments were repeated 3times, here data is presented in a comparative manner.

Mealybugs feeding on SGC and DGC plants did not show any significant change during first 24 h feeding. After 48 h toxins were proved to be little toxic to the pests. Almost 28% of mealybugs (nymphs) feeding on DGC 6 and 15 and 10% on DGC 5 and 9 were dead. But no death was observed on SGC plants. After 72 h approximately 65% mealybugs on DGC 6 and 15, approximately 35% insects on DGC 5 and 9 were found dead. After 96 h 100% (15/15) deaths of mealybugs feeding on plants DGC 6 and 15 were observed as compared to the approximately 80% mortalities on DGC 5 and 9 and 30% mortalities on SGC plants. After 120 h, 100% mealybugs (15/15) were found dead on DGC 5 and 9 as compared to approximately70% deaths on SGC plants.

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While rest of mealybugs were dead after 144 h. ANOVA and Tukey’s analysis shows data are highly significant with p-value <0.01 (Fig. 4-10 & 4-11).

Mealybugs fed upon the toxic plants showed symptoms of interest as browning of insect bodies and loss of body mass. The young individuals also multiplied on few plants with a normal ovisac. But the young ones could not grow to the next instar and found dead while feeding on toxin proteins. The dead ones found on the plants expressing single gene had erected bodies and their legs were stretched outwards while those found on the plants expressing two toxins had inwardly folded bodies or legs were folded inside.

In case of healthy plants, all the insects feeding on them were quite happy. They gained body mass and multiplied enormously. The young ones/ nymphs were grown to the next instar.

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Figure 4-10 Graph representing % mean mortality of mealybugs with time by the effect of Hvt and lectin toxins

Figure 4-11 Bioassay performed with mealybugs (a) plant expressing single protein Hvt (b) plant expressing two toxin proteins (c) multiplying on healthy N. tabacum

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4.8.2 Aphids (Myzus persicae) In case of aphids, 25 aphids were exposed to the transgenic plants. Data was again collected on the basis of 24 h. During first 24 h, aphids feeding on transgenic plants expressing toxin proteins, did not show any remarkable effect of toxins. Studies done after 48 h reveal almost 80% of individuals (mostly nymphs) were found dead on DGC 6 and 15 and DGC 5 and 9 showed about 40% mortalities. While very few deaths were observed on SGC plants i.e. approximately 5%. After 72 h, almost 100% individuals (22/25) were found dead on the plants DGC 6 and 15. While approximately 70% and approximately 30% deaths were observed on plants DGC 5 and 9 and SGC plants, respectively. After 96 h, approximately100% individuals (23/25) were dead on DGC 5 and 9 in comparison with 65% deaths on SGC plants. After 120 h, 100% aphids (23/25) were dead on SGC plants. ANOVA and Tukey’s analysis shows data is highly significant with p-value <0.01 (Fig. 4-12 & 4-13).

Dead insects exhibit the symptoms of blackening of body, loss of body mass and stiffness in body. The insects killed by up taking a single toxin had stretched bodies and legs were stretched outwards while those had been killed by two toxin proteins were not so stretched and the legs were folded inwards.

Insects feeding on healthy controls were grown normally. They multiplied in a healthy way and body mass was increased. The aphids on initial instars were moved to the next growth phase.

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Figure 4-12 Graph representing % mean mortality of aphids with time by the effect of Hvt and lectin toxins

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Figure 4-13 Bioassay performed with aphids (a) plant expressing single protein Hvt (b) plant expressing two toxin proteins (c) alive and multiplying on healthy N. tabacum (d) exoskeletons of aphids on healthy plants

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4.8.3 Whiteflies (Bemisia tabaci) Twenty whiteflies, on each plant, were exposed to the transgenic plants. Once again data were collected with the intervals of 24 h. The insects showed promising results after initial 24 h. More than 50% insects were found dead on the plants DGC 15 followed by more than 40% mortalities in DGC 6 plants. While the insects on other plants were found lazy and had some abnormal movements. After 48 h, DGC 6 and 15 approximately 100% individuals were dead i.e. 18/20, a few were escaped. Whilst DGC 5 and 9 and SGC plants showed approximately 50% and approximately 56% mortalities, respectively. But after 72 h feeding, approximately 100% whiteflies (17/20) were dead on DGC and SGC (18/20) plants. ANOVA and Tukey’s analysis shows data is highly significant with p-value <0.01 (Fig. 4-14 & 4-15).

The dead insects showed important physical characters. Initially, the insects have shown jerky and shaky movements of the body and sometime they fold their bodies inside while feeding on DGC plants. But, the insects fed on SGC plants showed only jerky and shaky movement in the body. Like other insects, whiteflies dead by single toxin protein had stretched bodies and legs were stretched outwards while those found on DGS expressing proteins had legs folded inwards wrapped around the body as clearly shown in Figure 4-15b.

The behavior of the insects feeding on healthy plants was normal in all aspects. They fed on plants normally and some of them also laid eggs on the plants.

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Figure 4-14 Graph representing % mean mortality of whiteflies with time by the effect of Hvt and lectin toxins

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4. Expression of Insecticidal Genes under Phloem Specific Promoter

Figure 4-15 Bioassay performed with whitefly fed on toxin proteins (a) plant expressing single protein Hvt (b) plant expressing two toxin proteins (c) whitefly feeding on healthy N. tabacum (d) whiteflies laid eggs on healthy N. tabacum

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4.9 Real-time PCR By using the gene specific primers (Table 4.2) real-time PCR experiment was carried out. The whole experiment was performed in a triplicate manner.

A single peak of melt curve was obtained as a result of qPCR for the gene of interests Hvt and lectin, individually. It indicates the specificity of the experiment that any nonspecific amplification was not observed as a result of PCR as shown in the Fig. 4-16 and 4-18.

As this experiment was performed in a relative manner, interesting results were produced, comparable to bioassay results. In case of Hvt gene, all the plants showed good expression of protein but in case of DGC 15 (1.000), it is almost 2-fold higher than the DGC 6 (0.704) and SGC 22 (0.682) while in comparison with expression level of Hvt DGC 5 (0.237), 9 (0.288) and SGC 21 (0.265) was almost 4-fold higher. Where the gene was expressed at very low level and expression was almost same as shown in the Figure. 4.17. While the control healthy plants (C & H) did not show any expression.

In case of lectin, the highest level of expression was observed in DGC 6. The lectin expression in DGC 6 (1.000) was almost 2-fold higher in comparison with other lines DGC 5 (0.609), DGC 9 (0.666) and DGC 15 (0.662), making it more toxic towards the insect pests. While the healthy controls and Hvt transgenic plants 21 and 22 did not show any expression. The gene expression can be observed easily from the qPCR graph shown in the Figure: 4.19.

ANOVA and Tukey’s analysis shows data are highly significant with p-value <0.01.

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Figure 4-16 Melt Curve Analysis for Hvt

Figure 4-17 Normalized gene expression ratio for Hvt as analyzed by real-time PCR

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4. Expression of Insecticidal Genes under Phloem Specific Promoter

Figure 4-18 Melt Curve Analysis for lectin

Figure 4-19 Normalized gene expression ratio for lectin as analyzed by real-time PCR

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4 Expression of Insecticidal Genes under Phloem specific Expression

4.10 Discussion The data presented the results for the expression of two insecticidal proteins in a phloem specific manner. The most important thing regarding promoter is expression. It expresses only in the vascular cells and does not affect dicots as it has been derived from a monocot infecting virus promoter. This approach was specifically used to control the sap sucking insects. So, when insects feed on phloem sap they also engulf toxin proteins. In the case of a constitutive promoter, like CaMV 35S promoter, it expresses almost all cells of the plant but its expression is rather low in the phloem cells as compared to the other tissues of plant and as well as to the expression of the phloem specific promoter. It’s a drawback for such kind of promoters as they cannot efficiently control the phloem feeding insect pests. Another major drawback of such promoters is that, because of continuous and over expression of toxins under such promoters produces resistance in insects against toxins as many of the insects have been developed resistance against Bt toxins [21]. So, it’s very important to express the toxins in the target tissue for target insects to avoid resistance in insects against toxins and also to secure non-target insects.

Gene pyramiding is an efficient technique to limit the escape of insects for their effective control. Most promising results were revealed by whiteflies feeding on the transgenic plants. Whiteflies feeding on the transgenic plants took very small time to get stabilize on the plants and show the effect of toxins. Insects killed by single protein (Hvt, a neurotoxin) had slow mortality rate, as they were examined with 24 h interval. Dead insects showed low body mass (lack of feeding) and browning of bodies along with their legs were stretched outwards. While the insects feeding on DGC plants were killed more efficiently and they exhibited different physical characters as their legs were folded inward (as shown in the Figures) but they exhibited rapid decrease in body mass and browning of bodies. It was assumed that these physical changes of dead insects as legs stretched inwards or outwards are due to the effect of toxins. In case of a single protein (Hvt), a neurotoxin, it affects or kills nervous system of the insect which lead to the stretchiness in the insect body. While in case of double protein, a neurotoxin along with lectin, here lectin acts upon the digestive tract of the insect, so along the jerky and shaky movement of insect it was also observed that insects wrap their legs around their stomach and their body is folded inside. That’s why in this case the legs of dead insects were found folded inwards the body. Insect’s fecundity was also affected

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4 Expression of Insecticidal Genes under Phloem specific Expression by the toxins as they were observed being multiplied on the control plants. Some of the whiteflies laid eggs on controls while aphids and mealybugs were found to be transformed into the next instar.

Detached leaf assays performed with Hvt gene expressed under CaMV 2X35S promoter and phloem specific promoter rolC has been proved effective against H. armigera and S. littoralis larvae [180] and S. frugiperda [187], respectively. Plant with high level of gene expression has also proved to be more toxic to the insect pests, experienced by the qPCR results [187]. Purified Hvt also caused the death of H. virescens and S. littoralis larvae while expressed under 2X 35S promoter in tobacco and cotton plants. But they did not negatively affect the non-target insect C. carnea and C. septempunctata were found normal in all aspects as larval and pupal development and body mass. Worker bees were also found to be safe when they were allowed to feed on a diet containing 40 µg/mL of Hvt [201].

Growth retardation, decrease in fecundity and decrease in the survival rate of nymph aphids (Myzus N. e) was observed when allowed to feed on the transgenic plants expressing ASAL [188]. Transgenic line of rice expressing ASAL under 35S promoter had a remarkable effect on the growth of sap sucking insects viz. green leafhopper (GLH), brown planthopper (BPH) and white backed planthopper (WBPH). The growth rate of the insects was reduced to 83%, 84% and 77%, respectively as compared to the control plants. Developmental delay, loss of fecundity and loss of feeding ability was also observed in the insects (GLH, BPH and WBPH) as compared to those feeding on control plants [71]. In planta expression of ASAL under 35S promoter was proved to be toxic against green leafhopper (GLH) and brown planthopper (BPH). ASAL increased the nymph mortality rate, decreased body mass and reduced the fecundity of GLH and BPH [202].

Transgenic rice plants expressing multiple genes Cry1Ac, Cry2A and GNA were proved to be resistant against rice leaf folder, yellow stem borer and brown planthopper with minimum plant damage as compared to the control plants where they destroyed the whole plant [17]. Cry1Ac and Cry1Ie were found to be more toxic than Cry1Ac or Cry1Ie alone when transgenic leaf discs expressing the toxins were exposed to cotton bollworm larvae. The growth of cotton bollworm larvae feeding on the leaves expressing both genes (Cry1Ac and Cry1Ie) was halted and dead bodies were found

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4 Expression of Insecticidal Genes under Phloem specific Expression earlier than those of feeding on leaves expressing Cry1Ac and Cry1Ie alone [203]. H. armigera larvae could not survive while feeding on transgenic tobacco lines expressing protease inhibitor (CeCPI) and sporamin Brown dead bodies of larvae were collected with loss of weight as compared to those feeding on control plants [204]. Larvae of Plutella xylostella were found dead (approximately 75-80%) when fed on transgenic Brassica napus L. expressing chitinase and sporamin with decrease in body mass. The plants were also proved to be resistant against fungal attack [205]. Transgenic rice lines expressing ASAL and GNA (F4, F5 stage) were proved to be toxic for sap sucking insects BPH, GLH and WBPH as compared to the plants expressing ASAL or GNA, individually. Survival rate of insects on plants expressing individual gene was quite high as compared to that in the pyramided plants. A level of 20-40% more developmental arrest, decrease in fecundity and decrease in feeding was observed in pyramided plants as compared to the plants expressing single gene [206].

Hence, it is proven that expression of genes in target tissues and gene pyramiding are quite efficient techniques to control the insect pests. In future, other promoters and genes can be identified, characterized and used for pest control.

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5.1 Introduction By the use of chemical insecticides, it is possible to increase the agricultural yields by controlling insect pest but wide use of these nonspecific chemicals causes destruction of non-target insects, development of resistance in plants and environmental contamination. Due to this fact, since 1960s, bioinsecticide have received considerable attention as environment friendly and highly desirable alternatives [207, 208].

For the long term and efficient control of herbivores, transgenic plants are being produced with a capability of producing a combination of insecticidal proteins. Successful integration of multiple transgenes to produce pest resistance by gene stacking has been reported. Different strategies as conventional crosses between the lines having transgene or co-integration of multiple gene cassettes are in practice to achieve gene stacking.

It has also been proposed to produce a fusion protein from two different proteins and express it under single promoter (6, 25). Unlike gene stacking, this allows one step expression of transgenes with recombinant traits, multiple T-DNA and promoter insertions and coordinated expression of different recombinant proteins.

To create resistance in plants against pathogens and pests, different fusion proteins have been constructed. Usually fusion constructs comprise of δ-endotoxins from B. thuringiensis and/or other plant defense related proteins [6, 14]. A fusion protein integrating the Bt toxins Cry1B and Cry1Ab, for instance, was engineered to broaden the insecticidal spectrum of Bt toxin expressing lines derived from tropical maize varieties [7].

The venom isolated from a range of arachnids consists of a cocktail of biologically active substances including toxins. Spider venom consists of a variety of toxic polypeptides and paralyzes the prey feeding upon them. These polypeptide neurotoxins target neuronal ion channels and to a lesser extent neuronal receptors and 73

5. Production of Hybrid Proteins to Control Sucking Pests

Presynaptic membrane proteins [209, 210]. Neurotoxins isolated from the venoms of H. curta and P. tristis block neuromuscular transmission in Drosophila larvae by their action on calcium channels [35, 211, 212].

GNA is a plant lectin and shows its limited insecticidal activity for lepidopteron larvae. It has been shown in artificial diet and transgenic plant studies that GNA reduced larval weight gain and slowed down the developmental rate of first stadium Lacanobia oleracea larvae [76, 213]. It is still unclear that how GNA affects insect pests but it involves binding of the lectin to the glycoproteins of gut epithelium surface [214].

It has been studied previously that GNA is resistant to gut proteolysis and can be found in the hemolymph of insects if are orally exposed [103]. The ability of GNA to cross the gut epithelium gives this protein the potential to act as a carrier to deliver fused peptides to the circulatory system of target insect species. It has been recently demonstrated that GNA delivers a fused insect neuropeptide (Manduca sexta allatostatin: Manse-AS) to the hemolymph of lepidopteron larvae following oral administration [215]. A fusion protein containing GNA with the Manduca sexta allatostatin sequence fused to the C-terminus caused a significant reduction in the growth and feeding of fifth stadium L. oleracea larvae when incorporated into artificial diet and showed insecticidal activity to neonates.

Here, the construct was produced with the fusion of Australian funnel web spider neurotoxin Hvt and onion leaf lectin to produce a recombinant protein for a broad range of pest control. Fusion of two different genes caused different changes into the resultant protein including the conformational changes/tertiary structure of protein product. The possible structures of the proteins produced as a result of Hvt and lectin fusion are also proposed. Two proteins were designed keeping Hvt at N-terminus in one protein and Lectin at N-terminus in other. Protein models were proposed by using I-TASSER [225-228]. A PVX expression system was used to express the translationally fused proteins.

5.1.2 Potato virus X (PVX) Multiple RNA and DNA viruses have been modified and being used as vectors for overexpressing and/or silencing the genes in plants. Some virus based vectors used to produce heterologous proteins or peptides including vaccine antigens and antibodies in

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5. Production of Hybrid Proteins to Control Sucking Pests plant cell with commercial importance [141-143, 215, 225]. It is easy and cost effective to overexpress the genes in plants through virus based vectors rather than stable transformation [144-149]. Potato virus X (PVX) is among the top 10 most important plant viruses based on its economical and scientific importance [150]. PVX is used as a model to study the plant-virus interactions and to study the mechanisms of viral propagation as viral replication, cell to cell movement and suppression of post- transcriptional gene silencing (PTGS). PVX has a single stranded RNA genome of about 6430 nt [216]. PVX genome consists of five open reading frames (ORFs). ORF1 encodes 167 kDa RNA-dependent RNA polymerase (RdRp). Three partially overlapped ORFs encode movement proteins of 25 kDa TGBp1, 12 kDa TGBp2 and 8 kDa TGBp3 (Triple Gene Block). The last ORF5 encodes coat protein (CP) of 25 kDa. These genes are required for viral replication, encapsulation and cell to cell movement [151].

Figure 5-1 PVX Genome Organization

TGB protein is the largest and multifunctional protein. TGBp1 (a 25kDa protein) is essential to build PVX replication complexes [152] initiation of PVX virion translation and for PVX movement [153]. TGBp1 was first discovered RNA gene silencing viral suppressor [154]. It has been reported recently that TGBp1 can interact with AGO proteins and lead to the AGO1 degradation through proteasome pathway [217]. It is also capable to modify the plasmodesmata (PD) aperture to establish the transportation of PVX ribonucleoprotein complexes to the neighbor cells [156]. TGBp1 “gating” activity, i.e. its propensity to expand the plasmodesmata size to the exclusion limit (SEL), is presumably precede or concomitant with its accumulation at pitfield PD. TGBp1 functional characterization reveals that its PTGS suppressor activity can be impaired by mutation showing PVX movement is dependent on its PTGS activity while silencing suppression is not sufficient to allow the virus movement between the cells [157].

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To serve as expression vector PVX genome was cloned behind a T7 RNA polymerase promoter or 35S promoter derived from cauliflower mosaic virus (CaMV) [158]. This vector has been successfully used to express the foreign genes in planta [158-161]. A PVX based vector was used to express fusion gene construct.

5.2 Methodology Two translationally fused protein constructs of Hvt and lectin were designed in such a way that the in one construct Hvt was kept at N-terminus and lectin at C-terminus removing the stop codon of Hvt while in other construct lectin was kept at N-terminus and Hvt at C-terminus by removing the stop codon of lectin (Fig. 5-2 & Fig. 5-3). This strategy was planned to check which construct/gene orientation behaves better against the sucking pests.

Figure 5-2 Hvt at N-terminal and lectin at C-terminal

Figure 5-3 Lectin at N-terminal and Hvt at C-terminal

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5.3.1 Cloning of Fusion Gene Constructs Both of the translationally fused gene constructs were artificially synthesized by GenScript and received in pUC57 vector. The genes were restricted from pUC57 by using restriction endonucleases Sal I and Sma I. The destination/expression vector was also digested by the same enzymes. Digestion was confirmed by gel electrophoresis and fusion genes were ligated into the PVX after cleaning by phenol-chloroform extraction by using the method mentioned in Chapter 2. Final clones were confirmed by doing restriction of plasmid with the respective enzymes.

5.3.2 Agrobacterium Transformation After getting pure and confirmed clones of fusion genes, the final constructs were subjected to transform in A. tumefaciens competent cells. GV3101, a strain of Agrobacterium, was used for the transformation and transient expression in plants. Electro-competent cells of GV3101 were prepared and the constructs were transformed via electroporation. Colonies were picked after transformation and confirmed for the presence of construct by doing PCR using gene specific primers. PCR positive colonies were picked and cultured in 50 mL LB along with required antibiotics and incubated at 28°C and 180 rpm. This culture was further used for plants infiltration.

5.3.3 Plant Infiltration Culture was taken out from incubator and centrifuged into 50 mL polypropylene tubes at 12000 rpm for 10 min. Supernatant was discarded and pellet was re-suspended in 10 mM MgCl2. After resuspension acetosyriyngone was added in a concentration of 100 mM/mL. Cultures were kept at room temperature for 2-3 h and then used to infiltrate plants.

N. tabacum (a model plant) was chosen to carry out all the transient assays. Plants were grown in the plant room at 26 ± 2°C and 16 L/8 D. Plants were infiltrated at the 3-4 leaf stage with the help of syringe and again kept in the plant room under controlled conditions.

5.3.4 Insect Rearing and Bioassay Insects, mealybugs, were reared in a separate glass house on cotton plants. A few mealybugs were collected from the field and left on cotton plants in the glass house and

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5. Production of Hybrid Proteins to Control Sucking Pests allowed to multiply. Mealybugs multiplied happily on cotton plants and used afterwards for bioassay to check the effect of fusion protein toxins.

PVX symptoms appeared so clearly in N. tabacum plants 12 dpi. At this stage plants were kept in the cages and allowed to feed by mealybugs. For this purpose, four large cages were used each carrying 10 plants sets. One cage had plants inoculated with N—Hvt-lectin—C construct while second carried N—lectin-Hvt—C inoculated plants. Rest of the two cages had control plants to compare the effect of fusion protein toxins. One had simply PVX inoculated plants while other was with healthy plants N. tabacum without any kind of inoculation. All of the plants in four cages were allowed to feed by equal no. of mealybugs under controlled conditions of plant room i.e. 26 ± 2°C and 16 L/8 D. Plants were checked on the basis of every 24 h to collect the mortality data of mealybugs.

5.4 Data Analysis The data was analyzed statistically using ANOVA and Tukey’s test.

5.5 Computational Study of Recombinant Proteins The sequence of the proteins was subjected to be analyzed by using computational biology tools. This study was carried out to see what kind of conformational/structural modifications are being produced as a result of the recombination of two different proteins.

5.6 Results 5.6.1 Bioassay Experiments were conducted in triplicate, mean values of the data are being presented here.

Plants infiltrated with the fusion protein constructs along with the control ones were kept in cages and allowed to feed by mealybugs. Mealybug is a sucking pest and sucks plant’s phloem sap by inserting its stylet to the phloem cells. Fifteen individuals of mix population mealybugs were allowed to feed on each plant. Plants were visited and checked every 24 h to see the effect of fusion protein toxins upon mealybugs. Initially mealybugs take time to get adjust on plants. During initial 24 h, no distinct change/mortality in the mealybugs was observed. After 48 h mealybugs were appeared

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5. Production of Hybrid Proteins to Control Sucking Pests to be lazy more importantly the nymphs showed the early effects of the toxin proteins. They started to lose their body mass and some jerky and shaky movements. After 72 h nymphs were found to be dead on the plants, the no. of dead individual was counted and % age mortality was taken. Initially 35% individuals were found dead on the plants having HL (Hvt-lectin) construct and 26% LH (Lectin-Hvt) on plants. From the initial results, it was obvious that the construct having Hvt at N-terminal is more effective against the pests than the construct having Hvt at C-terminal.

Mealybugs in the growing stages uptake more food/phloem sap from the plant as compared to the adults. Adults can store food in their body so the initial results were collected on the basis of nymph mortalities rather than adult individuals. Adults starts to be effected very slowly as they are less dependent on plant food.

After 4 days, more mortalities were observed, almost 57% of the individuals were dead on the HL plants followed by 40% mortalities on LH plants. 52% mortalities were observed on LH plants as compared to the high rate of mortalities on HL plants i.e. 71%, after 5 days. While maximum no. of mortalities was observed after 6 days i.e. 83% and 65% on HL plants and LH plants, respectively. Some of the adult individuals multiplied on the plants but the young ones could not grow or moved to the next instar due to the effect of toxin protein. The ovisac of the adult multiplying mealybugs was found to be normal. The dead insects showed the symptoms of browning of bodies and loss of body mass.

Whilst, in case of healthy control and PVX control, the mealybugs have grown in a normal manner and multiplied enormously. Young nymphs were developed into adults and adult individuals multiplied with true ovisac. All the symptoms were normal as body mass gain and multiplication etc. A few of the individuals were found to be escaped from the plants so 100% mortality data cannot be generated.

ANOVA and Tukey’s analysis showed that data are highly significant with p- value <0.01 (Fig. 5-4).

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5. Production of Hybrid Proteins to Control Sucking Pests

Figure 5-4 Mortality rate of mealybugs feeding on transiently expressed fusion proteins

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5. Production of Hybrid Proteins to Control Sucking Pests

Figure 5-5 Mealybug Bioassays for fusion protein (a) Hvt (b) lectin (c) Hvt-lectin (d) lectin-Hvt (e) health control (f) PVX control

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5. Production of Hybrid Proteins to Control Sucking Pests

5.6.2 Computational Biology Studies Computational biology tools reveal that when two different proteins fuse translationally to make a single protein it brings drastic conformational changes. I-TASSER [225-228] results show both recombinant proteins excel different confirmations in comparison with one another and with parent proteins too (Fig. 5-6).

Figure 5-6 Predicted models of proteins using I-TASSER (a) Hvt (b) lectin (c) Hvt-lectin (d) lectin-Hvt

The structural studies proof that both recombinant proteins show different confirmations. This could help to resolve the mystery that why one recombinant protein (HL) is more toxic to insects as compared to the other (LH). HL shows more compact and stable confirmation as compared to that of LH.

5.6 Discussion Mealybug, a sucking pest, exposed to feed on plants expressing fusion toxin proteins showed interesting results. Nymphs were first to be effected by the toxin proteins. It happens, because nymphs (1st and 2nd instar) depend on plant food for their growth and suck phloem sap to fulfill their needs while adults store some food in their body and not thoroughly dependent on plant for their survival. Because of this reason adults are effected later than the nymphs by toxin proteins.

To cope with the issue of sucking pest control it is mandatory to find out some broad-spectrum solution. Recently, it has been necessitated to construct different translationally fusion proteins to control the insect pest and to enhance the effect of a single protein. The effect of HVIa, Pl1a and GNA toxicity against Myzus persicae have already been studied separately and also constructed fusion proteins [1, 2]. Fusion proteins were proved to be more toxic to aphids than the single protein. The Pl1a/GNA

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5. Production of Hybrid Proteins to Control Sucking Pests fusion protein killed all the individuals of 4106A strain in 7-days when fed on 1 mg/mL concentration In diet. While for Hv1a/GNA recombinant protein 100% mortality was observed after 6-days at same concentration i.e. 1 mg/mL in diet. Promising results were collected when fourth stadium larvae of L. oleracea were fed on SMI1/GNA fusion protein. Larvae injected with low concentration of SMI1/GNA were paralyzed and 40% mortality was observed after 72 h (i.e. 18-25 µg/g insect) while 100% mortality was observed after 48 h at high concentration (60-90 µg/ g insect). In case of oral intake, 100% mortality rate was found in fifth stadium tomato moth larvae when fed on 2.5% fusion protein containing diet [218]. Butal T/GNA recombinant fusion protein was proved to be toxic against N. lugens (rice brown plant hopper). 92% mortalities were recorded after 6-days of fusion protein uptake as compared to 64% mortalities on feeding GNA alone [219].

Two different proteins Hvt (a spider venom toxin) and onion leaf lectin were fused together to get a broad-spectrum resistance against insect pests. Both possible fusion constructs were prepared and expressed transiently in model plant system (N. tabacum). As it has already been mentioned that plants expressing the fusion protein having Hvt at N-terminal proved to be more effective against mealybugs than the fusion protein having Hvt at C-terminal with 83% mortality rate.

From the proposed models of the both fusion protein construct shows drastic change in the confirmation/tertiary structure of proteins, as shown in the figures. Just changing the position of two proteins make a big difference in their structure. It would be a strong reason for one protein product to be more active against insect pests rather than other.

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6. General Discussion

6.1 Can We Grow Cotton and Other Crops without Pesticides? Pakistan is the fifth largest cotton producing country. Cotton is the backbone of Pakistan’s economy. Billion $ pesticides are being purchased annually to control insect pests. Introduction of in the field helped to overcome the threat of chewing insects upto a certain limit. Bt toxins are engulfed by the insect, dissolved in the gut and cause the destruction of insect gut lining which lead to death of the insect [204, 220]. Now, the major threat to cotton and other vital crops is sap-sucking pests e.g. aphids, whiteflies, jassids, thrips, mirids, hoppers etc. These pests are also potent vectors of different viruses which infect plant and cause decrease in yield [139].

All this work was done to find and/or evaluate some effective strategy to control sucking pests. Two toxin proteins, other than Bt toxins, Hvt and lectin were expressed in a phloem specific manner (expression under phloem specific promoter).

In the initial trials, the construct expressing both genes was proved to be quite effective against multiple sucking pests (mealybugs, aphids and whiteflies). These toxins halted the growth of sucking pests when allowed to feed on transgenic plants expressing toxins in targeted tissues. While in case of fusion proteins, the combination HL was proved to be more effective (≈ 85%) against sucking pests. This toxin can be proved effective against target pests after more trials and stable transformation in vital crops e.g. cotton, wheat, maize etc.

Pesticides are extensively being used in the field, not only to control the insect pests but also to get rid of many kinds of herbs and weeds. These, herbs and weeds, not only compete with the crop for food and vital nutrients but also serve as alternate host for a number of viruses [139]. To overcome these problems pesticides are being used since decades. Probably, it may not be possible to eradicate pesticides 100% from agriculture but using different technologies can minimize the use of pesticides.

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6.2 Biosafety Status of Hvt and Lectins As different transgenic plants expressing different genes are being cropped on thousands of hectares of land they interact with many of the abiotic and biotic factors of the environment. It is highly recommended to do biosafety studies to check their impact on ecosphere. Especially in case of the transgenic plants expressing insecticidal toxins, it is obvious to study the biosafety impact of those toxins.

Bt is being used widely in many crops (edible and non-edible) against the control of chewing pests. Bt toxins found to be very successful to control many of the lepidopteron insects but no adverse effects have been observed on other organisms including vertebrates [14].

Transgenic plants expressing ω-Hv1a spider venom toxin were found to be effective against a wide range of insects belonging to the order Coleoptera, Diptera, Hemiptera, Lepidoptera, ticks etc. [221]. Hvt proved to be safe for vertebrates as evaluated at high concentration in the diet of larvae of C. septempunctata, C. arena and A. colemani. All of the individuals were observed to be negatively affected by Bt and Hvt toxins [221].

Lectins are mainly glycoproteins found abundantly in all life kingdoms including planta. They are naturally present in the plants of daily use onion, garlic, tobacco and many more etc. Being already a part of the plant family lectins don’t exhibit any adverse effects on the other organisms including humans and considered to be safe to use even in edible crops against pest control. In fact, lectins are considered to be masters of programmed cell death and used in therapy against cancer [54, 94, 222, 223].

6.3 RNA Based Strategies versus Protein Based Strategies The most common strategy has been applied to protect crops from pest attack is application of pesticides and still being applied. Due to the hazardous effects of these pesticides on other organisms including human being [224], scientists are trying to find out some effective eco-friendly strategy to counter the pest attack, either protein toxin based or RNA based.

In case of proteins, as discussed earlier, the toxin peptides are engulfed by the insects when they feed on the toxin carrying plants. These toxin proteins effect insects

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6. General Discussion in different ways. Sometimes they attack the intestinal lining of the insect e.g. Bt and lectins while in some other cases they target the nervous system of the insect by blocking the different ion channels e.g. spider toxins. These toxins also cause fecundity in the insects as studied. Mode of action of the toxin proteins almost remains the same among the insects belonging to one order [221]. For the control of insects belonging to different order, different proteins can be expressed in combination or there is also another approach known as the expression of hybrid proteins. In case of hybrid proteins, two different proteins belonging to two different families are fused translationally and the resulting protein (single hybrid protein) is supposed to be either more effective against particular pests or effective against a wide range of insects [220]. Some other proteins can also be used for this purpose effecting some other pathways of the insects. The most important thing for the toxin proteins is, they should be safe for other organisms from biosafety point of view so that ecosystem would not suffer.

In case of RNA based technologies, either miRNA or siRNA, any of the pathway of insect can be blocked by producing short RNAs. Short RNAs are produced inside plant by stable transformation. When insects feed on the transgenic plants, they uptake shRNAs along with the food and block the said gene. By using this technology, the genes can be silenced before being expressed. It includes silencing of many metabolic pathway, silencing of genes involved in reproduction or growth stages of insects. For the control of multiple insects, different construct needed to be designed or it is highly recommended to find some conserved sequence among the insects of different order to design a single construct to control the insects. The most advantageous point for RNA based technologies is, they are free of biosafety issues.

Either protein based or RNA based technology is adopted, does not matter. The important thing is the expression of toxin proteins and shRNAs should be in the targeted tissues where insects feed. For this purpose, a wide variety of promoters is available and being studied for their tissue specific expression. As, for the control of phloem feeders, it is needed to express toxin proteins or shRNAs in a phloem specific manner. For this purpose, phloem specific promoters isolated from BBTV components were used, as discussed in Chapter 3.

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6. General Discussion

6.4 Double Gene versus Fusion Genes? Gene pyramiding/gene stacking, is a technique, is being used since last decade in different fields. In case of resistance producing resistance against insects or other pathogens, its purpose is to introduce multiple barriers. This is being done due to the high rate of resistance breakdown by pathogens. By adding multiple barriers in the plants, it becomes difficult for the pathogen to invade the crop. In case, if pathogen crosses one barrier there should be another or multiple barrier to stop the pathogen to move on.

Here, a comparison for two techniques is elaborated in this manuscript, double gene and fusion gene. Which one would be more effective? In my understanding, double gene would be more effective and lasting long as compared to the fusion/hybrid gene. Double gene is a profound example of gene pyramiding/gene stacking. As discussed earlier, insect pest would be in trouble while crossing two barriers because two toxins are being produced independently in the phloem of the plant by the expression of phloem specific promoter. It would give a tough time to the sucking pests to overcome the barriers, one is neurotoxin (Hvt) and other is a glycoprotein (lectin) attacks intestinal lining [218].

While, in case of hybrid protein, one translationally fused gene is produced. This hybrid protein probably may be very different as compared to the native toxins in both structure and function. The bioassay results of hybrid protein revealed it to be more toxic towards insects as compared to the native toxins [110]. But, as far as resistance breakdown is concerned, it’s easy for insects to overcome the effects of single protein as compared to the two or more proteins as insects got resistance against Bt being a single protein [111].

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