title page

감사의 글

ABSTRACT

Contents

Abbreviations 17

Overview 18

1. Pepper plants 19

2. Pepper mottle virus (PepMoV) 20

2.1. The family potyviridae and the genus potyvirus 24

2.2. Potyvirus genome structures and expressions 27

3. Cucumber mosaic virus (CMV) 37

3.1. Genome structure 38

3.2. Protein functions 44

CHAPTER 1. Generation of a novel plant viral vector based on highly infectious and stable Pepper mottle virus-Vb1 cDNA clone 47

1.1. Abstract 48

1.2. Introduction 50

1.3. Materials and methods 55

1.3.1. Sources of plants 55

1.3.2. Virus source 55

1.3.3. Bacterial strains and plasmids 56

1.3.4. Construction of full-length cDNA of PepMov-Vb1 56

1.3.4.1. A full-length cDNA copy of PepMoV-Vb1 RNA with SP6 promoter 56

1.3.4.2. A full-length cDNA copies of PepMoV-Vb1 RNA with CaMV 35S promoter 59

1.3.5. Construction of GFP expression vector base on pSP6PepMoV-Vb1 60

1.3.6. Assessments of infectivity of pSP6PepMoV-Vb1 and pSP6PepMoV-Vb1/GFP 63

1.3.6.1. In vitro transcription and infectivity test 63

1.3.6.2. Reverse transcription- polymerase Chain reaction (RT-PCR) 64

1.3.6.3. Western blot analysis 64

1.3.6.4. Detection of GFP expression 65

1.3.7. Analysis of stability and Passage experiments of pSP6PepMoV-Vb1/GFP 65

1.4. Results 67

1.4.1. Complete nucleotide sequencing of PepMoV-Vb1 67

1.4.2. Sequence alignments and phylogenetic analyses of PepMoV-Vb1 71

1.4.3. Cloning of full-length cDNA of PepMoV-Vb1 75

1.4.4. Infectivity assay of in vitro infectious SP6PepMoV-Vb1 clone 82

1.4.5. Confirmation of infectivity of SP6PepMoV-Vb1 clone 85

1.4.6. Construction of the a novel viral vector based on SP6PepMoV-Vb1 genome 88

1.4.7. Infection of N. benthamiana with pSP6PepMoV-Vb1/GFP and systemic expression of GFP gene 91

1.4.8. Host range study of pSP6PepMoV-Vb1/GFP 98

1.4.9. Analysis of stability and passage experiments of pSP6PepMoV-Vb1/GFP 99

1.5. Discussion 104

CHAPTER 2. Application of virus-induced gene silencing in N. benthamiana for virus-resistant plant development using ihpHC-Pro RNA of PepMoV-Vb1 109

2.1. Abstract 110

2.2. Introduction 112

2.3. Materials and methods 118

2.3.1. Virus infection, virus-induced RNA silencing and plant maintenance 118

2.3.2. Construction of intron-hairpin HC-Pro (ihpHC-Pro) vectors 118

2.3.3. Agrobacterium tumefaciens-mediated transient expression 120

2.3.4. GFP monitoring 121

2.4. Results 122

2.4.1. pSP6PepMoV-Vb1/GFP trigger virus induced gene silencing (VIGS) in 16c N. benthamiana 122

2.4.2. ihpHC-Pro causes efficiently inhibition of PepMoV-Vb1 infection 127

2.5. Discussion 134

CHAPTER 3. Identification and sequence Analysis of a Resistance-Breaking Isolate of Cucumber mosaic virus on Capsicum annuum L. 138

3.1. Abstract 139

3.2. Introduction 141

3.3. Materials and methods 145

3.3.1. Source of viruses and virus inoculation. 145

3.3.2. Restriction analysis of CP gene of the virus. 145

3.3.3. Western blot analysis 146

3.3.4. Full-length cDNA cloning of Ca-P1-CMV RNAs 146

3.3.5. Determination of sequence of the virus RAN and phylogenetic analysis. 147

3.3.6. RT-PCR-RFLP of Ca-P1-and Fny-CMV co-infection 149

3.3.7. Pseudorecombinant combination of Ca-P1- and Fny-CMV 149

3.4. Results 152

3.4.1. Isolation and pathogenicity of P0 resistance-breaking isolate of CMV from pepper plants(이미지참조) 152

3.4.2. Construction and complete nucleotide sequencing of full-length cDNA clones of Ca-P1-CMV RNAs 156

3.4.3. Ca-P1-CMV belongs to subgroup IB 158

3.4.4. Symptom comparison of Ca-P1-CMV on zucchini squash plants 172

3.4.5. Restriction enzyme analysis 172

3.4.6. Analysis of Ca-P1-CMV and Fny-CMV co-infection 175

3.4.7. Analyses of infectivity test and resistance-breaking determinant of Ca-P1-CMV 179

3.5. Discussion 181

REFERENCES 185

Appendix 207

Appendix 1. The complete nucleotide sequence of the PepMoV-Vb1 RNA genome. The nucleotide sequence of the plus-strand of the cDNA is given. 212

Appendix 2. The complete nucleotide sequence and the encoded amino acid sequence of the Ca-P1-CMV genomic RNA 1. 214

Appendix 3. The complete nucleotide sequence and the encoded amino acid sequence of the Ca-P1-CMV genomic RNA 2. 216

Appendix 4. The complete nucleotide sequence and the encoded amino acid sequence of the Ca-P1-CMV genomic RNA 3. 217

Appendix 5. Sequence comparison of Ca-P1-CMV 1a proteins with different CMV strains. IA, CMV subgroup IA ; IB, CMV subgroup IB ; II, CMV subgroup II 222

Appendix 6. Sequence comparison of CA-P1-CMV 2b proteins with different CMV strains. 223

국문 요약 224

Overview 16

Table 1. Pepper infecting viruses 21

Table 2. Pepper mottle virus-resistance germplasm sources 25

Table 3. Functions of potyvirus gene products 30

Table 4. Cucumber mosaic virus-resistance germplasm sources 39

CHAPTER 1. Generation of a novel plant viral vector based on highly infectious and stable Pepper mottle virus-Vb1 cDNA clone 16

Table 1-1. Primers used for construct and sequencing of the PepMoV-Vb1 full-length clone 58

Table 1-2. Primers used to fusion PCR for SP6PepMoV-Vb1/GFP construction 62

Table 1-3. The position of amino acid residues in the putative cleavage sites of PepMoV-Vb1 polyprotein and amino acid sequences adjacent to the cleavage site 70

Table 1-4. Percentage sequence identity of PepMo V-Vb1 compared with other potyviruses that infecting Solanaceous species 73

Table 1-5. Host range and symptomatology of SP6PepMoV-Vbl/GFP 102

CHAPTER 2. Application of virus-induced gene silencing in N. benthamiana for virus-resistant plant development using ihpHC-Pro RNA of PepMoV-Vb1 16

Table 2-1. Sequence of the nucleotide primer for ihpHC-Pro RNA vector construction 119

Table 2-2. Interference with PepMoV-Vb1 infection by ihpRNA in N. benthamiana 133

CHAPTER 3. Identification and Sequence Analysis of a Resistance-Breaking Isolate of Cucumber mosaic virus on Capsicum annuum L. 16

Table 3-1. Primers for RT-PCR amplification of Ca-P1-CMV genomic RNAs 148

Table 3-2. RT-PCR primers for restriction enzyme analysis 150

Table 3-3. Sequence comparisons of RNA1 and RNA2 of Ca-P1-CMV with those of other subgroup I and II strains 162

Table 3-4. Sequence similarity (%) of nucleotide (nt) and amino acid (aa) between Ca-P1-CMV and other known strains of CMV 163

Overview 13

Fig. 1. Schematic of genome organization and polyprotein processing of potyvirus. The primary events are probably co-translational and autocatalytic yielding precursors and mature products.... 29

Fig. 2. Schematic genome organization of Cucumber mosaic virus (CMV). Modified from Roossinck (2002) and Moury (2004). Nucleotide (nt) numbers and the sizes for encoded proteins are given for the Fny isolate.... 43

CHAPTER 1. Generation of a novel plant viral vector based on highly infectious and stable Pepper mottle virus-Vb1 cDNA clone 13

Fig. 1-1. Schematic diagram of work flow steps involved in the full-length cloning and infectious clone screening of PepMoV-Vb1. 57

Fig. 1-2. Schematic representation of complete genome sequencing strategy of PepMoV-Vb1 (A) and RT-RCR products of PepMoV-Vb1 (B). 68

Fig. 1-3. Phylogenetic relationships among 15 species of the potyvirus infecting solanacea based on multiple alignment of complete polyprotein (A), P1 (B) and CP (C) sequences. 74

Fig. 1-4. Putative full-length RT-PCR product of PepMoV-Vb1 cDNA. M. 1 kb DNA ladder, F and arrowhead indicates PCR product, approximately 9.6kb. 76

Fig. 1-5. Construction procedure of the pSP6PepMoV-Vb1 vector with bacteriophage SP6 RNA promoter and RT-PCR products of PepMoV-Vb. N-PepMoV-Vb, RT-PCR product of N-terminus region (∼4.4 kb) ; C-PepMoV-Vb ; RT-... 78

Fig. 1-6. Comparison of restriction fragment length polymorphism (RFLP) pattern of full-length cDNA of pSP6PepMoV-Vb1 based on the nucleotide sequence of PepMoV-Vb1.... 79

Fig. 1-7. Construction procedure of a full-length cDNA copy of PepMoV-Vb1 RNA downstream from CaMV 35S promoter. The final plasmid was named p35SPepMoV-Vb1 and represents a complete DNA copy of the PepMoV-Vb1... 80

Fig. 1-8. Comparison of restriction fragment length polymorphism (RFLP) pattern of putative full-length cDNAs of p35SPepMoV-Vb1, RT-PCR analysis (A) and corresponding restriction enzyme map or primer position used PCR analysis (B). 81

Fig. 1-9. In vitro transcription products pSP6PepMoV-Vb1 analyzed on 1% agarose gel. Lane M, 1kb+ DNA ladder, Lane1, transcripts of SP6PepMoV-Vb1 ; Lane2, transcripts derived from putative full-length RT-PCR product.(이미지참조) 83

Fig. 1-10. Symptoms on N. benthamiana induced by mechanical inoculation with wild type PepMoV-Vb and with in vitro transcript synthesized from pSP6PepMoV-Vb1.... 84

Fig. 1-11. RT-PCR (A) and western blot (B) analyses of N. benthamiana infected with pSP6PepMoV-Vb1 infectious clone. Lane 1, leaf extract from a plant infected with wild type PepMoV-Vb; Lanes 2, Healthy plant as a negtive control; Lnae 3-9,... 86

Fig. 1-12. Symptoms on C. annuum ECW inoculated with pSP6PepMoV-Vb1. Picture A,C, E and G. wild type PepMoV-Vb/sap; B,D,F and H. pSP6PepMoV-Vb1/sap. 87

Fig. 1-13. Detection of a virus-specific gene or protein from N. benthamiana plants inoculated with in vitro transcripts from pSP6PepMoV-Vb1. Infectivity confirm of pSP6PepMoV-Vb1 on N. benthamiana plants by RT-PCR with CP primer (A) and... 89

Fig. 1-14. Construction procedure of the pSP6PepMoV-Vb1/GFP. GFP gene inserted between the NIb and CP cistron in pSP6PepMoV-Vb1. A NIb/CP recognition peptide was introduced between NIb and CP. 90

Fig. 1-15. In vitro transcription products of pSP6PepMoV-Vb1/GFP analyzed on 1% agarose gel. M1, 1 kb+ DNA ladder~100ng; M2, 1kb+ DNA ladder~300ng; i-T, SacII linearized pSP6PepMoV-Vb1 template; i-I, in vitro transcript of... 93

Fig. 1-16. Visualization under UV light of green fluorescent protein (GFP) expression in pSP6PepMoV-Vb1/GFP infected N.benthamiana. Pictures were taken at 4, 5, 7, 10 and 14 dpi. 94

Fig. 1-17. Green fluorescence emitted by GFP under UV-light in flowers (A) and roots (B) of N. benthamiana infected with pSP6PepMoV-Vb1/GFP at 30 dpi. 95

Fig. 1-18. RT-PCR analysis of RNA extracted from N. benthamiana pIant infected with pSP6PepMoV-Vb1/GFP (A) and primer position in the viral genome (B). The specific primers used to amplify PepMoV-Vb1-CP (~820 bp) and GFP (~700 bp).... 96

Fig. 1-19. Western blot analysis of PepMoV-Vb coat protein and turboGFP in systemically infected tobacco leaves 14 days post inoculation. M, protein marker, lane 1, Healthy plant : lane 2, pSP6PepMoV-Vb1 infected plant ; lane 3,... 97

Fig. 1-20. Green fluorescence on inoculated and systemic leaves of pepper plants. Pepper plants were inoculated on their first true leaves with progeny virus derived from N. benthamiana plants infected with the pSP6PepMoV-V1/GFP.... 101

Fig. 1-21. Analysis of the stability of pSP6PepMoV-Vb1/GFP through passage (P) experiment in systemically infected leaves of N. benthamiana. Primers used for the RT-PCR amplification of PepMoV-Vb1-CP and GFP.... 103

CHAPTER 2. Application of virus-induced gene silencing in N. benthamiana for virus-resistant plant development using ihpHC-Pro RNA of PepMoV-Vb1 14

Fig. 2-1. Effects of GFP silencing N. benthamiana line 16c plants by SP6PepMoV-Vb1/GFP(A,B) and comparison symptom severity between SP6PepMoV-Vb1/GFP and SP6PepMoV-Vb1 in N. benthamiana line 16c plant (B).... 123

Fig. 2-2. Comparison of nucleotide and amino acid sequences between turboGFP and other GFPs. 126

Fig. 2-3. Schematic drawing of the ihpHC-Pro RNA construct (A) and nucleotide sequence of ihpHC-Pro-N, M and C-term, respectively (B). RB right border ; LB left border ; P35S Cauliflower mosaic virus 35S promoter ; T35S Cauliflower mosaic... 128

Fig. 2-4. ihpHC-Pro RNA expression interfere with SP6PepMoV-Vb1/GFP. Schematic representation of N. benthamiana plants were first infiltrated with A. tumefaciens containing N-term, middle and C-tern of HC-Pro RNA-expressing... 131

Fig. 2-5. Agrobacterium-mediated transient expression of HC-Pro ihpRNA interferes with SP6PepMoV-Vb1/GFP infection. Leaves of N. benthamiana plants were initially infiltrated with A. tumefaciens cultures carrying N-term, Middle and C-term... 132

CHAPTER 3. Identification and Sequence Analysis of a Resistance-Breaking isolate of Cucumber mosaic virus on Capsicum annuum L. 15

Fig. 3-1. Severe mosaic symptom on cultivated hot pepper crops in 2004. 153

Fig. 3-2. Pathogenicity of Ca-P1-CMV as compared to Fny-CMV on 4 different Capsicum annuum plants. Infections of viruses were determined by RT-PCR and back inoculation methods. 154

Fig. 3-3. Confirmation of pathogenicity of Ca-P1-CMV using RT-PCR (A) and western blot analysis (B) on 4 different Capsicum annuum plants. M, 1 kb DNA marker (A), protein marker (B) ; (+), Fny-CMV infected plant ; (-) Mock ; 1 ;... 155

Fig. 3-4. RT-PCR analysis for full-length cDNAs of Ca-P1-CMV RNAs cloning M, 1 kb DNA marker ; lane1, expected RNA3 RT-PCR product ; lane2, expected RNA2 RT-PCR product ; lane3, expected RNA1 RT-PCR product ; arrowheads indicate... 157

Fig. 3-5. Phylogentic analyses of Ca-P1-CMV and representative strains CMV and a species of the genus Cucumovirus based on amino acid sequences of 1a ORF. The horizontal branch lengths are proportional to the genetic distance, and the numbers at... 165

Fig. 3-6. Phylogentic analyses of Ca-P1-CMV and representative strains of CMV and a species of the genus Cucumovirus based on amino acid sequences of 2a (A) and 2b (B). The horizontal branch lengths are proportional to the genetic distance, and the numbers... 166

Fig. 3-7. Phylogentic analyses of Ca-P1-CMV and representative strains of CMV and a species of the genus Cucumovirus based on amino acid sequences of MP (A) and CP (B).... 167

Fig. 3-8. Phylogenic tree of CMV-isolates based on their 5’NTR of RNA3 sequences. 169

Fig. 3-9. Phylogenic tree of CMV-isolates based on their 3’NTR of RNA3 sequences. 170

Fig. 3-10. Nucleotide sequence comparison of the 5' NTR of Ca-P1-CMV with other subgroups. (A) Alignment of the 5'NTRs of 25 strains. Boxes (a) and (f) are conserved in all strains. Boxes (b), (c) and (e) are variable. Boxes (d1) and (d2) are direct repeats conserved in all subgroup I strains.... 171

Fig. 3-11. Systemic symptoms on zucchini squash plants inoculated with Fny, Pf and Ca-P1-CMA strains at 10 dpi. 173

Fig. 3-12. Comparison of restriction pattern of RNA3 between Ca-P1-CMV and 3 other strains of CMVs by using RT-PCR amplicons of CP genes. Lane M1 : 1 kb+ DNA ladder ; lane 1 : RT-PCR product (uncut) ; 2 : HindIII ; 3 : SacII ; 4 : XhoI ; M2 : 100... 174

Fig. 3-13. Western blot analysis of CMV coat protein in systemically infected tobacco leaves 8 days post inoculation. M, Protein marker ; lane 1, Ca-P1-CMV inoculated plant ; lane2, Fny-CMV inoculated plant ; lane 3, Ls-CMV inoculated... 176

Fig. 3-14. RFLP analysis of RT-PCR amplified products of co-infection (Ca-P1-CMV and Fny-CMV). Schematic restriction maps in Ca-P1-CMV and Fny-CMV RNAs (A).... 177

Fig. 3-15. Symptoms on 4 different Capsicum annuum plants. Pepper plants were infected with RNAs of Fny-, Ca-P1-CMV and pseudorecombinant F1C2F3 RNA at 6 dpi. 180