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Genome-based identification of Pvr4 conferring resistance against potyvirus in Capsicum annuum

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dc.contributor.advisor최도일-
dc.contributor.author김샛별-
dc.date.accessioned2017-07-13T17:39:19Z-
dc.date.available2017-07-13T17:39:19Z-
dc.date.issued2015-08-
dc.identifier.other000000067531-
dc.identifier.urihttps://hdl.handle.net/10371/121009-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 식물생산과학부, 2015. 8. 최도일.-
dc.description.abstractPvr4 is a resistance gene showing broad-spectrum resistance against multiple potyviruses, including Pepper mottle virus (PepMoV), Pepper severe mosaic virus (PepSMV), and Potato virus Y (PVY). Capsicum annuum landrace CM334 is known to have Pvr4, but it has not been cloned and the mechanism of resistance is unknown. To identify the avirulence factor in potyviruses corresponding to Pvr4, a total of eleven viral cistrons of PepMoV were expressed into potyvirus-resistant (Pvr4) and -susceptible (pvr4) pepper plants. Hypersensitive response (HR) was observed only when a RNA-dependent RNA polymerase (NIb) of PepMoV was expressed in Pvr4-haboring pepper leaves in a genotype-specific manner. In addition, the over-expression of NIb proteins of other potyviruses including PepSMV, PVY also induced HR in Pvr4-harboring pepper plants. These results indicate that NIbs of PepMoV, PepSMV, and PVY may play important roles as avirulence factors for Pvr4 in pepper plants. To identify Pvr4 resistance gene against potyvirus in pepper, genome-based cloning with two populations including BC1F3 and F2 populations was performed. High-density molecular markers including 32 co-dominant markers were developed in the TG420 marker region using tomato and pepper genome. Three SNP markers showed a co-segregation with Pvr4 in two populations and Pvr4 is located within 350 kb region containing sixteen annotated genes. Among them, eight genes were coiled-coil (CC) nucleotide-binding site leucine-rich repeat (NB-LRR) and they were clustered in this region. Transient over-expression of the eight NB-LRR type genes and NIb of PepMoV in susceptible pepper (C. annuum Jupiter) and Nicotiana benthamiana leaves revealed that only one gene (CA10g21170) induced HR at 2 dpi. The relative amounts of PepMoV RNA transcripts and protein were significantly suppressed in the leaves that transiently over-expressed of CA10g21170 after 3 dpi. Furthermore, CA10g21170 showed resistance against other potyviruses including PepSMV and PVY. Consequently, these results prove that CA10g21170 is indeed Pvr4 leading to recognize NIb and suppress PepMoV, PepSMV and PVY replication. Pvr4 consisted of seven exons and encodes a CC-NB-LRR type protein with 1746 amino acids. Genomic region of Pvr4 including exon and intron is 13,870 bp. In planta assays using the TRV-based gene silencing revealed that silencing of HSP90, SGT1 and RAR1 in N. benthamiana suppressed HR induced by Pvr4 and NIb of PepMoV. To confirm the heterologous in planta expression of Pvr4 in other plant, Pvr4-harboring transgenic potato were generated. Inoculation of virus validated that the replication of PVY-O was significantly suppressed in the transgenic upper leaves. Taken together, the cloned potyvirus resistance gene, Pvr4 could provide information for the application of broad-spectrum potyvirus resistance in crop breeding, as well as for understanding potyvirus resistance mechanisms in plants.-
dc.description.tableofcontentsABSTRACT…………………………………………………………………i
CONTENTS………………………………………………………………iv
LIST OF TABLES………………………………………………………viii
LIST OF FIGURES…………………………………………………………ix
LIST OF ABBREVIATIONS……………………………………………xii

GENERAL INTRODUCTION……………................................................1

CHAPTER 1. Identification of RNA-dependent RNA polymerase (NIb) as the avirulence factor of potyvirus in Pvr4-bearing pepper plants
ABSTRACT……………………………………………….………………13
INTRODUCTION…………………………………………………………14
MATERIALS AND METHODS……….………………………………….18
Plant materials……………………………………………...….………18
Application of Pvr4-linked CAPS marker for identification of pepper genotype………………………………………………….………19
Cloning of potyvirus cistrons for in planta expression………………19
In planta expression assay in pepper plants……….……………..…24
Immunodetection of PepMoV-encoded proteins………………..……25
RESULTS…………………………………………………………...……26
Genotypes and PepMoV accumulation in pepper plants ………….…26
Identification of NIb as the avirulence factor of PepMoV in Pvr4-bearing pepper plants……………………………………………………...29
NIb proteins of other potyviruses as avirulence factors in Pvr4-mediated resistance …………………………………………………………38
DISCUSSION……………………………………………………………42
REFERENCES……………………………………………..……………46

CHAPTER 2. Genome-based cloning of Pvr4 conferring multiple potyvirus resistance from Capsicum annuum CM334
ABSTRACT………………………………………………………………52
INTRODUCTION…………………………………………………………54
MATERIALS AND METHODS…………………………………………58
Plant materials ………………………………………………………58
Phenotypic evaluation against potyviruses……………………………58
Development of Cleaved Amplified Polymorphic Sequences (CAPS) markers…………………………………………………………59
BAC library screening and sequence analysis…………………………61
Cloning of Pvr4 candidates by ligation-independent cloning (LIC)…61
Transient over-expression of Pvr4 candidates and NIbs of potyviruses..63
Phenotypic evaluation of Pvr4 in N. benthamiana………………….....64
Construction of the TRV-Pvr4 vectors and VIGS in pepper…………65
Construction of the signaling components for VIGS assays in N. benthamiana……………………………………………………66
Analysis of transcript levels by reverse transcription polymerase chain reaction (RT-PCR)………………………………………………66
Transformation of potato plants and selection of transgenic potato plants……………………………………………………………67
Phenotypic evaluation of Pvr4 in transgenic potato plants……………68
RESULTS…………………………………………………………………69
Genetic analysis of Pvr4 resistance to PepMoV ……………................69
Comparative mapping of the Pvr4 locus in tomato and pepper………73
Development of Pvr4-linked markers…………………………………75
Candidate gene annotation in the Pvr4 locus…………………………78
Identification of Pvr4 function by transient co-expression assay……80
Specific interaction of Pvr4 and PepMoV-NIb………………………80
Sequence analysis of the Pvr4 and pvr4 genes…………………………85
Resistance of Pvr4 against potyviruses in N. benthamiana……………87
The nature of the Pvr7 gene present in C. chinense PI159236……….92
Virus-induced gene silencing (VIGS) of Pvr4…………………………97
Signaling pathway of Pvr4-mediated HR…………...…………………99
Resistance of transgenic potato harboring Pvr4……………………102
DISCUSSION……………………………………………………………105
Genome-based mapping of the Pvr4 gene……………………………105
Identification of the Pvr4 gene function by transient co-expression assay……………………………………………………………….....107
Sequence analysis of the Pvr4 and pvr4 genes………………………108
Resistance of the Pvr4 gene against potyviruses in N. benthamiana….109
Signaling pathway of Pvr4-mediated HR…………………………….110
The nature of the Pvr7 gene present in C. chinense PI159236………111
Resistance of transgenic potato harboring Pvr4………………………112
REFERENCES…………………………………………………………113
ABSTRACT IN KOREAN………………………………………………122
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dc.formatapplication/pdf-
dc.format.extent4032813 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectPepper-
dc.subjectPotyvirus-
dc.subjectResistance gene-
dc.subjectPvr4-
dc.subjectPepMoV-
dc.subject.ddc633-
dc.titleGenome-based identification of Pvr4 conferring resistance against potyvirus in Capsicum annuum-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pagesxii,124-
dc.contributor.affiliation농업생명과학대학 식물생산과학부-
dc.date.awarded2015-08-
Appears in Collections:
College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Plant Science (식물생산과학부)Theses (Ph.D. / Sc.D._식물생산과학부)
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