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Characterization of resistance-related chloroplast proteins of soybean against soybean mosaic virus infection and replication : 콩 모자이크 바이러스 감염 및 복제에 대한 저항성 관련 엽록체 단백질의 특성 규명
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | Kook -Hyung Kim | - |
| dc.contributor.author | 존 | - |
| dc.date.accessioned | 2023-11-20T04:29:49Z | - |
| dc.date.available | 2023-11-20T04:29:49Z | - |
| dc.date.issued | 2023 | - |
| dc.identifier.other | 000000178426 | - |
| dc.identifier.uri | https://hdl.handle.net/10371/196690 | - |
| dc.identifier.uri | https://dcollection.snu.ac.kr/common/orgView/000000178426 | ko_KR |
| dc.description | 학위논문(박사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2023. 8. Kook -Hyung Kim . | - |
| dc.description.abstract | 콩 모자이크 바이러스 감염 및 복제에 대한 저항성 관련 엽록체 단백질의 특성 규명
존브왈야 국문초록 엽록체 유전자가 식물-바이러스 상호작용에 관여한다는 증거는 점점 증가하는 반면 광합성 유전자와 바이러스 저항성의 관련은 거의 연구되지 않았다. Rsv3 저항성 유전자를 보유한 콩 재배종 L29의 RNA-Seq 데이터를 분석한 결과, 콩 모자이크 바이러스(SMV) 중 비병원성 변이G5H의 감염시 여러 엽록체 관련 유전자가 강하게 유도되었으나, 병원성 변이 G7H에는 약하게 유도된 것으로 나타났다. 이중 광계 I 구성 유전자 PSaC와 ATP-합성효소 복합체의 일부인 ATP-합성효소 α-subunit (ATPsyn-α)로 추가 분석을 진행하였다. rsv3-null 감수성 품종인 Lee74에서 위의 유전자를 함께 발현하는 G7H는 야생형에 비해 훨씬 낮은 감염성을 띠었다. 같은 결과가 두 유전자를 발현한 담배 식물에서 G7H 감염시켰을 때도 확인되었다. 두 단백질 모두 엽록체 외피와 핵, 세포질에서 발견되었다. 엽록체는 방어 관련 호르몬의 초기 생합성 부위이기 때문에 ATP syn-α- 및 PSaC 매개 방어에 호르몬 관련 유전자가 관여하는지 여부를 판단했다. 흥미롭게도, ATP 합성-α를 발현하는 G7H변이에 감염된 식물에서는 여러 호르몬의 생합성에 관여하는 유전자 발현이 증가하였으나, PSaC를 발현하는 SMV-G7H에 감염된 후 자스몬산 및 살리실산 생합성 유전자 발현만 향상되었다. 두 키메라 모두 여러 항바이러스 RNA 침묵기작 유전자의 발현을 유도하였으며, 이는 이러한 저항성이 부분적으로 RNA 침묵 경로를 통해 이뤄질 수 있음을 의미한다. PSaC와 ATP 합성효소-α는 각각 NIb와 NIa-Pro와 상호작용한다는 것이 공동 면역 침전(Co-IP)에 의해 확인되었다. G7H 유전체로 PSaC 또는 ATP 합성효소-α의 C 말단 부위를 과발현시키면 감수성 품종 Lee74에서 바이러스 축적 및 전신 감염이 줄어든다. 본 연구 결과는 PSaC와 ATP syn-α 유전자가 SMV 복제 및/또는 감염된 식물에서 이동하는 동안 NIb와 NIa-Pro의 기능에 영향을 줌으로써 SMV 감염에 대한 저항성을 조절한다는 것을 시사한다. 주요어: 콩모자이크바이러스, 콩, 기주인자, 저항성 학번: 2019-34734 | - |
| dc.description.abstract | John Bwalya
Major in Plant Microbiology Department of Agricultural Biotechnology The Graduate School of Seoul National University GENERAL ABSTRACT Accumulating evidence attests that chloroplast-related genes are involved in plant–virus interactions. However, the involvement of photosynthesis-related genes in plant immunity is largely unexplored. Analysis of RNA-Seq data from the soybean cultivar L29, which carries the Rsv3 resistance gene, showed that several chloroplast-related genes were strongly induced in response to infection with an avirulent strain of soybean mosaic virus (SMV), G5H, but were weakly induced in response to a virulent strain, G7H. For further analysis, we selected the PSaC gene from the photosystem I and the ATP-synthase α-subunit (ATPsyn-α) gene whose encoded protein is part of the ATP-synthase complex. Overexpression of either gene within the G7H genome reduced virus levels in the susceptible cultivar Lee74 (rsv3-null). This result was confirmed by transiently expressing both genes in Nicotiana benthamiana followed by G7H infection. Both proteins localized in the chloroplast envelope as well as in the nucleus and cytoplasm. Because the chloroplast is the initial biosynthesis site of defence-related hormones, we determined whether hormone-related genes are involved in the ATPsyn-α- and PSaC-mediated defense. Interestingly, genes involved in the biosynthesis of several hormones were up-regulated in plants infected with SMV-G7H expressing ATPsyn-α. However, only jasmonic and salicylic acid biosynthesis genes were up-regulated following infection with the SMV-G7H expressing PSaC. Both chimeras induced the expression of several antiviral RNA silencing genes, which indicate that such resistance may be partially achieved through the RNA silencing pathway. PSaC and ATPsyn-α proteins interacted with NIb and NIa-Pro of SMV respectively. These interactions were confirmed by co-immunoprecipitation (Co-IP). Overexpression of the C-terminal either from PSaC or ATPsyn-α in the SMV-G7H genome reduced viral accumulation and systemic infection on susceptible cultivar. Our findings suggest that PSaC and ATPsyn-α genes modulate resistance to SMV infection by affecting the function of NIb and NIa-Pro in SMV replication and movement. These findings highlight the role of photosynthesis-related genes in regulating resistance to viruses. Keyword: Soybean mosaic virus, Glycine max, Nicotiana benthamiana, Viral replication complex, Chloroplast-virus interaction, Viral replication Student Number: 2019-3473 | - |
| dc.description.tableofcontents | CONTENTS
GENERAL ABSTRACT i TABLE OF CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii CHAPTER I. The Crucial Role of Chloroplast-Related Proteins in Viral Genome Replication and Host Defense against.Positive-Sense Single-Stranded RNA Viruses ABSTRACT 2 INTRODUCTION 3 I. Chloroplasts and chloroplast-related proteins facilitate the viral replication cycle of plant (+) ss RNA viruses 5 II. Chloroplast-related proteins interact with viral components during virus replication 11 III. Chloroplast plays a vital role in plant antiviral defense 16 CONCLUSIONS AND FUTURE PERSPECTIVES 18 LITERATURE CITED 20 CHAPTER II. Photosynthesis-related genes induce resistance against soybean mosaic virus: Evidence for involvement of the RNA silencing pathway ABSTRACT 34 INTRODUCTION 35 MATERIALS AND METHODS I. Plant materials, growth conditions, and virus infections 39 II. Silencing ATPsyn-α and PSaC in soybean plants 42 III. PRNA extraction and RT-qPCR 42 IV. Statistical analysis 43 V. Western protein blot 43 VI. Phylogenetic analysis 43 VII. Gene description, function, and pathways 44 VIII. Subcellular localization of ATPsyn-α and PSaC proteins 44 IX. Visualization of GFP expression and localization of the target proteins in plants 44 RESULTS I. Chloroplast-related genes are induced in the resistant cultivar L29 in response to SMV-G5H infection 46 II. GmPSaC and GmATPsyn-α genes are induced in cultivars with different resistance backgrounds 56 III. Overexpression of ATPsyn-α and PSaC induces resistance against G7H in the susceptible cultivar Lee74 60 IV. Knockdown of ATPsyn-α and PSaC increased Lee74 susceptibility against G7H infection 65 V. Localization of ATPsyn-α and PSaC in N. benthamiana and their effects on N. benthamiana resistance against SMV-G7H 69 VI. Involvement of defence-related hormones in ATPsyn-α and PSaC- mediated resistance 72 VII. Antiviral RNA silencing genes are regulated in ATPsyn-α and PSaC- mediated resistance 76 DISCUSSION 80 LITERATURE CITED 85 CHAPTER III. Chloroplast- related host proteins interact with NIb and NIa-Pro of soybeans mosaic virus and induce resistance in the susceptible cultivar ABSTRACT 91 INTRODUCTION 93 MATERIALS AND METHODS I. Plant materials, growth conditions, and virus infections 96 II. RNA extraction and real-time quantitative PCR (RT-qPCR) 96 III. Plasmid construction 97 IV. Western blotting 98 V. Co-Immunoprecipitation (Co-IP) assay 98 VI. Statistical analysis 99 VII. Subcellular localization 99 VIII. Yeast two-hybrid and X-α-Gal assays 100 RESULTS I. Soybean proteins ATPsyn-α and PSaC interact with NIa and NIb, respectively 103 II. Analysis of in planta interactions in Nicotiana benthamiana 104 III. The C-terminal region of either ATPsyn-α or PSaC is crucial for protein-protein interaction 106 IV. The C-terminal of either ATPsyn or PSaC is required for resistance 108 V. Predicted crucial amino residues for resistance to SMV infection 111 DISCUSSION 114 LITERATURE CITED 119 ABSTRACT IN KOREAN 127 LIST OF TABLES CHAPTER I Table 1 Chloroplast factors and their cellular localization during virus replication 11 Table 2 Summary of viral protein-chloroplast factor interactions 14 CHAPTER II Table 1 List of primers used in this study 41 Table 2 Functional analysis and gene ontology of the photosynthesis-related genes regulated by SMV infection 50 CHAPTER III Table 1 List of primers used in this study 101 LIST OF FIGURES CHAPTER I Fig. 1. Schematic representation of necessary steps for positive-sense single-stranded RNA [(+)ss RNA] virus genome replication. Following the entry into host cells 10 Fig. 2. Schematic representation of the important events during chloroplast host factors and potyvirus interactions in a plant cell. 15 CHAPTER II Fig. 1. Expression of photosynthesis-related genes in response to soybean mosaic virus (SMV) infection 48 Fig. 2. Domains and phylogenetic analyses of GmPSaC and GmATPsyn-α 54 Fig. 3. Soybean susceptibility to infection by SMV-G7H 58 Fig. 4. Effect of overexpressing ATPsyn-α and PSaC on resistance against G7H in the susceptible cultivar Lee74 63 Fig. 5. Effect of silencing GmATPsyn-α and GmPSaC on soybean susceptibility to SMV-G7H infection. Lee74 plants were silenced in GmATPsyn-α and GmPSaC using BPMV silencing vector 67 Fig. 6. Localization and effects of GmATPsyn-α and GmPSaC on resistance to SMV-G7H in Nicotiana benthamiana leaves 70 Fig. 7. Expression levels of key genes of defence-related hormones in Lee74 plants in response to SMV-G7H expressing ATPsyn-α and PSaC genes.Relative expression levels in Lee74 plants of salicylic acid-related gene 74 Fig. 8. Expression levels of RNA silencing genes in Lee74 plants in response to SMV-G7H expressing ATPsyn-α and PSaC genes 78 Fig. 9. Expression levels of RNA silencing genes in Nicotiana benthamiana in response to infection with chimeras of pSMV-G7H::eGFP 79 CHAPTER III Fig. 1. SMV Viral proteins and Soybeans Chloroplast- related proteins interactions 103 Fig. 2. Co-expression of PSaC/ ATPsyn-α with NIb/Nia-pro the Nicotiana benthamiana cell 105 Fig. 3. Analysis of SMV NIb/ NIa-pro interaction with GmPSaC/GmATPsyn-α deletions 107 Fig. 4. Experimental design, Chimera constructions, and Effect of GmPSaC full length and deletion mutants 119 Fig. 5. Experimental design, Chimera constructions, and effect of GmATPsyn-α full length and deletion mutants 110 Fig. 6. Analysis of predicted crucial amino residues 112 Fig. 7. Multiple sequences alignment of PSaC and ATPsyn-α C-terminal with other plant homologous proteins 113 | - |
| dc.format.extent | xi. 128 | - |
| dc.language.iso | eng | - |
| dc.publisher | 서울대학교 대학원 | - |
| dc.subject | Keyword | - |
| dc.subject | Soybean mosaic virus | - |
| dc.subject | Glycine max | - |
| dc.subject | Nicotiana benthamiana | - |
| dc.subject | Viral replication complex | - |
| dc.subject | Chloroplast-virus interaction | - |
| dc.subject | Viral replication | - |
| dc.subject.ddc | 630 | - |
| dc.title | Characterization of resistance-related chloroplast proteins of soybean against soybean mosaic virus infection and replication | - |
| dc.title.alternative | 콩 모자이크 바이러스 감염 및 복제에 대한 저항성 관련 엽록체 단백질의 특성 규명 | - |
| dc.type | Thesis | - |
| dc.type | Dissertation | - |
| dc.contributor.AlternativeAuthor | John Bwalya | - |
| dc.contributor.department | 농업생명과학대학 농생명공학부 | - |
| dc.description.degree | 박사 | - |
| dc.date.awarded | 2023-08 | - |
| dc.contributor.major | 식물미생물학 | - |
| dc.identifier.uci | I804:11032-000000178426 | - |
| dc.identifier.holdings | 000000000050▲000000000058▲000000178426▲ | - |
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