Classification of two criniviruses in Taiwan and functional analysis of their RNA silencing suppressors

粉蝨所傳播的長線狀病毒 (criniviruses) 為線型病毒Closteroviridae科，長線狀Crinivirus屬的病毒，已逐漸在許多經濟作物上造成嚴重的危害，因此受到關注。目前，在台灣已被報導的兩種線狀病毒，瓜類褪綠黃化病毒 (cucurbit chlorotic yellows virus，CCYV) 和番茄褪綠病毒 (tomato chlorosis virus，ToCV) 分別感染葫蘆科和茄科作物。線狀病毒的基因組成是由兩條不同正極性單股RNA所構成，分別被命名為RNA-1和RNA-2，其3′端不具有聚腺苷酸化 (poly A-tail)，且在5′端可能帶有封端 (capped)，RNA-1上生產病毒複製相關的蛋白，而RNA-2上則產生病毒移動與包裹病毒顆粒的相關蛋白。本研究旨在探討台灣的CCYV和ToCV之分離株的分子特性以做為這兩種病毒的分類依據。 本論文第一章為 「前人文獻與研究目的」，主要彙整與本研究相關的參考文獻並概述本論文之目的與內容。 本論文第二章為 「台灣具遺傳特異的番茄褪綠病毒株系之分子特性探討與檢測分析」，在台灣所發現的XS分離株，對其進行全長度基因組序列之解序與分析，發現XS的RNA-1和RNA-2核苷酸序列與其他已知ToCV分離株，分別僅有77.8-78%和78-78.1%的相同度 (identity)。雖然核苷酸序列相同度低，但是其RNA依賴RNA聚合酶 (RNA-dependent RNA polymerase，RdRp)、熱休克同源性蛋白 (heat shock protein 70 homolog，Hsp70h) 和主要外鞘蛋白 (minor capsid protein，CP) 與其他ToCV分離株的相同度為88.3-96.2%，親緣演化分析顯示，目前已知的ToCV分離株都非常相近，但XS則獨立在單一演化分支上，此結果顯示XS為ToCV之獨立的株系 (strain)。為了瞭解XS在田間的分布情形，從XS的基因組序列設計專一性引子對，利用反轉錄聚合酶鏈鎖反應 (reverse transcription polymerase chain reaction，RT-PCR) 的方法，檢測受ToCV感染的番茄作物和粉蝨。2013年至2017年的田間調查結果顯示，174個的番茄樣品中，ToCV的檢出率高達60.9%，177個粉蝨樣本檢出率為28.8%，表明ToCV已成為台灣番茄生產的新威脅。此章節已被國際期刊「Plant Disease」接受。 本論文第三章為 「台灣的瓜類褪綠黃化病毒分離株之分子特性探討」，針對台灣所發現的CCYV分離株LB，進行全長度基因組序列之解序與分析，顯示CCYV-LB的RNA-1和RNA-2核苷酸序列與其他日本與中國的CCYV分離株，分別為具有99.5-100%和99.6-99.8%的相同度。而在Crinivirus屬中分析各病毒的RdRp、Hsp70h和CP之親緣演化關係，發現CCYV與 菜豆黃化退化病毒 (bean yellow disorder virus，BYDV)、瓜類黃色矮化退化病毒 (cucurbit yellow stunting disorder virus，CYSDV)、萵苣黃化病毒 (lettuce chlorosis virus，LCV) 和白屈菜葉脈褪綠病毒 (tetterwort vein chlorosis virus，TVCV) 在同一個演化分支上。此章節已發表於國內「植物醫學期刊」。 本論文第四章為 「CCYV P22蛋白對病毒病原性和RNA沉默抑制作用的功能分析」，本實驗室先前已構築的矮南瓜黃化嵌紋病毒 (zucchini yellow mosaic virus，ZYMV) 的突變株ZAC，具有經定點突變的協同性蛋白 (helper component-protease，HC-Pro) 基因，可於受感染的天然宿主矮南瓜上產生輕微的斑駁病徵，且病徵可恢復；而在單斑寄主奎藜上則喪失誘導過敏反應的能力。本研究乃利用ZAC載體來表現CCYV-LB的各個基因以探討CCYV-LB的可能致病因子，結果發現CCYV-LB RNA-1上的P22可以補償ZAC HC-Pro基因缺失的功能，且在矮南瓜和奎藜上造成比ZYMV野生型更為嚴重的病徵，顯示P22與病原性相關，進一步對P22進行一系列的定點突變，發現L127胺基酸與CCYV-LB的致病能力有關。此外，利用農桿菌注射法證實CCYV-LB P22亦具有RNA沉默抑制作用，其抑制了宿主合成雙股RNA過程中的相關因子，也可透過與短片段 (21個核苷酸) 和長片段 (267個核苷酸) 的RNA結合達到抑制基因沉默作用。 本論文透過研究CCYV和ToCV的分子特性和流行病學做為擬定防治此二種病毒策略之參考。Whitefly-borne criniviruses (genus Crinivirus, family Closteroviridae) have been considered as important plant viruses due to their rapid widespread and destructive nature for many economic crops. Currently, two criniviruses, cucurbit chlorotic yellows virus (CCYV) and tomato chlorosis virus (ToCV), have been reported infecting cucurbits and tomatoes, respectively, in Taiwan. Criniviruses have a bipartite genome consisting of two positive-sense ssRNA segments, named RNA-1 and RNA-2. The 3′ ends of criniviral RNA segments are not polyadenylated and the 5′ ends are likely to be capped. The RNA-1 encodes proteins for virus replication, while the RNA-2 encodes proteins for movement and assembly of virus particles. In present study, the complete genomic sequences of the Taiwanese isolates of CCYV and ToCV were determined to demostrant their classification status. Chapter I, “Literature review and research objectives” describes references relevant to this study and objectives for the investigations. Chapter II, “Molecular characterization and detection of a genetically distinct Tomato chlorosis virus strain from Taiwan”. The whole genome sequence of a virus isolate XS was determined. The nt sequences of RNA-1 and RNA-2 of XS sharing lower identities of 77.8-78% and 78-78.1%, respectively, with those of all other known ToCV isolates. The RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (Hsp70h) and major capsid protein (CP) of XS share 88.3-96.2% aa identities with those of other ToCV isolates. Phylogenetic analyses of RdRp, Hsp70h and CP revealed that all other geographic ToCV isolates are closely related and clustered in the same clade, whereas the XS isolate is distinct to form a unique branch. Our results suggest that XS is a distinct strain of ToCV. A one tube-based method of reverse transcription-polymerase chain reaction (RT-PCR) using specific primers designed from the genomic sequence of the XS was able to detect ToCV in infected tomato plants and individual whiteflies. The field survey during 2013 to 2017 disclosed a high ToCV rate of 60.9% from 174 symptomatic tomato samples and 28.8% from 177 whitefly samples, demonstrating that ToCV has become an emerging threat for tomato production in Taiwan. This has been accepted for publication in “Plant Disease” journal. Chapter III, “Molecular characterization of Cucurbit chlorotic yellows virus from Taiwan”. The whole genome sequence of CCYV-LB was also determined. Sequence analysis revealed that CCYV-LB is closely related to other CCYV isolates from Japan and China, sharing 99.5-100% and 99.6-99.8% nt identities in RNA-1 and RNA-2, respectively. Phylogenetic analyses of the RdRp, Hsp70h and CP among Crinivirus species indicated that CCYV is closedly related to bean yellow disorder virus (BYDV), cucurbit yellow stunting disorder virus (CYSDV), lettuce chlorosis virus (LCV) and tetterwort vein chlorosis virus (TVCV). This has been published in “Journal of Plant Medicine”. Chapter IV, “Functional analyses of P22 protein of Cucurbit chlorotic yellows virus for viral pathogenicity determinant and RNA silencing suppression”. An attenuated zucchini yellow mosaic virus (ZYMV) mutant, denoted ZAC, with a site-directed mutated helper component-protease (HC-Pro) gene, inducing mild mottling then recovery on its natural host zucchini squash and losing the ability of local lesion induction on inoculated Chenopodium quinoa leaves, was used as a vector to express individual coding sequences of CCYV-LB for identifying possible pathogenicity determinant of CCYV in planta. The RNA1-encoded P22 of CCYV-LB compensated for the dysfunctional HC-Pro of ZAC that induced severe symptoms on both zucchini squash and C. quinoa, reflecting its pathogenicity-related role. Alanine substitution assays of the CCYV-LB P22 indicated that the amino acid 127 leucine residue is indispensable for the pathogenicity function. Furthermore, the RNA-silencing suppression function of CCYV-LB P22 was verified by agroinfiltration assay. The P22 inhibited the function of host factors for the synthesis of dsRNA, a critical step to initiate RNA silencing signaling. Moreover, the binding ability of P22 with the 21-nt short and 267-nt long ssRNA and dsRNA molecules was further verified. Understanding of the molecular characteristics and epidemiology of CCYV and ToCV helps us to formulate an efficient strategy for controlling these viruses.中文摘要........................................ i Abstract.......................................iii 目錄............................................vi 第一章 前人文獻與研究目的........................1 Chapter I Literature review and research objectives.......1 一、 長線狀病毒 (CRINIVIRUS) 屬之相關研究.................2 1. 線型病毒 (Closteroviridae) 科的分類史................2 2. 長線狀病毒 (Crinivirus) 屬之生物特性.................3 3. 長線狀病毒 (criniviruses) 與其媒介昆蟲...............4 4. 粉蝨傳播長線狀病毒 (criniviruses) 之特性.............6 5. 長線狀病毒 (criniviruses) 之危害及發生情形...........7 6. 長線狀病毒 (criniviruses) 的防治....................8 二、 病毒之基因沉默抑制子 (RNA SILENCING SUPPRESSOR，RSS) ..........................................................12 1. 基因沉默 (gene silencing) 之發現...................12 2. 基因沉默之機制.....................................13 3. 病毒RNA沉默抑制子 (RNA silencing suppressor，RSS) 之介紹 ..........................................................17 4. 病毒的RNA沉默抑制子與致病因子之關係..................21 5. 病毒的RNA沉默抑制子與致病因子之探討方法...............22 三、 本論文之研究目的....................................24 1. 台灣的ToCV之分子特性分析與病毒檢測...................24 2. 台灣的CCYV-LB分離株之分子特性分析和致病因子與RNA沉默抑制子之研究.....................................................24 四、 參考文獻...........................................27 第二章 台灣具遺傳特異的番茄褪綠病毒株系之分子特性探討與檢測分析 ...........................................................47 Chapter II Molecular characterization and detection of a genetically distinct Tomato chlorosis virus strain in Taiwan ...........................................................47 ABSTRACT...................................................48 INTRODUCTION...............................................49 MATERIALS AND METHODS......................................52 RESULTS....................................................59 DISCUSSION.................................................63 REFERENCES.................................................68 TABLES AND FIGURES.........................................74 第三章 台灣的瓜類退綠黃化病毒之分子特性探討...................91 Chapter III Molecular characterization of Cucurbit chlorotic yellows virus from Taiwan..................................91 ABSTRACT...................................................92 INTRODUCTION...............................................93 MATERIALS AND METHODS......................................96 RESULTS....................................................100 DISCUSSION.................................................103 REFERENCES.................................................107 TABLES AND FIGURES.........................................114 第四章 CCYV P22蛋白對病毒病原性和RNA沉默抑制作用的功能分析....123 Chapter IV Functional analyses of p22 protein of Cucurbit chlorotic yellows virus for viral pathogenicity determinant and RNA silencing suppression..............................123 ABSTRACT...................................................124 INTRODUCTION...............................................126 MATERIALS AND METHODS......................................130 RESULTS....................................................140 DISCUSSION.................................................145 REFERENCES.................................................152 TABLES AND FIGURES.........................................16