Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89343
標題: 番茄班萎病毒基因靜默抑制子非結構蛋白功能區段、病原性及其自我交互作用分析
Functional motifs of tospoviral NSs protein for RNA silencing suppression, pathogenicity and self-interaction
作者: Chung-Hao Huang
黃崇豪
關鍵字: 西瓜銀斑病毒
基因靜默抑制子
非結構蛋白
功能區段
病原性
自我交互作用
Watermelon silver mottle virus
nonstructural protein S
gene silencing suppressor, functional motifs
pathogenicity
self-interaction
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摘要: 薊馬傳播之番茄斑萎病毒屬(Tospovirus)之病毒為世界性重要植物病毒。台灣之西瓜銀斑病毒(Watermelon silver mottle virus, WSMoV)對葫蘆科作物危害極為嚴重。病毒非結構蛋白NSs為抑制植物防禦機制-基因靜默作用(gene silencing)之重要因子,且與病毒致病性相關。本實驗室已製備WSMoV的NSs蛋白之單株抗體,可以廣泛地偵測到全部亞洲型的tospoviruses,該抗體所辨識之NSs蛋白表體抗原(epitope)是為位於NSs蛋白第98-120個胺基酸的高度保留區段(稱之為WNSscon)。在本篇研究中,我們證實WNSscon對於NSs蛋白抑制基因靜默作用的重要性。另外也發現其他影響NSs蛋白的基因靜默抑制功能、病原性及自我交互作用之胺基酸區段。本論文總共分成三個章節,分述如下: 本論文第一章「前人文獻與研究目的」,主要蒐集整理與本研究相關之參考文獻並概述本論文之目的與內容。 本論文第二章「西瓜銀斑病毒非結構蛋白NSs上兩個新的功能區段負責其抑制基因靜默及病原性功能」,本章實驗比對番茄斑萎病毒屬之NSs胺基酸序列,並將其高度保留區段突變,以農桿菌暫時表現系統在菸草上分析基因靜默抑制功能。我們發現位於亞洲型番茄斑萎病毒屬高度保留之共同抗體決定位(common epitope, CE)( 109KFTMHNQIF117)的H113和位於C端假定(putative) β平板(397IYFL400)結構的Y398為本實驗新發現之功能性區段,其機制分別影響NSs 訊息RNA及蛋白質穩定性。進一步實驗,我們也發現其他六個高度保留區段(CR1-6)及位於高度保留區段的五個胺基酸(Y15、Y27、G180、R181和R212)影響NSs蛋白基因靜默抑制功能。其中除了Y398A NSs突變蛋白外,其餘NSs突變蛋白及其訊息RNA皆可因共同表現馬鈴薯Y病毒屬的基因靜默抑制子協同蛋白酶(HC-Pro)而恢復其表現量。利用輕症矮南瓜黃化嵌紋病毒載體分析這些胺基酸皆與NSs蛋白在矮南瓜的病原性有關,推測可能透過抑制寄主基因靜默作用。 本論文第三章「西瓜銀斑病毒非結構NSs蛋白位於C端區段中假定螺旋構造對於自我交互作用與基因靜默功能之重要性」,本章實驗利用試管內免疫沉降法、酵母菌二元雜交法和雙分子螢光互補法證明NSs蛋白的自我交互作用。經過進一步分析我們推測一個位於C端區段中第八個α螺旋構造(胺基酸位置338-360定義為H8)和NSs蛋白的穩定性與交互作用有關。將H8中的三個胺基酸突變(Y338/H350/F353, triple-point-mutation, TPM)會破壞NSs蛋白的穩定性導致喪失自我交互功能。進一步實驗,我們利用農桿菌暫時表現系統於菸草上分析H8缺失的NSs突變蛋白和TPM蛋白基因靜默抑制功能較為弱,但是經過西方墨點法分析後發現是因為此二突變蛋白在菸草中表現不穩定所致。因此從我們的結果推測NSs單體或雙體蛋白皆有基因靜默抑制功能,但是NSs單體蛋白極為不穩定。所以我們推測NSs蛋白的自我交互功能可能和其在植物中的穩定性有關。
The thrips-borne species of the genus Tospovirus seriously limit the production of many economically important crops worldwide. Watermelon silver mottle virus (WSMoV) is the one of most severe tospoviruses threatening cucurbits in Taiwan. The nonstructural NSs protein is a RNA silencing suppressor of tospovirus, antagonizing host defense reaction and responsible for viral pathogenicity. The monoclonal antibodies (MAbs) previously generated in our laboratory against the NSs protein of WSMoV react strongly not only with the NSs protein of WSMoV, but also with those of all Asia-type serogroup members. The MAb-recognized common epitope WNSscon of these tospoviruses locates in the consensus aa 98-120 of NSs proteins of Asia-type tospoviruses. In this study, our results reveal that WNSscon plays an important role in RNA silencing suppression function. We also found other highly conserved regions of NSs protein are crucial for its RNA silencing suppression function, pathogenicity and self-interaction. This dissertation is divided into three chapters as described below: Chapter I, 'Literature review and research objectives' describes references relevant to this study and the objectives for the investigations. Chapter II, 'Two novel motifs of Watermelon silver mottle virus NSs protein are responsible for RNA silencing suppression and pathogenicity' In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) (109KFTMHNQIF117), which is highly conserved in Asia-type tospoviruses, and the benzene ring of Y398 at the C-terminal β-sheet motif (397IYFL400) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism. Chapter III, 'The importance of putative helix at C-terminal region of Watermelon silver mottle virus nonstructural NSs protein for self-interaction and RNA silencing suppression' In this study, in vitro co-imunoprecipitation (Co-IP) and in vivo bimolecular fluorescence complement (BiFC) assays were further used to analyze the self-interaction capability of NSs protein. We identified a putative α-helix at C-terminal region of the NSs protein (aa 338-360, denoted as H8) involves in its protein stability and self-interaction. Mutations at the three aa residues within the H8 helix, Y338/H350/F353 (denoted as triple-point-mutation, TPM), disrupted the stability of NSs protein and caused dysfunction in self-interaction. Furthermore, as assayed by agroinfiltration we found the mutated proteins of ΔH8 and TPM are unstable in infected cells of Nicotiana benthamiana plants and thus result in weaker RNA silencing suppression activity. Taken together, here our results suggest that both monomeric and dimeric NSs proteins have RNA silencing suppression function. Presumably, self-interaction may help prevent NSs protein degradation in planta.
URI: http://hdl.handle.net/11455/89343
文章公開時間: 2018-05-14
Appears in Collections:植物病理學系

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