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標題: 馬鈴薯Y群病毒之基因沉寂抑制子HC-Pro對植物自發性防禦反應干擾機制之探討
Interference of Potyviral Gene Silencing Suppressor Hc-Pro with Innate Defense Response of Plants
作者: 葉錫東
關鍵字: 植物保護類;商品化
寄主植物對病毒的過敏性反應 (Hypersensitive response, HR) 係由活性氧誘發細胞死亡與單斑形成,以防止病毒擴散。而植物病毒則有與之抗衡的蛋白,使病毒能在寄主內系統性感染。我們先前的研究指出基因沉寂抑制子HC-Pro 影響奎藜過敏反應的形成, HC-Pro 點突變的弱系矮南瓜黃化嵌紋病毒 (ZYMV) 及蕪菁嵌紋病毒 (TuMV)皆喪失誘發奎藜產生HR 的能力。本計畫以酵母菌雙雜合系統 (Y2H) 鑑定出之HC-Pro交互作用的奎藜蛋白,包含奎藜碳酸酐酶 (CqCA) 及阿拉伯芥之同源蛋白AtCA1 和AtCA2 為研究對象。AtCA1,即水楊酸結合蛋白,係誘發HR 防禦的關鍵因子。因此,藉由瞭解CA 和HC-Pro 間的作用,可知弱系病毒喪失引發奎藜產生HR 的原因。因奎藜尚未有功能性基因體的分析平臺,我們將以阿拉伯芥為研究平臺。以AtCA1 與AtCA2的基因剔除株、高表現株、基因剔除株之轉基因回復株、其它抗病相關之突變株、及ZYMV 與TuMV 及其突變株之為研究系統。再以Y2H 法研究HC-Pro 與AtCA1、AtCA2的交互作用、色層分析法研究其異寡聚合體形式及生體內、生體外的結合試驗分析HC-Pro 影響CA 與水楊酸結合的能力。HC-Pro 對葉綠體的結構與功能的影響則以顯微鏡與量測相關葉綠體酵素分析之。於各項分析中,將比較野生型與突變之HC-Pro 對實驗的反應差異。本研究對HC-Pro 與HR 的分子機制探討,將對植物的抗病反應提供新的見解及抗病毒的新策略。

Hypersensitive response (HR) of plants to virus is a reactive oxygen species(ROS)-mediated cell death, which prevents further spread of virus from the initial sites ofinfection. Viral proteins specialized for inhibiting HR and subsequent systemic acquiredresistance (SAR) can support the systemic spread of the virus in the host body. Our earlierstudies showed (i) the influence of the potyvirus gene silencing suppressor HC-Pro on HRresponse of Chenopodium quinoa to potyvirus infection and (ii) the inability of theattenuated mutants of Zucchini yellow mosaic virus (ZYMV) and Turnip mosaic virus(TuMV), which carry point mutations in the conserved motifs HC-Pro, to induce HR. Ourstudies using yeast two-hybrid system identified several HC-Pro interacting C. quinoa (Cq)proteins, including a putative carbonic anhydrase (putative CqCA), highly homologous toCA1 and CA2 of Arabidopsis thaliana (At). AtCA1, a salicylic acid bind protein, designatedSABP3, is one of the key proteins in the elicitation of HR. Thus, the changes in the bindinginteraction of potyvirus HC-Pro and putative CqCA may be the major underlying reason forthe inability of ZYMV and TuMV attenuated mutant viruses to induce HR in C. quinoa.Since C. quinoa does not have sufficient functional genomics tools, we plan to useArabidopsis system in our further investigation. In the present study, Atca1 and Atca2knock-out Arabidopsis mutant lines; AtCA1 and AtCA2 over-expressing transgenicArabidopsis lines; Atca1 and Atca2 knock-out mutant lines transgenically complementedwith AtCA1 and AtCA2; different defense-related Arabidopsis mutants and the wild typeZYMV and TuMV and the respective mutant viruses will be used for analyses. Theprotein-protein interaction studies will involve yeast 2-hybrid analysis of HC-Pro bindingwith AtCA1 and AtCA2, chromatography to analyze the stoichiometry of CA and HC-Prohetero-oligomeric complex, in vitro and in vivo binding studies to analyze the influence ofHC-Pro binding with CA on SA interaction with CA. The possible changes in chloroplaststructure and function caused by HC-Pro binding with AtCA1 and AtCA2 (affecting SAinteraction with CA) will also be studied by microscopy and by assaying enzymes reportingstructural integrity and functional viability of chloroplasts. In each experiment, along withwild type HC-Pro, the mutant HC-Pro proteins of the HR-deficient attenuated mutant viruseswill also be included to understand differential behavior. The present studies on themolecular mechanism of potyviral HC-Pro interference with HR will provide further insightsto understand plant defense and to devise new strategies to manage viral disease.
其他識別: NSC100-2313-B005-009-MY3
Appears in Collections:動物科學系

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