探討菸草鑲嵌病毒p126蛋白在RNA靜默機制中所扮演的角色

Abstract

轉錄後基因靜默（post-transcriptional gene silencing，PTGS），亦即RNA干擾現象 (RNAi)，會導致目標基因的mRNA被分解，是為一種調節基因表現的機制。近幾年來有許多研究顯示，此現象不但廣泛地存在於生物界，更為真核生物用以抵禦病毒侵染的一套固有防禦機制。然而，病毒也演化出另一種反防禦策略，亦即利用抑制子 (suppressor)，去干擾內生的基因靜默訊息傳遞路徑，以達到成功感染目的。本研究以煙草鑲嵌病毒 (TMV) 為模式，探討TMV p126蛋白如何參與對抗PTGS。首先以GFP煙草轉植株 (Nicotiana benthamiana, line 16c) 建立篩選平台，藉由農桿菌接種法 (agroinfiltration) 送入外來GFP，啟動PTGS機制導致16c中GFP mRNA量降低。我們首先觀察到當煙草株遭受TMV感染，或單獨表現p126蛋白時，GFP不僅可恆常表現，甚至有被助長表現之情況。進一步將p126蛋白依據其不同的功能性區塊 (domain) 進行擷取，共分為methyltransferase (MET)、non-conserved region (nonI)、non-conserved region II (nonII) 和helicase (HEL) 四個區塊。當個別表現MET、nonII和HEL於16c時，皆顯示其有抑制PTGS之功能。為了進一步釐清其參與機制，我們利用點突變破壞MET及HEL之酵素活性區，但結果顯示其酵素功能似乎不參與在抑制PTGS之機制中。令人驚訝的是，當我們替換nonII三個會影響病徵之胺基酸後，便失去抑制PTGS之功能。另外，我們也建立p126煙草轉殖株，期望找出病毒致病性與PTGS抑制功能兩者之間的相關連結，但若要明確了解整個機制則仍有待持續的研究與探討。

Post-transcriptional gene silencing (PTGS) is a conservative phenomenon across kingdoms. In plants, it has been demonstrated that PTGS functions as an intrinsic defense mechanism during viral infection. On the other hand, viruses have evolved counter-defense strategies by encoded suppressors to circumvent PTGS. In previous studies, Tobacco mosaic virus (TMV) encoded p126 protein has been exhibited as a suppressor of PTGS although its mechanism remains unknown. In this current study, we found that there are multiple suppressor domains/motifs within the p126 protein, including methyltransferase, helicase and non-conserved region II, implicating that these domains/motifs could function synergistically against PTGS. Nevertheless, these suppressor domains might exhibit differential consequences on local and/or systemic gene silencing. Moreover, the enzymatic activities of methyltransferase and helicase are dispensable in the suppression pathways. Interestingly, several amino acids within the non-conserved region II pivotal for pathological consequence seem to intimately correlate with extend of the ability of gene silencing suppression. Together, these results suggest that a role of gene silencing suppression of TMV determinates its susceptibility on host plants. Furthermore, the MAPK signal cascade(s) was involved in the functioning of the p126-mediated gene silencing in tobacco. Our findings will therefore help elucidate the function and evolution of different virus suppressor and lead to new crop protection strategies.