阿拉伯芥轉錄後基因靜默路徑相關基因研究

Abstract

轉錄後基因靜默作用 (PTGS) 是植物利用mRNA降解和轉譯抑制來抵抗病毒的重要機制。然而，馬鈴薯Y病毒如蕪菁嵌紋病毒 (TuMV) 的致病蛋白 P1/HC-Pro 可突破此防禦機制。在植物感染過程中，P1 蛋白與HC-Pro 分割且增強 HC-Pro 的作用，但其機制尚不清楚。先前研究表示三種馬鈴薯病毒的 P1，包括 TuMV、煙草蝕刻病毒 (TEV) 和矮南瓜黃化嵌紋病毒 (ZYMV) 在體內與VERNALIZATION INDEPENDENCE 3 (VIP3) 相互作用。VIP3 作為 RNA 胞泌體的組成蛋白，在阿拉伯芥中參與降解RNA誘導型緘黙化複合體 (RISC) 5' 端的切割片段。在這項研究中，我們透過 CRISPR/Cas9 基因編輯在阿拉伯芥中創造了 vip3ge 突變體。同時，我們產製 VIP3 特異性抗體來檢測不同突變體中的 VIP3 蛋白表現量。我們利用mRNA 北方墨點法分析 P1/HC-ProTu 和 vip3ge 突變株的5' 端切割片段的降解狀況。結果顯示CSD2 5'-切割片段在 vip3ge 突變體和 P1/HC-ProTu植物中累積。此外，我們還研究了 ARGONAUTE 2 (AGO2)，它是 Argonaute (AGO) 蛋白之一，可結合小 RNA 並進行切割。先前研究發現由miR403所標定的AGO2 mRNA 是由 AGO1 所調節。我們的結果顯示P1/HC-ProTu植物中 AGO1 蛋白表現量減少，AGO2 蛋白表現量上升，表明 AGO2 取代 AGO1 抵抗病毒的防禦作用。此外，我們產製 ATG8a 特異性抗體 (α-ATG8a 抗體) 來研究 P1/HC-ProTu植物中觸發AGO1 降解的自噬機制。 α-ATG8a 抗體能夠檢測 Col-0 和其他突變株的內源性 ATG8a。總體而言，這項研究有助於我們闡明阿拉伯芥中 PTGS 的相關基因如 VIP3、AGO2 及 ATG8a，以及病毒抑制因子 P1/HC-ProTu如何抑制 PTGS 。

Post-transcriptional gene silencing (PTGS) is an essential mechanism for plants to suppress viruses by mRNA decay and translational repression. However, potyvirus such as turnip mosaic virus (TuMV) encodes pathogenicity proteins, P1/HC-Pro that counteract this defence mechanism. During infection upon the plant, P1 protein cleaves itself from HC-Pro. Previous research showed that HC-Pro which has an FRNK motif triggers degradation of AGO1, while the function of P1 in enhancing HC-Pro remains unclear. It was demonstrated that P1 of three potyviruses, including TuMV, tobacco etch virus (TEV) and zucchini yellow mosaic virus (ZYMV) interacted with VERNALIZATION INDEPENDENCE 3 (VIP3) in vivo. VIP3 is a component of RNA exosome that is involved in the degradation of RNA-induced silencing complex (RISC) 5'-cleavage fragments in Arabidopsis. In this study, we created vip3ge mutants in Arabidopsis by CRISPR/Cas9 gene editing approach. We also generated a VIP3-specific antibody to detect VIP3 protein level in different mutants. The generated α-VIP3 antibody is able to detect endogenous VIP3 in Arabidopsis. We performed mRNA northern blot with P1/HC-ProTu and vip3ge mutants. The accumulation of CSD2 5'-cleavage fragments was shown in vip3ge mutants and P1/HC-ProTu plants. In addition, we also study on ARGONAUTE 2 (AGO2) which is one of the Argonaute (AGO) proteins that bind small RNAs and cleave at their target sites. Previous research showed that AGO2 mRNA is targeted by miR403 which is regulated by AGO1. Our result also showed that level of AGO1 decreased and AGO2 protein increased in P1/HC-ProTu plants, suggesting that AGO2 takes over the role of AGO1 in antiviral defence. Besides, we generated ATG8a-specific antibodies (α-ATG8a antibody) to investigate the mechanism of autophagic AGO1 degradation that is triggered in P1/HC-ProTu plants. The α-ATG8a antibody is able to detect endogenous ATG8a in the wild-type Col-0 and other transgenic lines. Overall, this study helps us elucidate the relevance of PTGS-related genes, including VIP3, AGO2, and ATG8a in Arabidopsis and how the viral suppressor P1/HC-ProTu disrupted the components of PTGS in the transgenic plants.