蕪菁嵌紋病毒及矮南瓜嵌紋病毒協同蛋白(HC-Pro)抑制HEN1甲基化能力及誘導AGO1細胞自噬作用降解之研究

Abstract

轉錄後基因靜默作用 (PTGS) 在植物中是抵抗病毒感染的重要防禦途徑。在參與 PTGS 的各種蛋白質中，HUA ENHANCER1 (HEN1) 負責 miRNA/miRNA* 雙鏈體的甲基化其隨後被加載到 ARGONAUTE1 (AGO1) 中，並形成功能複合物以切割目標 mRNA。然而，植物病毒一直在發展病毒抑制因子來預防 PTGS，例如已有廣泛研究的馬鈴薯Y病毒的 P1/HC-Pro 對 RNA 靜默的抑制。在這項研究中，我們研究了來自矮南瓜黃化嵌紋病毒 (ZYMV) 和蕪菁嵌紋病毒 (TuMV) 的 HC-Pros 在 RNA 沉默抑制中的作用，特別是在 AGO1 和 HEN1 抑制中的作用。為此目的，表現 TuMV 的 P1/HC-Pro (P1/HC-ProTu 植物) 或 ZYMV (P1/HC-ProZY 植物) 轉基因植物及重組 P1/HC-Pro 植物，包括 P1Tu/HC-ProZY 和 P1ZY/HC-ProTu 植物用於此研究中。所有植物均表現出相同的嚴重鋸齒狀捲曲葉表型。同時，P1和HC-Pro蛋白表現量可被穩定檢測。此外，表現HC-ProTu 的植物中的 AGO1 表現量顯著降低，表現 HC-ProZY 的植物中 AGO1 蛋白表現量略有降低，表明 AGO1 是 HC-ProTu 的主要靶標。此外，自噬相關基因 ATG8a 在 P1/HC-ProTu 和 P1/HC-ProZY 植物中的表現量增加，顯示 AGO1 的降解與 ATG8a 的表現相關。另一方面，在體內的甲基化檢測中，HC-ProTu 導致 miRNA 的非甲基化狀態，而 HC-ProZY 未在轉基因植物或 ZYMV 感染的筍瓜中顯示 miRNA 甲基化的負調控。我們還證明了 HEN1 與 miRNA 結合以進行甲基轉移。有趣的是，HC-ProZY 在體外顯示出對這種結合的部分抑制。相反，HC-ProTu 完全阻止了 HEN1及 miRNA 之間的反應，而 HC-ProTu-K 由於 FRNK 基序處的 Arg182Lys 取代而減弱了抑制。這提出了 FRNK 結構域對於 HC-Pro 和 HEN1 結合的重要性。儘管如此，我們的體外系統比體內系統更簡單，這可提供蛋白質抑制基因靜默最佳的條件，並瞭解 HC-ProZY 對 HEN1 甲基化過程的影響。總而言之，這項研究有助於我們更清楚地了解 HC-ProTu 和 HC-ProZY 對 PTGS 的抑制作用，讓我們更深入的發現病毒與宿主植物之間的防禦機制。

In plants, post-transcriptional gene silencing (PTGS) is an important defence pathway to counteract virus infection. Among various proteins involved in the PTGS, HUA ENHANCER1 (HEN1) is responsible for the methylation of miRNA/miRNA* duplex, which is subsequently loaded into ARGONAUTE1 (AGO1) to form a functional complex to cleave the targeted mRNAs. However, plant viruses have been developing several viral suppressors to prevent the PTGS. Typically, P1/HC-Pro of potyviruses has been studied widely on its inhibition activity towards RNA silencing. In this research, we study the effects of HC-Pros from turnip mosaic virus (TuMV) and zucchini yellow mosaic virus (ZYMV) in RNA silencing suppression, especially in AGO1 and HEN1 inhibition. For that purpose, transgenic plants which express P1/HC-Pro of TuMV (P1/HC-ProTu plant) or ZYMV (P1/HC-ProZY plant) and recombinant P1/HC-Pro plants, including P1Tu/HC-ProZY and P1ZY/HC-ProTu plants, were used in the study. All the plants showed the identical severe serrated-curling leaf phenotype; besides, P1 and HC-Pro protein levels could be detectable stably. Moreover, AGO1 level markedly decreased in plants expressing HC-ProTu and slightly reduced in plants expressing HC-ProZY, indicating that AGO1 is the prime target of HC-ProTu. In addition, the autophagic-related gene ATG8a expression was increased in the P1/HC-ProTu and P1/HC-ProZY plants, demonstrating that the degradation of AGO1 is correlated with the expression of ATG8a. On the other hand, in the in vivo methylation assay, HC-ProTu caused the unmethylation status of miRNA, while HC-ProZY did not show any negative regulation of the methylation of miRNA in the transgenic plants or ZYMV-infected zucchini plants. We also proved that HEN1 binds miRNA to perform its methyltransferase activity. Intriguingly, HC-ProZY showed partial inhibition to this binding in vitro. Oppositely, HC-ProTu completely prevented the reaction between HEN1 and miRNA, while HC-ProTu-K had attenuated repression due to the Arg182Lys substitution at the FRNK motif. This proposed the importance of the FRNK domain for the binding of HC-Pro and HEN1. Nonetheless, our in vitro system is simpler than the in vivo system, which might provide the proteins with the optimized conditions for their gene silencing suppression, leading to the different effects of HC-ProZY on the methylation process of HEN1. Collectively, this study helps us to understand more clearly the suppression activity of HC-ProTu and HC-ProZY on PTGS, which provides us with the background to discover deeper about the defence response between virus and host plants.