VIP3、TSN1/2、AGO2、CLF 和 LHP1 在阿拉伯芥 中病毒防禦與 RNA 靜默的潛在角色之研究

Abstract

RNA 靜默 (RNA silencing) 在調控植物基因表達中扮演關鍵角色，並作為對抗病毒感染的重要防禦機制。然而，許多病毒已演化出病毒 RNA靜默抑制子 (viral suppressor of RNA silencing) 來對抗 RNA 靜默。值得注意的是，蕪菁嵌紋病毒之編碼P1蛋白不僅能增強病毒抑制子 HC-Pro 的 RNA silencing 抑制能力，還能與植物的內源性蛋白質（如VIP3和TSN1/2）相互作用。然而，P1蛋白為何需要與這些內源性蛋白質相互作用的機制仍不清楚。此外，AGO2及多梳群蛋白（PcG proteins，如CLF和LHP1）也被認為可能參與RNA沉默途徑。這些發現引發了關於這些蛋白質如何影響RNA沉默並在抗病毒防禦機制中發揮作用的問題。本研究通過分析AGO1蛋白水平、miRNA靶向基因表達及降解組定序，探討了 VIP3、TSN1/2、AGO2、CLF和LHP1 在阿拉伯芥 (Arabidopsis thaliana) 中的抗病毒防禦及 RNA silencing 中的角色。我們發現 VIP3、TSN1/2 和 AGO2 是 AGO1 介導的切割活性的新型負調控因子，並將其定義為 RNA 靜默相關元件。相比之下，CLF 和 LHP1 可能不直接參與RNA沉默，但可能調控 PcG 靶向基因的 mRNA 降解。值得注意的是，tsn1/tsn2 和 vip3 突變株對 TuMV 感染表現出更高的抗性，其中 vip3 突變體對病毒累積的抑制效果最為顯著。因此，我們的結果表明，VIP3 和 TSN 作為 AGO1 活性的抑制子，而 P1 蛋白可能通過穩定 VIP3 或 TSN 來抑制病毒 RNA 的切割，從而促進 TuMV 感染。這些發現增進了我們對病毒如何利用宿主細胞機制來促進自身感染的理解。

RNA silencing plays crucial role in regulating plant gene expression and serves as an important defense mechanism against viral infections. However, many viruses have developed viral suppressors of RNA silencing (VSRs) to counteract RNA silencing. Notably, the potyvirus-encoded suppressor P1 enhances RNA silencing suppression of HC-Pro and interacts with plant endogenous proteins such as Vernalization Independence 3 (VIP3) and TUDOR-SN ribonucleases 1 and 2 (TSN1/2). However, the reason why the P1 protein needs to interact with those endogenous proteins remains unknown. AGO2 and Polycomb group (PcG) proteins, including Curly Leaf (CLF) and Like Heterochromatin Protein 1 (LHP1), have also been implicated in the RNA silencing pathway. These findings raise intriguing questions about how those proteins influence RNA silencing and play a role in viral defense mechanisms. Here, we investigate the roles of VIP3, TSN1/2, AGO2, CLF, and LHP1 in antiviral defense and RNA silencing in Arabidopsis thaliana by analyzing AGO1 protein levels, miRNA-ㄒ targeted gene expression, and degradome sequencing. We identified VIP3, TSN1/2, and AGO2 as novel negative regulators of AGO1-mediated cleavage activity and identified them as RNA silencing-related components. In contrast, CLF and LHP1 may not directly participate in RNA silencing but could regulate mRNA degradation of PcG- targeted genes. Strikingly, tsn1/tsn2ge and vip3ge mutants exhibited enhanced resistance to TuMV infection, with vip3ge mutants showing the strongest suppression of viral accumulation. Thus, our results demonstrate that VIP3 and TSN suppress AGO1 activity, suggesting that the P1 protein may stabilize VIP3 or TSN to inhibit the cleavage of viral RNA, thereby promoting TuMV infection. These insights enhance our understanding of how viruses exploit host cellular mechanisms to promote their own infection.