微型核酸註解及 P1/HC-Pro 參與植物病毒交互保護與基因靜默抑制作用

Abstract

植物基因靜默系統包含了兩種微小核酸，微型核酸 (microRNA; miRNA) 以及短片段干擾核酸 (short-interfering RNA; siRNA)，除了在基因表現、各式生理發育路徑扮演重要調控角色之外，也對病原菌如病毒等入侵提供了防禦系統。本篇論文中我們首先建立了 miRNA鑑定流程並且於演化地位重要的地錢 Marchantia polymorpha 中，整合並運用轉錄體，微小核酸轉錄體等大數據分析工具，並使用降解體來準確篩選，找到了包含保守種類共129種 miRNA。另外並利用病毒衍生出來抑制植物基因靜默抗性系統的植物病毒抑制子，蕪菁嵌紋病毒的P1/HC-Pro，研究基因靜默系統的分子機制，並同時找出P1/HC-Pro的作用位點。我們使模式植物Arabidopsis分別表現原型的 P1/HC-Pro (P1HCR) 以及 HC-Pro 上的 FRNK保守片段之R182K點突變HC-ProK (P1HCK)，發現P1HCR會造成嚴重 miRNA抑制並表現出畸形表型但P1HCK不會，顯示後者失去了抑制基因靜默的功能。我們並發現P1HCR中，基因靜默路徑下游的重要蛋白AGO1 會受到細胞自噬 (autophagy) 的降解。此外，FRNK中的 Arg residue會直接與 HEN1甲基酶交互作用並使 HEN1無法接觸原本的受質 miRNA，而使 miRNA甲基化受到抑制；失去甲基化的 miRNA 將對後續的 AGO1 loading 或 RISC (RNA-Induced Silencing Complex) 的組裝受到干擾，此時 autophagy 被誘導並將不完全組裝或失去功能的 AGO1及RISC 等降解，造成基因靜默抑制現象。我們並賦予了 miRNA 甲基化一樣新的定義：miRNA甲基化將是決定其能否 AGO1 loading 或 RISC 組裝之關鍵，在此之前科學家普遍認為 miRNA甲基化主要控制 miRNA是否穩定不被 uridylation 或降解。最後，回到病毒本身，我們詢問弱系病毒造成交叉保護 (cross protection) 的分子機制為何？ HC-Pro突變後的弱系病毒TuMVK 將作為很好的研究材料，結果發現除了已知的 protein-mediated resistance之外， RNA-mediated resistance 也有參與交叉保護，而初級免疫系統中的水楊酸 (SA) 路徑亦與交叉保護系統交互作用共同對抗並做出貢獻。

Plant gene silencing system that comprises two major small RNAs, microRNA (miRNA) and short-interfering RNA (siRNA), play a broad role in gene regulation, various biological or developmental processes, as well as stress response such as defense against virus. In this thesis, we first established an miRNA identification and characterization pipeline using the ContigView platform to manipulate the high throughput data through integrated bioinformatics strategies and successfully got an miRNA profile of a novel organism, liverwort Marchantia polymorpha. The investigation of viral suppressor not only helps understanding the virus-plant crosstalk but also uncovering the mechanism of hidden molecular gene silencing pathway. Being the first discovered suppressor, P1/HC-Pro of Potyvirus; however, is still remained mysterious. We built a transgenic Arabidopsis model separately expressing the wild-type P1/HC-Pro (P1HCR) from Turnip mosaic virus (TuMV) and the mutant form P1HCK with the Arg182Lys mutation on the conserved motif FRNK. The data suggested the HCK almost lost all capability to interfere with miRNA pathways. A genetic model of atg mutant was then used to demonstrate that HCR destabilizes Argonaute 1 (AGO1), a core component of RNA-Induced Silencing Complex (RISC), via autophagy pathway, which is a stress response and protein down-regulation machinery. Moreover, the FRNK motif on HCR plays a significant role in the methyltransferase HEN1-binding rather than small RNA-binding to inhibit HEN1 activity on miRNA 3'-end 2'-O-methylation, resulting in unmethylated miRNA abnormal accumulation as free-form status, which might lead to incomplete RISC assembly and the recruitment of autophagy system. Therefore, the autophagy pathway might serve as a surveillance system to clean up the incompletely assembled or dysfunctional RISC. All together, we provide the evidence to link autophagy and HCR-mediated gene silencing suppression pathways. Last but not least, using the in vivo model of P1HCR plant we found the 2'-O-methylation on miRNA is the determinant to load the miRNA into AGO1 or help assemble miRNA-RISC and this is going to be the first report of a novel function of miRNA methylation other than the protection aspect that was approved for decades. Finally, the Tu-GK mutant virus that carries HCK helps to establish a mild strain platform for understanding the molecular mechanism of cross protection. This attenuated virus confers complete cross protection against infection by a severe wild type strain in Arabidopsis thaliana. In addition to protein and RNA-mediated resistance, we revealed here that salicylic acid (SA) pathway takes part in cross protection mechanism