植物菌質體誘導葉狀花之研究和探討病毒的P1蛋白對HC-Pro抑制基因靜默機制的影響

Abstract

植物菌質體（phytoplasmas）之SAP54和PHYL1效應子，會造成植物花器葉化的症狀，以致大幅減少種子的形成。為了解決花器葉化的轉基因植物產生缺少種子的困境。本研究發展出一種病毒表現系統，來表現SAP54或PHYL1蛋白。我們使用蕪菁嵌紋病毒（TuMV）的弱系病毒株TuGK，作為病毒載體。此病毒載體能感染擬南芥植物，並且不會影響植物生長發育及能快速表現PHYL1或SAP54蛋白在植物器官中。我們成功地藉由此系統，獲得大量的葉狀花組織，來研究PHYL1或SAP54所引起的花器葉化機制。TuGK除了是一種合適的病毒載體外，它的另一功能是不會干擾植物的基因靜默機制（PTGS）。基因靜默機制是植物的抗病機制之一。植物利用此來防禦自然界的病毒。而病毒是透過抑制微型核糖核酸（miRNA）和小干擾RNA（siRNA）途徑來反制植物基因靜默機制。potyvirus屬之病毒之P1/HC-Pro是第一個被發現會抑制植物基因靜默機制，故稱之基因靜默抑制子。P1/HC-Pro可透過P1蛋白酶的功能，將P1/HC-Pro切割成P1和HC-Pro兩個蛋白，以利病毒感染植物。目前對於P1蛋白的功能仍不清楚。故此論文，利用蛋白質體學分析方法，探討病毒的P1是否會與植物的蛋白相互作用，並分析P1蛋白是如何抑制植物基因靜默機制。從轉錄體和基因網絡分析中，鑑定出多個植物基因，皆與病毒之P1/HC-Pro能夠抑制植物基因靜默機制有關。另外，也發現P1 / HC-Pro轉基因植物中的AGO1蛋白會被降解。而病毒的P1會與植物的VERNALIZATION ININDENDENCE 3 / SUPERKILLER（VIP3 / SKI8）蛋白相互作用。VIP3 / SKI8是一個RNA外泌體（exosome），它的功能是當RNA誘導沉默複合物（RNA induced silencing complex-RISC）切割完後的5端RNA片段，外泌體會去降解此片段。我們推測P1與VIP3 / SKI8互動，可能會因此而促進了HC-Pro抑制植物基因靜默機制的能力。另外，根據轉錄體和基因網絡分析，鑑定出多個植物基因，包含微型核糖核酸標靶（miRNA targets），鈣訊號相關基因，植物荷爾蒙激素相關基因（JA, ET, and ABA）和防禦反應有關的基因，這些基因皆與植物基因靜默機制有關。綜合以上結果，我們透過基因遺傳學（genetic）和生物體學（omic）的方法，發現了很多植物基因靜默機制的相關基因會與病毒之P1 / HC-Pro互相調控。

SAP54 and PHYL1 are orthologs of pathogen effectors from phytoplasmas. These effectors can cause plant leafy flowers symptoms. To study plant flowers become leafy flower, we develop a viral vector to overcome seedless problem in transgenic plants. TuGK vector from mild strain virus of turnip mosaic virus (TuMV) is a good viral vector for carrying PHYL1 or SAP54 gene. TuGK did not interfere with plant development and it can infect Arabidopsis with rapidly expressing PHYL1 or SAP54 protein. We successfully obtained large amounts of leafy flower tissues for PHYL1 or SAP54 studies from viral vector infected Arabidopsis plants. The TuGK is a suitable viral vector because it loses posttranscriptional gene silencing (PTGS) suppression ability. PTGS is one of the plant antivirus mechanism and to counteract PTGS, plant viruses have also encoded viral suppressors to interrupt PTGS by repressing the regulation of microRNA (miRNA) and short-interfering RNA (siRNA) pathways. In Potyvirus, P1/HC-Pro is the first identified viral suppressor. The HC-Pro protein gets released by the self-cleaving activity of P1. Though the entire function of P1 protein remained unclear. In this study, we investigated the mechanism of P1 enhancing PTGS suppression by identifying P1-interacting proteins using proteomic analysis approach. Moreover, we discovered several novel genes that might relate to P1/HC-Pro-mediated PTGS suppression through transcriptomes and comparative network analysis. To summaries the findings in this study, the AGO1 protein was found to be specifically degraded in turnip mosaic virus (TuMV) P1/HC-Pro transgenic plants (P1/HCTu plant). Also, it was interesting to find that P1 directly interacts with VERNALIZATION INDEPENDENCE 3/SUPERKILLER (VIP3/SKI8) that might enhance HC-Pro-mediated PTGS suppression. VIP3/SKI8 is an RNA exosome and can interfere with the degradation of RNA induced silencing complex (RISC) 5'-cleavage fragment. Furthermore, through the transcriptome approach, we discovered potentially critical genes in PTGS by the comparative network analysis, containing miRNA targets, calcium signaling genes, hormone (JA, ET, and ABA) signaling genes, and defense response genes. Taken together, through these genetic and omics methods, we exposed many critical genes related to PTGS. These new discoveries allow us to better understand viral suppressor P1/HC-Pro interaction with host PTGS mechanism.