新穎微管E3 ligase RED 基因群之功能分析

Abstract

泛素化系統在許多植物功能之調控路徑中具關鍵功能，而其中與目標蛋白質結合之E3泛素接合酶 (E3 ligase) 更扮演極重要之決定角色。我們先前在番茄及阿拉伯芥中發現一群功能未知之RING-H2 type E3 ubiquitin ligase，將之命名為RING-type E3 ubiquitin ligases with an uncharacterized DAR1 domain (REDs)，並確認番茄Sl_RED1是一坐落於細胞骨架微管上的泛素接合酶。本研究旨在探討番茄SlRED1及其同源基因之蛋白質特性及功能，並尋找REDs之交互作用蛋白質。結果顯示，SlRED1、SlRED2、AtRED1、AtRED2皆坐落於微管上，而SlRED1之胺基酸片段1-60為其坐落於微管之必要片段。此外，位於SlRED1 C端的RING domain與DAR1 domain則為與E2進行直接結合之關鍵片段。另外，過量表現SlRED1的菸草(Nicotiana benthamiana)轉殖株對青枯病菌（Ralstonia solanacearum）與細菌性軟腐病原細菌（Pectobacterium carotovorum subsp. carotovorum）的抗性提高，但不影響植物葉部氣孔密度與水份散失率。以上結果顯示，REDs為一群坐落於微管之新穎E3泛素接合酶，且SlRED1在植物對抗病原細菌具正向功能。目前正積極搜尋REDs之交互作用蛋白質，以期釐清RED之作用機制。

Ubiquitin E3 ligases are highly complex regulators and play a pivotal role in plant signaling transduction pathways switching extracellular signals into various cellular responses. In previous studies, we identified a group of plant RING-H2 type E3 ubiquitin ligases with an uncharacterized DAR1 domain (REDs) and our data indicated that SlRED1 is a microtubule-localized E3 ligase. The objective of this study was to characterize these REDs and identify their interacting proteins. The results show that SlRED1, SlRED2, AtRED1 and AtRED2 are co-localize on the microtubules and the N-terminal region (amino acid 1~60) is required for the microtubular localization of SlRED1. In addition, the RING and DAR1 domains are both important for the interaction between SlRED1 and E2 conjugating enzyme. Furthermore, the overexpression of SlRED1 in transgenic Nicotiana benthamiana plants led to enhanced resistance to the infection of R. solanacearum and Pectobacterium carotovorum pv. carotovorum, but had no effect on leaf stomata number and transpiration. Collectively, these results indicate that REDs define a new group of plant microtubule-localized E3 ligases and that SlRED1 plays a positive role in resistance to pathogenic bacteria.