TMLIP2在Tm-22介导的Tobamovirus属病毒抗性中的作用

Tobamoviruses, including Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV), cause serious disease and huge destruction in agriculture.The resitance gene Tm-22 confers durable resistance against both TMV and ToMV in Solanaceae species and became one essential target for crop-breeding in the control of Tobamovirus diseases. The recongnition of R-AVR and the subsequent elicition of immune reaction take central place in host-pathogen interaction. The Tm-22 protein and its correponding avirulent effector movement protein (MP) consist a type model for plant immunity research. Considering the broad application of Tm-22 in agricultre, dessecting the mechanism of Tm-22-MP interaction gains practical meanings and theoratial values as well. We screened yeast 2-hybrid library with MP and Tm-22 LRRs domain as a bait respectively, and found one protein which could interact with both MP and LRRs. This protein, named TMLIP2 (for Tm-22 LRRs interactin protein 2), was characterized as a J domain containg factor belonging to the C group of DnaJ family. TMLIP2 interated with MP and full length Tm-22 in yeast and could co-immunoprecipated with MP or Tm-22 in plants. Our preliminary data suggests that suppression of TMLIP2 with VIGS (virus induced gene silencing) method causes less infection foci and reduced cell-to-cell movement and systemic infection of TMV in Nicotiana benthamiana. While in Tm-22 transgenic plants, silencing of TMLIP2 led to compromised and even the loss of resistance against TMV. It was worth noting that TMLIP2 interacted with both the AVR effector MP and the R protein Tm-22 in vitro and in plant. The depletion of TMLIP2 in susceptible N. benthamiana reduced TMV infection and lagged TMV intercellular movement, while in Tm22 transgenic lines sequestered resistance against TMV. Those data suggested the role of TMLIP2 in Tm-22-MP interaction and plant immune responses. This project will investigate the role of TMLIP2 in Tm-22-MP system and explore the mechnism of TMLIP2 in regulating plant resistance, which will help unraveling mechanisms underlied in host-pathogen interaction and plant resistance responses.

烟草花叶病毒属病毒TMV、ToMV等对作物危害严重，介导TMV抗性Tm-22基因是培育抗病品种的重要位点。抗性蛋白(R)和无毒蛋白(AVR)之间识别和激发抗性反应在植物-病原互作中居于核心地位。Tm-22及其AVR TMV运动蛋白MP既可做为植物免疫研究的模式系统，研究成果又具有重要的实践价值。本课题分离得到了既与AVR蛋白MP互作，又与R蛋白Tm-22互作的蛋白TMLIP2。沉默TMLIP2既可以抑制本生烟草内TMV的侵染和运动；又可以导致Tm-22转基因本生烟草的TMV抗性严重抑制甚至丧失。因此TMLIP2在Tm-22植物上参与抗性反应，是植物抗病毒的重要因子；在易感本生烟草上，又可以作为病毒侵染和运动的必需因子，参与病毒侵染过程。研究TMLIP2在Tm-22-MP系统中的作用，分析TMLIP2在致病和抗性中的作用，可以深入了解植物-病原的互作机制，并为TMV抗性育种提供参考。

Tobamovirus属病毒如番茄花叶病毒(ToMV)和烟草花叶病毒(TMV)等是马铃薯、烟草、番茄等作物上的重要的病毒病害。CC-NBS-LRR类抗性蛋白(R蛋白) Tm-22蛋白能够识别ToMV/TMV的运动蛋白(MP)，进而诱导植物产生抗病反应。通过酵母双杂交系统筛选到与Tm-22相互作用的Ⅰ型J蛋白TMLIP2/NbMIP1。同源性比较显示本生烟草内存在7个TMLIP2/NbMIP1的同源基因。根据基因序列分为5组，并分别命名为NbMIP1.1a、NbMIP1.1b、NbMIP1.2、NbMIP1.3、NbMIP1.4a、NbMIP1.4b和NbMIP1L1。为避免命名混乱，将筛选得到蛋白因子TMLIP2/NbMIP1命名为NbMIP1。.NbMIP1s既能与Tm-22相互作用，又能与ToMV MP相互作用。NbMIP1蛋白C-端的Zinc Finger、CTD结构域负责与MP、Tm-22相互作用。病毒诱导的基因沉默(VIGS)实验显示，NbMIP1s沉默不仅能导致Tm-22介导的抗性丧失，还能延缓Tobamovirus的细胞间运动和系统运动，显著抑制病毒的侵染，说明NbMIP1s对寄主抗病反应和病毒的侵染都具有重要作用。沉默NbMIP1s能够导致MP和Tm-22蛋白水平的显著降低，却不影响MP和Tm-22的转录和蛋白的亚细胞定位，说明NbMIP1s通过调节蛋白的稳定性发挥生物学功能。.NbMIP1s能够与SGT1相互作用，沉默NbSGT1也能够降低Tm-22蛋白水平，并导致Tm-22介导的抗病反应的丧失。沉默NbMIP1s还能导致N、Rx-1、Cf9、Pto等R蛋白介导的抗性和非寄主免疫反应的抑制。提示NbMIP1s作为辅助伴侣分子(co-chaperone)与SGT1共同参与植物免疫反应。.构建了NbMIP1 hairpin RNAi转基因本生烟草，与对照植物相比，NbMIP1s RNAi植物能够有效的抑制TMV细胞间运动以及系统运动。.综上所述，NbMIP1s能够与ToMV MP、Tm-22、SGT1蛋白相互作用， NbMIP1s沉默在易感植株内可以抑制Tobamovirus病毒侵染，在抗性植株内可以导致Tm-22介导的抗病反应的丧失。阐明了NbMIP1s是维持MP、Tm-22蛋白的稳定性的重要因子，在植物抗病反应和病毒侵染中发挥功能。