番木瓜环斑病毒与寄主互作中蛋氨酸亚砜还原酶B1参与的病毒反防御和叶绿体抗氧化防护机制

Papaya ringspot virus (PRSV), a member of the aphid-transmitted genus Potyvirus , is the most widespread and destructive virus disease affecting papaya cultivation worldwide.Typically,in compatible virus-host plant interactions, one of the earliest responses of plant cells to pathogens is the production of reactive oxygen species (ROS) and chloroplasts can be a source of oxidative stress during viral disease development.Plant methionine sulfoxide reductase B1 (MsrB1) protects the photosynthetic apparatus from oxidative damage by scavenging ROS to repair Met-oxidized proteins. However, the chloroplast-localized papaya (Pa) MsrB1 interacts with the virus-encoded PRSV nuclear inclusion protein a protease (NIa-Pro), disturbing import of PaMsrB1 into chloroplasts. PRSV NIa-Pro contains 4 Met residues, one of which is predicted to be particularly prone to oxidation into Met sulfoxide (MetO). These findings led us to hypothesize that PaMsrB1 catalyzes the reduction of oxidized PRSV NIa-Pro by ROS, representing a novel mechanism of PRSV towards the host defense. In the proposed project, glutaredox (Grx), SDS-PAGE, and MALDI-TOF/TOF MS will be used to determine whether PaMsrB1 catalyzes reduction of oxidized PRSV NIa-Pro. Additionally, to understand the anti-oxidative defense mechanism of PaMsrB1 in chloroplasts during host-virus interaction, affinity chromatography, Nano LC-MS/MS, and biochemical analyses of activity of PaMsrB1 will be used to isolate and identify substrates of PaMsrB1. PRSV accumulation and infection, chlorophyll fluorescence parameters，ROS cellular localization and metabolic and signal transduction pathways involved in PaMsrB1 will be analyzed through the use of transgenic papaya plants in which the PaMsrB1 gene is over-expressed or silenced. The results of these studies will provide additional insight to the mechanisms of viral infection and host-defense in PRSV, paving the way for future investigation to PRSV prevention.

番木瓜环斑病毒(PRSV)是番木瓜种植业毁灭性病害。定位于叶绿体的蛋氨酸亚砜还原酶B1(MsrB1)能还原被ROS氧化蛋白中的Met,以保护植物光合代谢。前期证明PRSV多功能蛋白NIa-Pro与番木瓜MsrB1(PaMsrB1)互作干扰了PaMsrB1定位于叶绿体,PRSV NIa-Pro含易被氧化的Met。本项目拟用Grx和质谱验证这一互作是否与病毒利用PaMsrB1还原修复由于寄主防御而被氧化的NIa-Pro有关,这可能是一种新的病毒反防御机制。进而综合亲和层析、液质联用、生理生化、转基因、亚细胞定位和分子生物学方法分离鉴定PRSV与寄主互作中PaMsrB1抗氧化调节的蛋白底物及参与的相关信号和代谢途径，分析PaMsrB1基因表达变化对PRSV积累、叶绿素荧光动力学参数、ROS积累分布的影响，以揭示PaMsrB1参与的病毒反防御和叶绿体抗氧化防护机制，为防治PRSV新策略提供科学依据

番木瓜环斑病毒(PRSV)是威胁番木瓜种植业的重要病害。定位于叶绿体的蛋氨酸亚砜还原酶 B1(MsrB1)能还原被 ROS 氧化蛋白中的 Met,以保护植物光合代谢。前期证明 PRSV多功能蛋白 NIa-Pro 与番木瓜 MsrB1(PaMsrB1)互作干扰了 PaMsrB1 定位于叶绿体，PRSV NIa-Pro 含易被氧化的 Met。本项目通过原核表达和纯化系统获得了具有还原活性的重组PaMsrB1和具有DNA酶及特异的蛋白酶双重活性的重组PRSV-NIa-Pro。通过Grx还原系统 ，SDS-PAGE 和质谱方法体外证明了被氧化的PRSV-NIa-Pro可以作为PaMsrB1的底物被还原，因此，PaMsrB1与PRSV-NIa-Pro的互作与病毒利用PaMsrB1还原修复由于寄主防御而被氧化的 NIa-Pro 有关，这是一种新的病毒反防御机制。进一步利用亲和层析和质谱分离获得13个PaMsrB1候选底物蛋白，其中有8个候选底物蛋白定位于叶绿体，在叶绿体合成代谢中发挥重要作用。通过遗传转化分别获得了过量表达和沉默下调 PaMsrB1 的转基因番木瓜植株，PRSV攻毒实验结果显示，PaMsrB1的过表达减弱了病毒和细胞内 ROS 产生积累，增强了植物抗病能力，反之则病毒和ROS积累增加，植物抗病能力下降。综合这上述研究结果，本项目的实施对揭示PRSV 侵染或植物防御过程中PaMsrB1 参与的病毒反防御和叶绿体抗氧化防护机制提供了科学依据。