水稻黑条矮缩病毒非结构蛋白P7-1与水稻GAPDH互作机理

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, is propagatively transmitted by the small brown planthopper (Laodelphax striatellus Fallén) and causes rice black-streaked dwarf and maize rough dwarf diseases, which lead to severe yield losses of crops in China. Although several RBSDV proteins have been investigated in details, functions of the nonstructural protein P7-1 are still largely unknown. To investigate the role of the P7-1 protein in virus pathogenicity, transgenic Arabidopsis thaliana plants were generated in which the P7-1 transgene was expressed under the 35S promoter. The RBSDV P7-1-transgenic Arabidopsis were male sterility.Flowers and pollen from P7-1-transgenic plants were of normal size and shape as in wild type, and anthers developed to normal size but fail to dehisce. Yeast two hybrid assay indicated that P7-1 interacted with GAPA (glyceraldehyde-3-phosphate dehydrogenase) NAD binding domain. Rice GAPDH (Os04g38600,Os03g03720)mRNA transcripts were induced by inoculated with RBSDV, and rice GAPDH protein (Os04g38600,Os03g03720)interacted with RBSDV P7-1 in yeast two hybrid assay. In this project, RBSDV P7-1 interacted with rice GAPDH protein will be tested by yeast two hybrid, pull down and BiFC (bimolecular fluorescence complementation) assays. Function of virus P7-1 and rice GAPDH in plant development and virus resistance will be done by P7-1, GAPDH overexpression and RNAi silencing transgenic rice inoculated with RBSDV. The mechanism of rice GAPDH regulated by RBSDV P7-1 will be analyzed by GAPDH activity, mRNA transcription, protein expression and subcellular localization.The function of RBSDV P7-1 interacted with rice GAPDH revealed in this project, will provide a theoretical basis for the further understanding of viral pathogenesis.

水稻黑条矮缩病毒(RBSDV)侵染水稻导致植株矮化、育性下降，严重威胁我国水稻生产。但对病毒蛋白与寄主因子互作导致病害症状的机理还了解甚少。前期研究表明，拟南芥过量表达RBSDV P7-1引起雄性不育，P7-1与拟南芥GAPDH(甘油醛-3磷酸脱氢酶)互作；RBSDV侵染水稻诱导GAPDH表达，酵母双杂交表明病毒P7-1也与水稻GAPDH互作。本项目拟采用Pull down、BiFC进一步验证病毒P7-1与水稻GAPDH互作；构建P7-1过表达和基因沉默转基因水稻，研究P7-1在病毒致病过程中功能；构建GAPDH过表达、基因沉默转基因水稻及T-DNA突变体，研究GAPDH在育性和抗病毒过程中的作用；通过分析GAPDH酶活、mRNA转录、蛋白表达及亚细胞定位揭示病毒P7-1调控GAPDH机理。本项目所解析的RBSDV P7-1与水稻GAPDH互作机理，将为进一步理解病毒致病机理提供理论依据。

我们用激光共聚焦检测RBSDV P7-1，水稻GAPA1和GAPA2在细胞内定位。我们发现，当这些蛋白单独在烟草叶片表达时，RBSDV P7-1定位于细胞核和细胞质；水稻GAPA1只定位于叶绿体；水稻GAPA2定位于叶绿体和细胞质。当RBSDV P7-1与GAPA1（或GAPA2）在烟草叶片共表达时，P7-1与GAPA1(或GAPA2)共定位于细胞质和细胞核。我们构建并产生了RBSDV P7-1基因沉默（P7-1-Si）、水稻GAPA1过量表达（GAPA1-OE）、水稻GAPA2过量表达（GAPA2-OE）以及水稻GAPA2基因沉默（GAPA2-Si）转基因水稻植株。通过带毒灰飞虱接种RBSDV，水稻植株RBSDV抗性鉴定结果表明，P7-1基因沉默、水稻GAPA1过量表达和水稻GAPA2基因沉默转基因水稻能增强对病毒RBSDV的抗性。这些结果表明，GAPA1和GAPA2在水稻抗RBSDV中起重要作用。