水稻潜根线虫效应子基因Ho501的鉴定及功能解析

Rice root nematode (RRN, Hirschmanniella spp.) is the most important pathogen, which poses a serious threat to rice production in Jiangxi province. However, to date, little is known about the pathogenic factors and molecular pathogenic mechanisms of RRN. Based on our preliminary results, a differential expression candidate effector named HO501 was obtained through comparative the transcriptome analysis of H. mucronata in different developmental stage before and after infection. Ho501, first isolated from H. mucronata, is a novel plant-parasitic nematodes effector gene and its encoded protein had a N-signal peptide for secretion. The Agrobacterium-mediated transient expression analysis demonstated the effector protein HO501 could suppress the hypersensitive response. It is the first case to show that the effector protein HO501 play a crucial roles for RRN virulence via inhibition of ETI response during nematode parasitism. Gene knock-out of Ho501 via In vitro RNA interference (RNAi) caused reduced parasitism, indicating its contribution to the parasitic ability in H. mucronata. In this study, we try to investigate the Ho501 gene tissue distribution patterns via in-situ hybridization and the protein subcellular location, explore the biological function of the Ho501 gene by using in planta RNA interference and over expression experiments. Then the interactors in the host of HO501 will be identified through CO-IP. Furthermore, the molecular regulatory network and pathogenic mechanisms of the gene Ho501 will be analyzed via over expression and knock-out of the interactors gene. This study will provide the foundation and theoretical basis for developing novel plant parasite control targets.

潜根线虫对江西省双季稻生产造成严重威胁，目前对其致病因子及致病机理还不清楚。Ho501是本研究前期通过比较转录组获得的一个潜根线虫侵染水稻后上调表达基因，其编码蛋白具有信号肽且不含跨膜结构域，是一个功能未知的植物寄生线虫新效应子基因。农杆菌浸润法介导的基因瞬时表达证明该基因能抑制寄主的免疫反应ETI，离体RNAi沉默该基因后线虫寄生能力显著下降。在此基础上，本研究拟通过原位杂交验证该基因在线虫体内的组织表达定位，通过亚细胞及免疫荧光定位分析其在线虫侵染寄主过程中的靶向定位，从细胞水平洞察该基因作用于寄主的具体生理过程；通过植物介导的RNAi和过表达验证该基因对线虫寄生的影响；利用免疫共沉淀技术筛选与该基因互作的寄主靶标蛋白；通过基因敲除和过表达其互作蛋白基因，分析该基因致病途径和调控网络，阐明由效应子HO501介导的潜根线虫致病机理，为开发新的水稻线虫病防治策略提供理论依据。

潜根线虫对江西省双季稻生产造成严重威胁，对其致病因子及致病机理还不清楚。Ho501是本研究前期通过比较转录组获得的一个潜根线虫侵染水稻后上调表达基因，其编码蛋白具有信号肽且不含跨膜结构域，是一个功能未知的植物寄生线虫新效应子基因。农杆菌浸润法介导的基因瞬时表达证明该基因能抑制寄主的免疫反应ETI，离体RNAi沉默该基因后线虫寄生能力显著下降。在此基础上，本研究通过原位杂交验证该基因在线虫食道腺内表达定位，通过35s增强启动子构建植物过表达载体，将Hm501基因与GFP标签融合表达于烟草表达系统。结果发现Hm501定位于内质网，； in planta RNAi发现RNAi转基因水稻能够有效沉默Hm501基因表达，降低水稻尖细潜根线虫侵染能力。利用免疫共沉淀技术筛选与该基因互作的寄主靶标蛋白；通过RNAi和过表达其互作蛋白基因，表明该基因调控硫氧化还原网络途径，阐明由效应子HO501介导的潜根线虫致病机理，为开发新的水稻线虫病防治策略提供理论依据。

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