叶蝉共生菌介导水稻矮缩病毒经卵传播的机制

The molecular mechanisms of transovarial transmission of plant viruses in their insect vectors are the hot and difficult spot of study. Rice dwarf virus (RDV) is transmitted by the green rice leafhopper Nephotettix cincticeps in a persistent-propagative manner and is transovarially transmitted. Our previous studies find that Rice dwarf virus (RDV) first enters the epithelial plug at the posterior end of the insect female’s ovariole, then invades the oocyte. Furthermore, the RDV particles are found on the membrane surface of two kinds of primary bacterial symbionts (Sulcia and Nasuia) in the ovariole. Thus, RDV may directly exploit the ancient entering oocyte pathway of symbionts in leafhoppers, allowing the virus to easily overcome the transovarial transmission barrier. In this project, we will use the methods including cell biology, protein interaction, antibody neutralization and inhibition of symbionts growth by the treatment of antibiotics or lysozyme, to investigate the transovarial transmission route of RDV in leafhopper, to identify the outer membrane proteins of bacterial symbionts (Sulcia and Nasuia) which could directly interact with the outer capsid proteins of RDV (P2 or P8) , and to elucidate the molecular mechanisms underling the symbiont-mediated transovarial transmission of RDV in leafhoppers. We also will study the relationships among the insect, symbionts and virus during the transovarial transmission of RDV. The results will reveal a new mechanism of transovarial transmission of a virus in its insect vector, and will provide the theoretical basis for explaining how the insect-transmitted viral diseases become serious under natural conditions, and for designing how to efficiently and persistently control them by the use of insect symbionts.

植物病毒的经卵传播至介体昆虫后代的分子机制是学科的热点和难点科学问题。水稻矮缩病毒（RDV）主要由介体黑尾叶蝉以持久增殖型方式传播的，并能经卵传播到介体后代。前期研究表明RDV是从介体叶蝉卵巢下端的上皮鞘侵入卵母细胞的，其在经卵传播过程中可直接附着在初生细菌类共生菌（Sulcia和Nasuia）外周，表明RDV可能是借用了古老的叶蝉体内共生菌的入卵通道，从而较为轻易地突破昆虫经卵传播屏障。本项目拟利用细胞生物学、蛋白互作、抗体中和作用和抗生素或溶菌酶抑制共生菌生长等手段，研究RDV经卵传播的途径，鉴定与RDV外壳蛋白（P2或P8）直接互作的共生菌外膜蛋白，解析共生菌介导RDV入卵的分子机制；同时，研究RDV经卵传播过程中形成的昆虫—共生菌—病毒三者间的互作关系。研究结果将揭示病毒的经卵传播新机制，为探索更为接近自然条件下的虫传病毒病致灾机制和利用共生菌设计高效、可持续的病害控制策略提供理论

针对近年来在我国发生的水稻病毒病，按计划开展了水稻病毒垂直传播机制的研究。经过4年的持续研究，揭示了水稻矮缩病毒（RDV）在介体黑尾叶蝉中借助介体初级共生菌Sulcia和Nasuia经卵传播的分子机制，描述了病毒粒体直接结合在共生菌外膜上由共生菌携带从黑尾叶蝉雌虫的血淋巴经后端卵巢的上皮鞘侵入卵母细胞的过程，发现病毒与共生菌结合是通过RDV次要外壳蛋白P2与Sulcia 外膜蛋白（OMP）直接互作以及RDV主要外壳蛋白P8与Nasuia孔蛋白（porin）直接互作介导的，通过抗生素处理、抗体阻断和RNA干扰等方法进一步证明了2类共生菌介导RDV从介体昆虫亲代垂直传递到子代的机制。研究结果首次揭示了昆虫共生菌与植物病毒在自然界中存在直接互作关系，解析了RDV在介体黑尾叶蝉中经卵传播过程中形成的昆虫—共生菌—病毒三者间存在互惠的多元互作关系，为探索更为接近自然条件下的虫传病毒病致灾机制和利用共生菌设计高效可持续的病害控制策略提供理论基础。本项目同时比较了与RDV同属与水稻呼肠孤病毒的水稻瘤矮病毒（RGDV）在介体中雌虫中的经卵传播的机制，描述了RGDV通过其编码的Pns11小管结构从介体电光叶蝉雌虫经卵巢滤泡细胞侵入卵母细胞的经卵传播的途径，证实了电光叶蝉共生菌Sulcia和Nasuia不能携带RGDV经卵传播。本项目研究成果在Nature Microbiology、Philosophical Transactions of Royal Society of London series B和Current Opinion in Virology等期刊发表5篇高质量的SCI 论文，负责人在项目执行期入选教育部长江学者特聘教授，培养3名博士和2 名硕士，其中1名博士获得福建省优秀博士论文。研究成果受到国内外同行的广泛认可，被多篇综述或研究论文引用并重点推荐。期间，负责人多次应邀在国际会议上做报告，在Current Opinion in Virology撰写2篇关于介体传播植物病毒的review，获得国家自然科学重点项目和国际(地区)合作与交流项目资助。