揭示植物叶际及介体昆虫肠道微生物组对植物病毒侵染的影响

Most plant viruses are transmitted by insect vector feeding on the aerial parts of plants (collectively called the phyllosphere). The phyllosphere and the insect gut are important sites for virus infection and transmission. Microbial inhabitants have been revealed to help their hosts physiologically and are believed to cope with the viral infection. With the emergence of high-throughput sequencing, cultivation-independent analyses have provided deep insights into the bacterial community composition of various host plants and animals. The past decade has witnessed the increasing application of the metagenomic sequencing of 16S rDNA amplicon in microbial community. However, the endophytic bacteria of rice plant have remained undefined by the above methodology. The extremely high sequence identities among bacterial 16S rDNA, rice mitochondrial 18S and chloroplast 16S rDNAs make it difficult to decipher the endophytic bacteria, without a clean bacterial 16S rDNA amplicon library. As a result, studies on the function of rice endophytic microbiome have been greatly hindered. We have previously established a novel protocol which can specifically amplify bacterial 16S rDNA without rice DNA contamination (Unpublished results). Combined with Illumina next generation sequencing, this novel method makes it practicable to study rice endophytic bacteria by high-throughput sequencing. In this proposal, we use Rice stripe virus (RSV)-small brown planthopper (SBPH)-rice plant tripartite interaction as a research model, to decipher the structure of the phyllosphere endophytic microbiota; establish the relationship between RSV infection and microbiota from SBPH gut and rice phyllosphere; create a bacterium-free RSV-SBPH-rice interaction system to address the potential antagonistic function of bacteria in RSV infection; identify novel functional genes of the core bacteria and explore the methodology for the prevention and control of viruses in both hosts. This study will fill the gaps in the research of rice endophytic microbiome; and will be at the forefront of the phyllosphere endophytic microbiome field that would provide novel insights to other vector-borne viruses of agricultural and medical importance.

大多数植物病毒通过介体昆虫取食植物叶片进行传播，植物叶片和昆虫肠道内定殖的微生物群落可影响宿主健康，并参与对病毒侵染的调控。微生物组的研究，自开展宏基因组学的探索以来，已经革新了人类对微生物在自然界中作用方式和程度的认知。然而对于重要的农作物水稻，其内生微生物群落的组成几近无法通过高通量测序的方法进行解析，极大地阻碍了对其各项功能研究的推进。本课题组在国际上率先建立了测定水稻内生细菌菌群结构的方法学（未发表结果）。本项目将以水稻条纹病毒-灰飞虱-水稻三者相互作用为研究体系，首先阐明水稻叶际菌群的基本结构，为互作体系内菌群的各项功能研究提供基础；进而解析菌群与病毒侵染的相关性；创建无菌传毒体系，探究核心菌与病毒的互作机制并发掘功能基因；开发通过核心菌对病毒进行防控的方法学。该研究有望填补水稻内生微生物组研究的空白，揭示水稻内生微生物组的生物学功能，并以此为靶标开发防治虫媒病害的新策略。

大多数植物病毒通过介体昆虫取食植物叶片进行传播，植物叶际和昆虫肠道微生物可影响宿主健康，并参与对病毒感染的调控。本项目以水稻条纹病毒（rice stripe virus, RSV）-灰飞虱-水稻三者互作为研究对象，系统研究了水稻和灰飞虱菌群对宿主健康的影响及其与RSV的相互调控。植物菌群方面，本项目建立了适用于植物内生细菌群落的16S二代测序方法，能对携带低丰度菌群的植物材料进行测序，据此解析了水稻根际、叶际、种子的菌群组成，并着重分析了种子菌群随发育的动态变化和功能。揭示了种子在开花期获得菌群并传播给种子，由种子附属结构携带，随种子萌发传播到下一代植株，在叶和根中进行结构重塑，并发挥促生长和预置免疫的功能。昆虫菌群方面，揭示了以Wolbachia和Hypomyces chrysospermus分别为主要共生细菌和真菌、而肠道菌丰度极低的菌群组成方式。在菌群-宿主-病毒三者互作方面，建立了无菌水稻培养体系并发现在RSV感染早期，菌群抑制病毒在植物中的增殖；而在灰飞虱体内，RSV抑制H. chrysospermus的增殖。其机制为：RSV通过两种不同途径调控Toll信号途径，从而调控H. chrysospermus增殖，其中RSV激活Toll途径关键转录因子Dorsal的表达，而灰飞虱Toll通路上游的丝氨酸蛋白酶Psh可与RSV互作并抑制RSV对Toll通路的激活。本项目的部分研究结果发表于Science China Life Sciences 并申请和获得授权专利6项。其中二代测序方法和无菌植物的培养体系适用于多种植物，水稻种子菌群的获取、携带和传播方式具有普遍性，这些研究可望为多种植物微生物群的研究提供指导。

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