青枯雷尔氏菌丝状噬菌体RSBg的广宿主分子机制

Bacterial wilt caused by Ralstonia solanacearum seriously affect the production of many important crops in China. Filamentous phage infects host bacteria permanently, which provides a new idea for the control of bacterial wilt. We previously found a filamentous phage RSBg infecting broad range of hosts, and the bioinformatics analysis revealed that the host range decision mechanism of this phage is different from the known mechanism. Firstly, this study will identify the key genes determining the host range of phage RSBg by gene shuffling between RSBg and phage infecting narrow-range hosts. The key genes of R. solanacearum in response to the infection of phage RSBg will also be identified using transcriptomics, transposon insertion sequencing and reverse genetics. The interaction between key proteins of phage and R. solanacearum will be determined by bacterial two-hybrid system and far-western blotting. Molecular mechanism for the broad-host-range of R solanacearum filamentous phage RSBg will finally be revealed combining all the results. This project will hopefully promote the study of the interaction between filamentous phage and host bacteria, and establish a theoretical foundation for the application of filamentous phage in the control of bacterial blight.

青枯雷尔氏菌(Ralstonia solanacearum)所引起的植物青枯病严重危害我国农业生产，丝状噬菌体持续感染宿主细菌，可为植物青枯病防治提供新思路。本项目前期发现一广宿主丝状噬菌体RSBg，分析发现该噬菌体的宿主决定机制不同于已知的机制。本项目将首先通过RSBg与窄谱噬菌体的基因互换，明确噬菌体RSBg广宿主谱的关键基因；其次通过转录组学、转座子插入测序和反向遗传学，筛选R. solanacearum应对噬菌体RSBg感染的关键基因；再进一步通过细菌双杂交以及far-western blotting等方法检测噬菌体与R. solanacearum关键蛋白的相互作用，最终揭示噬菌体RSBg广宿主的分子机制。本项目的有效开展将促进丝状噬菌体与宿主互作的研究，为丝状噬菌体在青枯病防治中的应用奠定理论基础。

植物青枯病是由青枯菌 (Ralstonia solanacearum) 所引起的毁灭性植物土传病害，是危害最严重的植物细菌性病害，被认为是植物的癌症。青枯病宿主多、分布广、危害大、防控难。噬菌体，即感染细菌的病毒，在青枯病防控中具有重要应用前景。丝状噬菌体是一类单链DNA病毒，为细长的丝状结构。相比于烈性有尾噬菌体，丝状噬菌体相对温和，通常不直接杀死宿主细菌，而处于持续感染状态。丝状噬菌体的特殊侵染特性使得其可能与烈性有尾噬菌体在青枯病噬菌体治疗中优势互补。项目通过基因组前噬菌体信息发掘，分离获得了丝状噬菌体RSCq，并解析了其完整的基因组序列。发明了一种通过体外转座子插入质粒拯救，工程丝状噬菌体的构建方法。通过该方法，分别构建了丝状噬菌体RSCq、RSBg、RSFm的工程噬菌体，并构建了彼此嵌合的工程噬菌体，发现三个丝状噬菌体所侵染的宿主谱并无明显差异。项目进一步研究了青枯菌与丝状噬菌体的共进化，结合高通量测序和遗传学方法，发现菌毛蛋白PilN是丝状噬菌体侵染青枯菌的关键靶标。综上所述，本项目建立了青枯菌丝状噬菌体的研究平台，为丝状噬菌体防控植物青枯病提供了技术基础；确定了丝状噬菌体侵染青枯菌的关键靶标，促进了青枯菌与丝状噬菌体间的相互作用研究。