噬菌体新穿孔素-内溶素双组分系统的鉴定及其裂解水稻细菌性褐条病菌的机制研究

In order to effectively control plant bacterial diseases using the bacteriophages that widely distributed in nature, it is very necessary to make clear the complex and diverse mechanism of bacteriophage lysis. The applicants firstly isolated and obtained lytic bacteriophage AP1 of rice bacterial brown stripe pathogen in the world, furthermore, a new two-component lysis system was successfully identified through complete genome sequencing, bioinformatics analyses and biological test. Prokaryotic expression revealed the lytic function of endolysin without signal peptide and the synergistic effect of holin, while the interaction between them was justified by bacterial two-hybrid analysis. The aim of this project is to clarify their biological function by isolating, purifying and characterizing the holin and endolysin of bacteriophage AP1; and then elucidate the role of holin and endolysin in the lysis of Acidovorax oryzae by comparing the lysis ability between the wild type and the constructed mutants; and finally determinate the mechanism of the new two-component system in the lysis of Acidovorax oryzae by identifying their synergistic effects in biology, cytology and co-expression levels, in combination with the interaction based on the analysis of BiFC and pull down. The result of this study will provide a theoretical basis for the use of the new lysis system in biological control of plant bacterial diseases.

为有效利用自然界广泛存在的细菌天敌-噬菌体来生态调控植物细菌病害，迫切需要搞清楚噬菌体复杂且多样化的裂解机制。申请者团队前期国内外首次分离获得水稻细菌性褐条病菌的烈性噬菌体AP1，全基因组测定及生信和生物学分析鉴定出一种具有新裂解机制的穿孔素-内溶素双组分系统，其无信号肽的内溶素单独具溶菌功能，且与不同于目前报道类型的穿孔素互作。本申请项目拟首先分离、纯化和特征化噬菌体AP1的穿孔素和内溶素，明确它们的生物学功能；然后利用全长基因的截短体分别构建穿孔素和内溶素的突变体并比较其和野生型的裂解能力，明确它们在噬菌体AP1裂解水稻褐条病菌中的功能；最后在生物学、细胞学和共表达水平上鉴定穿孔素和内溶素在裂解中的协同增效，结合pull down和BiFC互作分析，以最终明确噬菌体AP1新穿孔素-内溶素双组分系统裂解水稻褐条病菌的机制，为更好地利用这一新裂解系统来生物防治植物细菌病害提供理论依据。

为有效利用自然界广泛存在的细菌天敌-噬菌体来生态调控水稻细菌性褐条病等植物细菌病害，迫切需要搞清楚噬菌体的裂解机制，本项目在前期国内外首次分离获得水稻褐条病菌烈性噬菌体AP1，生信分析发现其存在新型穿孔素-内溶素双组分系统的基础上，首先利用原核表达结合生信分析等手段，分别研究了噬菌体AP1的穿孔素和内溶素，从实验层面明确了穿孔素HolAP定位在细胞膜上，单独表达HolAP不影响细菌生长，其无信号肽的内溶素LysAP单独具溶菌功能；然后构建了LysAP的C端跨膜区域缺失突变体，发现其对细菌生长无明显影响；表达纯化LysAP缺失突变体，发现其不能透过细胞膜到达周质以裂解细胞壁；叠氮化钠抑制菌体裂解证实了LysAP是通过Sec蛋白转运到内膜上，点突变发现保守位点之一的E点突变LysE15A显著影响LysAP的裂解，明确其在噬菌体AP1裂解水稻褐条病菌中的功能；接着鉴定出水稻褐条病菌中存在与LysAP高度同源的蛋白LysAo，通过构建lysAo过表达和缺失突变体，并与野生型对比发现虽然突变没有改变细菌的噬菌体敏感性，但LysAo具有裂解细菌活性，这一现象无疑凸显了水平转移的内溶素基因在噬菌体-宿主细菌互作中的重要性，为全面揭示噬菌体的裂解机制提供了新视野；最后过表达HolAP与LysAP并与单独表达对比，结合细菌双杂等互作分析，在生物学、细胞学和共表达水平上确定了穿孔素和内溶素在裂解细菌中的协同增效，最终明确了噬菌体AP1新穿孔素-内溶素双组分系统裂解水稻褐条病菌的机制，为更好地利用这一新裂解系统来生物防治植物细菌病害提供了理论依据。总之，本项目已顺利完成了各项研究内容和目标，在成功鉴定水稻褐条病菌噬菌体穿孔素-内溶素双组分系统的基础上，基本阐明了其裂解水稻细菌性褐条病菌的复杂机制，为更好地利用噬菌体来生态控制水稻细菌性褐条病提供了坚实的基础。