噬菌体内溶素抑制水稻细菌性褐条病菌细胞分裂的机制研究

Endolysin, the key lysozyme hydrolyze bacterial cell wall, is vital for bacteriophage AP1 to lyse the pathogen of rice bacterial brown streak, Acidovorax oryzae. In order to elucidate the function of endolysin in host lysis, we expressed the endolysin of phage AP1 heterogenously. Unexpectedly, bacteria number decreased significantly, and bacterial morphology changed from short rod to long filamentous. Here, we aim to elucidate the cytological basis about the significant inhibition on host bacterial division by endolysin. Firstly, the real time morphology changes of host bacteria will be monitored through overexpressing of AP1 endolysin using microscopic technique. Secondly, with overexpression of AP1 endolysin in host bacteria, various biological phenotypes will be determined and analyzed to clarify the bacteriological basis of endolysin inhibiting host cell division. Then, upon the overexpression of endolysin, RNA-seq and qPCR will be applied to detect the differential expression of host genes to determine the endolysin response genes. In addition, functional genes related to cell division in response to endolysin in A. oryzae will be identified through constructing mutants and complements. Accordingly, the molecular mechanism of endolysin inhibiting host cell division will be clarified, which will provide theoretical basis for the effective utilization of phage endolysin for green prevention and bio-treatment of rice bacterial brown streak.

内溶素是一种能够水解细菌细胞壁的溶壁酶，是噬菌体AP1能够裂解水稻细菌性褐条病菌的关键。为搞清楚内溶素在噬菌体裂解宿主中到底起到了何种功能，申请人异源表达了噬菌体AP1的内溶素，结果意外发现除菌体数量显著减少外，菌体形态也由短杆状变为长丝状。本申请项目拟在前期观察到内溶素显著抑制宿主细菌分裂这一重要生物学现象的基础上，首先通过显微观察来实时监测过表达AP1内溶素的宿主细菌的菌体形态，以明确内溶素抑制宿主分裂的细胞学基础；其次测定与分析过表达AP1内溶素后宿主细菌的各种生物学表型，明确内溶素抑制宿主分裂的细菌学基础；接着转录组和qPCR测定过表达内溶素后宿主基因的差异表达情况，鉴定出宿主体内的内溶素响应基因；最后通过构建突变体和回补体来鉴定水稻褐条病菌体内响应内溶素的细胞分裂相关基因，以最终明确内溶素抑制宿主细胞分裂的分子机制，为有效利用噬菌体的内溶素来绿色防治水稻细菌性褐条病提供理论依据。

噬菌体内溶素蛋白是参与宿主细胞裂解步骤的重要蛋白，可以特异性地水解细菌细胞壁的肽聚糖组分。在前期研究中，我们鉴定了噬菌体AP1的内溶素蛋白，并确认了其通过与穿孔素协同合作参与到细菌宿主Acidovorax oryzae RS-2的裂解中。在异源表达AP1内溶素时，发现细菌细胞明显变长，细菌细胞的分裂过程也明显受到了抑制。本项目在此基础上，通过将该蛋白在宿主体内成功表达，使用显微观察技术检测噬菌体AP1内溶素引起宿主细胞形态的特征，确认其的确影响了宿主细胞分裂而导致细胞形态变长。通过转录组鉴定出宿主在噬菌体内溶素表达过程中的基因表达特点，发现与宿主菌细胞膜蛋白相关功能的Novel00158（HlyD membrane-fusion）、peg.2947（Outer membrane factor lipoprotein）和Novel00157（AcrB/AcrD/AcrF family）为响应噬菌体AP1内溶素表达的核心基因。解析了AP1噬菌体内溶素的蛋白结构，通过点突变和缺失突变验证了C端跨膜区域的生物学功能。将AP1内溶素的跨膜域构建到不含跨膜结构的X2-Lys内溶素蛋白上，结果显示后者同样可以使细菌菌体形态变长，与AP1内溶素具有相似的表型。总之，噬菌体AP1内溶素可导致宿主细胞形态变长，宿主体内膜蛋白相关基因响应了内溶素的表达，并且内溶素蛋白的C端跨膜区域与细菌细胞形态变化密切相关。