中文摘要：侵染瓜类果斑菌（细菌性果斑病的病原体）的噬菌体是防治该病的有效手段。但噬菌体耐药菌的出现障碍了噬菌体生物防治，了解细菌的噬菌体耐药能力必不可少。本研究旨在研究瓜类果斑菌的噬菌体耐药性及噬菌体耐药性和致病性之间的关系，分离并鉴定与之相关的基因。通过连续斑点试验和噬菌斑试验，后采用种子包衣法进行致病性试验，从3264株瓜类果斑菌Tn5突变体中分离到一株噬菌体耐药的弱毒突变体AC-17-G1。突变体在铜蛋白基因中间整合了Tn5。该突变体通过与相应的野生型基因互补，恢复了其致病性和噬菌体敏感性。利用CRISPR/Cas9系统从野生型中定向突变该基因，使致病性丧失并获得噬菌体耐药性。AC-17-G1在King's B培养基中的生长远小于野生型，但在添加D-甘露糖6-磷酸或D-果糖6-磷酸的培养基中生长正常，表明铜蛋白具有磷酸甘露糖异构酶的功能。经十二烷基磺酸钠分析，发现从突变体中提取的脂多糖（LPS）小于从野生型中提取的。可见，铜蛋白是一种参与LPS合成的磷酸甘露糖异构酶，而LPS是瓜类果斑菌致病性和噬菌体敏感性的重要决定因素。
外文摘要：Bacteriophages infecting Acidovorax citrulli , the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.
外文关键词：Acidovorax citrulli；cupin；pathogenicity；phage resistance
作者：Rahimi-Midani, A；Kim, MJ；Choi, TJ
作者单位：Pukyong Natl Univ；Dong A Univ
期刊名称：PLANT PATHOLOGY JOURNAL
期刊影响因子：1.57
出版年份：2021
出版刊次：6
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