甜瓜细菌性果斑病菌致病性突变体筛选与hrcR基因的克隆

 细菌性果斑病(Bacterial Fruit Blotch,BFB)是西瓜和甜瓜的重要病害。本实验从发病甜瓜果实上分离得到致病菌株MH21,经鉴定为燕麦食酸菌西瓜亚种(Acidovorax avenae subsp. citrulli)。利用转座子Mini-Tn5构建MH21菌株的突变体库,Southern印迹杂交结果显示Mini-Tn5在菌株MH21染色体上可随机、单拷贝插入。通过浸种处理甜瓜种子进行突变体的致病性检查,筛选得到1株致病性完全丧失的突变体M543。对M543中转座子插入基因的克隆和测序表明其突变基因为燕麦食酸菌Ⅲ型分泌系统(TTSS)中的保守基因hrcR。推测菌株MH21中hrcR基因编码的蛋白定位于细菌内膜,是分泌通道主要成分之一。hrcR基因互补菌株的致病性检查结果显示其致病力恢复到野生型的84%。研究同时发现hrcR基因突变后MH21菌株丧失了在烟草上诱导过敏性坏死反应的能力。本研究从遗传学角度说明TTSS是西甜瓜果斑病菌致病性的重要因子。

Screening of the pathogenicity mutants of Acidovorax avenae subsp, citrulli and cloning of the hrcR gene

The Bacterial Fruit Blotch (BFB) caused by Acidovorax avenae subsp. citrulli is a serious disease on watermelon and melon. A strain MH21 was isolated from a diseased melon fruit, and was identified as A. ave-nae subsp. citrulli. In this study, the strain MH21 was mutated using transposon Mini-Tn5 to select the mu-tants defective in their pathogencity on melon seedling. Southern blot analysis revealed that transposon Tn5 generated the random and single-copy insertions in the chromosome of strain MH21. A mutant M543 lost its pathogenicity on melon seedling was selected and the gene disrupted by Tn5 was identified to be the homolog of hrcR gene, which coded a conserved inner-membrane protein of Type Ⅲ secretion system (TTSS) in some well-documented bacterial pathogens. The pot experiments revealed that pathogenicity of M543 could be com-plemented by plasmid-born wide-type hrcR gene. Additionally, mutant M543 defective in hrcR gene also lost its ability to trigger hypersensitive reaction (HR) on tobacco leaf. The genetics evidence in this report demon-strated that the TTSS is critical for the pathogenicity of A. avenae subsp. citrulli on melon.