Function Analysis of Flagellin Gene flgM in Acidovorax citrulli
YANG Bing-Ye, HU Fang-Ping, CAI Xue-Qing*
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection,Fujian Agriculture and Forest University, Fuzhou 350002, China
Abstract：Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is one of the most important destructive seed borne diseases on watermelon (Citrullus lanatus) and melon (Cucumis melo) in the world and the cause pathogen is a worldwide quarantine pest. This disease causes serious economic loss to watermelon production. In order to effectively control the disease, The pathogenicity mechanism of A. citrulli on watermelon is needed to know. Flagellum is a movement organ of bacteria and plays an important role in bacterial infection. Alterative anti-sigma factor flgM, required in the regulation of flagellar gene transcription, has been verified in different bacteria species, but its feature in A. citrulli is unclear. The objective of this study is to understand the function of gene flgM on flagellum formation and pathogenicity of A. citrulli. The flgM gene deletion mutant was generated by homologous recombination with the help of suicide plasmid pK8mobsacB. Morphological characteristics have been tested of the flagella, pathogenicity, hypersensitive response, motility, quorum sensing, biofilm formation, growth rate, twitching, etc. among the wild type, the mutant and the complementary strain. Moreover, a real-time quantitative PCR (qRT-PCR) was carried out to compare the expression of genes, flhD, fliE, fliC, flgK, flgA, fliS, fliD and fliA in the wild type strain, the deletion mutant strain and the complementary strain using glutathiamide synthetase gene (glnA) as a reference. The results showed that the deletion mutant FJAc01-flgM and complementary strain FJAc01-flgMhb were generated successfully after gentamicin resistant screening and verified by PCR. Compared to wild type strain, the deletion mutant did not grow flagella and greatly attenuated in biofilm, motility, colonial morphology and the virulence on watermelon, but significantly accelerated in growth rate, which could be restored in the complementary strain. After 5 d of stabbed inoculation on watermelon fruit and spray inoculation on watermelon seedling, the disease indexes were 74.67 and 26.39 respectively inoculated by wild type strain FJAc01, but the disease indexes were 46.00 and 2.78 respectively by the deletion mutant strain FJAc01-flgM, and the disease indexes were 63.85 and 20.83 respectively by the complementary strain FJAc01-flgMhb. Whereas, there are no significant changes of mutant in hypersensitive response on tobacco (Nicotiana benthamiana), quorum sensing and pathogenicity to melon compared to the wild type strain. The wild type strain could form typical haloes obviously which caused by bacteria migrating via twitching on NA medium, but the deletion mutant weakened this ability and the complementary strain recovered the ability partially. The results of qRT-PCR indicated that the expression of flgK, fliA, fliE genes were up regulated, whereas flhD, fliC, fliS genes were down regulated in the mutant strain, and the expressions of these genes were recovered in the complementary strain. However, the expressions of gene flgA and fliD were not significant changed in the mutant. In conclusion, the flagellar gene flgM could regulate the flagellum formation, pathogenicity, biofilm formation, motility, growth rate and colony morphology of A. citrulli.
杨丙烨, 胡方平, 蔡学清. 西瓜噬酸菌鞭毛基因flgM的功能分析[J]. 农业生物技术学报, 2019, 27(3): 504-515.
YANG Bing-Ye, HU Fang-Ping, CAI Xue-Qing. Function Analysis of Flagellin Gene flgM in Acidovorax citrulli. 农业生物技术学报, 2019, 27(3): 504-515.
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