Functional Analysis Of FliA And FliC In Acidovorax Citrulli

Bacterial fruit blotch(BFB),caused by Acidovorax citrulli,leads to serious damage to cucurbits worldwide.BFB can infect many cucurbits,such as watermelon,melon and pumpkin.A.citrulli infected leaves and fruits,seriously resulting in fruit rot and seeds with bacteria.And it also posed a serious threat to the healthy development of the western melon industry.Flagellar and chemotaxis were considered to be closely related to the adhesion ability of pathogens on the host surface.In order to study the effection of flagellin gene and chemotaxis core gene on Aac-5,we got the mutant strains ? cheA ? fliC,?fliC and their complementary strain by homologous recombination.In order to study flagellar synthesis gene fliA,we got the mutant strains ?fliA and its complementary ?fliA::fliA.The mutant strain and corresponding complementary strain obtained above by testing virulence,hypersensitive response(HR),twitching and so on.The study analyzed the different expression level of genes related to type ? secretion system,toxic protein quorum-sensing and ropN by qPCR.The results are as follows:1.Bioinformatics analysis of fliA and fliC in A.citrulliBioinformatics analysis showed that fliA gene regulates the synthesis of bacterial flagella,and fliC gene involved in the synthesis of bacterial flagellin.Through the construction of phylogenetic tree,the FliA protein of A.citrulli was closely related to the Burkholderia cenocepacia,and the FliC protein of it was closely related to the Xanthomonas oryzae pv.oryzae.2.The phenotype tests and construction of gene deletion mutant strains ?fliA,and corresponding complemented strains.The results showed that compared with wild-type strains Aac-5,the pathogenicity of ?fliA was significantly down,the flagellum was lack,abilities of mobility and growth were decreased,and the biofilm formation ability was significantly enhanced.But the complementary strain ? fliA::fliA was able to recover these abilities.3.The phenotype tests and construction of gene deletion mutant strains ? cheA? fliC,?fliC and corresponding complemented strains of themThe results showed that compared with wild-type strains Aac-5,the pathogenicity of ? cheA? fliC was significantly lower,the growth ability was decreased,the biofilm formation ability was significantly enhanced,the seed adhesion ability was significantly reduced,but the complementary strain ? cheA? fliC::cheA::fliC was able to recover these abilities.Compared with ?fliC and ?cheA,individually,the pathogenicity of ? cheA? fliC was significantly decreased,and the biofilm formation ability was significantly improved.The athletic ability of these strains was basically the same,which indicated that fliC is the same as cheA in affecting the athletic ability of watermelon.Flagella gene mutant strain ? fliC of A.citrulli type II strains is also consistent with those of M6 mutant of type I.4.The related core genes expression in ? cheA? fliC,?fliC and ?fliAThe expressions of genes such as hrpA,hrcU,trcC,virB,luxI,luxR,and ropN in the ?fliA mutant strain were significantly decreased,indicating those genes were positively regulated by fliA.The expression of hrcU,hrpA and hrpE related type III secretion system,trcC related toxic proteins,and fliM related flagellum were decreased in the ? cheA? fliC mutant strains,which was consistent with the trends of expression of these genes in ?fliC and ?cheA mutants.This phenomenon explained the pathogenicity and related phenotypes changed,and demonstrated that the chemotaxis core gene cheA regulated the flagellar gene fliM.