Functional Analyses Of HrcQ,abmR And AbmK In Acidovorax Citrulli

Bacterial fruit blotch(BFB)caused by the gram-negative bacteria Acidovorax citrulli,is a devastating disease that threats the melon industry in the world.It not only spreads quickly on watermelon and muskmelon,and causeing serious losses,but also harms other cucurbit crops,such as cucumber,pumpkin,etc.Type ? secretion system(T3SS)and two-component signal transduction system(TCSTS)are key virulence factors in plant-and animal-based gram-negative pathogens and can participate in a variety of physiological and biochemical reactions and life activities of cells.To clarify the effects of type ? secretion gene hrcQ and putative two-component system genes abmK and abmR on the pathogenicity of A.citrulli,we constructed three knockout vectors,and screened the corresponding mutant strains.To investigate the functions of these genes in A.citrulli,we determined the virulence,hypersensitive response(HR),twitching,biofilm formation and growth ability of wild-type strains,mutant strains and their complementary strains,and analyzed the expression level of related genes by qRT-PCR.The following results were obtained from this study:(1)Bioinformatics analysis and protein structure prediction of hrcQ,abmK and abmR.HrcQ contains FliMN domain,while AbmK contains TM,PAC,HisKA,HATPase and REC domains;AbmR contains REC,AAA+ and HTH domain.Hydrophobicity and transmembrane structure prediction showed that there is no transmembrane structure in HrcQ and AbmR,and there are two transmembrane structures in AbmK.The phylogenetic tree indicated that the HrcQ,AbmK and AbmR proteins in A.citrulli are closely related to the HrcQ,SsrA and AtoC proteins of Ralstonia solanacearum,respectively.The amino acid sequences of AbmK and AbmR are highly homologous to that of the two-component signal transduction system proteins in Xanthomonas and other pathogenic bacteria,suggesting that they have the feature of two-component system.(2)Construction of ?hrcQ,?abmK,and ?abmR mutant strains and their complementary strains and determination of pathogenic phenotypes.By constructing suicide recombinant plasmids and complementary vector plasmids,we screened mutant strains ?hrcQ,?abmK,and ?abmR,as well as their corresponding complementary strains.The results of pathogenic phenotypes indicated that ?hrcQ lost the ability to cause the HR on non-host,and the virulence on hosts.In addition,?hrcQ was caused significant reduction in twitching,biofilm formation and growth ability.Compared with the wild type,the virulence of ?abmK and ?abm R was significantly reduced,twitching and biofilm formation ability were decreased to varying degrees.The deletion of abmK and abmR did not affect HR and in vitro growth ability.Additionally,we found that the pathogenic phenotype of ?abmK reduced significantly more than that of ?abmR.(3)Knockout of hrcQ,abmK and abmR influenced the expression leves of pathogenicity-related genes.hrcQ,abmK,and abmR have regulatory relationships at the transcriptional level with T3 SS genes(hrpG,hrpX,hrpE,hrcR,and hrcJ,etc.),flagellar genes(fliA),chemotaxis genes(cheA),and quorum-sensing genes(luxR).While flagella,chemotaxis and quorum-sensing genes are closely related to the motility,biofilm formation capacity and pathogenicity of strains.It is speculated that hrcQ,abmK and abmR may affect the expression levels of these genes,in turn,affect the pathogenic phenotype.In summary,hrcQ determines the pathogenicity and HR of A.citrulli,abmK and abmR are associated with a variety of pathogenic phenotypes,both of which may be two-component system genes;hrcQ,abmK,and abmR have regulatory relationships with a variety of pathogenic-related genes.