Function Analysis Of Type ? Secretion System HpaB And HpaA In Acidovorax Citrulli

Bacterial fruit blotch(BFB)is an important quarantine and seed-borne disease around the world.The pathogen is gram-negative bacteria-Acidovorax citrulli,which mainly infects watermelon,melon and other cucurbit crops.BFB has the characteristics of fast speed,wide spread and severe disease,which seriously restricts the development of the melon industry and causes huge economic lossesType III secretion system(T3SS)is widely present in gram-negative bacteria.It is a key virulence factor in the interaction between bacteria and host plants,participates in regulating a variety of physiological and biochemical reactions in cells.The hpa(hrp-associated)gene is an important part of hrp gene cluster of the type III secretion system in plant pathogenic bacteria The secreted proteins of T3SS usually need to be mediated by proteins encoded by the hpa gene during secretion.These proteins are usually beneficial for plant pathogenic bacteria to exert their pathogenicity.In order to investigate the biological functions of the type III secretion system hpaB and hpaA in Acidovorax citrulli,this study takes the Aac5 strain as the research object,constructs the hpaB and hpaA deletion mutant strains AhpaB and AhpaA and their corresponding complementary strains AhpaBcomp and AhpaAcomp,test their pathogenicity and other pathogenicity-related phenotypes together with wild-type strain Aac5.And the expression of T3SS-related genes and pathogenicity-related genes are analyzed by quantitative real-time PCR.The results are as follows(1)hpaB and hpaA are closely related in bioinformaticsAfter bioinformatics analysis,hpaB(Aave 0446)and hpaA(Aave 0447)are closely related,there may be a certain regulatory relationship.On the whole genome map,the gene hpaA is located downstream of hpaB,which is consistent with the transcription direction of hpaB.The protein encoded by the hpaB gene is consisted of 151 amino acids.The functional annotation information is Tir chaperone family protein.The HpaB protein has no special domain;The protein encoded by the hpaA gene is consisted of 324 amino acids.The functional annotation information is a hypothetical protein,whose orthologous functional annotation information is a type ? secretion regulatory protein.The HpaA protein domain consists of a coiled coil and a low complexity domain.(2)The deletion genes of hpaB and hpaA,affect pathogenicity and other pathogenicity-related phenotypes.The target genes deletion mutant strains AhpaB and AhpaA and their corresponding complementary strains AhpaBcomp and AhpaAcomp are successfully constructed.Compared with the wild-type strain Aac5,phenotypes results showed that,the mutant strains AhpaB and AhpaA have significantly reduced pathogenicity to watermelon and melon of host plants,lost the ability to induce HR(hypersensitive response,HR)on Nicotiana tabacum,and its growth ability in the host and in vitro also decreased significantly.When the hpaB gene is deleted,the biofilm formation ability is enhanced,but when the hpaA gene is deleted,the biofilm formation ability is weakened.At the same time,when these two genes are deleted,the swimming ability are decreased.Complementary strains AhpaBcomp and AhpaAcomp are able to recover these capabilities during the assays.(3)The deletion genes of hpaB and hpaA,affect the expression of T3SS-related genes and pathogenicity-related genes.In AhpaB,the expression of T3SS genes(hpaA,hrpE,hrcR,hrpX,hrpG),virulence gene trbC and flagella gene fliC are all significantly increased,and the expression of T3SS hrcQ and quorum-sensing genes luxR are significantly decreased.In ?hpaA,the expression of T3SS genes(hpaB,hrpG,hrcV),virulence protein trbC,chemotaxis gene cheA and flagella gene fliC are significantly increased,while the expression of T3SS genes(hrcQ,hrpE)and pili genepilL are significantly decreased.Moreover,when the genes hpaB and hpaA are deleted from each other,the changes of gene expression are extremely significant,when the hpaA gene is deleted,the effect on the expression of hpaB gene is more significant.It is speculated that hpaB and hpaA have a certain regulatory relationship at the transcription level.They can regulate the expression of the above genes to varying degrees.In turn,both of them have impacts on pathogenicity and pathogenic phenotypes.