Functions Of MarR Family Transcription Factors In Acidovorax Citrulli

Bacterial fruit blotch（BFB）is a worldwide quarantine bacterial disease,which is mainly spread with seeds and damage cucurbit crops such as watermelon and melon.BFB has brought huge economic losses to the melon and melon industries worldwide.The pathogen of BFB is Acidovorax citrulli,which is gram-negative and has a single flagellum.The optimal growth temperature of A.citrulli is between 24～28℃.And effectors can be directly injected into host cells by the type Ⅲ secretion system（T3SS）in A.citrulli.Therefore,there is great significance to the breeding of BFB-resistant commercial disease-resistant varieties through exploring the regulation and pathogenic mechanism in A.citrulli.Multiple antibiotic resistance regulator（MarR）,is an important transcription factors family which can not only regulate gene expression but also control multiple antibiotic resistance as a transcriptional inhibitor.MarR is the key to responding to chemical signals and result in changes in gene activity in bacteria.Previous studies have shown that MarR family transcription factors play an important role in the regulation of biofilm formation,motility,antibiotic response and virulence gene expression.In this study,the bioinformatics analysis of MarR family genes was completed before the experiments.And MarR deletion mutant strains and complementary strains were constructed based on the bioinformatics analysis.We determined several pathogenic related phenotypes of wild-type strain Aac5 and strains above.Variability of transcriptional levels was determined in MarR mutant strains by qRT-PCR.In addition,we constructed GUS expression strains and tested the promoter activities to verify the effect caused by MarR of the T3SS key genes.The main results obtained in this study are as follows:1.Bioinformatics analysisThere are 7 MarR family transcription factors in A.citrulli.All transcription factors of MarR family have MarR type HTH domain,which can bind to DNA.2.Pathogenicity-related phenotype determination of MarR family transcription factors in A.citrulliThe pathogenicity of Aac5 and its ability of produce the HR on tobacco have not been affect by the deletion of Aave₀324,Aave₀795,Aave₃178 and Aave₃934.The ability of biofilm formation of ΔAave₀324,ΔAave₀795 and ΔAave₃934 was significantly decreased.Moreover,the swimming ability,growth ability in vitro and resistance to ampicillin of ΔAave₀795 was decreased.The deletion of genes Aave₃922,Aave₄271,Aave₄639 all weakened the pathogenicity and biofilm formation ability in A.citrulli.The mutant strainsΔAave₃922 and ΔAave₄639 have reduced in vivo colonization ability,ΔAave₄639 mutant have weakened exercise ability and in vitro growth ability,and mutantΔAave₄271 has slightly enhanced in vitro growth ability.In addition,the deletion of Aave₃922 significantly weakened the resistance to ampicillin in A.citrulli.But the mutant strains ΔAave₃922,ΔAave₄271 and ΔAave₄639 did not affect the ability to induce allergic necrosis in tobacco in A.citrulli.3.Transcriptional regulation of MarR factor in A.citrulliAt transcriptional level,Aave₀324,Aave₀795,Aave₃178 and Aave₃934 all have positive effects on the key genes of T3SS hrpX and hrcJ,and Aave₀795 can also affect hrpG.Among them,Aave₀324 only has negative regulatory effects on flagellar gene fliC and biofilm-related genes Aave₂620,while Aave₀795,Aave₃178 and Aave₃934 have negative regulatory effects on pathogenic genes.Transcriptional regulation analysis showed that Aave₃922 can positively regulate the expression of T3SS key genes hrpX and hrcJ,and positively regulate the flagellar gene fliC and biofilm related gene Aave₂620,Aave₄271 negatively regulated the expression of hrpX,hrcQ and hrcJ,and has a negative effects on chemotaxis gene cheA and biofilm related gene Aave₂620,Aave₄639 can positively regulate hrpG,but negatively regulate hrcJ,and positively regulate chemotaxis gene cheA and negatively regulate luxR.In addition,promoter activity test of GUS showed that,the promoter activity of hrpX gene was significantly decreased in ΔAave₃922,and the promoter activity of hrpX and hrcJ genes was enhanced in ΔAave₄271.The promoter activity of hrpG gene was decreased,while the promoter activity of hrcJ gene was increased in ΔAave 4639.