Regulatory Pathways Of Histidine Kinase BarA_Ac,and Identification And Functional Analysis Of SRNA In Acidovorax Citrulli

China is a large watermelon production country,and planting watermelon is also the main economic source of many melon farmers.The bacterial fruit blotch（BFB）caused by Acidovorax citrulli is a serious vegetable disease that would cause serious damage to the quality and yield of watermelon and melon,affecting the development of watermelon and melon industry of our country.In order to achieve effective prevention and control of BFB,it is necessary to study the pathogenic mechanism of A.citrulli.The expression deployment of the type Ⅲ secretion system（T3SS）is activated by HrpG and HrpX in A.citrulli.However,there is still a large gap in the virulence regulation network of A.citrulli.In order to explore the function of the histidine kinase BarA_Ac in the two-component regulatory system（TCS）in A.citrulli Aac5 strain,the deletion mutant and complement strains of barA_Ac were constructed.In this study,virulence and pathogenicity related phenotypes of the tested strains were measured,and their possible involvement in regulation was analyzed by RNA-seq.Point mutation,Western blot and GUS activity were used to verify and analyze the regulatory relationship between barA_Ac and T3SS regulatory genes hrpG and hrpX.In addition,in many pathogens,small non-coding RNAs（sRNAs）are also involved in the regulation of virulence.In order to search for possible sRNAs in A.citrulli,sRNA-seq was used to screen possible sRNAs in A.citrulli Aac5 strain,and the functions of two sRNAs in Aac5 strain were preliminatively studied through overexpression.The main research results are as follows:1.Domain analysis of BarA_Ac revealed that it has two transmembrane domains and one signal receiving domain located outside the membrane at the N-terminal,and a conserved BarA histidine kinase domain at the C-terminal.Through genetic experiments of gene deletion and complement,we found that after barA_Ac deletion,the virulence of A.citrulli Aac5 strain on host watermelon and its early colonization ability in seedling cotyledon were significantly enhanced,which accelerated the induction of hypersensitive response（HR）in non-host tobacco,while the biofilm formation ability and swimming motility were significantly reduced.RNA-seq analysis showed that a large number of genes involved in translation and peptide synthesis were down-regulated,while T3SS and the type Ⅲ effectors（T3Es）genes were up-regulated in mutant strain.By analyzing the expression of T3SS regulatory factors HrpG and HrpX,it was found that the expression of HrpG was significantly up-regulated in KB culture.The mRNA and protein expressions of hrpX were up-regulated,but the protein expression was not as up-regulated as that of HrpG.Based on the fact that HrpG can activate hrpX transcription,this study further carried out point mutations at the conservative aspartic acid（Asp）phosphorylation sites at HrpG 52^nd and 60^th in the form of plasmid and genome,and found that the promoter activity,mRNA,and protein expression level of hrpX was significantly down-regulated and the ability to induce HR in tobacco was lost.That is,BarA_Accan negatively regulate the expression of HrpG in A.citrulli Aac5 strain,thus affecting virulence,and the52nd and 60th Asp sites of HrpG are important sites for its transcriptional activation.2.In this study,A.citrulli Aac5 strain was cultured in KB and XVM2 medium for sRNA-seq.Analysis of differentially expressed genes showed that the transcription expression of hrpG did not respond to XVM2（a plant-like barren environment）.The response to different environments（in vitro and in vivo）might not occur at the transcription level,but at the protein level.By mapping sequencing data to the genome,487 sRNA candidates（SRCs）were detected in A.citrulli Aac5 strain.Among them,86.0%（431/487）of SRCs were less than 100 nt.Among all the SRCs obtained by screening,35.7%（174/487）of SRCs were located at the intergene region,46.0%（224/487）of SRCs were located at the gene coding region（32.1%of which were located at the non-template chain）,14.6%（71/487）of SRCs were located at 5′-untranslated region,and only 3.7%（18/487）of SRCs were located at 3′-untranslated region.3.Analysis of SRCs in T3SS cluster showed that sRA025,sRA026,sRA027 and sRA028 could be transcribed independently of upstream and downstream genes.It was found that the overexpression of sRA025 and sRA028 did not affect the ability of A.citrulli Aac5 strain to induce HR in tobacco,while the overexpression of sRA026 and sRA027 made Aac5 strain lose the ability to induce HR in tobacco.Further analysis of pathogenic phenotypes showed that the overexpressed strains of sRA026 and sRA027were weakened in seed to seedling transmission,spray inoculation virulence,and host watermelon cotyledon colonization,but did not affect the growth ability of in vitro culture（KB medium and XVM2medium）,and did not affect biofilm formation and swimming motility.In other words,sRA026 and sRA027 act as sRNAs that negatively regulate virulence in A.citrulli.In addition,when sRA026 and sRA027,which were predicted to bind to target mRNA,were replaced,overexpression did not affect the virulence of A.citrulli Aac5 strain and the colonization ability in host cotyledon.Previously,for the T3SS regulatory network in A.citrulli,only HrpG was known to be the highest regulator,which regulates the expression of T3SS and T3Es genes by regulating the transcription of hrpX.This study found that BarA_Ac is located in the upstream of HrpG and can negatively regulate its expression,thus affecting the expression of T3SS and T3Es genes and virulence in A.citrulli Aac5 strain.Moreover,the 52nd and 60th Asp of HrpG are necessary for its transcriptional activation.In addition,487 SRCs were identified,among which sRA026 and sRA027 in the T3SS cluster could negatively regulate the virulence of Aac5 strain.These results supplemented the virulence regulation network of A.citrulli,and provided a reference for further studies on the pathogenesis of A.citrulli.