Functional Characterization Of Viruence-related Aave＿0638 And Aave＿0274 Of Acidovorax Citrulli

Bacterial Fruit Blotch（BFB）,caused by seed-born pathogen Acidovorax citrulli,is a devastating disease of cucurbits.Since the disease was first discovered by Webb and Goth in United States in 1965,BFB has spread to many parts of the world,mainly via transport of contaminated seeds,causing huge economic losses to the global cucurbits production,especially watermelon and melon.Previous studies have shown that A.ctrulli infects host plants relying on functional type III secretory system（T3SS）which is responsible for transporting virulent effectors to cells to disorder or distory the host immunity or cellular metabolism and eliciting hypersensitive response（HR）in no-host plants.In order to excavate more pathogenicity-associated genes of A.citrulli and explore its seed dispersal mechanism,our study generated a mutant library using transposon Tn5 on the background of wild-type Xj L12,and found two mutants altering the the ability to elicit HR.The genes inserted with Tn5 in the mutant genome were identified by subcloning and knockouted to investigate the role of that in A.citrulli pathogencity.The mainly results are as follows:1.In this study,nearly 2000 mutants were screened,and two Tn5-insertion mutants,respectively named as SK24 and N38,with altered HR phenotype were obtained.Through subclone localization,the transposon sites in SK24 and N38 were identified as Aave＿0638encoding YggS family pyridoxal phosphate-dependent enzyme and Aave＿0274 encoding histidine kinase,respectively.The result of virulence assay showed that the absence of YggS significantly reduced the virulence of A.citrulli to melon,and weakened the colonization ability of A.citrulli in melon seeds and cotyledon,as well as seed-to-seedling transmission.The above result suggested that YggS is indispensable factor of A.citrulli virulence.2.To explore the effect of YggS on virulence-related factors and biochemical metabolism,we conducted a variety of biological character assays and RNA-seq.Compared with wild-type strain,the lack of ygg S delayed the growth.Δygg S showed reduced swimming while enhanced twitching,moreover overexpression of ygg S resulted in reduced swimming.Δygg S delayed the HR in the Nicotiana tabacum.Furthermore,the loss of ygg S increased sensitivity to H₂O₂,pyridoxine（PN）,and antibiotics BCDA,meanwhile exogenous add L/D-alanine suppressed the sensitivity to BCDA.The q PCR results showed that YggS negatively regulated the expression of some fimbrium and flagellum related genes in A.citrulli,such as pil A and flg M,while positively regulated the expression of partly tested T3SS genes including hrc C,hrc N,hrc J,hrp G and so on.3.RNA-seq date showed that YggS was involved in the regulation of A.citrulli secretion system,motility,quorum sensing,oxidative stress response and other virulence-related factors,among which the effect on T3SS was the most significant.In addition,based on GO and KEGG enrichment analysis,YggS is also involved in TCA cycle,PLP metabolism,amino acid and keto acid metabolism and other important biochemical reactions,and especially participates in all reactions related to molecular function as a negative regulator.4.A preliminary study on Aave＿0274 showed that the deletion of it weakened the pathogenicity of A.citrulli to melon cotyledons and reduced the ability of seed-to-seedling transmission.In addition,ΔAave＿0274 enhanced the ability of biofilm formation,decreased swimming,and lost the ability to stimulate HR on tobacco.Further studies showed that Aave＿0275,forming a two-component regulatory system with Aave＿0274,performed as a negative regulatory factor in the pathogenicity,and Aave＿0274 could inhibit this negative effect.In summary,our study identified an essential pathogenic factor YggS and pathogenicity-associated factor Aave＿0274 for A.citrulli by screening Tn5 insertion mutant library.YggS is involved in regulating secretion system,motility,oxidative stress response,PLP and amino metabolism.Importantly,the absence of ygg S attenuates A.citrulli virulence mainly due to impaired function of T3SS.In addition,Aave＿0274 involved in regulating A.citrulli biofilm,motility and the ability to induce HR,has a complex regulatory relationship with Aave＿0275.This study provides new insights into the pathogenesis of A.citrulli and the function of YggS.