Preliminary Functional Analysis Of T6SS Structural And Effector Genes In Pseudomonas Syringae Pv.actinidiae

Bacteria have many different types of secretory systems for effector secretion.The type VI secretion system（T6SS）is a newly discovered secretory system in gram-negative bacteria and plays an important role in human pathogenic bacteria,animal pathogens and plant pathogenic bacteria that significantly affected virulence of pathogens.Kiwifruit bacterial canker,which caused by Pseudomonas syringae pv.actinidiae（Psa）,has become a destructive disease of worldwide kiwifruit production.In order to find whether Psa had T6SS and the function of T6SS genes,the T6SS of Psa M228 were annotated by bioinformatics analysis.The function of T6SS gene was analyzed based on biolo gical function,colonization and expansion ability,virulence,relationship between T6SS gene cluster and type III secretion system（T3SS）gene expression.The detailed results listed below.1 Through bioinformatics analysis,we found that Psa M228 possesses all 13 essential structural genes for T6SS,but tssH and tssM had frameshift mutation.Besides,there are many different hcp and vgrG effector genes..By homologous recombination,we constructed 15mutants in this study:a T6SS gene cluster deleted mutant;structural gene tssB,tssC deleted mutants;structural gene tssH,tssM frameshift recovery mutants and their tssC gene deleted mutants;four hcp effector protein gene deleted mutants;4 vgrG effector protein gene deleted mutants.2 T6SS structural and effector genes affect the biological function of Psa.Except the T6SS gene cluster deleted mutant,the growth rate of other mutants during logarithmic growth was significantly reduced;there was no significant change in the motility of the mutants;the biofilm formation ability of the mutants M228Δhcp1-225 and M228ΔvgrG48 was significantly enhanced;different mutants showed different changes in the tolerance to high-concentration metal ions(Na⁺,K⁺,Cu²⁺)and weak acid or weak base conditions.3 M228ΔT6SS colonization ability significantly weakened and failed to expand in leaf veins;M228ΔtssC,M228tssH-r,M228tssM-r,M228Δhcp1-225,M228Δhcp3,M228ΔvgrG48,M228ΔvgrG181,with weakened expansion ability,were able to colonize in the veins but only expand in a small range.The results of stem virulence showed that the virulence of M228ΔT6SS was significantly reduced,while the remaining mutants with weakened colonization and expansion ability showed a decrease in stem virulence compared with M228,but there was no significant difference.4 The qRT-PCR technique was used to analyze the relationship between the T6SS gene cluster and T3SS-related gene expression.During the interaction between the mutant M228ΔT6SS and the host,the expression levels of the hrpR,hrpL,T3SS structural genes hrpC,hrpZ,hopP1,and T3SS effector genes hopM1 and hopH1 were significantly down-regulated.