Research On The Virulence Mechanism Of Type Ⅳ Secretory System In Rice Bacterial Brown Stripe Pathogen Acidovorax Avenae Subsp.avenae RS-2

Bacterial brown stripe（BBS）caused by the gram-negative bacteria Acidovorax avenae subsp.avenae（Aaa）is a serious disease in rice,which distributed widely in many countries around the world and results in heavy economic losses.Previous studies within our group has focused on bacterial pathogen identification,characterization,virulence mechanism investigation,etc.Whole genome sequence analysis revealed the existence of type I secretion system（T1SS）to type VI secretion system（T6SS）in both strains Aaa RS-1 and RS-2.The involvement of secretion system in Aaa bacteria virulence was supported by our studies on type VI secretion system（T6SS）.The revealing about the role of each secretion system of Aaa contributes to the understanding on the bacteria virulence mechanism of BBS.The aim of this study was to examine the role of T4SS in the pathogen of Aaa strain RS-2 through comparing bacterial growth,virulence to rice seedlings,biofilm formation,swimming motility,H2O2 tolerance,extracellular polysaccharide（EPS）production,extracellular enzymatic and phage infection activities between wild-type and the 9 T4SS mutants which were constructed using the method of homologous recombination.Results indicated that mutation of Acav₀607,Acav₀967,Acav₀971 and Acav₃3 78 genes not only significantly reduced bacterial virulence,but also caused by 20.14～62.01%reduction in biofilm formation and by 37.67～47.7%reduction in motility.The complemented strains of these 4 T4SS mutants restored similar virulence symptom as the wild type.However,the 4 T4SS genes have a differential effect on bacterial growth after 24 h of incubation and H2O2 tolerance,and only A cav 0967 show the resistance to phage infection.The motility related proteins are proposed to be involved in the phage infection process based on LC-MS/MS identification result.Our previous data shown that cellular motility may contribute to the bacterial pathogenicity,it is therefore not unexpected that the phage infection might be related to bacterial pathogenicity.Through comparing the WEGO and KEGG analysis result from LC-MS/MS identification,it is inferred that the phage may infect the host through interring the electron carrier,oxidation and translation regulation process.The immune system of mutant Acav₀967 may functions resist against phage infection to avoid cell death,which is proposed to be highly associated with the flagella assembly,two-component system,fatty acid biosynthesis,oxidative phosphorylation processes.In addition,no change was observed in bacterial virulence by mutation of the other 5 T4SS genes and all the 9 T4SS genes did not affect EPS production or extracellular enzymatic activities compared to the wild type.Taken the above results together,current study demonstrated the importance of T4SS genes in particular Acav₀607,Acav₀967,Acav₀971 and Acav₃378 genes in virulence of Aaa strain RS-2,which may be partially attributed to the reduction in bacterial motility and biofilm formation.However,mutation of Acav₀605,Acav₁461,Acav₁821,Acav₂102 and Acav₃595 genes do not significantly affect bacterial virulence but other phenotype.Totally,this study revealed the functional importance and diversity of T4SS genes involved in the virulence mechanism of Aaa,which provides a fundamental understanding on the study about the pathogenesis of this rice bacterial pathogen.It also helps to provide new strategies for disease control in rice field.