Fuctional Analysis Of Pathogenicity Related Genes Piln And Pdxj In Acidovorax Citrulli

Bacterial Fruit Blotch (BFB) is a serious disease worldwidely on plants of cucurbitaceae, caused by Acidovorax citrulli. So far, the research on this disease is mainly concentrated in the advances in the identification of pathogens, detection, prevention and the identification of cultivar resistance. Pathogenesis of pathogens is still poorly understood, and the methods of prevention don’t eradicate the disease completely but only reduce.In this study, we generated a transposon (Tn5) mutant library on the background of strain xjl12and screened it for reduced virulence by seed-transmission assays and injection inoculation with melons. Here, we got four strains with reduced virulence. One of these Tn5mutants is impaired in pilN, which is involved in fimbrial assembly family protein and play a part in the biosynthesis of type IV pili. Another Tn5mutant is impaired in pdxJ, which is involved in pyridoxal phosphate biosynthetic protein and directly affects the biosynthesis of vitamin B6. As we all know, pdx family in whole genome of Acidovorax citrulli contains two identical sequence genes——pdxJ and pdxA. The insertional mutant Δ pilNac and ApdxJ were respectively generated by biparental mating from the wild-type strain xjl12and swimming motility, pathogenicity in host, hypersensitive response (HR) in nonhost tobacco, etc are determined, in order to explain the pathogenic mechanism from the perspective of molecular genetics.Results by phenotype measurement showed that the pathogenicity on seed leaf were obviously attenuated in ApilNac compared to wild type. There was significant difference in swimming motility, production of exocellular cellulase and induction of HR between mutant (ΔpilNac) and wild type. In addition, these results of qRT-PCR displayed that the transcription levels of hrpG, hrcV and cel A were down-regulated in ΔpilNac. It is demonstrated that pilN gene plays an important role in surface adhesion, colonization, biofilm formation in many bacterials and also has a significant effect on type III secretion pathway, indicating that the biosynthesis of type IV pili is closely related to pathogenicity.Though measurement for pathogenicity, significant reductions in virulence and biofilm formation were observed in another insertional mutant ApdxJ. Meanwhile, there was a significant decline in growth, swimming motility as well as abolishment of flagella. By exogenous VB6the mutant could partially recover capacity for growth and pathogenicity. After disruption of pdxJ gene, the bacterial growth of the mutant is inhibited and couldn’t form the pathogenicity-related factors, resulting in loss of virulence. Therefore, this is a strong evidence to prove the close relationship between pyridoxal phosphate (commonly known as VB6) and pathogenicity.