Characterization Of Virulence Related Genes And The Regulatory Function Of TetR Under Oxidative Stress In Acidovorax Orvzae RS-1

Bacterial brown stripe is a devastating disease in rice. The Gram-negative bacterium Acidovorax oryzae is the causal agent of this disease. Although this worldwide rice disease has caused significant yield, quality losses and had huge influence on economy, currently little is known about this bacterial pathogen, especially at the molecular and cellular levels.Previous researches revealed the pathogenicity of A. oryzae strain RS-1is closely related with its Type IV pili (TFP) and Type six secretion system (T6SS), which has been identified and characterized recently. Gene pilP, which encodes a TFP assembly protein, is involved in several bacterial activities. The pilP-deficient mutant strain seriously affected the motility twitching ability, biofilm formation and virulence. Besides, based on genome-wide in silico analysis, our previous work showed there existed a T6SS in A. oryzae RS-1. In order to further elucidate T6SS core components and their gene functions, icmF and dotU single gene mutants were constructed by insertional mutagenesis. Virulence of mutant strains was decreased after inoculation on rice with icmF and dotU mutant. In addition, the interaction between IcmF and DotU was examined by both bacterial two-hybrid assay and bimolecular fluorescence complementary assay.Also, we characterized a novel gene cluster, including tetR, on the chromosome of A. oryzae RS-1by genome-wide comparing of A. oryzae RS-1, A. oryzae ATCC19860, and A. citrulli AAC00-1. Phylogenetic analysis demonstrated this gene cluster may be horizontally transferred from a Pseudomonas ancestor. TetR acted as a repressor in this gene cluster and the oxidative stress resistance was enhanced in tetR-deletion mutant strain. Moreover, the enzyme activity level of superoxide dismutase (SOD) and catalase was increased in mutant. Electrophoretic mobility shift assay (EMSA) demonstrated that TetR regulator bound directly to the promoter of this gene cluster. Consistently, the results of quantitative real-time PCR also showed alterations in expression of associated genes. Moreover, the proteins affected by TetR under oxidative stress were revealed by comparing proteomic profiles of wild-type and mutant strains. Totally there were111proteins showing difference between wild-type strain and mutant. After GOC enrichment analysis, it was found that these proteins are mainly associated with oxidative stress response, detoxification, and biofilm-formation. Taken together, our results demonstrated that this gene cluster including tetR helped to maintain cell balance under oxidative stress and this feedback mechanism was important in pathogen evolution.