Functional Analysis Of The Gene Cluster OxyR/ahpF/ahpC In H₂O₂Resistance And Full Virulence Oixanthomonas Oryzae Pv.Oryzae

In the long-term interaction between pathogens and hosts during the evolution，a complicatedinteraction mechanism has been formed. Oxidative burst is an important defense mechanism used byhost plants to prevent the invasion of pathogens. Plant host cells can generate high level of ROS rapidlyto kill the invading microbes. In order to survive and proliferate in the conditions with high level ofROS, pathogens must establish the anti-oxidative system. It has been known that the anti-oxidantsystem in bacteria is regulated by several major regulatory proteins including OxyR, OhrR, SoxR, andPerR. In the genome of Xanthomonas oryzae pv. oryzae （Xoo）, the causal agent of bacterial leaf blightof rice, the ahpC and ahpF genes are located next to each other and upstream of the transcriptionalfactor oxyR. We speculate that they may play an important role in the degradation of hydrogen peroxide.In this work, we have carried out the following experiments to elucidate the role of the gene cluster（oxyR/ahpF/ahpC） in the anti-oxidant pathway of Xoo.1. To characterize the function of OxyR, an oxyR gene deletion mutant was generated fromwild-type strain PXO99^A. Growth inhibition assays on the plates were carried out to evaluate itssensitivity to H₂O₂.The oxyR mutant demonstrated a bigger inhibition zone than the wild-type strainwhen exposed to similar amount of H₂O₂on the plates, suggesting it became hyper-sensitive to H₂O₂.We then determined whether the mutant had any defect in aerobic growth conditions. The amount ofwild type was about1.3times of the mutant, suggesting that the growth rate of the mutant is slower ormore cells are dying than the wild-type during the stationary phase. Moreover, We tested the virulenceof the oxyR mutant on susceptible rice variety Oryza sativa L. ssp. japonica by leaf clipping method.The mutant caused shorter disease lesions than the wild type strain. Therefore, we concluded that tOxyR is involved in degradation of H₂O₂and contributes to the virulence of Xoo.2. To study how the genes are transcribed in the ahpC/ahpF/oxyR gene cluster, we extractedRNA from PXO99^Aand performed reverse transcript PCR （RT-PCR）. The result showed that ahpC andahpF, but not oxyR, are transcribed as one mRNA. We then used lacZ report gene to detect the promoteractivity of ahpC. In the oxyR mutant, no LacZ activity was detected, suggesting that OxyR positivelyregulated the expression of ahpC/F.3. To elucidate the role of AhpC/F in H₂O₂resistance and full virulence of Xoo, we generatedsingle gene deletion mutant ΔahpC and ΔahpF, and a double gene deletion mutant ΔahpC/F, derivedfrom wild-type strain PXO99^A. The results showed that the catalase activities of ΔahpC, ΔahpF andΔahpC/F significantly increased4times in comparison with the wild type. And these mutants becamehyper-resistant to exogenous H₂O₂. These phenotypes might be caused by the compensatory expressionof catalase genes, which is supported by the high catalase activities in the mutants. Meanwhile, we alsoobserved a slow growth rate of the mutants during the exponential phase compared with the wild type. Ahigher level of endogenous peroxide was detected in the mutants, implying the major role of AhpC/F isto degrade endogenous H₂O₂generated during normal aerobic metabolism. In addition, the mutantscaused shorter disease lesions and grew less than the wild type strain in the susceptible rice leaves, suggesting AhpC/F contribute to the full virulence of Xoo.In conclusion, in Xoo strain PXO99^A, the alkyl hydroperoxide reductase genes ahpC and ahpFwere co-transcribed and together responsible for scavenging endogenous peroxide. Expression ofahpC/F is positive regulated by OxyR. AhpC/F were required for the normal growth of Xoo in themedia and in planta, suggesting AhpC/F are important enzymes to protect Xoo from oxidative stressduring aerobic growth conditions and during interactions with the host plant.