Sensitivity Regulation Mechanism Of Xanthomonas Oryzae Pv.oryzae And Xanthomonas Oryzae Pv.Oryzicola To Phenazine-1-carboxylic Acid

Rice bacterial leaf blight,caused by Xanthomonas oryzae pv.oryzae?Xoo? and rice bacterial leaf streak,caxxsed by Xanthomonas oryzae pv.oryzicola?Xoc?,are major diseases of rice.Phenazine-1-carboxylic acid?PCA? is a natural product that is isolated from Pseudomonas spp.and is used to control many important rice diseases in China.We previously reported that PCA disturbs the redox balance,which results in the accumulation of reactive oxygen species?ROS?in Xoo.In this study,we found that PCA significantly up-regulated the transcript levels of catB and katE,which encode catalases,and that PCA-sensitivity was reduced when Xoo and Xoc were cultured with exogenous catalase.Furthermore,catB deletion mutants of Xoo and Xoc showed dramatically decreased total catalase activity,increased sensitivity to PCA,and reduced virulence in rice.In contrast,deletion mutants of srpA and katG,which also encode catalases,exhibited little change in PCA sensitivity.The results indicate that catB in both Xoo and Xoc encodes a catalase that helps protect the bacteria against PCA-induced stress.A member of the adaptor protein family,Ankyrin protein,has been investigated largely in humans but rarely in plant-pathogenic bacteria.In this study,a novel ankyrin-like protein,AnkB,was identified in Xanthomonas oryzae pv.oryzae and Xanthomonas oryzae pv.oryzicola.The expression of ankB was significantly upregulated when these bacteria were treated with phenazine-l-carboxylic acid?PCA?.ankB is located 58 bp downstream of the gene.?which encodes a catalase?in both bacteria^ and the gene expression of catB and catalase activity were reduced following ankB deletion in X.oryzae pv.oryzae and X oryzae pv.oryzicola.Furthermore,we demonstrated that AnkB directly interacts with CatB by glutathione iS-transferase?GST?pulldown assays.Deletion of ankB increased the sensitivity of X.oryzae pv.oryzae and X oryzae pv.oryzicola to H₂O₂ and PCA,decreased bacterial biofilm formation,swimming ability,and exopolysaccharide?EPS?production,and also reduced virulence on rice.Together our results indicate that the ankyrin-like protein AnkB has important and conserved roles in antioxidant systems and pathogenicity in X. oryzae pv. oryzae and X. oryzae pv.oryzicola.OxyR and SoxR are two transcriptional regulators in response to oxidative stress in most bacteria,and SoxR is reported to be activated by the endogenous redox-cycling compound phenazines in phenazine-producing organisms.However,which transcriptional regulator is activated in pathogens treated with the antibiotic phenazine-l-carboxylic acid?PCA?has not been determined.In this study,we found that PCA treatment activated OxyR rather than SoxR in the phytopathogenic bacteria Xanthomonas oryzae pv.oryzae?Xoo?and Xanthomonas oryzae pv.oryzicola?Xoc?.We also found Xoo was much sensitive to PCA and H₂O₂ and had a defective antioxidant system,i.e.,less of total antioxidant capacity and total catalase activity IhanXoc,although Xoo and Xoc are very closely related.Based on KEGG sequences,OxyR differs in 10 amino acids in Xoo vs.Xoc.By exchanging OxyR between Xoo and Xoc, we elucidated that OxyR explained the differences in antioxidant capacity,total catalase activity,and sensitivity to PCA and H₂O₂.We also found that OxyR afifects Xoo and Xoc growth in a nutrient-poor medium,virulence on host plants?rice?,and the hypersensitive response?HR? on non-host plants ?Nicotiana benthamiana?.Thus,OxyR is a critical regulator that interprets the differences in anti-oxidative stress between and Jtbc and contributes to the differences in survival of them against oxidative stress.