Tolerance Mechanism Of Xanthomonas Spp.Pathogens To Phenazine-1-carboxylic Acid And Its Phenazine Homologues

Xanthomonas spp.,a class of bacterial plant pathogens,can infect over 400 kinds of plants causing severe yield and quality loss worldwide,including important cash crops and food crops such as rice,wheat,rape,citrus,banana and cassava.Phenazines are a class of antibiotics extracted from microorganisms,and well known for their potent antimicrobial activities to many organisms,including parasites,nematodes,fungi,and bacteria.Most phenazines are the derivative of Phenazine-1-carboxylic acid(PCA),and PC A has the dual effects of inhibiting plant pathogens and promoting plant growth.Presently,PCA has been registered to control controlling rice sheath blight caused by Rhizoctonia solani,cucumber downy mildew caused by Pseudoperonospora cubensis,fusarium head blight caused by Fusarium graminearum,and many other plant diseases in China.In our previous study,we found that the antioxidant system can regulate the PCA tolerance in Xanthomonas spp.,which exhibit different tolerance to PCA,and subsequent studies showed that some other mechanisms were involved with the tolerance of Xanthomonas spp.to PCA.In this study,Xanthomonas campestris pv.campestris(Xcc),with the highest tolerance to PCA,was investigated by constructing and screening a Tn5 insertion mutant library.Our results suggested that there are more important regulation mechanism of PCA and its phenazine homologues in Xanthomonas spp.than the antioxidant system.1.Verification of the genes involved in the PCA tolerance in Xcc.By constructing and screening the Xcc Tn5 transposon library,we obtained 5 PCA-sensitive insertion mutants,and their EC50(half maximal effective concentration)values decreased about 30 times.TAIL PCR and plasmid rescued method results suggested that the cytochrome c maturation(CCM)system and twin-arginine translocation(TAT)pathway were disrupted in these PCA sensitive insertion mutants.We also obtained 18 insertion mutants which their EC50 to PCA were decreased 2-3 time compared with WT.The insertion sites of the transposon in these mutants are mostly located in the gene cluster of toluene tolerance proteins.In consideration of the different PCA EC50 values of these mutants,we inferred that the CCM system and TAT pathway are more important in regulating PCA tolerance in Xcc,and focused on the CCM system and TAT pathway in the remainder of this study.2.The CCM system and TAT pathway regulate the tolerance to PCA and its phenazine homologues in Xcc by influencing the structure of the cytochrome bc1 complex.The characteristics of the CCM system deletion mutants verified the vital role of the CCM system in PCA tolerance.The CCM system also impacts the tolerance of Xanthomonas spp.to some other phenazines with similar structure to PCA,such as phenazine,1-OH-phenazine.The Dsb pathway,which is also involved in the maturation process of c-type cytochrome protein,was determined to regulate the PCA tolerance,subsequently.The results suggested that the defect of cytochrome c proteins was associated with PCA tolerance in Xcc more directly.In addition,studies on the TAT pathway showed that the CCM system and the TAT pathway may impact the PCA tolerance in Xcc through cytochrome bcl complex,which is maturated by the CCM system and TAT pathway together.The characteristics of the TAT pathway deletion mutants presented in the current study were similar to the CCM system defection mutants.Like the CCM system defection mutants,the TAT pathway mutants exhibit reduced fitness,decreased tolerance to phenazines but equally tolerance to kanamycin and captan,and similar accumulation of the oxidants to WT after treatment with PCA.The defect of cytochrome C4 could increase the PCA sensitivity,its influence to PCA sensitivity in Xcc is weaker than the defect of cytochrome bc1 complex.The The defect of cytochrome c552 could increase the H2O2 sensitivity,while the sensitivity to PCA was similar to WT.3.The cytochrome bcl complex regulates the the tolerance of Xanthomonas spp.to PCA and its phenazine homologues via NADH/NAD+.By construction of the deletion mutants cytochrome bcl complex in Xcc and Xanthomonas oryzae pv.oryzae(Xoo),we verified the critical role of cytochrome bc1 complex in the tolerance of Xanthomonas spp.to PCA and its phenazine homologues.Subsequent studies of the different subtypes of cytochrome c protein suggested that this tolerance mechanism involved with cytochrome bc1 complex was different from that the antioxidant system,and the cytochrome bcl complex played a more important role than the antioxidant system.The electron donor of cytochrome bcl complex,succinate dehydrogenase,has a great influence in the growth of Xanthomonas spp.;the defect of succinate dehydrogenase in Xcc caused destructive impact in growth.However,the defect of succinate dehydrogenase in Xcc had no significantly influence to the PCA tolerance.And the other electron donor of cytochrome bcl complex,NADH,are closely associated with the effect of PC A to Xcc.Defect of the cytochrome bc1 complex decreased intracellular degradation activity of NADH,causing NADH accumulation.The increased intracellular NADH/NAD+level promoted the PCA transforming from oxidized state to reduced state.Phenazine methylsulfate and 2,6-dichlorophenol indophenol,with higher binding activity with NADH than PCA,can competitively consume intracellular NADH and increase the tolerance of cytochrome bc1 complex defection mutants to PCA.The cytochrome bc1 complex inhibitors,such as azoxystrobin and kresoxim-methyl,have no inhibitory effect on Xanthomonas spp..With the determination on the fungal disease pathogen fusarium,we found that the defect of cytochrome bcl complex in fusarium also decreased the PCA tolerance.The effect of PCA on fusarium was significantly improved with the azoxystrobin treatment,while the control agent has no difference,indicating that the synergistic using of cytochrome bcl complex inhibitor and PCA was applicable.In conclusion,by constructing and screening of the Xcc Tn5 insertion mutant library,we found that the defect of CCM system and TAT pathway can cause significantly decrease tolerance of PCA and its phenazine homologues in Xcc.Subsequent studies suggested that the CCM system and TAT pathway regulate the PCA tolerance through the cytochrome bcl complex which is matured though the CCM system and TAT pathway.The defect of cytochrome bcl complex impacts the degradation of intracellular NADH,and the increased intracellular NADH/NAD+ levels reduced the oxidized PCA,causing cell damages.With the development of bacterial cytochrome bcl complex inhibitors,strategy of using the cytochrome bcl complex inhibitors and PCA together in controlling plant diseases must have a broad application prospect.