Screening Of Genes Essential For Phage Infection Into Xanthomonas Oryzae Pv. Oryzae And Their Roles In Pathogenicity

Plant pathogenic bacteria,bacteriophages and plants live together,interact and undergo co-evolution.Xanthomonas oryzae pv.oryzae（Xoo）is the most important bacterial pathogen to rice.Phage therapy may be a specific and environmental friendly method for control of the rice bacterial blight disease.This study established a Tn5 transposon mutant library of Xoo and aimed to find the genes essential for phage infection into Xoo and related to Xoo pathogenicity to rice by screening mutants resistant to phage infection and pathogenicity tests and to explore the mechanisms for Xoo interactions with natural enemy phages and host rice.In this study,4524 mutants of Xoo Zhejiang isolate C2 were obtained by the Tn5 transposon mutation technology.Thirty-six mutants resistant to phage infection were screened out by phage infection tests.Molecular techniques including reverse PCR and bioinformatics analysis identified 19 essential genes in Xoo for phage infection: 12 genes related to liposaccharide synthesis,which are cysB,metB,wxoD,rmlB,rmlA,rfb303,lpxH,a gene encoding nucleoside-diphosphate-sugar epimerase（C2₆8）,two genes encoding glycosyltransferase（C2₈01 and C2₁771）and two genes（C2₈03 and C2₈20）encoding hypothetical proteins;two genes related to biosynthesis: tagB and gene encoding NAD-dependent dehydratase（C2₇98）;one gene etfB related to cell metabolism;one gene encoding a Tat pathway signaling protein;one gene encoding a transposase;two genes（C2₇97 and C2₈02）encoding hypothetical proteins.Inoculation of Tn5 mutants on rice plants revealed rmlA,rfb303,a gene encoding glycosyltransferase（C2₁771）,and a gene encoding a hypothetical protein（C2₈03）containing a nucleotide-diphospho-sugar transferase motif are related to Xoo pathogenicity.Physiological and biochemical phenotype analyses found that lpxH is critical for Xoo growth in rich media,cysB,metB,wxoD and rmlB are related to bacterial motility,and tagB,a gene encoding glycosyltransferase（C2₈01）and two genes encoding hypothetical proteins（C2₈02 & C2₈03）are related to bacterial motility and biofilm formation.In order to verify whether the Tn5 inserted genes are essential for phage infection,eight mutants and complements of these genes（cysB,rmlB,rmlA,rfb303,gene encoding glycosyltransferase（C2₁771）,a gene encoding hypothetical protein containing a nucleotide-diphospho-sugar transferase motif（C2₈03）,tagB,a gene encoding Tat pathway signaling protein）were generated.They are mutants（35）794,（35）812,（35）813,（35）2234,（35）1771,（35）803,（35）601,and（35）3261 and the corresponding complements 794-C,812-C,813-C,2234-C,1771-C,803-C,601-C and 3261-C.Phage infection tests revealed that none of the mutants could be infected by the phage J2 while their complements restored the susceptibility to phage infection.Therefore,these eight genes are essential for phage infection.Moreover,the pathogenicity phenotypes of（35）803,（35）1771,and（35）2234 were consistent with the corresponding Tn5 mutants,confirming that the gene rfb303,the gene（C2₁771）encoding glycosyltransferases,and the gene（C2₈03）encoding hypothetical protein containing a nucleotide-diphospho-sugar transferase motif are essential for phage infection into Xoo and are virulence genes of Xoo.In conclusion,this study found that genes essential for phage infection into Xoo mainly participate in liposaccharide synthesis and determine the surface resistance of bacteria to phage.Some of the liposaccharide synthesis-related genes are Xoo virulence genes.The study also found some new genes essential for phage infection into Xoo.These findings lay the foundation for study of the interaction mechanism and co-evolution mechanism of Xoo with phages and rice.