The Helping Role And Mechanism Of Prophage And Non-host Phage During Phage Infection Of Xanthomonas Oryzae Pv.oryzae

Bacterial blight of rice caused by Xanthomonas oryzae pv.oryzae(Xoo)is the most important bacterial disease in rice,which leads to the decline of rice quality and yield.Resistant varieties and chemical pesticides are the main strategies for the prevention and control of this bacterial disease.As pathogens evolve and ecological sustainability becomes increasingly important,it is very necessary to develop a green alternative for the control of rice bacterial diseases.Phages that are widespread in nature are highly specific in targeting pathogenic bacteria without harming beneficial microbial communities,making them a promising option for plant disease management.However,phage application was greatly restricted by the ongoing arms race between plant pathogenic bacteria and phages.To solve the phage resistance problem,this study investigated the role of new phage endolysin,the released prophage,and non-host phage during phage infection of Xoo.The main results are summarized as follows:1.This study not only identified that the glycosyltransferase gene is crucial for understanding the resistance of Xoo to OP2-type phage infection,but also constructed the expression system of an endolysin capable of lysing Xoo strains that are resistant to phage infection on the basis of isolation and identification of an OP1-type phage.Through genomic resequencing and comparative analysis of Xoo＿C2 and a naturally occurring Xoo strain that is resistant to OP2-type phage infection(Xoo＿C2R),we identified mutations in the glycosyltransferase gene that affect the synthesis and structure of lipopolysaccharides on the cell surface.These mutations resulted in decreased phage adsorption and ultimately produced the phage resistance observed in Xoo＿C2R.Since Xoo＿C2R has extremely high resistance to OP2-type phage and its endolysin,a new Xoo phage ZP3 was successfully isolated from diseased rice leaves.The induced expression of endolysin Lys ZP from phage ZP3 can affect cell membrane integrity,causing bacterial death,which showed the antibacterial ability to Xoo＿C2R.The secretion of Lys ZP depends on the N-terminal transmembrane domain,which serves as a signal-arrest-release sequence and mediates the transport of Lys ZP to the cytoplasm.Unlike widely distributed OP2-type phages,ZP3 is an OP1-type Xoo phage through electron microscopy observation,one-step growth curve,multiplicity of infection,host spectrum,stability,and genome sequencing analysis,which provided new phage resources for preventing and controlling bacterial blight in rice.2.This study revealed the release and biological significance of prophage in Xoo strains as well as the mechanism underlying the phenomenon.Following the prophage prediction in genomes of 118 Xoo strains sequenced in our laboratory and downloaded from the database,a filamentous prophage RG2 was successfully isolated from 28 out of 56 Xoo strains preserved in our laboratory through double plate assay.Genome sequencing and PCR amplification showed that prophage RG2 had a linear-stranded DNA genome with 8202 bp,which was integrated into bacterial genome through TATACATTGCGAA sequence.Host spectrum analysis revealed that the released prophage RG2 not only lysed 41 out of 56 Xoo strains,but also lysed naturally resistant strain Xoo＿C2R.In addition,recombinase Xer C and Xer D were found to be closely related to the release of Xoo prophage by construction of Tn5 mutant library,gene knockout and complement,phenotype screening and RT-q PCR.Co-cultivation results showed that the released prophage could lyse the highly virulent strain C2 of Xoo,and the proportion of released strains in the mixed bacterial solution increased from 10% to 77%,gaining a competitive advantage in the ecology.This study provides a new “prophage release” strategy to effectively control the infection caused by Xoo strains,especially phage resistant strains,in rice plants.3.This study revealed the helping role and working model of non-host phage in highly efficient cell lysis of Xoo strains as well as the mechanism underlying the phenomenon.Although a wide variety of phages coexist in rice fields,comprising both lytic phages that infect their bacterial hosts and a significant number of non-host phages,it is still unclear about the role of non-host phages in phage-bacteria interaction.Here,we found a novel phenomenon that non-host phage AP1 and trace phage X2 alone was not able to lyse Xoo,but the combination of the two can efficiently infect Xoo.The adsorption results showed that non-host AP1 could adsorb onto the bacterial surface.Potassium efflux experiments indicated the injection of AP1 DNA into Xoo strain.Genomic analysis of the phage infection mixture revealed that AP1 integrated its DNA into bacterial genome,which caused sequence variation and genome rearrangement.Thus,we proposed the working model of non-host phage“adsorption-entry-integration-DNA rearrangement-sensitivity enhancement”.Following Eigen GWAS of Xoo population genomes(484 strains),transcriptome analysis,RT-q PCR validation and screening of Tn5 library(2746 mutants),transposases were found to be related to non-host phage assistance.This finding will be greatly helpful for us to use phages to control bacterial blight in rice.In summary,this study aimed at using phages to control rice bacterial blight and solving the problem of phage resistance by establishing an expression system of new endolysin with antibacterial activity against phage-resistant strains on the basis of isolation and identification of new phage resources in nature,and by identifying the prophage release and non-host phage help phenomenon of great biological significance,which not only enriched beneficial phage resources,but also contributed to a comprehensive understanding of phage-bacteria interactions.