Identification Of An Inducer For High-efficiency Prophage Release Of Xanthomonas Oryzae Pv. Oryzae And Functional Analysis Of Associated Transposases

Rice bacterial blight is one of the three serious diseases of rice,which is caused by Xanthomonas oryzae pv.oryzae?Xoo?.As the bacteria gene mutated frequently and the environment got worse by the use of pesticide,traditional disease control methods are not as effective as before.So the development of novel green biocontrol of rice disease is in need.Interestingly,prophage is widely predicted in bacteria pathogen like Xoo.Prophage release is an important biological phenomenon and it can contribute greatly to the biotrol of bacteria disease.Some researches showed that the prophages can be induced by UV,Mitomysine C,nalidixic acid or others that can cause DNA damage.However,it is still unclear which external stimuli can induce the prophage release of Xoo.In order to find the inducer which cause the prophage release of Xoo,we used different inducers including exogenous phage AP1?lytic phage isolated from Acidovorax oryzae,but cannot infect Xoo?,mitomysine C,norfloxacin,UV,NaCl and H₂O_2.Here,30 Xoo strains which were isolated from Guangdong,Zhejiang and Liaoning rice area in China and type strain PXO99 were tested.Prophage release was identified by using double-plate plaque experiment,restriction endonuclease pattern of phage genome analysis,and transmission electron microscopy identification.The results showed that the phage AP1 could induce the prophage release of 27 Xoo strains,while other traditional inducers could only induce the prophage release of 1,3,2,2 and 1Xoo strain.Accordingly,phage AP1 is proposed as a biological agent that can induce the high efficient prophage release of Xoo.In order to determine the conditions for AP1 to efficiently induce the prophage release of Xoo,we selected a representative Xoo strain C2,and investigated the conditions for the induction of AP1.We can get the conclution that high concentration of AP1?>10¹00 pfu/mL?can induce the prophage release of C2,and the optimal ratio is 1:100?v/v?.Moreover,in order to clarify the molecular mechanism of efficient inducing release of Xoo prophage,the whole genome sequence of C2 was characterized using high-throughput sequencing strategy.The full length of strain C2 is 4,941,727bp,GC content is 63.73%,and 11 prophage regions?6 intact prophages?were revealed by using PHASTER online prediction analysis.These intact prophages might be induced,because integrase and Att L/R sites are predicted in 3 of them.To find out where the induction occurs,on the surface of the bacterial cell or enter into the bacterial cell,phage adsorption experiments was carried out.As a result,AP1 can adsorb on the surface of C2.Then,K⁺concentration determination and phage fluorescent staining experiments confirmed the injection of AP1 DNA into C2 cells that results in the K⁺efflux change and the injection of phage AP1 into C2 cell?post 1 h incubation?.In summary,the successful entry of AP1into C2 cells may be a prerequisite for the efficient induction and release of prophage.Furthermore,to identify the genes involved in the high efficiency prophage release upon AP1injection,the Tn5 transposon was randomly inserted into the C2 genome by electro-transformation.About 2200 mutants were constructed and 76 mutants with no prophage release were screened by the agar double plate method.Then the 76 Tn5 insertion sites were determined by using reverse PCR and BLAST analysis.Results showed that the CDS corresponding to the insertion sites of the76 mutants are mostly located on different functional genes such as transposase,glycosyltransferase,acetyltransferase,and synthetase,among which the proportion of transposase family was the highest,nearly 1/3?23/76?,and the role of transposase in prophage induction has yet been detailed studied.The wild-type C2 and C2-Tn5 mutant strains were tested for growth and K⁺efflux cheanges,and it was found that the growth was not significantly affected in the transposase mutants.This excludes the possibility that the transposase gene interferes with prophage release by affecting AP1 adsorption or injection and bacterial growth.Finally,the mutants of two transposase encoding genes named?K4 and?K19 and their corresponding complements were constructed by gene knock-out technique.Prophage induction were lost in these two mutants,but can be recovered in the complemented strains.Thus,the important role of transposases was validated in prophage induction and highly efficient release.