Identification Of Housekeeping Genes And Pathogenicity Related Genes Of Xanthomonas Oryzae By Transposon Sequencing (Tn-seq)

Xanthomonas oryzae is a species of pathogenic bacteria that infect rice and could cause heavy yield lose and quality drop of rice.Xanthomonas oryzae pv.oryzae(Xoo)and Xanthomonas oryzae pv.oryzicola(Xoc)are causal agents of bacterial leaf blight and leaf streak of rice,respectively.Xoo and Xoc are important model bacteria in rice-pathogenic bacteria studies.The full genome sequences of may Xoo and Xoc strains have been avaible,however,only limited functional genomic study has been carried out and the housekeeping genes and the pathogenicity-related genes are yet to be identified.The conventional method for functional genomic studies generally relies on the construction of saturate mutant library using transpon insertion,or by one-byone gene knock out,followed by low-throughput phenotypic screening.These strategies are time-consuming,heavy labor and funding-dependent,thus the functional genomic analysis have become the bottle neck in correlating the genotypic and phenotypic studies.In the recent decade,transposon mutagenesis technology has been combined with high-throughput sequencing technology to overcome such drawbacks of conventional functional genomic analysis.Such methods are termed Transposon Sequencing(Tn-seq),which has greatly accelerated the functional genomic analysis on some animal and human pathogenes.In this study,a hyper-saturated mutant library by using random transposon insertion of strain Xoo K74 and strain Xoc GX01,and Tn-seq analysis were carried out on these libraries growing in rich medium PSA and in rice leaves,respectively.Tn-seq analysis performed in this study identified 348 and 339 potential housekeeping genes in Xoo K74 and Xoc GX01 for growing in rich media,respectively.These housekeeping genes are involved in basic life processes such as DNA replication,gene transcription and protein translation,oxidative phosphorylation,etc.The potential housekeeping genes of the two strains were searched against the housekeeping gene record in the DEG database,and revealed that 104 genes and 105 genes were unique for K74 and GX01,respectively.Moreover,additional 47 and 45 genes in K74 and GX01,respectively,were identified for potentially required for optimal growth in rice leaves.In order to further verify the accuracy of the results obtained by Tn-seq analysis,several potential housekeeping genes and pathogenicity-related genes of the two strains were selected for mutagenesis and phenotypic analysis.The results showed that GTK60＿12135 and XOCgx＿04200 are difficult to be deleted,suggest that they are most likely housekeeping genes.Five and eight potential pathogenicity-related genes from both K74 and GX01 were mutated.The pathogenicity of the five mutants of Xoo K74,NK5915,NK5955,PNK18190,NK18215,and NK18230,were reduced by 86.15%,67.6%,63.68%,95.95%,95.77%,respectively,and their pathogenicity was significantly reduced.The eight mutants of Xoc GX01,NK01720,NK03665,NK03970,NK04080,NK06720,NK07065,NK08800,and NK10200,have reduced pathogenicity by 59.66%,0.1%,31.78%,66.53%,53.08%,38.82%,69.36%,respectively.67.75%.Of the eight selected genes in GX01,XOCgx＿RS07065 and XOCgx＿RS06720(gsh B)are reported as pathogenicity-related genes for the first time.This study applied Tn-seq analysis on Xanthomonas oryzae for the first time,proved the feasibility of this technic.The Tn-seq pipeline designed in this study provides a quick and low-cost alternative for the identification of housekeeping genes and conditional essential genes of Xanthomonas oryzae.This pipeline may be easily adapted to other Xanthomonas species.The identification of the housekeeping genes and pathogenicity-related genes in this study could be helpful to better understand the pathogenic mechanism of the pathogen,and more importantly,could provide support for further antibacterial drug development.