Tn-seq Identification Of Genes Related To Migration And Infection Of Ralstonia Solanacearum In Tobacco Host And Functional Characterization

Ralstonia solanacearum is one of the ten most widely distributed,most costly and difficult plant pathogenic bacteria in the world.R.solanacearum has seriously affected the production of many important cash crops including tobacco,tomato,potato and ginger in the world.So far,there has been no effective control measures.So far,30 provinces and cities have reported the occurrence of bacterial wilt injury one after another,among which the Yangtze River basin and southern provinces and cities of tomato,tobacco wilt injury is particularly serious,many serious field plots of continuous years of vanishing,which constitutes a serious threat to our agricultural production.Identify genes related to environmental adaptability of R.solanacearum at the whole genome level through construction of high-frequency coverage mutant library and high-throughput Sequencing with Transposon Sequencing（Tn-seq）,so as to fully understand the detailed infection process of Ralstonia R.solanacearum on host plants.Based on this,the high frequency coverage mutant library of R.solanacearum OE1-1 was constructed based on Tn-seq technology.Genes related to environmental adaptability of OE1-1and genes related to migration and infection in tobacco plants were identified,and their functions were verified.The main research results are as follows:（1）Essential genes of R.solanacearum OE1-1:The mutant library of OE1-1 strain was constructed based on the transposon Mariner C9（p Mar C9-R6K）,with a capacity of 500,000 mutants.Sequencing library was constructed by Mme I enzyme digestion,joint fusion and high throughput PCR amplification.The results show that the transposon coverage frequency is about 71%,that is 7.1 insertions per 100 bases.Tn-seq analysis software（TSAS）identified 453essential genes in strain OE1-1,and their functions mainly focused on 18 categories,including translation,ribosome structure and biosynthesis,cell wall/membrane/capsule biosynthesis,nucleotide transport and metabolism.（2）Genes related to the adaptability of R.solanacearum minimal medium:The mutant library was screened under stress on minimal medium（Hogland nutrient solution with sucrose as the only carbon source）,and genomic DNA was extracted.Tn-seq analysis showed that 218 genes were associated with survival in OE1-1 minimal medium.The functions mainly focus on amino acid transport and metabolism,energy generation and conversion,cell wall/membrane/capsule biosynthesis and other 17 categories;Eight of these genes were selected,and the survival of OE1-1 in basic medium was confirmed by the construction of gene knockout mutants and functional complement experiments,indicating the feasibility of screening environmental adaptive genes based on Tn-seq.（3）Genes related to R.solanacearum migration and infection in tobacco:The mutant library was injected into the intercellular space of the leaves of Nicotiana tabacum with a needle-free syringe.When tobacco wilt appeared,the mutant library was recovered from the stem of tobacco,and genomic DNA was extracted.Tn-seq analysis showed that 2,202 genes might be related to the migration and infection of R.solanacearum in tobacco.Among them,the detected abundance of 2,118 genes in tobacco stem decreased significantly,indicating that these genes may be necessary for R.solanacearum migration and infection in tobacco.The abundance of 84 genes（such as Phc B and Phc A）in tobacco stem increased significantly,indicating that these genes may weaken the migration and infection of R.solanacearum in tobacco.Since Phc B and Phc A are necessary for R.solanacearum to infect host plants,this study focused on the specific function of Phc B and Phc A in R.solanacearum tobacco.Through the construction of phc B,phc A knockout mutant,functional complement,host infection,and in vivo competitive growth tests,the results showed that the phc B and phc A mutant completely lost the infection to the host plant and could not grow normally in the tobacco stem(the maximum density was about 10^5-6 cfu g^-1,The maximum density of the wild-type strain was 10¹⁰cfu g^-1),but the phc B mutant and phc A mutant were mixed with the wild-type strain,respectively,and infected tobacco plants by foliar inoculation.The phc A mutant recovered the same growth ability as the wild-type strain in the tobacco stem,with the maximum density of 10¹⁰ cfu g^-1,while the phc B mutant had the maximum density of 10¹¹ cfu g^-1 in the tobacco stem,and the growth ability was significantly better than that of the wild-type strain.These results indicated that although the phc B or phc A deletion mutants alone lost the ability to infect and colonize tobacco hosts,the impaired ability could be restored by the presence and influence of other strains in complex agricultural farming environments.Therefore,the development of single gene deletion mutants as candidates for targeted antibiotic drugs has to consider complex environmental factors.In conclusion,the application of Tn-seq strategy can systematically and rapidly identify essential genes and conditional essential genes of R.solanacearum.The essential genomes,auxotrophic-related genes and adaptation-related genes identified in this study can provide new theoretical reference for the understanding of the pathogenic mechanism of R.solanacearum and the prevention and control of bacterial wilt.