Research On The Mechanism Of Copper Resistance And Expression Regulation Of CopSRABCD Gene Cluster In Ralstonia Solanacearum

Bacterial wilt of plants,affected by plant wilt causing Ralstonia spp.?i.e.R.solanacearum,R.pseudosolanacearum and R.syryzii?,is a major soil-borne disease around the world.As an important bactericide to control bacterial diseases such as bacterial wilt,the widespread use of copper-based bactericides has led to the emergence of copper-resistant strains in a variety of plant pathogenic bacterial population.Genome-wide sequencing showed that the copper-resistant coding gene copSRABCD,homologous with Pseudomonas syringae,was carried on the megaplasmid of Po82 strain.Po82 strain of Ralstonia solanacearum was used as the research object.By means of reverse genetics strategy,copA,copD,copSR gene deletion and corresponding strains of Po82 were constructed.The relationship between cop gene and biological characteristics such as response to copper stress,metabolic activity,pathogenicity,and motility of Ralstonia solanacearum has been clarified.At the same time,the expression and regulation of copSRABCD gene cluster were also determined.RNA-seq transcriptome sequencing was used to determine the transcripts of Po82 strain and copABCD gene deletion strain in nutrient-rich culture conditions,copper-induced culture and host environment.The co-expression and differentially expressed genes under different culture conditions were explored in order to further clarify the function of copSRABCD gene cluster and discover new copper resistance and pathogenic genes.It will lay a foundation for further understanding the molecular mechanism of host and environmental adaptation evolution of Ralstonia solanacearum.1.Functional validation for candidate genes of copA,copD and copSRThe results of copper MIC analysis showed that the copper resistance MIC values of Po82?copA,Po82?copD and Po82?copSR strains were lower than the wild type strains.The results of virulence test showed that,compared with wild type strain,the virulence of Po82?copA strain decreased and Po82?copSR strain increased,but Po82?copD strain had no significant difference in virulence.The results of growth curve showed that copA and copD deletion strains affected the growth rate of bacteria under copper stress.The results of motility test showed that the copA gene was not related to the motility of Ralstonia solanacearum.The deletion of copD and copSR genes affected the sport ability of Ralstonia solanacearum.Moreover,The absence of copA resulted in a reduction of metabolic utilization rate of carbon sources such as?-D-glucose,D-trehalose and nitrogen sources such as L-alanine and glucuronide.2.Regulation of copper resistance gene cluster expressionqRT-PCR results showed that the expression of copSRABCD gene cluster was induced by copper ion.The expression of copSRABCD gene increased with the increase of CuSO₄ concentration.The gene cluster was induced expression.RT-PCR results showed that copABCD was co-transcription and copSR was co-transcription.The expression pattern of copABCD gene in copSR deletion strains was determined.The results showed that copSR could negatively regulate copABCD gene.3.Transcriptome of Ralstonia solanacearum in vitro and in vivo environmentIn Po82 and copABCD gene deletion strain,copper induced not only positively regulates copper resistance-related genes,but also significantly up-regulates the expression of some genes encoding pathogenic factors.And most of these up-regulated genes are hrp cluster genes and T3Es coding genes.After copper treatment,the expression of heavy metals and antibiotic regulatory genes such as czcABC,fur and marR also increased significantly.Copper induction also affects the metabolic pathway of Ralstonia solanacearum.In the host environment,copABCD gene cluster deletion strains up-regulates the expression of type IV pili coding genes and pathogenicity-related genes.The results of transcriptome sequencing laid a foundation for further clarifying the function of copSRABCD gene cluster and discovering new copper resistance and pathogenicity related genes.