| Tobacco bacterial wilt caused by Ralstonia solanacearum is an important bacterial disease on tobacco,bringing a huge loss to the tobacco industry,and with no effective control measures.As a bacterial virus,bacteriophage can effectively lyse R.solanacearum.But due to the high specificity of phage,it affects the effect of controlling bacterial diseases.Therefore,the study of the interaction between phage and bacteria,and the resistance mechanism of phage resistant strains,are of great significance for the use of bacteriophage to control crop bacterial wilt.In order to clarify the probable anti-phage mechanism in R.solanacearum,two R.solanacearum strains Tb15 and Tb1546 with different resistance to 6 phages were sequenced.The research of 5 database annotations,phylogenetic analysis,prediction and comparison of pathogenic factors,genomic island,prophage,differential gene and CRISPR were done.The type I R-M system and hsd S gene were knocked out to show the function on phage resistance.The results of genome sequencing show that the size of Tb15 is 5.83 Mb(3.82 Mb for chromosome,2.00 Mb for plasmid),GC content is 66.87 %,and the predicted gene is 5151;Tb1546 is 5.58 Mb(chromosome is 3.63 Mb,plasmid is 1.94 Mb),GC content is 67.20 %,and the predictive gene is 4925.Phylogenetic analysis based on 16 S r DNA and housekeeping genes indicates that both Tb15 and Tb1546 belong to phylotype I,which is more closely related to R.solanacearum strain CQPS-1 from Chongqing,China.Result of collinear shows that chromosomes of Tb15 and Tb1546 have the same directionality,but the plasmids are inconsistent.Tb15 and Tb1546 both have 20 genomic islands,respectively containing 6 and 4 complete prophage regions.But the genome of resistant strain Tb15 is larger,various types of virulence factors and CRISPR sequences are more.The gene difference shows that Tb15 contains 445 unique genes,of which Cro/CI type repressor is related to phage resistance.In addition,8 genes unique to COG defense mechanism contain complete type I R-M system and are also related to phage resistance.The knockout result of the R-M system shows that the mutants ΔRM and Δhsd S are sensitive to P7-1 but still showed resistance to other phages,demonstrating that there are other resistance mechanisms existed in Tb15.In addition,in order to clarify the function of orf30 gene in phage P574,the gene was cloned and transformed into R.solanacearum Tb1546 and Tb15 respectively.The growth rate,colony morphology,extracellular polysaccharide,cellulase,adsorption rate,efficiency of plating(EOP)and plaque size with different kinds of phage between transformed strain and wild strain were compared.q RT-PCR was used to analyze the gene expression.The results show that the orf30 gene belongs to the transcriptional regulator of the XRE family,and the transformed strains(orf30-Tb1546 and orf30-Tb15)loss of pathogenicity.The growth rate of the transformed strains in LB is accelerated,the morphology in the TTC medium becomes dry and deep red,and the extracellular polysaccharide and cellulase activity are decreased.The result of q RT-PCR shows that orf30 can regulate the phc system and multiple two-component systems,thus affecting the pathogenicity of R.solanacearum.The results of phage-related biological experiments show that the adsorption rate of R.solanacearum transformed with orf30 to phages is affected to varying degrees except phage P1556-1;orf30 enhancs the cleavage ability of Tb1546 to 6 kinds of phage.And the resistance of Tb15 to P7-1 and P1553 is changed to sensitivity.The EOP result shows that the plaques of other phages are not changed except that the plaque efficiency of P7-1 and P1553 are unchanged on orf30-Tb1546 plate.The efficiency decreases,with P1556-2 and P574 being the most significant. |
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