| Dickeya zeae is an important plant bacterial pathogen worldwide.It has a wide range of hosts and infects more than ten host plants such as rice,corn,potato and banana.Rice bacterial foot rot caused by D.zeae is one of the most important bacterial diseases,which occurs seriously in local rice areas in China and Southeast Asian countries,resulting in reduced yield of rice.At present,studies on pathogenicity of D.zeae and its mechanism of virulence regulation are not perfect,especially studies on disease-related s RNA are rare.In this paper,the D.zeae strain DZJS-4 was used to create the s RNA mutant library by the method of non-trace gene knockout,and the pathogenicity related s RNA was screened,and the role and function of the s RNA were clarified through transcriptomic sequencing analysis,so as to lay the foundation for further research on the molecular mechanism of s RNA regulation.43 sRNA were selected from the DZJS-4 genome,and 43 different s RNA mutants were obtained by gene cloning and knockout.Two s RNAs,Arc Z-Dz and Rsm B-Dz,were selected for this study.Phenotypic determination showed that,compared with wild-type strains,mutants△ArcZ-Dz and△RsmB-Dz did not produce toxin,reduced ability to form biofilm,decreased activity of protease,cellulase and pectinase,enhanced swimming ability in△Arc Z-Dz and weakened swimming in△Rsm B-Dz.In addition,the mutants△Arc Z-Dzand△Rsm B-Dz weakened HR（hypersensitive response）in tobacco,affected the inhibition of rice seed germination,and reduced the virulence on rice plants significantly,and their complementary strains could restore the corresponding functions.Illumina HiSeq200 sequencing platform was used to conduct transcriptome analysis of D.zeae strains of DZJS-4,△Hfq,△Arc Z-Dz and△Rsm B-Dz,and to compare their differences in gene transcription levels.Sequencing results showed that there were 1,875expressed differentially genes in△Hfq,including 922 up-regulated genes and 953down-regulated genes.A total of 1457 expressed differentially genes inΔArc Z-Dz,including 735 up-regulated genes and 722 down-regulated genes.A total of 232 expressed differentially genes inΔRsm B-Dz,including 87 up-regulated genes and 145 down-regulated genes,suggesting that Arc Z-Dz regulated more functions than Rsm B-Dz.GO functional enrichment these differentially expressed genes are mainly involved in biochemical processes,biological functions and cellular components.By comparing the expressed differentially genes for△Hfq,△Arc Z-Dz,and△Rsm B-Dz,it was found that 950 expressed differentially genes were the same in△Hfq and△Arc Z-Dz.Moreover,84 genes for|log2（Fold Change）|≧2 were the same in△Hfq and△Arc Z-Dz.However,147 expressed differentially genes were the same in△Hfq and△Rsm B-Dz,and 2 genes for|log2（Fold Change）|≧2 were the same between them,suggesting that Arc Z-Dz may require the involvement of Hfq.Further analysis of virulence related genes showed mainly expression genes in flagellum,chemotaxis and the secretion system.The expression of 26 flagellum cluster genes were affected in△Hfq and△Arc Z-Dz,the expression of 28 and 38 chemotropic genes were different from that of wild strain,respectively,15 genes were the same in these two mutants,but only one chemotropic gene was up-regulated in△Rsm B-Dz.The expression of type VI secretion system genes in△Hfq and△Arc Z-Dz strains were affected significantly.12 down-regulated genes were in△Arc Z-Dz,including icm F and vgr G genes.△Hfq had 11 down-regulated genes,while△Rsm B-Dz had only 2down-regulated genes.In the IV secretion system of bacteria,only the gene encoding Rhs protein was down-regulated in the three mutants.In addition,3-4 genes of type III secretion system were down regulated significantly in△Hfq and△Arc Z-Dz strains.The above results indicated that the two sRNA,ArcZ-Dz and Rsm B-Dz,from D.zeae were important pathogenicity regulators,and Arc Z-Dz had a wider range of regulatory effects,requiring the involvement of Hfq,while Rsm B-Dz had a narrow range of regulatory effects,which may not require the involvement of Hfq. |
PDF Full Text Request