Pathogenic Function Analysis Of Impg In Pectobacterium Carotovorum Subsp. Carotovorum And The Proteins Interaction With It

Pectobacterium carotovorum subsp.carotovorum?Pcc?is the crucial pathogenic bacterium that causes soft rot of crops.the hosts of Pcc distribute broadly,which will cause serious harm of agricultural production and huge economic losses.Pcc can produce plants cell wall degrading enzymes?PCWDEs?,such as gelatinase,cellulase,protease and other pathogenic factors.Studying its pathogenic mechanism can develop effective prevention countermeasures.The main pathogenic factors of Pcc extracellular secretion by T2SS,and the secretion of T6SS mainly involved in environmental competition.The preliminaryly study,we found that,impG,the constitutive gene of T6SS in PccSl was related to pathogenesis.In order to explore the mechanism of involved of impG,this study used the impG+FLAG tag to find the interacting of proteins with ImpG by Co-IP and bacterial double-hybrid assay,and exploring the pathogening mechanism of impG with interacted proteins.In this study,we tested 25 potential proteins may interact with ImpG were obtained:AlaS?DnaK?EttA?GlpK?HslU?MglA?MviM?OppD?OsmY?PrkA?PurA?RplA?RplE?RplF?RplP?RplV?RpsP?TolB?PC1₀243?PC1₀287?PC1₀296?PC1₀601?PC1₃387?PC1₃700?PC1₄255.Further analysis of the above interaction proteins revealed that most of the proteins interacting with ImpG were ribosomal proteins and proteins related to glucose metabolism and transport,which suggested that the ImpG protein may need to be stimulated by ribosomal proteins in T6SS,and in the same time it may need a lot of energy delivery.By discovered the mutations in 12 of these 25 genes,only the mutant ApurA pathogenicity was significantly reduced,and the mutations of genes corresponding to glucose metabolism and transport-related proteins did not significantly affect the pathogenicity of Chinese cabbage.PurA is an adenylate succinate synthase?ADSS?that participates in intracellular nucleotides in de novo synthesis pathway and salvage synthesis pathway.Therefore,it is believed that when ImpG protein participates in the normal function of T6SS,at the same time it may have effect on the de novo synthesis pathway of bacterias.Same as PurA,adenylate succinate lyase?PurB?is involved in the biosynthetic pathway.Comparing the gene sequence,it is found that there is an purB gene and 2 purB homologous genes in PccS1.In order to further understand the de novo synthesis pathway,this study constructed the ?purB mutant,and the results showed that deletion of purB would not produce significant changes in pathogeni-city.In this study,wild-type PccS1 and mutant ?impG were grown in MMX basal medium and MMX + tissue fluid?ZE?to OD600 = 1.0 for the relevant sequencing.The differences between the wild type and the mutant and its tissue fluid were compared by software analysis using the relevant database and bioinformatics,and the relevant growth transcription spectra were obtained.The results of the analysis showed that the number of down-regulated genes were more than up-regulated genes after adding the tissue fluid ZE in MMX,no matter it was wild-type PccSl or mutant AimpG,which indicated that the tissue fluid had positive effect on the induction of pathogens;The comparison of PccS1 and mutant AimpG and the comparison of the two additions of the histolytic fluid ZE showed that the up-regulated genes of PccS1 were always larger than that of down-regulated genes,indicating that it had negative effect without impG.Meanwhile,it had effect on the two-component system,amino acid metabolism,bacterial secretion system and energy or transport system both on wild-type PccS1 and mutant ?impG,among which the genes of differential expression grew more in tissue fluid ZE and compared with the mutant AimpG,12 in17 genes associated with T6SS of wild-type PccS1 are raised.Furthermore,in this sdudy,we also compared the protein's sequences of RplY and the other ribosomal protein's sequence,which interacting with ImpQ with seven different of published subspecies of soft rot fungi?involved:P.aroidearum PC1?P.carotovorum subsp.carotovorum PCC21?P.carotovorum WPP14?P.brasiliensis PBR1692?P.wasabiae WPP 163?P.wasabiae SCC3193?P.wasabiae CFBP3304 and PccS1,which were originally host monocotyledonous or dicotyledonous plants.From the monocotyledonous plants:PccS1 and PC1;from the dicotyledonous plants:PCC21?WPP14?PBR1692?WPP163?SCC3193?CFBP3304?.The results show that the sequence of RplY,RplA and RplF exactly the same from the monocotyledonous plants;It had regular differences from the dicotyledonous plants compared with hosts,which is monocotyledonous.In conclusion,the experiments of protein interaction and the result of transcriptome sequencing show:?1?ImpG plays an important role in T6SS composition;?2?purA gene is related to pathogenicity;?3?ImpG protein may require a lot of energy when it works.?4?Pathogen part of the ribosomal protein in hosts,which the monocotyledonous or dicotyledonous plants,may be difference and has the important roles of disease;furthermore,it could be make a preliminary judgments to find the original host.The far-reaching influence of ImpG protein and its interaction ribosomal protein and the specific pathogenesis of pur A gene need further exploration.