| Pseudomonas syringae includes a large numbers and more diversity of plant pathogenic bacteria.According to its host range,Pseudomonas syringae can be divided into more than 60 pathovars.Pseudomonas syringae pv.tabaci(synonym:P.amygdali pv.tabaci)belongs to pathovar tabaci of Pseudomonas syringae.It mainly infect tobacco(Nicotiana tabacum)and result in explosive and destructive wildfire disease.Our laboratory had found that a certain concentration of ammonium ions can aggravate the symptom of wildfire.In order to explore the mechanism that the exogenous ammonium ions increase the pathogenicity,this research explored the effects of exogenous ammonium ions on the bacterial behavior,the pathogenicity,the reproduction,and the bacterial gene expression in vitro were studied by use of transcriptome technique.At the same time,the relationship among the wildfire resistance of tobacco cultivars at mature stage and the activity of leaf nitrogen metabolism enzymes and the concentration of leaf apoplastic ammonium ions.The main results of this research are as follows:(1)Through the allergy(HR)and pathogenicity tests of 3 strains,it was found that strain BY23(P.s.pv.tabaci BY23)showed chlorosis symptoms at 3 days,indicating that strain BY23 is a compatible pathogen of tobacco and a weed The pathogen of wildfire;the strain DC3000(P.s.pv.tomato DC3000)cannot infect tobacco and is an incompatible pathogen of tobacco,while the strain HY(P.s.HY)has no allergic and pathogenic symptoms,right and wrong pathogenic bacteria.The wildfire pathogen BY23 can infect K326 in the range of OD600=0.001～0.01.When the inoculation concentration is OD600=0.004,the typical symptoms of wildfire disease can appear in 5 days.Therefore,OD600=0.004 is considered to be the most suitable inoculation concentration for studying the relationship between exogenous ammonium ions and the pathogenicity of wildfire pathogens.(2)In vitro culture experiments show that 10-7mol/L exogenous ammonium ions can promote the growth of the wildfire pathogen strain BY23,and the reproduction speeds up significantly after 3 to 4 hours.However,for the tomato bacterial spot pathogen(P.s.pv tomato)strain DC3000 and Pseudomonas saprophytic strain HY showed inhibition of reproduction;in addition,a certain concentration(10-6mol/L～10-8mol/L)of exogenous ammonium ions can significantly enhance the motility of strain BY23,Accelerates the formation of its biofilm,but has no effect on strains DC3000 and HY;however,exogenous ammonium ions have an effect on the production of extracellular polysaccharides and the activity of extracellular enzymes(extracellular protease,cellulase,amylase)of the three strains There was no obvious effect;in the BY23 culture filtrate test,it was found that the filtrate of a certain concentration of ammonium ions and pathogenic bacteria co-cultured for 48 hours can promote the opening of stomata,destroy the integrity of plant cells,and enhance the toxicity to the host plant.(3)In vivo inoculation test found that 10-7mol/L ammonium ions mixed with pathogenic bacteria can accelerate the infection of pathogenic bacteria after K326,and the colonization number of BY23 in the host is also enhanced,24 hours increased by 1.85 times,48 hours increased by 2.19 times.(4)The results of transcriptome sequencing showed that the gene expression,42 genes were significantly up-regulated and 183 genes were significantly down-regulated;at 3 hours of culture,7 genes were significantly up-regulated and 4 genes were downregulated.In order to further study the biological importance of differential gene expression,the differential expressed genes were compared with the reference genomes of Ps.pv.tabaci.The GO data enrichment analysis showed that most of the GO terms of the differentially expressed genes of BY23 were stimulated by exogenous ammonium ions.The change is classified into three functional categories:cellular component,biological process,and molecular function,mainly in cytoplasm andof BY23 in vitro can be regulated by exogenous ammonium ions(10-7mol/L).At 3 hours of culture cell membrane.After KEGG data analysis and annotation to 123 pathways,differentially expressed genes are mainly involved in bacterial chemotaxis,quorum sensing,nitrogen metabolism,flagella assembly,bacterial secretion system and other pathways,and bacterial chemotaxis,motility,amino acid metabolism,etc.The function is related,indicating that exogenous ammonium ions may be a kind of signal molecule that can regulate the conversion of pathogenic bacteria,nutrient absorption and the ability of infection to cause disease.(5)In order to verify the differentially expressed genes,12 significantly differentially expressed genes were selected,and their expression levels were verified by real-time fluorescent quantitative PCR.The verification results show that the relative expression levels of the 12 differentially expressed genes are basically consistent with the transcriptome sequencing results,indicating that the transcriptome data is reliable.Twelve BY23 pathogenic-related genes obtained by GO and KEGG analysis were selected for verification,and they were also consistent with the transcriptome results.Through comparison,it was found that these genes are mainly involved in the synthesis of flagellar matrix-related proteins and flagella movement,indicating that exogenous ammonium ions may be By promoting the formation of bacterial flagella and enhancing the motility of bacteria to improve their pathogenic ability.(6)By measuring the activity of carbon and nitrogen metabolism related enzymes,the level of apoplastic ammonium ion and the severity of wildfire disease,of tobacco cultivars under natural fields,the results indicate that the difference of tobacco resistance to wildfire show most significant correlation with glutamine synthetase(GS)activity and the accumulation of apoplastic ammonium ionsThe above results show that the presence of exogenous ammonium ions can not only affect the in vitro pathogen reproduction,motility,and biofilm formation,but also affect the colonization and pathogenicity in host tissues.Transcriptome analysis further found that exogenous ammonium ions regulate the biacterial motility and increase the pathogenicity by up-regulating the expression of the bacterial flagellar synthesis and chemotaxis-related genes.Therefore,it can be considered that exogenous ammonium ions are likely to be an important signal substance that initiates the early infection of wildfire bacteria.This conclusion is also supported by the measurement results of the apoplast ammonium ions of resistant and susceptible varieties in the field. |
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