| Fungal diseases account for more than 80% of plant diseases,and are extremely harmful.Biological control is one of the important way to promote green control of crop diseases.The key to achieve biocontrol is to screen good biocontrol engineering bacteria and reveal biocontrol mechanism.In the early stage,Bacillus subtilis J-15 was screened from cotton rhizosphere soil,which can secrete secondary metabolites against various fungi,such as Verticillium dahliae,Botrytis cinerea,fusahum graminearum and other harmful bacteria.It shows good stability and antagonistic activity,and the secondary metabolites secreted by the strain have lethal effect on the spores of Verticillium dahliae,but the antifungal mechanism is still unclear.In this paper,the cytotoxic mechanism of the secondary metabolite to Saccharomyces cerevisiae(S.cerevisiae)was studied from the cellular level,and the differential expression genes and differential expression proteins of J-15 secondary metabolite(SMs)lethal S.cerevisiae were identified by transcriptome(RNA-Seq)and quantitative proteomics(TMT),the obtained proteome and transcriptome data were correlated analysis,to provide the basis for elucidating the antifungal mechanism of J-15 SMs.(1)MTT method was used to verify that the J-15 SMs can significantly reduce the cell activity of S.cerevisiae in a time-dependent manner.Annexin V-FITC/Pi double staining flow cytometry showed that the J-15 SMs caused S.cerevisiae cell necrosis in time and concentration dependence.Treatment of S.cerevisiae with J-15 secondary metabolites results in rupture of the nuclear membrane and diffusion of nucleic acids.(2)RNA-Seq technique was used to analyze the transcriptional profile of S.cerevisiae induced by J-15 SMs,and 6502 genes were identified.Compared with the non treatment group,667 genes were differentially expressed(332 genes were up regulated,335 genes were down regulated)after the treatment 12 h by J-15 SMs,and 164 genes were differentially expressed(93 genes were up regulated,71 genes were down regulated)after the treatment 24 h.By analyzing the differentially expressed genes,it is speculated that the J-15 SMs inhibited the key enzymes of TCA cycle,glycolysis and other important pathways of fungi,damaged the cell translation,destroied the nutrient intake system,caused intracellular oxidative damage,and inhibited cell growth and promoted cell autophagy,Among them,the expression of autophagy related gene Atg1 was significantly different,suggesting that Atg1 may play a key role in inducing autophagic death.(3)TMT technology was used to analyze the protein expression of S.cerevisiae induced by J-15 SMs,and 4053 proteins were identified.Compared with the non treatmentgroup,after 12 h treatment with J-15 SMs,56 proteins were differentially expressed(31proteins were up regulated,25 proteins were down regulated).After 24 h treatment,76 proteins were differentially expressed(54 proteins were up regulated,22 proteins were down regulated).The differential protein analysis showed that the J-15 SMs inhibited the expression of key proteins in glycolysis,TCA cycle,pyruvate metabolism and other important metabolic pathways,caused cell stress response,inhibited cell translation,inhibited cell growth and reduced cytotoxicity.In addition,the expression of autophagy related protein ATG5 was significantly increased.Therefore the J-15 SMs may cause fungal cell death through the action of ATG5 and other death related proteins.(4)Association analysis of differential genes and differential proteins identified by treated J-15 SMs in S.cerevisiae for 12 and 24 h.The correlation coefficients were 0.6651 and 0.5091,and associated with 23 and 13 genes with significant differences in mRNA and protein levels,respectively,which laid a foundation for further revealing the molecular mechanism of J-15 SMs antagonistic fungi.In this paper,the model fungus S.cerevisiae was taken as the research object.It was preliminarily revealed that the J-15 SMs could kill fungal cells by destroying nuclear membrane,inhibiting glycolysis and inducing autophagy,which laid a foundation for elucidating the molecular mechanism of J-15 antagonism to fungi,and also provided important theoretical guidance for the development and utilization of new biological pesticides for biological control. |
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