| Cotton is an important cash crop,cotton Verticillium wilt seriously affects cotton yield and quality and has become one of the major limiting factors for cotton production.Verticillium dahliae Kleb.,a soil-borne fungus,is the primary pathogen causing cotton Verticillium wilt.With the increasing awareness of environmental protection,more and more attention has been paid to efficient,green,and environmentally friendly biological control techniques to overcome the limitations of traditional control methods.Bacteria have become the focus of biological control of cotton Verticillium wilt due to their fast growth,easy cultivation,and regulatory mechanisms for host defense against pathogens.Previous studies in our laboratory found Bacillus sp.T6 was isolated from contaminated Verticillium dahliae,which can inhibit the growth of V.dahliae,and its main virulence factor is the volatile substance styrene.Still,the molecular mechanism of its antagonism to V.dahliae must be more precise.In this study,we investigated the molecular mechanism of the action of T6 on V.dahliae by transcriptomic analysis,molecular docking,and q PCR and found that its virulence factor styrene might promote the lysis of V.dahliae by acting on the lysozyme of V.dahliae.The heterologous expression of lysozyme was further validated,and a plant-inducible peptide engineering strain was constructed to improve the efficacy of Bacillus T6 prevention.The main results of this study are as follows:1.Microscopic observation and transcriptomic analysis of the styrene effect on Verticillium dahliae.Microscopic observation revealed that the mycelium of V.dahliae became thin and lysed after the addition of styrene.Based on this experiment,the transcriptome of V.dahliae was sequenced after 2 h,2.5 h,and 3 h of styrene induction and normal culture,the transcriptome sequencing results showed that 9111 genes were detected,of which 2200 were changed at the transcriptional level.927 genes were altered at the transcriptional level after 2 hours of styrene induction,of which 282 genes were down-regulated at the transcriptional expression level and 645 genes were up-regulated at the transcriptional level;at 2.5 hours,1156 genes changed their transcription levels,including 259 genes whose transcription expression levels were down-regulated and 897 genes whose transcription levels were up-regulated;at 3 hours,1304 genes had their transcription levels changed,including 641 genes whose transcription expression levels were down-regulated and663 genes whose transcription levels were upregulated.Upregulated genes are significantly enriched in three categories: biological processes,molecular functions,and cellular components,which are associated with metabolic enzymes,stress response proteins,regulatory factors,and membrane component proteins.Compared to the control group,the genes downregulated during styrene treatment mainly involve transport,catabolism,cell growth,and biosynthesis,especially peptidases,lipases,proteases and chitinases.2.Molecular docking analysis and functional verification of styrene and potential molecular target proteins.According to the results of transcriptome analysis,screen the candidate gene of V.dahliae VDAG＿03038(peripheral trehalase),VDAG＿05117(surface protein),VDAG＿04085(sphingolipids long chain alkali reactive protein pil1),VDAG＿00706(bli-3),VDAG＿01167(multiple drug resistance protein CDR1),VDAG＿08124(sugar transporter family proteins),VDAG＿01467(glucose inhibitory protein),VDAG＿06032(Cytochrome P450 71B13),VDAG＿00511(glucan 1,3-β-Glucosidase),VDAG＿09554(Lysozyme),VDAG＿02913(Brefeldin A Resistance Protein),VDAG＿07280(proteins containing LEA domains),VDAG＿07197(ethanol dehydrogenase),VDAG＿02980(Cell Wall Protein Phi A)and VDAG＿07553(NAD dependent epi isomerase/dehydratase family protein),combined with the genome information of V.dahliae,the corresponding protein sequence was obtained through comparative analysis.Based on the three-dimensional structure in the protein database,using software such as Discovery Studio and Auto Dock Tools,molecular docking of potential target proteins with styrene was performed,and other information was combined to screen out the potential receptor protein lysozyme for styrene acting on V.dahliae.The molecular docking energy of lysozym with styrene is-4.53 kcal/mol,and Ile(isoleucine)at 142 positions,Trp(tryptophan)at 171 positions,Lys(lysine)at 173 positions,and Leu(leucine)at 182 positions on the A-chain can be docked with styrene.The results of q PCR showed that after 2.5 h induction by 100 μL styrene,the relative expression of the lysozyme gene was up-regulated by 91.33 times compared with the control group.This result was consistent with the results of transcriptome sequencing,indicating that the lysozyme gene plays a crucial role in the dissolution process of V.dahliae.3.Expression,purification,and antibacterial activity detection of lysozyme genes.The total length of the VDAG＿09554(lysozyme)gene in V991 was 666 bp,encoding 188 amino acids.The cloned gene sequence was used to construct the heterogenic expression plasmid using the p ET30 a expression vector.After sequencing verification,it was transferred to Escherichia coli BL21 for heterogenic expression to obtain Lys-30 a.The induction conditions were 0.2 mmol/L IPTG at 16℃ and 180 rpm for 12 h,and then Ni-NTA was used to separate the recombinant protein with His label for purification to obtain 22 KDa products.Finally,the purified protein was detected by SDS-PAGE electrophoresis.The activity of recombinant lysozyme was determined to be 1.257 U/m L using chitosan as a substrate.At the same time,lysozyme had a specific inhibitory effect on decreasing the number of spores of V.dahliae,which proved the vital role of lysozyme in the control of Verticillium wilt of cotton by Bacillus T6.4.Construction and activity detection of plant induced peptide engineering strains.Based on the literature review,we found that the presence of plant inducible peptides in V.dahliae can significantly improve the antibacterial activity of plants.The cloned gene sequences of plant inducible peptide genes Vd PEL1 and Pev D1 in V.dahliae V991 were selected,and the heterologous expression plasmid was constructed using the p HT43 expression vector.After sequencing and validation,the recombinant strains V3-T and P18-T were transferred to Bacillus T6 for heterologous expression.The induction conditions were 0.2 mmol/L IPTG induction at 16 ℃ and 180 rpm for 12 hours,obtaining the engineered strains V3-T and P18-T.The results of active oxygen detection showed that the increase in active oxygen content of engineering strain V3-T was 1.44 times that of the wild-type strain;the increase in active oxygen content of engineering strain P18-T was 2.61 times that of the wild-type strain,and there was significant difference between the two strains.In the pot experiment,the engineered strain V3-T experimental group’s disease index was reduced by 21.43%,the incidence rate was decreased by 7.07%,and the control impact was 21.45% when compared to the blank control group.The outcomes demonstrated that there was no discernible difference between the engineered strain V3-T’s control effect and that of the wild strain T6’s control effect.In addition,the control effect of the experimental group using the engineered strain P18-T was improved to 78.85%,while the disease index was decreased by 79.03%,and the incidence rate was decreased by 70.71%,compared with the blank control group.The findings were superior to the wild strain T6 control effect,suggesting that the strain has excellent resistance to cotton Verticillium wilt and can be used as a biocontrol agent to control cotton Verticillium wilt.In summary,this study initially clarified the molecular mechanism of Bacillus T6 antagonism against V.dahliae,screened the potential target protein lysozyme for heterologous expression and validated it,successfully constructed a plant-inducible peptide engineering strain and tested its activity,which improved the biological control effect of Bacillus T6,and provided experimental data and a theoretical basis for the subsequent biological control of cotton Verticillium wilt. |
