Study On The Regulation Mechanism Of Vfr On 2,4-DAPG Synthesis In Biocontrol Agent FD6 Against Tomato Gray Mold Disease

Pseudomonas protegens FD6 isolated from the canola rhizosphere in Fujian Province showed an excellent biocontrol effect against tomato gray mold.The control effect of Botrytis cinerea on tomato fruits in the greenhouse showed that the control effect against P.protegens FD6 reached 72.60%,which was significantly higher than that of Bacillus amyloliquefaciens W10 and the chemical agent Pythromycin.The major antagonistic activity produced by P.protegens FD6 corresponded to 2,4-diacetylphloroglucinol and pyoluteorin.There is an inverse interaction between 2,4-DAPG and PLT.The production of antibiotics is often regulated by many genes involved in their synthesis pathway including some transcription factors.Previous study showed that Vfr negatively regulates the production of 2,4-DAPG and PLT,Based on this result,this study will further explore the regulation mechanism of transcription factor Vfr on 2,4-DAPG biosynthesis through ChIP-seq,bacterial one-hybrid,ChIP-PCR and EMSA.A bacterial strain for Vfr-Flag fusion expression was constructed using a 3×FLAG as a fusion tag,then we used ChIP-seq to identify direct regulatory targets of Vfr.The results of data analysis showed that Vfr directly regulates the expressions of 90 genes.These 90 genes can be divided into 17 categories,including secondary metabolites,transcription factors,two-component system,pentose phosphate pathway,biofilm formation,bacterial chemotaxis and motility,secretion system,etc.Then qRT-PCR results indicated that the transcription levels of 7 genes increased(>2 times),and the transcription level of 21 genes decreased(>2 times).The transcription level of the vfr decreased by 5.5 times.The consensus Vfr-binding DNA motifs contain sequences similar to[A/G]TCACA[T/G/C][G/A/C/T].We constructed the prokaryotic expression vector for Vfr expression,and the VfrHis protein was expressed successfully in Escherichia coli BL21 in both soluble protein and inclusion body.The molecular weight is about 25 kDa.The Vfr protein was most expressed under induction by 0.5 mM IPTG,at 20℃ for 8 h.Purified protein can be obtained by elution with 150 mM imidazole on a nickel column.The protein concentration is 685 ng/μL after concentration.Finally,we compared the gene expression changes between FD6 and Δvfr,and the phlF,phlG,hcnB and vfr involved in the synthesis of 2,4-DAPG and HCN were chosen for further research.The results revealed that the transcription factor Vfr can directly bind to the upstream regions of vfr,phlF,phlG and hcnB.In summary,there are about 30 genes directly controlled by Vfr in P.protegens FD6.The transcriptional regulator Vfr is autoregulated and affects the synthesis of both antimicrobial compounds by directly binding to phlF,phlG and hcnB.