Molecular Mechanism Of TetR Family Transcription Factor BcPDR1 Regulating Pathogenicity In Botrytis Cinerea

Previous studies in our laboratory obtained the pathogenic gene BcPDR1 of Botrytis cinerea and identified this gene is a TetR transcription factor family gene,which positively regulated the pathogenicity of Botrytis cinerea.However,the mechanism of BcPDR1 regulating fungal pathogenicity is not clear.In this study,we identified the important functional regions and key functional sites of BcPDR1 protein using bioinformatics technology combined with gene reverse expression technology and gene site-directed mutation technology.The interaction protein of BcPDR1 was screened and identified by using cDNA library of Botrytis cinerea and yeast two-hybrid(Y2H)technology.The ChIP-Seq technique,yeast one-hybrid(Y1H)technique,and double luciferase reporter system(LUC)were used to screen and identify the target genes regulated by BcPDR1,which confirmed that BcPDR1 acted as a transcription factor of the TetR family to regulate pathogenicity,providing a new theoretical basis for the research on the molecular mechanism of pathogenicity of Botrytis cinerea.The main research results are as follows:1.Identification of important functional regions of Botrytis cinerea BcPDR1 protein.To identify the important functional regions of Botrytis cinerea BcPDR1 protein,the BcPDR1 protein was divided into three regions in the experiment:BcPDR1-1(1-240 bp),BcPDR1-2(241-363 bp)and BcPDR1-3(364-597 bp).Three regional reverse mutants were constructed based on the gene knockout mutant ΔBcpdr1 of BcPDR1 using gene complementary recovery technology.The phenotypes and pathogenicity of the three regional reverse mutants were analyzed,and the results showed that the colony morphology,hyphal morphology,growth rate,and infection structure of the three regional reverse mutants were basically the same as those of mutant ΔBcpdr1.The mutant ΔBcpdr1 produced obvious disease spots on tomato fruits and tobacco leaves,and the disease spot of the reverse mutant BcPDR1-3 was the most obvious.It indicated that BcPDR1-3(364-597 bp)was an important functional region of BcPDR1 protein.2.Identification of key amino acid sites of Botrytis cinerea BcPDR1 protein.Using bioinformatics technology,the conservative domain and key amino acids of BcPDR1 protein were analyzed,and four key sites were selected for site-directed mutagenesis.Based on the mutant ΔBcpdr1,the corresponding mutants BcPDR1-M1,BcPDR1-M2,BcPDR1-M3 and BcPDR1-M4 were constructed.The phenotype and pathogenicity of the mutants were analyzed,and the results showed that the colony morphology,hyphal morphology and growth rate of the mutants were basically the same as those of mutantΔBcpdr1.The mutant could produce obvious lesions in tomato fruits and tobacco leaves,especially in BcPDR1-M4,which indicated that the BcPDR1-M4(189 Ile)locus was the key site of BcPDR1 protein.3.Screening and identification of protein-protein interaction protein of Botrytis cinerea BcPDR1.In this study,yeast two-hybrid screening was performed using a cDNA library of Botrytis cinerea and a candidate interaction protein for BcPDR1 was obtained.Candidate interacting proteins were identified using bioinformatics analysis and yeast two-hybrid assay.The results showed that the yeasts co-transformed with the combination of AD-BcPDR1+BD-Bcin15g02120 and BD-BcPDR1+AD-Bcin15g02120 could grow normally on SD/-T/-L/-H and SD/-T/-L/-H/-A medium containing 3-AT,while the yeasts co-transformed with the negative control combination could not grow,indicating that BcPDR1 and Bcin15g02120 could directly interact with each other in yeast cells.4.Screening and identifying the regulatory target genes of Botrytis cinerea BcPDR1.In the early stage of our study,we screened and obtained the candidate target genes of BcPDR1 using the ChIP-Seq technology.In this study,yeast one-hybrid assay(Y1H)and double luciferase reporter system(LUC)were used to identify candidate target genes regulated by BcPDR1.The results showed that BcPDR1 could bind to the promoter regions of Bcin05g07320,Bcin09g00190,Bcin09g00920(BcATF)and Bcin06g05900(Bcbub2).These results indicated that Botrytis cinerea BcPDR1 might regulate Bcin05g07320,Bcin09g00190,Bcin09g00920(BcATF)and Bcin06g05900(Bcbub2).