Molecular Mechanism Of BcPDR1 Gene In Regulating Pathogenicity Of Botrytis Cinerea

In our preliminary study,the pathogenic gene BcPDR1 was obtained and identified that it positively regulated the growth,development and pathogenicity of Botrytis cinerea.In order to clarify the molecular mechanism of the BcPDR1 gene in regulating pathogenicity of B.cinerea,PCR and Western blot techniques were used to determine the coding function of the BcPDR1 gene.Pull down and yeast two-hybrid techniques were used to screen and identify interaction proteins of the BcPDR1 protein.Digital gene expression profiling technique,qRT-PCR and cell wall degrading enzymes activity detection kit were used to screen and identify differentially expressed peroxidase family genes and cell wall degrading enzyme genes affected by BcPDR1.Using qRT-PCR and double gene mutation technique,the relationship between BcPDR1 and cAMP and MAPK signaling pathways in B.cinerea was clarified.The main results were as follows:1.Study on the coding function of BcPDR1 gene in B.cinerea.PCR amplification was performed on cDNA and genomic DNA of B.cinerea wild-type strain using BcPDR1 specific primers.Single bands were obtained,and the transcription function of BcPDR1 was preliminarily determined.The GST Tag antibody,GFP Tag antibody,BcPDR1 antibody were used to detect the expression of BcPDR1-GST protein,the total protein of the complementing mutant CE,the total protein of the wild type strain BC22 and the complementing mutant CE using Western blot technique.The results showed that all the tested samples obtained a single positive band,which was consistent with the size of the target protein,indicating that BcPDR1 had a protein coding function.2.Screening and identification of B.cinerea BcPDR1 interacting protein.216 candidate interacting proteins of BcPDR1 protein were obtained by Pull down technique.12 candidate interacting proteins,PPDK,MDAR1,MDAR2,IMD1,PDC2,NADP-ME2,EDA9,SHM2,AIP12,U88A1,2AB2 B and RAD2 C,were obtained by bioinformatics analysis.The yeast two-hybrid technique was used to identify the candidate interacting proteins of BcPDR1.It was found that yeast cells transformed with AD-BcPDR1+BD-IMD1,AD-BcPDR1+BD-SHM2,AD-BcPDR1+BD-AIP12,BD-BcPDR1+AD-IMD1,BD-BcPDR1+AD-SHM2,and BD-BcPDR1+AD-AIP12 grew well in the two-deficient media(-Leu/-Trp),three-deficient media(-Leu/-Trp/-His)and four-deficient media(-Leu/-Trp/-His/-Ade),indicated that BcPDR1 protein could directly interact with IMD1,SHM2,and AIP12 in yeast cells.Yeast cells co-transformed with BcPDR1 and other candidate interaction proteins grew well on the two-deficient media(-Leu/-Trp),but it could not grow in the three-deficient media(-Leu/-Trp/-His)and four-deficient media(-Leu/-Trp/-His/-Ade),indicated that they could not directly interact with other candidate interaction proteins in yeast cells.3.Screening and identification of differentially expressed genes of BcPDR1 in B.cinerea.933 differentially expressed genes were obtained by digital gene expression profiling technology.The results of GO enrichment analysis of differentially expressed genes showed that differentially expressed genes were mainly expressed in in the growth(GO: 0040007)of biological process and signaling(GO: 0023052),transporter(GO: 0005215),nucleic acid binding transcription factor(GO: 0001071),enzyme regulator(GO: 0030234)in translation regulator(GO: 0045182)of molecular function.Using the expression profile data,the expression levels of 14 cell wall degrading enzymes and 29 peroxidase genes were analyzed.It was found that the expression levels of Bcpg5,Bcpgx1,Bcpg4,Bcams1,and Bcmnl1 in wild-type BC22 and complementing mutant CE were significantly higher than those of mutants BCt89 and ?BcPDR1,but the expression levels of Bcpme5,Bcpg3,Bcpme1,Bcpg6,and Bcpg1 in mutants BCt89 and ?BcPDR1 were significantly higher than those in wild-type BC22 and complementing mutant CE.The expression levels of BccatA,Bcprd8,Bccat3,BcppoA90,Bcprd7,Bcprd11,Bcprx3,Bccat4,Bcprd2,Bcprd5,Bcprx7 in wild-type BC22 and complementing mutant CE were significantly higher than those in mutants BCt89 and ?BcPDR1,but the expression levels of Bcprx9,Bcprd1,Bcprd4,Bcprx8,Bcprd9,Bcccp2,Bcprd10 in mutants BCt89 and ?BcPDR1 were significantly higher than those in wild-type BC22 and complementing mutant CE.Real-time PCR technology was used to detect the expression levels of 14 cell wall degrading enzyme genes in wild-type BC22 and BcPDR1 gene mutants.it was found that the expression levels of Bcpg5,BccutA,Bcpgx1,Bcams1,Bcmnl1,and BccutB in wild-type BC22 and complementing mutant CE were significantly higher than those of mutants BCt89 and ?BcPDR1,but the expression levels of Bcmns1,Bcrot2,Bcpme2,Bcpg1,Bcpg3,Bcpme1,Bcpg6 and Bcpg4 in mutants BCt89 and ?BcPDR1 were significantly higher than those in wild-type BC22 and complementing mutant CE.These results were basically consistent with the results of digital expression profiles.The activity of cell wall degrading enzymes in the wild-type BC22 and BcPDR1 gene mutants were determined by using an enzyme activity assay kit.It was found that the activity of polygalacturonase(PMG),pectinase(PG),cellulase(CX),polygalacturonic acid trans-eliminating enzyme(PGTE),and pectinmethyltrans-eliminating enzyme(PMTE)in wild-type BC22 and complementing mutant CE were significantly higher than those in mutants BCt89 and ?BcPDR1.These results demonstrated that BcPDR1 was up-regulated the activity of cell wall degrading enzymes in B.cinerea.Real-time PCR technology was used to detect the expression levels of 29 peroxidase genes in wild-type BC22 and BcPDR1 gene mutants.It was found that the expression levels of Bcprx4,Bccat5,Bcgpx3,Bcprd3,Bcprx9,Bcprd1,Bcprx1,Bcccp1,Bcprd4,Bcprx8,Bcprd9,BcnoxA,Bcprd6,Bcccp2,Bccat2,Bccat6,BcppoA80,Bcprd10 in mutants BCt89 and ?BcPDR1 were significantly higher than those in wild-type BC22 and complementing mutant CE,but the expression levels of BccatA,Bcprd8,BcppoA90,Bcprd7,Bcprd11,Bccat4,Bcprd2,Bcprx7 in wild-type BC22 and complementing mutant CE were significantly higher than those in mutants BCt89 and ?BcPDR1.The results were basically consistent with the results of digital expression profiles.4.Study on the relationship between BcPDR1,cAMP and MAPK signaling pathways in B.cinerea.SQ22536 and U0126,cAMP and MAPK signaling pathway inhibitors,were used to detect the sensitivity of wild-type BC22,mutant BCt89,?BcPDR1 and complementing mutant CE to the signaling pathway inhibitors.It was found that mutants BCt89 and ?BcPDR1 were not sensitive to SQ22536 and U0126,and the inhibition rates were significantly lower than that of the wild-type BC22 and the complementing mutant CE.The content of cAMP in wild-type BC22 and BcPDR1 gene mutants were detected by using HPLC assay.It was found that the cAMP content of the mutants BCt89 and ?BcPDR1 were significantly higher than that of wild-type BC22 and complementing mutant CE.Real-time PCR technology was used to detect the expression levels of key genes of cAMP and MAPK signaling pathways in wild-type BC22 and BcPDR1 gene mutants.The results showed that the key genes of cAMP signaling pathways,bcg1,bcg2,bcg3,BAC,BcpkaR,Bcpka1,Bcpka2,in mutants BCt89 and ?BcPDR1 were significantly higher than those in wild-type BC22 and complementing mutant CE.The expression levels of key genes of MAPK signaling pathway,Btp1,BOS1,BcCDC4,BcRAC,bmp1,bmp3,BcSaK1 and BAP1,were significantly higher than those in wild-type BC22 and complementing mutant CE,while the expression level of key gene BcATF1 in MAPK signaling pathway was significantly lower than that wild-type BC22 and complementing mutant CE.These results demonstrated that BcPDR1 down-regulated the cAMP,MAPK signaling pathway key genes bcg1,bcg2,bcg3,BAC,BcpkaR,Bcpka1,Bcpka2,Btp1,BOS1,BcCDC4,BcRAC,bmp1,bmp3,BcSaK1 and BAP1,BcPDR1,and up-regulated the key gene BcATF1 of the MAPK signaling pathway.Real-time PCR technology was used to detect the expression levels of BcPDR1 gene in RNAi mutants of key genes of cAMP and MAPK signaling pathways.The results showed that the expression levels of BcPDR1 gene in RNAi mutants of key genes pka1,pka2,pkaR,bcg2 and bcg3 of cAMP signaling pathway were significantly lower than wild-type BC22,the expression levels of BcPDR1 gene in the RNAi mutants of the key genes of the MAPK signaling pathway,bmp1 and bmp3,were significantly lower than wild-type BC22.These results demonstrated that the key genes pka1,pka2,pkaR,bcg2 and bcg3 of the cAMP signaling pathway up-regulated the expression of BcPDR1 gene,and the key genes of MAPK signaling pathways bmp1 and bmp3 up-regulate the expression of BcPDR1 gene.The double gene mutation technique was used to obtain the double gene mutants of BcPDR1 and cAMP pathway key genes Bcpka1,Bcpka2,BcpkaR,bcg2,bcg3 and MAPK pathway key genes bmp1,bmp3.The phenotype and pathogenicity of double gene mutants were analyzed.Compared to ?BcPDR1,the phenotype and pathogenicity of ?BcPDR1/Bcpka1-OE,?BcPDR1/Bcpka2-OE,?BcPDR1/BcpkaR-OE,?BcPDR1/bcg2,?BcPDR1/bcg3,?BcPDR1/bmp1-OE,?BcPDR1/bmp3-OE were complemented to different degrees,indicating that BcPDR1 was involved in the cAMP and MAPK signaling pathways.