灰葡萄孢丝裂原活化蛋白激酶编码基因bmp1和bmp3的功能
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摘要
【背景】植物病原真菌丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号途径参与病菌有性生殖、细胞壁完整、菌丝侵染、致病力、胁迫响应等过程,灰葡萄孢MAPK信号途径参与病菌生长发育、致病力以及胁迫响应,但MAPK信号途径基因在灰葡萄孢中的功能尚未完全阐明,该信号途径对灰葡萄孢的生长发育和致病力的调控机制尚不明确。【目的】明确灰葡萄孢MAPK编码基因bmp1、bmp3在病菌生长发育、致病力以及氧化胁迫响应过程的功能,为进一步阐明MAPK信号途径调控灰葡萄孢生长发育和致病力的分子机制奠定基础。【方法】利用RNAi技术构建灰葡萄孢MAPK编码基因bmp1和bmp3的RNAi突变体,并以野生型BC22菌株为对照,对bmp1和bmp3基因的RNAi突变体的表型、致病力以及对氧化胁迫的敏感性进行分析。【结果】灰葡萄孢bmp1和bmp3基因的RNAi突变体其菌落形态、菌丝形态均与野生型BC22菌株没有明显差别;bmp1基因的RNAi突变体生长速率明显减慢,分生孢子产量明显降低;bmp3基因的RNAi突变体的生长速率与野生型BC22菌株没有明显差别,不能产生分生孢子。bmp1和bmp3基因的RNAi突变体在番茄果实的表面均不能产生明显的致病症状,而且不能穿透玻璃纸。bmp1基因的RNAi突变体在含有H_2O_2的培养基上受抑制的程度显著低于野生型,而在含甲萘醌的培养基上受抑制的程度显著高于野生型;bmp3基因的RNAi突变体在含有H_2O_2和甲萘醌的培养基受抑制的程度均显著高于野生型。【结论】灰葡萄孢bmp1基因正调控病菌生长、分生孢子形成、致病力和穿透能力,参与调控病菌对氧化胁迫的响应;灰葡萄孢bmp3基因正调控病菌分生孢子形成、致病力、穿透能力以及对氧化胁迫的响应。
[Background] Mitogen-activated protein kinase(MAPK) signaling pathway of plant pathogenicfungi is involved in sexual reproduction, cell wall integrity, mycelial infection, pathogenicity, stressresponse and other processes. The MAPK signaling pathway is involved in the growth, development,pathogenicity and stress response in Botrytis cinerea. However, the function of MAPK signaling pathwaygene has not been fully elucidated in B. cinerea. [Objective] The objective of this study is to analyze thefunction of B. cinerea MAPK encoding genes bmp1 and bmp3 in growth, development, pathogenicity andresponse to oxidative stress, and to lay a foundation for clarifying the molecular mechanism of the MAPKsignaling pathway in growth, development and pathogenicity in B. cinerea. [Methods] RNAi mutants ofbmp1 and bmp3 genes were successfully constructed using RNAi technology. Compared with thewild-type strain BC22(WT), phenotype, pathogenicity, and sensitivity to oxidative stress of bmp1 andbmp3 genes RNAi mutants were analyzed. [Results] The colony morphology and mycelia morphology ofbmp1 and bmp3 gene RNAi mutants showed no obvious difference with WT. The bmp1 gene RNAimutants grew slowly and produce fewer conidial. The growth rate of bmp3 gene RNAi mutants showed noobvious difference with WT. The bmp3 gene RNAi mutants did not produce conidia. RNAi mutants ofbmp1 and bmp3 genes showed no pathogenicity and penetrating ability. RNAi mutants of bmp1 gene weresignificantly less inhibited than those of WT in the media containing H_2O_2, while the degree of inhibitionwas significantly higher in the media containing menadione than that of WT. RNAi mutants of bmp3 genewere significantly inhibited in H_2O_2 and menadione media than that of WT. [Conclusion] The bmp1 genepositively regulated growth, conidial formation, pathogenicity and penetrability, and was involved in theregulation of the response to oxidative stress in B. cinerea. The bmp3 gene positively regulates conidialformation, pathogenicity, penetrability, and response to oxidative stress in B. cinerea.
[Background] Mitogen-activated protein kinase(MAPK) signaling pathway of plant pathogenicfungi is involved in sexual reproduction, cell wall integrity, mycelial infection, pathogenicity, stressresponse and other processes. The MAPK signaling pathway is involved in the growth, development,pathogenicity and stress response in Botrytis cinerea. However, the function of MAPK signaling pathwaygene has not been fully elucidated in B. cinerea. [Objective] The objective of this study is to analyze thefunction of B. cinerea MAPK encoding genes bmp1 and bmp3 in growth, development, pathogenicity andresponse to oxidative stress, and to lay a foundation for clarifying the molecular mechanism of the MAPKsignaling pathway in growth, development and pathogenicity in B. cinerea. [Methods] RNAi mutants ofbmp1 and bmp3 genes were successfully constructed using RNAi technology. Compared with thewild-type strain BC22(WT), phenotype, pathogenicity, and sensitivity to oxidative stress of bmp1 andbmp3 genes RNAi mutants were analyzed. [Results] The colony morphology and mycelia morphology ofbmp1 and bmp3 gene RNAi mutants showed no obvious difference with WT. The bmp1 gene RNAimutants grew slowly and produce fewer conidial. The growth rate of bmp3 gene RNAi mutants showed noobvious difference with WT. The bmp3 gene RNAi mutants did not produce conidia. RNAi mutants ofbmp1 and bmp3 genes showed no pathogenicity and penetrating ability. RNAi mutants of bmp1 gene weresignificantly less inhibited than those of WT in the media containing H_2O_2, while the degree of inhibitionwas significantly higher in the media containing menadione than that of WT. RNAi mutants of bmp3 genewere significantly inhibited in H_2O_2 and menadione media than that of WT. [Conclusion] The bmp1 genepositively regulated growth, conidial formation, pathogenicity and penetrability, and was involved in theregulation of the response to oxidative stress in B. cinerea. The bmp3 gene positively regulates conidialformation, pathogenicity, penetrability, and response to oxidative stress in B. cinerea.
引文
[1]Hamel LP,Nicole MC,Duplessis S,et al.Mitogen-activated protein kinase signaling in plant-interacting fungi:distinct messages from conserved messengers[J].The Plant Cell,2012,24(4):1327-1351
[2]Chen RE,Thorner J.Function and regulation in MAPKsignaling pathways:lessons learned from the yeast Saccharomyces cerevisiae[J].Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,2007,1773(8):1311-1340
[3]Brefort T,Doehlemann G,Mendoza-Mendoza A,et al.Ustilago maydis as a pathogen[J].Annual Review of Phytopathology,2009,47:423-445
[4]Zhao XH,Kim Y,Park G,et al.A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea[J].The Plant Cell,2005,17(4):1317-1329
[5]Leroch M,Mueller N,Hinsenkamp I,et al.The signalling mucin Msb2 regulates surface sensing and host penetration via BMP1 MAP kinase signalling in Botrytis cinerea[J].Molecular Plant Pathology,2015,16(8):787-798
[6]Zheng L,Campbell M,Murphy J,et al.The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea[J].Molecular Plant-Microbe Interactions,2000,13(7):724-732
[7]Rui O,Hahn M.The Slt2-type Map kinase Bmp3 of Botrytis cinerea is required for normal saprotrophic growth,conidiation,plant surface sensing and host tissue colonization[J].Molecular Plant Pathology,2007,8(2):173-184
[8]Heller J,Ruhnke N,Espino JJ,et al.The mitogen-activated protein kinase BcSak1 of Botrytis cinerea is required for pathogenic development and has broad regulatory functions beyond stress response[J].Molecular Plant-Microbe Interactions,2012,25(6):802-816
[9]Segmüller N,Ellendorf U,Tudzynski B,et al.BcSAK1,a stress-activated mitogen-activated protein kinase,is involved in vegetative differentiation and pathogenicity in Botrytis cinerea[J].Eukaryotic Cell,2007,6(2):211-221
[10]Michielse CB,Becker M,Heller J,et al.The Botrytis cinerea Reg1 protein,a putative transcriptional regulator,is required for pathogenicity,conidiogenesis,and the production of secondary metabolites[J].Molecular Plant-Microbe Interactions,2011,24(9):1074-1085
[11]Yan LY,Yang QQ,Sundin GW,et al.The mitogen-activated protein kinase kinase BOS5 is involved in regulating vegetative differentiation and virulence in Botrytis cinerea[J].Fungal Genetics and Biology,2010,47(9):753-760
[12]Schamber A,Leroch M,Diwo J,et al.The role of mitogen-activated protein(MAP)kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea[J].Molecular Plant Pathology,2010,11(1):105-119
[13]Doehlemann G,Berndt P,Hahn M.Different signalling pathways involving a Gαprotein,cAMP and a MAP kinase control germination of Botrytis cinerea conidia[J].Molecular Microbiology,2006,59(3):821-835
[14]Choquer M,Fournier E,Kunz C,et al.Botrytis cinerea virulence factors:new insights into a necrotrophic and polyphageous pathogen[J].FEMS Microbiology Letters,2007,277(1):1-10
[15]Nakajima M,Akutsu K.Virulence factors of Botrytis cinerea[J].Journal of General Plant Pathology,2014,80(1):15-23
[16]Zhao W,Panepinto JC,Fortwendel JR,et al.Deletion of the regulatory subunit of protein kinase A in Aspergillus fumigatus alters morphology,sensitivity to oxidative damage,and virulence[J].Infection and Immunity,2006,74(8):4865-4874
[17]Temple MD,Perrone GG,Dawes IW.Complex cellular responses to reactive oxygen species[J].Trends in Cell Biology,2005,15(6):319-326
[18]Sun JS,Tsuang YH,Huang WC,et al.Menadione-induced cytotoxicity to rat osteoblasts[J].Cellular and Molecular Life Sciences,1997,53(11/12):967-976
[19]Viaud M,Fillinger S,Liu WW,et al.A class III Histidine Kinase acts as a novel virulence factor in Botrytis cinerea[J].Molecular Plant-Microbe Interactions,2006,19(9):1042-1050
[20]Hong JK,Kim HJ,Jung H,et al.Differential control efficacies of vitamin treatments against bacterial wilt and grey mould diseases in tomato plants[J].The Plant Pathology Journal,2016,32(5):469-480
[2]Chen RE,Thorner J.Function and regulation in MAPKsignaling pathways:lessons learned from the yeast Saccharomyces cerevisiae[J].Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,2007,1773(8):1311-1340
[3]Brefort T,Doehlemann G,Mendoza-Mendoza A,et al.Ustilago maydis as a pathogen[J].Annual Review of Phytopathology,2009,47:423-445
[4]Zhao XH,Kim Y,Park G,et al.A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea[J].The Plant Cell,2005,17(4):1317-1329
[5]Leroch M,Mueller N,Hinsenkamp I,et al.The signalling mucin Msb2 regulates surface sensing and host penetration via BMP1 MAP kinase signalling in Botrytis cinerea[J].Molecular Plant Pathology,2015,16(8):787-798
[6]Zheng L,Campbell M,Murphy J,et al.The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea[J].Molecular Plant-Microbe Interactions,2000,13(7):724-732
[7]Rui O,Hahn M.The Slt2-type Map kinase Bmp3 of Botrytis cinerea is required for normal saprotrophic growth,conidiation,plant surface sensing and host tissue colonization[J].Molecular Plant Pathology,2007,8(2):173-184
[8]Heller J,Ruhnke N,Espino JJ,et al.The mitogen-activated protein kinase BcSak1 of Botrytis cinerea is required for pathogenic development and has broad regulatory functions beyond stress response[J].Molecular Plant-Microbe Interactions,2012,25(6):802-816
[9]Segmüller N,Ellendorf U,Tudzynski B,et al.BcSAK1,a stress-activated mitogen-activated protein kinase,is involved in vegetative differentiation and pathogenicity in Botrytis cinerea[J].Eukaryotic Cell,2007,6(2):211-221
[10]Michielse CB,Becker M,Heller J,et al.The Botrytis cinerea Reg1 protein,a putative transcriptional regulator,is required for pathogenicity,conidiogenesis,and the production of secondary metabolites[J].Molecular Plant-Microbe Interactions,2011,24(9):1074-1085
[11]Yan LY,Yang QQ,Sundin GW,et al.The mitogen-activated protein kinase kinase BOS5 is involved in regulating vegetative differentiation and virulence in Botrytis cinerea[J].Fungal Genetics and Biology,2010,47(9):753-760
[12]Schamber A,Leroch M,Diwo J,et al.The role of mitogen-activated protein(MAP)kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea[J].Molecular Plant Pathology,2010,11(1):105-119
[13]Doehlemann G,Berndt P,Hahn M.Different signalling pathways involving a Gαprotein,cAMP and a MAP kinase control germination of Botrytis cinerea conidia[J].Molecular Microbiology,2006,59(3):821-835
[14]Choquer M,Fournier E,Kunz C,et al.Botrytis cinerea virulence factors:new insights into a necrotrophic and polyphageous pathogen[J].FEMS Microbiology Letters,2007,277(1):1-10
[15]Nakajima M,Akutsu K.Virulence factors of Botrytis cinerea[J].Journal of General Plant Pathology,2014,80(1):15-23
[16]Zhao W,Panepinto JC,Fortwendel JR,et al.Deletion of the regulatory subunit of protein kinase A in Aspergillus fumigatus alters morphology,sensitivity to oxidative damage,and virulence[J].Infection and Immunity,2006,74(8):4865-4874
[17]Temple MD,Perrone GG,Dawes IW.Complex cellular responses to reactive oxygen species[J].Trends in Cell Biology,2005,15(6):319-326
[18]Sun JS,Tsuang YH,Huang WC,et al.Menadione-induced cytotoxicity to rat osteoblasts[J].Cellular and Molecular Life Sciences,1997,53(11/12):967-976
[19]Viaud M,Fillinger S,Liu WW,et al.A class III Histidine Kinase acts as a novel virulence factor in Botrytis cinerea[J].Molecular Plant-Microbe Interactions,2006,19(9):1042-1050
[20]Hong JK,Kim HJ,Jung H,et al.Differential control efficacies of vitamin treatments against bacterial wilt and grey mould diseases in tomato plants[J].The Plant Pathology Journal,2016,32(5):469-480