GCK家族蛋白激酶MoSOK1调控稻瘟病菌的生长发育与致病性
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摘要
稻瘟病菌致病相关基因功能的研究有助于揭示其致病机理,为稻瘟病防治提供理论依据。本研究利用基因替换技术对稻瘟病菌SOK1同源基因(Mo SOK1)进行了分析。Mo SOK1编码GCK(germinal center kinase)家族的Ste20蛋白激酶,其与全蚀病菌中对应蛋白具有最高同源性。本研究表明,Mo SOK1基因在附着胞分化关键期上调表达;与野生型菌株相比,Mo SOK1基因缺失突变体菌落颜色加深,气生菌丝减少,生长速度变慢,产孢量下降,分生孢子萌发延迟,致病性降低。另外,交配实验表明,Mo SOK1基因缺失突变体能够进行交配,但产子囊壳能力降低。本研究初步表明,Mo SOK1基因参与稻瘟病菌生长发育和致病过程。
SOK1 is a Ste20 protein kinase of the germinal center kinase( GCK) that is activated by oxidant stress and chemical anoxia. It is unknown so far whether SOK1 play roles in regulation of the fungal pathogenicity. Herein,we functionally characterized the SOK1 homologue in the rice blast fungus Magnaporthe oryzae( Mo SOK1). Mo SOK1 was up-regulated during conidial germination and appressorial formation. Targeted gene replacement showed that Mo SOK1 acts as a regulator to fungal development and virulence in M. oryzae. Δmosok1 mutants showed decreased vegetative growth and conidiation,delayed conidial germination and a significant reduction in virulence. Mo SOK1 was also found related to the sexual reproduction of the fungus. These data implicate that SOK1 signaling is required for fungal development and pathogenicity in phytopathogenic fungi.
SOK1 is a Ste20 protein kinase of the germinal center kinase( GCK) that is activated by oxidant stress and chemical anoxia. It is unknown so far whether SOK1 play roles in regulation of the fungal pathogenicity. Herein,we functionally characterized the SOK1 homologue in the rice blast fungus Magnaporthe oryzae( Mo SOK1). Mo SOK1 was up-regulated during conidial germination and appressorial formation. Targeted gene replacement showed that Mo SOK1 acts as a regulator to fungal development and virulence in M. oryzae. Δmosok1 mutants showed decreased vegetative growth and conidiation,delayed conidial germination and a significant reduction in virulence. Mo SOK1 was also found related to the sexual reproduction of the fungus. These data implicate that SOK1 signaling is required for fungal development and pathogenicity in phytopathogenic fungi.
引文
[1]EBBOLE D J.Magnaporthe as a model for understanding hostpathogen interactions[J].Annual Review of Phytopathology,2007,45:437-456.
[2]DEAN R A,TALBOT N J,EBBOLE D J,et al.The genome sequence of the rice blast fungus Magnaporthe grisea[J].Nature,2005,434(7036):980-986.
[3]LIU X H,LU J P,ZHANG L,et al.Involvement of a Magnaporthe grisea serine/threonine kinase gene,Mg ATG1,in appressorium turgor and pathogenesis[J].Eukaryotic Cell,2007,6(6):997-1005.
[4]WANG J,ZHANG Z,WANG Y,et al.PTS1 peroxisomal import pathway plays shared and distinct roles to PTS2 pathway in development and pathogenicity of Magnaporthe oryzae[J].PLo S One,2013,8(2):e55554.
[5]LI L,WANG J,ZHANG Z,et al.Mo Pex19,which is essential for maintenance of peroxisomal structure and woronin bodies,is required for metabolism and development in the rice blast fungus[J].PLo S One,2014,9(1):e85252.
[6]PARK G,BRUNO K S,STAIGER C J,et al.Independent genetic mechanisms mediate turgor generation and penetration peg formation during plant infection in the rice blast fungus[J].Molecular Microbiology,2004,53(6):1695-1707.
[7]BOURETT T M,HOWARD R J.In vitro development of penetration structures in the rice blast fungus Magnaporthe grisea[J].Canadian Journal of Botany,1990,68(2):329-342.
[8]HOWARD R J,VALENT B.Breaking and entering:host penetration by the fungal rice blast pathogen Magnaporthe grisea[J].Annual Review of Microbiology,1996,50:491-512.
[9]HOWARD R J,FERRARI M A,ROACH D H,et al.Penetration of hard substrates by a fungus employing enormous turgor pressures[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(24):11281-11284.
[10]DE JONG J C,MCCORMACK B J,SMIRNOFF N,et al.Glycerol generates turgor in rice blast[J].Nature,1997,389(6648):244-244.
[11]LEE Y H,DEAN R A.Hydrophobicity of contact surface induces appressorium formation in Magnaporthe grisea[J].FEMS Microbiology Letters,1994,115(1):71-75.
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[13]XIAO J Z,WATANABE T,KAMAKURA T,et al.Studies on cellular-differentiation of Magnaporthe grisea-physicochemical aspects of substratum surfaces in relation to appressorium formation[J].Physiological and Molecular Plant Pathology,1994,44(3):227-236.
[14]LIU S,DEAN R A.G protein alpha subunit genes control growth,development,and pathogenicity of Magnaporthe grisea[J].Molecular Plant-Microbe Interactions,1997,10(9):1075-1086.
[15]BOLKER M.Sex and crime:heterotrimeric G proteins in fungal mating and pathogenesis[J].Fungal Genetics and Biology,1998,25(3):143-156.
[16]XU J R,HAMER J E.MAP kinase and c AMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea[J].Genes&Development,1996,10(21):2696-2706.
[17]ZHAO X,KIM Y,PARK G,et al.A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea[J].Plant Cell,2005,17(4):1317-1329.
[18]LEE Y H,DEAN R A.c AMP regulates infection structure formation in the plant pathogenic fungus Magnaporthe grisea[J].Plant Cell,1993,5(6):693-700.
[19]ZHOU X,ZHAO X,XUE C,et al.Bypassing both surface attachment and surface recognition requirements for appressorium formation by overactive ras signaling in Magnaporthe oryzae[J].Molecular Plant-Microbe Interactions,2014,27(9):996-1004.
[20]BROACH J R,DESCHENES R J.The function of ras genes in Saccharomyces cerevisiae[J].Advances in Cancer Research,1990,54:79-139.
[21]MITCHELL T K,DEAN R A.The c AMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea[J].Plant Cell,1995,7(11):1869-1878.
[22]XU J R,URBAN M,SWEIGARD J A,et al.The CPKA gene of Magnaporthe grisea is essential for appressorial penetration[J].Molecular Plant-Microbe Interactions,1997,10(2):187-194.
[23]TODA T,CAMERON S,SASS P,et al.SCH9,a gene of Saccharomyces cerevisiae that encodes a protein distinct from,but functionally and structurally related to,c AMP-dependent protein kinase catalytic subunits[J].Genes&Development,1988,2(5):517-527.
[24]WARD M P,GARRETT S.Suppression of a yeast cyclic AMP-dependent protein kinase defect by overexpression of SOK1,a yeast gene exhibiting sequence similarity to a developmentally regulated mouse gene[J].Molecular&Cellular Biology,1994,14(9):5619-5627.
[25]POMBO C M,BONVENTRE J V,MOLNAR A,et al.Activation of a human Ste20-like kinase by oxidant stress defines a novel stress response pathway[J].EMBO Journal,1996,15(17):4537-4546.
[26]POMBO C M,TSUJITA T,KYRIAKIS J M,et al.Activation of the Ste20-like oxidant stress response kinase-1 during the initial stages of chemical anoxia-induced necrotic cell death.Requirement for dual inputs of oxidant stress and increased cytosolic[Ca2+][J].Journal of Biological Chemistry,1997,272(46):29372-29379.
[27]MARTINDALE J L,HOLBROOK N J.Cellular response to oxidative stress:signaling for suicide and survival[J].Journal of Cellular Physiology,2002,192(1):1-15.
[28]EGAN M J,WANG Z Y,JONES M A,et al.Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(28):11772-11777.
[29]SKAMNIOTI P,HENDERSON C,ZHANG Z,et al.A novel role for catalase B in the maintenance of fungal cell-wall integrity during host invasion in the rice blast fungus Magnaporthe grisea[J].Molecular Plant-Microbe Interactions,2007,20(5):568-580.
[30]HUANG K,CZYMMEK K J,CAPLAN J L,et al.HYR1-mediated detoxification of reactive oxygen species is required for full virulence in the rice blast fungus[J].PLo S Pathogens,2011,7(4):e1001335.
[31]HUANG K,CZYMMEK K J,CAPLAN J L,et al.Suppression of plant-generated reactive oxygen species is required for successful infection by the rice blast fungus[J].Virulence,2011,2(6):559-562.
[32]TALBOT N J,EBBOLE D J,HAMER J E.Identification and characterization of MPG1,a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea[J].Plant Cell,1993,5(11):1575-1590.
[33]李海娇,卢建平,刘小红,等.适用于稻瘟病菌基因敲除、过表达和荧光融合蛋白表达载体的构建和使用[J].农业生物技术学报,2012,20(1):94-104.LI H J,LU J P,LIU X H,et al.Vectors building and usage for gene knockout,protein expression and fluorescent fusion protein in the rice blast fungus[J].Journal of Agricultural Biotechnology,2012,20(1):94-104.(in Chinese with English abstract)
[34]WANG J,LI L,ZHANG Z,et al.One of three Pex11 family members is required for peroxisomal proliferation and full virulence of the rice blast fungus Magnaporthe oryzae[J].PLo S One,2015,10(7):e0134249.
[2]DEAN R A,TALBOT N J,EBBOLE D J,et al.The genome sequence of the rice blast fungus Magnaporthe grisea[J].Nature,2005,434(7036):980-986.
[3]LIU X H,LU J P,ZHANG L,et al.Involvement of a Magnaporthe grisea serine/threonine kinase gene,Mg ATG1,in appressorium turgor and pathogenesis[J].Eukaryotic Cell,2007,6(6):997-1005.
[4]WANG J,ZHANG Z,WANG Y,et al.PTS1 peroxisomal import pathway plays shared and distinct roles to PTS2 pathway in development and pathogenicity of Magnaporthe oryzae[J].PLo S One,2013,8(2):e55554.
[5]LI L,WANG J,ZHANG Z,et al.Mo Pex19,which is essential for maintenance of peroxisomal structure and woronin bodies,is required for metabolism and development in the rice blast fungus[J].PLo S One,2014,9(1):e85252.
[6]PARK G,BRUNO K S,STAIGER C J,et al.Independent genetic mechanisms mediate turgor generation and penetration peg formation during plant infection in the rice blast fungus[J].Molecular Microbiology,2004,53(6):1695-1707.
[7]BOURETT T M,HOWARD R J.In vitro development of penetration structures in the rice blast fungus Magnaporthe grisea[J].Canadian Journal of Botany,1990,68(2):329-342.
[8]HOWARD R J,VALENT B.Breaking and entering:host penetration by the fungal rice blast pathogen Magnaporthe grisea[J].Annual Review of Microbiology,1996,50:491-512.
[9]HOWARD R J,FERRARI M A,ROACH D H,et al.Penetration of hard substrates by a fungus employing enormous turgor pressures[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(24):11281-11284.
[10]DE JONG J C,MCCORMACK B J,SMIRNOFF N,et al.Glycerol generates turgor in rice blast[J].Nature,1997,389(6648):244-244.
[11]LEE Y H,DEAN R A.Hydrophobicity of contact surface induces appressorium formation in Magnaporthe grisea[J].FEMS Microbiology Letters,1994,115(1):71-75.
[12]GILBERT R D,JOHNSON A M,DEAN R A.Chemical signals responsible for appressorium formation in the rice blast fungus Magnaporthe grisea[J].Physiological and Molecular Plant Pathology,1996,48(5):335-346.
[13]XIAO J Z,WATANABE T,KAMAKURA T,et al.Studies on cellular-differentiation of Magnaporthe grisea-physicochemical aspects of substratum surfaces in relation to appressorium formation[J].Physiological and Molecular Plant Pathology,1994,44(3):227-236.
[14]LIU S,DEAN R A.G protein alpha subunit genes control growth,development,and pathogenicity of Magnaporthe grisea[J].Molecular Plant-Microbe Interactions,1997,10(9):1075-1086.
[15]BOLKER M.Sex and crime:heterotrimeric G proteins in fungal mating and pathogenesis[J].Fungal Genetics and Biology,1998,25(3):143-156.
[16]XU J R,HAMER J E.MAP kinase and c AMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea[J].Genes&Development,1996,10(21):2696-2706.
[17]ZHAO X,KIM Y,PARK G,et al.A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea[J].Plant Cell,2005,17(4):1317-1329.
[18]LEE Y H,DEAN R A.c AMP regulates infection structure formation in the plant pathogenic fungus Magnaporthe grisea[J].Plant Cell,1993,5(6):693-700.
[19]ZHOU X,ZHAO X,XUE C,et al.Bypassing both surface attachment and surface recognition requirements for appressorium formation by overactive ras signaling in Magnaporthe oryzae[J].Molecular Plant-Microbe Interactions,2014,27(9):996-1004.
[20]BROACH J R,DESCHENES R J.The function of ras genes in Saccharomyces cerevisiae[J].Advances in Cancer Research,1990,54:79-139.
[21]MITCHELL T K,DEAN R A.The c AMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea[J].Plant Cell,1995,7(11):1869-1878.
[22]XU J R,URBAN M,SWEIGARD J A,et al.The CPKA gene of Magnaporthe grisea is essential for appressorial penetration[J].Molecular Plant-Microbe Interactions,1997,10(2):187-194.
[23]TODA T,CAMERON S,SASS P,et al.SCH9,a gene of Saccharomyces cerevisiae that encodes a protein distinct from,but functionally and structurally related to,c AMP-dependent protein kinase catalytic subunits[J].Genes&Development,1988,2(5):517-527.
[24]WARD M P,GARRETT S.Suppression of a yeast cyclic AMP-dependent protein kinase defect by overexpression of SOK1,a yeast gene exhibiting sequence similarity to a developmentally regulated mouse gene[J].Molecular&Cellular Biology,1994,14(9):5619-5627.
[25]POMBO C M,BONVENTRE J V,MOLNAR A,et al.Activation of a human Ste20-like kinase by oxidant stress defines a novel stress response pathway[J].EMBO Journal,1996,15(17):4537-4546.
[26]POMBO C M,TSUJITA T,KYRIAKIS J M,et al.Activation of the Ste20-like oxidant stress response kinase-1 during the initial stages of chemical anoxia-induced necrotic cell death.Requirement for dual inputs of oxidant stress and increased cytosolic[Ca2+][J].Journal of Biological Chemistry,1997,272(46):29372-29379.
[27]MARTINDALE J L,HOLBROOK N J.Cellular response to oxidative stress:signaling for suicide and survival[J].Journal of Cellular Physiology,2002,192(1):1-15.
[28]EGAN M J,WANG Z Y,JONES M A,et al.Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(28):11772-11777.
[29]SKAMNIOTI P,HENDERSON C,ZHANG Z,et al.A novel role for catalase B in the maintenance of fungal cell-wall integrity during host invasion in the rice blast fungus Magnaporthe grisea[J].Molecular Plant-Microbe Interactions,2007,20(5):568-580.
[30]HUANG K,CZYMMEK K J,CAPLAN J L,et al.HYR1-mediated detoxification of reactive oxygen species is required for full virulence in the rice blast fungus[J].PLo S Pathogens,2011,7(4):e1001335.
[31]HUANG K,CZYMMEK K J,CAPLAN J L,et al.Suppression of plant-generated reactive oxygen species is required for successful infection by the rice blast fungus[J].Virulence,2011,2(6):559-562.
[32]TALBOT N J,EBBOLE D J,HAMER J E.Identification and characterization of MPG1,a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea[J].Plant Cell,1993,5(11):1575-1590.
[33]李海娇,卢建平,刘小红,等.适用于稻瘟病菌基因敲除、过表达和荧光融合蛋白表达载体的构建和使用[J].农业生物技术学报,2012,20(1):94-104.LI H J,LU J P,LIU X H,et al.Vectors building and usage for gene knockout,protein expression and fluorescent fusion protein in the rice blast fungus[J].Journal of Agricultural Biotechnology,2012,20(1):94-104.(in Chinese with English abstract)
[34]WANG J,LI L,ZHANG Z,et al.One of three Pex11 family members is required for peroxisomal proliferation and full virulence of the rice blast fungus Magnaporthe oryzae[J].PLo S One,2015,10(7):e0134249.