大丽轮枝菌VdSCH9基因的功能研究
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摘要
大丽轮枝菌是一种土传性植物病原真菌,可侵染多种植物并引发黄萎病。目前,人们关于大丽轮枝菌的侵染和致病机制的了解还很不深入。本文通过敲除大丽轮枝菌编码丝氨酸/苏氨酸的蛋白激酶基因VdSCH9,阐明了其在大丽轮枝菌生长发育及致病过程中的作用。SCH9基因在酵母中的表达与c AMP-PKA途径和TOR信号通路相关,对酵母的生长、压力响应和寿命等有重要作用。大丽轮枝菌VdSCH9敲除突变体的生长速率显著下降,菌落边缘菌丝更为稀疏,菌丝分枝减少,对棉花植株为害的平均病情指数为56.6,显著低于野生型和互补突变体的平均病情指数90.5和82.8,对茄子植株为害的平均病情指数为65.9,也显著低于野生型和互补突变体的平均病情指数91.1和89.8。另外,敲除突变体对于高渗透压、氧化还原压力、细胞膜和细胞壁完整性等压力条件的敏感性增强。因此,VdSCH9对于大丽轮枝菌的生长、压力响应及致病力均有重要作用。
Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillum wilt with a broad range of host plants. At present the molecular infection mechanisms of the pathogen are poorly understood. The authors explored the function of gene VdSCH9 encoding serine/threonine protein kinase in colony growth and pathogenicity by knocking out VdSCH9 in the fungus.The gene SCH9 is found to be involved in the c AMP-PKA and TOR pathway in the yeast. It also plays an important role in colony growth, stress response and longevity of yeast. The ΔVdSCH9 mutant was significantly reduced in growth rate, and it was also defective in aerial hyphal growth and hyphal branching. The cotton pathogenicity assay shows that the disease index of ΔVdSCH9 mutant was 56.6, significantly lesser than that of wild type and complement mutant, being 90.5 and 82.8 respectively. The similar result was acquired for the VdSCH9-deleted mutant in pathogenicity index on eggplant plants, being 65.9 for ΔVdSCH9 strain, and 91.1 and 89.8 for wild type and complement mutants. The gene-deleted mutants were more sensitive under stresses of oxidation, hyperosmotion, and cell wall and membrane integrity. These results suggest that the gene VdSCH9 is evidently involved in colony growth, stress response and pathogenesis in V. dahliae.
Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillum wilt with a broad range of host plants. At present the molecular infection mechanisms of the pathogen are poorly understood. The authors explored the function of gene VdSCH9 encoding serine/threonine protein kinase in colony growth and pathogenicity by knocking out VdSCH9 in the fungus.The gene SCH9 is found to be involved in the c AMP-PKA and TOR pathway in the yeast. It also plays an important role in colony growth, stress response and longevity of yeast. The ΔVdSCH9 mutant was significantly reduced in growth rate, and it was also defective in aerial hyphal growth and hyphal branching. The cotton pathogenicity assay shows that the disease index of ΔVdSCH9 mutant was 56.6, significantly lesser than that of wild type and complement mutant, being 90.5 and 82.8 respectively. The similar result was acquired for the VdSCH9-deleted mutant in pathogenicity index on eggplant plants, being 65.9 for ΔVdSCH9 strain, and 91.1 and 89.8 for wild type and complement mutants. The gene-deleted mutants were more sensitive under stresses of oxidation, hyperosmotion, and cell wall and membrane integrity. These results suggest that the gene VdSCH9 is evidently involved in colony growth, stress response and pathogenesis in V. dahliae.
引文
Buchner V,Burstein Y,Nachmias A,1989.Comparison of Verticillium dahliae-produced phytotoxic peptides purified from culture fluids and infected potato stems.Physiological&Molecular Plant Pathology,35(3):253-269
Carder JH,Hignett RC,Swinburne TR,1987.Relationship between the virulence of hop isolates of Verticillium albo-atrum and their in vitro secretion of cell-wall degrading enzymes.Physiological&Molecular Plant Pathology,31(3):441-452
Chen D,Wang Y,Zhou X,Xu JR,2014.The Sch9 kinase regulates conidium size,stress responses,and pathogenesis in Fusarium graminearum.PLo S One,9(8):e105811
Cooper RM,Wood RKS,1980.Cell wall degrading enzymes of vascular wilt fungi.III.Possible involvement of endo-pectin lyase in Verticillium wilt of tomato.Physiological Plant Pathology,16(2):285
Fabrizio P,Pozza F,Pletcher SD,Gendron CM,Longo VD,2001.Regulation of longevity and stress resistance by Sch9 in yeast.Science,292(5515):288-290
Gu Q,Zhang C,Yu F,Yin Y,Shim W,Ma Z,2015.Protein kinase fgsch9 serves as a mediator of the target of rapamycin and high osmolarity glycerol pathways and regulates multiple stress responses and secondary metabolism in Fusarium graminearum.Environmental Microbiology,17(8):2661
Heinz R,Lee SW,Saparno A,Nazar RN,Robb J,1998.Cyclical systemic colonization in Verticillium-infected tomato.Physiological&Molecular Plant Pathology,52(6):385-396
Huber A,Bodenmiller B,Uotila A,Stahl M,Wanka S,Gerrits B,Aebersold R,Loewith R,2009.Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9is a central coordinator of protein synthesis.Genes&Development,23(16):1929-1943
Isaac I,2003.Speciation in Verticillium.Annual Review of Phytopathology,5(1):201-222
Kim JE,Lee HJ,Lee J,Kim KW,Yun SH,2009.Gibberella zeae chitin synthase genes,Gz CHS5 and Gz CHS7,are required for hyphal growth,perithecia formation,and pathogenicity.Current Genetics,55(4):449-459
Klosterman SJ,Atallah ZK,Vallad GE,Subbarao KV,2009.Diversity,pathogenicity,and management of Verticillium species.Annual Review of Phytopathology,47(1):39-62
Liu SY,Chen JY,Wang JL,Li L,Xiao HL,Adam SM,Dai XF,2013.Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae.Gene,529(2):307-316
Liu W,Zhao J,Li X,Li Y,Jiang L,2010.The protein kinase Ca Sch9p is required for the cell growth,filamentation and virulence in the human fungal pathogen Candida albicans.FEMS Yeast Research,10(4):462-470
Liu XT,Song XX,Guo JC,1998.Studies and advances on cotton Verticillium wilt.Acta Gossypii Sinica,10(1):6-13(in Chinese)
Luo X,Xie C,Dong J,Yang X,Sui A,2014.Interactions between Verticillium dahliae and its host:vegetative growth,pathogenicity,plant immunity.Applied Microbiology&Biotechnology,98(16):6921-6932
Maruthachalam K,Klosterman SJ,Kang S,Hayes RJ,Subbarao KV,2011.Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis.Molecular Biotechnology,49(3):209-221
Mol L,Scholte K,Vos J,2010.Effects of crop rotation and removal of crop debris on the soil population of two isolates of Verticillium dahliae.Plant Pathology,44(6):1070-1074
Mullins ED,Chen X,Romaine P,Raina R,Geiser DM,Kang S,2001.Agrobacterium-mediated transformation of Fusarium oxysporum:an efficient tool for insertional mutagenesis and gene transfer.Phytopathology,91(2):173-180
Pascual Ahuir A,Proft M,2007.The Sch9 kinase is a chromatin-associated transcriptional activator of osmostress responsive genes.Embo Journal,26(13):3098-3108
Paz Z,García-Pedrajas MD,Andrews DL,Klosterman SJ,Baeza-Monta?ez L,Gold SE,2011.One step construction of Agrobacterium-recombination-ready-plasmids(OSCAR),an efficient and robust tool for ATMT based gene deletion construction in fungi.Fungal Genetics&Biology,48(7):677-684
Pegg GF,1984.The impact of Verticillium disease in agriculture.Phytopathologia Mediterranea,23(2/3):176-192
Powers T,2007.TOR signaling and S6 kinase 1:yeast catches up.Cell Metab,6(1):1-2
Proctor RH,Hohn TM,Mccormick SP,1995.Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene.Molecular Plant-Microbe Interactions,8(4):593-601
Rolke Y,Liu S,Quidde T,Williamson B,Schouten A,2004.Functional analysis of H2O2-generating systems in Botrytis cinerea:the major Cu-Zn-superoxide dismutase(BCSOD1)contributes to virulence on French bean,whereas a glucose oxidase(BCGOD1)is dispensable.Molecular Plant Pathology,5(1):17-27
Roosen J,Engelen K,Marchal K,Mathys J,Griffioen G,2005.PKA and Sch9 control a molecular switch important for the proper adaptation to nutrient availability.Molecular Microbiology,55(3):862-880
Schneider E,Ghillebert R,Wilms T,Winderickx J,2013.Molecular mechanisms linking the evolutionary conserved TORC1-Sch9 nutrient signalling branch to lifespan regulation in Saccharomyces cerevisiae.FEMSYeast Research,14(1):17-32
Shang WJ,Bai YW,Chen T,Yang JR,Hu XP,2013.Germination condition and lethal temperature for microsclerotia of Verticillium dahliae.Mycosystema,32(6):986-992(in Chinese)
Tzima A,Paplomatas EJ,Rauyaree P,Kang SC,2010.Roles of the catalytic subunit of c AMP-dependent protein kinase A in virulence and development of the soilborne plant pathogen Verticillium dahliae.Fungal Genetics&Biology,47(5):406-415
Urban J,Soulard,Alexandre H,2007.Sch9 is a major target of TORC1 in Saccharomyces cerevisiae.Molecular Cell,26(5):663
Wang YL,T ian L,Xiong D,Klosterman SJ,Xiao S,T ian C,2016.The mitogen-activated protein kinase gene,Vd Hog1,regulates osmotic stress response,microsclerotia formation and virulence in Verticillium dahliae.Fungal Genetics&Biology,88(3):13-23
Wullschleger S,Loewith R,Hall MN,2006.TOR signaling in growth and metabolism.Cell,124(3):471
Yorimitsu T,Zaman S,Broach JR,Klionsky DJ,2007.Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae.Molecular Biology of the Cell,18(10):4180
Zhang A,Shen Y,Gao W,Dong J,2011.Role of Sch9 in regulating Ras-c AMP signal pathway in Saccharomyces cerevisiae.FEBS Letters,585(19):3026-3032
刘学堂,宋晓轩,郭金城,1998.棉花黄萎病菌的研究及最新进展.棉花学报,10(1):6-13
商文静,陈婷,白应文,杨家荣,胡小平,2013.大丽轮枝菌微菌核的萌发条件及致死温度.菌物学报,32(6):986-992
Carder JH,Hignett RC,Swinburne TR,1987.Relationship between the virulence of hop isolates of Verticillium albo-atrum and their in vitro secretion of cell-wall degrading enzymes.Physiological&Molecular Plant Pathology,31(3):441-452
Chen D,Wang Y,Zhou X,Xu JR,2014.The Sch9 kinase regulates conidium size,stress responses,and pathogenesis in Fusarium graminearum.PLo S One,9(8):e105811
Cooper RM,Wood RKS,1980.Cell wall degrading enzymes of vascular wilt fungi.III.Possible involvement of endo-pectin lyase in Verticillium wilt of tomato.Physiological Plant Pathology,16(2):285
Fabrizio P,Pozza F,Pletcher SD,Gendron CM,Longo VD,2001.Regulation of longevity and stress resistance by Sch9 in yeast.Science,292(5515):288-290
Gu Q,Zhang C,Yu F,Yin Y,Shim W,Ma Z,2015.Protein kinase fgsch9 serves as a mediator of the target of rapamycin and high osmolarity glycerol pathways and regulates multiple stress responses and secondary metabolism in Fusarium graminearum.Environmental Microbiology,17(8):2661
Heinz R,Lee SW,Saparno A,Nazar RN,Robb J,1998.Cyclical systemic colonization in Verticillium-infected tomato.Physiological&Molecular Plant Pathology,52(6):385-396
Huber A,Bodenmiller B,Uotila A,Stahl M,Wanka S,Gerrits B,Aebersold R,Loewith R,2009.Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9is a central coordinator of protein synthesis.Genes&Development,23(16):1929-1943
Isaac I,2003.Speciation in Verticillium.Annual Review of Phytopathology,5(1):201-222
Kim JE,Lee HJ,Lee J,Kim KW,Yun SH,2009.Gibberella zeae chitin synthase genes,Gz CHS5 and Gz CHS7,are required for hyphal growth,perithecia formation,and pathogenicity.Current Genetics,55(4):449-459
Klosterman SJ,Atallah ZK,Vallad GE,Subbarao KV,2009.Diversity,pathogenicity,and management of Verticillium species.Annual Review of Phytopathology,47(1):39-62
Liu SY,Chen JY,Wang JL,Li L,Xiao HL,Adam SM,Dai XF,2013.Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae.Gene,529(2):307-316
Liu W,Zhao J,Li X,Li Y,Jiang L,2010.The protein kinase Ca Sch9p is required for the cell growth,filamentation and virulence in the human fungal pathogen Candida albicans.FEMS Yeast Research,10(4):462-470
Liu XT,Song XX,Guo JC,1998.Studies and advances on cotton Verticillium wilt.Acta Gossypii Sinica,10(1):6-13(in Chinese)
Luo X,Xie C,Dong J,Yang X,Sui A,2014.Interactions between Verticillium dahliae and its host:vegetative growth,pathogenicity,plant immunity.Applied Microbiology&Biotechnology,98(16):6921-6932
Maruthachalam K,Klosterman SJ,Kang S,Hayes RJ,Subbarao KV,2011.Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis.Molecular Biotechnology,49(3):209-221
Mol L,Scholte K,Vos J,2010.Effects of crop rotation and removal of crop debris on the soil population of two isolates of Verticillium dahliae.Plant Pathology,44(6):1070-1074
Mullins ED,Chen X,Romaine P,Raina R,Geiser DM,Kang S,2001.Agrobacterium-mediated transformation of Fusarium oxysporum:an efficient tool for insertional mutagenesis and gene transfer.Phytopathology,91(2):173-180
Pascual Ahuir A,Proft M,2007.The Sch9 kinase is a chromatin-associated transcriptional activator of osmostress responsive genes.Embo Journal,26(13):3098-3108
Paz Z,García-Pedrajas MD,Andrews DL,Klosterman SJ,Baeza-Monta?ez L,Gold SE,2011.One step construction of Agrobacterium-recombination-ready-plasmids(OSCAR),an efficient and robust tool for ATMT based gene deletion construction in fungi.Fungal Genetics&Biology,48(7):677-684
Pegg GF,1984.The impact of Verticillium disease in agriculture.Phytopathologia Mediterranea,23(2/3):176-192
Powers T,2007.TOR signaling and S6 kinase 1:yeast catches up.Cell Metab,6(1):1-2
Proctor RH,Hohn TM,Mccormick SP,1995.Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene.Molecular Plant-Microbe Interactions,8(4):593-601
Rolke Y,Liu S,Quidde T,Williamson B,Schouten A,2004.Functional analysis of H2O2-generating systems in Botrytis cinerea:the major Cu-Zn-superoxide dismutase(BCSOD1)contributes to virulence on French bean,whereas a glucose oxidase(BCGOD1)is dispensable.Molecular Plant Pathology,5(1):17-27
Roosen J,Engelen K,Marchal K,Mathys J,Griffioen G,2005.PKA and Sch9 control a molecular switch important for the proper adaptation to nutrient availability.Molecular Microbiology,55(3):862-880
Schneider E,Ghillebert R,Wilms T,Winderickx J,2013.Molecular mechanisms linking the evolutionary conserved TORC1-Sch9 nutrient signalling branch to lifespan regulation in Saccharomyces cerevisiae.FEMSYeast Research,14(1):17-32
Shang WJ,Bai YW,Chen T,Yang JR,Hu XP,2013.Germination condition and lethal temperature for microsclerotia of Verticillium dahliae.Mycosystema,32(6):986-992(in Chinese)
Tzima A,Paplomatas EJ,Rauyaree P,Kang SC,2010.Roles of the catalytic subunit of c AMP-dependent protein kinase A in virulence and development of the soilborne plant pathogen Verticillium dahliae.Fungal Genetics&Biology,47(5):406-415
Urban J,Soulard,Alexandre H,2007.Sch9 is a major target of TORC1 in Saccharomyces cerevisiae.Molecular Cell,26(5):663
Wang YL,T ian L,Xiong D,Klosterman SJ,Xiao S,T ian C,2016.The mitogen-activated protein kinase gene,Vd Hog1,regulates osmotic stress response,microsclerotia formation and virulence in Verticillium dahliae.Fungal Genetics&Biology,88(3):13-23
Wullschleger S,Loewith R,Hall MN,2006.TOR signaling in growth and metabolism.Cell,124(3):471
Yorimitsu T,Zaman S,Broach JR,Klionsky DJ,2007.Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae.Molecular Biology of the Cell,18(10):4180
Zhang A,Shen Y,Gao W,Dong J,2011.Role of Sch9 in regulating Ras-c AMP signal pathway in Saccharomyces cerevisiae.FEBS Letters,585(19):3026-3032
刘学堂,宋晓轩,郭金城,1998.棉花黄萎病菌的研究及最新进展.棉花学报,10(1):6-13
商文静,陈婷,白应文,杨家荣,胡小平,2013.大丽轮枝菌微菌核的萌发条件及致死温度.菌物学报,32(6):986-992