title: The Adh1 gene of the fungus Metarhizium anisopliae is expressed during insect colonization and required for full virulence.
abstract: Zymography of alcohol dehydrogenase (ADH) activity in the entomopathogenic fungus Metarhizium anisopliae grown under various conditions revealed that micro-aerobic growth was associated with increased ADH activity. The major ADH protein, AdhIp, was purified to homogeneity by affinity chromatography and has an estimated molecular weight of 41kDa and an isoelectric point (pI) of 6.4. Peptide mass fingerprint analysis allowed the identification and cloning of the gene that encodes this protein, Adh1, as annotated in the M. anisopliae genome database. AdhIp is related to the medium-chain dehydrogenase/reductase (MDR)/zinc-dependent alcohol dehydrogenase-like family and contains conserved ADH sequence motifs, such as the zinc-containing ADH signature, the FAD/NAD binding domain and amino acid residues that are conserved in most microbial ADHs. Semi-quantitative RT-PCR analysis revealed that Adh1 gene expression occurs at low levels during early Plutella xylostella infection and that the Adh1 gene was primarily expressed at larval death and as mycelia emerge from the insect cuticle before conidiation. Antisense-RNA experiments indicated that NAD(+)-dependent ADH activity was diminished by 20-75% in the transformants, and the transformants that had lower ADH activity showed allyl alcohol resistance, which indicates that reduction in ADH activity also occurs in vivo. Bioassays performed using antisense adh1 transformants, which have lower ADH activity, showed that LC50 values were two to five times higher than the wild-type, indicating that AdhIp is required for full capability of the fungus to penetrate and/or colonize the insect.
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Campus Guanajuato Noria Alta s/n, Colonia Noria Alta Guanajuato, Gto, CP 36050, Mexico(1). Electronic address: oacn72@hotmail.com.
authors.forename: Olga Alicia
authors.initials: OA
authors.lastname: Callejas-Negrete
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Campus Guanajuato Noria Alta s/n, Colonia Noria Alta Guanajuato, Gto, CP 36050, Mexico(1). Electronic address: torguz@ugto.mx.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Torres-Guzmán
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Campus Guanajuato Noria Alta s/n, Colonia Noria Alta Guanajuato, Gto, CP 36050, Mexico(1). Electronic address: isen28@hotmail.com.
authors.forename: Israel Enrique
authors.initials: IE
authors.lastname: Padilla-Guerrero
authors.affiliation: Present address: Facultad de Ciencias Naturales, Universidad Autónoma de Queretaro Centro Universitario, Cerro de las Campanas s/n, Santiago de Queretaro, Qro, CP 76010, Mexico(2). Electronic address: ulises_esquivel200@yahoo.com.mx.
authors.forename: Ulises
authors.initials: U
authors.lastname: Esquivel-Naranjo
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Campus Guanajuato Noria Alta s/n, Colonia Noria Alta Guanajuato, Gto, CP 36050, Mexico(1). Electronic address: marifa_s@hotmail.com.
authors.forename: Maria Fernanda
authors.initials: MF
authors.lastname: Padilla-Ballesteros
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Campus Guanajuato Noria Alta s/n, Colonia Noria Alta Guanajuato, Gto, CP 36050, Mexico(1). Electronic address: adriana_gt@ugto.mx.
authors.forename: Adriana
authors.initials: A
authors.lastname: García-Tapia
authors.affiliation: Departamento de Biología Molecular e Biotecnología, Centro de Biotecnología, Universidade Federal do Rio Grande do Sul, Campus do Vale Av. Bento Gonçalves 9500, Agronomía, 91501970, Porto Alegre, Brazil(3). Electronic address: aschrank@cbiot.ufrgs.br.
authors.forename: Augusto
authors.initials: A
authors.lastname: Schrank
authors.affiliation: Departamento de Agronomía, División de Ciencias de la Vida, Universidad de Guanajuato, Campus Irapuato-Salamanca, Carr. Irapuato-León Km. 9, Irapuato, Gto, CP 36820, Mexico(4). Electronic address: salazare@ugto.mx.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Salazar-Solís
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Campus Guanajuato Noria Alta s/n, Colonia Noria Alta Guanajuato, Gto, CP 36050, Mexico(1). Electronic address: felixg@ugto.mx.
authors.forename: Félix
authors.initials: F
authors.lastname: Gutiérrez-Corona
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Campus Guanajuato Noria Alta s/n, Colonia Noria Alta Guanajuato, Gto, CP 36050, Mexico(1). Electronic address: gonzang@ugto.mx.
authors.forename: Gloria Angélica
authors.initials: GA
authors.lastname: González-Hernández
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D003001:Cloning, Molecular; D020869:Gene Expression Profiling; D020868:Gene Silencing; D007526:Isoelectric Point; D007814:Larva / Q000382:microbiology / Q000502:physiology; D007915:Lepidoptera / Q000382:microbiology* / Q000502:physiology; D052981:Metarhizium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D008970:Molecular Weight; D060888:Real-Time Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D016019:Survival Analysis; D014774:Virulence
chemical_list: D000426:Alcohol Dehydrogenase
keywords: Alcohol dehydrogenase; Entomopathogenic fungus; Metarhizium; Metarhizium anisopliae; Pest control