title: Purification and characterization of AsES protein: a subtilisin secreted by Acremonium strictum is a novel plant defense elicitor.
abstract: In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2(˙)) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.
authors.affiliation: Instituto Superior de Investigaciones Biológicas, Consejo Nacional de Investigaciones Científicas y Técnicas, and Instituto de Química Biológica "Dr. Bernabé Bloj," Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, T4000ILJ San Miguel de Tucumán, Argentina.
authors.forename: Nadia R
authors.initials: NR
authors.lastname: Chalfoun
authors.affiliation: Instituto Superior de Investigaciones Biológicas, Consejo Nacional de Investigaciones Científicas y Técnicas, and Instituto de Química Biológica "Dr. Bernabé Bloj," Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, T4000ILJ San Miguel de Tucumán, Argentina.
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Grellet-Bournonville
authors.affiliation: Instituto Superior de Investigaciones Biológicas, Consejo Nacional de Investigaciones Científicas y Técnicas, and Instituto de Química Biológica "Dr. Bernabé Bloj," Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, T4000ILJ San Miguel de Tucumán, Argentina.
authors.forename: Martín G
authors.initials: MG
authors.lastname: Martínez-Zamora
authors.affiliation: Unidad de Química y Bioquímica, Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28040 Madrid, Spain.
authors.forename: Araceli
authors.initials: A
authors.lastname: Díaz-Perales
authors.affiliation: Sección Biotecnología, Estación Experimental Agroindustrial Obispo Colombres, T4101XAC Las Talitas, Tucumán, Argentina.
authors.forename: Atilio P
authors.initials: AP
authors.lastname: Castagnaro
authors.affiliation: Instituto Superior de Investigaciones Biológicas, Consejo Nacional de Investigaciones Científicas y Técnicas, and Instituto de Química Biológica "Dr. Bernabé Bloj," Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, T4000ILJ San Miguel de Tucumán, Argentina. Electronic address: juan@fbqf.unt.edu.ar.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Díaz-Ricci
mesh_terms: D000164:Acremonium / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism; D001483:Base Sequence; D001681:Biological Assay; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D060467:Disease Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D031985:Fragaria / Q000276:immunology / Q000382:microbiology*; D005656:Fungal Proteins / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology*; D057865:Plant Immunity; D017382:Reactive Oxygen Species; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020860:Subtilisin / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism; D014357:Trypsin; D014462:Ultrafiltration
chemical_list: D018076:DNA, Complementary; D005656:Fungal Proteins; D017382:Reactive Oxygen Species; D013381:Subtilisins; D014357:Trypsin; D020860:Subtilisin
keywords: Acremonium strictum; Elicitor; Fragaria x ananassa; Mass Spectrometry (MS); Plant Defense; Protein Purification; Protein Sequence; Serine Protease; Subtilisin; cDNA Sequencing