Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor

Autores
Chalfoun, Nadia Regina; Grellet Bournonville, Carlos Froilan; Martinez Zamora, Martin Gustavo; Díaz Perales, Araceli; Castagnaro, Atilio Pedro; Diaz Ricci, Juan Carlos
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
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.
Fil: Chalfoun, Nadia Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología; . Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Grellet Bournonville, Carlos Froilan. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Martinez Zamora, Martin Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Díaz Perales, Araceli. Universidad Politécnica de Madrid. Escuela Técnica Superior de Ingenieros Agrónomos de Madrid; España; . Universidad Politécnica de Madrid. Escuela Técnica Superior de Ingenieros Agrónomos de Madrid. Departamento de Biotecnología. Unidad de Química y Bioquímica; España;
Fil: Castagnaro, Atilio Pedro. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino; Argentina
Fil: Diaz Ricci, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Materia
SUBTILISIN
SERIN-PROTEASE
ACREMONIUM STRICTUM
PROTEIN PURIFICATION
PROTEIN SEQUENCE
MASS SPECTROMETRY
cDNA SEQUENCING
ELICITOR
PLANT DEFENSE
FRAGARIA x ANANASSA
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/1681
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/1681
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitorChalfoun, Nadia ReginaGrellet Bournonville, Carlos FroilanMartinez Zamora, Martin GustavoDíaz Perales, AraceliCastagnaro, Atilio PedroDiaz Ricci, Juan CarlosSUBTILISINSERIN-PROTEASEACREMONIUM STRICTUMPROTEIN PURIFICATIONPROTEIN SEQUENCEMASS SPECTROMETRYcDNA SEQUENCINGELICITORPLANT DEFENSEFRAGARIA x ANANASSAhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1https://purl.org/becyt/ford/4.4https://purl.org/becyt/ford/4In 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.Fil: Chalfoun, Nadia Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología; . Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Grellet Bournonville, Carlos Froilan. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Martinez Zamora, Martin Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Díaz Perales, Araceli. Universidad Politécnica de Madrid. Escuela Técnica Superior de Ingenieros Agrónomos de Madrid; España; . Universidad Politécnica de Madrid. Escuela Técnica Superior de Ingenieros Agrónomos de Madrid. Departamento de Biotecnología. Unidad de Química y Bioquímica; España;Fil: Castagnaro, Atilio Pedro. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Diaz Ricci, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaAmerican Society for Biochemistry and Molecular Biology2013-03-25info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/1681Chalfoun, Nadia Regina; Grellet Bournonville, Carlos Froilan; Martinez Zamora, Martin Gustavo; Díaz Perales, Araceli; Castagnaro, Atilio Pedro; et al.; Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry; 288; 20; 25-3-2013; 14098-141130021-9258enginfo:eu-repo/semantics/altIdentifier/doi/doi:10.1074/jbc.M112.429423info:eu-repo/semantics/altIdentifier/url/http://www.jbc.org/content/288/20/14098.full.pdf+htmlinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:02:14Zoai:ri.conicet.gov.ar:11336/1681instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 10:02:15.073CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor
title Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor
spellingShingle Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor
Chalfoun, Nadia Regina
SUBTILISIN
SERIN-PROTEASE
ACREMONIUM STRICTUM
PROTEIN PURIFICATION
PROTEIN SEQUENCE
MASS SPECTROMETRY
cDNA SEQUENCING
ELICITOR
PLANT DEFENSE
FRAGARIA x ANANASSA
title_short Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor
title_full Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor
title_fullStr Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor
title_full_unstemmed Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor
title_sort Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor
dc.creator.none.fl_str_mv Chalfoun, Nadia Regina
Grellet Bournonville, Carlos Froilan
Martinez Zamora, Martin Gustavo
Díaz Perales, Araceli
Castagnaro, Atilio Pedro
Diaz Ricci, Juan Carlos
author Chalfoun, Nadia Regina
author_facet Chalfoun, Nadia Regina
Grellet Bournonville, Carlos Froilan
Martinez Zamora, Martin Gustavo
Díaz Perales, Araceli
Castagnaro, Atilio Pedro
Diaz Ricci, Juan Carlos
author_role author
author2 Grellet Bournonville, Carlos Froilan
Martinez Zamora, Martin Gustavo
Díaz Perales, Araceli
Castagnaro, Atilio Pedro
Diaz Ricci, Juan Carlos
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv SUBTILISIN
SERIN-PROTEASE
ACREMONIUM STRICTUM
PROTEIN PURIFICATION
PROTEIN SEQUENCE
MASS SPECTROMETRY
cDNA SEQUENCING
ELICITOR
PLANT DEFENSE
FRAGARIA x ANANASSA
topic SUBTILISIN
SERIN-PROTEASE
ACREMONIUM STRICTUM
PROTEIN PURIFICATION
PROTEIN SEQUENCE
MASS SPECTROMETRY
cDNA SEQUENCING
ELICITOR
PLANT DEFENSE
FRAGARIA x ANANASSA
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
https://purl.org/becyt/ford/4.4
https://purl.org/becyt/ford/4
dc.description.none.fl_txt_mv 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.
Fil: Chalfoun, Nadia Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología; . Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Grellet Bournonville, Carlos Froilan. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Martinez Zamora, Martin Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Díaz Perales, Araceli. Universidad Politécnica de Madrid. Escuela Técnica Superior de Ingenieros Agrónomos de Madrid; España; . Universidad Politécnica de Madrid. Escuela Técnica Superior de Ingenieros Agrónomos de Madrid. Departamento de Biotecnología. Unidad de Química y Bioquímica; España;
Fil: Castagnaro, Atilio Pedro. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino; Argentina
Fil: Diaz Ricci, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
description 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.
publishDate 2013
dc.date.none.fl_str_mv 2013-03-25
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/1681
Chalfoun, Nadia Regina; Grellet Bournonville, Carlos Froilan; Martinez Zamora, Martin Gustavo; Díaz Perales, Araceli; Castagnaro, Atilio Pedro; et al.; Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry; 288; 20; 25-3-2013; 14098-14113
0021-9258
url http://hdl.handle.net/11336/1681
identifier_str_mv Chalfoun, Nadia Regina; Grellet Bournonville, Carlos Froilan; Martinez Zamora, Martin Gustavo; Díaz Perales, Araceli; Castagnaro, Atilio Pedro; et al.; Purification and Characterization of AsES Protein: a subtilisin secretedby Acremonium strictum is a novel plant defense elicitor; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry; 288; 20; 25-3-2013; 14098-14113
0021-9258
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/doi:10.1074/jbc.M112.429423
info:eu-repo/semantics/altIdentifier/url/http://www.jbc.org/content/288/20/14098.full.pdf+html
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.13397

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