INVESTIGADORES
DIAZ RICCI Juan Carlos
artículos
Título:
Purification and Characterization of AsES Protein. A Subtilisin Secreted by Acremonium strictum is a Novel Plant Defense Elicitor
Autor/es:
CHALFOUN, NADIA REGINA; GRELLET BOURNONVILLE, CARLOS F.; MARTÍNEZ ZAMORA, MARTÍN G.; DÍAZ PERALES, ARACELI; CASTAGANARO, ATILIO P; DÍAZ RICCI, JUAN C.
Revista:
JOURNAL OF BIOLOGICAL CHEMISTRY
Editorial:
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Referencias:
Lugar: Bethesda, Maryland; Año: 2013 vol. 288 p. 14098 - 14113
ISSN:
0021-9258
Resumen:
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, is reported. The defense-eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cut-off 30 kDa), anionic exchange (Q sepharose, pH 7.5) and hydrophobic interaction (Phenyl sepharose) chromatographies. 2D-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 with the expression of defense-related genes (i.e. PR1, Chi2-1). Accumulation of ROS (e.g. H2O2, O2.-) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial aminoacid sequences and RACE reactions, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced aminoacid 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 of 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 defense-eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants, by activating its innate immunity.