Characterization and cDna Sequencing of a Novel Fungal Subtilisin with Defense liciting. Activity

CHALFOUN, NADIA REGINA; GRELLET BOURNONVILLE, CARLOS F.; MARTINEZ ZAMORA, GUSTAVO; CASTAGNARO, ATILIO P.; DÍAZ RICCI, JUAN C.

Potrero de los Funes. San Luis

Congreso; 47º Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB).; 2011

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB).

One of the possible disease biocontrol strategies in crops consists in activating the plant innate immunity by using defense elicitor molecules. In this work the purification and characterization of an extracellular elicitor protein produced by the pathogen Acremonium strictum is reported. Culture filtrate was fractioned by ultrafiltration (cut-off 30 kDa), followed by anionic exchange (Q sepharose, pH 7.5) and hydrophobic interaction (Phenyl Superose) chromatographies. 2D-SDS/PAGE of the active fraction revealed a single spot of 34 kDa and pI 8.8. Phytopathogenicity tests confirmed that the purified protein induced total protection against anthracnose in strawberry plants. Based on the use of degenerate primers designed from the partial aminoacid sequences and RACE technology, the complete nucleotide sequence of the elicitor-encoding cDNA of 1167 nucleotides was obtained. The deduced aminoacid sequence showed significant identity with fungal serine proteinases of the subtilisin family, suggesting that elicitor is synthesized as a larger precursor that contains in addition to the 283-residue of mature protein, a 15-residue secretory signal peptide, and a 90-residue peptidase inhibitor domain I9. The elicitor exhibited proteolytic activity in vitro and was inhibited by PMSF but not by TPCK. This is the first report of a fungal subtilisin that shows defense-eliciting activity in plants.