Recombinant Snakin-1 (SN1r) antifungal activity in vitro involves membrane permeabilization

MUÑOZ FERNANDO; ALMASIA, NATALIA INÉS; PAGANO M; DALEO G; VAZQUEZ-ROVERE, CECILIA; GUEVARA MARÍA G.

Congreso; XLIV REUNIÓN ANUAL DE LA SAIB; 2008

Snakin1 (SN1) is a cysteine-rich peptide isolated from potato tubers with a MW of 6,922 kDa. Previous reports show that SN1 has antimicrobial activity on fungal and bacterial plant pathogens. In order to analyze in depth the antimicrobial mechanism of action, we have successfully subcloned the SN1 cDNA into pQE70 vector, expressed into M15 cells, induced with 1mM IPTG after 2 h. and purified in Ni-agarose column. Recombinant SN1 (StSN1r) was able to inhibit germination of F. solani spores and growth of hyphae at the same concentrations reported for SN1wt in vitro. When F. solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of at the same concentrations reported for SN1wt in vitro. When F. solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of able to inhibit germination of F. solani spores and growth of hyphae at the same concentrations reported for SN1wt in vitro. When F. solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of at the same concentrations reported for SN1wt in vitro. When F. solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of StSN1r) was able to inhibit germination of F. solani spores and growth of hyphae at the same concentrations reported for SN1wt in vitro. When F. solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of at the same concentrations reported for SN1wt in vitro. When F. solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of F. solani spores and growth of hyphae at the same concentrations reported for SN1wt in vitro. When F. solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of wt in vitro. When F. solani hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of hyphae and spores were incubated with different concentrations of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of StSN1r, membrane permeabilization of these structures was observed, as shown by the uptake of the fluorescent dye SYTOX Green. These results show that, as reported for other plants antimicrobial cysteine-rich peptides, cytotoxic activity of StSN1 involves plasma membrane permeabilization.SN1 involves plasma membrane permeabilization.