Fungal strains, growth conditions and preparation of culture filtrates
The wild type Trichoderma atroviride strain P1 [45] and its ech42 gene (encoding CHIT42 endochitinase) disruption mutant [10] were maintained at 25°C on potato dextrose agar (PDA) and as spore suspension in 10% glycerol at -40°C. The Botrytis cinerea strain 319 was isolated from tobacco, grown at 25°C on malt extract agar and kept as a spore suspension in 10% glycerol at -40°C. Fungal starter cultures were obtained in potato dextrose broth at 25°C, 150 rpm for 3 days with light, collected by centrifugation, rinsed with sterile distilled water, and used to inoculate a salt medium [46] containing 0.1 % (w/v) sucrose and 0.1 % (w/v) peptone. The cultures were grown at 25°C, 150 rpm, with light, for 3 days. Culture filtrates and the substrate alone, used as a control, were filter sterilized (0.22 μm), concentrated by roto-evaporation approximately 20-fold and fractionated with YM-3 MW (3000 Da cut-off) (Amicon Centriprep, Millipore) at 4000 rpm 6°C. The samples used as metabolite mixtures were: the whole concentrated filtrate, the fraction >3000 Da and that Trichoderma, Δech42 mutant, Botrytis), plus the extracts of P1, or the Δech42 mutant, grown in the presence of Botrytis. For plant cell treatments, fungal culture filtrates were lyophilized and resuspended in plant cell culture medium. The final dose applied to cells corresponded to 4-fold concentrated fungal medium.
Plant cell cultures
Cell suspension cultures of soybean (Glycine max L., line 6.6.12) stably expressing cytosolic aequorin were maintained as described by [34]. Cell treatments with fungal culture filtrates were performed two weeks after reinoculation, during the exponential growth phase of the cells.
Aequorin-dependent Ca2+ measurements
In vivo reconstitution of aequorin and Ca2+ measurements were carried out as previously described [18].
Intracellular ROS detection
Intracellular production of reactive oxygen species (ROS) was measured according to [12], by loading the cells with 15 μM 2',7'- dichlorodihydrofluorescein diacetate (H2DCF-DA, Molecular Probes, Leiden, The Netherlands). This non polar compound is actively taken up by cells and converted by esterases in H2DCF, a non fluorescent molecule, which is rapidly oxidized to the highly fluorescent DCF by intracellular peroxides. Treatments with fungal culture filtrates were carried out 10 min after dye loading and extensive washing. DCF was excited at 488 nm and emitted fluorescence was detected through a 520 bandpass filter. Cells were observed within 10 min.
Cell viability
Cell viability was determined, after 30 min treatment with the fungal culture filtrates, by the Evans Blue method [47].
Caspase 3-like activity
Caspase 3-like activity was measured, after 30 min cell treatment, using the "caspase-3 colorimetric activity assay kit" (Chemicon International, Inc., Temecula, CA), as previously described [26] by quantification of free p-nitroaniline (pNA) released by the enzymatic cleavage of the caspase 3 synthetic substrate Ac-DEVD-pNA.
Hoechst 33342 (HO) and Propidium Iodide (PI) staining
After 30 min treatment with the different fungal culture filtrates, cells were incubated for 10 min in darkness with 8 μg/ml HO and 5 μg/ml PI (Sigma-Aldrich, St. Louis, USA) at room temperature. Cells were observed using a fluorescence microscope with an excitation light of 350 nm and 570 nm for HO and PI, respectively.
Transmission electron microscopy
Cells were collected after 15 min treatment and processed as previously described [48].
Statistical analysis
Data were expressed as mean ± S.D. The statistical significance of differences (P t test.
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