FIGURE 5.

Production of superoxide in response to extracellular ATP (eATP). (A) Detection of superoxide. WT, Δnox1, Δnox2, and ΔnoxR strains were incubated with ATP (0.1 mM), followed by incubation in a 0.3 mM NBT solution and examined by bright-field microscopy (BF). The blue/purple coloration indicates the production of superoxide (formazan generation). The scale bar = 10 μm. (B) eATP-induced conidiation. The WT strain was treated with ATP (0.1 mM), or a combination of NAC (60 mM) and ATP, or NAC and injured with a scalpel. The Δnox1, Δnox2, and ΔnoxR mutants were induced with ATP (0.1 mM). (C) The Δnox1 mutant was injured and mycelial samples collected at the indicated times. Proteins were extracted, separated by SDS-PAGE, and used for immunoblotting with anti-Tmk1 (anti-p42/p44), Tmk1-P (anti-Phospho-p42/p44), anti-Tmk3 (antip38) and Tmk3-P (anti-Phospho-p38) antibodies. Mycelium from an undamaged colony was included as control (C). Arrows indicate the bands corresponding to Tmk1 or Tmk3. The experiments were repeated two times with similar results.