Figure 1.
The oxalic acid (OA) deficient mutant strain (A2) induces an HR-like response.
Agar plugs containing actively growing cultures of Sclerotinia sclerotiorum were inoculated onto tomato leaves. Wild-type (A) was pathogenic and induced runaway cell death; however, growth of the A2 strain (B) was restricted by the plant. Pictures were taken 48 hours post-inoculation. Markers associated with the HR were observed specifically following mutant, but not wild type inoculations. Using DAB staining, the oxidative burst was visible in response to the A2 strain (D), but not the wild-type (C) 8 hours post-inoculation. Unlike the wild-type (E), the A2 strain also induced callose deposition (F). Callose deposition was evaluated after 2 days. Both callose deposition and DAB staining are shown at the leading edge of the lesions.
Figure 2.
Wild-type Sclerotinia, but not the OA-deficient (A2) mutant, reduces the host cellular environment.
Transgenic Nicotiana benthamiana leaves containing the redox sensitive GFP (roGFP) cassette were investigated by confocal microscopy using (A) 410 nm and (B) 474 nm filters for observation of roGFP under oxidizing and reducing conditions, respectively. Two lines were chosen (line 2 shown here) for further analysis and inoculated with agar plugs containing actively growing (C) Wild-type Sclerotinia, (D) oxalate deficient A2 mutant, (E) nox1 mutant, (F) nox2 mutant, (G) sod mutant, and (H) wild-type Botrytis cinerea. Eight hours post-inoculation leaves were visualized under a 474 nm filter for observation of the reduced form of the roGFP.
Figure 3.
Oxalic acid modulates the host redox environment.
Transgenic redox sensitive GFP Nicotiana benthamiana leaves from line 2 were infiltrated by a needle-less syringe with (A) 10 mM KOA pH 7, (B) 10 mM KOA pH 3, (C) 10 mM HCl, or (D) 10 mM DTT. Four hours post-infiltration, leaves were visualized under a 474 nm filter for observation of the reduced form of the roGFP.
Figure 4.
Artificial induction of reducing conditions reverts the A2 phenotype.
Tomato leaves were pre-infiltrated with H20 (left), and either 10 mM KOA pH 7 (right) or 10 mM DTT (middle). The H2O, KOA, and DTT infiltrated regions were inoculated with agar plugs containing actively growing OA-deficient A2 strain (top panel). Additionally, trypan blue was used to stain fungal mycelia within the infiltrated area (bottom panel). The circles represent a close up of the stained area showing trypan blue stained mycelia.
Figure 5.
Oxalic acid induces reducing conditions that correlates with the dampening of the host oxidative burst.
Detached leaves of roGFP plants were infiltrated with either H2O or 10 mM KOA buffered to pH 7, and were examined for GFP fluorescence (Top two panels) over a time-course (0, 3, 6, and 12 hours). The same leaves were also stained for superoxide production (Bottom two panels) with nitro-blue tetrazolium (NBT).