PMC

HR-like reaction of sitiens epidermal cells at 12 hpi. Cells near the site of fungal penetration with this reaction showed intracellular DAB accumulation (A), cytoplasmic aggregation (B), and green to yellow autofluorescence after UV excitation (C). Scale bar = 50 μm. [See online article for color version of this figure.]

Extracellular peroxidase activity in wild-type and sitiens leaf discs infected with B. cinerea. Leaf discs were inoculated with two 5-μL droplets of B. cinerea (infected) or with the control (mock) solution and fixed in ethanol at the different time points. Peroxidase activity was measured at 654 nm after addition of TMB and 0.03% H₂O₂. The mean and se of the absorbance of the incubation solution from three discs of different plants are presented.

Effect of ascorbate and catalase treatment on epidermal cell wall modification in sitiens tomato inoculated with B. cinerea. The sitiens leaf discs that were floated in a solution of ascorbate or catalase and infected with B. cinerea were fixed in ethanol at 16 hpi and stained with safranin-O. Ascorbate treatment completely removed the cell wall staining, whereas catalase treatment resulted in fainter accumulation of the stain compared to the control treatment. For each treatment, at least three discs were examined and gave the same pattern of cell wall modification. Scale bar = 50 μm.

Temporal evolution of H₂O₂ accumulation in wild-type and sitiens tomato after infection with B. cinerea. DAB staining of leaf discs infected with two 5-μL drops of a conidial suspension was performed at different time points post inoculation (4, 8, 12, 16, 20, 24, 48, and 72 hpi). One representative disc out of three replicates is shown for each time point. [See online article for color version of this figure.]

Disease symptoms on wild-type and sitiens tomato leaf discs after infection with two 5-μL drops of a B. cinerea conidial suspension. A, Macroscopic disease symptoms. In wild-type leaf discs, spreading gray disintegration of the host tissue is visible and in sitiens discs necrotic spots at the site of the infection droplet are observed. B, Close up of macroscopic disease symptoms. In sitiens, primary necrotic lesions contain a high number of distinct dark spots at 48 and 72 hpi. In wild type, primary necrotic spots at 48 hpi are typically larger, less numerous, have a paler shade of brown, and more often develop into spreading water-soaked lesions.

Schematic model of the location and sequence of fungal growth, H₂O₂ accumulation, and cell wall modification in wild-type and sitiens leaf tissue infected with B. cinerea. Wild-type tissue displays only very few zones with H₂O₂ accumulation or cell wall modification until 24 hpi. Between 24 and 48 hpi, strong H₂O₂ accumulation appears mainly in the mesophyll cell layer and is associated with cell death and fungal spreading. In sitiens, H₂O₂ accumulation and modifications coincide in a first phase (around 8 hpi) mainly in the anticlinal walls of epidermal cells. At later time points (24 and 48 hpi), fungal growth inside sitiens is blocked by these modifications and is thereby limited to the epidermal outer periclinal wall. Intracellular H₂O₂ is mainly restricted to HR-like epidermal cells. co, Conidium; cu, cuticula; ep, epidermal cell layer; me, mesophyll cell layer; pe, point of penetration.

Effect of ABA, ascorbate, catalase, and DPI treatment on H₂O₂ accumulation and symptom development in wild-type and sitiens tomato leaf discs after inoculation with two 5-μL droplets of B. cinerea conidial suspension. A, DAB staining at 20 hpi. One representative disc out of three replicates is presented. B, Micrograph of sitiens H₂O₂ accumulation at 8 hpi. C, B. cinerea symptom development. For each treatment, at least 18 leaf discs from at least five plants were infected and the number of spreading lesions was evaluated at 4 dpi. Data were statistically analyzed using binary logistic regression. Bars with different letters are significantly different at P < 0.05. Profuse pathogen development is visible on discs treated with ascorbic acid at 4 dpi. All experiments were repeated with similar results.

Cell wall modifications in wild-type and sitiens tomato inoculated with B. cinerea. In sitiens, cell wall modifications were first detected at the sites of H₂O₂ accumulation and were generally present from 8 hpi beneath the inoculation droplet in the epidermal anticlinal cell walls, with an increase in staining intensity at subsequent time points. In wild-type tomato, only few cells had limited cell wall modifications. Cell wall modifications were visualized with Coomassie Blue staining after SDS denaturation to detect protein cross-linking (dark blue; A) with safranin-O (red-pink; B) and with toluidine blue to detect phenolic compounds (turquoise), whereas pectic fragments stain purple (C). For each time point and stain, infection sites on at least three leaf discs from independent sitiens and wild-type plants were examined and gave the same pattern of cell wall fortification. Scale bar = 50 μm.

Effect of ABA on H₂O₂ accumulation in epidermal cells after infection with B. cinerea. A, Association of DAB accumulation with B. cinerea conidia. In sitiens, H₂O₂ was located at the site of penetration and in some parts of the anticlinal wall of the penetrated cell, whereas in wild type and in sitiens supplemented with ABA, no H₂O₂ accumulation was detected. Germinating conidia were classified in two groups based on the presence or absence of associated DAB accumulation in the epidermal cells, whose percentage is shown. B, DAB accumulation in epidermal cell walls. In sitiens, DAB staining was general in the entire outline of the anticlinal cell wall, whereas in wild type and sitiens supplemented with ABA, no H₂O₂ accumulation was detected at 8 hpi (top). The restriction of sitiens DAB accumulation to the epidermal layer was confirmed on cross sections (bottom). Epidermal cells were classified in two groups based on the presence or absence of DAB accumulation in the anticlinal walls, whose percentage is shown in the anticlinal walls at 8, 16, and 20 hpi. C, Intracellular DAB accumulation in epidermal cells showing an HR-like reaction. At 16 hpi, wild-type and ABA-supplemented sitiens epidermal cells did not accumulate intracellular DAB, whereas in sitiens, groups of HR-like cells with intracellular DAB accumulation were present near the site of fungal penetration. Epidermal cells were classified in two groups based on the presence or absence of intracellular DAB accumulation and the percentage is shown at 12, 16, and 20 hpi. In all graphs, bars represent the means and the sds of data from six inoculation droplets originating from three plants. In each inoculation droplet, at least 50 conidia (A) or 300 epidermal cells (B and C) originating from representative zones within each inoculation droplet were counted. Data from one experiment is presented. The experiment was repeated with similar results. Scale bar = 50 μm.