Figure 1.
Expression of ced-9 in Arabidopsis inhibits wild type infection but does not affect the A2 phenotype.
Agar plugs containing actively growing cultures of wild type S. sclerotiorum (strain 1980) and the OA deficient A2 mutant were inoculated onto Col-0 and ced-9 expressing Arabidopsis leaves. (A) Wild type inoculations onto Col-0 plants resulted in typical lesions for this pathogen including a rapid, spreading cell death; however, infection was completely suppressed in ced-9 expressing plants. The expression of this gene had no effect on the A2 phenotype. (B) Trypan blue staining indicates the extent of cell death for each genotype/strain combination, including an agar plug control. All images were recorded 48 hours post inoculation.
Figure 2.
Microscopic examination of cross sections of tomato leaves at the leading edge of the lesion following fungal inoculation.
S. sclerotiorum A2 (A) and wild type (B) strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were post-fixed in osmium tetroxide, and embedded in Spurr's epoxy resin. A microtome was used to cut 400 nm sections. Toluidine blue stain was used to reveal fungal hyphae. H = hyphae. The dotted line represents the leading edge of the visible lesion. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227.
Figure 3.
S. sclerotiorum A2 strain induces autophagic structures in plants.
S. sclerotiorum wild type and A2 strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were stained with 100 µM final concentration of MDC (Sigma) in PBS for 30 min. Fluorescence was visualized using an Olympus IX81 inverted fluorescence confocal microscope (Olympus systems, Germany), with an excitation wavelength of 335 nm and an emission wavelength of 508 nm. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227. Scale bar = 10 µm.
Figure 4.
Transmission Electron Microscopy (TEM) fungal inoculated tomato leaves.
Representative TEM images from four independent experiments. (A, G); Healthy non-inoculated leaf tissue. (B–F) Tomato leaves inoculated with the OA deficient A2 strain. (H,I) Tomato leaves inoculated with wild type S. sclerotiorum. Arrows, autolysosomal/autophagosomal-like structures; C, chloroplast; V, vacuole; N, nucleus; Circle, active dismantlement of chloroplast; Rectangle, chromatin condensation within the nucleus. Black scale bars = 2 µm, white scale bars = 1 µm. Sections were examined with a Phillips Morgagni 268 transmission electron microscope at an accelerating voltage of 80 kV. Digital images were recorded with a MegaViewIII digital camera operated with iTEM software.
Figure 5.
Inhibition of autophagy restores A2 pathogenicity.
(A,B) Agar plugs containing actively growing cultures of the OA deficient A2 mutant were inoculated onto leaves of Arabidopsis Col-0 and select Arabidopsis autophagy mutant plants. These mutants showed enhanced susceptibility to the normally non-pathogenic A2 strain. Lesion diameter was monitored over time and all images were recorded 48 hours post inoculation. (C) Tomato leaves were either pre-infiltrated with water (control) or autophagy inhibitors Wortmannin, LY294002, Chloroquine (CQ), and 3-methyladenine (3-MA). Agar plugs containing actively growing A2 were placed on the infiltrated leaves to initiate infection. (B) 48 hours post inoculation; Trypan blue was used to determine the extent of fungal colonization and cleared with acetic acid and ethanol (1: 3, v/v). Images were taken 48 hours post inoculation.
Figure 6.
Oxidant accumulation in wild type and atg mutant plants.
(A) NBT treated Arabidopsis (Col-0 and two independent atg8a mutant lines) following agar plug inoculation with the A2 mutant. Images were collected 48 hours post inoculation. Dotted lines represent the edge of the observable legion. (B) RT-PCR was used to evaluate the transcript levels of three catalases (CAT1, 2, and 3) and three superoxide dismutases in Col-0 plants following inoculation with wild type S. sclerotiorum (black bars) and the A2 mutant strain (grey bars). *>2 fold change, **>5 fold change.
Figure 7.
Oxalic acid is multifunctional.
OA is a pathogenicity determinant in Sclerotinia that has a number of functions that facilitate fungal pathogenicity. OA inhibits plant defense responses (eg callose deposition) and modulates the host redox environment by blocking the host oxidative burst and creating reducing environment. OA also suppresses autophagy. At later stages, OA accumulation lowers the pH, activates cell wall degrading enzymes and a MAP kinase required for pathogenic sclerotial development. This process culminates in OA induced ROS leading to elicitation of apoptotic cell death and disease. (For further details see Dickman, 2007; Williams et al., 2011).