Responses of the Necrotrophic Fungus Alternaria brassisicola to the Indolic Phytoalexin Brassinin

Abstract

Alternaria brassicicola causes black spot disease in Brassicaceae. During host infection, this necrotrophic fungus is exposed to various antimicrobial compounds, such as the phytoalexin brassinin which is produced by many cultivated Brassica species. To investigate the cellular mechanisms by which this compound causes toxicity and the corresponding fungal adaptive strategies, we first analyzed fungal transcriptional responses to short-term exposure to brassinin and then used additional functional approaches. This study supports the hypothesis that indolic phytoalexin primarily targets mitochondrial functions in fungal cells. Indeed, we notably observed that phytoalexin treatment of A. brassicicola disrupted the mitochondrial membrane potential and resulted in a significant and rapid decrease in the oxygen consumption rates. Secondary effects, such as Reactive oxygen species production, changes in lipid and endoplasmic reticulum homeostasis were then found to be induced. Consequently, the fungus has to adapt its metabolism to protect itself against the toxic effects of these molecules, especially via the activation of high osmolarity glycerol and cell wall integrity signaling pathways and by induction of the unfolded protein response.

Keywords: brassinin; ergosterol; fungus; mitochondria; necrotroph; phytoalexin; signaling pathways.

FIGURE 1

Susceptibility of A. brassicicola to brassinin. (A) Growth of the wild-type strain for 33 h at 24°C. The unit of the Y-axis corresponds to the relative nephelometric unit (RNU). Microplate wells containing standard PDB medium supplemented either with DMSO or various concentrations of brassinin were inoculated with a wild-type conidial suspension (10⁵ conidia/mL, final concentration). Fungal growth was recorded using a nephelometric reader. Each condition was conducted in triplicate and the experiments were repeated twice. Areas under the growth curves were used to calculate MGI₅₀, i.e., the phytoalexin concentration for which 50% of mycelium growth inhibition was observed. (B) Effect of brassinin (100 μM) on viability of germinated A. brassicicola conidia. Conidia (10⁵ mL^{– 1}) were germinated for 24 h, incubated in PDB for various times in the presence of brassinin at the desired concentration. After centrifugation, the pellets containing the germinated conidia were re-suspended in 200 μL, which were applied on the Petri dishes containing PDA medium. Colonies were visualized after 48 h of incubation. Control plates were prepared with conidia incubated for up to 24 h with DMSO (1% v/v final concentration).

FIGURE 2

Venn diagram of overlapping and non-overlapping genes with significantly regulated expression levels in brassinin-treated cultures compared to DMSO-treated cultures. The number of induced or repressed genes are indicated with black or gray numbers, respectively. Probes with a P ≤ 0.01and a log ratio ≥ 1 or ≤-1 were considered as differentially expressed.

FIGURE 3

Growth inhibition rates of the wild-type strain and AbErg5 and AbAp1 deficient mutants (two transformants per genotype) exposed to 100 μM brassinin. The results show the percentages of inhibition in treated samples compared to the control. Fungal growth was recorded using a nephelometric reader from microplate wells containing standard PDB medium supplemented with brassinin or DMSO and inoculated with conidia (10⁵ mL^{– 1}). Each genotype was analyzed in triplicate and the experiments were repeated three times per growth condition. Error bars indicate standard deviations. Asterisks indicate a significant difference between the mutant and the parental isolate (Student test, P < 0.01).

FIGURE 4

Assessment of changes in mitochondrial membrane potentials within A. brassicicola cells exposed for 30 min to 100 μM brassinin or to DMSO, using the fluorescent potentiometric dye JC-1. The top pictures correspond to light-field microscopy while the other pictures correspond to fluorescence microscopy. Scale bars = 20 μm.

FIGURE 5

Impact of brassinin on A. brassicicola respiration. The results are expressed as the inhibitory effects (% of the control) on the respiration rate of germlings, following 1 h-exposure to 100 and 200 μM brassinin or 0.5 mM KCN. SD is indicated.

FIGURE 6

Assessment of oxidative stress within A. brassicicola cells exposed for 30 min to 100 μM brassinin or to DMSO. The fluorescent dyes H₂DCFDA and DHE were used to detect the accumulation of ROS within hyphae of germlings treated for 1 h with DMSO (control) or 100 μM brassininin. For each panel, the bottom part corresponds to fluorescence microscopy and the top part to light-field microscopy. Scale bars = 20 μm.

FIGURE 7

Cellular localization of the AbHOG1-GFP fusion protein in hyphae exposed to either DMSO (control) or 100 μM brassininin for 20 min and observed using confocal microscopy. Bars = 25 μm. A double-labeled strain expressing AbHOG1-GFP and mCherry-NLS was used. In smaller panels located under the pictures is presented the phosphorylation of the HOG1-like MAPK in A. brassicicola wild-type after exposure to brassinin. Total protein extracts were blotted with either anti-Hog1 C-terminus antibody or anti-dually phosphorylated p38 antibody.

FIGURE 8

Cellular localization of the AbAP1-GFP fusion protein in the wild-type background and Δabhog1 background. Double-labeled strains expressing mCherry-NLS and AbAP1-GFP were exposed to either DMSO (control) or 100 μM brassininin for 0.5 h. Bars = 25 μm.

FIGURE 9

Brassinin activation of the UPR signaling pathway. Quantitative RT-PCR results for the expression of UPR target genes (AbBipA, AbPdiA, and AbEroA) and the spliced AbHacA mRNA (AbHacAⁱ) in an A. brassicicola wild-type strain during brassinin exposure (200 μM) for 10, 20, and 30 min, 1, 2, and 6 h. For each target, expression induction is represented as a ratio (studied gene transcript abundance/actin transcript abundance) of its relative expression in each brassinin-treated sample to its relative expression in DMSO-treated cultures. The data are means of three repetitions. Error bars indicate standard deviations and asterisks indicate a relative expression significantly different from 1 (Student test, P < 0.01).

FIGURE 10

Model illustration of the fungal response induced by brassinin. The indolic phytoalexin primarily targets mitochondrial functions in fungal cells, inducing secondary effects such as ROS production and alteration of lipid homeostasis (1). Then, ROS production elicits AbHOG1 and AbAP1-mediated responses in order to reinforce antioxidant defenses and limit their deleterious effects (2). Changes in lipid homeostasis affect the activity of cell wall synthesis enzymes located in the plasma membrane and synthesis of the GPI anchor, leading to activation of the cell wall integrity pathway (3). Perturbations in membrane and cell wall homeostasis trigger UPR induction (4).