Fig 1.
The Alternaria alternata major facilitator superfamily transporter (AaMFS19) confers resistance to xenobiotics.
(A) Images of the wild-type (WT), two ΔAaMFS19 deletion mutants (D27 and D63), and the CP3 complementation strain grown on potato dextrose agar (PDA) amended with 2-chloro-5-hydroxypyridine (CHP), 2,3,5-triiodobenzoic acid (TIBA), fungicides and other chemicals as indicated. Only representative replicates are shown. (B) Quantitative analysis of chemical sensitivity. Sensitivity indicated by percentage growth reduction was calculated as a cumulative percentage of growth of wild-type and AaMFS19 mutant strains grown on the same plate. The data presented are the mean and standard deviation of two independent experiments with four biological replicates.
Fig 2.
The Alternaria alternata major facilitator superfamily transporter (AaMFS19) confers resistance to oxidants and cell wall destructing agents.
(A) Images of the wild-type (WT), two ΔAaMFS19 deletion mutants (D27 and D63), and the CP3 complementation strain grown on potato dextrose agar (PDA) amended with hydrogen peroxides (H2O2 and cumyl H2O2), potassium superoxide (KO2), single oxygen-generating compounds, eosin Y, rose Bengal, hematoporphyrin (HP), toluidine blue (TB), methylene blue (MB), and cercosporin, and the cell wall-destructing agent, Congo red. Only representative replicates are shown. (B) Quantitative analysis of chemical sensitivity. Sensitivity indicated by percentage growth reduction was calculated as a cumulative percentage of growth of wild-type and AaMFS19 mutant strains grown on the same plate. The data presented are the mean and standard deviation of two independent experiments with four biological replicates.
Fig 3.
Expression of the AaMFS19 gene encoding a major facilitator superfamily transporter in Alternaria alternata.
(A) Northern blot analysis of AaMFS19 in response to 2-chloro-5-hydroxypyridine (CHP), 2,3,5-triiodobenzoic acid (TIBA), vinclozolin (Vin), clotrimazole, and H2O2. The wild-type strain was grown on a layer of cellophane overlaid on PDA for 3 days and shifted to PDA amended with chemicals as indicated for an additional 24 h. Fungal RNA was purified, electrophoresed on a formaldehyde-containing gel, blotted to membrane, and hybridized with an AaMFS19-specific probe. Ethidium bromide (EtBr)-stained rRNA is shown to ensure equal loading of the samples. (B) Semi-quantitative reverse transcription PCR analysis of AaMFS19, the actin and β-tublin-coding genes, and the GPx3 gene encoding a glutathione peroxidase. PCR products were visualized on 1.2% agarose gel staining with EtBr.
Fig 4.
Northern blot analysis of AaMFS19 in the wild-type (WT) and mutant strains of Alternaria alternata.
(A) RNA was purified from WT, the mutant strains lacking Yap1 transcription regulator (Δyap1-D1 and D2), and the YCp1 strain re-acquiring and expressing a functional copy of Yap1. (B) RNA was purified from fungal strains impaired for a Fus3 MAP kinase (Δfus3), a Hog1 MAP kinase (Δhog1), a histidine kinase (Δhsk1), a Skn7 response regulator, and a Slt2 MAP kinase (Δslt3). Fungal strains were grown on a layer of cellophane overlaid on PDA for 3 days. RNA was purified, electrophoresed on a formaldehyde-containing gel, blotted to membrane, and hybridized with an AaMFS19-specific probe. Ethidium bromide (EtBr)-stained rRNA is shown to ensure equal loading of the samples.
Fig 5.
Virulence assays on detached calamondin leaves.
Conidial suspension (104 conidia/ml) collected from the wild type, the strains lacking AaMFS19 (D27 and D63), and the CP3 strain expressing a copy of AaMFS19 were sprayed uniformly onto detached calamondin leaves. The mock controls were treated with water only. The leaves were incubated in a moisture chamber for lesion development. Photos were taken 2 days post inoculation (dpi). Experiment was repeated three times with at least 10 leaves. Only representative replicates are shown.
Fig 6.
Toxicity of 2-chloro-5-hydroxypyridine (CHP) and 2,3,5-triiodobenzoic acid (TIBA) to Alternaria alternata.
Wild-type was grown on PDA amended with (A) CHP and (B) TIBA at different concentrations and colony diameter was measured 5 days post inoculation. (C) Wild-type was cultured on PDA amended with 0.1% dimethyl sulfoxide (DMSO), 2.5 mM CHP, or 6 mM TIBA for 5 days and conidia were harvested and examined microscopically. (D) Conidia were germinated on glass slide, incubated in a moist chamber for 6 h, and examined microscopically. The data presented are the mean and standard deviation of two independent experiments with two biological replicates.
Fig 7.
2-chloro-5-hydroxypyridine (CHP) and 2,3,5-triiodobenzoic acid (TIBA) affects hyphal elongation of Alternaria alternata.
Wild-type was grown on (A) PDA, (B) (C) PDA amended with CHP, and (D) with TIBA for 3 days. Hyphae were examined microscopically.