Fig 1.
The A. alternata SSK1 homolog (AaSSK1) is required for conidial germination and differentiation.
(A) Percentage of conidial germination of the wild-type (WT) and the Δssk1 deletion mutants (T5 and T11) on 96-well microtiter plates for 6 h. (B) Images of germinated conidia (top panel) and quantitative analysis of the length of germ tubes on glass slides (bottom panel). (C) Production of single or multiple germ tubes on glass slides. All (100%) conidia prepared from WT and Δssk1 germinated on glass slides at 6 h. The data presented are the mean and standard deviation of two independent experiments with at least three replicates.
Fig 2.
SSK1 is required for oxidative stress resistance in A. alternata.
(A) Images of the wild-type (WT) and the Δssk1 deletion mutants (T5 and T11) grown potato dextrose agar (PDA) amended with hydrogen peroxide (H2O2, 5 mM), tert-butyl-hydroxyperoxide (t-BHP, 2.6 mM) and menadione (MND, 1 mM) for 4 to 5 days. (B) Quantitative comparison of Δssk1 with different mutant strains—Δhsk1 impaired for histidine kinase, Δhog1 for HOG1 MAP kinase, and Δskn7 for SKN7 response regulator—in the presence of oxidative stress. Percentage change in radial growth was calculated by dividing a percentage of colony diameters of the deletion mutants over those of wild-type grown on the same plate. The data presented are the mean and standard deviation of two independent experiments with at least three replicates.
Fig 3.
SSK1 plays a vital role in osmotic stress resistance in A. alternata.
(A) Images of the wild-type (WT) and the Δssk1 deletion mutants (T5 and T11) grown potato dextrose agar (PDA) amended with sugars or salts for 4 to 5 days. (B) Quantitative comparison of Δssk1 with different mutant strains—Δhsk1 impaired for histidine kinase, Δhog1 for HOG1 MAP kinase and Δskn7 for SKN7 response regulator—in the presence of osmotic stress (each at 1 M). The data presented are the mean and standard deviation of two independent experiments with at least three replicates.
Fig 4.
SSK1 is required for multi-drug resistance in A. alternata.
(A) Images of the wild-type (WT) and the Δssk1 deletion mutants (T5 and T11) grown potato dextrose agar (PDA) amended with 2-chloro-5-hydroxypyridine (CHP) or 2,3,5-triiodobenzoic acid (TIBA), each at 5 mM. (B) Quantitative analysis of the wild-type, Δssk1, Δhog1, and Δhsk1 strains in the presence of CHP or TIBA.
Fig 5.
The A. alternata mutants impaired for SSK1 are defective in the ability to detoxify H2O2.
(A) The wild-type (WT) and the Δssk1 mutant strains (T5 and T11) grown on agar plates were flooded with 30 mM H2O2 and stained with 3,3’-diaminobenzidine. (B) Detoxification of H2O2 by A. alternata strains was determined by monitoring a decrease of absorbance at 240 nm over time. Five agar plugs (2 mm in diam.) containing fungal conidia/hyphae were cut from fungal colonies grown on PDA for 3 to 5 days, placed in solution, and incubated at ~25°C. The control treatment contained no fungal hyphae. The data presented are the mean and standard deviation of three independent experiments with three replicates.
Fig 6.
The A. alternata mutants confer resistance to fungicides.
(A) Growth of the wild-type (WT), the strains lacking SSK1 (Δssk1-T5 and Δssk1-T11), the histidine kinase (Δhsk1), the HOG1 MAP kinase (Δhog1), and the SKN7 response regulator (Δskn7) on PDA amended with vinclozolin (10 μg/ml), fludioxonil (0.1 μg/ml) or iprodione (10 μg/ml) dissolved in 1% DMSO for 3 to 5 days. (B) Quantitative analysis of fungal radial growth. The Δskn7 Δhog1 strain was impaired for both SKN7 and HOG1. Percentage change in radial growth was calculated by dividing a percentage of colony diameters of the deletion mutants over those of wild-type grown on the same plate. The data presented are the mean and standard deviation of two independent experiments with at least three replicates. Top Brackets indicate significant differences between treatments.
Fig 7.
SSK1 plays a crucial role in A. alternata pathogenesis in citrus.
(A) Development of necrotic lesions on detached calamondin leaves inoculated with conidial suspension (104 conidia per ml) prepared form the wild-type (WT) and the Δssk1 mutant strains (T5 and T11). The mock controls were treated with water only. Δssk1 mutants occasionally induced visible lesions (indicated by arrows). (B) Induction of necrotic lesions by A. alternata strains on detached calamondin leaves that were wounded prior to inoculation. Inoculated leaves were kept in plastic boxes for 5 days. Wild-type produced aerial hyphae and conidia on necrotic areas (indicated by arrows).
Fig 8.
Schematic illustration of signaling pathways leading to osmotic and oxidative stress resistance and fungicide sensitivity in A. alternata.
A. alternata deploys the HSK1- and the SKN7-mediated phosphorelay signaling pathways between histidine (His) and asparate (Asp) to cope with sugar-induced osmotic stress. SSK1 plays a minor role in sugar-induced osmotic stress resistance. However, SSK1 and HOG1 mediated by unknown kinase sensors confer resistance to oxidative and salt-induced osmotic stress. Low-level H2O2 produced by NADPH oxidases (NOX) is proposed to activate SSK1, HOG1, YAP1, and SKN7, all implicated in ROS resistance. HSK1, SSK1, HOG1, SKN7, but not YAP1 and NOX are involved in fungicide sensitivity.