The MAP Kinase Kinase Gene AbSte7 Regulates Multiple Aspects of Alternaria brassicicola Pathogenesis

Abstract

Mitogen-activated protein kinase (MAPK) cascades in fungi are ubiquitously conserved signaling pathways that regulate stress responses, vegetative growth, pathogenicity, and many other developmental processes. Previously, we reported that the AbSte7 gene, which encodes a mitogen-activated protein kinase kinase (MAPKK) in Alternaria brassicicola, plays a central role in pathogenicity against host cabbage plants. In this research, we further characterized the role of AbSte7 in the pathogenicity of this fungus using ${\Delta}AbSte7$ mutants. Disruption of the AbSte7 gene of A. brassicicola reduced accumulation of metabolites toxic to the host plant in liquid culture media. The ${\Delta}AbSte7$ mutants could not efficiently detoxify cruciferous phytoalexin brassinin, possibly due to reduced expression of the brassinin hydrolase gene involved in detoxifying brassinin. Disruption of the AbSte7 gene also severely impaired fungal detoxification of reactive oxygen species. AbSte7 gene disruption reduced the enzymatic activity of cell walldegrading enzymes, including cellulase, ${\beta}$-glucosidase, pectin methylesterase, polymethyl-galacturonase, and polygalacturonic acid transeliminase, during host plant infection. Altogether, the data strongly suggest the MAPKK gene AbSte7 plays a pivotal role in A. brassicicola during host infection by regulating multiple steps, and thus increasing pathogenicity and inhibiting host defenses.

Fig. 2. Relative expression levels of the BHAb gene in AbSte7 disruption mutants M1 and M2 at different time points during infection. The expression levels of BHAb in WT, M1, and M2 were compared to the expression levels of the gene encoding actin for normalization. Relative expression levels were calculated by dividing the expression levels of M1 or M2 by those of WT. The expression levels were calculated using the comparative ΔΔCt method. The data are displayed as mean ± standard error for three independent experiments.

Fig. 3. Spectrophotometric analysis of H₂O₂ detoxification by A. brassicicola WT and ΔAbSte7 mutants. The absorbance at 240 nm was measured for a 30 mM H₂O₂ solution following the addition of mycelia plugs of A. brassicicola WT or ΔAbSte7 mutants M1 and M2 or agar plugs lacking mycelia (control) and incubating for 75 min. The ΔAbSte7 mutants had a severely impaired ability to detoxify H₂O₂. The data are presented as mean ± standard error for three independent experiments.

Fig. 5. Enzymatic activity of cellulase (Cx, A), β-glucosidase (B), polygalacturonase (PG, C), pectin methylesterase (PME, D), polygalacturonic acid trans-eliminase (PGTE, E) and pectin methyl-trans-eliminase (PMTE, F) in infected cabbage leaves after inoculation with the A. brassicicola WT or ΔAbSte7 M1 mutant strains. Bars represent standard errors of the mean. Means with an asterisk (*) indicate a significant difference at P ≤ 0.05.

Fig. 1. Phytotoxicity assays of crude broth extracts from different strains. (A) Crude broth extracts from different Alternaria brassicicola strains were inoculated on detached cabbage leaves. WT: wild-type of A. brassicicola; M1 and M2: AbSte7 disruption mutants; C1: ΔAbSte7 complementation strain; MA: 5% methanol control. (B) Lesion size following infection with different A. brassicicola strains was recorded at 5 days post-inoculation. Each column contains the mean ± standard error of three independent experiments with three replicates. The different letters in the column indicate significant differences as determined by Student’s ttest (P < 0.05).

Fig. 4. (A) Carbohydrate utilization by A. brassicicola WT and ΔAbSte7 mutant strains. The ΔAbSte7 mutant M1 and M2 and WT strains were grown on minimal media supplemented with the carbon sources xylan, cellulose, and pectin at 25℃ for 7 d. (B) The carbohydrate utilization and hydrolytic enzyme production in ΔAbSte7 and WT strains were determined by the growth rates in minimal medium supplemented with diverse carbon sources. The growth rates (D) were calculated as: D = D1-D2. D1 and D2 were the colony diameter measured at 7 days after incubation and the diameter of the agar block for inoculation (8 mm), respectively. Each column represents the mean ± SE of the three independent experiments with three replicates.

Table 1. Effects of brassinin on the colony growth of ΔAbSte7 mutants and wild-type strains of Alternaria brassicicola on potato dextrose agar plates