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Abstract
Methionine is a unique sulfur-containing amino acid, which plays an important role in biological protein synthesis and various cellular processes. Here, we characterized the biological functions of AaMetB, AaMetC, and AaMetX in the tangerine pathotype of Alternaria alternata Morphological analysis showed that the mutants lacking AaMetB, AaMetC, or AaMetX resulted in less aerial hypha and fewer conidia in artificial media. Pathogenicity analysis showed that AaMetB, AaMetC, and AaMetX are required for full virulence. The defects in vegetative growth, conidiation and virulence of ΔMetB, ΔMetC, and ΔMetX can be restored by exogenous methionine and homocysteine, indicating that AaMetB, AaMetC, and AaMetX are required for methionine biosynthesis. However, exogenous cysteine only restored the growth and virulence defects of ΔMetR but not ΔMetB/C/X, suggesting that AaMetR is essential for cysteine biosynthesis. Oxidant sensitivity assay showed that only ΔMetR is sensitive to H2O2 and many ROS-generating compounds, indicating that AaMetR is essential for oxidative tolerance. Interestingly, fungicides indoor bioassays showed that only the ΔMetR mutants are susceptive to chlorothalonil, a fungicide that could bind to the cysteine of glyceraldehyde-3-phosphate dehydrogenase. Comparative transcriptome analysis showed that the inactivation of MetB, MetC, MetX, or MetR significantly affected the expression of methionine metabolism-related genes. Moreover, the inactivation of AaMetR significantly affected the expression of many genes related to glutathione metabolism, which is essential for ROS tolerance. Taken together, our study provides genetic evidence to define the critical roles of AaMetB, AaMetC, AaMetX, and AaMetR in cysteine and methionine metabolism, fungal development and virulence of Alternaria alternata IMPORTANCE The transcription factor METR regulating methionine metabolism is essential for reactive oxygen species (ROS) tolerance and virulence in many phytopathogenic fungi. However, the underlying regulatory mechanism of METR involved in this process is still unclear. In the present study, we generated AaMetB, AaMetC and AaMetX deletion mutants and compared these mutants with AaMetR disrupted mutants. Interestingly, we found that AaMetB, AaMetC and AaMetX are required for vegetative growth, conidiation, and pathogenicity in Alternaria alternata, but not for ROS tolerance and cysteine metabolism. Furthermore, we found that METR is involved in the biosynthesis of cysteine, which is an essential substrate for the biosynthesis of methionine and glutathione. This study emphasizes the critical roles of MetR, MetB, MetC, MetX in the regulation of cysteine and methionine metabolism, as well as the cross-link with glutathione-mediated ROS tolerance in phytopathogenic fungi, which provides a foundation for future investigations.
Highlights
Methionine is a unique sulfur-containing amino acid which plays an important role in biological protein synthesis and various cellular processes
Our results indicate that the loss of pathogenicity of DMetB, DMetC, and DMetX mutants is caused by the deficiency of methionine biosynthesis, which indicates that methionine biosynthesis regulated by AaMetB, AaMetC, and AaMetX is required for the full virulence of A. alternata tangerine pathotype
The bZIP transcription factor MetR plays an important role in the regulation of methionine metabolism, reactive oxygen species (ROS) tolerance, and pathogenicity in Magnaporthe oryzae [42], Aspergillus fumigatus [29], A. alternata [16], and Cryptococcus neoformans [18]
Summary
Methionine is a unique sulfur-containing amino acid which plays an important role in biological protein synthesis and various cellular processes. Our study provides genetic evidence to define the critical roles of AaMetB, AaMetC, AaMetX, and AaMetR in cysteine and methionine metabolism, fungal development, and virulence of Alternaria alternata. We found that AaMetB, AaMetC, and AaMetX are required for vegetative growth, conidiation, and pathogenicity in Alternaria alternata but not for ROS tolerance and cysteine metabolism. This study emphasizes the critical roles of MetR, MetB, MetC, and MetX in the regulation of cysteine and methionine metabolism, as well as the cross-link with glutathione-mediated ROS tolerance in phytopathogenic fungi, which provides a foundation for future investigations. Homocysteine can be generated through the methionine cycle, which makes it an important metabolic intermediate as it is required for the biosynthesis of sulfur-containing amino acids. For many antifungal drugs, such as ebelactone A and the antibiotic azoxybacillin, the methionine biosynthesis pathway has been chosen as a common target due to its absence in the animal kingdom [25,26,27]
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