The critical role of MetR/MetB/MetC/MetX in cysteine and methionine metabolism, fungal development and virulence of Alternaria alternata

Methionine is a unique sulfur-containing amino acid, which plays an important role in biological protein synthesis and various cellular processes. Here, we characterize the biological functions of AaMetB, AaMetC, and AaMetX in the tangerine pathotype of Alternaria alternata. Morphological analysis showed that mutants lacking AaMetB, AaMetC, or AaMetX resulted in less aerial hypha and fewer conidia in artificial media. The virulence assays revealed that AaMetB, AaMetC, and AaMetX are required for full virulence. The defects of MetB, MetC, and MetX in vegetative growth, conidiation and virulence can be restored by exogenous methionine and homocysteine, indicating that AaMetB, AaMetC, and AaMetX are required for methionine biosynthesis. The defects of MetR in vegetative growth and virulence can be restored by exogenous cysteine, indicating that AaMetR is essential for cysteine biosynthesis. The oxidant sensitivity assay showed that only MetR is sensitivity to H2O2 and many ROS-generating compounds, which indicates that AaMetR is essential for oxidative tolerance. Interestingly, indoor bioassays of these mutants on fungicides showed that only the MetR mutants are susceptive to chlorothalonil, which can interact with 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 many genes related to methionine metabolism. Moreover, the inactivation of AaMetR significantly affected the expression of many genes related to glutathione metabolism, which is required for ROS tolerance. In conclusion, our study provides genetic evidence to define the critical roles of AaMetB, AaMetC, AaMetX, and AaMetR in the metabolism of cysteine and methionine, fungal development and virulence of Alternaria alternata.