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 <em>AaMetB</em>, <em>AaMetC</em>, and <em>AaMetX</em> in the tangerine pathotype of <em>Alternaria alternata</em>. Morphological analysis showed that mutants lacking <em>AaMetB</em>, <em>AaMetC</em>, or <em>AaMetX</em> resulted in less aerial hypha and fewer conidia in artificial media. The virulence assays revealed that <em>AaMetB</em>, <em>AaMetC</em>, and <em>AaMetX </em>are required for full virulence. The defects of <em></em><em>MetB</em>, <em></em><em>MetC</em>, and <em></em><em>MetX</em> in vegetative growth, conidiation and virulence can be restored by exogenous methionine and homocysteine, indicating that <em>AaMetB</em>, <em>AaMetC</em>, and <em>AaMetX </em>are required for methionine biosynthesis. The defects of <em></em><em>MetR </em>in vegetative growth and virulence can be restored by exogenous cysteine, indicating that <em>AaMetR</em> is essential for cysteine biosynthesis. The oxidant sensitivity assay showed that only <em></em><em>MetR</em> is sensitivity to H₂O₂ and many ROS-generating compounds, which indicates that <em>AaMetR</em> is essential for oxidative tolerance. Interestingly, indoor bioassays of these mutants on fungicides showed that only the <em></em><em>MetR </em>mutants<em> </em>are susceptive to chlorothalonil, which can interact with the cysteine of glyceraldehyde-3-phosphate dehydrogenase. Comparative transcriptome analysis showed that the inactivation of <em>MetB</em>, <em>MetC</em>, <em>MetX</em>, or <em>MetR</em> significantly affected the expression of many genes related to methionine metabolism. Moreover, the inactivation of Aa<em>MetR</em> 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 <em>AaMetB</em>, <em>AaMetC</em>, <em>AaMetX</em>, and <em>AaMetR</em> in the metabolism of cysteine and methionine, fungal development and virulence of <em>Alternaria alternata.</em>