Critical Role of MetR/MetB/MetC/MetX in Cysteine and Methionine Metabolism, Fungal Development, and Virulence of Alternaria alternata

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 hyphae 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 MetB, MetC, and MetX deletion mutants (hereafter called DMetB, DMetC, and DMetX) can be restored by exogenous methionine and homocysteine, indicating that AaMetB, AaMetC, and AaMetX are required for methionine biosynthesis. However, exogenous cysteine restored the growth and virulence defects of DMetR but not DMetB, DMetC, and DMetX, suggesting that AaMetR is essential for cysteine biosynthesis. Oxidant sensitivity assay showed that only DMetR is sensitive to H2O2 and many reactive oxygen species (ROS)-generating compounds, indicating that AaMetR is essential for oxidative tolerance. Interestingly, fungicide indoor bioassays showed that only the DMetR mutant is susceptive to chlorothalonil, a fungicide that could bind to the cyste-ine of glyceraldehyde-3-phosphate dehydrogenase. Comparative transcriptome analysis showed that the inactivation of MetB, MetC, MetX, and MetR significantly affected the expression of methionine metabolism-related genes. Moreover, the inactivation of AaMetR significantly affected the expression of many genes related to glutathi-one 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.