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 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 restored the growth and virulence defects of ΔMetR but not ΔMetB, ΔMetC, and ΔMetX, suggesting that AaMetR is essential for cysteine biosynthesis. Oxidant sensitivity assay showed that only ΔMetR 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 ΔMetR mutant is 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, 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 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 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, 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.

Complementary Biotransformation of Antimicrobial Triclocarban Obviously Mitigates Nitrous Oxide Emission toward Sustainable Microbial Denitrification

“…In addition, the synthesis of l -cystathamine was also inhibited due to the downregulated expression of cystathionine γ-synthase MetB ( P < 0.01). TCC further affected the metabolism of cysteine and methionine, which play an intermediate role in the ROS detoxification …”

Fungal consortium of two Beauveria bassiana strains increases their virulence, growth, and resistance to stress: A metabolomic approach

“…Another molecule found in the consortium, L-homocystine, participates in the cysteine and methionine metabolic pathway ( Table 2 ). This pathway responds to environmental variations, due to its enormous importance in cell physiology; moreover, proteins derived from this metabolic pathway, including thioredoxin and glutathione, play a key role in cellular protection against oxidative stress [ 34 , 37 – 40 ]. These molecules are essential for maintaining cellular homeostasis and detoxifying ROS, singlet oxygen molecules, hydrogen peroxide, superoxide anions, and hydroxyl radicals.…”

Histone Methylation Is Required for Virulence, Conidiation, and Multi-Stress Resistance of Alternaria alternata

“…Tested compounds included H 2 O 2 (10 mm), sorbitol (1 M), KCl (1 M), NaCl (1 M), CaCl 2 (250 mm), CuSO 4 (1 mm), sodium dodecyl sulfate (SDS, 0.01%), Congo red (CR, 0.2 mg/ml), chlorothalonil (10 mg/l), Mancozeb (2 mg/l), Boscalid (3 mg/l), hydroxyurea (HU, 5 mm), or Camptothecin (CPT, 5 μm). To examine the carbon utilization ability, mutants and wild type were inoculated on MM medium ( Gai et al, 2021 ) using glucose (20 g/l), sucrose (10 g/l), starch (10 g/l), or lactose (10 g/l) as sole carbon source. All tests were repeated at least twice with three replicates of each treatment.…”

“…CBS often causes symptoms such as defoliation, shoot blight, and fruit-drop, which pose a serious threat to the citrus industry ( Huang et al, 2015 ). Current research shows that deficiency of oxidative stress resistance, ACT-toxin biosynthesis, or vegetative growth will cause the virulence reduction of A. alternata ( Gai et al, 2019 , 2021 ; Ma et al, 2019 , 2021 ; Chung et al, 2020 ). According to previous studies shown above, histone methylation is critical for the secondary metabolite biosynthesis, multi-stress resistance, vegetative growth, fungal development, and pathogenicity in other fungi.…”