Enzymatic regulation of methionine/homocysteine levels by the folate pathway dictates broad-spectrum defense in Arabidopsis.

Folate-dependent one‑carbon (1C) metabolism is vital for plant development and stress responses, but its specific role in immunity remains unclear. Arabidopsis Methylenetetrahydrofolate reductase 2 (MTHFR2), a prominent enzyme in 1C metabolism, is crucial in regulating methionine biosynthesis and controlling broad-spectrum immunity. Here, we show by HRMS profiling that loss-of-function mutation in MTHFR2 disturbs methionine and homocysteine concentrations, which lead to differential immune responses against a broad-spectrum of pathogens, including Magnaporthe oryzae, Rhizoctonia solani, and Pseudomonas syringae. The untargeted metabolomic study revealed notable alterations in key 1C metabolites and a toxic buildup of homocysteine thiolactone that worsened stress regulation in mthfr2 mutants. Further, we observed that exogenous methionine application dampened fungal growth, indicating its bi-functional role in immunological signaling and direct protection. Moreover, the synergistic effects of MTHFR2 and nonhost resistance genes like HEAT SHOCK PROTEIN 90 (HSP90), SUPPRESSOR OF BIR1 (SOBIR1), and RESPIRATORY BURST OXIDASE HOMOLOGY F (AtRBOHF) significantly enhanced ROS signaling and defense gene expression. These results indicate that metabolic imbalances impact salicylic acid- and jasmonic acid-mediated pathways, thereby alerting immune responses. Our study demonstrates that Arabidopsis MTHFR2, a key enzyme in folate-dependent 1C metabolism, regulates methionine biosynthesis and is indispensable for the plant's broad-spectrum immunity. By linking folate metabolism to the plant's immune cascade, we uncover a crucial biochemical mechanism that strengthens the plant's resistance to pathogens.