Conserved structural dynamics of MTHFR2 link one-carbon metabolism to metabolite-based plant immunity

Methylenetetrahydrofolate reductase 2 (MTHFR2) plays a vital role in the one-carbon (1C) pathway, mediating plant immunity in Arabidopsis against rice blast, a role conserved across the plant kingdom. To understand the structural, conformational dynamics, evolutionary modifications and molecular activity of MTHFR2, we identified that the ^A55_V substitution in an EMS mutant of Arabidopsis which diminishes disease resistance against rice blast. We hypothesized about its key role in regulating the enzyme activities involved in the 1C metabolic pathway. Molecular dynamic simulations, demonstrated that the ^A55_V mutation induces structural alterations and instability at the enzyme active site, affecting its function. Ramachandran plot analysis revealed that the mutant had a slight reduction in favored conformations (94.097% vs. 94.662%). The root mean square deviation analysis exhibited 48.7% and 183% increase in structural deviation and conformational variability, respectively, in the mutant suggesting lower stability. Root mean square fluctuation analysis showed a 39.8% increase in the flexibility of residues in the NADH binding pocket, indicating impaired ligand recognition, altering the resultant product (5-CH₃-THF). Differences in global compactness (radius of gyration) and solvent exposure (solvent-accessible surface area) were minimal, yet localized instability was apparent. Evolutionary analysis revealed that MTHFR2 was highly conserved among plant species and soybean orthologs had 81% similarity, therefore exhibit nonhost resistance. In vitro functional assays showed that soybean extracts suppressed Magnaporthe oryzae conidia germination and development, implying a conserved metabolite-based defense mechanism. Collectively, our findings establish a structural and functional framework for understanding MTHFR2's conserved role and lay the groundwork for future research aiming at connecting enzyme function to metabolite-based immunity in plants.