Proteomic, Transcriptomic, Mutational, and Functional Assays Reveal the Involvement of Both THF and PLP Sites at the GmSHMT08 in Resistance to Soybean Cyst Nematode

The serine hydroxymethyltransferase (SHMT; E.C. 2.1.2.1) is involved in the interconversion of serine/glycine and tetrahydrofolate (THF)/5,10-methylene THF, playing a key role in one-carbon metabolism, the de novo purine pathway, cellular methylation reactions, redox homeostasis maintenance, and methionine and thymidylate synthesis. GmSHMT08 is the soybean gene underlying soybean cyst nematode (SCN) resistance at the Rhg4 locus. GmSHMT08 protein contains four tetrahydrofolate (THF) cofactor binding sites (L129, L135, F284, N374) and six pyridoxal phosphate (PLP) cofactor binding/catalysis sites (Y59, G106, G107, H134, S190A, H218). In the current study, proteomic analysis of a data set of protein complex immunoprecipitated using GmSHMT08 antibodies under SCN infected soybean roots reveals the presence of enriched pathways that mainly use glycine/serine as a substrate (glyoxylate cycle, redox homeostasis, glycolysis, and heme biosynthesis). Root and leaf transcriptomic analysis of differentially expressed genes under SCN infection supported the proteomic data, pointing directly to the involvement of the interconversion reaction carried out by the serine hydroxymethyltransferase enzyme. Direct site mutagenesis revealed that all mutated THF and PLP sites at the GmSHMT08 resulted in increased SCN resistance. We have shown the involvement of PLP sites in SCN resistance. Specially, the effect of the two Y59 and S190 PLP sites was more drastic than the tested THF sites. This unprecedented finding will help us to identify the biological outcomes of THF and PLP residues at the GmSHMT08 and to understand SCN resistance mechanisms.