Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most damaging pathogen of soybean (Glycine max (L). Merr), causing an estimated $1.5 billion in annual yield losses in the United States. Planting SCN-resistant soybean cultivars is the most effective control strategy, with two major resistant types widely deployed in commercial cultivars. PI 88788-type resistance requires high copy rhg1-b, while Peking-type resistance, as in cultivar Forrest, requires low copy rhg1-a and Rhg4. Rhg4 encodes a serine hydroxymethyltransferase (GmSHMT08-a) differing by two amino acids from the susceptible version (GmSHMT08-b) that impact the enzyme’s ability to bind folate. SHMTs are critical for 1-C metabolism in all organisms, catalyzing the interconversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene-THF. The soybean genome harbors two cytosolic SHMT genes, GmSHMT08 and GmSHMT05. However, the molecular basis of GmSHMT08-a mediated resistance remains unclear. Here, we leveraged a unique set of soybean Gmshmt08-a mutants to pinpoint GmSHMT08-a dependent gene expression changes in response to SCN via transcriptomic profiling. Findings suggest that GmSHMT08-a enhances the expression of genes involved in cysteine-methionine metabolism and redox homeostasis, activating plant defense pathways linked to jasmonic acid and ethylene signaling with potential roles in triggering the degeneration of the nematode’s feeding site. To investigate the roles of these genes further, we optimized Rhizobium rhizogenes-mediated hairy root transformation with CRISPR/Cas9 gene knockout, streamlining high-throughput nematode phenotyping assays in a greenhouse setting. This protocol overcame space limitations, increased transformation efficiency, and reduced time and resources. To examine potential defense-growth trade-offs of GmSHMT08-a, we analyzed the role of cytosolic SHMTs in soybean growth and development using structural biochemistry, field phenotyping of Gmshmt08 mutants, and CRISPR/Cas9 editing. Analysis of phenotypic traits of Gmshmt08-a under field conditions revealed a negative influence of GmSHMT08-a on pod and seed number demonstrating a trade-off in soybean growth and development with SCN resistance. Recent studies emphasized the involvement of additional genes in Peking-type resistance, extending beyond rhg1-a and Rhg4 to provide broad-spectrum resistance. In this study, we characterized GmSNAP02, a novel gene providing SCN resistance through a loss-of-function. Together, the findings have advanced our understanding of GmSHMT08-a mediated resistance and its implication for soybean growth and development, and identified a novel gene in resistance, both with immediate impacts for breeding SCN-resistant cultivars.