Evaluation of Chemical-Inducible Gene Expression Systems for Beet Cyst Nematode Infection Assays in Arabidopsis thaliana.

Cyst nematodes co-opt plant developmental programs for the establishment of a permanent feeding site called a syncytium in plant roots. In recent years, the role of plant developmental genes in syncytium formation has gained much attention. One main obstacle in studying the function of development-related genes in syncytium formation is that mutation or ectopic expression of such genes can cause pleiotropic phenotypes making it difficult to interpret nematode-related phenotypes, or in some cases, impossible to carry out infection assays due to aberrant root development. Here, we tested three commonly used inducible gene expression systems for their application in beet cyst nematode infection assays of the model plant Arabidopsis thaliana. We found that even a low amount of ethanol diminished nematode development, deeming the ethanol-based system unsuitable for use in cyst nematode infection assays; whereas treatment with estradiol or dexamethasone did not negatively affect cyst nematode viability. Dose and time course responses showed that in both systems, a relatively low dose of inducer (1 µM) is sufficient to induce high transgene expression within 24 hours of treatment. Transgene expression peaked at 3-5 days post induction and began to decline thereafter, providing a perfect window for inducible transgenes to interfere with syncytium establishment while minimizing any adverse effects on root development. These results indicate that both estradiol- and dexamethasone-based inducible gene expression systems are suitable for cyst nematode infection assays. The employment of such systems provides a powerful tool to investigate the function of development essential plant genes in syncytium formation.

Development of a Standardized Soybean Cyst Nematode Screening Assay in Pennycress and Identification of Resistant Germplasm.

The prospect of incorporating pennycress as an oilseed cover crop in the Midwest's corn-soybean rotation system has drawn researcher and farmer attention. The inclusion of pennycress will be beneficial as it provides an excellent soil cover to reduce soil erosion and nutrient leaching while serving as an additional source for oilseed production and income. However, pennycress is an alternative host for soybean cyst nematode (SCN), which is a major biological threat to soybean that needs to be addressed for sustainable pennycress adoption into our current production systems. To develop a standardized SCN resistance screening strategy in pennycress, we tested and optimized five parameters: (i) germination stimulants, (ii) inoculation timing, (iii) inoculation rate, (iv) experimental incubation time, and (v) susceptible checks. The standardized SCN resistance screening protocol includes the following: (i) treating pennycress seeds with gibberellic acid for 24 h, (ii) transplanting seedlings 12 to 15 days after initiating germination and inoculating 10 to 12 days after transplantation, (iii) inoculating at a rate of 1,500 eggs/100 cc soil (1,500 eggs per plant), (iv) processing roots at 30 days after inoculation, and (v) using susceptible pennycress accession Ames 32869 to calculate the female index. The standardized protocol was used to quantify the response of a diverse set of pennycress accessions for response against SCN HG type 1.2.5.7 and HG type 7. While there were no highly resistant pennycress lines identified, 15 were rated as moderately resistant to HG type 1.2.5.7, and eight were rated moderately resistant to HG type 7. The resistant lines identified in this study could be utilized to develop SCN-resistant pennycress cultivars. The study also opens a new avenue for research to understand SCN-pennycress interactions through molecular and genomic studies. This knowledge could aid in the successful inclusion of pennycress as a beneficial cover/oilseed crop in the United States Midwest.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Structural and functional analysis of two SHMT8 variants associated with soybean cyst nematode resistance.

Two amino acid variants in soybean serine hydroxymethyltransferase 8 (SHMT8) are associated with resistance to the soybean cyst nematode (SCN), a devastating agricultural pathogen with worldwide economic impacts on soybean production. SHMT8 is a cytoplasmic enzyme that catalyzes the pyridoxal 5-phosphate-dependent conversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate. A previous study of the P130R/N358Y double variant of SHMT8, identified in the SCN-resistant soybean cultivar (cv.) Forrest, showed profound impairment of folate binding affinity and reduced THF-dependent enzyme activity, relative to the highly active SHMT8 in cv. Essex, which is susceptible to SCN. Given the importance of SCN-resistance in soybean agriculture, we report here the biochemical and structural characterization of the P130R and N358Y single variants to elucidate their individual effects on soybean SHMT8. We find that both single variants have reduced THF-dependent catalytic activity relative to Essex SHMT8 (10- to 50-fold decrease in kcat /Km ) but are significantly more active than the P130R/N368Y double variant. The kinetic data also show that the single variants lack THF-substrate inhibition as found in Essex SHMT8, an observation with implications for regulation of the folate cycle. Five crystal structures of the P130R and N358Y variants in complex with various ligands (resolutions from 1.49 to 2.30 Å) reveal distinct structural impacts of the mutations and provide new insights into allosterism. Our results support the notion that the P130R/N358Y double variant in Forrest SHMT8 produces unique and unexpected effects on the enzyme, which cannot be easily predicted from the behavior of the individual variants.

Cataloging SCN resistance loci in North American public soybean breeding programs.

Soybean cyst nematode (SCN) is a destructive pathogen of soybeans responsible for annual yield loss exceeding $1.5 billion in the United States. Here, we conducted a series of genome-wide association studies (GWASs) to understand the genetic landscape of SCN resistance in the University of Missouri soybean breeding programs (Missouri panel), as well as germplasm and cultivars within the United States Department of Agriculture (USDA) Uniform Soybean Tests-Northern Region (NUST). For the Missouri panel, we evaluated the resistance of breeding lines to SCN populations HG 2.5.7 (Race 1), HG 1.2.5.7 (Race 2), HG 0 (Race 3), HG 2.5.7 (Race 5), and HG 1.3.6.7 (Race 14) and identified seven quantitative trait nucleotides (QTNs) associated with SCN resistance on chromosomes 2, 8, 11, 14, 17, and 18. Additionally, we evaluated breeding lines in the NUST panel for resistance to SCN populations HG 2.5.7 (Race 1) and HG 0 (Race 3), and we found three SCN resistance-associated QTNs on chromosomes 7 and 18. Through these analyses, we were able to decipher the impact of seven major genetic loci, including three novel loci, on resistance to several SCN populations and identified candidate genes within each locus. Further, we identified favorable allelic combinations for resistance to individual SCN HG types and provided a list of available germplasm for integration of these unique alleles into soybean breeding programs. Overall, this study offers valuable insight into the landscape of SCN resistance loci in U.S. public soybean breeding programs and provides a framework to develop new and improved soybean cultivars with diverse plant genetic modes of SCN resistance.

Loss-of-function of an α-SNAP gene confers resistance to soybean cyst nematode.

Plant-parasitic nematodes are one of the most economically impactful pests in agriculture resulting in billions of dollars in realized annual losses worldwide. Soybean cyst nematode (SCN) is the number one biotic constraint on soybean production making it a priority for the discovery, validation and functional characterization of native plant resistance genes and genetic modes of action that can be deployed to improve soybean yield across the globe. Here, we present the discovery and functional characterization of a soybean resistance gene, GmSNAP02. We use unique bi-parental populations to fine-map the precise genomic location, and a combination of whole genome resequencing and gene fragment PCR amplifications to identify and confirm causal haplotypes. Lastly, we validate our candidate gene using CRISPR-Cas9 genome editing and observe a gain of resistance in edited plants. This demonstrates that the GmSNAP02 gene confers a unique mode of resistance to SCN through loss-of-function mutations that implicate GmSNAP02 as a nematode virulence target. We highlight the immediate impact of utilizing GmSNAP02 as a genome-editing-amenable target to diversify nematode resistance in commercially available cultivars.

Discovery of Novel Effector Protein Candidates Produced in the Dorsal Gland of Adult Female Root-Knot Nematodes.

Root-knot nematodes (RKN) (Meloidogyne spp.) represent one of the most damaging groups of plant-parasitic nematodes. They secrete effector proteins through a protrusible stylet to manipulate host cells for their benefit. Stylet-secreted effector proteins are produced within specialized secretory esophageal gland cells, one dorsal gland (DG) and two subventral glands (SvG), whose activity differ throughout the nematode life cycle. Previous gland transcriptomic profiling studies identified dozens of candidate RKN effectors but were focused on the juvenile stages of the nematode, when the SvGs are most active. We developed a new approach to enrich for the active DGs of M. incognita adult female RKN for RNA and protein extraction. Female heads were manually cut from the body, and a combination of sonication and vortexing was used to dislodge contents inside the heads. DG-enriched fractions were collected by filtering, using cell strainers. Comparative transcriptome profiling of pre-parasitic second-stage juveniles, female heads, and DG-enriched samples was conducted using RNA sequencing. Application of an established effector mining pipeline led to the identification of 83 candidate effector genes upregulated in DG-enriched samples of adult females that code for proteins with a predicted signal peptide but lack transmembrane domains or homology to proteins in the free-living nematode Caenorhabditis elegans. In situ hybridization resulted in the identification of 14 new DG-specific candidate effectors expressed in adult females. Taken together, we have identified novel candidate Meloidogyne effector genes that may have essential roles during later stages of parasitism. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Soybean microbiome composition and the impact of host plant resistance.

Microbial communities play an important role in the growth and development of plants, including plant immunity and the decomposition of complex substances into absorbable nutrients. Hence, utilizing beneficial microbes becomes a promising strategy for the optimization of plant growth. The objective of this research was to explore the root bacterial profile across different soybean genotypes and the change in the microbial community under soybean cyst nematode (SCN) infection in greenhouse conditions using 16S rRNA sequencing. Soybean genotypes with soybean cyst nematode (SCN) susceptible and resistant phenotypes were grown under field and greenhouse conditions. Bulked soil, rhizosphere, and root samples were collected from each replicate. Sequencing of the bacterial 16S gene indicated that the bacterial profile of soybean root and soil samples partially overlapped but also contained different communities. The bacterial phyla Proteobacteria, Actinobacteria, and Bacteroidetes dominate the soybean root-enriched microbiota. The structure of bacteria was significantly affected by sample year (field) or time point (greenhouse). In addition, the host genotype had a small but significant effect on the diversity of the root microbiome under SCN pressure in the greenhouse test. These differences may potentially represent beneficial bacteria or secondary effects related to SCN resistance.

Characterization and response of two potato receptor-like kinases to cyst nematode infection.

Plant-parasitic cyst nematodes (Heterodera and Globodera spp.) secrete CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) effector proteins, which act as ligand mimics of plant CLE peptides to promote successful nematode infection. Previous studies of the Arabidopsis-beet cyst nematode (BCN; H. schachtii) pathosystem showed that Arabidopsis CLE receptors including CLAVATA1 (CLV1), CLV2, and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) are required for BCN CLE signaling. Studies further revealed that nematode CLE signaling through GmCLV2 and StCLV2, an Arabidopsis CLV2 orthologue from soybean (Glycines max) and potato (Solanum tuberosum), respectively, is required for the soybean cyst nematode (SCN; H. glycines) and the potato cyst nematode (PCN; G. rostochiensis) to induce disease in their respective host plant. In this study, we identified and characterized two additional potato receptors, StRPK2 and StCLV1, homologues of Arabidopsis RPK2 and CLV1, for a role in PCN parasitism. Using promoter-reporter lines we showed that both StRPK2 and StCLV1 are expressed in the potato root but vary in their spatial expression patterns. Interestingly, StRPK2 but not StCLV1 was found to be expressed and upregulated at PCN infection sites. Nematode infection assays on StRPK2-knockdown lines revealed a decrease in nematode infection. Collectively, our results suggest that parallel CLE signaling pathways involving StCLV2 and StRPK2 are important for PCN parasitism and that manipulation of nematode CLE signaling may represent a viable means to engineer nematode resistance in crop plants including potato.

A novel sugar beet cyst nematode effector 2D01 targets the Arabidopsis HAESA receptor-like kinase.

Plant-parasitic cyst nematodes use a stylet to deliver effector proteins produced in oesophageal gland cells into root cells to cause disease in plants. These effectors are deployed to modulate plant defence responses and developmental programmes for the formation of a specialized feeding site called a syncytium. The Hg2D01 effector gene, coding for a novel 185-amino-acid secreted protein, was previously shown to be up-regulated in the dorsal gland of parasitic juveniles of the soybean cyst nematode Heterodera glycines, but its function has remained unknown. Genome analyses revealed that Hg2D01 belongs to a highly diversified effector gene family in the genomes of H. glycines and the sugar beet cyst nematode Heterodera schachtii. For functional studies using the model Arabidopsis thaliana-H. schachtii pathosystem, we cloned the orthologous Hs2D01 sequence from H. schachtii. We demonstrate that Hs2D01 is a cytoplasmic effector that interacts with the intracellular kinase domain of HAESA (HAE), a cell surface-associated leucine-rich repeat (LRR) receptor-like kinase (RLK) involved in signalling the activation of cell wall-remodelling enzymes important for cell separation during abscission and lateral root emergence. Furthermore, we show that AtHAE is expressed in the syncytium and, therefore, could serve as a viable host target for Hs2D01. Infective juveniles effectively penetrated the roots of HAE and HAESA-LIKE2 (HSL2) double mutant plants; however, fewer nematodes developed on the roots, consistent with a role for this receptor family in nematode infection. Taken together, our results suggest that the Hs2D01-AtHAE interaction may play an important role in sugar beet cyst nematode parasitism.

At the molecular plant-nematode interface: New players and emerging paradigms.

Plant-parasitic nematodes (PPNs) secrete an array of molecules that can lead to their detection by or promote infection of their hosts. However, the function of these molecules in plant cells is often unknown or limited to phenotypic observations. Similarly, how plant cells detect and/or respond to these molecules is still poorly understood. Here, we highlight recent advances in mechanistic insights into the molecular dialogue between PPNs and plants at the cellular level. New discoveries reveal a) the essential roles of extra- and intracellular plant receptors in PPN perception and the manipulation of host immune- or developmental pathways during infection and b) how PPNs target such receptors to manipulate their hosts. Finally, the plant secretory pathway has emerged as a critical player in PPN peptide delivery, feeding site formation and non-canonical resistance.

Epistatic interaction between Rhg1-a and Rhg2 in PI 90763 confers resistance to virulent soybean cyst nematode populations.

KEY MESSAGE: An epistatic interaction between SCN resistance loci rhg1-a and rhg2 in PI 90763 imparts resistance against virulent SCN populations which can be employed to diversify SCN resistance in soybean cultivars. With more than 95% of the $46.1B soybean market dominated by a single type of genetic resistance, breeding for soybean cyst nematode (SCN)-resistant soybean that can effectively combat the widespread increase in virulent SCN populations presents a significant challenge. Rhg genes (for Resistance to Heterodera glycines) play a key role in resistance to SCN; however, their deployment beyond the use of the rhg1-b allele has been limited. In this study, quantitative trait loci (QTL) were mapped using PI 90763 through two biparental F3:4 recombinant inbred line (RIL) populations segregating for rhg1-a and rhg1-b alleles against a SCN HG type 1.2.5.7 (Race 2) population. QTL located on chromosome 18 (rhg1-a) and chromosome 11 (rhg2) were determined to confer SCN resistance in PI 90763. The rhg2 gene was fine-mapped to a 169-Kbp region pinpointing GmSNAP11 as the strongest candidate gene. We demonstrated a unique epistatic interaction between rhg1-a and rhg2 loci that not only confers resistance to multiple virulent SCN populations. Further, we showed that pyramiding rhg2 with the conventional mode of resistance, rhg1-b, is ineffective against these virulent SCN populations. This highlights the importance of pyramiding rhg1-a and rhg2 to maximize the impact of gene pyramiding strategies toward management of SCN populations virulent on rhg1-b sources of resistance. Our results lay the foundation for the next generation of soybean resistance breeding to combat the number one pathogen of soybean.

Pattern-triggered immunity against root-knot nematode infection: A minireview.

Pattern-triggered immunity (PTI) is the basal level of defense a plant has against pathogens. In the case of root-knot nematodes (RKN), PTI relies on the recognition of nematode-associated molecular patterns (NAMPs) for activation. Nematodes have successfully overcome PTI many times by evolving effector proteins to combat PTI responses. As a result, much study has focused on effector-triggered immunity (ETI). Here, we highlight recent advances in our understanding of PTI against RKN. A new interest in understanding PTI in response to RKN infection shows that understanding the basal defense responses RKN have overcome provides critical insight into what mechanisms the effectors have evolved to target in the host plant.

Peptide Effectors in Phytonematode Parasitism and Beyond.

Peptide signaling is an emerging paradigm in molecular plant-microbe interactions with vast implications for our understanding of plant-nematode interactions and beyond. Plant-like peptide hormones, first discovered in cyst nematodes, are now recognized as an important class of peptide effectors mediating several different types of pathogenic and symbiotic interactions. Here, we summarize what has been learned about nematode-secreted CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptide effectors since the last comprehensive review on this topic a decade ago. We also highlight new discoveries of a diverse array of peptide effectors that go beyond the CLE peptide effector family in not only phytonematodes but in organisms beyond the phylum Nematoda.

Esophageal Gland RNA-Seq Resource of a Virulent and Avirulent Population of the Soybean Cyst Nematode Heterodera glycines.

The soybean cyst nematode Heterodera glycines is the most economically devastating pathogen of soybean in the United States and threatens to become even more damaging through the selection of virulent nematode populations in the field that can overcome natural resistance mechanisms in soybean cultivars. This pathogen, therefore, demands intense transcriptomic/genomic research inquiries into the biology of its parasitic mechanisms. H. glycines delivers effector proteins that are produced in specialized gland cells into the soybean root to enable infection. The study of effector proteins, thus, is particularly promising when exploring novel management options against this pathogen. Here, we announce the availability of a gland cell-specific RNA-seq resource. These data represent an expression snapshot of gland cell activity during early soybean infection of a virulent and an avirulent H. glycines population, providing a unique and highly valuable resource for scientists examining effector biology and nematode virulence.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Resistance Gene Pyramiding and Rotation to Combat Widespread Soybean Cyst Nematode Virulence.

Soybean cyst nematode (SCN) is an important pathogen of soybean causing >$1 billion in yield losses annually in the United States. Planting SCN-resistant soybean cultivars is the primary management strategy. Resistance genes derived from the plant introduction (PI) 88788 (rhg1-b) and PI 548402 (Peking; rhg1-a and Rhg4) are the main types of resistance available in commercial cultivars. The PI 88788 rhg1-b resistance allele is found in the majority of SCN-resistant cultivars in the north central United States. The widespread use of PI 88788 rhg1-b has led to limited options for farmers to rotate resistance sources to manage SCN. Consequently, overreliance on a single type of resistance has resulted in the selection of SCN populations that have adapted to reproduce on these resistant cultivars. Here we evaluated the effectiveness of rotating soybean lines with different combinations of resistance genes to determine the best strategy for combating the widespread increase in virulent SCN and limit future nematode adaptation to resistant cultivars. Eight SCN populations were developed by continuous selection of a virulent SCN field population (Heterodera glycines [HG] type 1.2.5.7) on a single resistance source or in rotation with soybean pyramiding different resistance gene alleles derived from PI 88788 (rhg1-b), PI 437654 (rhg1-a and Rhg4), PI 468916 (cqSCN-006 and cqSCN-007), and PI 567516C (Chr10). SCN population densities were determined for eight generations. HG type tests were conducted after the eighth generation to evaluate population shifts. The continued use of rhg1-b or 006/007 had limited effectiveness for reducing SCN type 1.2.5.7 population density, whereas rotation to the use of rhg1-a/Rhg4 resistance significantly reduced SCN population density but selected for broader SCN virulence (HG type 1.2.3.5.6.7). A rotation of rhg1-a/Rhg4 with a pyramid of rhg1-b/006/007/Chr10 was the most effective combination at both reducing population density and minimizing selection pressure. Our results provide guidance for implementation of a strategic SCN resistance rotation plan to manage the widespread virulence on PI 88788 and sustain the future durability of SCN resistance genes.

Phytonematode peptide effectors exploit a host post-translational trafficking mechanism to the ER using a novel translocation signal.

Cyst nematodes induce a multicellular feeding site within roots called a syncytium. It remains unknown how root cells are primed for incorporation into the developing syncytium. Furthermore, it is unclear how CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide effectors secreted into the cytoplasm of the initial feeding cell could have an effect on plant cells so distant from where the nematode is feeding as the syncytium expands. Here we describe a novel translocation signal within nematode CLE effectors that is recognized by plant cell secretory machinery to redirect these peptides from the cytoplasm to the apoplast of plant cells. We show that the translocation signal is functionally conserved across CLE effectors identified in nematode species spanning three genera and multiple plant species, operative across plant cell types, and can traffic other unrelated small peptides from the cytoplasm to the apoplast of host cells via a previously unknown post-translational mechanism of endoplasmic reticulum (ER) translocation. Our results uncover a mechanism of effector trafficking that is unprecedented in any plant pathogen to date, andthey illustrate how phytonematodes can deliver effector proteins into host cells and then hijack plant cellular processes for their export back out of the cell to function as external signaling molecules to distant cells.

Targeted suppression of soybean BAG6-induced cell death in yeast by soybean cyst nematode effectors.

While numerous effectors that suppress plant immunity have been identified from bacteria, fungi, and oomycete pathogens, relatively little is known for nematode effectors. Several dozen effectors have been reported from the soybean cyst nematode (SCN). Previous studies suggest that a hypersensitive response-like programmed cell death is triggered at nematode feeding sites in soybean during an incompatible interaction. However, virulent SCN populations overcome this incompatibility using unknown mechanisms. A soybean BAG6 (Bcl-2 associated anthanogene 6) gene previously reported by us to be highly up-regulated in degenerating feeding sites induced by SCN in a resistant soybean line was attenuated in response to a virulent SCN population. We show that GmBAG6-1 induces cell death in yeast like its Arabidopsis homolog AtBAG6 and also in soybean. This led us to hypothesize that virulent SCN may target GmBAG6-1 as part of their strategy to overcome soybean defence responses during infection. Thus, we used a yeast viability assay to screen SCN effector candidates for their ability to specifically suppress GmBAG6-1-induced cell death. We identified several effectors that strongly suppressed cell death mediated by GmBAG6-1. Two effectors identified as suppressors showed direct interaction with GmBAG6-1 in yeast, suggesting that one mechanism of cell death suppression may occur through an interaction with this host protein.

Screening soybean cyst nematode effectors for their ability to suppress plant immunity.

The soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive pathogens of soybeans. SCN is an obligate and sedentary parasite that transforms host plant root cells into an elaborate permanent feeding site, a syncytium. Formation and maintenance of a viable syncytium is an absolute requirement for nematode growth and reproduction. In turn, sensing pathogen attack, plants activate defence responses and may trigger programmed cell death at the sites of infection. For successful parasitism, H. glycines must suppress these host defence responses to establish and maintain viable syncytia. Similar to other pathogens, H. glycines engages in these molecular interactions with its host via effector proteins. The goal of this study was to conduct a comprehensive screen to identify H. glycines effectors that interfere with plant immune responses. We used Nicotiana benthamiana plants infected by Pseudomonas syringae and Pseudomonas fluorescens strains. Using these pathosystems, we screened 51 H. glycines effectors to identify candidates that could inhibit effector-triggered immunity (ETI) and/or pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We identified three effectors as ETI suppressors and seven effectors as PTI suppressors. We also assessed expression modulation of plant immune marker genes as a function of these suppressors.

Impaired folate binding of serine hydroxymethyltransferase 8 from soybean underlies resistance to the soybean cyst nematode.

Management of the agricultural pathogen soybean cyst nematode (SCN) relies on the use of SCN-resistant soybean cultivars, a strategy that has been failing in recent years. An underutilized source of resistance in the soybean genotype Peking is linked to two polymorphisms in serine hydroxy-methyltransferase 8 (SHMT8). SHMT is a pyridoxal 5'-phosphate-dependent enzyme that converts l-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. Here, we determined five crystal structures of the 1884-residue SHMT8 tetramers from the SCN-susceptible cultivar (cv.) Essex and the SCN-resistant cv. Forrest (whose resistance is derived from the SHMT8 polymorphisms in Peking); the crystal structures were determined in complex with various ligands at 1.4-2.35 Å resolutions. We find that the two Forrest-specific polymorphic substitutions (P130R and N358Y) impact the mobility of a loop near the entrance of the (6S)-tetrahydrofolate-binding site. Ligand-binding and kinetic studies indicate severely reduced affinity for folate and dramatically impaired enzyme activity in Forrest SHMT8. These findings imply widespread effects on folate metabolism in soybean cv. Forrest that have implications for combating the widespread increase in virulent SCN.