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1.
FEBS J ; 291(2): 323-337, 2024 01.
Article in English | MEDLINE | ID: mdl-37811683

ABSTRACT

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.


Subject(s)
Cysts , Nematoda , Animals , Glycine max/genetics , Glycine Hydroxymethyltransferase/chemistry , Nematoda/metabolism , Folic Acid , Plant Diseases
2.
Mol Plant Pathol ; 21(9): 1227-1239, 2020 09.
Article in English | MEDLINE | ID: mdl-32686295

ABSTRACT

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.


Subject(s)
Arabidopsis/immunology , Gene Expression Regulation, Plant , Glycine max/genetics , Plant Diseases/immunology , Plant Proteins/metabolism , Tylenchoidea/physiology , Animals , Arabidopsis/genetics , Arabidopsis/parasitology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Death , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Plant Diseases/parasitology , Plant Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/physiology , Glycine max/parasitology
3.
J Biol Chem ; 295(11): 3708-3718, 2020 03 13.
Article in English | MEDLINE | ID: mdl-32014996

ABSTRACT

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.


Subject(s)
Disease Resistance , Folic Acid/metabolism , Glycine Hydroxymethyltransferase/metabolism , Glycine max/enzymology , Nematoda/physiology , Plant Diseases/parasitology , Plant Proteins/metabolism , Animals , Binding Sites , Conserved Sequence , Glycine Hydroxymethyltransferase/chemistry , Kinetics , Ligands , Models, Biological , Models, Molecular , Plant Proteins/chemistry , Pyridoxal Phosphate/metabolism , Static Electricity , Structural Homology, Protein , Tetrahydrofolates/chemistry , Tetrahydrofolates/metabolism
4.
Plant Physiol ; 175(3): 1370-1380, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28912378

ABSTRACT

Rhg4 is a major genetic locus that contributes to soybean cyst nematode (SCN) resistance in the Peking-type resistance of soybean (Glycine max), which also requires the rhg1 gene. By map-based cloning and functional genomic approaches, we previously showed that the Rhg4 gene encodes a predicted cytosolic serine hydroxymethyltransferase (GmSHMT08); however, the novel gain of function of GmSHMT08 in SCN resistance remains to be characterized. Using a forward genetic screen, we identified an allelic series of GmSHMT08 mutants that shed new light on the mechanistic aspects of GmSHMT08-mediated resistance. The new mutants provide compelling genetic evidence that Peking-type rhg1 resistance in cv Forrest is fully dependent on the GmSHMT08 gene and demonstrates that this resistance is mechanistically different from the PI 88788-type of resistance that only requires rhg1 We also demonstrated that rhg1-a from cv Forrest, although required, does not exert selection pressure on the nematode to shift from HG type 7, which further validates the bigenic nature of this resistance. Mapping of the identified mutations onto the SHMT structural model uncovered key residues for structural stability, ligand binding, enzyme activity, and protein interactions, suggesting that GmSHMT08 has additional functions aside from its main enzymatic role in SCN resistance. Lastly, we demonstrate the functionality of the GmSHMT08 SCN resistance gene in a transgenic soybean plant.


Subject(s)
Disease Resistance , Glycine Hydroxymethyltransferase/genetics , Glycine max/enzymology , Glycine max/parasitology , Mutagenesis/genetics , Plant Diseases/immunology , Plant Diseases/parasitology , Tylenchoidea/physiology , Animals , Genetic Complementation Test , Genetic Testing , Glycine Hydroxymethyltransferase/chemistry , Models, Molecular , Mutation/genetics , Plants, Genetically Modified , Glycine max/immunology , Tylenchoidea/pathogenicity , Virulence
5.
Nat Commun ; 8: 14822, 2017 03 27.
Article in English | MEDLINE | ID: mdl-28345654

ABSTRACT

Two types of resistant soybean (Glycine max (L.) Merr.) sources are widely used against soybean cyst nematode (SCN, Heterodera glycines Ichinohe). These include Peking-type soybean, whose resistance requires both the rhg1-a and Rhg4 alleles, and PI 88788-type soybean, whose resistance requires only the rhg1-b allele. Multiple copy number of PI 88788-type GmSNAP18, GmAAT, and GmWI12 in one genomic segment simultaneously contribute to rhg1-b resistance. Using an integrated set of genetic and genomic approaches, we demonstrate that the rhg1-a Peking-type GmSNAP18 is sufficient for resistance to SCN in combination with Rhg4. The two SNAPs (soluble NSF attachment proteins) differ by only five amino acids. Our findings suggest that Peking-type GmSNAP18 is performing a different role in SCN resistance than PI 88788-type GmSNAP18. As such, this is an example of a pathogen resistance gene that has evolved to underlie two types of resistance, yet ensure the same function within a single plant species.


Subject(s)
Disease Resistance/genetics , Genes, Plant , Glycine max/genetics , Glycine max/parasitology , Nematoda/physiology , Soybean Proteins/genetics , Alleles , Animals , Cloning, Molecular , DNA, Plant/genetics , Genetic Complementation Test , Haplotypes , High-Throughput Nucleotide Sequencing , Host-Parasite Interactions , INDEL Mutation , Models, Genetic , Polymorphism, Single Nucleotide
6.
BMC Res Notes ; 6: 255, 2013 Jul 06.
Article in English | MEDLINE | ID: mdl-23830484

ABSTRACT

BACKGROUND: Bean pod mottle virus (BPMV) based virus-induced gene silencing (VIGS) vectors have been developed and used in soybean for the functional analysis of genes involved in disease resistance to foliar pathogens. However, BPMV-VIGS protocols for studying genes involved in disease resistance or symbiotic associations with root microbes have not been developed. FINDINGS: Here we describe a BPMV-VIGS protocol suitable for reverse genetic studies in soybean roots. We use this method for analyzing soybean genes involved in resistance to soybean cyst nematode (SCN). A detailed SCN screening pipeline is described. CONCLUSIONS: The VIGS method described here provides a new tool to identify genes involved in soybean-nematode interactions. This method could be adapted to study genes associated with any root pathogenic or symbiotic associations.


Subject(s)
Comovirus/metabolism , Gene Silencing , Genetic Vectors , Glycine max/genetics , Glycine max/parasitology , Nematoda/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/parasitology , RNA Interference , Animals , Gene Expression Regulation, Plant , Host-Parasite Interactions/genetics , Plant Roots
7.
Curr Opin Plant Biol ; 16(4): 457-63, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23890967

ABSTRACT

Sedentary plant-parasitic nematodes (PPNs) establish specialized feeding cells within roots to maintain long-term relationships with their hosts. However, feeding cells degenerate prematurely in plants that harbor resistance (R) genes against these parasites reducing their life span and ability to reproduce. Recognition of the nematode, mediated directly or indirectly by plant R proteins, occurs via nematode secreted effectors and evokes a resistance response, which is referred to as effector-triggered immunity (ETI). Recent breakthroughs in nematode effector biology shed new light on key players mediating ETI and have identified those involved in plant defense suppression as novel targets for engineering resistance in transgenic plants. Advances in plant genetics and genomics has facilitated the discovery of R genes to nematodes. Nevertheless, underlying resistance mechanisms remain poorly understood and are confounded by recently identified R genes that do not fit previously proposed paradigms. Thus, there is still much to be learned about how plants fight off underground attacks from PPNs. In coming years, we can expect breakthroughs in our understanding of the nature and mechanisms of plant resistance and nematode virulence as we explore these novel R genes.


Subject(s)
Embryophyta/physiology , Helminth Proteins/genetics , Nematoda/physiology , Plant Proteins/genetics , Animals , Embryophyta/genetics , Food Chain , Helminth Proteins/metabolism , Nematoda/genetics , Plant Immunity , Plant Proteins/metabolism , Rubber
8.
Nature ; 492(7428): 256-60, 2012 Dec 13.
Article in English | MEDLINE | ID: mdl-23235880

ABSTRACT

Soybean (Glycine max (L.) Merr.) is an important crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (Heterodera glycines Ichinohe) is a microscopic roundworm that feeds on the roots of soybean and is a major constraint to soybean production. This nematode causes more than US$1 billion in yield losses annually in the United States alone, making it the most economically important pathogen on soybean. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Here we report the map-based cloning of a gene at the Rhg4 (for resistance to Heterodera glycines 4) locus, a major quantitative trait locus contributing to resistance to this pathogen. Mutation analysis, gene silencing and transgenic complementation confirm that the gene confers resistance. The gene encodes a serine hydroxymethyltransferase, an enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Alleles of Rhg4 conferring resistance or susceptibility differ by two genetic polymorphisms that alter a key regulatory property of the enzyme. Our discovery reveals an unprecedented plant resistance mechanism against a pathogen. The mechanistic knowledge of the resistance gene can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop.


Subject(s)
Glycine max/genetics , Glycine max/parasitology , Host-Parasite Interactions , Nematoda/physiology , Plant Proteins/genetics , Plant Proteins/metabolism , Amino Acid Sequence , Animals , DNA Mutational Analysis , Gene Order , Gene Silencing , Genetic Complementation Test , Glycine Hydroxymethyltransferase/genetics , Glycine Hydroxymethyltransferase/metabolism , Haplotypes , Models, Molecular , Molecular Sequence Data , Plant Proteins/chemistry , Polymorphism, Genetic/genetics , Protein Structure, Tertiary , Quantitative Trait Loci/genetics , Glycine max/enzymology
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