Systematic Mutagenesis of Serine Hydroxymethyltransferase Reveals an Essential Role in Nematode Resistance

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.

Figure 1.

Characterization of GmSHMT08 complemented transgenic soybean plants. A, SCN phenotype of T1 plants. Each dot corresponds to the number of cysts on an individual plant. B, SCN phenotype of plants in the T2 generation. The seeds from T1 plants P2, P10, and P12 were used in the greenhouse assay. Each dot corresponds to the number of cysts on an individual plant.

Figure 2.

GmSHMT08 mutants identified by forward genetic screening. SCN phenotyping of EMS-mutagenized soybean cv Forrest identified GmSHMT08 mutants exhibiting a loss of resistance. A, Schematic depicting the positions of GmSHMT08 mutations within the GmSHMT08 protein sequence. The mutant line name and the amino acid change (parentheses) are shown. Mutants F6266 and F6756 were discovered previously by Liu et al. (2012). The red arrowheads correspond to amino acid polymorphisms in GmSHMT08 between resistant cv Forrest and susceptible cv Essex. B, The SCN FI of newly identified GmSHMT08 mutants in comparison with resistant cv Forrest. Each dot corresponds to the FI of an individual plant.

Figure 3.

Characterization of GmSHMT08 mutants. A, SCN phenotype of GmSHMT08 mutants in comparison with resistant (cv Forrest and EXF67) and susceptible (cv Essex, EXF63, and EXF50) soybean lines. Each dot corresponds to the number of cysts on an individual plant. Asterisks denote statistically significant differences in resistance in comparison with cv Forrest (paired Student’s t test, P < 0.01, red; P < 0.0001, black). B, Rhg4 expression in cv Forrest and the F234 mutant. Root cDNA was used as a template for qPCR. C, E. coli complementation assay using mutant GmSHMT08 proteins. The proteins were expressed under the control of the IPTG-inducible T7 promoter in E. coli shmt mutant GS245 pLysS strain. Proteins were induced with 0.25 mm IPTG at 37°C. The absorbance of bacterial cultures at 600 nm was measured at the time intervals shown and plotted.

Figure 4.

HG type tests of selected nematode populations MM1 and MM2. The nematode populations MM1 and MM2 were derived by mass selection of PA3 for more than 60 generations on RILs EXF63 (MM1) and EXF67 (MM2). The FI values on each resistance indicator line with respect to susceptible soybean cv Lee74 were plotted. The dotted line denotes the 10% FI used as the cutoff value in determining resistance to SCN.

Figure 5.

Computation modeling and mapping of mutants on the GmSHMT08 structure. A, Mutation positions on or in close proximity to the dimer binding sites (solid gray and solid light blue) for both interacting subunits. Shown in red (with blue labels) are mutation positions for the first subunit and in navy (with red labels) for the second subunit. B, Mutation positions on or in close proximity to the tetramer binding sites (solid yellow and solid green) for both interacting subunits. Shown in orange (with blue labels) are mutation positions for the first subunit and in purple (with red labels) for the second subunit.

Figure 6.

Computation modeling and mapping of mutants on the GmSHMT08 structure. A, Mutation positions on or in close proximity to the functional sites (THF, MTHF, and FTHF in light pink, PLP-serine/PLP-glycine (PLS/PLG) in light orange, and Gly in light purple) for both interacting subunits. Shown in magenta are mutation positions for the first subunit and in green for the second subunit. B, GmSNAP18 protein-binding sites mapped on the GmSHMT08 tetramer together with its surface mutations. Shown are the surface mutations mapped on only one of the dimer subunits. The two GmSHMT08 dimers are colored gray and light blue. The two putative GmSNAP18 binding sites for each dimer are shown in black and navy. Shown in red (with blue labels) are mutation positions for one monomer of the first dimer and in magenta (with red labels) for a monomer of the second dimer.