Soybean crops are becoming increasingly important throughout the world as a source of renewable oil as well as protein. However, they are also a crop that poses many great challenges during production. Perhaps the most destructive of these, a pest called the cyst nematode (Heterodera glycines Ichinohe), has been controlled using resistant soybean plants for many years. However, very little is known about the actual mechanism behind this resistance, and new understanding is desperately needed as old resistant strains of soybean lose their effectiveness. Liu et al.(2012) set out to clone the specific gene that provides resistance to cyst nematodes, and find what proteins it produces to accomplish this feat. The researchers used complicated methods to isolate a gene within the Rhg4 (resistance to Heterodera glycines 4) section of soybean DNA as the gene of interest, and determine that it is responsible for the production of an enzyme that interconverts serine and glycine, two different amino acids. In the future, this knowledge will lead to genetically modified soybean strains and increased production.—Chad Redman
Liu, S., Kandoth, P. K., Warren, S. D., Yeckel, G., Heinz, R., Alden, J., Yang, C., Jamai1, A., El-Mellouki, T., Juvale, P. S., Hill, J., Baum, T. J., Cianzio, S., Whitham, S. A., Korkin, D., Mitchum, M. G., Meksem, K., 2012. A Soybean Cyst Nematode Resistance Gene Points to a New Mechanism of Plant Resistance to Pathogens. Nature 492, 256–262.
Liu et al. investigated the genes responsible for making certain strains of soybean resistant to Heterodera glycines (rgh), a prevalent and damaging pest commonly known as the cyst nematode. While the general locations, known as quantitative trait loci, of these genes have been mapped for many years, the genes themselves have not before been precisely defined, cloned, or examined for their specific functions. The genes in question have been tracked down to chromosomes 18 (rgh1) and 8 (Rgh4) in the soybean genome, with Rgh4 being the more significant and important. Therefore, the team of researchers began by breeding a variety of soybean called Forrest, which exhibits resistance to cyst nematodes thanks to rgh1 and Rgh4 working in tandem. Because the resistance alleles of the mystery genes are not completely dominant, the lines of Forrest soybean had to be inbred for several generations, and comparing the resistant and nonresistant offspring that were produced gave Liu et al. the opportunity to track down exactly what the differences were. In short, by breeding nematode resistant soybean plants, researchers could define exactly what genes at the rgh1 and Rgh4 quantitative trait loci were causing certain plants to exhibit resistance.
After bounding the gene that they suspected to be linked to nematode resistance, Liu et al. devised several ways of confirming that they had identified the correct gene. First, they used a technique called TILLING, which essentially searches for mutations in DNA strands. Through this technique, the researchers were able to identify the absolute pinpoint differences between the genes of interest in resistant versus nonresistant plants.
After using TILLING, Liu et al. sequenced the gene which they suspected contributed cyst nematode resistance to soybean plants in 81 fully distinct varieties of soybean. They were testing to find out if the lines which exhibited a resistant phenotype also possessed the gene with the correct mutations that had been revealed through the use of TILLING. Simillarly, they examined the nonresistant plants to test for the opposite allele of their gene suspect.
In order to further strengthen the connection between the identified gene and soybean resistance to cyst nematodes, the researchers implemented a technique known as virus-induced gene silencing. Fortunately, this name says it all; a virus is designed to effectively “turn off” the targeted gene. Liu et al. infected soybean plant tissue with this virus and exposed previously resistant plants to nematodes.
Next, researchers wanted to confirm that they had found a gene that was acting from the Rgh4 site of the soybean genome. This was done relatively simply; a nematode-susceptible plant had the Rgh4 section of DNA from a resistant plant introduced into its gnome, and then its resistance was measured relative to its original level of resistance.
As a final step in their procedure, Liu et al. sought a picture of what protein the gene they had isolated was coding for. Using yet another complicated technique called homology modeling, researchers were able to determine what protein was being produced and what its function was. Moreover, they ascertained how proteins varied from resistant to nonresistant soybean plants. Once a suspect protein was recognized, the different forms of this protein were injected into E. coli strains that needed the same protein to survive. The functionality of these E. coli was telling of the function served by the protein in question.
Certainly, Liu et al. conducted a procedure with many complicated facets. However, they remained wonderfully focused and their results are easily summarized, as they all point to the same conclusions. The early work produced lines of resistant and nonresistant soybean which exhibited one difference in particular that interested researchers; a gene called SHMT was found to have five point mutations that differentiated resistant and nonresistant soybean plants. From here, the team of researchers was able to prove that this gene was causing nematode resistance using TILLING, which allowed them to identify the precise difference between the two gene alleles. What this allowed for was the testing of each specific mutation for its effect on resistance. Additionally, by sequencing SHMT in 81 different varieties of soy bean, Liu et al.found that resistant strains had the SHMTallele which they had predicted would lead to resistance from the TILLING method. Still more, silencing the SHMT gene removed nematode resistance from Forrest soybean plants, indisputable proof of its role in providing such resistance. In their attempt to show that SHMT is in the Rgh4 locus, the researchers succeeded, finding that transplanting this section of DNA from a resistant plant to a nonresistant plant would transfer resistance over. Essentially, this finding merely affirms the importance of Rgh4 over rgh1. And finally, Liu et al. uncovered just what SHMT is responsible for producing. They found that it codes for an enzyme which reversibly converts glycine to serine. In fact, they suspect the resistant allele of this protein interrupts this process, a surprising finding given that this protein is conserved across a huge number of species, including humans, and is essential to metabolism. However, there was some uncertainty as to whether or not there may be other mechanisms being affected that were not described. In sum, what this enlightening paper found was that SHMT produces a protein which inhibits the ability of the cyst nematode to feed off the roots of soybean plants, potentially solving a huge problem for the agricultural industry.