Engineered Resistance to Soybean Cyst Nematodes via Induced Gene Silencing (RNAi)
by Harold N. Trick, Director of KSU Plant Transformation Lab, Kansas State University
SCN females and cysts. Photo: ISU
RNA interference (RNAi) is a biological process in which certain RNA molecules inhibit gene expression. In the free-living nematode Caenorhabditis elegans
, which serves as an experimental model for the soybean cyst nematode (SCN), specific genes can be selectively turned off in the nematode by either injecting or feeding small interfering RNA molecules (siRNA) to the worms. We are using this approach, also called gene silencing
, by introducing specific siRNAs into soybean so that the soybean cyst nematode will take up these small, selectively lethal molecules upon feeding.
With funding from the Kansas Soybean Commission, United Soybean Board, and the North Central Soybean Research Program, our group of researchers in Kansas, Ohio, Georgia, and Illinois have identified six different nematode genes as potential targets for gene silencing — all of which are required for either the overall fitness of the nematode or for nematode reproduction. By expressing specific DNA sequences in soybean, we have created transgenic lines that produce siRNAs targeting these nematode genes.. Our goal is to provide soybean breeders with lines containing a new set of durable biotech traits for nematode resistance.
In the past year, we have selected four transgenic soybean lines that have shown consistent improved resistance to SCN. Two lines that target a specific nematode gene were able to reduce the number of SCN cysts by 50-60% and the number of SCN eggs by 55-70%, compared to the control. Two lines targeting a second gene were able to reduce both cyst and egg densities by 50 to 70% compared to the control. These four transgenic lines have been increased for more seeds for future studies.
Three bioassays have been conducted over the past year to confirm the resistance of transgenic lines targeting these two genes. The results from these independent experiments confirmed that stable transgenic lines significantly reduce the survival rate of SCN Race 3 on host soybeans.
We also measured the expression levels of target genes within eggs that were isolated from cyst feeding on our transgenic lines. RNA was isolated from eggs derived from two separate transgenic soybean lines and we found that the expression levels of the two targeted SCN parasitism genes were significantly suppressed compared to the controls (SCN eggs recovered from non-transgenic plants). The reduction rates ranged from 3.5 to 4.8-fold for one gene and 4.2 to 5.5- fold for the second, compared to controls. This finding is additional evidence that the host-induced RNA interference can regulate the gene expression of the soybean cyst nematode and reduce the survival rate of the SCN population.
From the results of RNA-sequencing analysis, it appears that the resistance resulting from siRNAs expression is dependent upon the amount of siRNA molecules in the host plant and that there is a certain threshold expression needed to elicit a resistant phenotype. We have sequenced two of our target genes from a number of HG types and have demonstrated that the genes are highly conserved, which tells us we are working with genes that are unique and essential, and not likely to change.
We are preparing a manuscript on these latest results and will post a link on this website when available. An earlier research publication, Host-derived suppression of nematode reproductive and fitness genes decreases fecundity of Heterodera glycines
, has been published in Planta
Read more about soybean cyst nematode
Read more about SCN-resistant soybean varieties