Objective 1: Enhance Soybean Cyst Nematode (SCN) resistance in transgenic soybean by modifying gene silencing strategies.
For the past few years we have been evaluating the effectiveness of traits to provide resistance to soybean cyst nematodes (SCN). Many of these traits have been designed to silence specific genes within the nematode and we have demonstrated a reduction in cyst numbers on these transgenic lines. We can further increase the resistance level by 1) using alternative gene sequences of these genes and 2) increasing the levels of siRNA produced by the plant. We have been targeting approximately 200-300 nucleotides of a given nematode gene with our current gene silencing approach. This is approximately 10 to 30% of the entire sequence of most target genes. Although we have demonstrated the effectiveness of this method, targeting alternate sequences of a particular gene may improve the silencing effect. We propose to take two of the genes previously used (one high and one low cyst/egg reduction from the bioassay) and target alternative sequences of the genes for gene silencing. Such a study will provide us with critical data in regards to the selection of future target sequences.
In general, the RNAi mechanism for gene silencing is based on a large (exponential) amplification of small interfering RNA (siRNA) molecules that bind to a specific gene sequence. Many laboratories including our own use this approach to effectively silence the plant’s own genes. For endogenous plant genes, the RNAi mechanism will produce siRNA molecules that recognize the total gene sequence, even if only 10% of the entire gene sequence is targeted, which in turn will cause a very high degree (possibly complete) of gene silencing. Our current methodology produces only siRNAs that correspond to the specific sequence (200 to 300 bp) fragment found in our DNA construction. The quantity of siRNA species does not increase exponentially because the nematode gene target is not found in the plant. We propose to over-express the targeted nematode gene sequence (either in the sense or antisense orientation) together with the RNAi vector construction. This approach should allow the exponential accumulation of siRNA species in the transgenic soybean plants thereby allowing a greater number of siRNA molecules to be ingested by the feeding nematode. This increase in siRNA ingested by the nematode should translate into increased SCN resistance. We have alternative transgenic approach to reduce SCN reproduction that is ongoing and an update will be given at the formal proposal presentation in December.
To assess the effectiveness of the above strategies greenhouse SCN bioassays on composite plants or transgenic soybean lines, as well as negative controls, will be performed. Lines will be planted into SCN infected soil (~6000 eggs/100 cm3) and grown in the greenhouse for five weeks. Soybean roots will then be washed free of soil and debris, SCN cysts removed from each plant and the number of cysts, eggs and root weight data will be collected for each replicate. Data collected from each bioassay will be examined by analysis of variance with the GLM procedure in SAS. Many of the transgenic lines made for SCN control have sequences similar enough to RKN genes so these will also be tested to see if they provide cross protection (i.e. resistance to both SCN and RKN). Additionally we will field test this material in 2019.
Transgenic lines generated from this research project will be incorporated into elite Kansas lines under the KSC funded project “Breeding and Management of Soybean for Improved Performance”. Where intellectual property rights are involved, the Kansas State University Research Foundation will be advised and they will assist us in the transfer of technology to third parties.
Objective 2. Transgenic approaches for increased fungal resistance with emphasis on SDS.
Sudden Death Syndrome (SDS) is caused by Fusarium virguliforme, a soil-borne fungus. Disease symptoms have been attributed to specific toxins produced by the fungus. One study indicated that when the fungal toxin gene FvTox1 was turned off in the pathogen by mutations, no symptoms developed on infected soybeans (Pudake et al., 2013). Our previous work using a gene silencing strategy targeting SCN genes is showing promising results and would serve as a model silencing the FvTox1 gene in F. virguliforme. With Chris Little we have successfully established a seedling assay for testing our transgenics using a hairy root bioassay. We have created silencing vectors for the FvTox1 gene, begun engineering soybean cultures, and plan to challenge the transgenic material with F. virguliforme. A positive result would be indicated by inhibition of fungal growth and absence of the disease. In addition to the FvTox1 gene we will look at other targets to silence in the fungus.
Additionally, we will investigate a separate approach to produce fungal resistance. Defensins and their relatives are peptides or small proteins that can inhibit antimicrobial growth (De Lucca and Walsh, 1999). These peptides are present in plants, insects, and vertebrates. We have selected five peptides from various sources, optimized expression for soybean, created expression vectors, and transformed soybean cultures. Seeds recovered from transgenics will be used first evaluate growth inhibition on F. virguliforme (SDS) but will screen other pathogens such as Macrophomina phaseolina (charcoal rot). For bioassays we will cooperate with Dr. Chris Little, KSU’s row crop pathologist.
Objective 3: Improve drought tolerance in soybean by manipulating drought tolerance-associated genes.
Drought is one of critical abiotic stresses limiting soybean production in Kansas. Changing expression patterns in drought related genes may increase tolerance. The transcriptional factors (proteins that regulates gene expression) belonging to the NAC (NAM, ATAF and CUC) gene family are closely related to drought-responsive genes in plants. Many members of NAC family enhanced drought tolerance have been reported. For example, the alteration of root architecture by osNAC9 in rice improved plant drought resistance and grain yield (Redillas et al., 2012). In recent analysis of soybean NAC gene family related to drought tolerance, several specific genes were identified in drought tolerant cultivars (Hussain et al., 2017). Our goal with this objective is to overexpress and/or down regulate selected transcription factors in hairy roots and evaluated root architecture and their response to drought conditions. Any genes that show potential we will then produce stable transgenic lines for further evaluations.
Objective 4. Evaluation of potential transgenic solutions to stem borer.
Recent findings made under the KSC funded project “Development of soybean host plant resistance and other management options for the soybean stem borer” (C.M. Smith, PI) have demonstrated the potential for stem borer development and viability by gene silencing. Similar to our SCN work we propose to engineer soybean with these gene-silencing constructs and then provide C.M. Smith these transgenic cultures and lines for his KSC funded program. In the FY2019 we are making the vectors for hairy root analysis and for stable transgenic plants. In FY2020 we will continue with bioassays and generate transgenic lines of the appropriate vectors.