Decreasing yield loss and increasing the value of soybeans is part of KSU’s mission to improve Kansas’ agriculture. Our proposal is taking a genetic engineering approach to this mission allowing us to utilize traits outside the scope of conventional breeding. The four objectives of this project are to introduce and evaluate new traits into soybean for increased SCN resistance, increased fungal resistance, improved drought tolerance or resistance to Dectes stem borer.

Fungal pathogens and parasitic nematodes are important, persistent problems that cause large economic losses across the Midwest. For example, the total estimated loss for the US in 2010 due to SCN was 118 million bushels or $1.25 billion. Root Knot Nematodes is also a major factor in soybean yield loss in the southern US and has the potential to become a problem for Kansas producers. Charcoal rot is the major fungal disease in the state of Kansas and losses in 2002 were estimated at 9%. Phytophthora root rot and Fusarium virguliforme (Sudden Death Syndrome, SDS) are other fungal pests that are beginning to make their presence in Kansas (SDS was at record levels in the 2004 growing season). It is timely to find methods to efficiently control to these pathogens, as there is little or no natural sources of resistance found in our germplasm. Dectes stem borer is becoming an increasing problem in the state with the potential for significant economic loss. Drought is also a major environmental stress that limits production. Novel approaches such as using antimicrobial peptides have merit and should be explored. Finding transgenic solutions to soybean diseases and environmental stresses would complement the efforts of the conventional breeding program by adding additional sources of resistance.

1. Enhance Soybean Cyst Nematode (SCN) resistance in transgenic soybean by modifying current strategies.
2. Transgenic approaches for increased fungal resistance with emphasis on SDS resistance.
3. Improve drought tolerance in soybean by manipulating drought tolerance-associated genes.
4. Evaluation of potential transgenic solutions to stem borer.

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 are also evaluating combining traits to see if there are any synergistic enhancements to SCN resistance. Two of our lines (Prp-17 and Y-25) have been crossed and we will plan to perform both greenhouse bioassays and field tests, comparing these lines to the single expressing lines and controls plants.
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). Field test in 2019 has shown encouraging results and we plan field test additional lines in 2022 (FY2023).
Transgenic lines generated from this research project will be incorporated into elite Kansas lines under the KSC funded project “Develop valuable soybean varieties and germplasm for use as genetic resources for companies and for direct on-farm production”. 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. We have created silencing vectors for the FvTox1 gene, engineered soybean cultures, and plan to challenge the transgenic material with F. virguliforme. In the FY2019/21 funding cycles we have recovered 5 putative positive lines and have regenerated plants from these cultures. Currently we are advancing these lines to the next generation. For bioassays, we will cooperate with Dr. Chris Little, KSU’s row crop pathologist who has developed an effective seedling bioassay.

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. Currently we have one gene cloned and transformed into soybean. We are collecting seeds from these transgenic lines and plan to perform drought experiments in FY2023.

Objective 4. Evaluation of potential transgenic solutions to Dectes 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. Since Dr. Smith’s retirement Brian McCornack has taken over that project. Similar to our SCN work we proposed to engineer soybean with these gene-silencing constructs and then perform bioassays to determine the effectiveness of these lines. In the FY2019 we made the vectors for hairy root analysis and for stable transgenic plants. In FY2020 we generated transgenic lines for three constructions and are regenerated plants from these lines. In the FY2022 funding cycle we have continued to regenerate plants form these lines and perform a greenhouse bioassay. In Fy2023 we plan to perform field trials at the North farm of K-State.

Update:
With the SCN objective we have several lines which we are evaluating and crossing. Field test results of SCN trials show one of our lines ("Y23") resulted in a 34% reduction in both of cyst /gm root and eggs/gm root. Two other lines did not show a significant reduction on SCN.

We have identified six separate lines with the fungal resistance project and they have been advanced to the third generation. We have identified homozygous lines and they are waiting to go into SDS resistance trials.

Field trails for the Dectes stem borer project were completed this past summer and data was collected. We evaluated three different constructs and looked at ovapositioning scars, measured tunneling and presence of larva at the crown. We did not see any significance in ovaposition scaring on petioles but we did see reduced tunneling in all transgenic lines compared to the controls. All control plants contained larval tunneling to the crown compared to a 33-66% reduction for the transgenic lines. Even though tunneling was observed in all control plants, only 58% of the plants observed to contain larva at the crown. All transgenic lines had significant number of larva present in the crown ranging from only 8-33% of the larva present in the crown. We also observe larval mortality in the transgenic lines. Of the larva present in the crowns, all the the control plants contained live larval whereas the transgenic lines had between 33 and 100% mortality of the larva found in the crowns of the transgenic material.
In our best event we observed a 66% reduction on the number of tunneling reaching the crown and only 8% of the plants having larva present in the stems and these larva were dead. We have one more line we need to evaluate but are waiting on molecular analysis to be complete.

Update:
A number of our transgenic soybean lines continue to exhibit partial nematode resistance with an approximate 20-35% reduction in both of cyst/gm root and eggs/gm root. We are in the process of making crosses between most effective lines to stack the transgenes in one background. We have also made crosses between our "Y23" line and Kansas adaptive lines that are either susceptible and moderately resistant to SCN. We would like to test whether our transgenes have a synergistic effect with conventional genetic basis of SCN resistance.

For fungal resistance we have produced six lines to downregulate the FvTOX1 gene in Fusarium virguliforme through RNA interference. Molecular analysis via RT-PCR confirmed all T3 transgenic lines are expressing the transgene and seeds were given to our collaborator for a challenge study. Further molecular analysis such as Southern blotting and qRT-PCR to check for transgene copy number and expression levels are ongoing. Results from the bioassay should be available in the next quarter.

With our drought tolerance project we are looking at specific genes within the NAC gene family which encode transcription factors involved in the control of plant morpho-physiology and stress responses. We have produced two transgenic soybean lines that have knockdown expression of NAC177. Molecular analysis show these lines are expressing the silencing construct and we have advanced these lines to the T2 generation and bulking up seed for bioassays. Additionally we are developing a gene editing protocol in soybean using Gm NAC177 and GmNAC174NAC genes as targets. The advantage of these lines would be they would not be considered transgenic and therefore not regulated articles.

For stem borer project our field trials had mixed levels of tolerances. In our best event we observed a 66% reduction on the number of tunneling reaching the crown and only 8% of the plants having larva present in the stems and these larvae were dead. However in most of our lines larval tunneling and presence in the crown were highly variable. This was caused most likely by differences in transgene expression among the individual samples and the stem thickness of the transgenic lines. We are currently selecting homozygous lines and selecting for the highest level of transgene expression. We are also crossing these lines into Kansas adapted environments.

This project is using tools of biotechnology to provide novel means of resistance or tolerance to diseases or environmental stresses. Specifically, 1) resistance to Soybean Cyst nematodes; 2) resistance to Sudden Death Syndrome (SDS); 3) tolerance to drought ; and, 4) resistance to the detectes stem borer. All of these projects are ongoing. We have created transgenic lines containing traits targeting the nematodes, pathogens, insects or drought stress, performed molecular analysis to identify lines which are stably express the traits, and advance these lines to homozygosity or bred these lines into Kansas adapted lines. We have good evidence that particular targets like our Y25 transgenic lines for nematode resistance and a couple lines expressing traits for stem borer resistance are providing increased levels of resistance. Additional assays are needed as well as a more complete molecular analysis.

This project will benefit farmers by providing new transgenic sources of resistance/tolerances to soybean diseases, insects and environmental stresses. This research would complement the efforts of conventional breeding programs by adding additional component to their breeding strategies.