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 three objectives of this project are to introduce and evaluate new traits into soybean for increased SCN resistance, increased fungal resistance, improved 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. 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. 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 and 2022 have shown encouraging results.
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: 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 field trials were performed at the North farm of K-State and results were in mixed due to sample size.

Update:
We continue to advance our transgenic soybean lines which exhibit partial nematode resistance. These lines show a 20-35% reduction in both of cyst/gm root and eggs/gm root compared to control plants. We are in the process of making crosses between most effective lines to stack the transgenes in one genetic background. We have also made crosses between our "Y23" line and Kansas adaptive lines that are either susceptible and moderately resistant to SCN. These lines are being backcrossed into the KS adapted genetic backgrounds. We would like to test whether our transgenes have a synergistic effect with conventional genetic basis of SCN resistance. Last, we have produced lines in the transformation pipeline which have three transgenes stacked in one locus to simplify breeding efforts. These cultures are currently under selection.

For the fungus resistance objective, previously, we have identified several FvTox1 RNAi lines using gDNA PCR, and using RT-PCR, we identified RNAi lines with high levels of transgene transcript expression. We challenged these lines with Fusarium virguliforme in collaboration with Dr. Rodrigo’s lab, but the assay was not successful as F. virguliforme failed to develop SDS symptoms even in the in the wild-type controls, suggesting that the infectivity assay needed to be optimized. Prior to another assay we needed to increase our seed supply of the transgenic lines. These plants are maturing in our greenhouses. As soon and collection is complete we will attempt more bioassays. Our future plan is to optimize the infectivity assay of F. virguliforme in the wild-type soybean plants first and then perform the infectivity assays in FvTox1 RNAi lines. Additionally, we are developing additional lines with a stronger promoter to boost production of the RNAi molecules in the stems of the soybean.

For soybean stem borer objective our past 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. In the past six months we have focused on advancing three lines to homozygousity and crossing these lines into Kansas adapted lines. We have collected seeds from these lines and confirmed homozygous lines by qualitative RT-PCR analysis. The crossing into adaptive lines is still in progress. For the next six months we intend continue to backcross our transgenic lines into KS adapted cultivars, to bulk up our seeds and initiate our next round of field trials on stem borers.

Updated August 15, 2024:
We continue to advance our transgenic soybean lines which exhibit partial nematode resistance. These lines show a 20-35% reduction in both of cyst/gm root and eggs/gm root compared to control plants. We are in the process of making crosses between most effective lines to stack the transgenes in one genetic background. We have also made crosses between our "Y23" line and Kansas adaptive lines that are either susceptible and moderately resistant to SCN. These lines have been backcrossed into the KS adapted genetic backgrounds and currently at the BC4 generation. We would like to test whether our transgenes have a synergistic effect with conventional genetic basis of SCN resistance. Last, we have produced lines in the transformation pipeline which have three transgenes stacked in one locus to simplify breeding efforts. These cultures are currently regenerating and we have identified three events which have the traits stacked which target multiple target genes.

For the fungus resistance objective, previously, we have identified several FvTox1 RNAi lines using gDNA PCR, and using RT-PCR, we identified RNAi lines with high levels of transgene transcript expression. We challenged these lines with Fusarium virguliforme Mont1 isoloate in collaboration with Dr. Rodrigo’s lab, but the assay was not successful as F. virguliforme failed to develop SDS symptoms even in the in the wild-type controls, suggesting that the infectivity assay needed to be optimized. Transgenic lines which were in the greenhouse in the winter have been harvested and we have increased our seeds for four events expressing the FV-Tox RNAi trait. We have also isolated an additional F. virguliforme strain from field collected material. We isolated DNA and cloned the FvTox gene from this strain to verify it has an identical FVTox sequence as the F. virguliforme Mont-1 isolate. As this is a Kansas isolate it will be more applicable our environment. The infectivity assay is currently being optimized with this isolate with wild type (non-transgenic ) plants. Additionally, we are developing additional lines with a stronger promoter to boost production of the RNAi molecules in the stems of the soybean.

For soybean stem borer objective our past 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. In the past six months we have focused on advancing three lines to homozygousity and crossing these lines into Kansas adapted lines. We have collected seeds from these lines and confirmed

This project is using tools of biotechnology to provide novel means of resistance or tolerance to diseases and insects. Specifically, 1) resistance to Soybean Cyst nematodes; 2) resistance to Sudden Death Syndrome (SDS) and: 3) 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. We also have developed soybean lines that have the potential for resistance to Sudden death syndrome. 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 and insects. This research would complement the efforts of conventional breeding programs by adding additional component to their breeding strategies.