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:
Objective #2. Transgenic approaches for increased fungal resistance with emphasis on SDS

RNAi-FvTox1 lines are being developed for SDS resistance. Several T1 lines were tested for the presence of transgene using gDNA as template and PCR positive lines were further tested using RT-PCR to determine the expression of transgene. Only the RT-PCR positive transgenic lines were transferred to either growth chambers or green house for seed collection.
The data for gDNA PCR and RT-PCR is summarized below
Line RT-PCR data Generation Expression level Seeds available
572-Fount-1(2)-4 Positive T3 Low
572-Fount-1(2)-5 Positive T3 Low
572-Fount-1(2)-6 Positive T3 Low

572-Fount-1(4)-3 Positive T3 Moderate 1
572-Fount-1(4)-4 Positive T3 Moderate 2
572-Fount-1(4)-6 Positive T3 Moderate 57
572-Fount-1(4)-8 Positive T3 Moderate 40

572-Fount-C-1 Positive T2 Moderate 10
572-Fount-C-3 Positive T2 Moderate 7
572-Fount-C-4 Positive T2 Moderate 3

572-Figure-C-4 Positive T2 Moderate 3

Objective #3. Improve drought tolerance in soybean by manipulating drought tolerance-associated genes. Knocking down transcription factors GmNAC177, GmNAC174has been attempted. T0 seeds of putative transgenic lines 592 Nadir-1 and 592 Nadir -2 have been collected and seedlings were tested for the presence of transgene using gDNA PCR with transgene specific primers. Transgenic lines positive for gDNA PCR were further analyzed using RT-PCR to determine the transgene expression. RT-PCR positive transgenic plants were transferred to green house for the collection of seeds.

Objective 4. Evaluation of potential transgenic solutions to Dectes stem borer. A greenhouse bioassay was attempted. Field collected Dectes were placed on caged soybeans plants in the greenhouse. Ovipositioning was observed as well as larva migration down the petiole into the stem.Some of the larva appeared to be less developed than expected. Results were inconclusive which may be due to the high temperatures in the greenhouse at the time of assay. It was determined that a field experiment is necessary to evaluate these lines.

Update:
Objective 1: Enhance Soybean Cyst Nematode (SCN) resistance in transgenic soybean by modifying gene silencing strategies.

We continued with crosses between our "Y25" line and “Prp17” lines with two different Kansas adaptive lines, which are either has mild tolerance to SCN HG type7 (K11-2363) or mild tolerance to SCN HG Type 6 (K12-2333). These are to compare whether we have synergistic effects between the transgene phenotypes and the natural resistance genes. We have also made reciprocal crosses between the Y25 and Prp17 transgenic to combine these traits into one line.

Crossing (Female X pollinator) Crossing results
K11-2363 X Y25 BC1, F1 seeds available
K11-2363 X Prp17 BC1, F1 seeds available
K12-2333 X Y25 BC1, F2 seeds available
K12-2333 X Prp17 BC1, F2 seeds available
Y25 X Prp17 F2 homozygous seeds to test
Prp17 X Y25 F1 seeds to test
* K11-2363B (mild tolerance to SCN HG type7)
* K12-2333 (mild tolerance to SCN HG Type 6)

We are also in conversations with a small biotech startup company to evaluate our lines in their research plots.

Objective 2. Transgenic approaches for increased fungal resistance with emphasis on SDS.
RNAi-FvTox1 lines are being developed for SDS resistance. Several T1 lines were tested for the presence of transgene using gDNA as template and PCR positive lines were further tested using RT-PCR to determine the expression of transgene. RT-PCR positive transgenic lines were transferred to green house for seed collection. We have identified several plants that are homozygous for the transgene at the T2 stage and are planting seeds to harvest the third generation. We plan to provide these lines to our collaborator to test for Fusarium virguliforme resistance.

Objective #3. Improve drought tolerance in soybean by manipulating drought tolerance-associated genes.

We have initiated several bombardments with a genome editing cassette to silence the NAC177 and the NAC174 gene. These experiments are going through selection and the tissue culture regeneration process. The NAC 177 experiments are the furthest along and we currently have 17 events identified. These lines went through the regeneration process and seeds have been collected from approximately half of these lines. The other half are maturing in the greenhouse.

Objective 4. Evaluation of potential transgenic solutions to Dectes stem borer.
After the greenhouse assay was inconclusive due in part by the high greenhouse temperatures we filed for an AHPHIS permit to put these lines in the field. This experiment is still ongoing but we do have evidence that 1) the field does have a number of adult Dectes present and 2) initial observations indicate we are seeing ovipositioning occurring on petioles of both transgenic and control plants. We will need to wait for late August and Early September before we can break down our field experiments.

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 thevdetectes 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.