Project Details:

Title:
Enhancement of Soybean Through Genetic Engineering

Parent Project: Enhancement of soybean through genetic engineering
Checkoff Organization:Kansas Soybean Commission
Categories:Breeding & genetics, Soybean diseases
Organization Project Code:1914
Project Year:2019
Lead Principal Investigator:Harold Trick (Kansas State University)
Co-Principal Investigators:
William Schapaugh (Kansas State University)
Tim C. Todd (Kansas State University)
Keywords: SCN, SDS, drought resistance, stem borer, genetic engineering

Contributing Organizations

Funding Institutions

Information and Results

Click a section heading to display its contents.

Project Summary

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.

Project Objectives

1. Enhance Soybean Cyst Nematode (SCN) resistance in transgenic soybean by modifying current strategies. Test the effectiveness of gene silencing constructions for root knot nematode resistance using RKN genes homologous to effective SCN genes.
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.

Project Deliverables

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).

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 over-express 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.

Progress of Work

Updated September 14, 2018:
The F2 seeds from crossing events (Prp17 X K11, Prp17 X K12, and Y25 X K12) have been harvested. Seeds from this generation have been planted to test for presence and expression of the transgene. The back crossing experiments will be set up at the same time.

The T3 seeds of “400Awash” are available for bioassay test. Bioassays to test the resistance to SMV, and analyze the expression level of the target eIF gene will be set up in the next quarter.

Dectes texanus is an important pest on soybean crops. When feeding with medium containing In Vitro RNAi, D. texanus showed the symptoms with expected phenotypes. Together with C.M. Smith’s groups we are constructing the RNAi vectors for stable expression in soybean hairy roots. Once completed we will try to use the transgenic hairy roots to deliver RNAi targeting specific genes in D. texanus. Two target genes in D. texanus have been identified by C.M. Smith’s group. We have cloned these two fragments into pENTR-D vectors recently, and the vector plasmids were extracted for sequencing confirmation. Once the sequence is verified by DNA sequencing we intend to use the gateway clone method to sub-clone the sequence into our RNAi expression vector, pANDA35HK for hairy root transformation.

In recent analysis of soybean NAC gene family related to drought tolerance, several specific genes were identified in drought resistance cultivars. To improve the soybean tolerance to drought condition we are screening several GmNAC genes for overexpression and/or down regulation by RNAi or artificial microRNA in hairy roots, for example, GmNAC004, and GmNAC021. Then we will test if the transgenic chimeric plants have improved drought resistance.

Updated November 15, 2019:
We have done back crosses with K11-2363B (partially resistance to SCN HG type7) and K12-2333 (partially tolerant) separately as parents for positive F2 plants, and seeds from these crosses. Currently we just harvested a lot of F2 seeds from these three lines (K11XPrp17, K12XPrp17, and K12XY25) for analyses. Below are the available seeds we have from crossing experiment.

Crossing (Female X pollinator) = Crossing results
K11-2363B X Y25 = Crosses unsuccessful
K11-2363B X Prp17 = Successful
K12 X Y25 = Successful
K12X Prp17 = Successful
Y25 X Prp17 = Crosses unsuccessful
Prp17 X Y25 = Crosses unsuccessful

Additional crosses will be set up to duplicate the crosses that weren't successful.

Research on the Dectes texanus continues. Three target gene fragments (named as DtChitin, DtPilot, and DtCP8E2) were successfully constructed into RNAi vector pANDA35HK individually for hairy root transformation. Three independent experiments of hairy root transformation have been done recently, and chemic plants (about 70 plants) are growing in Magenta boxes. The putative positive hairy roots are going to transferred into the plates with only roots, and partial roots are going to be extracted gDNA for PCR test. The positive ones will be maintaining in the tissue culture medium for feeding assay later on. We will continue additional hairy root transformations for these three genes to maintain enough tissue for in vitro feeding.

Field test was performed last summer with transgenic soybean lines expressing putative SCN resistance genes. Overall nematode population was too low to determine if the transgenic lines had improved resistance compared to non-transgenic controls. This may have been due to the abnormal weather conditions experienced this past season. However, the field test did allow us to increase seed number for additional bioassays scheduled for next year.

Updated November 15, 2019:
We have done additional crosses with K11-2363B (partially resistance to SCN HG type7) and K12-2333 (partially tolerant) separately as parents for positive F2 plants, and seeds from these crosses were obtained. We are continuing to analyze progeny for transgene and transgene expression. Below are the available seeds we have from crossing experiment.

Crossing (Female X pollinator) = Crossing results:
K11-2363B X Y25 = F1 seeds obtained for testing
K11-2363B X Prp17 = F2 seeds obtained
K12 X Y25 = F2 seeds obtained
K12X Prp17 = F2 seeds obtained
Y25 X Prp17 = F1 seeds obtained for testing
Prp17 X Y25 = F1 seeds obtained for testing

Research on the Dectes texanus continues. Three target gene fragments (named as DtChitin, DtPilot, and DtCP8E2) were successfully constructed into RNAi vector pANDA35HK individually for hairy root transformation. The initial experiments of hairy root transformations resulted in a few transgenic roots but the roots did not grow well in media. We have repeated these experiments and are in the process of regenerating roots expressing the RNAi vectors.

Field test was performed last summer with transgenic soybean lines expressing putative SCN resistance genes. Overall nematode population was too low to determine if the transgenic lines had improved resistance compared to non-transgenic controls. This may have been due to the abnormal weather conditions experienced this past season. However, the field test did allow us to increase seed numbers for additional bioassays scheduled for this summer year. An APHISpermit (notification) for field release was submitted.

Started antimicrobial peptide production for microbial inhibition assays for two different AMP’s. We see good efficiency for production of one peptide so far and need to scale-up reaction. Started production of limiting reagent TEV protease, procedure working well.

Updated November 15, 2019:
Host-derived silencing for SCN:

Again this year we have field tests with our transgenic lines. Due to the wet spring, planting was delayed until the beginning of June. At the end of the funding cycle we observed germination and plant growth in all of our entries. SCN counts are planned for mid-July.

We have conducted crossing and backcrossing from the RNAi transgenic lines to two Kansas cultivars: K11-2363B and K12-2333, respectively. So far we have done three rounds crossing and one round backcrossing. The F2 seeds from crossing events (Prp17 X K11, Prp17 X K12, and Y25 X K12) have been increased and harvested. For K11XY25 and stacking two RNAi constructs (Y25 X Prp17, and Prp17 X Y25) into one transgenic line, we only have some F1 seeds ready, and need to be tested. Seeds availability is shown in the following table.

K11-2363B (mild resistance to SCN HG type7) and K12-2333 (mild tolerance)
Crossing (Female X pollinator) = Crossing results
K11 X Y25 = More F1 seeds to test
K11X Prp17 = F2 available
K12 X Y25 = F2 available
K12X Prp17 = F2 available
Y25 X Prp17 = More F1 seeds to test
Prp17 X Y25 = More F1 seeds to test

We have done one round of backcross with either K11 or K12 as parent for positive F2 plants, and get some pods with seeds. We need to grow these seeds and PCR test if the backcross is successful or not.

Currently, we will continue to set up crossing and backcrossing assays with available seeds, and will try to set up some available seeds for SCN bioassays in the greenhouse. It is anticipated that these lines could be used in field tests next year.

Host-derived RNAi silencing for the stem borer in soybean:

Dectes texanus is an important pest on soybean crops. When feeding with medium containing In Vitro RNAi, D. texanus showed the symptoms with expected phenotypes. Therefore, we have constructed three RNAi vectors targeting three different bug genes. They have been used for stable transformation in soybean, and for expression in hairy roots, named p35HK-DtPilot, p35HK_DtChtn, and p35HK_DtCP8E2, respectively.

Some putative tissues from stable transformation have been developed and selected, and we are going to extract gDNA for PCR confirmation.

For hairy root systems, we had generated a few chimeric plants, but not enough for bioassay test. Currently, the summer intern undergrad student and I try to establish a new protocol for generating transgenic hairy roots for these three RNAi constructs.

Improving soybean drought tolerance through genetic engineering GmNAC (NAM, ATAF and CUC transcription factors) gene family:

From analyses of soybean GmNAC gene family related to drought tolerance, we had selected several candidates for genetic engineering. Currently, we are working on four candidates on the list: GmNAC004, GmNAC174, GmNAC177 and GmNAC021. The gene fragments of GmNAC174 and GmNAC177 have been cloned, and we are going to make RNAi constructs to knocking down these two genes by stable transformation as well as in chemic hairy root plants. Those transgenic chemic plants will be tested for drought tolerance improvement.

Final Project Results

Benefit to Soybean Farmers

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.

Performance Metrics

Project Years