Project Details - Full Facts for Selected Year

Parent Project: Enhancement of soybean through genetic engineering
Checkoff Organization:Kansas Soybean Commission
Categories:Breeding & genetics, Soybean diseases
Project Title (This Year):Enhancement of Soybean through Genetic Engineering
NCSRP, USB, QSSB Project Code:1814
Project Year:2018
Lead Principal Investigator:Harold Trick (Kansas State University)
Co-Principal Investigators:
Keywords:

Contributions

Contributing OrganizationAmount
Kansas Soybean Commission $75,162.00

Funding

Funded InstitutionAmount
Kansas State University $75,162.00

Information and Results

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Project Summary

We anticipate the continued recovery of transgenic soybean plants expressing genes for fungal resistance proteins and nematode resistance. We expect to find some level of resistance to F. virguliforme (SDS), charcoal rot, and other fungal pathogens. However, the level of resistance of these plants will be unknown until these plants are challenged with the pathogens. Likewise we anticipate the recovery of nematode resistant lines of transgenic soybean. All projects above have the potential to reduce the negative impacts caused these pests and pathogens have on the soybean yield across the state and to increase the overall value of soybean.
The long-term outcome of this research will be the integration of these disease resistant traits into the soybean breeding program at KSU. 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.

Project Objectives

1. Enhance Soybean Cyst Nematode (SCN) resistance in transgenic soybean by modifying current silencing strategies.
2. Test the effectiveness of gene silencing constructions for root knot nematode resistance using RKN genes homologous to effective SCN genes.
3. Transgenic approaches for increased fungal resistance with emphasis on SDS resistance.

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.
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.
We have a second transgenic approach to reduce SCN reproduction that is ongoing and an update will be given at the formal proposal presentation in December.
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: Test the effectiveness of gene silencing constructions for root knot nematode resistance using RKN genes homologous to effective SCN genes.
Root-knot nematodes, particularly Meloidogyne incognita, pose an additional risk to soybean production in the United States, accounting for 127,000 tonnes in yield losses annually (Wrather et al., 2006). Although predominantly found in the southern soybean-producing states, M. incognita increasingly is recognized as a threat to soybean production in the Midwest (Allen at al., 2005; Kruger et al., 2008), and periodically is associated with stunted soybean plants in the Kansas River Valley. The nematode causes extensive galling of soybean roots, disrupting root function and resulting in seed yield losses up to and exceeding 50% in infested areas (Allen at al., 2005). Resistant varieties are used to manage M. incognita in the southern U.S., but availability of adapted resistant cultivars is limited for Kansas and the Midwest.
Target genes for RNA silencing will be selected based on research performed by our group evaluating this phenomenon in the soybean/SCN interaction. Genes showing a greater than 40% reduction in cyst or eggs in the soybean system will be our primary targets for the root knot nematode. One of our stable lines demonstrating reduce SCN eggs (containing the prp-17 vector) has also shown a reduction in RKN. 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).

Objective 3. 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, 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.
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 three 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.

Progress of Work

Updated March 5, 2018:
We have crossed transgenic soybean with hpRNAi_Y25 and Prp17, which were transformed in JackX cultivar as background, to two KS cultivars, K11-2363B (mild resistance to SCN HG type7) and K12-2333 (mild tolerance) separately. In addition, transgenic soybean containing hpRNAi_Y25 and Prp17 transgenes were crossed with each other to stack two RNAi constructs together. All hpRNAi_Y25 or Prp17 transgenic plants were tested by PCR to confirm the presence of GOI. Our goal is to cross approximately 50 plants for each cross. Currently these plants are in the greenhouse maturing or in the process of being crossed. After harvesting the seeds we will need to germinate seeds, test for transgenes by PCR and continue to introgress into the Kansas adapted lines.
We are evaluating whether some of our transgenic soybean lines have resistance to Soybean Mosaic Virus. (SMV). A large scale experiment on SMV-G7 infection was conducted with different cultivars, ‘Jack’, ‘Chapman’, and ‘William82’, each have at least 12 plants for inoculation. With ELISA and RT-PCR test, all ‘Chapman’ plant were negative to our SMV_G7 isolate, and ‘William82’ and ‘Jack’ cultivars we have 90% and 70% infection efficiency, respectively. The ‘Jack’ plant with infected SMV_G7 were kept for further bioassay. It is likely that ‘Chapman’ cultivar is resistant to SVM_G7 we have. We had harvested more seeds from ‘400Awash’ (Jack background) for T2 generation. One bioassay has been inoculated with T2 seedlings of ‘400Awah’ transgenic plant, symptomless transgenic plants were observed. We will test samples with ELISA to detect if SMV is present and keep the plant tissue for real time PCR to determine the quantity of virus and eIF gene expression level. More bioassay will be conducted for inoculation with T2 seeds harvested.

The host derived RNAi constructs for FvTox1 gene were used for stable transformation in soybean, currently 8 positive putative plants were identified in tissue culture and we are regenerating these lines.

Updated March 5, 2018:
We have completed the first set of crossing our hpRNAi_Y25 and Prp17 transgenic soybean events to two KS commercial cultivars, K11-2363B (mild resistance to SCN HG type7) and K12-2333 (mild tolerance) separately. We also crossed these lines with each other to stack two RNAi constructs together. All hpRNAi_Y25 or Prp17 transgenic plants were tested by PCR to confirm the presence of GOI. A total of approximately 50 plants were done with crossing about 1-3 times on each plant. We have harvested about half of the crossed lines and have begun to plant the progeny for a second round of backcrossing to Kansas lines. We are confirming presence of the transgenes among our backcrossed lines by PCR and rt-PCR.

As an alternative approach to SCN resistance we are continuing to transform soybean cultures with two genes that together should disrupt SCN mating. We have confirmed additional events for both constructs and are currently regenerating the cultures. we are also exploring the possibility to introduce these genes into a bacteria as an alternative delivery method.

Work with the defensin genes was focused on generating plasmids for Agrobacterium transformations. These will be used for hairy root assays and to transform Arabidopsis as a more rapid evaluation of the defensing genes. Currently we have three of the seven constructs made and sequenced verified.

We have harvested 4 plants of T2 “400Awash” and 2 plants from T3 generation. Currently, we have planted two sets of seeds for GOI test, and segregation of the transgene was still observed. To consider more specific editing for eIF gene, we have constructed a binary vector of CRISPR/Cas9 for Agrobacterium transformation. The vector is based on pCambia1301 backbone, and containing a Cas9 gene driven by CaMV35S promoter, and sgRNA for targeting. It also contains a GUS reporter, and Hygromycin resistance gene for selection. The enzyme digestion assay of the plasmid showed the expected digestion pattern, and we have sent the plasmid off for sequencing for confirmation.

Updated April 16, 2018:
We have performed a second round of crossing with our transgenic soybean with hpRNAi_Y25 and Prp17 to K11-2363B and K12-2333 separately is underway . Approximately 20 plants were used in the crossings and currently pods are forming in these plants. A third round of crossing has just been initiated.
For the first round of crossing, all seeds (about 150 seeds) were planted and are being tested for GOI by PCR, currently gDNA from half of plants were extracted and tested by PCR. There was one plant (Prp17X K11) positive for GOI, which contained intact expression cassette. Additonal K11-2363B seedlings were planted for back crossing with this F1 positive plant. There are about 40 seedlings left to test for K12XPrp17, K12XY25, and Rrp17 X Y25 crosses which is currently underway.

As an alternative approach to SCN resistance we are continuing to transform soybean cultures with two genes that together should disrupt SCN mating. We were having initial difficulty in recovering events with one of the genes and suspected that the gene may have a lethal phenotype. To investigate this potential problem we introduced these constructs into Arabidopsis. Two Arabidopsis thaliana events were confirmed to contain the genes and demonstrated that the gene was not lethality to plants. Since then we have confirmed six of our soybean events to contain both constructs and we are currently regenerating these cultures. We are also continuing exploring the possibility to introduce these genes into a bacterium as an alternative delivery method. We are working out the transformation and expression methodologies for this bacterium with our two genes.

Work with the defensin genes was focused on generating plasmids for Agrobacterium transformations. These will be used for hairy root assays and to transform Arabidopsis as a more rapid evaluation of the defensing genes. Constructs were verified by sequencing.

For the eIF knocking down project, more T3 seeds of “400Awash” are available for bioassay test with soybean mosaic virus infection. A bioassay will be set up to test if the transgenic lines have any improved resistance to SMV.

Final Project Results

Benefit to Soybean Farmers

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.
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 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. Novel approaches such as using antimicrobial peptides have merit and should be explored. Finding transgenic solutions to soybean diseases would complement the efforts of the conventional breeding program by adding additional sources of resistance.

Performance Metrics

Project Years

YearProject Title (each year)
2019Enhancement of Soybean Through Genetic Engineering
2018Enhancement of Soybean through Genetic Engineering
2017Enhancement of soybean through genetic engineering
2016Enhancement of soybean through genetic engineering
2015Enhancement of soybean through genetic engineering
2014Enhancement of soybean through genetic engineering
2013Enhancement of soybean through genetic engineering
2012Enhancement of soybean through genetic engineering
2011Enhancement of soybean through genetic engineering
2010Enhancement of soybean through genetic engineering
2009Enhancement of soybean through genetic engineering