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 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 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 will be discussed 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. In FY2014 we are continuing with greenhouse bioassays. One of our stable lines demonstrating reduce SCN eggs (containing the prp-17 vector) have also shown a 64% reduction in RKN. Composite plants made with other constructions and are also being screened for RKN resistance in the greenhouse. Plants will be grown for 1-2 months and the roots will be rated for the amount of galling using a standard gall index. The amount of nematode reproduction will be determined by extracting infective juveniles from the roots. In this next funding cycle we will continue produce transgenic lines and evaluate their effectiveness on 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 are showing promising results and would serve as a model silencing the FvTox1 gene in F. virguliforme. We propose to create silencing vectors for the FvTox1 gene, create hairy roots expressing these silencing constructs, and challenge the transgenic material with the fungus. A positive result would be indicated by inhibition of fungal growth and absence of the disease.

Additionally, we will investigate 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. Initially we have selected four peptides from various sources and created expression vectors. We will use bacterial expression systems to first characterize fungal inhibition in either in vitro or detached leaf assays. We will 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. Genes from the effective peptides will be engineered into soybean cultures for in planta evaluations.

Update:
In our first approach for increased SCN resistance transgenic events transformed with the hpRNAi-Y25 and -Prp17 vectors continue to show about a 60-80% reduction in cysts and eggs compared to controls in greenhouse bioassays. We also looked at SCN development on these plants at different time points at 3, 7, 14, 20, and 30 days post inoculation by Acid Fuchsin staining. The preliminary results observed of that Prp17 transgenic plants were likely disturbed the establishment of J2 entry into roots, and/or development of juvenile nematodes because much less juveniles were observed in Prp17 transgenic plants at all stages compared to the susceptible JackX cultivar. The Y25 transgenic plants had no obviously different numbers of juveniles compared to the control but overall less cysts. This may suggest that the suppressed Y25 gene might have affected on reproduction stages of SCN.
We have begun backcrossing transgenic soybean with hpRNAi_Y25 and Prp17, which were transformed to JackX cultivar as background into Kansas adapted lines. Both transgenic lines were crossed with cultivar K11-2363B (mild resistance to SCN HG type7) and K12-2333 (mild tolerance) separately. In addition, transgenic soybean with hpRNAi_Y25 and Prp17 were crossed with each other to stack two RNAi constructs together. All hpRNAi_Y25 or Prp17 transgenic plants were tested by PCR to confirm GOI. These two homozygous lines identified previously were all positive in the test too. Currently, a total of approximately 50 plants were used for crossing and are in the greenhouse growing to maturity. hpRNAi_Y25 events with high expression of GOI (tested by qRT-PCR) were also increased for seeds and at the end of the quarter we have begun to harvest these seeds. We plan use these lines to test the durability of SCN resistance along with testing their effectiveness with different SCN populations.
In a second approach for nematode resistance we are attempting to modify a biochemical pathway in soybean to produce a compound that will affect nematode reproduction. Two genes for shunting a biochemical pathway in soybean towards the production of this compound were placed in vector constructs to express these genes independently and together and were also used to transform soybean cultures. We have identified seven positive events from one gene and 1 event that contains both transgenes. These cultures were regenerated and plants have been transferred to the green house for further testing and seed production.
The root knot nematode (RKN) bioassays with Prp17 and Y25 transgenic lines were performed. The number of galls was recorded by two methods, one quick evaluation scale used in the Todd lab and one developed by Bridge et al. (Bridge and Page, Tropical Pest Management 26, 296-298, 1980). No visual differences were observed among the galls from the transgenic lines and susceptible controls. After one-week incubation in flask, RKN juveniles were counted for each sample and the number was normalized by root weight. The mean number of RKN Juveniles on Prp17 transgenic line was reduced only by about 24% compared to the susceptible controls, and Y25 transgenic lines obtained similar densities compared to control. These results may be due to the gene similarity between the SCN and RKN, or some gene silenced transgenic plants merged to bias the mean density in the transgenic lines. We will to perform additional assays and include other lines to better evaluate RKN resistance.
To increase SDS tolerance, the hpRNAi vector for the FvTox gene has been completed and used to transform soybean cultures. The host derived RNAi constructs for FvTox1 gene were used for stable transformation in soybean, 5 positive putative plants were identified in tissue culture. These plants have been hardened off and are beginning to be transferred to the greenhouse for seed production.

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