The SCN establishes a feeding site, called syncytium, in soybean roots. The syncytium is the sole source of food and energy and is essential for the nematode's development; i.e., demise of the syncytium means death to the SCN. The proposed project aims to create transgenic soybean plants that express a ‘kill gene’ that causes cell death specifically at the site of syncytium formation, thereby triggering a breakdown of the developing syncytium. Without its syncytium, the infecting nematode will also die and the soybean plant becomes resistant. There already are mechanisms available to kill the syncytium. However, there are no mechanisms in hand to exclusively trigger the kill gene only in the syncytium. Thus, attempts to control SCN by killing the syncytium have failed because these attempts caused excessive and damaging cell death in other soybean plant tissues as well (Baum laboratory unpublished data). The breakthrough advancement of this proposal is to devise a strategy to activate the kill mechanism only in the syncytium. For this purpose, we have devised a scheme in which the kill gene activation is reliant on two or even three separate syncytium activation events. Only when all activation steps are triggered, does the kill gene become effective and cell death occurs. This strategy prevents kill gene activation elsewhere in the soybean plant in non-target cells.

Our studies will rely on technology that we generated and established in prior work in our laboratory, namely the use of composite soybean plants as our experimental organism (Baum laboratory unpublished data). Composite soybean plants consist of transgenic roots and wild-type shoots and provide a relatively easy and fast experimental subject to create transgenic soybean roots for promoter analyses and SCN infection. The goal of this project is to generate soybean plants that are resistant to nematode infection without affecting growth or yield. Controlling the SCN is a pressing matter, and developing new methods to fight this devastating pest is crucial.

Objectives: The proposed work can be articulated in a series of discrete specific objectives.

Objective 1) Test promoters and verify their reported specificities:
A number of soybean promoters have been shown to become strongly active in the SCN syncytium. Furthermore, literally hundreds of additional soybean genes have been shown to be activated in the syncytium and, therefore, their promoters are promising candidates for the work proposed here. We will test a large number of such promoters in our hairy root-composite plant system to identify pairs and triplets of promoters that are co-activated in the syncytium but whose expression patterns do not overlap elsewhere in the soybean roots. From our preliminary work we already know that such promoter pairs and triplets exist. We will use this specific objective to identify the best possible promoter combinations.

Objective 2) Reconstitution of inactive halves of proteins:
The proposed resistance engineering relies on a published molecular system to functionally reconstitute two inactive protein halves into one active combined protein. We will adopt this available tool to the pairs of protein halves that are needed in our strategy. We do not envision problems with this step because the technology has been proven reliable in other systems.

Objective 3) Assembling gene constructs in soybean roots to engineer SCN resistance:
Using cloning vectors that our lab has generated, we will assemble the requisite gene constructs in hairy root transformation vectors for hairy root generation. We have extensive experience with all aspects of hairy root technology and the generation of composite plants. Composite plants harboring the test constructs as well as control plants will be inoculated with SCN and assessed for their resistance. We will also conduct cytology and microscopy experiments to follow the SCN infection progress.

Objective 4) Choose promising constructs and initiate the preparation of whole transgenic soybean plants:
We expect to identify several gene construct assemblies to provide control of SCN in hairy root-composite plants. We will choose 1 to 3 successful gene cassettes to generate whole transgenic soybean plants with the goal to test such plants in greenhouse experiments for SCN resistance. Given the time required for this effort of more than a year, we expect to start this process at the end of the proposed funding period but to receive the transgenic plant material only after this current project period has ended. Therefore, no funding for soybean transformation is requested in this proposal.

Project Metrics, Economic Analysis and Performance Measures, Milestones, Deliverables and Outcomes – KPIs/Performance Metrics, Economic Impact/Significance, Timelines and Milestone Deliveries to extend state-of-the-art:
Year 1
· Verify activity characteristics of promoters.
· Assemble promoters into pairs and triplets based on their activity strength and spatial and temporal activity in hairy roots.
Year 2
· Construct the gene cassettes to split proteins and allow reconstitution.
· Begin generating hairy roots and composite plants harboring test resistance constructs.
· Write news releases.
Year 3
· Extensive testing of resistance gene constructs.
· Selection of candidate constructs for whole soybean transformation.
· Discussions with ISA and industry partners about possible commercialization of successful constructs.
· Writing of peer-reviewed publications and news releases.

Updated September 11, 2025:
Brief Description of Accomplishments as of March, 2025

This project seeks to express in plants a multipartite cell toxin kill gene under the control of separate nematode-responsive promoters with the goal to render these plans resistant to nematode infection. In order to activate the expression of the kill gene under the control of two or three promoters specifically in the syncytium, one of the required strategies is to split gene sequences in two inactive components and then use available technologies to reconstitute the gene function in cells where both halves are present. The project, thus, is progressing along the two objectives of (1) Testing soybean promoters for nematode responsiveness and (2) Reconstituting the cell toxin in planta.

Objective 1) Test promoters and verify their reported specificities
From the published data from other labs, we are testing various syncytium specific promoters via promoter-GUS expression in transgenic tobacco and soybean hairy roots and subsequent soybean cyst nematode infection to select our potential candidates. In the last report, we reported that we have finished testing two published promoters in tobacco and soybean hairy roots. Testing of more such promoters is underway. We need these promoters later on to drive the expression of kill gene halves.

Objective 2) Reconstitution of inactive halves of the kill gene
We have finalized the delineation of gene fragments (protein halves) of one candidate kill gene. Currently we have ten gene fragments cloned into a vector, driven by a dexamethasone- inducible promoter. These gene fragment constructs have been confirmed through whole plasmid sequencing.

These constructs have been transformed into Agrobacterium tumefaciens for testing in tobacco plants. Next, we will assess whether these gene fragments are forming proteins using western blotting and then will determine if these fragments do no longer elicit any hypersensitive response (HR) defense. Following this, we will test the same constructs also in soybean. We are also creating additional gene cassettes for testing. So far, we have optimized the Agroinfiltration protocol in tobacco with the full-length kill gene to induce HR and are ready to test kill gene fragments for their ability to elicit HR.

View uploaded report

Prior investment of check-off, state, federal, and industry funds has created the required genomic, molecular and technical knowledge to now devise novel control mechanisms against SCN. It is envisioned that the investment of check-off dollars in this project will translate basic data into tangible grower tools to control the #1 soybean yield robber. It can be envisioned that industry partners would indeed adopt this technology and bring it to the market for soybean farmers to benefit from their investment.