Years of basic research have created a wealth of fundamental knowledge about the #1 soybean yield robber, the soybean cyst nematode (SCN). Similarly, the basic plant biology and molecular biology research communities at large have created a body of knowledge with direct relevance to controlling soybean diseases. We are now able to draw from many of these discoveries to devise novel strategies to control the SCN. The development and testing of one promising new strategy is proposed here. There is a pressing need to translate prior basic research investments into tangible solutions in the fight against SCN. The research of this proposal seeks to develop new gene constructs that can be incorporated into soybean plants to render them resistant to SCN infection. Successful completion of the proposed work will provide soybean breeders with a simple gene cassette that is relatively easily inserted into existing soybean cultivars without the need of decades of conventional breeding approaches.

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

We will be testing the expression of many syncytium promoters using soybean composite plants to decipher the best combination of promoters to avoid overlap outside the syncytium. We will use vectors devised by us (Baum laboratory unpublished data) to develop constructs that are able to deliver and express this split-recombinase system and test the resultant constructs in composite soybean plants. Once a good combination of promoters is identified, we will produce stable soybean transgenics and test their ability to control the nematode in greenhouse experiments. 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.

Update:
There is no research progress to report yet. However, we have been able to identify a postdoctoral researcher through a open search who will start work on the project within the next three months at the latest. It is our hope that work permit paperwork will be obtained much faster.

Update:
Since the last progress report, we were waiting on the processing of immigration paperwork to hire our new postdoctoral researcher, Dr Chunoti Changwal. On March 25, 2024 Dr. Changwal was finally able to join our group and has been fully on-boarded. Before her start date, I had assembled the requisite information about this ISA-funded project and had informed Dr. Changwal about the details pertaining to the required soybean promoters, the ‘Intein’ RNA recombination system, and the soybean hairy root technology that we have established in our laboratory. Dr. Changwal is now working through all this information and is starting actual bench work towards implementing the missing techniques and resources and to perform the first of the proposed experiments. The project will now pick up speed and is expected to move along on schedule.

Updated October 29, 2024:
Since the last progress report, postdoctoral researcher Dr Chunoti Changwal was able to join our group and has been fully on-boarded. Because she essentially started her work only in April 2024, we have requested a no-cost extension, and this report is not a true final report but a first 6-months report.
As a first action, Dr. Changwal worked through the available literature with the requisite information for this ISA-funded project, particularly pertaining to the required soybean promoters, the ‘Intein’ recombination system and the soybean hairy root technology that we have established in our laboratory. Dr. Changwal has been working through all this information and made significant progress towards the proposed experiments.
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 the kill gene sequences in two inactive components and then use available technologies to reconstitute the protein 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 laboratories, we are testing various promoters that have been reported as syncytium-responsive. We are using promoter-GUS reporter gene expression analyses in soybean hairy roots and subsequent soybean cyst nematode infection to study these candidate promoters. To date, we have finished testing two published promoters in soybean hairy roots. Testing of other such promoters is underway. We will assemble promoter expression specifics in order to use these promoters later on to drive kill gene halves.
Objective 2) Reconstitution of inactive halves of proteins
We have finalized the delineation of gene fragments (protein halves) of one candidate kill gene after thorough literature search and discussions with other labs. We currently are cloning these fragments to make gene cassettes for testing in tobacco and soybean plants.
We are expecting that both lines of inquiry will continue to steadily advance in the next reporting period.

Updated October 29, 2024:

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.