The soybean cyst nematode (SCN) is the most serious pathogen problem of US soybean production. This nematode is an obligate parasite that becomes sedentary inside the soybean root and induces hundreds of soybean cells to completely change their morphology and function to form a novel plant organ, the syncytium, which nourishes the parasite.
Morphological and physiological changes in the soybean root and the developing syncytium are conditioned by specific gene expression profiles in each of the affected cells. So far, researchers have been able to assess gene expression levels as averages over many cells that are harvested together. However, if one wants to assess the true mechanisms governing syncytium formation and successful nematode infection, one needs to assess gene expression profiles in a cell-specific manner. In other words, in order to fully understand SCN infection and to develop novel control strategies, the scientific community needs to resolve gene expression changes to the single-cell level in infected soybean roots. Similarly, we need to assess gene expression profiles in a single-cell specific manner also in the infecting nematode, particularly in the gland cells producing disease-inducing effector proteins. The exciting news is that all these questions now are answerable in our laboratory using technology and know-how we have established or easily can establish if funded.

Our enabling preliminary discoveries are that we have established a soybean hairy root composite plant system and an extensive vector repertoire, with which we will be able to swiftly express gene constructs in transgenic soybean roots. Second, we have been using cell sorting technology to sort plant and nematode nuclei by size and appearance and will be able to perform transcriptomic analyses of sorted nuclei. While the exact experimental approach is subject to change and adjustments as new techniques become available, the following two objectives will be pursued.
Objective 1) We will drive a nuclearly-localized green fluorescent protein (gfp) variant using a soybean promoter that becomes active in the syncytium. Consequently, syncytial nuclei will harbor gfp and will fluoresce when excited. When subjected to cell sorting, we will be able to purify populations of syncytial soybean cells at different time points during nematode infection. We will then use these nuclei fractions to perform transcriptomic analyses to answer the questions mentioned above.
Objective 2) We have demonstrated that we can purify nematode gland cells through cell sorting based on size and appearance. Again, as a result, we are able to purify populations of SCN gland cell nuclei at different time points during nematode infection and from different strains of SCN as well as from nematodes that have been feeding on different soybean cultivars. We will then use these nuclei fractions to perform transcriptomic analyses to answer the questions mentioned above.

Year 1
Milestones:
• Finalize inoculation and preparation procedure
• Perform first experiment
o SCN inoculated soybean roots over time
o Perform single-cell transcriptomics
• Finalize experimental procedure for SCN nuclei harvest
• Perform first experiment
o SCN life stages
o Perform single-cell transcriptomics

Year 2
Milestones:
• Perform gene expression analyses; cluster analyses; network analyses
• Design and perform next soybean transcriptomic experiment using different R genetics and nematode populations
• Perform gene expression analyses; cluster analyses; network analyses
• Design and perform next SCN transcriptomic experiment using different HG types and soybean resistance genes

Year 3
Milestones:
• Perform gene expression analyses; cluster analyses; network analyses

The research ideas raised here are of primary scientific and academic interest but are uncannily directly relevant to future projects to target SCN by new molecular approaches as it will uncover key aspects of infection biology. The research idea proposed here will lead to powerful preliminary data, which will enable the submission of competitive research proposals to federal funding outlets. Successful completion of the proposed work will set in motion the development of a completely novel omics research field in the analyses of nematode pathosystems. Moderate ISRC seed funding will be eminently important to jump-start this research avenue.