Research Project: Leveraging Crop Genetic Diversity and Genomics to Improve Biotic and Abiotic Stress Tolerance in Soybean

Location: Corn Insects and Crop Genetics Research

2025 Annual Report

Objectives
Objective 1. Conduct research to identify and characterize novel genes, markers, and molecular networks in the NPGS soybean collection that contribute to increased abiotic stress tolerance, and work with other researchers to develop soybeans with improved yield through tolerance to traits such as iron and phosphate deficiency. Sub-Objective 1A. Conduct comprehensive phenotyping of a soybean iron stress panel. Sub-Objective 1B: Conduct whole genome expression analyses of the soybean iron stress panel. Sub-Objective 1C: Build gene regulatory network (GRN) for iron stress. Sub-Objective 1D: Characterize iron stress regulators using VIGS and genome editing. Sub-Objective 1E: Couple grafting with RNA-seq to study iron stress root and shoot signaling. Objective 2: Conduct research to identify and characterize novel genes, markers, and molecular networks in the NPGS soybean collection that confer or enhance disease resistance, and work with other researchers to use the information to develop soybeans with improved resistance or tolerance to diseases such as Asian soybean rust, Phytophthora rot, and brown stem rot. Sub-Objective 2A: Conduct whole genome expression analyses of a Rpp (Resistance to P. pachyrhizi) panel. Sub-Objective 2B: Conduct whole genome expression analyses of candidate effector overexpression transgenic lines. Sub-Objective 2C: Build gene regulatory network (GRN) for resistance to P. pachyrhizi. Sub-Objective 2D: Characterize P. pachyrhizi defense and immunity regulators using VIGS and genome editing.

Approach
Improving crop yields and mitigating losses to biotic and abiotic stress is critical to global food security. While crop production must sustain population growth, we must minimize our dependence on supplemental nutrients and reduce the impact of pathogens on crop quantity and quality. The overarching goal of this project is to develop gene regulatory networks for soybean abiotic and biotic stress responses, using our long history of research in iron deficiency stress and Phakopsora pachyrhizi disease resistance as models. By leveraging the soybean germplasm collection, extensive phenotyping, gene expression and protein interaction studies, we will identify the major signaling genes regulating these networks. Virus induced gene silencing and genome editing will be used to characterize their function and the networks they control. Successful completion of the objectives will result in validated genes and markers for improving soybean stress responses. Results will be added to publicly available databases, for use by the legume research community. The knowledge generated will accelerate breeding programs and enable the engineering of new and improved traits for soybean.

Progress Report
The project team is conducting research to identify and characterize novel soybean genes in the National Plant Germplasm Soybean collection that contribute to increased stress tolerance (Objective 1) or confer disease resistance (Objective 2). Soybean constitutes the largest segment of global agricultural trade, with soybean exports from the United States valued at $18.7 billion. However, many soybeans in the upper Midwestern United States suffer from iron deficiency, resulting in over $260 million in yield losses across the Midwest. Similarly, soybean diseases cost U.S. growers over $4 billion dollars a year. To address these problems, we conducted research on multiple fronts as described below: - Characterizing plant signaling in response to stress. Soybean iron deficiency can result in yield losses of approximately 20%. In previous experiments, we grafted shoots from an iron stress susceptible line to roots from an iron stress tolerant line (and vice versa). Following recovery, grafted plants were exposed to iron sufficient or iron deficient conditions. Roots and shoots of grafted plants were exposed to short or long term iron deficiency. The study demonstrated the iron stress tolerant line Clark recognizes and signals iron deficiency stress from the roots. Roots from the iron stress susceptible line Isoclark were unable to signal iron stress. This finding was novel as studies in model species suggest iron stress signaling is controlled by the shoots. To better understand iron stress signaling between roots and shoots, we have repeated this experiment, focusing on additional timepoints. To identify the genes responding to iron stress, we are examining the activity of each gene in the soybean genome (whole genome expression analyses). In addition, we repeated the experiment a third time, placing grafted plants in the field for the 2024 field season. This data will be used to examine how grafting impacts iron stress responses in the subsequent generations. To determine if root control of iron stress signaling is specific to Clark or general to soybean, we have modified our grafting protocol to use Fiskeby III (iron stress tolerant) and Mandarin (iron stress susceptible). While iron stress tolerance in Clark is mapped to soybean chromosome Gm03, iron stress tolerance in Fiskeby III has been mapped to chromosome Gm05. We are in the process of characterizing grafted Fiskeby III/Manderin plants to determine if Fiskeby III also uses root to shoot signaling to regulate iron stress responses. - Characterizing the role of a candidate gene in the Fiskeby III soybean iron stress response. Iron deficiency is one of the leading causes of yield loss in the upper Midwest. Soybean line Fiskeby III exhibits high tolerance to a multitude of stresses, including iron deficiency. In previous work with collaborators at the University of Minnesota, we combined genetic mapping, whole genome expression analyses and knock down of gene activity to identify a transporter gene associated with iron stress tolerance in Fiskeby III. Whole genome expression analysis showed knocking down the activity of this gene activates a gene responsible for regulating the activity of other genes. To determine how these genes work together to regulate iron stress tolerance, we have knocked down their activity simultaneously. We are now studying how these plants respond to iron stress, to confirm their role in iron stress tolerance. - Understanding the iron stress response across multiple soybean lines. In previous research, we used whole genome expression analyses to characterize iron stress responses across 20 diverse soybean genotypes. This analysis was limited to leaves and roots at 1 hour after iron stress treatment. To build on this research, we need a sufficient seed supply to conduct whole genome expression studies at additional time points and to test the function of candidate iron stress response genes. Therefore, we are conducting a seed increase in summer 2025 in the hopes of generating sufficient seed from as many lines as possible. - Identification of candidate genes involved in both the iron stress response and resistance to root rot caused by Fusarium graminearum. F. graminearum is the most prevelant form of Fusarium in Iowa soybean fields. Soybean plants with iron deficiency symptoms often display root rot symptoms, but to date there has been no formal association between the two. In collaboration with researchers at Iowa State University, we used whole genome expression analyses of leaves and roots of plants exposed to either iron deficiency, F. graminearum infection, or both. We are in the process of analyzing data to identify genes unique to each stress and genes differentially expressed in response to both iron deficiency and F. graminearum to determine if there is a common response to both stresses. Collectively, the experiments under Objective 1 have helped fill the knowledge gap on novel crop responses to iron stress. The research has identified key genes regulating iron stress signaling. These datasets have identified genes for improving crop responses to abiotic stress conditions that will decrease our dependence on costly iron applications and improve soybean yield and quality for farmers and consumers. - Characterization of the soybean rust resistance gene Rpp1b in soybean. Soybean rust is a threat to soybean production worldwide. Identification and characterization of resistance genes is essential for improving commercial cultivars and protecting application of soybean to food, feed and industrial uses. In a previous collaboration with ARS researchers in Ft. Detrick, Maryland, we identified candidate genes for Rpp1 using genetic mapping, cloning, gene knockdowns and whole genome expression analyses. Rpp1 confers plant immunity, characterized by the inability of the fungus to colonize the plant (no symptoms). Surprisingly, knocking down Rpp1 resulted in a resistance response characterized by localized cell death, suggesting the line carrying Rpp1 is carrying a second soybean rust resistance gene, described in the literature as Rpp1b. To identify Rpp1b we sequenced the region corresponding Rpp1/Rpp1b in a soybean line that only carries Rpp1b. We are now using gene knock down experiments to identify Rpp1b. - Identification of candidate genes for Rpp7-mediated resistance to soybean rust. Soybean rust reduces soybean yields by as much as 80%. Previous screening of the soybean germplasm collection identified seven other soybean rust resistance genes. Rpp7 is the only resistance gene that has maintained its effectiveness. In collaboration with ARS researchers in Ft. Detrick, Maryland, we are sequencing the region corresponding to Rpp7 and are conducting knock-down analyses of candidate resistance genes. - Characterizing Resistance to Phytopthora sojae 2 (Rps2) mediated signaling in soybean. Phytophthora is the second most damaging disease of soybean. We previously sequenced the genomic region corresponding to Rps2 in the resistant line L76-1988, identifying 25 candidate resistance genes. As part of a research team funded by the United Soybean Board, we have developed and tested 25 gene knock downs to help identify Rps2. To help identify additional genes contributing to resistance, we have conducted whole genome expression analyses. This will characterize downstream defense responses and identify resistance genes responding to pathogen infection. Collectively, the experiments under Objective 2 have identified and characterized novel resistance genes for soybean rust, Fusarium rot and Phytophthora root rot, providing new avenues for improving disease resistance in soybean. Breeders and scientists can use the genes identified by this project to incorporate resistance into improved commercial cultivars, benefitting farmers, producers and consumers.

Accomplishments
1. Discovered new ways to protect soybean plants from heat stress using soil microbes. Heat stress in soybeans occurs when plants are exposed to high temperatures for extended periods of time, which can lead to altered growth and decreased yields. Heat stress events are increasing in frequency and duration, causing significant economic losses to farmers. Microorganisms in the soil can interact with plant roots to help soybeans tolerate heat, but the mechanism of this effect is mostly unknown. To better understand this interaction, ARS scientists in Ames, Iowa, and collaborators at Iowa State University examined gene expression from soybean plants with differing heat stress tolerance grown in either in Iowa field soil (containing native soil microbes) or sterile soil. They identified genes that helped plants survive periods of heat stress and confirmed soil bacteria help provide relief from heat stress. This information provides new avenues for improving soybean production for farmers and producers.

2. Investigating the activity of iron stress genes in a stress resilient soybean line. Genes involved in iron deficiency responses have been well characterized in model plant species, but their roles in crop species have not been well explored. Fiskeby III (PI 438471) is a soybean line from Sweden that demonstrates high levels of resiliency to numerous stresses. ARS scientists in Ames, Iowa, were interested in determining if iron stress tolerance genes identified from model species contribute to iron stress tolerance in Fiskeby III or if Fiskeby III utilizes novel stress tolerance genes. The researchers coupled gene knock downs with whole genome expression analyses to examine the function of these genes. Knocking down the activity of these genes altered general stress responses, but did not impact iron deficiency tolerance, confirming Fiskeby III utilizes novel mechanisms to tolerate iron deficiency stress. This research provides new avenues for improving iron deficiency tolerance in soybean, benefitting farmers, growers and consumers.

Review Publications
Bredow, M., Khwanbua, E., Chicowski, A., Qi, Y., Breitzman, M., Holan, K.L., Liu, P., Graham, M.A., Whitham, S.A. 2025. Elevated CO2 alters soybean physiology and defense responses, and has disparate effects on susceptibility to diverse microbial pathogens. New Phytologist. https://doi.org/10.1111/nph.20364.
O'Rourke, J.A., Graham, M.A. 2024. Investigating the role of known Arabidopsis iron genes in a stress resilient soybean line. International Journal of Molecular Sciences. 25(21). Article 11480. https://doi.org/10.3390/ijms252111480.
Kohlhase, D.R., O'Rourke, J.A., Graham, M.A. 2024. RNA-Seq of grafted near-isogenic soybean (Glycine max) lines reveals root genotype drives shoot responses to iron deficiency chlorosis. Plant Stress. https://doi.org/10.1016/j.stress.2024.100717.
Stupar, R.M., Locke, A.M., Allen, D.K., Stacey, M.G., Ma, J., Weiss, J., Nelson, R., Hudson, M.E., Joshi, T., Li, Z., Song, Q., Jedlicka, J., Macintosh, G.C., Grant, D., Parrott, W.A., Clemente, T.E., Graham, M.A., O'Rourke, J.A., Stacey, G., An, Y., Aponte-Rivera, J., Bhattacharyya, M.K., Baxter, I., Bilyeu, K.D., Campbell, J.D., Cannon, S.B., Clough, S.J., Mcgrinn, M., Curtin, S.J., Diers, B.W., Dorrance, A.E., Gillman, J.D., Graef, G.L., Hancock, N., Hudson, K.A., Hyten, D.L., Kachroo, A., Koebernick, J., Libault, M., Lorenz, A.J., Mahan, A.L., Massman, J.M., Meksem, K., Okamuro, J.K., Pedley, K.F., Rainey, K.M., Scaboo, A.M., Schmutz, J., Song, B., Steinbrenner, A.D., Stewart-Brown, B.D., Toth, K., Wang, D., Weaver, L., Zhang, B. 2024. Soybean genomics research community strategic plan: a vision for 2024-2028. The Plant Genome. https://doi.org/10.1002/tpg2.20516.