Location: Corn Insects and Crop Genetics Research
2024 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 genes, markers, and molecular networks in the National Plant Germplasm System soybean collection that contribute to increased abiotic stress tolerance (Objective 1) or confer or enhance disease resistance (Objective 2). Progress has been made in multiple areas including:
Characterizing novel root to shoot iron stress signaling mechanisms in soybean. Iron deficiency chlorosis significantly impacts soybean yield and quality. In previous gene expression experiments we demonstrated that the soybean line Clark signals iron deficiency stress from root to shoot, in contrast to signaling reported in model species. This research leveraged two nearly identical soybean lines, with contrasting iron stress tolerances.
Shoots from an iron stress susceptible line were grafted to roots from an iron stress tolerant line (and vice versa). Building on this research the scientists have developed an improved grafting protocol that achieves >90% survival rate of grafted plants in both soil and hydroponics (up from 50%). Grafted plants will be moved to the field to monitor iron stress tolerance throughout the growing season and collect seed to determine if grafting impacts the iron stress tolerance of the next generation. Additionally, the scientists are repeating an earlier experiment using grafted plants in hydroponic solutions. Following grafting, plants will be maintained in iron sufficient media for two weeks to allow grafts to heal. Plants will then be transferred to either fresh iron sufficient media or iron deficient media for 14 days. Grafted plants will be evaluated for their iron stress tolerance. To generate tissue for whole genome expression analysis, the experiment will be repeated, and leaf and root tissue will be harvested at 30, 60, and 120 minutes, two days and four days after transfer to either iron sufficient or iron deficient media. This research will identify knowledge gaps between crop and model species and key genes involved in legume iron stress tolerance signaling networks from roots to shoots. These datasets will identify genes and markers for improving crop responses to abiotic stress conditions.
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 abiotic stresses, including iron deficiency. In previous work with collaborators at the University of Minnesota, we combined fine-mapping, whole genome expression analyses and virus induced gene silencing (VIGS) to identify a multidrug and toxic compound extrusion (MATE) transporter associated with iron stress tolerance in Fiskeby III. Whole genome expression analysis showed silencing of the MATE candidate gene induces a gene responsible for interacting with transcription factors bound to the DNA; directly regulating transcription. Further, expression of this gene is required to induce a plant defense gene that modulates iron homeostasis in leaves and subsequent responses in roots. In the current work, we have designed VIGS constructs to simultaneously silence both the MATE gene and the gene responsible for interacting with transcription factors. Phenotypic analyses of these plants in iron sufficient and deficient hydroponics is currently underway. Given the unique stress resiliency of Fiskeby III, the ARS scientists have also designed VIGS constructs to silence canonical iron tolerance genes to determine their role in the Fiskeby III stress tolerance response. These research projects will provide new targets for improving soybean’s response to abiotic stress.
Understanding the iron stress response across multiple genotypes. In previous research with collaborators from Iowa State University, we used whole genome expression analyses to characterize iron stress responses across 20 diverse soybean genotypes. To build on this research, we had planned to conduct field seed increases for Iowa maturity groups in the summer of 2023 and greenhouse seed increases for early and late maturity groups in fall of 2023. However, asbestos contamination forced the closing of the USDA greenhouse in Ames, Iowa. Therefore, we are conducting a second field season for summer 2024 in the hopes of generating sufficient seed from as many lines as possible. Seeds from these seed increases will be used in a hydroponics system to examine phenotypic and gene expression differences in response to iron stress.
Characterization of the Resistance to Phakospora Pachyrhizi (Rpp) 1b allele in soybean. Asian soybean (ASR) rust is a threat to soybean production worldwide. Identification of resistance/defense genes is essential for improving commercial cultivars. Rpp1 and Rpp1b are thought to be tightly linked genes or slightly different versions of the same gene found in different soybean lines. While Rpp1 confers plant immunity, characterized by inability of the fungus to colonize the plant (no symptoms), Rbb1b confers resistance, characterized by localized cell death, which limits fungal growth and infection. In a previous collaboration with ARS researchers in Ft. Detrick, Maryland, we identified candidate genes for Rpp1 using map-based cloning, gene expression analyses, VIGS and whole genome expression. The research team expected that silencing Rpp1-mediated immunity would result in susceptibility to ASR. Instead, Rpp1 silencing resulted in a resistant response. This suggests that Rpp1 and Rpp1b are distinct genes, but the resistant response of Rpp1b is masked by Rpp1-mediated immunity. Therefore, we are sequencing the Rpp1b region from a different soybean line that exhibits Rpp1b-mediated resistance, but not immunity, to ASR. Based on preliminary sequence analyses, the team has developed new VIGS constructs to characterize Rpp1b, these studies are currently underway. Breeders and scientists can use the markers and genes developed by this project to incorporate resistance to ASR into improved commercial cultivars.
Identification of candidate genes for Rpp7-mediated resistance to ASR. ASR can reduce soybean yields by as much as 80%. While fungicides can be used to manage ASR, high costs and limited effectiveness highlight the need for durable sources of resistance. Screening of the soybean germplasm collection has only identified eight dominant Rpp genes (Rpp1/Rpp1b, Rpp2-Rpp7, and Rpp6907). Rpp7 is of particular interest as there are no reports of ASR isolates able to overcome Rpp7-mediated resistance. In collaboration with ARS researchers in Ft. Detrick, Maryland, we used a bioinformatic approach to identify the region corresponding to Rpp7 in the genome sequence of the ASR susceptible line Williams 82. The sequences of the resistance genes in this region were used to develop a VIGS construct that silenced Rpp7 in the resistant soybean line PI 605823. We are planning on using a map-based cloning approach to sequence the region corresponding to Rpp7 in PI 605823. This research will aim to identify molecular markers more tightly linked to Rpp7 to facilitate development of ARS resistant cultivars.
Characterizing Resistance to Phytopthora sojae 2 (Rps2) mediated signaling in soybean. Phytophthora is the second most damaging disease of soybean. In research funded by the United Soybean Board and in collaboration with The Ohio State University researchers, we previously sequenced the Rps2 locus in the resistant line L76- 1988. We identified 25 candidate resistance genes. We attempted to develop four different VIGS constructs that would silence most of the candidate resistance genes in this region. While one VIGS construct was able to constitutively induce resistance responses, no VIGS construct was able to silence resistance. We have now developed VIGS constructs for all 25 candidate genes. Approximately eight have been screened to date. While we have successfully silenced candidate resistance genes for diverse fungal pathogens, it is possible the VIGS viral vector may induce defense responses that inhibit follow-up infection with P. sojae. To try and optimize VIGS for use with P. sojae, we have also developed a VIGS construct targeting the soybean trehalose-6-phosphate synthase 6 gene, which is associated with susceptibility to P. sojae. This will allow us to optimize the VIGS/P. sojae system with a gene with a known phenotype. Characterizing these novel resistance genes will provide new avenues for crop improvement.
Accomplishments
1. A cluster of iron stress tolerance genes are conserved across crop lineages. Iron deficiency chlorosis (IDC) is an abiotic stress that can cause significant decreases to crop yield. When grown under iron stress conditions, IDC susceptible soybean cultivars yield 35% less than IDC tolerant cultivars. ARS scientists in Ames, Iowa, and collaborators at Iowa State University coupled virus induced gene silencing and whole genome expression analyses in soybean to demonstrate that GLUTAMATE SYNTHASE 1 (GmGLU1) and RESPONSE REGULATOR 4 (GmRR4) contribute to iron stress tolerance. GmGLU1 and GmRR4 are co- located with GmbHLH300, a previously identified iron stress responsive transcription factor, in the historical IDC quantitative trait locus on soybean chromosome 3. Comparative genomic analyses of soybean, Arabidopsis, tomato, and agronomically important crop legumes peanut, chickpea, alfalfa and common bean, revealed conservation of this important gene cluster across multiple crop species. Therefore, this gene cluster could be used as a tool by breeders and other scientists to identify high priority candidate iron stress tolerance genes across a broad range of crop species. Once sequences are identified within a species of interest, sequence differences could be used to generate markers for use in marker-assisted selection.
2. Multiple receptor-like proteins (RLPs) are required for resistance to brown stem rot (BSR) in soybean. BSR, caused by the soil borne fungal pathogen Phialophora gregata, is one of the top ten yield reducing pathogens for soybeans grown in the Northern United States. Different genetic studies have suggested BSR resistance could be controlled by a single gene or multiple linked genes, making it difficult to develop resistant germplasm in a breeding program. ARS scientists in Ames, Iowa, used virus induced gene silencing to target six different RLP clusters in plants containing Resistance to Brown stem Rot (Rbs) genes Rbs1, Rbs2 or Rbs3. If a RLP cluster was required for BSR resistance, silencing would result in susceptibility to BSR. Two clusters required for Rbs1-mediated resistance were identified, however, silencing these clusters in Rbs2 or Rbs3 containing plants had no impact on BSR resistance. This suggests additional RLPs confer resistance to Rbs2 and Rbs3. Improved understanding of BSR resistance mechanisms will enable faster identification of novel resistant germplasm and easier integration of resistance into elite soybean germplasm by breeders and scientists.
3. Atmospheric carbon dioxide (CO2) levels impact plant defense responses in soybean. Atmospheric CO2 levels are rising at an unprecedented rate and will likely impact crop productivity and health. While elevated CO2 levels result in higher photosynthesis rates in some species, they may also alter resistance to stress, pathogens and insect pests. ARS scientists in Ames, Iowa, and collaborators at Iowa State University examined the effect of elevated CO2 on soybean defense responses to diverse foliar and soil-borne pathogens. Elevated CO2 suppressed defense responses to bean pod mottle virus, soybean mosaic virus, and sudden death syndrome. In 2019, soybean viruses and sudden death syndrome reduced yield by 21 million bushels in the U.S. and Ontario, Canada. Interestingly, elevated CO2 enhanced resistance to Psuedomonas syringae species by priming some stress and defense responses, even in the absence of the pathogen. This work provides a foundation for understanding of how future CO2 levels could impact particular soybean/pathogen interactions differently. This information can be leveraged by breeders and scientists to develop climate resilient plants.
4. Identifying genes controlling flowering time in mungbean. Controlling flowering time is crucial to optimize plant growth in crops. Plants that mature (flower) too early or too late exhibit reduced yield. Mung bean (Vigna radiata (L.) Wilczek) is an important crop world-wide and is gaining popularity in the U.S., but relatively little is known about the genetics of this species. To investigate the genetics underlying flowering time, ARS researchers in Ames, Iowa, and Iowa State University collaborators grew 482 diverse mungbean accessions in Boone, Iowa, over two years, with the onset of flowering noted for each accession. These data, coupled with genetic markers, were used to conduct a genome wide association that identified two markers that account for 25% of the variability in flowering time. Genes associated with the markers are similar to known flowering genes from soybean and other species. The E3 protein delays flowering and maturity in soybean and the FERONIA protein regulates flowering time in Arabidopsis. Thirteen copies of FERONIA were associated with one marker. This study highlights the utility of comparative genomics to identify important candidate genes in species with limited genetic and experimental resources. Breeders working in species with limited genomic resources can leverage soybean tools for crop improvement.
Review Publications
Bish, M.D., Ramachandran, S.R., Wright, A.L., Lincoln, L.M., Whitham, S.A., Graham, M.A., Pedley, K.F. 2024. The soybean Rpp3 gene encodes a TIR-NBS-LRR protein that confers resistance to Phakopsora pachyrhizi. Molecular Plant-Microbe Interactions. 37:561-570. https://doi.org/10.1094/MPMI-01-24-0007-R.
Chiteri, K.O., Rairdin, A., Sandu, K., Redsun, S., Farmer, A., O'Rourke, J.A., Cannon, S.B., Singh, A. 2024. Combining GWAS and comparative genomics to fine map candidate genes for days to flowering in mung bean. BMC Genomics. 25. Article 270. https://doi.org/10.1186/s12864-024-10156-x.
McCabe, C.E., Lincoln, L.M., O'Rourke, J.A., Graham, M.A. 2024. Virus induced gene silencing confirms oligogenic inheritance of brown stem rot resistance in soybean. Frontiers in Plant Science. 14. https://doi.org/10.3389/fpls.2023.1292605.
Kohlhase, D.R., O'Rourke, J.A., Graham, M.A. 2024. GmGLU1 and GmRR4 contribute to iron deficiency tolerance in soybean. Frontiers in Plant Science. 15. https://doi.org/10.3389/fpls.2024.1295952.