Location: Corn Insects and Crop Genetics Research
2023 Annual Report
Objectives
Objective 1: Identify and characterize genes, markers, and molecular networks contributing to yield, resistance to pathogens, and nutrient stress tolerance in soybean and other legumes, and work with researchers to use the information in crop improvement by conventional breeding and gene editing technology.
Sub-objective 1.A. Identify and characterize legume gene expression and epigenetic networks that control nutrient homeostasis, generating information for improving resistance or tolerance to abiotic stress.
Sub-objective 1.B. Identify and characterize soybean disease resistance loci and defense gene expression and epigenetic networks, generating information for improving resistance or tolerance to pathogens that cause economic loss in soybeans.
Approach
The United States leads world soybean production, contributing over 40 billion dollars to the economy in 2014. However, nutrient, disease and pest stresses limit agricultural production. The overarching goal of this project is to provide data and resources that will increase soybean (Glycine max (L.) Merrill) production by mitigating losses due to abiotic and biotic stresses. To study nutrient deficiency we will use iron deficiency chlorosis as a model. To study disease resistance responses we will use a variety of pathogens including Phakopsora pachyrhizi (Asian soybean rust), Phialophora gregata (brown stem rot), Phytophthora sojae (Phytophthora rot), and the insect pest Aphisglycines (soybean aphid). Regulation of abiotic and biotic stress responses requires constant signaling, likely controlled by gene expression and epigenetic changes. Further, a single stress exposure likely primes subsequent plant stress responses. To characterize the genes and networks involved in these responses we will couple RNA-seq, Methyl-seq and Virus Induced Gene Silencing. Finally, we will use RNA-seq data to characterize resistance loci and downstream defense responses. Successful completion of this project will result in genes, gene networks and validated markers that can be used to breed soybean germplasm with durable resistance to abiotic and biotic stress. This project will provide valuable resources to public and private soybean breeders, scientists and growers.
Progress Report
In support of Subobjective 1.A, Identifying gene expression networks contributing nutrient stress tolerance:
Characterizing the speed and diversity of the soybean iron stress response. Iron deficiency chlorosis (IDC) is a global crop production problem, significantly impacting yield. While various genetic approaches have been used to identify genes involved in iron stress tolerance from model species, few studies have focused on agronomically important crop species. We designed a series of whole genome expression studies to explore the speed and diversity of the soybean iron stress response and find new avenues for crop improvement. In the first study, we identified over 10,000 genes whose activity was changed in response to iron stress at 30, 60 or 120 minutes after the onset of iron stress in the iron stress tolerant line Clark. In the second study, we examined iron stress responses in Clark (IDC tolerant) and a nearly genetically identical line IsoClark (IDC susceptible) at two and ten days after iron stress. These analyses identified thousands of genes responding to iron stress response. Genes involved in iron uptake, defense and regulation of cell replication are hallmarks of the soybean iron stress response and their expression is tightly controlled across time. Further, findings suggest soybean uses a novel root to shoot signal to initiate early iron stress responses, not described or characterized in model species. These findings improve our understanding of the genes and networks underlying plant stress tolerance which can be leveraged by researchers to improve iron stress tolerance in soybean and other important crop species.
Deconstructing the genetic architecture of iron deficiency chlorosis in soybean. IDC, caused by calcareous soils or high soil pH, can limit iron availability, negatively affecting soybean yield. In collaboration with Iowa State University, we used a genome wide association study of 460 diverse soybean plant introduction lines to identify significant markers and 69 genomic regions associated with IDC tolerance. Cluster analysis of significant markers across the historical QTL on chromosome Gm03 split the region into four regions, each containing candidate genes for IDC tolerance. This study was the basis for two subsequent studies. In the first study, we used virus induced gene silencing (VIGS) to demonstrate that silencing of three different genes in the historical QTL altered iron stress responses. In the second study, in collaboration with Iowa State University, we selected 18 soybean lines from the genome wide association study above, for whole genome expression analyses 60 minutes after iron stress. Multiple lines responded to iron stress within 60 minutes, much faster than observed in model species. Further, iron stress responses were first detected in the roots, again suggesting soybean uses novel root to shoot signaling mechanisms. Finally, the results suggested multiple mechanisms for iron stress tolerance within the soybean germplasm collection. These studies highlight the need for conducting research in diverse, agronomically important crop species to aid plant breeders in developing soybean lines with improved stress tolerance and greater yield.
Identifying gene expression changes in response to micro- and macro- nutrient deficiencies in soybean. Given that iron deficiency is a perennial problem in the soybean growing regions of the US and phosphate deficiency looms as a limitation to global agricultural production, nutrient stress studies in crop species are critically important. We directly compared whole-genome expression responses of leaves and roots to iron (a micronutrient) and phosphate (a macronutrient) deficiency at 24 hours. The study revealed an interesting contrast, soybean responds to iron deficiency and phosphate resupply. Though the timing of responses was different, both nutrient stress signals used the same molecular pathways. We also examined gene expression changes in response to repeated iron or phosphate stress, identifying 3,375 genes only differentially expressed after repeated stress. In a subsequent study, we measured gene expression in soybean in response to iron deficiency stress followed by exposure to phosphate deficiency stress, to more closely mimic different stress conditions experienced by field grown plants. These analyses determined that sequential stress induces a unique suite of genes not differentially expressed under repeated stress conditions. These findings improve our understanding of soybean responses to range of stress exposures. Understanding the molecular underpinnings of these responses in crop species could have major implications for improving stress tolerance and preserving yield.
Characterizing novel regions of the soybean genome for tolerance to iron deficiency. Fiskeby III is a soybean cultivar with a high level of resistance to multiple abiotic stresses, including iron deficiency. Conversely, Mandarin (Ottawa) suffers severe yield loss when exposed to iron stress. In a project funded by the United Soybean Board (USB), and working with collaborators at the University of Minnesota, we used whole genome expression analyses to compare the Fiskeby III and Mandarin (Ottawa) responses to iron stress conditions. Combining previous mapping data with the expression analyses identified 15 candidate genes within a genomic region associated with iron stress tolerance on Fiskeby III chromosome Gm05. Virus induced gene silencing (VIGS) was used to evaluate each gene for its contribution to iron stress tolerance. Silencing of a multidrug and toxic compound extrusion (MATE) transporter gene resulted in iron stress symptoms, even though plants were grown in iron sufficient conditions. These analyses confirmed the identify of a new iron stress response gene. Given the unique Fiskeby III stress tolerance, known iron stress genes originally identified through studies in model species have been silenced in Fiskeby III. Whole genome expression analyses are being used to determine if these genes play a role in Fiskeby III iron stress responses. These findings can be leveraged by plant breeders to improve abiotic stress tolerance in soybean and other crop species.
In support of Subobjective 1.B, Identifying gene expression networks contributing to defense against pathogens:
Leveraging genomics to determine the molecular effects of combining multiple resistance genes to combat Aphids in soybean. Plant breeders often incorporate multiple resistance genes into a single line, offering robust and durable resistance responses. In soybean, aphid resistance is conferred by Rag genes. Plants containing both the Rag1 and Rag2 genes are significantly more resistant to aphid attack than plants containing either gene alone. To better understand the impact of combining resistance genes, in collaboration with Iowa State University, we conducted whole genome expression analyses of four soybean lines (aphid-susceptible, Rag1, Rag2 and a line containing Rag1 and Rag2), collecting samples after infestation with soybean aphids. Approximately 1,000 genes were specifically expressed in response to aphids in the line containing Rag1 and Rag2, but not in either of the single resistance lines. Many of the genes unique to the Rag1 and Rag2 line had functions related to defense including modification of cell walls, detection of pests and pathogens and defense signaling. Understanding how combining multiple resistance genes results in enhanced resistance is essential for future crop improvement, helping preserve yield for farmers and growers.
Understanding disease resistance signaling from root to shoot. Brown stem rot (BSR), caused by Phialophora gregata, reduces soybean yield by up to 38%. BSR leaf symptoms are often misdiagnosed as other soybean diseases or nutrient stress, making novel sources of BSR resistance difficult to identify in breeding programs. To shed light on the genes and networks contributing to resistance,we used funding from the National Institute of Food and Agriculture and conducted whole genome expression analyses of infected and mock-infected root, stem, and leaf tissues of a BSR resistant soybean at 12, 24 and 36 hours. Gene networks associated with defense, photosynthesis, nutrient regulation, DNA replication and growth are the hallmarks of BSR resistance. P. gregata infection cannot be detected visually for five weeks after infection. However, our data suggests resistance and susceptibility can be detected molecularly, hours after infection. In follow up experiments funded by the USB, we are working to identify and characterize BSR resistance genes. We identified five clusters of receptor-like proteins associated with the mapped locations of the BSR resistance genes Rbs1, Rbs2 and Rbs3. Using VIGS to silence the clusters individually failed to compromise resistance in resistant genotypes. Therefore, the VIGS protocol was modified to silence two clusters at a time. We identified two clusters that when silenced together, knocked down BSR resistance. The genes and networks described here will be used to develop novel diagnostic tools to facilitate expedited breeding and screening for BSR resistance.
Accomplishments
1. Soybean: a new model to study iron deficiency chlorosis (IDC) in plants. IDC negatively affects crop quality and yield. Studies from model species have demonstrated shoot control or influence of iron uptake in roots. However, these studies were conducted days and weeks after the onset of iron stress. ARS scientists in Ames, Iowa, used grafting of nearly identical soybean lines Clark and IsoClark (iron stress tolerant and susceptible, respectively), to demonstrate the Clark rootstock can drive iron stress tolerance in IsoClark leaves. In contrast, IsoClark rootstock is unable to confer iron stress tolerance in Clark leaves. RNA-seq analyses of grafted plants 30 and 120 minutes after iron stress identified 518 and 846 differentially expressed genes in leaves and roots, respectively. Grafts with a Clark rootstock induced genes involved in iron uptake and utilization at 30 minutes in the root and by 120 minutes in the leaves, regardless of the leaf genotype. This suggests an unknown mobile signal, initiated in roots, regulates iron stress responses in soybean leaves. Better understanding of the complex differences between crop and model species will aid in the development of crops with improved IDC tolerance, reducing farmer losses and increasing yield.
2. Identification of Rpp3, a nucleotide binding site-leucine rich repeat (NBS-LRR) protein that confers resistance to Phakopsora pachyrhizi in soybean. Soybean rust, caused by the fungus Phakopsora pachyrhizi, is an economically important disease that negatively impacts soybean production throughout the world. Yield losses as high as 80% have been reported. While most soybean germplasm is susceptible, seven genetic regions have been identified that provide resistance to P. pachyrhizi (Rpp1 to Rpp7). Rpp3 was first discovered and characterized in the soybean accessions PI 462312 and PI 506764. To identify Rpp3, ARS scientists from Ames, Iowa, and Fort Detrick, Maryland, in collaboration with Iowa State University, amplified and sequenced five Rpp3 candidate genes from each resistant line. Conserved regions from the ten genes were used to develop virus induced gene silencing constructs that were able to knock down expression of the candidate genes and compromise resistance to P. pachyrhizi. Gene expression analysis and sequence comparisons of the Rpp3 candidate genes in PI 462312 and PI 506764 suggest that a single candidate gene, Rpp3C3, is responsible for Rpp3-mediated resistance. This research will allow breeders to rapidly differentiate between known and potentially novel Rpp3 alleles, facilitating the breeding of durable resistance.
3. Iron stress tolerant soybean line, Fiskeby III, utilizes novel genes and pathways in response to iron deficiency stress. Iron deficiency negatively impacts plant yield, so to improve plant tolerance, new sources of stress tolerance need to be identified and integrated into breeding programs. Fiskeby III is a soybean line resilient to multiple abiotic stresses, including iron deficiency. Building on previous research, ARS scientists in Ames, Iowa, silenced a novel gene associated with iron deficiency stress tolerance and subjected silenced and control Fiskeby III plants to stress conditions for either one day or seven days. Analysis of whole genome expression profiling revealed Fiskeby III responds quickly to iron deficiency stress, inducing phosphate deficiency responses to re-establish nutrient homeostasis within the plant. Fiskeby III induces the canonical soybean iron deficiency responses, but utilizes different molecular pathways, shifts the timing of the responses, and employs genes unique to Fiskeby III. Identifying and characterizing these genes and pathways in Fiskeby III provides novel targets for breeding elite soybean lines with improved stress tolerance which will increase yield and farmer profits.
Review Publications
Karhoff, S., Vargas-Garcia, C., Lee, S., Mian, R.M., Graham, M.A., Dorrance, A., McHale, L. 2022. Identification of candidate genes for a major quantitative disease resistance locus from soybean PI 427105B for resistance to phytophthora sojae. Frontiers in Plant Science. 13. Article 893652.
O'Rourke, J.A., Graham, M.A. 2022. Coupling VIGS with short and long-term stress exposure to understand the Fiskeby III iron deficiency stress response. International Journal of Molecular Sciences. 24(1).Article 647. https://doi.org/10.3390/ijms24010647.
