PI: Steven A. Whitham (Iowa State University) CoPI: Thomas Baum (Iowa State University) CoPI: John H. Hill (Iowa State University) CoPI: Melissa G. Mitchum (University of Missouri)
Soybean is the second most economically important row crop species in the United States, but very little information is available on the specific genes that regulate soybean defenses. The soybean crop is perennially subjected to attack by a variety of pathogens. Studies involving model plants, such as Arabidopsis, have yielded much information on plant defense genes and have shown that there is general conservation of defense related pathways across species. Knowledge of defense-related genes in model plants combined with the extensive sequence information available to soybean provides the opportunity to transfer research from model systems to soybean. The overall goal of this project is to interrogate the functions of soybean genes in disease resistance pathways. This will be accomplished by incorporating information from soybean gene expression, resistance gene mapping studies, and soybean homologs of key genes that regulate defense responses in Arabidopsis. In each of the three specific objectives of this project, high throughput virus-induced gene silencing (VIGS) assays will be used to investigate the roles of the candidate genes in resistance to the important soybean pathogens Soybean mosaic virus (SMV), Asian soybean rust (ASR), and soybean cyst nematode (SCN). This research will provide key knowledge needed to unravel defense signaling networks of soybean, which is a requirement for modulating plant resistance in the future. In soybean, there is a critical need to develop methods for analyzing the functions of genes on a scale that allows researchers to exploit the available soybean sequence information and knowledge gained from model systems. The implementation of the VIGS assays will demonstrate proof of concept that high-throughput gene function studies are feasible in soybean and can be applied to other important traits. The broader impacts of this project address both scientific and societal needs. It is expected that this research will be exploited to improve soybean cultivars for disease resistance by accelerating breeding programs and by enabling the engineering of novel resistance traits. The investigators will make the vectors, VIGS constructs, and methods developed in this project available to the soybean research community. The soybean sequences inserted into the VIGS constructs will be made available through the Soybase database (http://soybase.agron.iastate.edu/). The participation of 7th-12th grade teachers and undergraduate students in this project is expected to further broaden the impact by encouraging young people with aptitude and interest to pursue careers in the biological sciences. The teachers will participate in the NSF Research Experience for Teachers program that is held each summer for seven weeks at Iowa State University. The teachers receive training in molecular biology techniques, curriculum development, and engage in independent research projects in the investigators? laboratories. The undergraduates will participate directly in the research project throughout the year under the mentorship of graduate students or postdoctoral scientists. Finally, scientific exchange will be enhanced through collaboration with Brazilian scientists interested in applying fundamental research to the problem of ASR.
The soybean genome encodes approximately 50,000 genes, but only a few of them have been assigned functions. Knowledge of the functions of genes in crop plants, such as soybean, will make it possible to more rapidly improve soybean to meet growing demands for food, feed, and other uses. One approach to understanding gene function is to systematically knock out or mutate genes and test the effects of the loss of genes on the organism. However, there are technical and other barriers that have prevented the generation of large collections of soybean mutants. In this project, Bean pod mottle virus (BPMV) was employed as a vector to rapidly and transiently silence the expression of soybean genes to increase the capability of researchers to assess gene function. This process is known as virus-induced genes silencing (VIGS) in which short fragment of selected soybean genes approximately 300 base pairs in length were inserted into the viral genome. Each insertion directs silencing of the corresponding plant gene during infection by the recombinant virus clone. The BPMV VIGS system was used to demonstrate that large-scale gene function studies are feasible in soybean. The system can be used to identify genes involved in a variety of traits expressed in shoots and roots. The major focus of this project was the application of the BPMV VIGS technology to identify genes required for recognition of viruses, fungi, nematodes, and abiotic stress. In addition, soybean genes including kinases and transcription factors were identified that are necessary for the activation of defense responses that protect soybean plants against these pathogens. A library of BPMV VIGS clones was designed and constructed to silence soybean target genes to test their functions in defense and stress responses. The target sequences, primers, and images of plants are available at the following web page (www.soybase.org/SoyVIGS/). The target genes were selected based on three major criteria: 1) candidate disease resistance genes homologs, 2) expression of genes that were induced during soybean defense responses, and 3) soybean homologs of Arabidopsis genes involved in defense signaling. These BPMV VIGS clones were utilized in screens to identify genes controlling resistance to soybean cyst nematode, soybean rust, downy mildew, Soybean mosaic virus, and iron deficiency chlorosis. This project contributed to discovery of a novel type of nematode resistance gene that encodes a serine hydroxymethyltransferase, essential for one-carbon metabolism. A novel role was identified for the DNA replication gene, GmRPA3, that resulted in improved tolerance to iron deficiency chlorosis when silenced. Large-scale VIGS screens led to a genetic framework for a network of transcription factors, kinases, enzymes, and other signaling genes required for defense against diverse pathogens. During the course of the project high school science teachers received training in the Research Experience for Teachers (RET) program at Iowa State University. The project facilitated training of undergraduate students, graduate students, and postdoctoral scientists, and it supported interaction with Brazilian scientists who had interests in soybean functional genomics through a developing country collaboration. The project demonstrated the feasibility of rapid and/or large-scale studies for discovering gene functions in soybean, and standardized methods for silencing target genes in soybean using BPMV VIGS were developed and made available.
