Co-PIs: Wenhao (David) Dai (North Dakota State University); Matias Kirst (University of Florida (UF)
As sedentary organisms, plants cannot avoid or escape biotic (e.g., herbivorous insects and opportunistic pathogens) and abiotic (e.g., drought and extreme temperature) stresses in their local environment. Instead, plants have evolved an enormous diversity of anatomical structures and chemical defenses to protect themselves. In response to attack by feeding insects, plants deploy multiple defense mechanisms that are tightly regulated at the molecular level. One of the most successful approaches to identify genes responsible for variation in a trait of interest is to produce mutants that are then screened for alterations in the traits of interest. In previous studies, several dozen mutant lines in poplar trees (Populus spp.) were identified as resistant to feeding by defoliating insects. In this project, the modified gene in at least 10 of these insect resistant lines will be identified. The roles these genes play in mediating insect resistance will be systematically examined in poplar through both gene knock-down and over-expression studies. Furthermore, mutant plants will be subject to thorough phenotypic characterization that includes evaluation of global changes in gene expression and measurement of insect feeding performance and larval development. It is expected that these studies will provide new insight into the genes and pathways that enhance resistance to feeding insects. Identification of specific insect resistance genes will facilitate breeding of improved tree varieties in the future.
This project will be performed by a team of scientists, graduate students and undergraduates from three universities. Students will receive intensive training in genomics, bioinformatics and biotechnology through participation in all aspects of the research. A new computer lab course in genomics will be developed at the University of North Dakota that will introduce undergraduate and graduate students to hands-on training in bioinformatic analysis of microarray data. These invaluable experiences will prepare students for future careers as professional biologists and educators. The long term goals of this project are to elucidate genetic mechanisms underlying insect resistance in perennial plants, and to incorporate this knowledge into existing tree breeding programs for better forest health. A detailed description of the results and protocols derived from this project can be viewed at www.und.edu/dept/biology/ralph/ralph.htm. All DNA sequences will be deposited in GenBank and microarray data will be accessible through the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/projects/geo/) and the PopGenIE database (www.popgenie.db.umu.se/popgenie/). Biological resources generated in this study will be publicly available upon request.
Principle Investigator Dr. Steven Ralph, University of North Dakota Co-PI Dr. Matias Kirst, University of Florida Co-PI Dr. Wenhao Dai, North Dakota State University As sedentary organisms, plants cannot avoid or escape biotic (e.g., herbivorous insects and opportunistic pathogens) and abiotic (e.g., drought and temperature) stresses in their local environment. Instead, plants have evolved physiological processes, anatomical structures and chemical defenses to protect themselves. In response to attack by feeding insects, plants deploy multiple defense mechanisms that are tightly regulated at the molecular level. One of the most successful approaches to identify genes responsible for variation in a trait of interest is to produce mutants that are then screened for alterations in such traits (i.e., forward genetics). Researchers at the University of North Dakota, the University of Florida, and North Dakota State University have used a forward genetics strategy to unravel the genetic architecture underlying defense against insect pests in forest trees. Forestry in the United States is a $200 billion per year industry that directly employs over a million people. Native and exotic forest insect pests threaten this economic resource, as well as the ecological and aesthetic value of our forests. Identification of specific insect resistance genes in this project will facilitate breeding of improved tree varieties for our future forests. Furthermore, knowledge gained from this study may provide insights into resistance mechanisms in other agricultural crops. Thanks to recent advances in biotechnology, forward genetics can now be applied to poplar trees (Populus spp.), a model system for tree biology. During this project, Dr. Ralph and his team screened nearly 1,400 mutant lines for resistance to feeding by defoliating white-marked tussock moth (Orgyia leucostigma) larvae using a variety of bioassays. This screen identified multiple poplar lines containing mutations that confer resistance to insect feeding. Together with Dr. Kirst and Dr. Dai, the research team has mapped the location of several of the genes influenced by these mutations. Ongoing research aims to determine the possible roles of these genes in plant defense using a variety of strategies that include gene over-expression, biochemical assays, and measuring global changes in transcript abundance. Collectively, the results from this research have been disseminated to the scientific community and the public through two scientific journal publications and 38 conference presentations. In addition to advancing our understanding of the genetic pathways that influence plant defense, this project also provided extensive training opportunities for nine graduate (eight PhD and one MS) and 17 undergraduate students. Throughout the project, these students learned about plant care and propagation, insect care, plant tissue culture, a variety of molecular biology techniques, bioassays for plant-insect interactions, as well as bioinformatic methods. The majority of these students also presented their research as first authors at regional, national and/or international scientific conferences. Several of these students received national or international awards in recognition of the high quality of their research projects. Nearly all of these undergraduate students have successfully graduated and many are now pursuing graduate or professional degrees. An additional 47 students also received enhanced training in genomics and bioinformatics through Dr. Ralph's Molecular Biology Techniques BIO410 course. Students in this class conducted experiments to map the affected gene in insect-resistant mutants identified in Dr. Ralph's laboratory. With support from this NSF project, students had the chance to learn many new techniques that are not normally offered as part of this course including constructing and screening genomic DNA libraries, gene cloning, measuring transcript abundance, and mapping mutations in the Populus trichocarpa genome sequence. In summary, this project has identified several genes in poplar trees that may contribute to defense against defoliating insect pests. Continuing research will be needed to determine if these genes are suitable candidates to be incorporated into tree breeding programs to produce superior genetic stocks of poplar species for the next generation of forests. Furthermore, this project provided students from the Upper Midwest, many of whom are from underserved rural areas, in-depth training in plant biology, genomics and bioinformatics. Many of the students who participated in the project are now continuing to pursue careers in science.
