1a. Objectives (from AD-416)
Objective 1: Discover and characterize plant and pathogen genes important for resistance or pathogenicity at the molecular level with special emphasis on, but not limited to soybean interactions with soybean rust and soybean cyst nematode. Hypothesis: There are detectable gene and protein differences between uninfected and pathogen-infected plants and between susceptible and resistant plants, and there are pathogen virulence factors critical to pathogen infection, development and survival. Objective 2: Determine modes of action for plant disease resistance genes, pathogen virulence factors and molecular signals responsible for host-parasite interactions through analysis and characterization of genetic, molecular, protein and metabolite networks. Hypothesis: Examination of the many genes involved in plant-pathogen interactions will reveal critical molecular networks with specific modes of action that are essential to resistance in soybean and to virulence in soybean pathogens. These networks may share commonalities to networks in other plants and pathogens. Objective 3: Engineer and evaluate new methods for obtaining resistance, such as gene silencing, over-expression and protein antagonism, and chemical inhibition of host and pathogen processes, with special emphasis on soybean rust and the soybean cyst nematode. Hypothesis: Expression of gene silencing constructs or of proteins inhibitory to important aspects of pathogen infection, development or maintenance can result in increased tolerance or resistance to a particular pest or pathogen.
1b. Approach (from AD-416)
We have soybean genotypes resistant to one or more rust isolates, but susceptible to all others. These soybean genotypes will be challenged with specific pathogen isolates to study the resistance and susceptible response. Gene and protein expression in both plants and pathogens will be monitored using microarrays, membrane arrays, expressed sequence tag analysis, in situ hybridization, and RT-PCR. Proteins will be detected by liquid chromatography-tandem mass spectrometry (LCMS/MS). Cell fractionation, laser capture microdissection, and subtractive hybridization will be used to isolate specific tissues, organelles, or materials involved in disease processes or responses. Other methods such as antibody localization, gene silencing, plant hairy root transformation (for SCN studies) and mutant analysis will be used to determine the function of genes and proteins and to evaluate their importance in resistance and susceptibility. The disease and pest resistance responses to infection in soybeans will be elucidated systematically using microarrays, proteomics and metabolomics to resolve the biological network evoked. A comparison of differential gene expression and protein accumulation in the resistant and susceptible response of soybean to pathogens will identify components of the network. These networks will be built, examined, and perturbed to confirm function of components using an array of tools, including bioinformatics, yeast two-hybrid screens, mutation analysis, immunolocalization, affinity purification, immuno-precipitation, protein tagging, gene over-expression, phage library display, and other methods that will resolve proteinprotein interactions and interactions among molecules. Based on these data, we can identify candidate members of pathways and networks involved in signaling and evoking the resistance response. Other plant systems, including common bean and Medicago truncatula, will be used as needed in parallel investigations studying host-pathogen responses and interactions to take advantage of the knowledge and specific traits of the resistance response in these systems. Approaches for achieving pathogen control include engineering transgenic plant tissue and organs to express genes that boost the natural defense system of the plant or to provide the plant with a new trait that confers resistance by blocking pathogen attack or survival. Genes shown to have important roles in plant defense may be over-expressed in transgenic plants. Likewise genes that are critical to survival of the SCN in the host or that make the plant susceptible to SCN may be silenced in transgenic roots using hairy root transformation techniques. Additionally, genes that express antibodies or protein antagonists will be engineered into soybean to block the survival and development of the pest or pathogen.
3. Progress Report
Using next-generation DNA sequencing tools, we obtained over 2 billion nucleotides of DNA sequence information on soybean and rust genes expressed during rust invasion of leaves of rust susceptible soybean at different time points during infection. This will allow us to identify genes that we will over express or silence in an effort to identify those genes that increase resistance of plants to the rust fungus. A similar approach was used to identify candidate soybean and nematode genes that may be candidates for manipulation to improve resistance of plants to nematodes. Ethylene is an important gas that induces fruit ripening and plant development. To find new proteins involved in ethylene responses, seedlings of the model mustard plant Arabidopsis thaliana were exposed to ethylene gas, and mass spectrometry was used to identify thousands of proteins by their mass. Proteins were identified that help the plant transmit the ethylene signal, produce more ethylene, resist stress and undergo cellular growth. These results provide new insight into ethylene-controlled regulatory mechanisms in plant cells. These data are most likely to influence scientists who are trying to better control fruit ripening and crop development of more agriculturally relevant crops like tomatoes and soybeans. Soybean cyst nematode (SCN) induces the formation of its feeding structure by breaking down the walls between root cells to form a much larger cell. We previously demonstrated that the SCN induced breakdown of cell walls is accomplished in a manner similar to the cell wall breakdown that occurs during separation of leaves and flowers from the parent plant. A protein named IDA is an important inductive signal for cell wall breakdown in other plants. We examined the role of IDA in soybean leaf drop and SCN infection of roots. IDA was clearly an important signal for soybean leaf drop. However, the role of IDA in SCN infection was less certain but is still of interest for further study. A better understanding of how SCN induces the formation of a functional feeding structure in roots will greatly improve the ability of scientists and industrial partners to control SCN infection of soybean.
1. Soybean genes that retard cyst nematode development. Soybean cyst nematodes attack the roots of soybean plants and cause approximately $1 billion in damages each year to U.S. soybeans. In an effort to improve resistance in soybeans to the soybean cyst nematode, ARS researchers at Beltsville, MD, selected 30 soybean genes for properties of protecting the soybean plant from cyst nematodes. Soybean plants were then genetically engineered to express greater amounts of these genes in their roots. Several genes delayed the development of 50% of the female cyst nematodes. These genes will potentially be useful to soybean breeders wanting to broaden resistance to the cyst nematode.
2. Targeting expression of resistance proteins to the site of pathogen invasion. Soybean cyst nematode, a pathogenic worm, attacks the roots of soybean plants causing approximately $1 billion in damages each year to the U.S. crop. In order for the nematode to absorb nutrients from the plant, the nematode manipulates genetic material in the plant so that the walls that surround root cells are broken down. ARS researchers at Beltsville, MD, discovered a short regulatory DNA sequence in many of the plant genes involved in this process. This sequence may be one of the genetic sites that the nematode controls to help it infect plants. With this information in hand, scientists can now use this regulatory sequence against the nematode by replacing the part of the gene that normally degrades cell walls and benefits nematode growth with a protein that can kill the nematode.
3. Soybean gene regulatory proteins linked to soybean rust resistance. Soybean rust disease is caused by a fungus, and there are only a few known soybean plants that resist rust infection. These resistant plants were studied by ARS researchers at Beltsville, MD, using mass spectrometry, a method that allows the direct identification of proteins by their mass. Several proteins that function in regulation of gene expression were found to be associated with resistance. The discovery of proteins that are important for disease resistance to fungal pathogens like soybean rust has potential to help improve susceptible soybean cultivars through breeding or via the creation of genetically modified soybeans that contain these genes.
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