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,immuno-precipitation, affinity purification, 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. We identified genes that were overexpressed or decreased in expression. Six genes that were overexpressed during the soybean–rust interaction were cloned and their DNA sequence was determined. They are being studied to determine their role in resistance of soybean to the rust fungus. A similar approach was used to identify soybean and nematode genes that may be candidates for manipulation to improve resistance of plants to nematodes. In a separate study, we concentrated on two economically important rust pathogens Phakopsora pachyrhizi and Uromyces appendiculatus, the causative agents of soybean rust and common bean rust, respectively. Haustoria, the rust organs that invaginate leaf cells and interact most directly with the host, were isolated from infected soybean and bean leaves. High throughput DNA sequencing technology was used to identify haustoria expressed genes and comparative DNA sequence analysis techniques used to classify the genes and identify those that may encode secreted proteins that interact with the host. These comprehensive sets of haustoria related genes help define the different pathogenic rust fungi and opens the door for interrupting the development or function of the haustoria to prevent the spread of infection. Soybean cyst nematode (SCN), a destructive pest that infects soybean roots, 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 this process is accomplished in a manner similar to the cell wall breakdown that occurs during abscission, the separation of leaves and flowers from the plant. A protein named IDA is an important inductive signal for cell wall breakdown in other plants. In soybean, the messenger RNA that codes for IDA increases 100-fold during abscission of leaves from the plant. However, recent work indicates IDA may play a non-essential, indirect role in leaf and flower abcission. Nevertheless, of particular interest to our mission, we identified an IDA mimic in root-knot nematodes (RKN), which we hypothesize plays a role in RKN development on soybean roots. A better understanding of how SCN and RKN induce 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 causing more damage than any other soybean pest in the U.S. In an effort to improve resistance in soybeans to the soybean cyst nematode, ARS researchers at Beltsville, MD selected approximately 100 soybean genes for properties of protecting the soybean plant from cyst nematodes. These genes were incorporated or “transformed” into the roots of soybean in order to express greater amounts of these genes in the roots. Ten genes that were over-expressed decreased the number of mature female nematodes by 50%. Four overexpressed genes increased the number of mature females 2-fold or greater. The ten genes that delayed the development of the cyst nematode may be useful to soybean breeders wanting to broaden resistance to nematodes.
2. Proteins controlled by ethylene during seedling development. 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 a technique called 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. Some proteins appeared to be differentially phosphorylated (biochemically modified) after ethylene treatment. The appearance/disappearance of these phosphate molecules suggests a biochemical mechanism to control the activity of these proteins during the ethylene response. A mutant plant missing one of the phosphorylated proteins did not develop normally in the presence of ethylene, thus proving the importance of this protein in seedling development. 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 agriculturally relevant crops like tomatoes and soybeans.
Cooper, B., Chen, R., Garrett, W.M., Chang, C., Tucker, M.L., Bhagwat, A.A. 2012. Proteomic pleiotropy of OpgGH, an operon necessary for efficient growth of Salmonella enterica serovar Typhimurium under low-osmotic conditions. Journal of Proteome Research. 11:1720-1727.