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 (LC-MS/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, immuno-localization, immuno-precipitation, affinity purification, protein tagging, gene over-expression, phage library display, and other methods that will resolve protein-protein 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
Identified genes and cloned twenty-five cDNA encoding soybean genes that may be candidates for developing resistance to nematodes through over-expression. Identified and cloned four fragments of genes from nematodes that may be candidates for developing resistance to these pests through gene silencing. Several of these plasmid constructs were used to transform soybean roots in composite plants. Two constructs decreased nematode development more than 70%.

Three promoter: GUS (Beta-Glucuronidase) constructs had been prepared and were inserted into soybean plants. The resulting transgenic plants were examined for tissue-specific and Soybean Cyst Nematode (SCN)-induced expression of the GUS reporter gene. In addition, two constructs were prepared and transformed into soybean that were designed to inhibit ethylene action and synthesis, respectively. Initial screens were preformed to identify transgenic lines that demonstrated reduced ethylene responsiveness. Genes for the regulatory proteins IDA (Inflorescence Deficient in Abscission) and a gene encoding a receptor kinase (HAESA) were identified in soybean and their expression monitored in SCN infected roots and other processes that involve major disruption and degradation of the cell wall.

Using mass spectrometry, identified proteins from soybeans that protect them from rust fungi. Several hundred proteins from the nucleus were shown to be under control of a soybean rust resistance gene. Individual genes from soybean are being cloned for further testing for specific roles in disease resistance.

4.Accomplishments
1. Genes identified that may be involved in resistance to nematodes. Nematodes are common pests of plants and cause billions of dollars in damage worldwide. Soybean cyst nematode causes approximately $1 billion in damages each year in the U.S. We cloned DNA fragments from eight different nematode genes and used them to silence the counterpart gene in the nematode. Two fragments decreased the number of nematodes developing to maturity by over 70%. Some of these gene constructs may be useful to broaden resistance of soybean to the nematode in genetically transformed plants.

Review Publications
Tremblay, A., Li, S., Scheffler, B.E., Matthews, B.F. 2009. Laser capture microdissection (LCM) and expressed sequence tag analysis of uredinia formed by Phakopsora pachyrhizi, the causal agent of Asian soybean rust. Physiological and Molecular Plant Pathology. 73(6):163-174.