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 proteins from SCN and from soybean cells and tissues infected with nematodes that may be candidates for developing resistance to these pests. From a set of 37,500 gene probes on microarrays, we identified those genes expressed in SCN at 12 hours, 3 days and 8 days post inoculation at the nematode feeding site of susceptible and resistant plants.

The plant hormone ethylene is necessary for successful colonization of soybean roots by the soybean cyst nematode. We identified 17 genes involved in ethylene biosynthesis and determined their expression patterns in non-infected and infected roots. Moreover, we quantified the concentration of the immediate precursor to ethylene (ACC) in infected and non-infected root pieces to ascertain that ethylene is specifically synthesized where the nematode is feeding. In addition, gene constructs were prepared to target expression of mRNAs that inhibit ethylene synthesis in the SCN infection site. We have obtained second-generation stably transformed soybean seeds for these constructs and are in the process of amplifying the seed number and screening for expression patterns.

Identified genes and proteins from soybeans and beans that protect them from rust fungi. 1,500 proteins from dry beans were shown to contribute to disease resistance and at least 90 overlapped with genes in soybeans that also contributed to disease resistance to soybean rust. Individual genes from the bean rust fungus are being tested in plants to determine how they affect the plant defense system.

Genes important to the life cycle of rust fungi need to be identified so that targets can be selected to develop soybean resistant to fungi. We analyzed 22,000 sequences collected from bean rust cDNA libraries and 1000 sequences from cDNA libraries of Asian soybean rust. We identified and began studies on a subset of fungal proteins that are good candidates for being secreted from the fungus into the plant host. These abilities likely help improve a germling’s ability to quickly infect plants.

4.Accomplishments
1. Identification of plant genes and proteins associated with disease resistance to rust fungi. Discovery of proteins that are important for disease resistance to rust fungi may help improve common bean cultivars through breeding or transgenic technology. Comparison between common bean plants that are naturally resistant and plants naturally susceptible revealed 1,500 proteins that contribute to the resistance response. At least 90 of the common bean proteins can be found as versions in soybean and help provide soybeans with resistance to the soybean rust fungus. Similar types of genes and proteins have also been identified in the model plant Arabidopsis thaliana. This association demonstrates that the defense systems between plants are very similar. These results helped to identify disease resistance proteins that might eventually be used to protect susceptible plants.

2. Identification of proteins in plant root cells. Soybean roots have millions of single cell hairs on them that increase surface area and enhance water uptake. These root hairs are also the sites of Rhizobium bacterial infection that allows the plant to produce its own nitrogen and live without added nitrogen fertilizer. It is believed that a set of proteins makes root hairs unique from other parts of the plant such as a leaf, but it is unknown what these unique proteins are. Experiments identified proteins from single cell root hairs. Some of these proteins may serve as water channels and appear to be unique to root hairs.

3. Genes identified that may be involved in resistance to nematodes. Soybean cyst nematode causes approximately $1 billion in damages each year in the U.S. We identified genes whose expression significantly increases in cells where the nematode is feeding and compared these genes in resistant and susceptible soybean varieties. Some of these genes may be useful to broaden resistance of soybean to the nematode in genetically transformed plants.

6.Technology Transfer

Number of New CRADAS	1
Number of Invention Disclosures Submitted	1

Review Publications
Thibivilliers, S., Joshi, T., Campbell, K., Scheffler, B., Borerma, R., Xu, D., Cooper, B., Nguyen, H.T., Stacey, G. 2009. Generation of Phaseolus vulgaris ESTs and investigation of the regulation upon Uromyces appendiculatus infection. Biomed Central (BMC) Plant Biology. 9:46.