Lab Research:
    Genomic and proteomic-based research on molecular biology and genetics of host resistance to emerging or potential soybean pathogens with a current emphasis on soybean rust and the soybean rust/soybean interaction.
Soybean Rust:
    The soybean rust fungus, Phakopsora pachyrhizi, is ranked #22 of the top 100 most dangerous and exotic pests and diseases and is currently listed as a biological agent determined to have the potential to pose a threat to plant health or plant products UreMelPp502.pdf; Public Health Security and Bioterrorism Preparedness and Response Act of 2002, Select Agents 7 CRF part 331). Soybean rust is a devastating disease. USDA-APHIS reports that soybean rust has caused losses of 60-80% in Africa and 80-100% in China. Soybean rust is not present in the continental US, and soybean growers fear the arrival of P. pachyrhizi spores through trade routes, winds or deliberate introduction. To compound the fears, soybean cultivars show little promise for genetic resistance or tolerance. There are only four known resistance genes for soybean, but all can be overcome by at least one of the many P. pachyrhizi isolates. In a search for genetic resistance, 940 soybean cultivars currently grown in the US and 12,000 soybean accessions from the USDA germplasm collection were evaluated in seedling assays against soybean rust at the BSL-3 containment greenhouse at Ft. Detrick, MD (Glen Hartman, University of Illinois, 2004 Proceedings of the Southern Soybean Disease Workers in St. Louis). None were found to be resistant and fewer than 100 showed any promise for disease tolerance. According to the APHIS Strategic Plan to Minimize the Impact of the Introduction and Establishment of Soybean Rust on Soybean Production in the United States, there will be little chance for eradication once the fungus is introduced because of the wind-borne nature of the fungal spores sbrplan12-03.pdf.; Only two fungicides are approved for soybean rust control and the effectiveness of these fungicides during an epidemic is unknown. Federal, state, academic researchers are investigating the parameters necessary for the control of rust with these fungicides. Because of the threat of the development of fungicide resistance, states are trying to get emergency exemptions for pesticides that are currently prohibited in the US. In light of the obvious vulnerability to the US soybean crop, APHIS forecasts yield losses to be 50% with an economic impact of $7B/yr should soybean rust become established in North America.
    Plant rust fungi, such as P. pachyrhizi, are Basidiomycetes of the order Uredinales and, in general, are very specialized obligate parasites that can only attack specific hosts (Agrios, 1988). Macrocyclic rusts can overwinter as sexual teliospores, which germinate to form basidia and produce haploid basidiospores. Basidiospores form spermagonia whereby compatible mating types undergo plasmogamy, and the subsequent mycelium form aecia and aeciospores that develop into uredia. Uredia produce uredospores which can develop into telia and complete the rust lifecycle. For P. pachyrhizi, the basidia and aecia stages have not been observed and the telia stages are not commonly produced (Marchetti et al., 1975). Thus, P. pachyrhizi usually reproduces asexually through the uredia stage. The rate at which uredospores germinate and penetrate tissues depends on temperature and wetness duration (Melching et al., 1989), although experimental infections are maximized after placement of inoculated plants in a 20 C dew chamber overnight (Marchetti et al., 1976). Unlike most rusts that enter hosts through stomatal openings, P. pachyrhizi uredospore germlings produce an appressorium over the epidermis and directly penetrate the cuticle via a penetration hypha at 20 hrs post inoculation (Bonde et al., 1976; McLean, 1979; Koch et al., 1983; Koch and Hoppe, 1988). After penetration, additional hyphae spread through the apoplast and infect subdermal cells. Uredia form on the lower leaf epidermis, and erupt through the cells to release uredospores which are dispersed by wind. Uredospores can be released as soon as 9 days after initial infection for a period of 3 weeks (Marchetti et al., 1975). Aside from this general body of knowledge of the rust lifecycle and etiology, and some basic knowledge on compatible/incompatible interactions on different hosts, very little is known about the genetics and molecular biology of P. pachyrhizi.
Proteomics:
    As an alternative to separating proteins by 2-D gels, samples containing complex mixtures of peptides (>1000) can be separated by HPLC and then analyzed by MS/MS, a.k.a. MudPIT (Multidimensional Protein Identification Technology; Washburn et al., 2001). This method is preferable to 2-D gel separation because 2-D gels are comparatively slow and labor intensive. In MudPIT, total protein is analyzed and computing power is used to identify proteins. In brief, lyophilized protein is resolubilized in urea, cysteins are reduced and then alkylated in iodoacetamide. Proteins are first digested with endoproteinase Lys-C and then with trypsin. Mass spectrometry is performed with a Thermo-Finnigan ProteomeX LTQ workstation containing two quaternary HPLC pumps, an auto sampler, a 10-port low volume switching valve, and a Finnigan LTQ Deca XP quadrupole ion trap mass spectrometer. A 100 um i.d. capillary is packed with 9 cm of 5 um C18 reverse phase resin and 4 cm of 5 um strong cation exchanger resin. The sample is pressure-cell loaded onto the column. A 12-step separation procedure follows using Buffer A (5% acetonitrile/0.1% formic acid), Buffer B (80% acetonitrile/0.1% formic acid), and Buffer C (500 mM ammonium acetate/5%acetonitrile/0.1% formic acid) (Wu et al., 2003). The rate of flow is 200 nl/min. The HPLC column eluent is electrosprayed with a distally applied liquid junction spray voltage of 1.8 kV directly onto the ionization source of a Thermo-Finnigan LCQ Deca XP Plus ion trap mass spectrometer. Spectra are scanned over the range of 400–1400 mass units. Automated peak recognition, dynamic exclusion, and daughter ion scanning of the top two most intense ions is performed by the Xcalibur software provided with the ProteomeX workstation. MS/MS data are analyzed using SEQUEST or Mascot, computer programs that allow the correlation of experimental data with theoretical spectra generated from known protein sequences (fields.scripps.edu; www.matrixscience.com). A computer server jointly operated by PSI and ANRI is used to process data and is capable of evaluating up to 10,000 proteins per day.
Publications:
Identification of Rice (Oryza sativa) Proteins Linked to the Cyclin-Mediated Regulation of the Cell Cycle (2003). Bret Cooper, Don Hutchison, Sylvia Park, Sonia Guimil, Peter Luginbühl, Cinzia Ellero, Stephen A. Goff and Jane Glazebrook. Plant Molecular Biology. 53, 273-279

Investigative Proteomics: Identification of an Unknown Plant Virus from Infected Plants Using Mass Spectrometry (2003). Bret Cooper, Donna Eckert, Nancy L. Andon, John R. Yates III and Paul A. Haynes. Journal for the American Society for Mass Spectrometry 14, 736-741; published online before print May 23, 2003

A Network of Rice Genes Associated with Stress Response and Seed Development (2003). Bret Cooper, Joseph D. Clarke, Paul Budworth, Joel Kreps, Don Hutchison, Sylvia Park, Sonia Guimil, Molly Dunn, Peter Luginbühl, Cinzia Ellero, Stephen A. Goff, and Jane Glazebrook. Proc. Natl. Acad. Sci. USA 100, 4945-50; published online before print April 8 2003, 10.1073/pnas.0737574100

Diverse RNA Viruses Elicit the Expression of Common Sets of Genes in Susceptible Arabidopsis thaliana Plants (2003). Steven A. Whitham, Sheng Quan, Bret Cooper, Bram Estes, Hur-Song Chang, Tong Zhu, Xun Wang and Yu-Ming Hou. Plant Journal 33, 271-283.

A Draft Sequence of the Rice Genome (Oryza sativa L. ssp. japonica) (2002). Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, Hadley D, Hutchison D, Martin C, Katagiri F, Lange BM, Moughamer T, Xia Y, Budworth P, Zhong J, Miguel T, Paszkowski U, Zhang S, Colbert M, Sun WL, Chen L, Cooper B, Park S, Wood TC, Mao L, Quail P, Wing R, Dean R, Yu Y, Zharkikh A, Shen R, Sahasrabudhe S, Thomas A, Cannings R, Gutin A, Pruss D, Reid J, Tavtigian S, Mitchell J, Eldredge G, Scholl T, Miller RM, Bhatnagar S, Adey N, Rubano T, Tusneem N, Robinson R, Feldhaus J, Macalma T, Oliphant A, Briggs S. Science 296, 92-100.

Constitutive Salicylic Acid-Dependent Signaling in cpr1 and cpr6 Mutants Requires PAD4 (2001). Dayadevi Jirage, Nan Zhou, Bret Cooper, Joseph D. Clarke, Xinnian Dong and Jane Glazebrook. Plant Journal 26, 395-407.

Collateral Gene Expression Changes Induced by Distinct Plant Viruses During the Hypersensitive Resistance Reaction in Chenopodium amaranticolor (2001). Bret Cooper. Plant Journal 26, 339-349.

Genetic Mechanisms for Engineering Host Resistance to Plant Viruses (1999). Bret Cooper. In Handbook of Biological Control. pp. 557-574. Ed. T.S. Bellows and T.W. Fisher. Academic Press, San Diego.

Domains of the TMV Movement Protein Involved in Subcellular Localization (1998). Theodore W. Kahn, Moshe Lapidot, Manfred Heinlein, Christoph Reichel, Bret Cooper, Ron Gafny and Roger N. Beachy. Plant Journal 15, 15-25.

Defective Movement of Viruses in the Family Bromoviridae Is Differentially Complemented in Nicotiana benthamiana Expressing Tobamovirus or Dianthovirus Movement Proteins (1998). A.L.N. Rao, Bret Cooper, and Carl M. Deom. Phytopathology 88, 666-672.

Cell-to-Cell Transport of Movement-Defective Cucumber Mosaic and Tobacco Mosaic Viruses in Transgenic Plants Expressing Heterologous Movement Protein Genes (1996). Bret Cooper, Isabelle Schmitz, A.L.N. Rao, Roger N. Beachy, and J. Allan Dodds. Virology 216, 208-213.

Differences in Subcellular Localization of Cucumber Mosaic Virus and Tobacco Mosaic Virus Movement Proteins in Transgenic and Infected Plants (1995). Bret Cooper and J. Allan Dodds. Journal of General Virology 76, 3217-3221.

Functional Similarities between Plant Virus Movement Proteins and Approaches to Creating Transgenic Plants that Restrict Viral Movement (1995). Bret Cooper. Dissertation. University of California, Riverside.

A Defective Movement Protein of TMV in Transgenic Plants Confers Resistance to Multiple Viruses Whereas the Functional Analog Increases Susceptibility (1995). Bret Cooper, Moshe Lapidot, James A. Heick, J. Allan Dodds and Roger N. Beachy. Virology 206, 307-313.