John Labavitch - US grants

A number of plant pathogens produce pectolytic enzymes whose activity degrades polygalacturonic acid within the host cell wall during the process of infection. This pectolytic cell wall degradation may contribute to pathogenesis by allowing physical penetration of the invading pathogen and perhaps in providing monomeric sugars to support energy metabolism of the pathogen. Pectic fragments produced through the action of pathogen polygalacturonases may, in addition, elicit defense responses in the infected host. Plant-derived inhibitors of pathogen polygalacturonases have been identified in several plant tissues and their presence has been shown to be associated with resistance to pathogen attack. It has been proposed that the presence of these proteinaceous polygalacturonase inhibitors may directly inhibit pathogen colonization and indirectly function to enhance the host defense response. This research will develop the biochemical and molecular genetic basis for critically assessing the role of plant inhibitors of pathogen polygalacturonases in altering pathogen susceptibility in transgenic plants. The specific objectives are to: 1) purify polygalacturonase inhibitors from pear and tomato fruit, 2) raise antibodies to purified polygalacturonase inhibitors and determine N-terminal and internal amino acid sequences, 3) construct appropriate cDNA libraries from pear and tomato and use antibody and oligonucleotide probes to isolate cDNA clones encoding pear and tomato polygalacturonase inhibitors and, 4) characterize the polygalacturonase inhibitor cDNAs by sequence analysis and patterns of mRNA accumulation. The results of these studies will provide the basis for designing strategies of over-expression or anti-sense depression of the polygalacturonase inhibitor cDNAs in transgenic plants. Losses of agricultural products to plant pathogens are substantial. While losses can often be controlled through the application of fungicides, the enhancement of endogenous plant defense systems by molecular genetic means has provided the hope of reducing the dependence upon chemical control of pathogens. The possible role of plant-produced polygalacturonase inhibitors (PGIs) in pathogen defense is widely recognized but has received little attention in recent years. This research will examine the role of PGIs in pathogen resistance. If proven effective in enhancing plant pathogen resistance, molecular manipulation will provide a powerful tool for achieving pathogen resistant crops.

ABSTRACT
PI: Ann L.T. Powell
PROPOSAL # 0544504
TITLE: Systems biology approach to tomato fruit susceptibility to a necrotrophic fungus

The association between ripening tomato fruit and the fungal pathogen, Botrytis cinerea, will be studied by collaborating laboratories, which combine complementary expertise in plant biology, biochemistry, biotechnology, carbohydrate chemistry and pathology, and thus provide the skills and resources for an efficient integrative systems biology approach. B. cinerea is an important plant pathogen; it infects all the aerial parts of plants but is notoriously aggressive on fruit, causing grey mold and rotting that lead to significant losses of palatable and ripe fresh fruit and vegetables. The research will investigate the resistance and susceptibility of tomato fruit to the fungus. During ripening, fruit undergo a crucial developmental transition from being unripe, green, and resistant to or tolerant of the fungus, to being ripe, red, and susceptible to damage by the fungus. The central hypothesis is that green fruit respond uniquely to B. cinerea, perhaps differently from red fruit and from the leaf and stem parts of the plant, and that this response changes in ripe fruit to encourage the fungus to break down the fruit and permit dispersal of mature seeds. Insights from this research will contribute to the development of methods for producing attractive, safe, healthy fruit and vegetables for human consumption. The objectives are to identify (1) responses activated in fruit by B. cinerea, (2) ripening processes that facilitate rotting of fruit by the fungus, and (3) functions of B. cinerea that are influenced by ripening. This research will include training programs for PhD, Masters and undergraduate student researchers and will involve collaboration with researchers in South Africa. In cooperation with a community science center in an important region of US agriculture, an interactive display will be developed to communicate directly to the public, including elementary school students, results of the research using agriculturally significant transgenic plants.

The metabolome is the sum total of the products of chemical reactions in the cell; it comprises a diverse set of chemicals that is species-specific and that is strongly influenced by the environment. Thus, despite decades of biochemical research, metabolomics is a field that is still in development, and no comprehensive database captures the complement of species-dependent metabolites. This project will capture the metabolome of 10 photosynthetic microorganisms (algae) by identifying all detectable metabolites that are synthesized as building blocks during their growth under conditions with variable sources of carbon (for example carbon dioxide and other organic compounds, including those found in wastewater). These data will greatly increase our knowledge of algal metabolism and carbon utilization. A major goal of this project is to integrate and improve existing metabolomic databases and libraries and to build new tools for the fast identification of metabolites. The resources resulting from this project will be made freely available to foster collaborations across multiple disciplines including engineering, analytical chemistry and bioinformatics. This project will facilitate the development of more efficient technologies for biofuel production based on photosynthetic microorganisms.

Broader Impacts
This project, through collaborations with researchers at the University of Tokyo and Osaka Prefectural University, will integrate and freely disseminate information and tutorials about libraries of chemical data, chemistry software, laboratory protocols for metabolism studies and results from the algal metabolomic studies. Web-based access will enable researchers and others interested in using these tools to compare the biochemistry of different species under various environmental conditions. In addition to the international training and education of the next generation of researchers, there will be outreach by project participants to students and pupils in K-12 education through the NSF-REU and GK-12 programs. Project staff will participate in the BioTech SYSTEM biotechnology program for teachers by providing educational activities for high school students and by partnering with local schools with large populations of under-served and under-represented minority students.