Progress 10/16/06 to 10/16/11
Outputs
OUTPUTS: This project focused on development of defense peptides for control of Asian soybean rust, caused by the fungal pathogen, Phakopsora pachyrhizi. During the project period, the research team conducted experiments to address four specific objectives. These included: 1) characterization of the binding of combinatorial phage-displayed peptides to receptors of selected Phakopsora life stages, 2) characterization of pathogen developmental effects induced by affinity-selected phage-displayed peptides, 3) assessment of inhibitory potential of peptides when fused to a protein scaffold, and 4) characterization of the activity of peptide-scaffolds when expressed in plants. Over a series of experiments, we selected peptides from a combinatorial phage-display library that effectively bound to germinating urediniospores of P. pachyrhizi and assessed their abilities to inhibit germ-tube elongation. We then fused effective peptides to maize cytokinin oxidase/dehydrogenase (ZmCKX1), a naturally occurring protein that served as a scaffold for peptide display. ZmCKX1 is naturally secreted from plant cells to the intercellular space where contact with invasive germ tubes is possible. When scaffold-peptides were mixed with urediniospores and inoculated to soybean leaves, infection and pustule formation were eliminated or delayed significantly. Transformation of soybean with scaffold-peptides was initiated. These activities have led to new technologies that have generated interest in the plant pathology scientific community. This interest is reflected in two invited presentations during the project period. Reports of progress on technology development were also disseminated by posters presented at annual meetings of the American Phytopathological Society. These activities also led to three patents and two referred publications. PARTICIPANTS: Collaborators on the project included Frank Schmidt, Dept. Biochemistry, and James Schoelz Dept. Plant Sciences, both of the University of Missouri. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results of the project activities have led to a change in knowledge. Specifically, the project demonstrated that it is possible to select inhibitory peptides against P. pachyrhizi, the causal agent of Asian soybean rust. Selection of inhibitory peptides from combinatorial libraries has been important in the case of this pathogen because of limited disease resistance available in world soybean germplasm collections. Selection of inhibitory peptides by methods of this project may prove useful for many other pathogens of important crop plants for which resistance or genomic resources are limited. Changes in knowledge generated by the project were reported as journal articles, patents, and conference presentations.
Publications
- Journal articles: Smehilova, M., Galuszka, P., Sebela, M., English, J.T., Frebort, I., Jaworek, P., Sedlarova, M., and Bilyeu, K. 2008. Subcellular localization and biochemical comparison of cytosolic and secreted cytokinin dehyrodgenase enzymes from maize. J. Exp. Bot. 60:2701-2712.
- Z.D. Fang, J.J. Marois, G. Stacey, J. E. Schoelz, J. T. English, and F. J. Schmidt. 2010. Combinatorially Selected Peptides for Protection of Soybean Against Phakopsora pachyrhizi. Phytopathology 100:1111-1117.
- Patents: J.T. English, F.J. Schmidt, Z.D. Fang, J.E. Schoelz. 2010. Combinatorially selcted peptides for protection of soybean against Phakopsora pachyrhizi.
- J.T. English, F.J. Schmdit, S. Bishop-Hurley, R. O. Morris, G. P. Smith. 2010 Phage Display Selection of Anti-Fungal Peptides.. J.T. English, Z.D. Fang, F.J. Schmidt,G. Stacey. 2010. Method fo inducing resistance to fungal infection in transgenic plants using plant defense peptides.
- Conference presentations: Fang, Z.D., Schoelz, J.E., Stacey, G., Schmidt, F.J., and English, J.T. Combinatorially based selection of defense peptide directed against Phakopsora pachyrhizi. American Phytopathological Society. San Diego, CA. 2007
- Fang, Z.D., Schoelz, J.E., Stacey, G., Schmidt, F.J., and English, J.T. 2008. Selection of phage-displayed peptides that inhibit soybean rust. American Phytopathological Society. Minneapolis, MN. 2008.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We have continued to identify peptides from combinatorial phage-display peptide libraries that inhibit Phakopsora pachyrhizi, the fungus that causes Asian soybean rust. We selected phage-display peptide clones that inhibit pathogen germling development by in vitro assay, and tested them for their ability to inhibit soybean leaf infection. Two peptides were shown to significantly reduce infection and lesion formation. We also attempted to transform soybean plants for expression of these inhibitory peptides. However, transformation was not achieved because of toxic effects of the peptide-display scaffold protein that was used. We are now examining other proteins as possible peptide-display scaffolds for use in soybean transformation. We disseminated information on this and related peptide research on a regular basis via invited presentations at national and international research conferences. One presentation was made at the 2nd International Phytophthora capsici Conference, Duck Key, FL in December, 2009. A second presentation was made at the National Wheat Scab Forum, Orlando, FL, also in December 2009. We have also made information available to commodity interest groups. PARTICIPANTS: Individuals: James T. English TARGET AUDIENCES: plant pathologists; plant breeders; soybean producers PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Changes in knowledge are developing from this project: Based on research outcomes, we are moving ever nearer to the development of peptide selection and delivery technologies for designed protection against Asian soybean rust. The technologies developed in this project are attracting interest of plant pathologists seeking new technologies to solve other long-standing disease problems. Technologies developed in this research will complement proteomic technologies by enabling inhibition of defined protein targets that have critical influences on pathogen development and disease development. These technologies will also enhance the ability to respond to new forms of resistance that develop within ever-changing pathogen populations in the field. Changes in actions are also likely: Soybean and other commodity groups are interested in this research as a means of developing high value germplasm with new forms of disease resistance.
Publications
- Smehilova, M., Galuszka, P., Sebela, M., English, J.T., Frebort, I., Jaworek, P., Sedlarova, M., and Bilyeu, K. 2008. Subcellular localization and biochemical comparison of cytosolic and secreted cytokinin dehyrodgenase enzymes from maize. J. Exp. Bot. 60:2701-2712.
- Fang, Z.D., Marois, J.J., Stacey, G., Schoelz, J.E., English, J.T., and Schmidt, F.J. 2010. Combinatorially selected peptides for protection of soybean against Phakopsora pachyrhizi. Phytopathology. Accepted with revision.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We have identified peptides that inhibit Phakopsora pachyrhizi, the fungus that causes Asian soybean rust. We identified peptides by screening P. pachyrhizi germlings against highly diverse, combinatorial phage-display peptide libraries and selecting phage clones with affinity for binding to germling surface factors. We tested recovered phage-display peptide clones by in vitro assays for inhibition of germling development. Several clones were discovered that reduce the rate of spore germination and germ tube elongation. Some clones have been tested initially for their ability to inhibit soybean leaf infection and pustule formation. To date, several clones have been identified that may negatively affect these processes. We have also begun to evaluate the expression of protein-peptide scaffolds in leaves with a focus on secretion into the apoplast. This is a step required for placement of defense peptides in proximity with penetrating fungal germ tubes. We have disseminated
information from this work on a regular basis via poster presentations at scientific conferences, both regionally and nationally. As an example, we presented a research update at the annual meeting of the American Phytopathological Society held in San Diego in August, 2007. We have also made information available to commodity interest groups.
PARTICIPANTS: Individuals: James T. English
TARGET AUDIENCES: plant pathologists; soybean producers
Impacts
Changes in knowledge are developing from this project: Based on experimental outcomes, we are moving nearer to the development of peptide selection and delivery technologies for designed protection against Asian soybean rust. The technologies developed in this project will also have application for numerous emerging pathogens for which resistance is unknown, and that lack genomic and proteomic profiles. These technologies will also enhance the ability to respond to new forms of resistance that develop within ever-changing pathogen populations in the field. Changes in actions are also likely: We hope to see broadened opportunities for development of new plant germplasm with improved disease protection and deployment by commodity organizations.
Publications
- Fang, D.F., Laskey, J.G., Huang, S., Bilyeu, K.D., Morris, R.O., Schmidt, F.J., and English, J.T. 2006. Combinatorially selected defense peptides protect plant roots from pathogen infection. PNAS 103:18444-18449.
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