Introduction and Objectives

Diseases cause $2.5 billion in losses annually to yield and quality of soybean production in the north central United States. One of these diseases, Phytophthora seedling and root rot (PRR), is ranked second in production losses valued at more than $400 million annually.

For many years, control of Phytophthora root rot has been based on resistance. Plant breeders continually screen and select soybean for resistance against P. sojae, the causal pathogen of PRR. In soybean fields, populations of P. sojae change in virulence over years of production, and eventually plant resistance fails. Current estimates are that any specific resistance gene lasts for only 5 to 7 years before new pathogen virulence races evolve to overcome its protective effect. There is an obvious need and market for alternative forms of soybean resistance, particularly those that are effective against multiple P. sojae races.

We have arrived at an era in which new formats of soybean disease resistance can be designed. New possibilities follow from recent advancements in combinatorial biochemistry, plant transformation technology, and molecular biology. Combinatorial biochemistry enables screening of diverse Image display libraries for peptides that bind to infective structures of P. sojae and disrupt their development before or during plant infection, regardless of race. Tools of molecular biology and plant transformation enable the construction of delivery systems to deploy disruptive peptides, as well as beneficial peptides within and around root systems.

In this project, we integrated these kinds of tools to pursue the development of (1) valued-added soybean with novel disease resistance capabilities, and (2) marketable, high-valued technologies for identifying and delivering defense peptides and other beneficial molecules in soybean, maize, or other crops.

Specific project objectives were (1) to test peptide-carrier protein constructs against P. sojae in vitro, and (2) to test peptide-carrier protein constructs against P. sojae in planta.

Context for Biotechnology Development

Pathogen development: Phytophthora sojae is an obligate parasite adapted to long-term survival in soil in the absence of soybean. Oospores enable survival in soil where they exist at low densities. In the presence of a susceptible soybean plant, the pathogen progresses rapidly through a series of finely mated developmental steps that produce cycles of infection and disease. These cycles depend on pathogen development from oospores through zoospore release, encystment, germination, and infection. Although the cycle appears straightforward at first glance, the procession of life stages is finely tuned to environmental signals, particularly those coming from soybean.

Zoospores are the life-stage of greatest importance for dispersal to root infection sites. A major susceptible site is located just behind the apical meristem of a root where cells are elongating. Exudates released from elongating cells serve as signals that direct chemotactic movement of zoospores toward the site (Carlile, 1983; Deacon and Donaldson, 1993).

Zoospores encyst as they approach the root surface in response to environmental signals. In the presence of an appropriate signal, zoospores encyst at the root surface with a specific orientation, so that a germ tube emerges towards susceptible tissue. If zoospores encyst before contact with the root, germ tubes emerge in any orientation and must reorient in order to locate the toot and infect. Cell surface receptors on the germ tube are involved in this root-orientation process. After infection, hyphae grow through plant tissue intercellularly and/or intracellularly (Stössel et al., 1980; Coffey and Wilson, 1983; Enkerli et al., 1997; Murdoch and Hardham, 1998), depending on the pathogen species. Haustoria are formed by P. sojae and other Phytophthora species (Coffey and Wilson, 1983; Jones et al., 1974; Stössel et al., 1980). Both hyphae and haustoria establish close contact with host cell walls and membranes. presumably, cell surface receptors are important in sensing plant signals, although direct evidence is lacking.

Phage-display peptides and cell-surface receptors: Phage-display random peptide libraries have proven useful for isolating ligands of importance to cell surface receptors of mammalian cells. They are appropriate here because of the importance of cell surface receptors in mediating pathogen development. Delivery of inhibitory peptides to sites of defense has not been readily achieved. For example, Hightower et al. (1994) studied the inhibitory effects of cecropin, a toxic peptide derived from Hyalophora cecropia pupae, on growth of Erwinia and Pseudomonas spp., in vitro and in planta. Cecropin effectively limited bacterial growth in vitro; however, when mixed with leaf extracts, the toxin was degraded rapidly. When cecropin was expressed in transgenic tobacco plants inoculated with the pathogenic bacteria, it did not reduce soft rot development. The authors suggested that toxin was degraded before secretion into the apoplast where it would interact with the pathogenic bacteria.

Peptide carriers: Fusion of a selected peptide to an appropriate carrier molecule should protect against rapid degradation in the plant or after secretion into the apoplast and rhizosphere. A number of carrier molecules have been examined for purposes of peptide display in mammalian systems (e.g. McConnell and Hoess, 1995), but none have been identified for use with plant defense peptides. We are focusing initially on cytokinin oxidase, an exportable protein that may provide stability to attached peptides during delivery to plant infection sites.

Results

In the first phase of this project, we (1) established a proprietary phage-display technology as an effective means of predicting bioactivity of synthesized peptides, (2) began to screen and select additional bioactive peptides, (3) developed peptide-carrier protein constructs, and (4) demonstrated that these peptide constructs can be secreted from Pichia (yeast) cells so vitro and are effective in disrupting Phytophthora development. Each accomplishment represents a major step toward developing marketable research tools and value-added soybean with enhanced disease resistance.

In this project we developed a proprietary library screening protocol (Bishop-Hurley et al., 2002) that is highly effective in predicting the inhibitory potential of peptides when synthesized as free molecules, independent of phage coat protein. We have filed for patent protection for this intellectual property (non-provisional patent application no. UMO 1521.1; filed 10 April, 2001). We are working with the MU Office of Technology and Special Projects (OTSP) to license the technology.

We developed this protocol using P. capsici as a model organism because of the ease of manipulating its development. Of particular importance is the ease of producing and releasing P. capsici zoospores. Using the protocol, we successfully screened diverse phage-display libraries and identified peptides that induce premature zoospore encystment (Table 1 and Appendix I; Figure 1). At this point, we shifted screening to focus on peptides that induce early encystment of P. sojae (Table 1). Early screening results indicated similar success in identifying peptides that induce early encystment (Appendix 1; Fig. 2). We have continued (beyond the giant period) to expand our collection and testing of selected phage-displayed peptides that bind to P. sojae cysts (Appendix 1; Fig. 3) and hyphae. All libraries in this project were provided by our collaborator, Dr. George P. Smith (University of Missouri).

Table 1. Examples of Phage-displayed random peptide libraries screened against P. capsici and P. sojae zoospores.

Library	Peptide Display Protein	Type of Insert	Number of Inserts Per phage
f8-1	pVIII	8-mer	3900
f88-4/15-mer	pVIII	15-mer	390
FUSE5	pIII	15	3-5

In subsequent experiments, we found that many phage-display peptides, when synthesized as free molecules independent of phage coat protein, also induced early zoospore encystment. This significant observation boosts our confidence in one ability to rapidly select bioactive defense peptides. Results of this initial research on methods development and peptide selection have been published (Bishop-Hurley et al., 2002).

We have also had significant success in developing peptide protein carriers. Our initial peptide protein-carrier construct is based on modifications of cytokinin oxidase, a known secretable and stable plant protein. After expression of career proteins in yeast, each peptide protein was secreted into the growth medium, collected and purified, and assessed for inhibition of zoospore motility and chemotaxis The inhibitory ability of die peptides was dramatic Proteins carrying inserts; of a test peptide (two tested in this grant period) induced 80-98% P. capsici zoospore encystment. In water controls, Phytophthora zoospore encystment was 25% or less. In ongoing experiments, we are inserting peptide selected against P. sojae into the carrier protein for similar expression in yeast and testing for zoospore encystment.

The research in this grant period (and continuing) has been performed partially in collaboration with Dr. Kristin Bilyeu (USDA Plant Genetics Unit, University of Missouri) and Dr. Ivo Frebort (Dept. Biochemistry, Palacky University, Czech Republic). Dr. Frebort has expertise in structural biochemistry and interests in structural aspects of cytokinin oxidase function. Among several activities, we are refining our peptide protein-carrier constructs to eliminate oxidative activity, and thus establish the protein as strictly an inert carrier molecule.

In the next research phase, we are developing means of transforming hairy root cultures with carrier proteins for in planta testing. This work is being conducted in collaboration with Dr. Chris Taylor of the Danforth Plant Science Center. When peptide efficacy is demonstrated in the hairy root system, we will transform and regenerate soybean plants that express defense peptides. We also intend to pursue patenting and commercialization of cytokinin oxidase-based peptide carriers and other exportable proteins as they are discovered.

Activities in this project have led to expanded, complementary basic studies of the mechanisms by which defense peptides alter the development and behavior of Phytophthora For example, Dr. Adrienne Hardham (Australian National University), an international expert on Phytophthora developmental biology, has kindly provided zoospore monoclonal antibodies that will assist in understanding the specificity of defense peptide function. We are seeking funding to allow research exchanges between our laboratories to expand the collaboration. Dr. Hardham is also a collaborator on our pending NSF proposal. We are also collaborating with Dr. Howard Berg (Danforth Plant Science Center) to visualize pathogen binding by peptides and monoclonal antibodies using fluorescence and electron microscopy. Dr. Berg is also a collaborator on our pending NSF proposal.

Summary of Products

• Proprietary protocol for predicting and selecting bioactive defense peptides
• Catalogue of proprietary defense peptides
• Proprietary peptide-protein delivery system (in development)

Impacts on Agricultural Biotechnology

This project is beginning to have impacts in the area of alternative disease resistance strategies. Our efforts are the first to show utility of combinatorial chemistry as an approach to develop defense peptides against pathogenic fungi. Interest in our work is evident from the early support received from Monsanto and other companies, and invited national and international presentations. Phage display technology has been used previously for diagnostics in the case of plant virus diseases, but it is now being considered as a means of generating new forms of virus resistance.

Evidence of emerging impact comes from an invitation to J. English to serve on the inaugural steering committee of the NSF-funded Phytophthora Molecular Biology Network. We expect to gain wider exposure and to have greater influences in the direction of new research as a result of this participation.

Impacts on Attitudes

Our work has piqued interest among colleagues who study plant microbe interactions and among industry contacts. The research is of particular interest because we are able to bypass some aspects of genomics to directly identify unique pathogen developmental factors that can be manipulated to created innovative forms of disease resistance.

Future Research

Application to other soybean diseases: Based on encouraging early successes in this IMBA project, we suggest expanding combinatorial approaches to selection of defense peptides against additional hinting pathogens of soybean. This includes development of defense peptides against Heterodera glycines, the soybean cyst nematode, and Fusarium solani, the fungal pathogen that causes sudden death syndrome. Each of these pathogens has major impacts on soybean production in Missouri, Illinois, and the midwestern U.S. For each pathogen, limited options exist for disease control, beyond traditional resistance breeding in the case of soybean cyst.

Defense peptide delivery system: It is also be important to emphasize the development of innovative strategies for delivering useful peptides and other molecules to points of function within plants. Given directed resources, it should be possible to develop an expanded catalogue of candidate molecules with utility for delivery of defense peptides (and other bioactive factors) to specific tissue sites. This will also require development of an expanded catalogue of promoters that enable design of tissue-specific production of fusion peptide-protein molecules. This critical technological area requires independent support — development cannot be earned out effectively as a component of another project.

We are ready to pursue these avenues of research to broaden the applicability of out technologies. We are already pursuing alternative sources of funding for more basic studies of mechanisms by which defense peptide provide protection from plant pathogens (see collaborators below).

Bibliography of Project-Related Publications

Bishop-Hurley, S.L., Mounter, S.A., Laskey, J., Morris, R.O., Elder, J., Roop, P., Rouse, C., Schmidt, F.J., and English, J.T. 2002. Phage-display peptides as developmental agonists for Phytophdiora capsici zoospores. Appl. Env. Microbiol. 68:3315-3320.

International Symposium on Stress Responses in Biological Systems. Chonnam National University; Kwangju, Korea. 2000. Selection of Phage-Display Peptides that Induce Encystment of Phytophthora capsici Zoospores.

American Phytopathological Society, New Orleans, LA. 2000. Bishop-Hurley, S.L., Schmidt, F.J., Smith, G.P., and English, J.T. Selection of phage-display peptides that induce encystment of Phytophthora capsici zoospores in vitro. Phytopathology 90:S7.

*American Phytopathological Society, Salt Like City, UT. 2001. Laskey, J. 0., Bishop-Hurley, S.L., Mounter, S.A., English, J.T., and Schmidt, F.J. Phage-display peptides that disrupt developmental progression of Phytophthora species. Phytopathology 91: S53.

*American Society for Microbiology, Orlando, FL. 2001. Bishop-Hurley, S.L., Laskey, J.G., Mounter, S.A., Schmidt, F.J., and English, J.T. Phage-display peptides that disrupt developmental progression of Phytophthora species. Proceedings American Society for Microbiology.

Literature Cited

Bishop-Hurley, S.L., Mounter, S.A., Laskey, J., Morris, R.O., Elder, J., Roop, P., Rouse, C., Schmidt, F.J., and English, J.T. 2002. Phage-display peptides as developmental agonists for Phytophthora capsici zoospores. Appl. Env. Microbiol. 68:3315-3320

Carlile, M. 1983. Mother, taxis and tropism in Phytophthora. Pages 95-107 in: Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology. D.C. Erwin, S. Bartnicki-Garcia, and P.H. Tsao, eds. APS Press, St. Paul, MN.

Coffey, M.D., and Wilson, U.E. 1983. Histology and cytology of infection and disease caused by Phytophthora Pp289-301 in: Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology. D.C. Erwin, S. Bartnicki-Garcia, and PH. Tsao, eds. APS Press, St. Paul, MN.

Deacon, J.W., and Donaldson, S.P. 1993. Molecular recognition in the homing responses of zoosporic fungi, with special reference to Pythium and Phytophthora. Mycol. Res. 97:1153-1171.

Enkerli, K., Hahn, M.G., and Mims, C.W. 1997. Ultrastructure of compatible and incompatible interactions of soybean roots infected with the plant pathogenic oomycete Phytophthora sojae. Can. J. Bot. 75:1493-1508.

Hightower, R., Baden, C., Penzes, E., and Dunsmuir, P. 1994. The expression of cecropin peptide in transgenic tobacco does not confer resistance to Pseudomonas syringae pv tabaci. Plant Cell Rept. 13:295-299.

Jones, D.R., Graham, W.G., and Ward, E.W.B. 1974. Ultrastructural changes in pepper cells in a compatible interaction with Phytophthora capsici. Phytopathology 64:1084-1090.

McConnell, S.J., and Hoess, R.H. 1995. Tendamistat as a scaffold for conformationally constrained phage peptide libraries. J. Mot. Biol. 250:460-470

Murdoch, E.G., and Hardham, A.R. 1998. Components in the haustorial of the flax rust fungus, Melampsora lini, are labelled by three anti-calmodulin monoclonal antibodies. Protoplasma 201:180-193.

Stössel, P., Lazarovits, G., and Ward, E.W.B. 1980. Penetration and growth of compatible and incompatible races of Phytophthora megasperma var. sojae in soybean hypocotyl tissues differing in age. Can. J. Bot. 58:2594-2601.

Involvement of Collaborators

The following are collaborators in our IMBA activities or are involved in our pending NSF proposal directed at basic studies of defense peptides and zoospore encystment.

Dr. Howard Berg (NSF)
Danforth Plant Science Center

Dr. Adrienne Hardham (NSF)
Australian National University

Dr. Kristin Bilyeu (IMBA and NSF)
University of Missouri

Dr. Chris Taylor (IMBA)
Danforth Plant Science Center

Dr. Ivo Frebort (NSF)
Palacky University
Czech Republic

Dr. George Smith (IMBA)
University of Missouri

Leveraged Grants

We have obtained funding or submitted proposals as a direct result of support from IMBA.

Current grant:

NSF — Integrated Plant Biology. 2002-2003. $33,000. Defining Catalytic Mechanism of Cytokinin Oxidase/Dehydrogenase. J.T. English and K. Bilyeu. (International collaboration with Dr. Ivo Frebort)

Pending grants:

NSF — Integrative Biology and Neuroscience, Plant and Microbial Development. 2003-2006. $374,275. Characterization of Receptor-Mediated Development in the Oomycete, Phytophthora capsici. J.T. English and F.J. Schmidt. (Collaboration with Drs. Adrienne Hardham and Howard Berg)

USDA-NRI. In preparation ($275,000 estimated) 2003-2005. Receptor-Mediated Development in Phytophthora. J. T. English and F. J. Schmidt. (Collaboration with Drs. Adrienne Hardham and Howard Berg)

Appendix I

Fig. 1. Phage-display peptide clones effectively predict the ability of corresponding synthesized peptides to induce P. capsici zoospore encystment. (Bishop-Hurley et al., 2001).

Fig. 2. Phage-display peptides selected from screened libraries induce encystment of P. sojae, as shown for this early set of test clones, Soj2-2 through Soj2-4. Zoospore encystment in water controls is less than 20%.

Fig. 3. Typical stage of germlings at nine of library screening. Germlings are the structural stage of P. sojae that penetrates a root and initiates infection. Germlings are about 50-100 μm in length at screening.