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 phage 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 complete selection of defense peptides that inhibit seedling and root disease caused by P. sojae and 2) to confirm the effectiveness of defense peptides delivered by carrier proteins in hairy roots.

Results

In this project, we 1) completed phage-peptide library screens against germlings and zoospores of P. capsici and P. sojae, 2) refined constructs for delivery of peptides based on cytokinin oxidase (CKX), 3) tested efficacy of CKX-peptide constructs in vitro, and 4) initiated hairy root and plant transformation experiments to test efficacy of CXK-peptide constructs in plant tissues. These accomplishment have brought us closer to a useable system of disease control based on deployment of defense peptides.

By the end of this project, we completed screens of three peptide libraries against three infective structures of P. sojae and P. capsici (Table 1). The libraries were designed to display peptides composed of 15 random amino acid (f88-4/15), 8 amino acids(f8), or 6 amino acids (f5/6). Each library screen included three or four rounds of selection and amplification, thus generating collections of peptides with high binding affinity for zoospores, cysts, and germlings. Screenings generated 20 to more than 40 useable peptides from each library to test for pathogen disruption We will continue to assess these collections for inhibitory effects beyond the project period.

Table 1. Peptide libraries screened against infective structures of P. sojae and P. capsici.

Species	Infective Structure	Library
P. sojae	zoospores	f88-4/15
	zoospores	f5/6
	cysts	f88-4/15
	cysts	f5/6
	germlings	f5/6
P. capsici	zoospores	f8
	germlings	f88-4/15

We also refined and expanded constructs of select candidate defense peptides in CKX as a means of delivery in plant tissue. CKX is a naturally occurring, secretable protein produced by plants. By incorporating peptides as part of this molecule, we optimized opportunities for delivery of peptides to the intercellular space and rhizosphere where they interact with Phytophthora.

Test Constructs included peptides pc87 and pc42 that, in phage-display format, are known to induce zoospore encystment. Constructs were made to express either single or triplicate copies of each peptide on the exposed terminal end of CKX. Each of these constructs was secreted from transformed yeast (Pichia), and each was able to induce high levels of zoospore encystment of P. capsici. Control treatment of zoospores with non-modified CKX or with a random peptide did not induce significant encystment. We are continuing to evaluate the impact of CKX-peptide constructs on P. sojae zoospores, cysts, and germlings.

Finally, we are working through the technical details of expressing these same CKX-peptide constructs in hairy root cultures of a tomato variety susceptible to P capsici. To create a hairy root culture, we moved the CKX-peptide gene into Agrobacterium rhizogenes, a bacterium that is able to infect roots and transmit DNA to plant tissue. Once transferred to the plant, the gene coding for CKX-peptide is expressed along with genes from A. rhizogenes that induce proliferation of root tissues.

In initial experiments, hairy roots expressing CKX-pc87 or CKX-pc42 induced high levels of zoospore encystment at a distance from the root surface. In contrast, roots expressing CKX alone or CKX with a random peptide insert did not induce zoospore encystment at a distance; rather encystment occurred at the root surface as it occurs in non-modified roots. These experiments demonstrated that CKX-peptide constructs are secreted from roots and at high enough concentrations to diffuse from the root surface and disrupt Phytophthora. Encystment at this distance reduces the potential for zoospores to germinate and locate susceptible root tissue. This result is very important to this project since it confirms the concept of peptide delivery for modification of pathogen behavior.

Finally, we have modified the CKX-peptide constructs to transform whole plants using A. tumefaciens as vector for gene delivery. This work is continuing beyond the project period. The advantage of whole plant transformation is that we will produce whole plants that produce seed, thus allowing us to maintain modified plants for continued experimentation.

Project Outcomes and Impacts

Our project has generated data to support three patent applications that are working their way through the U.S. patent examiner's office. The first application describes our peptide screening protocol. The second application describes the actual defense peptides that have utility for pathogen modification. The third application describes the use of CKX as a means of delivering the peptides within plant tissue.

Our proof of concept for defense peptide technology has led to great interest and initial funding to apply our technology for control of soybean rust, caused by the fungus, Phakopsora pachyrhizi. Given the lack of major gene resistance to rust in soybean. Defense peptides are considered by experts to be one of the most promising biotechnological approaches to disease control. Our work with this pathogen has received attention recently in Science (see attached PDF; page 2, bottom of first column). Our initial funding for work on soybean rust has come from the Missouri Soybean Merchandising Council. We also have rust proposals pending with USB and Monsanto.

A final major outcome of this project has been the creation of a startup biotechnology company, Hawthorn Biotechnologies LLC. Founders include J. T. English, F.J. Schmidt, G. Stacey, and M. Kerley. The intention is to use this company as a means of accelerating the movement of peptide and related technologies to commercial use. The technologies have application not only for plant protection, but also protective and therapeutic applications for control of animal and human intestinal pathogens. We are now creating a business plan and meeting with various companies to examine possibilities for commercialization of our technologies. We expect to receive our first contract for biotechnology research related to soybean in the very near future.