Project Objectives
- Evaluate utility for defense peptide delivery in soybean.
- Construct peptide delivery molecules based on either naturally occurring cell-wall proteins or apoplast proteins for delivery of peptides to points of interaction with P. sojae during colonization of root cortical and vascular tissues.
Executive Summary
This project focused on the final component step of a proprietary strategy for designed resistance against diseases of soybean. Designed resistance is based on applications of defense peptides that disrupt development of a target pathogen. Our initial focus has been on protection of roots against infection by Phytophthora. Technologies have been developed that will be useful for rapid development of resistant germplasm in response to threats from newly introduced pathogens. For instance, we have begun to apply this defense peptide strategy to Asian soybean rust. Finally, the technologies will enable development of germplasm with resistance against indigenous pathogens that have long been recalcitrant to control.
To complete this final step (Objective 1), we 1) tested defense peptide- display scaffolds constructed from cytokinin oxidase, 2) confirmed the stability and function of the peptide-scaffolds when secreted from plant roots where they contact infectious spores of Phytophthora, and 3) established that the peptides defend roots from Phytophthora infection. Studies of alternative scaffold proteins and their design remain in progress (Objective 2).
Biotechnology industries and agribusiness are showing escalating interest in our peptide products. Most recently, representatives from Syngenta and BASF visited our lab to discuss possible applications. In addition to our progress in research and development, we initiated a startup company as a means of moving project technologies towards commercialization. We established an agreement for forming and operating Hawthorn Biotechnologies LLC. We continue to develop business plans and strategies to acquire funding from SBIR and industrial sources.
Project Highlights
- Zoospores of Phytophthora normally are able to detect and swim towards susceptible plant roots, a crucial step required for infection and root rot development in soybean and many other crop species. We completed the design of scaffolds to display defense peptides selected for their ability to induce premature encystment of infectious zoospores and thus halt movement to roots. Our scaffold is based on a naturally occurring plant protein, cytokinin oxidase (CKX). This protein is highly stable in extreme environments, and it is naturally secreted from plant cells into the intercellular space and into soil surrounding the root (the rhizosphere). We attached the defense peptides (identified with previous IMBA funding) to the CKX terminus. At this location, peptides are exposed for interaction with zoospores.
- We showed that our peptides induce zoospore encystment and halt movement when secreted as part of the CKX scaffold. We tested and confirmed the effectiveness of our scaffold-peptides in a model yeast organism that can be grown easily in the lab.
- We also transformed tomato roots with the gene that produces the scaffold-peptide complex. Tomato is easier to manipulate than soybean and served as a model for evaluating our defense peptide in plants. When we tested the transgenic tomato roots, we confirmed that the scaffold-peptide was secreted into the rhizosphere and accumulated to sufficient concentration to induce a halo around a root that was devoid of infectious zoospores (Fig. 1). We found that these roots were protected from infection by encysted zoospores.
- Our experiments provided proof of concept for the third and final component step of a defense peptide strategy for designed disease resistance. The first two steps (completed with previous IMBA funds) included 1) methods for selecting candidate defense peptides and 2) assessment of candidate peptides for their abilities to disrupt pathogen development. We have moved beyond our tomato model system and are continuing to apply this strategy to defend soybean from Phytophthora and Asian soybean rust
- Having proven the functionality of peptides displayed by a protein scaffold, we believe we are now able to design resistance against new pathogens that threaten economically sustainable production of soybean, maize, and other crops. Our strategy provides the means to shorten response times to these pathogens compared to conventional breeding methods that involve extensive searches for resistance genes.
Figure 1. Secretion of CKX-peptides from transgenic roots induces zoospores to encyst at a distance from the root surface and limits infection (A). Normal encystment of zoospores around roots that do not express a defense peptide leads to 100% infection (B).
(A)
(B)
Project Impacts
- Completion of this final component step of our plant defense strategy places us very close to the development of new soybean germplasm with designed resistance against Phytophthora and other pathogens that impact soybean producers.
- This project has contributed to intellectual property related to designed disease resistance. Patent applications supported by results from this project, either directly or indirectly, are indicated later in the report.
- This project has escalated interest within major biotechnology and agribusinesses. For instance, in the summer 2006, we were visited by research and development representatives from Syngenta and BASF. These companies are interested in our defense peptide products for value-added protective seed treatments and for detection of pathogen occurrence in production areas to assist in determining need for protective biocide applications.
- We initiated a startup company as a means of moving project technologies towards commercialization. We established an agreement for forming and operating Hawthorn Biotechnologies LLC. We are continuing to develop a business plan and strategies to acquire funding from SBIR and industrial sources.
Presentations, Abstracts, and Patents
Poster Presentation and Abstract:
Protein scaffold design for delivery of peptides that induce encystment of Phytophthora capsici zoospores. Zhiwei Fang, Sarah Mounter, Michael Simon, Frank Schmidt, and Jim English. NSF Phytophthora Molecular Biology Workshop, New Orleans, May 2004. (P. capsici is used as a model organisms for technology development and proof of concept)
Patent:
A patent application was filed to protect the use of cytokinin oxidase as a peptide-delivery protein. Patent title: Phage Display Selection of Antifungal Peptides (US Patent Application serial no. 09/829,549).
A patent application was filed to protect the use of peptides that halt growth of Uromyces and Phakopsora, the rust fungi that cause common bean rust and Asian soybean rust, respectively. Patent title: Phage-display plant defense peptides directed against Phakopsora pachyrhizi and Uromyces appendiculatus.
Publications:
Fang, Z.D., Laskey, J.G., Huang, S., Bilyeu, K.D., Morris, R.O., Schdmit, F.J., and English, J.T. 2006. Combinatorially selected defense peptides protect plant roots from pathogen infection. Proceedings of the National Academy of Sciences. In press.
Equipment Purchases:
None
