THE ENZYMATIC DEGRADATION OF QUORUM-SENSING SIGNALS TO DECREASE BACTERIAL PHYTOPATHOGENICITY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 1003010
• Grant No.: 2014-33610-21890
• Cumulative Award Amt.: $100,000.00
• Proposal No.: 2014-00233
• Project Start Date: Jun 1, 2014
• Project End Date: Jan 31, 2015
• Grant Year: 2014
• Program Code: [8.2]- Plant Production and Protection-Biology

Recipient Organization
PHYLLOTECH, LLC
505 S ROSA RD STE 102
MADISON,WI 537191262

Performing Department
(N/A)

Non Technical Summary
Quorum-sensing (QS) allows bacterial populations to coordinate gene expression in a cell-density dependent manner and plays a significant role in plant pathogenesis. In gram-negative bacteria, QS is mediated by signaling molecules called acyl-homoserine lactones (AHLs). Our technological innovation is the use of an AHL-degrading enzyme to inactivate the AHL signals of bacterial phytopathogens and reduce their abilities to cause plant disease. Our first objective is to produce the enzyme in both bacterial and plant systems and test its efficacy against signal molecules. Our second objective is to test the ability of the enzyme to reduce disease caused by numerous phytopathogens, either through exogenous application of the heterologous protein or through the use of a producing bacterial strain as a biocontrol agent. Our goals are to demonstrate that the enzymatic degradation of AHLs can be utilized for the control of diseases and to understand the importance of quorum-sensing signal degradation in bacterial infection.

Animal Health Component

100%

Research Effort Categories

Basic

0%

Applied

100%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
215	1499	1100	100%

Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
1499 - Vegetables, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

acyl-homoserine lactone

acylase

bacterial phytopathogen

quorum sensing

Goals / Objectives
Our main goal of this project is to develop an enzymatic method for disrupting the intercellular communication of bacterial plant pathogens and reduce disease. Our milestones will include:1. The addition of a bacterial secretion signal to an enzyme that degrades bacterial signaling molecules.2. The scalable production of the enzyme in both a bacterial system and a plant system.3. The utilization of the enzyme to control bacterial disease in on-plant disease assays.

Project Methods
Standard methodologies for molecular biology, and bacterial and plant transformation will be utilized to generate the plant line and bacterial strain that produces the signal-degrading enzyme. Both chemical-based and bioreporter-based assays will test the ability of the enzyme to degrade signaling molecules. Plant-based disease assays and epiphytic fitness assays will determine if the enzyme can control plant disease caused by bacterial phytopathogens.

Progress 06/01/14 to 01/31/15

Outputs
Target Audience: The primary target audience for this research is customers who wish to purchase and utilize an environmentally-friendly product to control diseases caused by bacterial phytopathogens. Potential customers include greenhouse producers, researchers, and large industrial partners wishing to expand their product portfolio. The technology developed during this phase I project was introduced to potential customers at the 2014 Annual Meeting of the American Phytopathological Society (Minneapolis, MN) and during onsite, invited visits to three large distributors of biopesticide and agricultural products. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During this Phase I SBIR project, two technologies based on AHL-degrading enzymes were developed that have great potential to be used to manage bacterial plant pathogens. These technologies are: 1) nonpathogenic Pseudomonasfluorescens strains which secrete AHL-degrading enzymes and that can be used as biocontrols and 2) the exogenous AHL-degrading enzymes that can be applied directly to crops. Two P. fluorescens strains which produce and secrete dissimilar, active AHL-degrading enzymes were developed. These strains, when co-inoculated with the plant pathogen Pectobacterium carotovorum, significantly reduced tissue maceration of potato tubers, caused by the pathogen. To assess the efficacy of exogenous application of the AHL-degrading enzymes directly, Pseudomonas syringae strains were engineered to overexpress the enzymes. The exogenous application of purified AHL-degrading enzymes reduced the disease severity of P. carotovorum, as well as brown spot disease on bean pods, caused by P. syringae. To produce large quantities of the AHL-degrading enzymes, the generation of N. tabacum lines that produced the enzymes in glandular secreting trichomes was initiated. The results of this project demonstrate that AHL-degrading enzymes can be utilized for the control of bacterial plant diseases, and also indicate a novel way to achieve disease control without the use of toxic chemicals or techniques that induce high selective pressure on the pathogen. PhylloTech's future efforts will focus on commercializing further these technologies, thus adding effective ancillary weapons to the current disease control arsenal that will benefit the agricultural industry.

Publications