INACTIVATION OF THE TRICHOTHECENE MYCOTOXINS ASSOCIATED WITH FUSARIUM HEAD BLIGHT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006542
• Project No.: WIS01867
• Project Start Date: Oct 1, 2015
• Project End Date: Sep 30, 2020

Project Director
Rayment, I.

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
Biochemistry

Non Technical Summary
The goal of this research proposal is to isolate, characterize, and optimize enzymes that can inactivate trichothecene mycotoxins associated with Fusarium head blight (FHB). This is a major plant disease of small grains that has caused devastating crop losses for wheat and barley (~$2.7 billion between 1998-2001), where most of the damage has occurred in the Midwest. At the present time there is no satisfactory way of controlling FHB or inactivating the mycotoxins associated with this plant disease. Thus, there is a compelling need to find new solutions to this agricultural problem.Trichothecenes are remarkably stable to both temperature and changes in pH and as such persist through milling, baking, and brewing. This problem would be resolved in part if enzymes could be identified that irreversibly inactivate the trichothecenes. Excitingly, bacterial isolates have been discovered that contain several unknown activities that lead to destruction of the trichothecenes. The nature of the enzyme(s) responsible for inactivation of trichothecenes in these organisms is unknown, but clearly they have potential to revolutionize the treatment of contaminated grain and the development of new resistance traits in wheat and barley. The goal of this study is to identify enzymes that deepoxidate the trichothecenes implicated in FHB.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	2499	1000	50%
712	2499	1000	50%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

fungal head blight

mycotoxins

trichothecenses

food safety

pathogen inactivation

Goals / Objectives
Fungal head blight (FHB) and its associated trichothecene mycotoxins are major source of crop losses in N. America. They not only reduce the yield, but also generate grain that is unsuitable for milling, brewing, or livestock. Thus far there is no satisfactory way to control FHB or reduce the toxicity of contaminated grain. The goal of this proposal is to develop improved enzymes for the inactivation and degradation of fungal mycotoxins associated with FHB. The current enzymatic methods for reducing the toxicity of trichothecenes focus on modification by either acetylation or glycosidation. Although these show some promise for controlling FHB they suffer from the fundamental weakness that the modification is reversible so that the toxin is not truly inactivated. This problem would be resolved in part if enzymes could be identified that irreversibly inactivate the trichothecenes. The obvious target for irreversible chemical modification is their 12,13 epoxide group which is necessary for substantial toxicity. Excitingly, bacterial isolates have been discovered that contain several unknown activities that lead to destruction of the epoxide moiety in trichothecenes. The focus here is identification and optimization of the enzymes responsible for degradation of fungal mycotoxins as a method for controlling the consequences of FHB.The goal of this study is to identify enzymes that deepoxidate the trichothecenes implicated in FHB. Thus the specific aims are:1. to identify the enzymes responsible for the deepoxidation activity in the six bacterial species that have been shown to exhibit this activity.2.to determine the biochemical strategy by which the toxins are inactivated.3.to optimize the enzymatic activity by biological selection and structural design.

Project Methods
The bacterial enzymes responsible for deepoxidation will be identified in a yeast screen that has been developed that is sensitive to low levels of mycotoxin. The genomic DNA for six aerobic bacterial species that have been demonstrated to deepoxidate trichothecenes has been provided by Dr. Zhou Ting at Agriculture and Agri-Food, Guelph, Ontario, Canada. It is planned to search for the source of the deepoxidase activity by screening genomic fragment plasmid libraries in S. cerevisiae. This search for activity has to be performed in a eukaryotic organism because trichothecenes do not inhibit bacterial growth. S. cerevisiae is the best system in which to perform this screen because of powerful array of genetic tools that provide a facile way to connecting biology to function. This is a proven approach for finding resistance genes.Once the enzymes have been identified, they will be optimized by biological selection and structural design. The genes identified in the initial screen will serve as a template for the generation of an improved enzyme. The initial goal will be to increase the enzymatic activity of the enzyme(s) and broaden the specificity. This will be an iterative process. The first step will be to create a mutant library from which improved variants can be selected with enhanced activity. A wide variety of methods have been developed to create mutant libraries including error prone PCR (epPCR), chemical mutagenesis, mutator strains, and site-saturation mutagenesis. Each of these methods has its advantages. In the first instance epPCR will be used to generate primarily single point mutations with a combined Taq and Mutazyme based protocol to generate unbiased libraries. These will be selected for improved activity. The main concern is whether sufficient sequence space can be explored to find a better gene, however there are numerous examples where this approach has been successful. Strong candidates will be passed iteratively through cycles of mutagenesis and selection seeking to find the best combination of mutations.Three dimensional structures of candidate enzymes will be determined to form the foundation for a rational optimization of the enzyme activity.

Progress 10/01/15 to 09/30/20

Outputs
Target Audience:Plant breeders and researchers focused on solving the problem of fungal head blight. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project trained a graduate student. How have the results been disseminated to communities of interest?Publication of literature articles and Ph.D. thesis. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Fusariumhead blight is a devastating fungal disease that infects cereal crops like wheat and barley by producing harmful toxins and reducing crop yields. In addition to helpingFusariumfungi infect crops, these toxins are a major health concern for animal and human consumers.Fusariumhead blight results in billions of dollars in lost profits for farmers each year in the Midwest and around the world. Thus far there are limited ways of controlling this problem. The goal here is to develop an enzyme that will completely detoxify the fungal toxins so that they can never enter the food chain. Previous methods led to masked toxins that in principle can be reactivated after ingestion. A screen for detecting enzymes with the ability to detoxify trichothecenes has been developed. This is a major accomplishment since it allows enzymes that show limited resistance to be improved by random mutagenesis and protein engineering. This sets the stage for completion of goal 3. The absence of a screen was a major stumbling block. The previous screens exhibited a very high background so that small improvements could not be detected. This is a particular problem when starting from a weak enzyme where its protection against the trichothecene is very small. In addition, the specificity in a Trichothecene UDP-glucosyltransferase from Oryza sativa was expanded. Family 1 UDP-glycosyltransferases in plants (UGTs) primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, where this is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins on account of its ability to glycosylate DON, nivalenol and hydrolyzed T-2 toxin (HT-2). In this study the specificity of the enzyme was expanded to cover all of the commonly encountered trichothecenes in the USA. As such this creates a target protein for genetic modification of barley and wheat.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Wetterhorn, K.M., Enzymatic Inactivation of Trichothecene Mycotoxins Associated with Fusarium Head Blight, University of Wisconsin, 2018

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Investigators interested in improving resistance to fungal head blight. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has facilitated the training of two graduate students. One had joined a biopharmaceutical company in Boston. The other is a postdoctoral fellow at the University of Michigan. How have the results been disseminated to communities of interest?The patent noted in last years' report has been approved and made public. This provides an opportunity for the methods described here to be used for the benefit of a large community. What do you plan to do during the next reporting period to accomplish the goals?The screen that has been developed will be used to improved trichothecene epoxide hydrolases. Engenous proteins from wheat have been identified by Dr. Gerhard Adam, that shows limited activity. Our screen can now be applied to improve that activity. The main benefit of these enzymes is that they belong to yeast so that we are simply seeking a better mutant protein.

Impacts
What was accomplished under these goals? Fusariumhead blight is a devastating fungal disease that infects cereal crops like wheat and barley by producing harmful toxins and reducing crop yields. In addition to helpingFusariumfungi infect crops, these toxins are a major health concern for animal and human consumers.Fusariumhead blight results in billions of dollars in lost profits for farmers each year in the Midwest and around the world. Thus far, there are limited ways of controlling this problem. The goal here is to develop an enzyme that will completely detoxify the fungal toxins so that they can never enter the food chain. Previous methods led to masked toxins that in principle can be reactivated after ingestion. A screen for detecting enzymes with the ability to detoxify trichothecenes has been developed. This is a major accomplishment since it allows enzymes that show limited resistance to be improved by random mutagenesis and protein engineering. This sets the stage for completion of goal 3. The absence of a screen was a major stumbling block. The previous screens exhibited a very high background so that small improvements could not be detected. This is a particular problem when starting from a weak enzyme where its protection against the trichothecene is very small.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience: My research targets all individuals that are interested in finding a solution to Fungal head blight. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The graduate student involved in this work has developed an independent development plan as required by the Department of Biochemistry and University of Wisconsin. Through his PhD program, he has developed teaching and speaking skills that will be essential in his future career. How have the results been disseminated to communities of interest? The results have been recently published in the Biochemistry journal. What do you plan to do during the next reporting period to accomplish the goals? Next year, we wil continue to complete the studies and evolve a trichothecene deepoxidase.

Impacts
What was accomplished under these goals? The support from this research have been used to develop a new enzyme for inactivating a broad range of trichothecene mycotoxins. In the first instance, we determined the three dimensional structure of a glucosyltranferase from rice that we had previously been shown to inactivate deoxynivalenol. Next, we broadened its activity to cover all of the major trichothecene mycotoxins found in Europe, Asia, and North and South America through protein engineering and site-directed mutagenesis. This class of enzymes has been shown to provide resistance towards specific toxins such as deoxynivalenol that are generated by common strains of Fusarium. Unfortunately, the earlier enzymes would only inactivate a limited number of toxins and hence had a limited range of action. Our work has generated a single enzyme that we will expect to provide resistance to FHB worldwide. Efforts to incorporate this into transgenic grains are in progress. This past year, we engineered the UDP glucosyltransferase from rice (Os79) to have a broad specificity towards both DON and T-2 toxin. The protein is stable and suitable for generating a genetically modified cereal with resistance to a wide range of trichothecene mycotoxins. In addition, we developed a screen for detecting enzymes that are capable of permanently inactivating trichothecene mycotoxins.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wetterhorn KM, Gabardi K, Michlmayr H, Malachova A, Busman M, McCormick SP, Berthiller F, Adam G, Rayment I. Determinants and Expansion of Specificity in a Trichothecene UDP-Glucosyltransferase from Oryza sativa. Biochemistry. 2017 Dec 19;56(50):6585-6596. doi: 10.1021/acs.biochem.7b01007. Epub 2017 Nov 30. PubMed PMID: 29140092.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:My research targets all individuals that are interested in finding a solution to Fungal head blight. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The graduate student involved in this work has developed an independent development plan as required by the Department of Biochemistry and University of Wisconsin. Through his PhD program, he has developed teaching and speaking skills that will be essential in his future career. How have the results been disseminated to communities of interest?The results have been recently published in the Biochemistry journal. What do you plan to do during the next reporting period to accomplish the goals?Next year, we wil continue to complete the studies and evolve a trichothecene deepoxidase.

Impacts
What was accomplished under these goals? Impact- Fungal Head Blight (FHB) is a devastating agricultural disease that affects wheat and barley worldwide, but has a particular impact in the upper Midwest. It degrades both the quality and quantity of the grain as well as introducing a wide range of mycotoxins. Of these mycotoxins, the trichothecenes are particularly troublesome where these lead to internal bleeding and vomiting in livestock. For this reason, strict limits have been imposed on the acceptable levels of trichothecenes in grains. The financial costs of this plant disease are devastating in years when an outbreak occurs. Unfortunately, it has been difficult to develop grains that exhibit resistance to the Fusarium species that underlie FHB. The goal of the current proposal is to develop enzymatic approaches to inactivating trichothecene mycotoxins. The impact of these studies will be to eventually create transgenic plants that exhibit resistance to FHB and are free of trichothecene mycotoxins. Results The support from this research have been used to develop a new enzyme for inactivating a broad range of trichothecene mycotoxins. In the first instance, we determined the three dimensional structure of a glucosyltranferase from rice that we had previously been shown to inactivate deoxynivalenol. Next we broadened its activity to cover all of the major trichothecene mycotoxins found in Europe, Asia, and North and South America through protein engineering and site-directed mutagenesis. This class of enzymes has been shown to provide resistance towards specific toxins such as deoxynivalenol that are generated by common strains of Fusarium. Unfortunately, the earlier enzymes would only inactivate a limited number of toxins and hence had a limited range of action. Our work has generated a single enzyme that we will expect to provide resistance to FHB worldwide. Efforts to incorporate this into transgenic grains are in progress. This past year, we engineered the UDP glucosyltransferase from rice (Os79) to have a broad specificity towards both DON and T-2 toxin. The protein is stable and suitable for generating a genetically modified cereal with resistance to a wide range of trichothecene mycotoxins.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wetterhorn KM, Newmister SA, Caniza RK, Busman M, McCormick SP, Berthiller F, Adam G, Rayment I. Crystal Structure of Os79 (Os04g0206600) from Oryza sativa: A UDP-glucosyltransferase Involved in the Detoxification of Deoxynivalenol. Biochemistry. 2016 Nov 8;55(44):6175-6186. Epub 2016 Oct 26. PubMed PMID: 27715009.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Scientists and stake holders in finding a solution to combatting fungal head blight of wheat and barley. Researchers focussed on understanding glycosyl transferases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The graduate student involved in this work has developed an independent development plan as required by the Department of Biochemistry and University of Wisconsin. He has also fulfilled his requirement of a first author publication. Through his PhD program he has developed teaching and speaking skills that will be essential in his future career. 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?Apply the selection protocol described above to locate an epoxide hydrolase that recognizes trichothecenes.

Impacts
What was accomplished under these goals? As a first step in accomplishing the goals we have developed a more sensitiveselection and screen for enzymes that provide protection against trichothecenes. This sets the stage for finding the epoxidehydrolases in bacterial isolates.In addition, we have determined the structure of a glucosyltransferase from rice that looks promising for providing protection against FHB.This work was recently published.We have also determined the kinetic activity of this enzyme against a wide range of trichothecenes and have gained an understanding of those factors that control specificity in this class of enzyme. This is significant since this group of enzymes has been shown to provide resistance to some forms of FHB.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wetterhorn KM, Newmister SA, Caniza RK, Busman M, McCormick SP, Berthiller F, Adam G, Rayment I. Crystal Structure of Os79 (Os04g0206600) from Oryza sativa: A UDP-glucosyltransferase Involved in the Detoxification of Deoxynivalenol.Biochemistry. 2016 Nov 8;55(44):6175-6186. PubMed PMID: 27715009.