USING SYSTEMS BIOLOGY TO UNRAVEL GENE AND METABOLITE NETWORKS INVOLVED IN MYCOTOXIN PRODUCTION IN THE FUSARIUM GRAMINEARUM-CORN INTERACTION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0229731
• Grant No.: 2012-67012-19810
• Cumulative Award Amt.: $130,000.00
• Proposal No.: 2012-01325
• Project Start Date: Aug 1, 2012
• Project End Date: Jul 31, 2014
• Grant Year: 2012
• Program Code: [A7201]- AFRI Post Doctoral Fellowships

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Plant Biology

Non Technical Summary
From an agricultural standpoint, fungal diseases such as ear and stalk rot of corn are primary factors limiting corn production in many parts of the U.S. Thus, understanding the genetic basis for the F. graminearum-corn interaction and the mechanisms affecting mycotoxin production in the host is key to the long-term improvement of this industry. In the U. S., the corn industry occupies approximately 92.3 million acres, with an estimates market value of ~$77 billion. In recent years however, the high content of mycotoxins in food, and ethanol byproducts used for feed has raised significant concerns on the corn industry. The F. graminearum-corn pathosystem provides a unique and timely opportunity for investigating transcriptional and metabolite changes in pathogen and host during the progression of ear and stalk rot. First, the release and publication of the corn and F. graminearum genomes provides a wealth of underexploited genomic potential for understanding the pathogen-host interaction and regulation and production of mycotoxins. Second, working with a crop and pathogen of significant agricultural importance such as corn and F. graminearum allows bypassing the use of traditional lab models such as Arabidopsis, and learn directly from the crop to translate this knowledge into solutions for disease and mycotoxin management, not only in corn, but cereal crops in general. This project will characterize differential responses of maize germplasm to F. graminearum infection and determine throughout the disease cycle, not only the types and levels of mycotoxin production in both infected stalks and ears but also gene expression and metabolite profiles in both corn and the pathogen thereby providing the first data on how host processes affect mycotoxin production by the pathogen while causing disease. These data will form a systems biology approach to identify key fungal and host genes and mechanisms involved in mycotoxin accumulation, a first step in developing control measures to reduce the impact of the pathogen and its toxins on corn-derived food and feed.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	1510	1080	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1510 - Corn;

Field Of Science
1080 - Genetics;

Keywords

corn

maize

fusarium graminearum

fusarium

mycotoxins

ear rot

stalk rot

food safety

fungal diseases

plant pathogen

zea mays

systems biology

metabolomics

transcriptomics

Goals / Objectives
The general objective of this proposal is to use systems biology approaches that combine metabolomics and transcriptomics to study the F. graminearum-corn interaction in planta and further our understanding of mycotoxin accumulation in the disease process. In addition, we will use this information to begin to understand mechanisms of resistance in corn. Specifically we aim to: 1. Survey a panel of maize germplasm with different levels of susceptibility to F. graminearum for metabolite production during ear and stalk rot disease. We will use a F. graminearum wild type and mycotoxin mutant strains to obtain metabolite data from infected corn ears and stalks. This experiment will expand our knowledge of host factors that affect mycotoxin production, and provide information about host resistance mechanisms. 2. Characterize gene expression and metabolite production during the F. graminearum-corn interaction as ear and stalk rot disease progresses. We will use F. graminearum wild type and mycotoxin mutant strains to obtain metabolite and transcriptome data from infected corn samples at successive time points with the goal to identify host and pathogen genes important for disease, mycotoxin production and host response. 3. Build gene-metabolite association networks to identify candidate genes host and pathogen involved in modulation of mycotoxin production throughout disease. Gene association networks can reveal relationships between genes in biochemical pathways as well as relationships between regulatory genes and their targets. Use of a systems biology approach in combination with the mycotoxin mutants will enable the identification of genes and metabolites relevant to infection, mycotoxin production and host resistance. These experiments will also reveal commonalities and differences in metabolite production and transcriptional changes in stalk rot versus ear rot.

Project Methods
We will perform a survey of metabolites produced during ear and stalk rot of corn caused by F. graminearum in a panel of 20-24 corn varieties. We will grow corn plants in the greenhouse and inoculate their stalks with a wild type and two mycotoxin (DON and ZEN) mutant strains of F. graminearum. For ear rot experiments, plants will be grown in the field and ears will be collected to perform detached ear inoculations in humid chambers in the lab with F. graminearum. We will perform disease ratings to determine the susceptibility of corn varieties to F. graminearum and we will collect infected tissue at four time points for metabolite analysis. We will generate a rich dataset of metabolite profiles across a panel of corn varieties with different levels of tolerance to F. graminearum. We expect to detect corn varieties with resistance to disease or mycotoxin accumulation, as well as host and pathogen metabolites induced in resistant vs. susceptible varieties. We will perform metabolite and transcriptomics analysis on a resistant to moderately resistant and a susceptible corn variety identified in the first set of experiments. We will sample at four time points during ear and stalk rot disease development caused by the wild type F. graminearum and two mycotoxin mutants. We will inoculate stalks (greenhouse) and ears (detached and field-grown) of these varieties with F. graminearum wild type, a DON, and a ZEN mutant. We will perform disease ratings and collect infected tissue at four time points for transcriptome and metabolome profiling. These data will allow us to characterize host and pathogen gene and metabolite expression as disease progresses, differential expression of genes and metabolites in stalk vs. ear rot as well as in wild type F. graminearum vs. DON or ZEN mutants. Using systems biology approaches, we will integrate metabolome and transcriptome data to construct gene-metabolite association networks. We expect to identify host and pathogen networks affecting mycotoxin production during stalk vs. ear rot, when wild type vs. mutant strains cause disease, and at specific time points during disease. This detailed characterization of the F. graminearum-corn interaction and of genes and metabolites affecting mycotoxin production will provide new insight about how mycotoxins accumulate in corn tissues, which is required information to develop control measures.

Progress 08/01/12 to 07/31/14

Outputs
Target Audience: Plant pathologists, plant breeders, genomicists, plant biologists, extension personnel, growers Changes/Problems: The project was initiated on August 1st, 2012 and terminated on February 28, 2013. The PD Lina Quesada was offered a position as an Assistant Professor at North Carolina State Universy that started on March 1st, 2013. The PD requested to finish the project once relocated at NCSU but the request was denied, thus, the award was closed on February 28. Progress made until that point is outlined in this report. What opportunities for training and professional development has the project provided? Project Director: Lina Quesada, Michigan State University, performed field, greenhouse and laboratory experiments, as well as oversight and management to the project, she was trained in field pollinations with corn, laboratory infections of corn ears, greenhouse infections of corn stalks, growing and maintaining corn in the field and greenhouse, handling and maintenance of fungal pathogens, pathogen isolation from infected corn tissue, liophylization, RNA extractions, RNA sequencing, bioanalyzer analysis, targetted metabolomics, non-targeted metabolomics, bioinformatics and systems biology methods; Mentor: C. Robin Buell, Michigan State University, provided oversight to the project; Collaborator: F. Trail, Michigan State University, provided oversight to the project; Professional Aide: Jameed Al-Haddad, Michigan State University, was trained in liophylization, RNA extractions, RNA sequencing, bioanalyzer analysis, targetted metabolomics, and non-targeted metabolomics; Professional Aide: Emily Crisovan, Michigan State University, was trained in field pollinations with corn, laboratory infections of corn ears, greenhouse infections of corn stalks, growing and maintaining corn in the field and greenhouse, handling and maintenance of fungal pathogens. How have the results been disseminated to communities of interest? Resuls have been disseminated through oral presentations of research results and publication of abstracts in conference proceedings. What do you plan to do during the next reporting period to accomplish the goals? The project was initiated on August 1st, 2012 and terminated on February 28, 2013. The PD Lina Quesada was offered a position as an Assistant Professor at North Carolina State Universy that started on March 1st, 2013. The PD requested to finish the project once relocated at NCSU but the request was denied, thus, the award was closed on February 28. Progress made until that point is outlined in this report.

Impacts
What was accomplished under these goals? We identified corn varieties resistant to Fusarium ear and stalk rot; we developed a targeted metabolomics method to identify and measure deoxynivalenol and zearalenone in pathogen and infected host tissue (ear and stalk); we generated metabolomic and transcriptomic profiling data for host and pathogen to characterize infection by wild-type Fusarium strains in corn stalks and ears, as well as by toxin-deficient Fusarium strains; finally we characterized the metabolome and transcriptome in Fusarium wild-type and toxin-decifient strains in vitro.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Quesada-Ocampo L.M., Al-Haddad J., Trail F., and Buell C.R. (2013) Susceptibility of corn to stalk rot caused by Fusarium graminearum and mycotoxin mutants. Phytopathology 153-O

Progress 08/01/12 to 07/31/13

Outputs
Target Audience: Plant pathologists, plant breeders, genomicists, and plant biologists Changes/Problems: The project was initiated on August 1st, 2012 and terminated on February 28, 2013. The PD Lina Quesada was offered a position as an Assistant Professor at North Carolina State Universy that started on March 1st, 2013. The PD requested to finish the project once relocated at NCSU but the request was denied, thus, the award was closed on February 28. Progress made until that point is outlined in this report. What opportunities for training and professional development has the project provided? Project Director: Lina Quesada, Michigan State University, performed field, greenhouse and laboratory experiments, as well as oversight and management to the project, she was trained in field pollinations with corn, laboratory infections of corn ears, greenhouse infections of corn stalks, growing and maintaining corn in the field and greenhouse, handling and maintenance of fungal pathogens, pathogen isolation from infected corn tissue, liophylization, RNA extractions, RNA sequencing, bioanalyzer analysis, targetted metabolomics, non-targeted metabolomics, bioinformatics and systems biology methods; Mentor: C. Robin Buell, Michigan State University, provided oversight to the project; Collaborator: F. Trail, Michigan State University, provided oversight to the project; Professional Aide: Jameed Al-Haddad, Michigan State University, was trained in liophylization, RNA extractions, RNA sequencing, bioanalyzer analysis, targetted metabolomics, and non-targeted metabolomics; Professional Aide: Emily Crisovan, Michigan State University, was trained in field pollinations with corn, laboratory infections of corn ears, greenhouse infections of corn stalks, growing and maintaining corn in the field and greenhouse, handling and maintenance of fungal pathogens. How have the results been disseminated to communities of interest? Resuls have been disseminated through oral presentations of research results and publication of abstracts in conference proceedings. What do you plan to do during the next reporting period to accomplish the goals? The project was initiated on August 1st, 2012 and terminated on February 28, 2013. The PD Lina Quesada was offered a position as an Assistant Professor at North Carolina State Universy that started on March 1st, 2013. The PD requested to finish the project once relocated at NCSU but the request was denied, thus, the award was closed on February 28. Progress made until that point is outlined in this report.

Impacts
What was accomplished under these goals? We identified corn varieties resistant to Fusarium ear and stalk rot; we developed a targeted metabolomics method to identify and measure deoxynivalenol and zearalenone in pathogen and infected host tissue (ear and stalk); we generated metabolomic and transcriptomic profiling data for host and pathogen to characterize infection by wild-type Fusarium strains in corn stalks and ears, as well as by toxin-deficient Fusarium strains; finally we characterized the metabolome and transcriptome in Fusarium wild-type and toxin-decifient strains in vitro.

Publications

• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2013 Citation: Quesada-Ocampo L.M., Al-Haddad J., Trail F., and Buell C.R. Susceptibility of corn to stalk rot caused by Fusarium graminearum and mycotoxin mutants. Phytopathology