Principal Investigator: Jennifer Geddes-McAlister and Rebecca Shapiro
Research Institution: University of Guelph (U of G)
Timeline: May 2019 – April 2022
- Define mechanisms of plant resistance towards mycotoxin accumulation in FHB of wheat and barley.
- Identify and characterize plant pathways responsible for the response to mycotoxins (i.e., mycotoxin degradation).
- Quantify metabolites produced upon mycotoxin exposure and assess their impact on plant health.
- Develop a platform to validate mycotoxin-degradation pathways.
- The identification of candidate genes for breeding of pathogen and mycotoxin-resistant wheat and barley varieties will allow farmers to grow higher yielding and improved quality crops.
- The identification of candidate factors that may serve as targets for novel treatment strategies against mycotoxin accumulation for wheat and barley crops will encourage industry-based partnerships for the development of new bio-fungicides in promotion of environmental sustainability.
- Our research determined the global protein level changes, driving plant defense towards mycotoxins.
- We identified over 100 plant proteins associated with response to the mycotoxin and developed a platform to test each candidate for survival in the presence of the mycotoxin.
For some of Ontario’s most popular crops (e.g., corn and wheat), Fusarium infection can lead to losses of over $200 million/year in epidemic years through yield reduction and diminished crop quality. In cereal crops, Fusarium head blight (FHB) is associated with mycotoxin production (e.g., deoxynivalenol (DON)), which can have severe consequences for the swine and poultry industries, as well as human health (Health Canada regulates the level of the mycotoxin allowed in food). This proposal aimed to address the needs of farmers and industrial end-users by using systems biology to define and characterize mechanisms of mycotoxin degradation within wheat and barley. Ultimately, we aimed to identify specific resistance-associated genetic markers for breeders that would facilitate development of varieties more resistant to Fusarium and DON accumulation.
Using a systems biology approach, we set out to: 1) Look at the global makeup of the cell in terms of protein and genetic transcripts to identify key plant pathways responsible for mycotoxin accumulation and degradation; 2) Analyze metabolites produced by plants following exposure to mycotoxins to identify those metabolites specifically generated upon mycotoxin processing (e.g., degradation); and 3) develop an in vitro assay to assess the role of proteins for degradation of mycotoxins. Our long-term goal (follow-up implementation plan for 2 years) is to generate seed lines possessing mycotoxin-degrading traits and perform large-scale field trials at the Elora Research Station. The resultant seed product line will have commercialization potential with reduced mycotoxin levels and improved biosafety.
We performed point inoculation experiments in the FHB-resistant (i.e., Sumai#3) and susceptible (i.e., Norwell) cultivars with 15ADON-producing Fusarium graminearum (to focus on the impact of the mycotoxin in Ontario). These experiments were performed over a time course of infection with infected and mock-inoculated heads collected at 24 and 120 h post inoculation. These experiments were performed with 10 biological replicates per condition. We optimized our protein extraction protocols to identify both host and fungal proteins produced during infection and quantify changes in host defense responses between the two cultivars over the time course of infection. The protein samples were measured on a state-of-the-art orbitrap mass spectrometer using new instrumentation at the University of Guelph – Advanced Analysis Centre, Mass Spectrometry Facility. We also developed a proteomics workflow for evaluating phosphorylation events occurring during infection, which provided insight into signal transduction cascades important for host defense and fungal virulence. The proteomics samples were processed using our in-house bioinformatics pipeline consisting of MaxQuant and Perseus to identify and quantify plant and fungal proteins differentially changing in abundance during infection.
We performed an untargeted metabolomics extraction protocol on the partnered spikelets and sent the samples for processing to Agriculture and Agri-Food Canada with Dr. David Overy. Test and optimization experiments were performed, followed by all sample measurements. MS1 was performed on the samples, which was followed by extensive bioinformatics training to identify metabolite features of interest. The top 100 features were selected and samples corresponding to these features were selected and measured for MS2. The students participated in a bioinformatics workshop with Dr. Overy for approximately 8 weeks to learn how to process and analyze the metabolomics datasets. The metabolomics datasets will be combined with the proteomics datasets (Objective 3).
For this objective we identified key plant defense response proteins that change in abundance throughout infection and that differ between the cultivars. We performed in silico characterization to determine potential roles in fungal virulence and mycotoxin production. All students involved in the research program have undergone a four-week bioinformatics workshop (led by Dr. Geddes-McAlister) to support analysis of the proteomics data. Next, proteomics and metabolomics data were integrated to identify and prioritize candidates. Genes encoding for the top three proteins of interest, along with a housekeeping gene and a positive control, were selected to establish the mycotoxin assessment platform. We established collaborations with Dr. Stephen Seah (U of G) and Dr. Rod Merrill (U of G) to amplify the genes of interest from wheat and assess their in vitro ability to degrade DON by cloning the gene products into a Saccharomyces cerevisiae expression system with a copper-inducible promoter. We also performed checkerboard assays with varying concentrations of copper and DON to assess degradation. Within the scope of the current project, we assessed the role of three candidate proteins in DON degradation, which we will expand upon in our next proposed project to evaluate an additional 24 proteins. Moreover, our next project will also measure and identify metabolites produced during the degradation of DON in the in vitro assays.
Our results support our deliverables by generating comprehensive datasets of proteins and metabolites produced during infection. We are using these results to build publicly available datasets as a step towards sharing our findings with the scientific community and end users. Our research has identified novel plant proteins with potential roles in the degradation of DON. We are testing these roles now and, if successful, we will recommend new genes for targeted breeding strategies to improve DON degradation in the field and increase quality and yield of crops.
We have also presented our findings in a variety of formats and seek further opportunities. In addition, we have met with representatives of OMAFRA to discuss our progress and plans, participated in the GFO DON working group meeting in fall 2020, established numerous collaborations to further our research, as well as a scientific advisory committee to support our program.
External Funding Partners:
The project was funded in part by the Ontario Agri-Food Innovation Alliance, a collaboration between the government of Ontario and the University of Guelph.
Project Related Publications:
Liu, B., Stevens-Green, R., Johal, D., Buchanan, R., Geddes-McAlister, J. 2021. Fungal pathogens of cereal crops: Proteomic insights into fungal pathogenesis, host defense, and resistance. Journal of Plant Physiology (Invited submission).
Liu, B., Johal, D., Ball, B., Serajazari, M., Geddes-McAlister, J. 2022. Label-free quantitative proteomic profiling of Fusarium head blight in wheat. Methods Mol Biol: Proteomics in Systems Biology (Invited submission; Book Chapter).
Buchanan, R., Serajazari, M., Geddes-McAlister, J. (In Press). Proteomic profiling of host response in the cereal crop Triticum aestivum to the mycotoxin, 15-acetyldeoxynivalenol, produced by the fungal pathogen, Fusarium graminearum. Methods Mol Biol: Plant-Pathogen Interactions (Invited submission; Book Chapter).
Liu, B., Johal, D., Buchanan, R., Ball, B., Serajazari, M., Geddes-McAlister, J. (In Press). Quantitative phosphoproteome analysis of a cereal crop fungal pathogen’s interaction with the host. Methods Mol Biol: Plant-Pathogen Interactions (Invited submission; Book Chapter).