Location: Cereal Disease Lab
2024 Annual Report
Objectives
Objective 1: Investigate the biology of FHB infection, mycotoxin accumulation, and pathogenesis in the barley-Fusarium and related pathosystems. (NP303, C2, PS2A). This will include screening wheat lines for infection by Fusarium graminearum and accumulation of mycotoxins as well as detailed analysis of pathogen infection and process of toxin accumulation.
• Sub-objective 1.A. Determine the protein content, spatial architecture, and functional significance of the toxin biosynthetic apparatus in Fusarium graminearum.
• Sub-objective 1.B. Test for differentiation of the endoplasmic reticulum for specific primary and secondary terpenoid metabolite pathways upon trichothecene induction.
Objective 2: Characterize pathogen diversity by studying natural fungal populations.
• Sub-objective 2.A. Test for population subdivision among strains of F. graminearum isolated from native grasses versus those collected as pathogens on wheat or barley.
Objective 3: Develop novel strategies for disease resistance in durum wheat and barley.
• Sub-objective 3.A. Characterize the gene expression pattern changes in FHB resistant durum cultivars produced by removal of CG methylation.
• Sub-objective 3.B. Characterize the genetic transmission of mutated loci and develop molecular markers for use in cultivar improvement.
Approach
Understanding how pathogens produce toxins and cause disease on different hosts can lead to improved management strategies for disease control. Specific approaches include: 1) Protein tagging, advanced microscopy, and protein-protein interaction techniques will be used to characterize multi-enzyme complexes involved in toxin biosynthesis and fungal pathogenesis; 2) FHB levels, strain diversity, and the nature of associated fungal communities, will be monitored by population genetic and metagenomic approaches improving the ability to forecast the economic impact and the design of effective management strategies; and 3) Novel sources of FHB resistance and mycotoxin tolerance will be developed and characterized for crop plants.
Progress Report
In support of Objective 1, research continued on investigating the biology of Fusarium head blight (FHB) infection, mycotoxin accumulation, and pathogenesis on barley-FHB and related pathosystems. For Sub-objective 1.A., transgenic F. graminearum with TRI4, TRI5, cytosolic GFP, and endoplasmic reticulum-localized GFP C-terminal translational fusions with the ultraID biotin ligase were validated via southern blotting, western blotting, and mycotoxin analyses. These validations confirmed single-event genomic insertions, expression of the target proteins, and preservation of mycotoxin biosynthesis in the transgenic strains. Using these results, single transformants were selected to move forward with proximity-dependent biotinylation (PDB) labeling experiments. Method development for PDB has been ongoing. Variables tested for PDB optimization include: 1) incubation time in toxin-inducing media for expression of TRI enzymes, 2) amount of biotin added to the fungal growth media, 3) incubation time with biotin to ensure adequate labeling, 4) protein extraction buffer and conditions, and 5) conditions for affinity capture of biotinylated proteins using streptavidin beads. We expect to have initial TRI4 and TRI5 PDB mass spectrometry results by the end of the fiscal year. For Sub-objective 1.B., multiomics sample processing is ongoing. For all treatment and control spikes across the infection time-course, samples were ground to a fine powder in liquid nitrogen. Separate sample aliquots were saved for DNA, RNA, protein, and metabolite extractions. Total DNA was extracted and quantitative real-time PCR was performed to estimate fungal biomass in all samples. As expected, fungal biomass increased over the course of infection. Sample processing for RNA and protein extractions is underway. We expect RNA samples to be submitted for sequencing before the end of the fiscal year. Protein samples will be analyzed in-house using an Exploris 480 mass spectrometer.
In support of Objective 2, research continued on characterizing FHB pathogen diversity by studying natural pathogen populations. Our past successful annotation of the Cereal Disease Lab’s culture collection allowed us to select 60 Fusarium spp. Isolates that were isolated from independent wild grasses across the state of Minnesota over the course of the last decade. This sampling scheme greatly increases the chances of finding unique and novel genotypes that will aid our understanding of host range. The inferences that we can draw from these natural isolates have been bolstered by two separate analyses of agriculture-related Fusarium isolates, including a global sampling in collaboration with the ARS Culture Collection (NRRL, Northern Regional Research Laboratory) and a CDL-led effort to understand the 2022 outbreak of FHB in Eastern Africa. These analyses have clarified gene flow between countries, implicating global trade as a significant driver of disease spread, especially between certain countries. Results from these efforts also clarify how these different isolates interact to cause disease with natural co-contaminating species like Epicoccum that were commonly found on plants afflicted with FHB. These analyses have also revealed previously undescribed plant-pathogenic lineages in the Fusarium graminearum species complex.
In support of Objective 3, research continued on developing novel strategies for disease resistance in durum wheat. For Sub-objective 3.A., RNA-seq analysis was performed on mutant and parental durum wheat lines at 12- and 48- hours post-inoculation with F. graminearum. Comparison of 4th generation mutant lines (M4) with the parental lines provided significant details on the acquired resistance. However, the three M4 lines (41708-72, E.25.10, and E.25.23) showed distinct mechanisms of acquired resistance. This shows the epigenetic modifications and mutations were random and each line has a different mutation. Upon analysis, we found several regulatory genes such as WRKY, PHD, C3H, CAMTA, bHLH, C2H2, NAC, and MYB were differentially expressed and might be contributing to resistance. For Sub-objective 3.B., five FHB resistant mutant lines were crossed with two advanced durum varieties lines ('ND Grano', and 'ND Stanley'). These F1 lines were then backcrossed again to the advanced durum varieties to generate BC1F1 lines. We selected 10 backcross derived seeds per population to advance to the next generation. The plan is to select 1 plant (based on agronomic characteristics and seed yield) per cross and advance at least 200 of its progenies to the next generation by selfing (corresponding to about 2,000 plants total). The remaining seeds from other plants will be stored in case of future need to increase population sizes.
Accomplishments
1. New high-throughput diagnostic assays for FHB pathogen monitoring. Fusarium Head Blight (FHB), or scab, is a devastating disease of wheat, barley, and other cereals and has great impacts on U.S. Food Security and Food Safety. In addition to causing losses in grain yield and quality, the FHB fungus also contaminates grain with mycotoxins that are harmful to human and animal health. Based on the type of mycotoxin produced, fungal isolates are categorized into different “chemotypes” and there is a need for high-throughput and robust diagnostic assays to rapidly identify different fungal chemotypes from pure fungal cultures and infected plant samples. ARS researchers in St. Paul, Minnesota developed a new assay based on High Resolution Melting (HRM) curve analysis to identify the NX-2 chemotype, which was discovered in 2014 and previously misidentified using assays that existed at the time. The new HRM assay was validated using DNA from 75 fungal isolates representing the four major chemotypes. The researchers further demonstrated the utility of the assay in detecting the NX-2 chemotype directly from DNA isolated from infected plant tissue, even when the fungal DNA was present at extremely low levels. This new and robust diagnostic assay for the NX-2 chemotype can be used for high-throughput molecular surveillance of FHB pathogen populations in grower’s fields, as well as inform disease management and mycotoxin monitoring efforts.
Review Publications
Li, Z., Velásquez-Zapata, V., Elmore, J.M., Li, X., Xie, W., Deb, S., Tian, X., Banerjee, S., Jørgensen, H.J., Pederson, C., Wise, R.P., Thordal-Christensen, H. 2024. Powdery mildew effectors AVRA1 and BEC1016 target the ER J-domain protein HvERdj3B required for immunity in barley. Molecular Plant Pathology. 25(5). Article e13463. https://doi.org/10.1111/mpp.13463.
Hsin-Yen, L., Ai, Q., Teixeira, R., Nguyen, P., Song, G., Montes, C., Elmore, J.M., Walley, J.W., Yingshan Hsu, P. 2023. Improved super-resolution ribosome profiling reveals prevalent translation of upstream ORFs and small ORFs in Arabidopsis. The Plant Cell. 36(3):510-539. https://doi.org/10.1093/plcell/koad290.