Location: Cereal Disease Lab
2023 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., codon-optimized ultraID expression constructs were generated and used to transform F. graminearum. Genes encoding the trichothecene biosynthetic enzymes TRI4 and TRI5 were tagged with ultraID as C-terminal translational fusions at the native genomic loci. Cytosolic green fluorescent protein GFP-ultraID and endoplasmic reticulum-localized GFP-ultraID-HDEL expression constructs were also generated and transformed into F. graminearum. For all transformed strains, positive F. graminearum colonies were purified via single spore isolation and verified by polymerase chain reaction. Method development for proximity-dependent biotinylation assays is currently underway. For Sub-objective 1.B., plant growth and inoculation conditions were optimized to attain reliable infection of barley plants for multi-omics analyses. Dip inoculation using a F. graminearum macroconidia spore solution yields >50% infection of plant spikelets on individual spikes. Replicate experiments of the infection time course are underway. Infected spikes at individual timepoints have been collected and frozen for further processing. Lab personnel have been trained on processing samples for label-based quantitative proteomics including protein extractions, enzymatic digestions, chromatography-based sample clean-up, isobaric labeling, and phospho-peptide enrichment.
In support of Objective 2, research continued on characterizing FHB pathogen diversity by studying natural pathogen populations. We have successfully characterized many additional isolates for species-level identification and mycotoxin-producing ability from past surveys. We have also added hundreds of newly characterized isolates to the culture collection, including many that we have annotated with chemical and species-level identifications. Finally, we have successfully integrated dozens of separate datafiles into a comprehensive annotated culture collection representing thousands of isolates and housing their respective metadata that will facilitate the use of the culture collection.
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. Tissue was collected and RNA was extracted. Sequencing libraries were prepared and sequenced. Reads were mapped to the durum wheat genome and quantified. Differential gene expression analysis revealed substantial changes in the transcriptome of mutants relative to the wild-type parental lines. Importantly, there were also substantial differences between mutants, indicating that different mechanisms of disease resistance are operating in individual mutant lines. From the RNA-seq data, we generated preliminary gene regulatory networks that predict the major drivers of the observed transcriptome differences in mutants and parents. The network analysis is being used to prioritize genes for functional characterization. For Sub-objective 3.B., five FHB resistant mutant lines were crossed with two advance durum varieties lines ('ND Grano', and 'ND Stanley'). Seed set was low in three of the crossing sets, and those are being repeated to generate additional seeds. However, many are being advanced to generate nested association mapping panel as well as material for further selection by the breeding programs.
Accomplishments
1. Large-scale identification of isocyanide biosynthetic genes across the fungal kingdom to inform food safety and disease resistance efforts. Fungal secondary metabolites (SMs) enable virulence, persistence in environments, and are themselves dangerous mycotoxins that contaminate agricultural products. As such, research into these compounds can help protect food safety and security. However, identification of genes associated with these compounds has been slow. ARS researchers in Saint Paul, Minnesota, have recently developed a new algorithm capable of finding a new class of SM called isocyanides. Isocyanide compounds are produced by some grass-infecting species where these compounds can poison agricultural animals who consume infected plants. However, research into this group of compounds has been stymied by an inability to find them prior to this new finding. The researchers have mapped these genes across nearly 4,000 fungal genomes including 144 Fusarium genomes and made these results available through a web app (https://isocyanides.fungi.wisc.edu/index.html). This resource will allow scientists to target genes encoding isocyanides for disruption, enabling the characterization of resulting compounds and their importance in pathogenesis.
Review Publications
Montes, C.M., Wang, P., Liao, C., Nolan, T.M., Song, G., Clark, N.M., Elmore, J.M., Guo, H., Bassham, D.C., Yin, Y., Walley, J.W. 2022. Integration of multi-omics data reveals interplay between brassinosteroid and Target of Rapamycin Complex signaling in Arabidopsis. New Phytologist. 236(3):893–910. https://doi.org/10.1111/nph.18404.
O'Mara, S.P., Broz, K.L., Schwister, E.M., Singh, L., Dong, Y., Elmore, J.M., Kistler, H.C. 2023. The Fusarium graminearum transporters Abc1 and Abc6 are important for xenobiotic resistance, trichothecene accumulation, and virulence to wheat. Phytopathology. https://doi.org/10.1094/phyto-09-22-0345-r.
Drott, M.T., Chul Park, S., Wang, Y., Harrow, L., Keller, N.P., Pringle, A. 2023. Pangenomics of the death cap mushroom Amanita phalloides, and of Agaricales, reveals dynamic evolution of toxin genes in an invasive range. The ISME Journal: Multidisciplinary Journal of Microbial Ecology. https://doi.org/10.1038/s41396-023-01432-x.
Velásquez-Zapata, V., Elmore, J.M., Wise, R.P. 2023. Bioinformatic analysis of yeast two-hybrid next-generation interaction screen data. In: Mukhtar, S., editor. Protein-Protein Interactions, Methods and Protocols. Methods in Molecular Biology. 2690:223-239. https://doi.org/10.1007/978-1-0716-3327-4_19.
Elmore, J.M., Velásquez-Zapata, V., Wise, R.P. 2023. Next-generation yeast two-hybrid screening to discover protein-protein interactions. In: Mukhtar, S.,editor. Protein-Protein Interactions, Methods and Protocols. Methods in Molecular Biology. 2690:205-222. https://doi.org/10.1007/978-1-0716-3327-4_19.
Rush, T.A., Tannous, J., Lane, M.J., Gopalakrishnan Meena, M., Carrell, A.A., Golan, J.J., Drott, M.T., Cottaz, S., Fort, S., Ané, J., Keller, N.P., Pelletier, D.A., Jacobson, D.A., Kainer, D., Abraham, P.E., Giannone, R.J., Labbe, J.L. 2022. Lipo-Chitooligosaccharides induce specialized fungal metabolite profiles that modulate bacterial growth. mSystems. 7(6). Article e01052-22. https://doi.org/10.1128/msystems.01052-22.
