Location: Corn Insects and Crop Genetics Research
2024 Annual Report
Objectives
Objective 1. Develop an integrated network of barley-powdery mildew interactions as a model to dissect immune signaling in cereals.
Sub-Objective 1A. Identify protein-protein interactions (PPI) among host and pathogen via yeast two-hybrid (Y2H) next-generation-interaction screens (NGIS).
Sub-Objective 1B: Authenticate immune-active genes via phenotyping and functional assays.
Objective 2: Identify regulatory elements associated with immunity to exploit in modern breeding programs.
Sub-Objective 2A: Genome-wide prediction of transcription factor (TF) activation motifs.
Sub-Objective 2B: Validation of immune-active, cis-regulatory elements (CRE)-associated transcription factors (TFs) via highthroughput phenotyping and in-depth molecular characterization.
Approach
Large-scale sequencing of plant and pathogen genomes has provided unprecedented access to the genes and gene networks that underlie diverse outcomes in host-pathogen interactions. Determination of regulatory focal points critical to these interactions will provide the molecular foundation necessary to dissect important disease resistance pathways. This knowledge can be used to guide modern plant breeding efforts in response to pathogens that present diverse challenges to the host.
Progress Report
Plant pathogens are among the greatest threats to crop production worldwide, resulting in yield losses of 10 to 20% (= $100 to 200 billion) each year. Obligate biotrophic fungi, for example, mildews and rusts, require a living host to survive and cause some of the most destructive epidemics. A general view of the regulatory programs that render a plant resistant to pathogens is beginning to emerge in model organisms but is still in its infancy for the large-genome temperate cereals that are vital to feeding the world’s growing population. The interaction between barley (host), and the powdery mildew fungus (pathogen), is central to address this challenge. In this system, the outcome is determined largely by the plant’s response to secreted pathogen effectors. Disease is blocked by host immune receptors encoded by resistance (R) genes, designated by the prefix, Ml (for mildew resistance). The barley-powdery mildew interaction is a model for other large-genome Triticeae grain crops which includes wheat and rye. This research generates the knowledge base to drive scientific advances that will ultimately improve crop productivity, pest and disease resistance, and tolerance to climate change.
In support of Objective 1, Sub-Objective 1A, in collaboration with Iowa State University, Ames, Iowa, and the University of Copenhagen in Copenhagen, Denmark, we combined bioinformatic analysis with high-resolution microscopy to untangle critical interactions among host and pathogens. After genome-wide screening for protein-protein interactions (PPI) among the barley host and powdery mildew pathogen, results were integrated with a project-developed barley interactome, which enabled the assembly of a high-confidence host-pathogen network of 1085 proteins and 1497 interactions. Proteins need to be shipped, or “trafficked” to specific locations within the cell during the plant life cycle. For example, vacuolar transporters are part of a complex network that enables a plant to react to changing environmental conditions, such as pathogen attack. Research focused on characterization of PPI that localized to the endoplasmic reticulum (ER), and a set of ten powdery mildew effectors that differentially impact trafficking of proteins from the ER to the vacuole during powdery mildew infection were discovered. This critical knowledge step can be exploited for next-generation marker assisted selection (MAS) to breed for resistance to new and emerging pathogens.
For Objective 2, Sub-Objective 2A, in collaboration with Iowa State University, we conducted a genome-wide investigation of epistasis to decipher the gene-by-gene interactions that regulate barley immunity to disease. Epistasis can occur whenever two or more genes interact to create new phenotypes. This, in turn, impacts decisions made in modern breeding strategies. The nucleotide-binding leucine-rich-repeat (NLR) immune receptor encoded by Mildew locus a (MLA) is an ancestral protein required for protection against destructive cereal diseases, including powdery mildew, Ug99 stem rust, stripe rust, spot blotch, and rice blast. Following powdery mildew infection, the activity of all barley genes was investigated to infer diverse epistatic effects governed by the MLA immune receptor and two other host factors critical to disease defense, Bln1 (for Blufensin 1) and Sgt1 (for suppressor of G2 allele of skp1). From a total of 468 annotated barley NLRs, 366 were expressed and 115 of those grouped under six different epistasis effect models, i.e., additive, symmetric, masked, suppression, positive, and negative. Genes classified under these models localized to host chromosome hotspots, inferring that genome accessibility and recruitment of transcriptional machinery are contributing factors to resistance activation. These results connect host plant gene activity with pathogen development, signifying that disease is regulated by an inter-organismal network that diversifies the response.
Accomplishments
1. Endoplasmic reticulum protein required for disease resistance in barley. Powdery mildew fungi infect more than 9,500 agronomic and horticultural plant species. To prevent economic loss, plant breeders incorporate disease resistance genes into varieties grown for food, feed, fuel and fiber. One of these genes specifies instructions for assembly of the Mildew locus a (MLA) immune receptor, an ancestral protein that also provides recognition to stem rust, stripe rust, spot blotch and rice blast in small grain cereal crops, such as barley, wheat, and rye. In these systems, host proteins and pathogen effector proteins interact during fungal infection to initiate plant defense, often relying on basic cellular processes, such as the endoplasmic reticulum (ER) and protein quality control systems elsewhere in the cell. The ER is a specialized folding compartment which ensures that only correctly folded molecules proceed further into the secretory pathway to carry out their functions, whereas misfolded proteins are degraded. ARS researchers in Ames, Iowa, along with Iowa State University and University of Copenhagen scientists used computational and genomic methods to identify the barley J-domain protein, HvERdj3B, as a host target for two diverse powdery mildew effector proteins. Until now, almost all research has focused on the plant nucleus or cytosol as the main cellular location for immune function. For the first time, their analyses demonstrated that following infection, host target-effector protein complexes move from the plant cytosol into the ER, promoting disease resistance or susceptibility. These discoveries provide new insight into the molecular components that control disease defense, and thus, impact next-generation breeding practices to more effectively use disease resistance to produce better crops.
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.
Wise, R.P., Fuerst, G.S., Peters, N., Boury, N., McGhee, L., Greene, M., Michaelson, S., Gonzalez, J., Hayes, N., Schuck, R., Maffin, L., Hall, G., Hubbard, T., Whigham, E. 2024. iTAG: Interactive laboratory exercises to explore genotype and phenotype using Oregon Wolfe barley. The Plant Health Instructor. 24. https://doi.org/10.1094/PHI-E-2023-09-0009.
Boury, N., Van den Bogaard, M., Wlezien, E.B., Peters, N., Wise, R.P. 2024. The great petunia carnage of 2017: A clicker case study using petunias to describe effect of genetic modification on the biochemistry of flower color and phenotype in plants. CourseSource. https://doi.org/10.24918/cs.2024.15.