Location: Corn Insects and Crop Genetics Research
2023 Annual Report
Objectives
Objective 1. Discover diverse fungal disease resistance mechanisms in cereal (barley and maize) crops.
Sub-Objective 1A. Use expression quantitative trait locus (eQTL) analysis in combination with genome-wide promoter-motif enrichment strategies to discover master regulators of immunity.
Sub-Objective 1B: Identify host targets of pathogen effectors by next generation yeast-two-hybrid interaction screens.
Sub-Objective 1C: Identify and characterize the genetic and molecular pathological modes of action for isolate-specific and non-specific Quantitative Disease Resistance (QDR) mechanisms that protect corn plants against northern leaf blight.
Objective 2: Generate novel sets of disease defense alleles for mechanistic dissection and application to crop protection.
Sub-Objective 2A: Functional confirmation via integrated reverse genetic analysis.
Sub-Objective 2B: Evaluate yield and northern leaf blight resistance properties of QDR alleles in hybrid genetic contexts.
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
Fungal pathogens are among the greatest threats to cereal grain production worldwide. Effector proteins secreted by these pathogens manipulate host processes in order to create an ideal environment for colonization. To defend themselves, plants have evolved a battery of receptors that activate immune responses. This project has aimed to identify components of host disease defense and the pathogen signaling molecules that suppress them in two host-pathogen systems: barley and barley powdery mildew, and corn and northern leaf blight (NLB). An in-depth understanding of the molecular mechanisms underlying disease resistance and susceptibility enables geneticists and breeders to produce high-yield disease-resistant crops.
New insights into disease resistance signaling were obtained in FY2023. To support Objective 1, Sub-Objective 1A, in collaboration with bioinformatics colleagues at Iowa State University, and funded in part by the National Institute of Food and Agriculture – Plant Biotic Interactions program, we developed a genome-wide regulatory network of 1,214 transcription factors, 20,125 cellular protein targets, and 54,698 interactions to explore disease response in large-genome, cereal grains. At the center is the Mildew locus a (MLA) nucleotide-binding leucine-rich-repeat (NLR) immune receptor, an ancestral protein required for protection against destructive cereal diseases, including powdery mildew, Ug99 stem rust, stripe rust, and rice blast. These results will be leveraged to develop and extend resistance to new and emerging pathogens.
For Objective 1, Sub-Objective 1B, time-course gene expression of barley and the powdery mildew pathogen was used to infer diverse gene effects governed by the MLA immune receptor and two other host genes critical to disease defense, Blufensin1 (Bln1) and Required for Mla6 resistance3 (Rar3). Gene effect models revealed epistatic interactions between Mla6 and Bln1 (a situation where the expression of one gene is modified by the expression of other genes) and the impact of rar3 on the barley and powdery mildew transcriptomes. From a total of 468 barley NLRs, 366 were expressed and 115 of those were classified under different gene effect models, which clustered at several chromosome hotspots. Most plant resistance genes deployed in agriculture encode NLRs. However, the mechanisms by which NLR receptors impart critical functions to plant cells are often targets of pathogen effectors, thus, these discoveries provide a foundation for further research into the complex molecular interactions that control disease resistance in crops.
In contrast to barley, whose disease defense relies heavily on NLR genes that often provide qualitative resistance, maize relies more on quantitative disease resistance (QDR) mechanisms. This has a consequence to breeding approaches that should be used to prevent disease outbreaks as well as recovery from them after they occur. Notable accomplishments include establishing genetic dosage effects of the QDR alleles for Northern corn leaf blight (NLB) and development of a “maize silk expression atlas”, which provides the data to understand how silks function in diverse environments utilized in maize production. This research demonstrates for the first time the wide diversity of genes expressed during maize silk growth and function, including important roles in development, metabolism, physiology and abiotic- and biotic-defense.
Key progress from the first four years of the project (FY2018-FY2022) are summarized below:
Small RNA transcripts during infection by barley powdery mildew control transcriptional regulation and disease resistance signaling: Among the wide array of nucleic acids in eukaryotic cells, small RNAs (sRNAs) are important regulatory molecules for diverse physiological processes. ARS and Iowa State University scientists in Ames, Iowa, in collaboration with colleagues at the Danforth Center, St. Louis, Missouri, implemented a genome-wide investigation of sRNAs in the cereal grain crop barley, and its powdery mildew pathogen, and identified multiple roles in disease resistance and pathogen virulence. Using a sophisticated informatics approach, they demonstrated for the first time, that small RNAs are integral to gene regulation during infection by this destructive fungal pathogen. Knowledge from this research will impact how plant breeders select for disease resistance, one of the most important traits that affect crop yield, and thus food security.
Convergent evolution of a novel plant disease resistance locus: Plant disease resistance is often mediated by intracellular immune receptors known as nucleotide-binding leucine-rich-repeat proteins (NLRs). Most crop resistance genes deployed in agriculture encode NLRs. The primary function of NLRs is to detect the presence of pathogen-secreted effector proteins. ARS scientists in Ames, Iowa, collaborated with colleagues at Cornell University in Ithaca, New York, and Indiana University in Bloomington, Indiana, to discover that multiple barley varieties recognize and respond to a conserved protease activity mediated by the bean pathogen effector, AvrPphB. Response to AvrPphB was mapped to a single segregating locus identified as a novel NLR gene, designated AvrPphB Resistance 1 (Pbr1). In addition, it was shown that wheat varieties also recognize AvrPphB protease activity and harbor two copies of Pbr1, suggesting that this disease resistance system could be deployed in Triticeae grain crops. These results provide the first evidence that host targets of AvrPphB have essential immune functions in both monocot and dicot crops. This knowledge will be used to expand protease effector recognition, creating disease resistant crops.
Novel disease resistance in barley: ARS and Iowa State University scientists in Ames, Iowa, funded by the National Science Foundation-Plant Genome Research Program, used genomic methods to identify a novel variant of SGT1, a protein vital for all life, in barley. As published in the February 2021 issue of GENETICS, this variant contains a unique mutation in the structural region that helps to stabilize and activate other disease resistance proteins. In nature, mutations to SGT1 are usually lethal, but this research demonstrates for the first time a unique modification that delineates the requirement for some disease resistances, while unaffecting others as well as normal cell processes. This discovery can be used to predict regions by which pathogen effectors and host proteins interact with SGT1, facilitating precise editing of effector incompatible, disease resistant crops.
New software to detect protein-protein interactions: Organisms respond to their environment through networks of interacting proteins and other biomolecules. A technique termed yeast two-hybrid (Y2H) has been integrated with next generation sequencing (NGS) to approach protein-protein interactions on a genome-wide scale. The fusion of these two methods has been termed next-generation-interaction screening, abbreviated as Y2H-NGIS. However, the diverse data sets resulting from this technology have presented unique challenges to analysis. ARS researchers in Ames, Iowa, partnered with bioinformatics scientists at Iowa State University to optimize the computational and statistical evaluation of Y2H-NGIS and provide metrics to quantify high-confidence interacting proteins, utilizing the general principles of enrichment, specificity, and in-frame prey selection to accurately assemble interactome networks. They then showed how the pipeline works experimentally, by identifying and validating novel interactions between the barley powdery mildew resistance protein, MLA6, and proteins involved in signaling, transcriptional regulation, and intracellular trafficking. Y2H-SCORES is available at GitHub repository https://github.com/Wiselab2/Y2H-SCORES/tree/master/Software Application of Y2H SCORES will enable bench scientists to quickly put together statistically relevant biological networks to model cellular behavior.
Genome-wide analysis of protein-protein interactions that control disease resistance in barley: To prevent economic loss due to disease, plant breeders incorporate resistance genes into varieties that are grown for food, feed, fuel and fiber. One of these resistance genes provides instructions for assembly of the barley MLA immune receptor, an ancestral cereal protein that confers recognition to powdery mildew, stem- and stripe rust. To function properly, these immune receptors must interact with other helper proteins during the different stages of fungal infection and plant defense. As published in the June 2022 cover article for GENETICS, ARS scientists in Ames, Iowa, in collaboration with Iowa State University used custom Big Data methods to identify over 66,000 protein-protein interactions and model cellular response to powdery mildew infection. In particular, fifteen new MLA-interacting proteins were identified, predicted to localize to five diverse cellular locations over the course of infection. These results enable future work to understand key components of disease defense.
Accomplishments
1. Barley uses endoplasmic reticulum (ER) quality control protein to defend against powdery mildew attack. Most plant resistance genes deployed in agriculture encode a type of intracellular immune receptor known as nucleotide-binding leucine-rich-repeat proteins (NLRs). However, the mechanisms by which NLR receptors impart critical functions to plant cells are often blocked by pathogen effectors, which then allow the pathogen to evade host defenses and cause disease. The barley powdery mildew pathogen secretes hundreds of these effectors into host cells to suppress defense, resulting in yield losses of 10 to 20%. ARS researchers in Ames, Iowa, in collaboration with Iowa State University and the University of Copenhagen in Denmark, employed advanced genomic technology, known as yeast two-hybrid next-generation interaction screens, to discover that a pair of these effectors, designated AVRA1 and BEC1016, target the barley J-domain protein, HvERdj3B, in the endoplasmic reticulum (ER). HvERdj3B is an ER quality control protein and suppressing its function increased powdery mildew disease. Together, these results suggest that barley innate immunity, that is, preventing powdery mildew penetration into epidermal cells, is dependent on ER quality control, which in turn requires the J-domain protein HvERdj3B and is regulated by the two effectors. These results that identify new components of host disease defense and the pathogen effectors that suppress them will enable breeders and growers to more effectively use disease resistance to produce better crops.
Review Publications
Choquette, N.E., Holland, J.B., Weldekidan, T., Drouault, J., De Leon, N., Flint Garcia, S.A., Lauter, N.C., Murray, S., Xu, W., Wisser, R. 2023. Environment-specific selection alters flowering-time plasticity and results in pervasive pleiotropic responses in maize. New Phytologist. 238(2):737-749. https://doi.org/10.1111/nph.18769.
Baldwin-Kordick, R., De, M., Lopez, M.D., Liebman, M., Lauter, N.C., Marino, J., McDaniel, M.D. 2022. Comprehensive impacts of diversified cropping on soil health and sustainability. Agroecology and Sustainable Food Systems. 46(3): 331-363. https://doi.org/10.1080/21683565.2021.2019167.
Guo, J., He, K., Meng, Y., Hellmich II, R.L., Chen, S., Lopez, M.D., Lauter, N.C., Wang, Z. 2022. Asian corn borer damage is affected by rind penetration strength of corn stalks in a spatiotemporally dependent manner. Plant Direct. 6(2). Article e381. https://doi.org/10.1002/pld3.381.
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.
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.
