Location: Corn Insects and Crop Genetics Research
2018 Annual Report
Objectives
Objective 1: Discover and functionally characterize host genes and pathways that respond to pathogen infection in barley and maize.
Sub-Objective 1A: Identification of barley Blufensin1 (Bln1)-mediated response pathways.
Sub-Objective 1B: Functional confirmation via integrated reverse genetic analysis.
Sub-Objective 1C: Identify and functionally characterize mechanisms of defense against leaf blights with hemi-biotrophic and necrotrophic lifestyles in maize.
Objective 2: Identify and analyze the role of pathogen effectors that influence host response in barley and maize.
Sub-Objective 2A: Isolation of Blumeria graminis f. sp. hordei (Bgh) effectors to identify plant targets that promote or suppress defense in host and nonhost interactions.
Sub-Objective 2B: Identification of candidate effectors produced by S. turcica and C. heterostrophus during interactions with maize.
Approach
Large scale sequencing of pathogen genomes, as well as their plant hosts, has provided unprecedented access to the genes and gene networks that underlie host-pathogen interactions. Regulatory focal points critical to these interactions will be investigated. Determination of these focal points will enable the molecular dissection of important disease resistance pathways, as well as the creation of a molecular toolbox from which to apply modern plant breeding methodologies.
Progress Report
Fungal pathogens are 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. While many aspects of plant defense have been studied, a comprehensive view of the cellular changes that actually render a plant resistant to pathogens has been a major challenge for plant-microbe research. Using plant-pathogen interaction systems of barley and corn, this project aimed to identify both host disease defense components and pathogen signaling molecules that suppress them. By understanding how plants and pathogens manipulate each other during complex interactions, geneticists and breeders can tip the scales in favor of the crop plants to promote more stable and more efficient production.
ARS researchers contribute to barley genome sequence for crop improvement. ARS scientists partnered with the International Barley Genome Sequencing Consortium from Germany, Scotland, Japan, Finland, Australia, China, and the United States (www.barleygenome.org) to sequence the genome of cultivated barley, one of the world’s major cereal crops, and the largest plant genome sequenced at that time. As a diploid inbreeding temperate crop, barley has traditionally been considered a model for plant genetic research. This promotes new approaches to broaden the germplasm base, facilitate new breeding strategies and accelerate rates of genetic gain. This is significant because barley grain is particularly high in soluble dietary fiber, which significantly reduces the risk of human diseases, including type II diabetes, cardiovascular disease and colorectal cancer. The completed barley genome, also supported by USDA-NRI, USDA-AFRI, and the National Science Foundation-Plant Genome Research Program (NSF-PGRP), provides new knowledge of broad significance to plant scientists and breeders, enabling growers to produce nourishing, disease resistant, and higher yielding crops.
ARS researchers use genomics to defeat plant-pathogenic microbes. Crop loss caused by disease remains one of the greatest challenges in agriculture in developed and developing countries. Rusts and mildews, caused by obligate fungal pathogens, are a major threat to cereal grain production worldwide. Effector proteins secreted by these pathogens modulate, inhibit, or accelerate host processes to optimize nutrient acquisition and colonization. As such, effectors serve as optimal probes to decode host signaling pathways, particularly immunity. ARS researchers in Ames, Iowa, the Imperial College of London in the United Kingdom, and the Leibniz Institüt for Plant Genetic Research, Gatersleben, Germany leveraged the genome sequence of the barley powdery mildew fungus to perform a large-scale screen for effector proteins in this important cereal pathogen. Several novel pathogen proteins were discovered that manipulate the host cell to cause disease. This finding, like similar discoveries in human medicine, will be used to develop new ways to combat diseases of crop plants, most notable cereal grains, which are our most important food sources. This discovery was also supported by NSF-PGRP.
Novel regulators of plant cell death. Pathogenic fungi, viruses, bacteria, insects, and nematodes parasitize agronomic and horticultural crops, as well as commercial and recreational forests. Commonly, pathogens use small molecules, called effectors, to suppress, modify, or evade host defense. ARS researchers in Ames, Iowa partnered with scientists at Iowa State University to demonstrate that a barley microRNA is regulated by a resistance protein, plus additional members of its interacting complex. This unique regulatory element controls the chloroplast copper/zinc superoxide dismutase protein, which then influences barley cell death to halt progression of disease. This discovery establishes a previously unrecognized role for microRNAs as regulators of plant defense.
A broadly conserved fungal effector enhances pathogen virulence in powdery mildew of barley. The interaction of barley with the powdery mildew fungus is a well-developed model to investigate the molecular communications among plants and plant pathogens. Scientists at USDA-ARS in Ames, Iowa led an International team from the USA (ARS, Iowa State University, Cornell University and Indiana University), and the United Kingdom (Imperial College and Royal Holloway University of London ) to uncover the mysteries of how pathogens colonize their hosts to cause disease. Novel gene-silencing and overexpression technologies were developed to demonstrate that a protein from the powdery mildew fungus promotes pathogen virulence in the grain crop, barley. Homologs encoding this particular protein are present in 96 of 241 sequenced fungal genomes representing plant pathogens, as well as human and insect pathogens. This broadly conserved protein family provides the opportunity to investigate mechanisms and fungal pathogenesis that are important in both medicine and agriculture, which will lead to preventative treatments. This discovery, establishes a previously unrecognized role for pathogen effectors as broadly conserved regulators of plant defense.
The iTAG Barley STEM training enables hands-on science for high-school students. ARS researchers in Ames, Iowa developed and implemented iTAG Barley (Inheritance of Traits and Genes), a grade 7-12 STEM outreach program, to help students to understand the relationship between genotype and phenotype. Using the diverse Oregon Wolfe Barley population as the model, teacher iTAG training enables their students to learn concepts in plant development, phenotypic diversity, genetics, and genomics. iTAG Barley is available with teacher and student versions in PDF or digital textbook format (iTAG for iPad), and includes thermal cyclers, microcentrifuges, gel boxes, transilluminators, pipetteman, seed, and reagents. Between 2010 and 2018, 40 teachers (mainly in Iowa and Alabama) have implemented iTAG Barley in 186 classrooms, impacting 4,516 students across urban and rural communities, 1/3 of whom are racially or ethnically underrepresented in STEM.
A corn gene conferring multi-disease resistance offers a new kind of crop protection. Several economically important diseases of corn that specifically attack leaves offer a unique multi-disease system in which to discover genes that confer broad-spectrum disease resistance in plants. ARS Researchers in Raleigh, North Carolina and Ames, Iowa led a multi-institutional, multi-disciplinary team in characterizing the function of a corn gene in resistance to both gray leaf spot and southern leaf blight. Research showed that increases in the expression of this gene are associated with increased production of the structural organic polymer lignin, as well as with increased resistance to both diseases. The findings are significant because they broaden and deepen our collective understanding of both quantitative disease resistance and multiple disease resistance, which are expected to be more evolutionarily durable against pathogen populations always seeking new ways to defeat plant defenses and cause infections. Continued deployment of such genetic mechanisms in crop protection has considerable societal and economic benefits because these mechanisms protect crop yields while reducing the need for costly aerial applications of environmentally hazardous fungicidal chemicals.
A new genome-wide approach, designated Next Generation Interaction Screening , has been implemented by ARS researchers in Ames, Iowa, where secreted effector proteins from the pathogen were utilized as ”bait” to fish for interacting targets in the barley host. Briefly, this approach combines a protein-protein interaction screen in yeast with next generation DNA sequencing. The outcome is a series of high-confidence host targets that can be used to create a network of interacting proteins, with key intersections (termed ‘Nodes’) that are candidates for molecular breeding programs. Several new barley receptor-like protein kinases and GTPase-activating proteins were identified that play key roles in host resistance to pathogens.
In experiments conducted using corn and its foliar pathogens, new findings suggest that genes thought to function in primary metabolism also have specific roles in defense. To study defense gene expression changes regulated by primary metabolism genes, ARS scientists in Ames, Iowa used a pair of lines that differed by a handful of genes, including an amino acid synthesis gene. The gene was strongly differentially expressed between lines and the version of the gene that confers quantitative disease resistance also caused a cascade of defense gene expression. Notably, fungal gene expression in the diseased tissue samples also differed, providing functional insights into how the corn gene is mounting the defense. However, it is unclear if these changes are due to direct manipulation of fungal signaling by the plant. Transgenic studies on this gene were conducted and have already validated the genetic results. This past year, we initiated transcriptomic experiments involving the transgenically manipulated amino acid synthesis gene, offering opportunities to functionally elucidate the mechanistic basis of this defense response.
Accomplishments
1. Master regulators of powdery mildew resistance. Understanding how plant pathogens manipulate their hosts will enable geneticists and breeders to promote more stable and more efficient production. ARS researchers in Ames, Iowa, partnered with bioinformatics scientists at Iowa State University to discover master regulators of plant disease resistance. Two of these regulators control the output of 961 and 3,296 “worker” genes, respectively. Moreover, of the 961 genes regulated during the early stages of attack, >30% of these are repurposed as infection progresses. Thus, these genes are part of an immune complex activated by multiple signals and encode proteins that function in concert to achieve immunity in response to different pathogen isolates or infection stages. This discovery of a conserved core of genes that can be activated by known molecular signals offers new mechanisms to deploy in crop protection.
2. Unveiling genetic properties associated with long-term crop improvement. A challenge for breeders is to assure predictability from season to season while not stifling the flexibility required by plants to adjust to their environment. ARS Scientists in Ames, Iowa and five other locations worked collectively within the Genomes-to-Fields Initiative, in order to investigate system properties of United States corn germplasm pertaining to performance across our nation’s arable landscape. The team discovered that modern corn breeding has favored stability by purging some genetic variation that encodes environmentally responsive mechanisms. While this current system has produced remarkable gains in yields, it has done so without an understanding of how fundamental system properties were changing. Without value judgement, these findings represent important new knowledge that will inform how to meet the age-old and ongoing breeding challenge of maintaining both stability and flexibility.
Review Publications
Mistry, D., Wise, R.P., Dickerson, J. 2017. DiffSLc: A graph centrality method to detect essential proteins of a protein-protein interaction network. PLoS One. https://doi.org/10.1371/journal.pone.0187091.
Yang, Q., He, Y., Kabahuma, M., Chaya, T., Kelly, A., Borrego, E., Bian, Y., El Kasmi, F., Yang, L., Teixeira, P., Kolkman, J., Nelson, R., Kolomiets, M., Dangl, J., Wisser, R., Caplan, J., Li, X., Lauter, N.C., Balint Kurti, P.J. 2017. A maize caffeoyl-CoA O-methyltransferase gene confers quantitative resistance to multiple pathogens. Nature Genetics. 49:1364-1372.
Gage, J., Jarquin, D., Romay, M., Lorenz, A., Buckler IV, E.S., Kaeppler, S., Alkhalifah, N., Bohn, M., Campbell, D., Edwards, J.W., Ertl, D., Flint Garcia, S.A., Gardiner, J., Good, B., Hirsch, C., Holland, J.B., Hooker, D., Knoll, J.E., Kolkman, J., Kruger, G., Lauter, N.C., Lawrence-Dill, C., Lee, E., Lynch, J., Murray, S., Nelson, R., Petzoldt, J., Rocheford, T., Schnable, J., Schnable, P., Scully, B.T., Smith, M., Springer, N., Srinivasan, S., Walton, R., Weldekidan, T., Wisser, R., Xu, W., Yu, J., De Leon, N. 2017. The effect of artificial selection on phenotypic plasticity in maize. Nature Communications. 8:1348. https://doi.org/10.1038/S41467-017-01450-2.
Alkhalifah, N., Campbell, D., Falcon, C., Miller, N., Romay, M., Walls, R., Walton, R., Yeh, C., Bohn, M., Buckler IV, E.S., Ciampitti, I., Flint Garcia, S.A., Gore, M., Graham, C., Hirsch, C., Holland, J.B., Hooker, D., Kaeppler, S., Knoll, J.E., Lauter, N.C., Lee, E., Lorenz, A., Lynch, J., Moose, S., Murray, S., Nelson, R., Rocheford, T., Rodriguez, O., Schnable, J., Scully, B.T., Smith, M., Springer, N., Thomison, P., Tuinstra, M., Wisser, R., Xu, W., Ertl, D., Schnable, P., De Leon, N., Spalding, E., Edwards, J.W., Lawrence-Dill, C. 2018. Maize genomes to fields: 2014 and 2015 field season genotype, phenotype, environment, and inbred ear image datasets. Biomed Central (BMC) Plant Biology. 11:452. https://doi.org/10.1186/s13104-018-3508-1.
Surana, P., Xu, R., Fuerst, G.S., Chapman, A., Nettleton, D., Wise, R.P. 2017. Inter-chromosomal transfer of immune regulation during infection of barley with the powdery mildew pathogen. Genes, Genomes, and Genomics. https://doi.org/10.1534/g3.117.300125.
