2009 Annual Report
1a.Objectives (from AD-416)
Apply functional genomics tools and resources available to barley, rice, and the model dicot Arabidopsis thaliana, to accelerate comparative analysis of cereal disease defense pathways and associate newly discovered genes with biological function.
Characterize diversity of global gene expression in cereal crops as a baseline to determine effects of transgene insertion and assess risk of developing improved crop products by bioengineering or marker-assisted breeding approaches.
1b.Approach (from AD-416)
Utilize allelic variability inherent to barley Mla, Rar1, and Rom1 as a switch to discover new pathways involved in both Rar1-dependent and Rar1-independent plant disease resistance. High-throughput barley dsRNAi and Arabidopsis T-DNA reverse genetic systems will be used to functionally validate candidate genes in resistance pathways of both monocot and dicot plants.
Determine the global transcript profiles of a diverse set of barley germplasm and specific sets of transgenic lines and their progenitors. Integrative computational approaches will be used to establish whether or not differences in gene expression can be used as a predictor for genetic anomalies associated with transgenic crops.
Obj. 1a. Comparative analysis of global gene expression in disease response pathways.
Barley has a complex interaction with powdery mildew that begins with early recognition of the pathogen. GeneChip transcriptome profiling was used to investigate 22,000 genes simultaneously during barley-powdery mildew interactions. New networks and regulators of genes associated with innate immunity, sugar signaling, biotrophy, ribosomal RNA processing and resistance-gene-independent cell death were identified.
Obj. 1b. Integrated reverse genetic analysis to establish gene function.
Gene candidates were tested via Transient Induced Gene Silencing, Virus Induced Gene Silencing, Overexpression, and/or GFP-tagged cellular localization. Several candidates displayed a functional phenotype, facilitating further hypothesis development. A significant outcome in 2008-2009 was the discovery and functional characterization of a new monocot-specific family of cysteine-rich peptides, which negatively impact plant defense. One of these, BLN1, is highly induced during Bgh infection and is similar to knottins, a family of small cysteine-rich peptides.
Obj. 1c. Integrate expression profiling, phenotypic and pathway data into PLEXdb.
The experiment normalization process in PLEXdb was improved and several new quality metrics were added. Model Genome Interrogator (MGI) and Microarray Platform Translator (MPT) were released, adding significant comparative analysis functionalities that will have a strong impact by making existing datasets more valuable for addressing new questions.
Obj. 2a. Analysis of pathogen-responsive promoters.
Based on similar 32 hour time-course profiles following powdery mildew inoculation in nine barley genotypes, 33 coexpressed clusters of genes were identified. The rice gene orthologues for the several hundred barley genes were identified and subjected to promoter analysis to search for potential cis-regulatory elements. Of the statistically significant promoter elements predicted, more than twenty appear to be novel. Confirmation that this resourceful approach works was obtained when a PCR (Polymerase Chain Reaction) amplicon was recovered for BLN1 (discussed elsewhere) in barley using a forward primer designed from the putative regulatory element identified in rice.
Obj. 2b. Comparative analysis of parallel gene expression in transgenic lines and their progenitors.
The bioinformatic analysis of evidence-based and ab initio predicted gene models for maize reference cultivar B73 was completed in collaboration with other ARS scientists in order to create a new maize microarray that will allow four times more maize genes to be profiled in a single experiment. New database and analysis tools have been built so that analysis can proceed expeditiously once the data for final experiments proposed under this sub-objective become available.
Devised new statistical methods to improve scientific reporting of Quantitative Trait Loci (QTL) mapping results. QTL mapping is the most effective way to discover and dissect the natural genetic variation that is needed for genetic improvement of plant and animal commodities. As genetic resolution has improved, a trend toward overextending low confidence results has led to inaccurate reporting because alteration of statistical practices was not adjusted commensurately. ARS researchers identified theoretical deficiencies in the commonly used methods and subsequently devised and validated two updated methods that more carefully limit false-positive reporting for QTL detection and confidence interval estimation. The updates are procedural corrections to approaches shown to be appropriate for standard interval mapping, but which have been widely misapplied to interpret composite interval mapping results in nearly 900 publications over the past decade. QTL analysis is a widely used and indispensible tool for researchers that identify useful genetic variation to improve crops and livestock. Limiting the error-rate for reporting new discoveries better focuses research expenditures targeted at validating preliminary results, making agricultural research considerably more efficient.
Novel regulator of cereal disease defense characterized from barley. Plant diseases are among the greatest deterrents to crop production worldwide. Pathogenic fungi, viruses, bacteria, insects, and nematodes impact agronomic and horticultural crops, as well as commercial and recreational forests. ARS researchers have isolated a novel regulator of disease defense. The new monocot-specific gene encodes a family of small cysteine-rich peptides, designated blufensins. BLUFENSIN1 (BLN1) is highly induced during infection by pathogenic fungi and contains both structural and sequence similarities to knottins, a diverse family of small disulfide-rich proteins characterized by a unique "disulfide through disulfide knot." This discovery, also supported by the National Science Foundation-Plant Genome Research Program, establishes a previously unrecognized role for small peptides as negative regulators of plant defense. Because common themes govern all plant-pathogen interactions, this finding provides new knowledge of broad significance to plant scientists, and to growers who utilize disease resistance to protect their crops.
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Hu, P., Meng, Y., Wise, R.P. 2009. Functional Contribution of Chorismate Synthase, Anthranilate Synthase, and Chorismate Mutase to Penetration Resistance in Barley-Powdery Mildew Interactions. Molecular Plant-Microbe Interactions. 22(3):311-320.
Druka, A., Druka, I., Centeno, A.G., Li, H., Sun, Z., Thomas, W.T., Bonar, N., Steffenson, B.J., Ullrich, S.E., Kleinhofs, A., Wise, R.P., Close, T.J., Potokina, E., Luo, Z., Wagner, C., Schweizer, G.F., Marshall, D.F., Kearsey, M.J., Williams, R.W., Waugh, R. 2008. Towards Systems Genetic Analyses in Barley: Integration of Phenotypic, Expression and Genotype Data into GeneNetwork. BioMed Central (BMC) Genetics. 9:73.
Meng, Y., Moscou, M., Wise, R.P. 2008. Blufensin1 Negatively Impacts Basal Defense in Response to Barley Powdery Mildew. Plant Physiology. 149(1):271-285.
Schulte, D., Close, T.J., Graner, A., Langridge, P., Matsumoto, T., Muehlbauer, G., Sato, K., Schulman, A.H., Waugh, R., Wise, R.P., Stein, N. 2009. The International Barley Sequencing Consortium — At the Threshold of Efficient Access to the Barley Genome. Plant Physiology. 149(1):142-147.
Moscou, M.J., Caldo, R.A., Lauter, N.C., Wise, R.P. 2008. Construction of Coexpression Networks to Explore Barley-Powdery Mildew Interactions. In: Lorito, M., Woo, S.L., Scala, F., editors. Biology of Plant Microbe Interactions: The Impact of "omics." Volume 6. St. Paul, MN: APS Press. p. 113-117.
Lauter, N.C., Moscou, M.J., Habiger, J., Moose, S.P. 2008. Quantitative Genetic Dissection of Shoot Architecture Traits in Maize: Towards a Functional Genomics Approach. The Plant Genome. 01(02):99-110.
Luo, Z.W., Potokina, E., Druka, A., Wise, R.P., Waugh, R., Kearsey, M.J. 2007. SFP genotyping from Affymetrix arrays is robust but largely detects cis-acting expression regulators. Genetics. 176:789-800.
Potokina, E., Druka, A., Luo, Z., Moscou, M., Wise, R.P., Waugh, R., Kearsey, M. 2008. Tissue Dependent Limited Pleiotropy Affects Gene Expression in Barley. Plant Journal. 56(2):287-296. DOI: 10.1111/j.1365-313X.2008.03601.x. Available: http://www3.interscience.wiley.com/cgi-bin/fulltext/120696653/HTMLSTART.
Schreiber, A.W., Sutton, T., Caldo, R.A., Kalashyan, E., Lovell, B., Mayo, G., Muehlbauer, G.J., Druka, A., Waugh, R., Wise, R.P., Langridge, P., Baumann, U. 2009. Comparative Transcriptomics in the Triticeae. BioMed Central (BMC) Genetics. 10:285.