2007 Annual Report
Obj. 2: 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.
Obj. 2: 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.
Regulation of gene expression in barley-powdery mildew interactions influences the establishment of fungal biotrophy and the development of host resistance. In 2006, we utilized the Barley1 GeneChip to analyze the transcript profile of 22,792 host genes throughout various time-course interactions among barley and the powdery mildew fungus, Blumeria graminis f. sp. hordei. Four-hundred thirty two Barley1 GeneChips, representing 144 replicated barley-powdery mildew interactions, were used to interrogate plants containing allelic variants and mutants of Mla, Rar1, and Rom1, a restorer of Rar1-independent, Mla12-specified resistance. In 2007, we initiated comparative bioinformatics approaches to delineate unique and conserved expression networks in barley-Fusarium, barley-Blumeria, and Arabidopsis-Erysiphe interactions.
Obj. 1b: Integrated reverse genetic analysis to establish gene function.
Assays that employ RNA interference (RNAi) offer several advantages for high-throughput investigations into the function of candidate genes in plants and other eukaryotes. Technical personnel visited IPK-Gatersleben, Germany for three months to train on the high-throughput RNAi system. This technology was then transferred to personnel on the project to assay candidate genes associated with host resistance. In addition, a high-throughput Virus Induced Gene Silencing (VIGS) assay based on biolistic transgene delivery has been developed. The single cell Transient Induced Gene Silencing (TIGS) and the whole leaf VIGS systems offer complementary approaches to dissect the various sub-networks and time-course interactions of disease defense pathways.
Obj. 1c. Integrate expression profiling, phenotypic and pathway data into BarleyBase, a MIAME-compliant database for cereal GeneChips.
With the advent of increasing numbers of DNA chips for many crop and crop pathogen species, we have developed BarleyBase/PLEXdb as a hypothesis building information resource to provide efficient access to highly parallel expression data with seamless portals to related genetic, physical, and trait data. In addition to the 432 Barley1 GeneChip hybridizations, representing 144 replicated barley-powdery mildew interactions that were uploaded to BarleyBase/PLEXdb in 2006, an additional 72 were added in 2007. The experiment normalization process in BarleyBase was improved and several new quality metrics were added to highlight experimental consistency and to help find problematic results.
Rice Genome Mapper v1 has been updated to "Model Genome Interrogator" v2. The new version provides supporting information from predicted Open Reading Frame (ORF) and full-length cDNAs and enables the batch mapping of wheat, barley, and maize probe sets onto rice, as well as soybean, Medicago, grape, and tomato onto Arabidopsis with subsequent retrieval of FASTA outputs of corresponding model genome sequence of 5’ and 3’ regulatory regions, or specific exons and introns. These FASTA outputs can be used for input into motif finding software.
To identify significant host expression responses to invasion by the barley powdery mildew fungus, 324 GeneChips were used to profile expression in inoculated vs. non-inoculated seedlings at hours 0, 8, 16, 20, 24, and 32 for each of nine genotypes that carry allelic variants of Mla, Rar1, and Rom1. 4,914 differentially expressed genes were identified in inoculated vs. non-inoculated seedlings from which a coexpression network was derived. Sub-networks containing genes involved in sugar transport, photosynthesis, WRKY signaling, secretion of PR proteins, signal peptide processing, and abiotic stress signaling were discovered. Functional analysis of the sugar transport sub-network indicate that obligate biotrophs co-opt monosaccharide transporters to enable nutrient acquisition while the host utilizes UDP-glucose dehydrogenase and invertases to partition sucrose during basal defense. These findings suggest that competing requirements of fungal biotrophy and host resistance interact to modulate gene expression in the sugar-transport sub-network.
This accomplishment addresses Component 1, Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement of National Program #302, Plant Biological and Molecular Processes - Problem Statement 1B: Applying Genomics to Crop Improvement.
Caldo, R.A., Nettleton, D., Wise, R.P. 2006. Interplay of gene-specific disease resistance, basal defense, and the suppression of host responses to barley powdery mildew. In: Sanchez, F., Quinto, C., Lopez-Lara, I., Geiger, O., editors. Biology of Plant Microbe Interactions. St. Paul, MN: International Society of Molecular Plant-Microbe Interactions. Vol. 5, p. 620-624. Kirst, M., Caldo, R., Casati, P., Tanimoto, G., Walbot, V., Wise, R.P., Buckler Iv, E.S. 2006. Contribution of genetic diversity to type 1 errors in short-oligonucleotide microarray analysis. Plant Biotechnology Journal. 4:489-498. Halterman, D.A., Wise, R.P. 2006. Upstream open reading frames of the barley Mla13 powdery mildew resistance gene function cooperatively to down-regulate translation. Molecular Plant Pathology. 7:167-176. Caldo, R.A., Nettleton, D., Peng, J., Wise, R.P. 2006. Stage-specific Suppression of Basal Defense Discriminates Barley Plants Containing Fast- and Delayed-acting Mla Powdery Mildew Resistance Alleles. Molecular Plant-Microbe Interactions. 19(9):939-947. Nettleton, D., Hwang, G., Caldo, R.A., Wise, R.P. 2006. Estimating the number of true null hypotheses from a histogram of p-values. Journal of Agricultural, Biological, and Environmental Statistics. 11:337-356. Rossi, C., Cuesta-Marcos, A., Vales, I., Gomez-Pando, L., Orjeda, G., Wise, R.P., Sato, K., Hori, K., Capettini, F., Vivar, H., Chen, X., Hayes, P. 2006. Mapping multiple disease resistance genes using a barley mapping population evaluated in Peru, Mexico, and the USA. Molecular Breeding. 18(4):355-366.