2008 Annual Report
1a.Objectives (from AD-416)
Effective genetic strategies are needed to address major crop loss due to pathogen disease and provide alternatives to current chemically based crop protection strategies that impose human health and environmental risks. The long-term objective of this project is to gain an improved understanding of the structure, function and evolution of key components of the plant innate immune system for strategic deployment of key components of this highly regulated system to reduce the threat of crop loss by pathogens. Our specific objectives are to use evolutionary models and molecular-genetic strategies to identify durable pathogen resistance genes, to use genomics and genetics to identify and understand functional and regulatory components of the plant innate immune system, and to use comparative structural and functional genomics to understand the mechanism of induction and function of cellular and systemic events that constitute effective induced defense responses and plant innate immunity. Identification of functional and regulatory components of the innate immune system, and understanding the process of induction of defense responses will provide the conceptual base for deploying strategies for broad-spectrum resistance.
1b.Approach (from AD-416)
1. Use an evolutionary model developed by this project, in combination with positional cloning, to isolate the PVX resistance gene, Nb.
Develop DNA markers for fine genetic mapping of the virus disease resistance locus Nb.
Develop PCR methods to isolate candidate disease resistance locus Nb. Perform complementation tests for functional confirmation of Nb isolation. Test performance of Nb in potato cultivars.
2. Identify and analyze transposable elements in the Solanaceae and assess their impact on gene expression in plant innate immunity.
Perform comparative analysis of Solanaceae genomic sequences to identify repetitive transposable element-derived sequences inserted within R-gene hotspots and other genes and intergenic regions for further exploration of their role in structural diversity and genome evolution.
Explore transcription of MITE-derive candidate regulatory sequences by mining Solanaceae EST databases for the presence of MITE sequences.
Explore functional role of candidate MITE sequences in biotic stress by: identifying MITE-derived small RNAs, studying their biogenesis and comparing expression of MITE small RNAs and their candidate targets in biotically challenged and unchallenged wild-type and RNAi plant lines.
3. Understand induced defense responses associated with plant innate immunity by studying host and pathogen effector interaction with cell proteins.
REPLACES: 5335-21000-006-00D (04/07).
We are focusing on application of an evolutionary model and molecular-genetic strategies to identify, isolate, verify, and fuctionally test cloned candidate cDNA's corresponding to the PVX resistance gene Nb. We are also investigating the role of abundant miniature inverted repeat transposable element (MITEs) in regulating genes with transposon insertions in promoters 5' and 3' UTRs, and introns in Solanaceae (tomato, potato, and tobacco). This addresses NP 303, Component 3.
Evolutionary model-based strategy for identification of new R-genes.
Refining and using R-gene evolutionary models for understanding the generation of new pathogen recognition specificities are among the current challenges for developing effective strategies for isolation of new resistance traits for crop protection. ARS scientists in the Plant Gene Expression Center in Albany, CA hypothesize that “slow evolving” Type II disease resistance loci are structurally and functionally conserved among different Solanaceae species and that sequences of isolated active R-genes with characteristic Type II structural properties can be used to isolate new R-gene orthologues from related Solanaceae species. Cloned candidate cDNAs corresponding to the PVX resistance gene Nb were isolated, mapped and functionally tested. One class of candidate Nb cDNAs conferred a hypersensitive response on tobacco leaves when co-inoculated with the PVX gene encoding the virus movement protein effector. The work suggests that a functional Nb cDNA has been cloned. The work also confirmed the hypothesized orthologous relationship between Type II R-gene sequences and established a new strategy based on a comparative genomics-based evolutionary model for isolation of Type II genes. The research in this project falls under NP 303, Component 3, "Plant Disease Resistance." The problem addressed is relevant to Component 3 Plant Disease Resistance, Problem Statement 3A: Mechanisms of Plant Disease Resistance and Problem Statement 3B: Disease Resistance in New Germplasm and Varieties.
Role of Solanaceae repetitive elements.
Understanding of the regulatory components for coordinate regulation of the innate immune system, and the process of induction of defense responses is needed to provide the conceptual foundation for deployment of components and strategies for broad-spectrum resistance. ARS scientists in the Plant Gene Expression Center in Albany, CA identified abundant transposable elements in the Solanaceae, many of which reside within or near genes. We postulated that the gene associated transposable elements (TEs) contribute to the regulation of genes via small RNA regulatory mechanisms. Postdoctoral scientists in the Baker lab, cloned and sequenced Solanaceae small RNAs and showed that they are derived from identified TEs. They also constructed “knock-down” RNAi lines to silence expression of genes required for biogenesis of small RNAs. These lines are being verified and used to determine if Solanaceae TEs serve as progenitors of siRNAs via biogenesis pathways similar to those in Arabidopsis that guide transcriptional and post-transcriptional silencing to genes and transposable elements. We will also test if pathogen infection affects the postulated siRNA-mediated gene regulation. These studies will make a significant contribution to understanding gene regulation in biotically challenged plants, an important issue for designing improved crops for American and world agriculture. The research in this project falls under NP 303, Component 3, "Plant Disease Resistance." The problem addressed is relevant to Component 3 Plant Disease Resistance, Problem Statement 3A: Mechanisms of Plant Disease Resistance and Problem Statement 3B: Disease Resistance in New Germplasm and Varieties.
5.Significant Activities that Support Special Target Populations