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United States Department of Agriculture

Agricultural Research Service


Location: Plant Gene Expression Center

2011 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.

3. Progress Report
ARS scientists in Albany, CA, have identified the progenitor genes (MIR), precursor transcripts (pre-miRNAs), and slicing function of two microRNA (miRNAs) nta-miR1 (22-nt) and nta-miR2 (21-nt) that post-transcriptionally target the tobacco mosaic virus resistance gene N and its homologues. Sequencing and bioinformatic analysis identified further N-gene-specific small RNAs and using RNAi knockdown lines in N. tabacum established that their biogenesis is RDR6- and DCL4-dependent, and that they are “in phase” with the 22-nt miRNA cleavage site. In-vivo transient assays showed that secondary siRNA production was dependent on cleavage by nta-miR1 (22-nt) but not nta-miR2 (21-nt) and that the 22-nt miRNA form arose from a fold-back precursor containing an asymmetric bulge but not from a symmetric fold-back structure. Further bioinformatic analyses identified several more families of R-gene miRNAs and associated secondary siRNAs in tobacco, tomato and potato. These candidate miRNAs are predicted to target transcripts expressed from three major classes of R-genes.

4. Accomplishments
1. Regulation of resistance genes by small RNAs. Each year, billions of dollars are lost by farmers worldwide due to plant disease. Application of the plant's own regulatory defense mechanisms can lead to more effective protection against plant pathogens. ARS scientists in Albany, CA, in collaboration with UC Berkeley scientists have discovered that small RNAs regulate the activities of plant resistance genes (R genes). Identification, isolation and characterization of components that regulate R gene expression in Solanaceae crops will yield powerful information and tools for manipulation of plant’s natural resistance responses to better suit the health and productivity of crops.

Last Modified: 2/23/2016
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