Location: Plant Gene Expression Center2012 Annual Report
1a. Objectives (from AD-416):
The overall aim of this project is to use genomic and molecular-genetic approaches to identify and characterize mechanisms that fine tune R-gene expression for informed deployment of resistance traits in crop protection strategies, Objective 1: Generate and use genome and transcriptome analyses tools for identification of candidate sequences involved in posttranscriptional regulation of R-gene expression. Objective 2: Use functional genomics to verify crop genome and transcriptome sequences that regulate R-gene expression post-transcriptionally. Objective 3: Use characterized properties of confirmed R-gene regulatory sequences to develop genomic resources for regulation of multiple R-genes in crops of the Solanaceae.
1b. Approach (from AD-416):
Objective 1: We will complete analysis of the ten MIR families we previously identified, update sequence databases and use updated databases and our established bioinformatic pipeline to identify potential additional MIR families. Objective 2: We will use 5’ RACE assays to verify miRNA sequence-specific targeting and cleavage of R and R-homologue transcripts using methods we established for validation of the activity of nta-miR1 and nta-miR2 that cleave the N transcripts. Objective 3: We will use three approaches to understand the potential impact of sRNA regulation on R-gene expression and resistance. The first approach will evaluate pathogen regulation of MIR precursor expression. The second approach will use R-gene miRNA and secondary siRNA sensors to determine the impact of different levels or activity of miRNAs on siRNA production and R-transcript levels. The third aim will determine if R-gene specific small RNAs impact pathogen resistance responses.
3. Progress Report:
This report documents progress for project 5335-22000-008-00D, which began April 23, 2012 and continues research from the project 5335-22000-007-00D entitled “Resources for Identification of Plant Disease Resistance Regulatory Sequences, Genes and Signaling Components for Crop Protection Strategies”, which terminated April 23, 2012. For additional information, see the report for Project Number 5335-22000-007-00D. Progress was made on all three objectives of project 5335-22000-008-00D. Under Objective 1 we made significant progress in generating and using genome and transcriptome sequences and analyses tools to identify candidate sequences involved in posttranscriptional regulation of R-gene expression. We identified new miRNA and siRNAs as candidates for R-gene regulation using 12 new small RNA sequence datasets from cultivated and wild Nicotiana and Solanum. We used our bioinformatic pipeline for predictions and to analyze the structures of candidate small RNAs and their precursors. Our analysis suggested a functional role for these RNAs in R-gene silencing. We identified novel and conserved siRNA loci predicted to regulate R-gene orthologues in tomato and potato. Progress was also made on Objective 2 using 5’ RACE assays to validate candidate miRNA and siRNA cleavage of R-genes in tobacco, tomato potato. These assays functionally verified candidate miRNAs and siRNAs predicted to regulate R-gene expression posttranscriptionally. Additionally, under objective 2 we constructed and used miRNA expression vectors to validate candidate miRNA cleavage of R-genes and we determined the requirement of 22-nt miRNAs for siRNA production from cleaved R-gene transcripts. We also established the structural requirements of miRNA progenitors for producing 22-nt miRNAs. We made progress under objective 3 aimed at evaluating pathogen regulation of miRNA precursor expression by generating vectors and transgenic lines tobacco lines expressing two different R-genes. Characterization of these lines showed that both R-genes are active. These lines will be used for continued investigation of pathogen impact on miRNA mediated R-gene regulation.
1. Identification of a conserved secondary siRNA locus that regulates the tomato bacterial resistance gene Bs4 and the potato virus resistance gene Nb. We showed that many Solanaceae R-genes are silenced by miRNA cleavage and that R-gene silencing is amplified through miRNA-triggered secondary siRNA production from cleaved R-gene transcripts. In contrast to these findings, we discovered that the tomato Bs4 gene and the recently isolated potato Nb gene both lack predicted secondary siRNAs. We used bioinformatic tools to identify candidate small RNAs targeting these genes for posttranscriptional silencing. We discovered both genes are regulated by a secondary siRNA generated from a novel and conserved locus producing highly abundant secondary siRNAs. The locus itself is derived form tow R-genes and transcripts from this locus are cleaved by miRNAs that also target R-genes. This finding is important because it identifies a new conserved locus and novel silencing mechanism for regulating conserved R-genes in crop species.
Li, F., Pignatta, D., Bendix, C., Brunkard, J., Cohn, M., Tung, J., Sun, H., Kumar, P., Baker, B.J. 2012. miRNA regulation of plant innate receptors. Proceedings of the National Academy of Sciences. 5:1790-1795.