Location: Plant Gene Expression Center2013 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:
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 siRNAs and miRNAs as candidates for R-gene regulation using 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.