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.
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.
Small RNA regulation of resistance genes. We discovered that the tomato bacterial spot resistance gene, Bs4 and the potato virus X (PVX) resistance, Nb, are orthologues and that these R-genes are regulated by trans-acting small interfering (tasi)RNAs derived from conserved progenitor TAS (trans-acting small RNA) loci in tomato and potato. We identified tasiRNAs that target Bs4 and Nb using bioinformatic prediction software and sequenced small RNA datasets generated in the lab. We confirmed the functional role of tasiRNAs in regulating the Nb-gene and showed that Nb mRNA transcripts are cleaved at predicted tasiRNA targete sites in potato. Bioinformatic analyses further suggest that TAS genes evolved from ancestral R-gene fragments and potentially target multiple members of different classes of R-genes. These results together with previous findings reveal an intimate association between R-gene evolution and RNA silencing loci and also uncover a possible evolutionary origin of TAS genes. These findings are important because they identify new conserved loci and a novel silencing mechanism for regulating conserved R-genes in crop species.
Identify miRNAs target. We identified a novel family of conserved miRNAs that target and potentially regulate the large family of R-genes encoding leucine rich repeat (LRR) receptor-like proteins (RLPs) including those for resistance to different strains of the fungal pathogen Cladosporium fulvum (Cf-genes). Structural analyses of miRNA precursor loci show they are derived from ancestral Cf-gene fragments arranged as long inverted repeats in the 3’ UTRs of four different and unrelated expressed genes. Further analyses is aimed at determining the mechanism of biogenesis and functional of these unique miRNAs. These results suggest that R-gene miRNAs can arise by the insertion of fragments derived from R-gene sequences into new sites in plant genomes, including genes. We speculate that miRNA silencing of R-gene expression co-evolved with R-gene loci and facilitates fine tuning of R-gene expression and the continuing amplification and diversification of resistance genes by reducing potential fitness costs associated with their evolution. These findings are critical for understanding how plants evade pathogens.