Location: Plant Gene Expression Center2017 Annual Report
1a. Objectives (from AD-416):
Under Objective 1 we will extend and use Solanaceae genomic, transcriptomic and genetic resources to identify and characterize miRNAs, miRNA precursors and predicted gene targets to determine miRNA origin, structure, biogenesis, and function in silencing of genes in innate immunity. We will focus on the DCL4-dependent class of miRNAs and their targets, which allow the use of combined genomic and genetic approaches to verify and characterize predicted miRNA structures and biogenesis. Our second objective is to validate miRNA function in regulating predicted target gene expression and encoded pathogen-triggered resistance responses to understand the potential impact of pathogen modulation of miRNA regulation in immunity. These experiments will provide a framework for evaluating the roles and mechanisms of pathogen effectors in regulating miRNA levels or activity and impact R-gene transcript levels. Finally, experiments under the third objective will use our combined genetic and genomic system to test pathogen modulation of silencing regulation of innate immunity genes. We will compare transcriptomes of virus infected resistant and susceptible Solanaceae model and crop species. Comparative analysis to identify differentially regulated miRNAs, innate immune gene targets, silencing pathway genes and other genes will be guided by our understanding of DCL4-miRNA regulation of specific gene targets. Objective 1: Develop genomic and genetic resources to monitor pathogen-induced changes in host RNA silencing and host innate immunity. Subobjective 1A: Identify and characterize conserved candidate DCL4-dependent miRNAs using bioinformatics pipeline and sequence additions to this pipeline for analysis of adaptive miRNA regulation of R-genes and innate immunity. Subobjective 1B: Generate genetic resources and use combined genetic and genomic approaches to validate DCL4-dependent miRNA biogenesis and function. Objective 2: Determine mechanisms of pathogen effector modulation of small RNA silencing and innate immune gene expression in the host. Subobjective 2A: Determine DCL4-miRNA regulation of target innate immune gene expression. Subobjective 2B: Determine DCL4-miRNA regulation of R-gene–mediated effector triggered immunity. Objective 3: Develop strategies for targeted regulation of R-genes and other innate immunity genes through RNA silencing for improved disease resistance. Subobjective 3A: Determine differentially expressed miRNAs and miRNA targets and other innate immunity genes in tobacco and tomato virus-infected resistant and susceptible tobacco and tomato.
1b. Approach (from AD-416):
Hypothesis 1A: A conserved class of DCL4-dependent 21-22 nt miRNAs target and silence genes in innate immunity. Approach: Use bioinformatics pipeline to analyze Solanaceae transcriptome sequences to identify candidate miRNAs and miRNA-target defense genes. Contingencies: Use bioinformatics pipeline to analyze new and updated Solanaceae transcriptome sequences. Hypothesis 1B: DCL4 produces miRNAs from long hairpin precursors that cleave predicted targets and can trigger secondary phasiRNAs. Approaches: Generate and characterize tobacco and tomato lines to identify and validate candidate DCL4-dependent miRNA biogenesis and in cleavage of predicted targets. Options: Use sRNA and mRNA transcriptome profiling to identify differentially regulated genes and sRNAs in uninfected and virus challenged plants and silencing lines for a broader sampling of miRNA and phasiRNAs and their regulated genes in innate immunity. Hypothesis 2A: DCL4-miRNAs silence innate immune gene expression and reduce levels of targeted transcripts in uninfected hosts. Approach: Compare expression levels of miRNA-targeted genes by RT-qPCR in wild type and silencing lines Contingencies: Use genome wide comparative analysis of sequenced transcriptomes of silencing lines and wild type plants to identify differentially expressed pairs of sRNA and mRNA targets in tomato and tobacco genomes. Hypothesis 2B: DCL4-miRNAs silence innate immune gene expression and reduce levels of RLP and other immunity proteins. Approaches: Use effector triggered immune assays to compare defense gene expression in wild type and silencing lines. Options: Use effector triggered immune assays to compare defense gene expression in wild type and viral suppressor lines. Hypothesis 3: Conserved silencing pathways and sRNAs adaptively regulate innate immunity genes in pathogen challenged tomato and tobacco. Approach: Use transcriptome profiling to compare miRNAs, target cleavage, secondary siRNA production and R-gene expression in healthy versus virus infected tobacco and tomato. Option: These studies will initially focus on established relationships between sRNAs and their immunity gene targets.
3. Progress Report:
This project continues work from project 2030-22000-008-00D, "Manipulation of Plant Disease Resistance Genes for Improved Crop Protection," which expired on April 22, 2017. We have begun to make progress on this new project. Under Subobjective 1A we have generated and are analyzing new tomato messenger (m)RNA and small (s)RNA transcriptome datasets from wild type and RNAi silencing lines to confirm earlier predictions and to identify new DICER-LIKE4 (DCL4)-dependent and other micro (mi)RNAs targeting innate immunity genes in the economically and nutritionally important tomato crop. We have initiated studies under Subobjective 1B to validate miRNA cleavage and secondary small interfering (si)RNA production from predicted mRNA targets. We have also initiated efforts under Subobjective 2A to determine DCL4-miRNA regulation of target innate immune gene expression.