Location: Plant Gene Expression Center
Project Number: 2030-22000-034-000-D
Project Type: In-House Appropriated
Start Date: Apr 23, 2022
End Date: Feb 24, 2023
Objective:
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.
Approach:
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.
