Location: Plant Gene Expression Center2018 Annual Report
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.
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.
Progress was made to generate genomic and genetic resources to understand pathogen regulation of plant immunity for application to fine-tune immunity gene expression and improve disease resistance for crop protection. Under Sub-objective 1A, ARS scientists, using bioinformatic analysis of new public and lab-generated Solanaceae transcriptome and genome datasets, identified and characterized conserved micro RNAs (miRNAs) that silence immunity genes. Under Sub-objective 1B ARS researchers generated tobacco and tomato genetic resources and used genetic, genomic and molecular approaches to validate DCL4-dependent miRNA biogenesis and function. Progress was made on Objective 2, which focused on determining the impact of pathogen effector modulation on small RNA silencing and innate immune gene expression in the host. For Sub-objective 2A scientists used quantitative analysis of miRNA and innate-immune gene RNA levels in wild type and silencing lines and confirmed that DCL4-miRNA regulates receptor-like protein (RLP) target innate immune gene expression. Researchers made progress under Sub-objective 2B and generated expression vectors and plant lines and determined that DCL4-miRNA regulates RLP-gene (Cf9)–mediated effector triggered immunity. Under Objective 3, scientists generated tobacco mosaic virus (TMV) infected N-gene resistant and n-susceptible tobacco and tomato samples for RNA transcriptome profiling to identify differentially expressed miRNAs and miRNA targets and other innate immunity genes in tobacco and tomato virus-infected resistant and susceptible tobacco and tomato to understand the role and mechanisms of silencing networks in plant innate immunity.
1. RNA decay and RNA silencing regulation of innate immunity. Although the activation and rate of messenger RNA (mRNA) synthesis has been the focus of studies on mechanisms controlling gene expression, recent work highlights the role of RNA decay in gene expression regulation. The RNA exosome complex degrades improperly translated mRNA or aberrant RNA cleaved by microRNA- and siRNA-directed endonucleases in a 3'-5' direction. The RNA exosome complex is emerging as a crucial component of plant immune systems; however, the necessary interacting partners, HBS1 and SKI7, have not been comprehensively defined in plants. To understand the role and interplay between RNA decay and RNA silencing in immunity, ARS scientists in Albany, California, investigated SKI7 (SUPPERKILLER 7) required to bridge stalled ribosomes to the RNA exosome for mRNA degradation. They identified the plant orthologues of HBS1/SKI7 and found that HBS1 and SKI7 are encoded by alternative splice forms of a single locus and that the SKI7 transcripts are subject to regulation by RNA decay. The significance of this work is that it shows that SKI7 is conserved across eukaryotes and indicates a broad coordination of nuclear and cytosolic RNA metabolism for regulation of gene expression and serves as a foundation to investigate RNA decay and RNA silencing interactions in regulating innate immune genes and innate immunity in crops.
2. Artificial microRNA-mediated resistance to Cucumber green mottle mosaic virus. Cucumber green mottle mosaic virus (CGMMV) infects cucurbit plants and causes severe economic losses to crop production. RNA silencing has been employed successfully to control several viral diseases, but has not yet been applied for management of CGMMV disease. ARS scientists in Albany, California, in collaboration with scientists at China Agricultural University, developed a CGMMV-Nicotiana benthamiana pathosystem to test if artificial microRNAs (amiRNAs) targeting CGMMV genes can protect plants from CGMMV disease. Artificial miRNAs targeting conserved sequences of CGMMV genes introduced into N. benthamiana and protection against viral disease was assessed by CGMMV infection of infiltrated plants. Results showed that high levels of amiRNAs targeting viral genes correlated with viral “resistance”, reduced viral replication, and reduced incidence of CGMMV disease compared to controls. This work demonstrates the successful application of amiRNA silencing for protection of N. benthamiana against virus replication and spread and it indicates that an amiRNA strategy is a promising approach for protecting crops against CGMMV disease.