Location: Plant Gene Expression Center2021 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.
In support of Objective 1, research continued on development of genetic and genomic resources to determine mechanisms of RNA silencing regulation of host defense gene expression and to fine tune host defense in healthy and pathogen-infected plants. Knowledge from these studies can be used to balance plant growth with pathogen defense for optimized crop yield and protection against pathogens. Under Sub-objective 1A, ARS scientists in Albany, California, used bioinformatic analysis of new public and lab-generated mRNA and sRNA datasets and identified a novel conserved small RNA pathway that is predicted to regulate plant immunity. Under Sub-objective 2A they used molecular genetic approaches to test the function of the novel small RNAs and identified other genes regulated by a similar and novel RNA silencing pathway. Under Sub-objective 1B, the lab generated new CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) gene-edited Nicotiana benthamiana (N. benthamiana) and tomato genetic lines to functionally test the candidate regulatory RNA. Progress was made on Objective 2 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, researchers used quantitative analysis of the novel sRNA innate-immune gene RNA levels in wild type and silencing lines to test the proposed role in immunity gene regulation. They identified other host gene targets of small RNA silencing that are potentially modulated by pathogen effectors. Progress was made on Sub-objective 2B, and researchers generated expression vectors and plant lines and newly identified host gene targets regulated by novel RNA silencing mechanisms. Under Objective 3, ARS scientists sequenced messenger RNA and small RNA to identify new host genes regulated by DCL4-miRNAs and other small regulatory small RNAs. Scientists are analyzing small RNA and mRNA from TMV (tobacco mosaic virus) infected Necrosis (N)-gene resistant and n-susceptible tomato, tobacco transgenic for the (Cf-9) Cladosporium fulvum-9 R-gene, N. benthamiana transgenic for the potato virus X (PVX) 25 kilodalton (kDa) protein and controls to identify differentially expressed miRNAs and siRNAs, and their target genes. Scientists generated webserver resources for comparative analysis of transcriptome profiles of lab-generated and publicly available RNA data from virus-infected and control uninfected plant lines to understand the role and mechanisms of silencing networks in plant innate immunity.
1. RNA silencing regulation of host defense gene expression. Small RNA silencing pathways play roles in regulating host genes for pathogen defense in Solanaceae crops. A better understanding of RNA silencing mechanisms regulating plant defense is needed. ARS researchers in Albany, California, generated a new genetic resource to test the function of a novel small RNA silencing pathway hypothesized to homeostatically regulate host gene expression and viral defense. The genetic resource provides a new resource to determine fundamental mechanisms of plant gene regulation in response to virus infection for optimized deployment of plant defense genes for crop protection.
2. Resistance to Cucumber green mottle mosaic virus is mediated by polycistronic artificial microRNA in cucumber. Cucumber green mottle mosaic virus (CGMMV) causes extensive loss in cucurbit crops, which are an important source of nutrients in the human diet. Environmentally safe approaches for cucurbit resistance to CGMMV are needed; however, genetic sources for resistance to CGMMV are limited. RNA interference-based artificial microRNA (amiRNA) technologies have been described for use in other crop-virus systems but have not been applied to curb CGMMV infection and control disease in cucurbits. ARS scientists in Albany, California, designed amiRNAs against conserved coding regions identified among 25 strains CGMMV. They identified anti CGMMV amiRNAs using a rapid Nicotiana benthamiana-based assay system and developed a rapid cucumber system to validate amiRNAs that effectively block the accumulation of CGMMV. They showed that infected cucumber plants transgenic polycistronic amiRNA had significantly lower levels of CGMMV and reduced virus disease symptoms, demonstrating that that polycistronic amiRNA technology can be used to develop effective immunity against CGMMV in cucurbits.