Research Project: Disease Resistance Gene Regulation through RNA Silencing for Improved Crop Protection

Location: Plant Gene Expression Center

2019 Annual Report

Objectives
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.

Progress Report
Progress was made on project 2030-22000-009-00D 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 in Albany, California, identified and characterized conserved microRNAs (miRNAs) that silence immunity genes using bioinformatic analysis of new public and lab-generated Solanaceae transcriptome and small regulatory RNA (sRNA) datasets. Under Sub-objective 1B, the lab generated new CRISPR/Cas9 gene-edited Nicotiana (N.) benthamiana and tomato genetic lines. They used genetic, genomic, molecular and bioinformatic approaches to validate DCL4-dependent miRNA biogenesis and function and identified new genes regulated by a DCL4-related RNA silencing mechanism. 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 miRNA and innate-immune gene RNA levels in wild type and silencing lines and confirmed DCL4-miRNA regulation of RLP (receptor-like protein) and other target innate immunity gene expression and effector induced immunity. They identified new host gene targets of small RNA silencing potentially modulated by pathogen effectors. Progress was made on Sub-objective 2B and researchers generated expression vectors and plant lines and investigated newly identified host gene targets regulated by DCL RNA silencing mechanisms. Under Objective 3, ARS scientists generated transgenic Cf9 tobacco lines deficient for RNA silencing and sequenced RNA and sRNA to identify new host genes regulated by DCL4-miRNA silencing. Scientists are sequencing RNA from TMV (tobacco mosaic virus) infected N-gene resistant and n-susceptible tobacco and tomato samples for transcriptome profiling to identify differentially expressed miRNAs and miRNA targets and other innate immunity genes. Scientists generated webserver resources for comparative analysis of transcriptome profiles to understand the role and mechanisms of silencing networks in plant innate immunity.

Accomplishments
1. Conserved and diverse microRNAs (miRNAs) fine-tune plant immunity. Innate immunity receptors, NLRs (nucleotide binding – leucine rich repeat receptors) and RLPs (receptor-like proteins), are encoded by large gene families that rapidly evolve and protect plants from diverse pathogens, but spurious activation of immune receptors can be deleterious to growth and crop yields. Conserved miRNAs fine-tune expression of NLR genes by guiding cleavage of NLR transcripts at consensus mRNA sequences, trigger amplified silencing and maintain low steady-state expression of entire NLR gene families. Similar conserved mechanisms for regulating expression of RLPs/RLKs, however, have not yet been discovered. ARS scientists in Albany, California, in collaboration with scientists at Huazhong Agricultural University in China, identified a new class of conserved of miRNAs that regulate expression of the large Cladosporium fulvum (Cf)-9-like family of RLPs in the Solanaceae. Compared to wild type plant mutants unable to synthesize these miRNAs, Cf-9 is more highly expressed, and the hypersensitive response is enhanced upon recognition of the Avr9 effector showing that these miRNAs fine-tune expression of the fast-evolving Cf-9-like family of RLPs. This work demonstrates that conserved miRNAs regulate expression of large immune receptor gene families and can be used to optimize deployment of NLR and RLP genes for balanced growth and defense in crops.

2. A model system for evaluating artificial microRNA-mediated resistance to plant viruses. Artificial microRNA (amiRNA) technology has been used to control viral diseases; however, an efficient screening process to identify amiRNAs active in protecting crops from viral disease has not been described. ARS scientists in Albany, California, in collaboration with scientists at China Agricultural University, developed a Nicotiana benthamiana system for rapid identification of artificial microRNAs to control virus disease. Scientists used the system to identify three artificial miRNAs targeting cucumber green mottle mosaic virus (CGMMV) RNA and showed that amiRNA expression reduced CGMMV replication and disease in virus infected plants. This work streamlines the process of generating amiR virus resistant crops and can be broadly applied to identify active antiviral amiRNAs against a broad spectrum of viruses to control disease in diverse crops.