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ARS Home » Pacific West Area » Albany, California » Plant Gene Expression Center » Research » Research Project #432708

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

Location: Plant Gene Expression Center

2022 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 Report
This is the final report for project 2030-22000-009-000D, which expired in April 2022 and has been replaced by project 2030-22000-034-000D. For additional information, see the report for the new project. To develop sustainable methods for disease control in crops, a better understanding of the mechanisms that regulate plant defense against pathogens is needed. ARS scientists in Albany, California, identified a class of conserved micro RNAs (miRNAs) that drive widespread repression of disease resistance genes for receptor-like genes and a Dicer-like (DCL) gene in the Solanaceae family, including the nutritionally important crop tomato and the model species, Nicotiana (N.) tabacum and N. benthamiana. Under Sub-objective 1A, ARS scientists and collaborators at Huazhong Agricultural University, China, used bioinformatic analysis of tomato and tobacco genome and transcriptome sequences and identified a new class of conserved 21-22 nt microRNAs in tomato and tobacco. They later identified the new class of miRNAs in N. benthamiana using resources described in Sub-objectives 1B and 2B. They predicted that miRNAs were processed from atypical long hairpin structures and that they belonged to a class of miRNAs whose production was dependent on Dicer-like 4 (DCL4) rather than DCL1. Two conserved miRNA families were predicted to target many members of the large family of leucine-rich-repeat receptor-like protein (LRR-RLP) resistance genes, including the important tomato resistance gene Cf-9, conferring resistance to the fungal pathogen Cladosporium fulvum (Cf), and a second conserved family predicted to target a gene in the RNA silencing pathway. The identification of these new miRNAs can be used by scientists to address innate immune gene regulation broadly in the Solanaceae and potentially in other plant families. Under Sub-objective 1B, ARS scientists made progress and generated new Solanaceae genetic resources to determine the structure and function of the predicted class of novel DCL4-miRNAs. They generated more than 20 tobacco lines carrying knockdown mutations in one of four RNA silencing pathway genes and lines carrying the N (Necrosis) tobacco mosaic virus resistance gene or the tomato Cf-9 fungal resistance gene. Tobacco N and Cf genes were introduced into RNA silencing knockdown lines. They used a tobacco mosaic virus (TMV)-resistant and an N line expressing a virus suppressor of RNA silencing, HC-Pro. They characterized four Nicotiana benthamiana lines carrying knockdown mutations in one more RNA silencing gene. Tomato lines with null mutations in one of four RNA silencing pathway genes were provided. Lines carrying null or knock-down mutations in RNA silencing genes were characterized for reduced expression of known miRNAs and small interfering RNAs produced by different RNA silencing pathways. Scientists investigating plant defense and resistance mechanisms and breeders of Solanaceae crops will benefit from the characterized genetic resources generated in Sub-objective 1B. Under Sub-objective 2A, scientists made progress and determined the mechanism of DCL4 miRNA production and miRNA silencing of predicted gene targets identified in Objective 1. They used several different approaches to determine the relative levels of predicted miRNAs and miRNA target gene expression in RNA silencing mutants and wild type lines. They compared levels of miRNAs, miRNA cleavage and expression of target genes in silencing mutants and wild type tobacco and tomato and showed that DCL4 is required for miRNA production, miRNA target gene cleavage and reduced target gene expression. They confirmed their results of DCL4-dependent miRNA production and silencing of gene targets using comparative transcriptome analysis of RNA isolated from RNA silencing mutant and wild type tobacco, tomato lines. They extended their gene expression analysis to N. benthamiana and identified conserved DCL4-miRNAs and receptor-like protein (RLP) and DCL2 target genes that supported their conclusions on DCL4-dependent biogenesis, and DCL4-miRNA regulation of targeted genes. The results of research under this objective determined the production and mechanism of a conserved miRNA that targets and silences expression of plant resistance and defense genes and will benefit plant scientists investigating mechanisms of plant defense. Under Sub-objective 2B, scientists made progress to understand the impact of DCL4-miRNA silencing on regulation of R gene and defense gene expression and induced resistance. ARS scientists in Albany, California, in collaboration with scientists at Huazhong Agricultural University in China, tested if conserved DCL4 miRNAs of tobacco regulate the expression of the tomato Cf-9 resistance gene and Cf-9 mediated hypersensitive response triggered by the Avr9 fungal effector. They introduced Cf-9 into wildtype tobacco and the RNA silencing mutant backgrounds and transiently expressed Avr9 by agroinfiltration in leaves of Cf-9 and Cf-9; dcl4 plants. They found that in Cf-9; dcl4 lines that fail to produce DCL4-miRNAs, Cf-9 is more highly expressed, and the hypersensitive response is enhanced upon recognition of the Avr9 effector, suggesting that that DCL4- miRNAs fine-tune expression of the fast-evolving Cf-9-like family of RLPs. ARS scientists further tested the impact of a conserved DCL4 miRNA on regulation of a predicted defense gene target. They tested the effect of HC-Pro (helper component proteinase), a viral suppressor of RNA silencing, on miRNA cleavage and expression of transcripts targeted by DCL4-dependent miRNA. They used transgenic tobacco plants expressing HC-Pro and observed reduced miRNA cleavage and strong induction of the DCL4 miRNA-targeted gene DCL2. Scientists will benefit from understanding the impact of DCL4 miRNA regulation on resistance and defense gene expression and pathogen defense established by this project. These resources can be used to address innate immune gene regulation broadly in the Solanaceae and in other plant families. Under Sub-objective 3A, ARS scientists made progress to determine the impact of virus infection on reprogramming of host gene expression. They are using gene expression analysis to determine differentially expressed miRNAs, miRNA targets and other innate immunity genes in virus-infected resistant and susceptible tomato. They will determine expression changes associated with TMV-induced resistance responses or TMV-induced disease to understand cellular pathways subject to viral reprogramming during infection, especially those guided by differentially controlled miRNAs and other small RNAs (sRNAs) targeting genes in innate immunity. Comparative expression analysis of sequenced transcriptomes is underway. These results will be of broader interest to researchers studying disease resistance, sRNA regulation, plant-microbe interactions, and more specifically to plant virologists interested in virus induced host processes.

1. Small RNAs with a big impact on plant immunity. Pathogen diseases are a major threat to sustainable and productive agriculture worldwide. Pathogen diseases are naturally controlled by resistance (R) receptors for pathogen recognition triggered resistance, whereas viral defense is primarily controlled by a host mechanism of small RNA guided degradation of viral RNA as well as resistance mechanisms. The discovery of plant small microRNAs targeting and regulating disease resistance receptors highlighted the regulatory relationship between these plant defense mechanisms. A better understanding of mechanisms regulating plant defense gene expression is needed. An ARS scientist in Albany, California, in collaboration with scientists at Huazhong Agricultural University, China, and the University of Wisconsin Madison, discovered a new miRNA mechanism that regulates expression of plant disease resistance genes and an RNA silencing pathway gene. The discovery of a new class of miRNAs that regulate disease resistance genes and a gene in RNA silencing advances understanding of innate immunity regulation and underscores the proposed evolutionary and regulatory relationship between silencing and innate immunity.

Review Publications
Miao, S., Liang, C., Li, J., Baker, B.J., Luo, L. 2021. Polycistronic artificial microRNA-mediated resistance to cucumber green mottle mosaic virus in cucumber. International Journal of Molecular Sciences. 22(22). Article 12237.