Location: Plant Gene Expression Center
2022 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
This report documents progress for project 2030-22000-034-000D, “Identification and Regulation of Plant Resistance and Defense Genes Using Genomic Resources for Improved Crop Protection Against Diseases,” which started April 23, 2022, and continues research from project 2030-22000-009-000D, “Disease Resistance Gene Regulation through RNA Silencing for Improved Crop Protection", which expired April 22, 2022.
Under Objective 1, ARS scientists generated new genetic resources for the economically and nutritionally important crop plant tomato and the model plant-microbe interaction species, N. benthamiana. Under Sub-objective 1A, ARS scientists constructed Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing vectors and used them to introduce mutations into tomato and N. benthamiana genes of the Dicer-like (DCL)4/DCL2/RDR6 network. Under Sub-objective 1B, the ARS researchers confirmed mutations were introduced in seven genes of the DCL4/DCL2/RDR6 network and that mutations were transmitted to progeny. They generated fours lines homozygous for edited genes in the RNA silencing pathway. Breeders of Solanaceae crops will benefit from genetic resources generated by this project to improve R-gene and defense gene deployment.
Under Objective 2, ARS scientists constructed and used Agrobacterium gene expression vectors in experiments to determine if micro RNAs (miRNAs) guide cleavage at predicted sites in target genes of the pathway for analysis to determine that plants homozygous for edited genes were null mutations in four genes of the DCL4/DCL2/RDR6 network. They further validated silencing mutations using comparative transcriptome analysis using messenger RNA (mRNA) and small RNA isolated from null, knock-down mutation and wild type lines. Comparative analysis is underway to test the DCL4/DCL2/RDR6 model. Basic knowledge of the mechanisms of plant disease resistance gene regulation will benefit scientists investigating resistance against myriad pathogens in evolutionarily diverse species.
ARS scientists made progress on Objective 3 to optimize virus infection procedures that will be used in comparative transcriptome analysis using RNA isolated from virus infected mutant and wild-type lines. They compared relative rates and timing, virus spread and disease severity of two viruses in two established mutant lines of the predicted silencing pathway. They will use the results of these experiments to optimize virus infection procedures for experiments to determine the impact of different virus suppressors of RNA silencing that serve as “counter defense” measures on the expression host genes of the proposed regulatory network. The results and information from these experiments will benefit scientists studying virus mechanisms and plant plant-microbe interactions to develop new defense strategies to protect plants from virus disease and reduce crop loss.
Accomplishments
1. Long noncoding RNA gets into the balancing act. Deeper understanding of the molecular mechanisms balancing plant growth and immunity are essential for a greener, more sustainable, and more reliable agricultural future. An ARS scientist in Albany, California, in collaboration with scientists at Huazhong Agricultural University, China, and the University of Wisconsin Madison, preview the recent discovery of a long noncoding RNA (lncRNA) that regulates plant immunity. The preview describes current understanding of two primary immune pathways, each based on characteristic proteins that recognize invading pathogens and trigger defense. The preview highlights the essential and unifying role the plant hormone, salicylic acid, required for activation of both immune pathways and sets the stage for introduction of a new player, long non-coding RNA, that protects plants by promoting growth in uninfected plants and unleashing defenses when pathogens attack by transcriptionally regulating salicylic acid biosynthesis. These findings provide plant researchers a new tool new to optimize plant growth and defense and will be used in strategies to develop sustainable crop defense.
Review Publications
Li, F., Brunkard, J., Baker, B.J. 2022. LncRNA gets into the balancing act. Cell Host and Microbe. 30(8):1061-1063. https://doi.org/10.1016/j.chom.2022.07.004.
