Location: Plant Gene Expression Center
2023 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 from April 23, 2022, to February 24, 2023, for project 2030-22000-034-000D, "Disease Resistance Gene Regulation through RNA Silencing for Improved Crop Protection”. This is the final report for the project, which was replaced by project 2030-12210-003-000D, "Enhancing Crop Resilience to Biotic and Abiotic Stress Through Understanding the Microbiome and Immune Signaling Mechanisms”. For additional information, see the report for the new project.
ARS scientists made progress under Objective 1 and 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 continued to transform plants with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing vectors and identified mutations in seven genes of the DCL4/DCL2/RDR6 network in tomato and N. benthamiana. Under Sub-bjective 1B, ARS scientists validated gene mutations and determined that mutations in six genes of the network were transmitted from transgenic T0 plants to T1, T2 and T3 progeny. They identified mutant lines homozygous for five genes and used Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), 5’-RACE and sequencing to identify null mutations in two genes. Homozygous mutant lines carrying homozygous mutations were crossed to lines carrying CRISPR/Cas9 or ethyl methanesulfonate mutations in different genes of the DCL4/DCL2/RDR6 pathway to generate double mutation lines to test the mechanism of the DCL4/DCL2/RDR6 pathway. Breeders of Solanaceae crops will benefit from the new genetic resources established by this project for improved resistance (R)-gene and defense gene deployment.
Under Objective 2, ARS scientists made progress and used their validated DCL4/DCL2/RDR6 mutant lines described in Objective 1 to test the role of different components of the DCL4/DCL2/RDR6 pathway. They prepared and sequenced 127 sRNA and mRNA samples from 12 independent single and double mutant lines of the pathway. They are using comparative transcriptome analysis of messenger RNA (mRNA) and small RNA (sRNA) samples to test the function of pathway mutations in the proposed regulatory pathway. They further tested the biogenesis and target specificity of a novel micro RNA (miRNA) of the pathway using transient expression assays in mutant and wildtype mutation lines. They also determined that artificial microRNA (amiRNA) vector constructions express active miRNAs for complementation analysis of microRNA (MIR) deletions lines. The results of these tests have validated the function of components of the DCL4/DCL2/RDR6 network and support the proposed role of the pathway in regulation defense gene expression. Basic knowledge of the mechanisms of plant disease defense and resistance gene regulation will benefit scientists investigating resistance against myriad pathogens in evolutionarily diverse species.
ARS scientists in Albany, California, made progress on Objective 3 to determine the impact of virus infection on expression of genes in the DCL4/DCL2/RDR6 network. They collected and analyzed publicly available sRNA, mRNA and degradome RNA (dRNA) transcriptome data from eight experiments and 90 samples of mock and virus infected tomato, and N. benthamiana plants. They are performing comparative analysis between RNA isolated from plants infected with five different viruses and the RNA isolated from mutant and wild-type lines of the DCL4/DCL2/RDR6 pathway. The results of these analyses provide understanding of the impact of different virus RNA silencing suppressors on the proposed regulatory pathway and guide optimization of virus infection and analyses of viral “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. A conserved transcriptional network regulates NLR-mediated immunity. Microbial pathogens cause 10-40 percent crop loss annually worldwide and are a threat to global food security. Plants are naturally protected from microbial disease by a major class of immune receptors, the intracellular nucleotide binding leucine rich repeat (NLR) proteins, which recognize pathogen effectors and trigger resistance responses. Although mechanisms of NLR-mediated pathogen effector recognition and NLR activation have been described, understanding the transcriptional regulation NLR resistance and downstream signaling components is lacking. An ARS scientist in Albany, California, in collaboration with scientists at Huazhong Agricultural University, China, and the University of California, Davis, found that the NLR gene, N (Necrosis), conferring resistance to tobacco mosaic virus (TMV) is transcriptionally regulated upon virus effector recognition and immune activation. They identified two transcription factors using combination of RNA transcriptome analysis and protein approaches, and identified two transcription factors that belong to different protein families that activate N in response to TMV activation. These findings provide plant researchers with new tools for regulated plant resistance and defense responses for application to development of sustainable crop defense.
2. High-quality assembly and annotation of Nicotiana benthamiana and N. tabacum genomes. Nicotiana benthamiana and N. tabacum are widely used models for understanding molecular mechanisms of plant-microbe interactions. They belong to the Solanaceae family, which includes important crops such as potato, tomato, and pepper, and which are threatened by devastating pathogens. An ARS scientist in Albany, California, in collaboration with scientists at Huazhong Agricultural University, China, and the University of California, Davis, generated chromosome-level reference genomes for N. benthamiana and N. tabacum. They used comparative structural and functional analysis to provide new insights on the parent origin and chromosome structural changes leading to the hybrid genome formation of each species. Based on their high-quality genome assemblies, they developed user-friendly web analysis tools for improved gene annotation and expression analysis. This study supports understanding of genetic mechanisms underlying plant-microbe interactions and contributes to crop improvement and biotechnological applications.
