skip to main content


The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 5:00 PM ET until 11:00 PM ET on Friday, June 21 due to maintenance. We apologize for the inconvenience.

Title: Phylogenetic analyses of seven protein families refine the evolution of small RNA pathways in green plants

Several protein families participate in the biogenesis and function of small RNAs (sRNAs) in plants. Those with primary roles include Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), and Argonaute (AGO) proteins. Protein families such as double-stranded RNA-binding (DRB), SERRATE (SE), and SUPPRESSION OF SILENCING 3 (SGS3) act as partners of DCL or RDR proteins. Here, we present curated annotations and phylogenetic analyses of seven sRNA pathway protein families performed on 196 species in the Viridiplantae (aka green plants) lineage. Our results suggest that the RDR3 proteins emerged earlier than RDR1/2/6. RDR6 is found in filamentous green algae and all land plants, suggesting that the evolution of RDR6 proteins coincides with the evolution of phased small interfering RNAs (siRNAs). We traced the origin of the 24-nt reproductive phased siRNA-associated DCL5 protein back to the American sweet flag (Acorus americanus), the earliest diverged, extant monocot species. Our analyses of AGOs identified multiple duplication events of AGO genes that were lost, retained, or further duplicated in subgroups, indicating that the evolution of AGOs is complex in monocots. The results also refine the evolution of several clades of AGO proteins, such as AGO4, AGO6, AGO17, and AGO18. Analyses of nuclear localization signal sequences and catalytic triads of AGO proteins shed light on the regulatory roles of diverse AGOs. Collectively, this work generates a curated and evolutionarily coherent annotation for gene families involved in plant sRNA biogenesis/function and provides insights into the evolution of major sRNA pathways.

more » « less
Award ID(s):
Author(s) / Creator(s):
; ;
Publisher / Repository:
Oxford University Press
Date Published:
Journal Name:
Plant Physiology
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    Abstract In monocots other than maize (Zea mays) and rice (Oryza sativa), the repertoire and diversity of microRNAs (miRNAs) and the populations of phased, secondary, small interfering RNAs (phasiRNAs) are poorly characterized. To remedy this, we sequenced small RNAs (sRNA) from vegetative and dissected inflorescence tissue in 28 phylogenetically diverse monocots and from several early-diverging angiosperm lineages, as well as publicly available data from 10 additional monocot species. We annotated miRNAs, small interfering RNAs (siRNAs) and phasiRNAs across the monocot phylogeny, identifying miRNAs apparently lost or gained in the grasses relative to other monocot families, as well as a number of transfer RNA fragments misannotated as miRNAs. Using our miRNA database cleaned of these misannotations, we identified conservation at the 8th, 9th, 19th, and 3′-end positions that we hypothesize are signatures of selection for processing, targeting, or Argonaute sorting. We show that 21-nucleotide (nt) reproductive phasiRNAs are far more numerous in grass genomes than other monocots. Based on sequenced monocot genomes and transcriptomes, DICER-LIKE5, important to 24-nt phasiRNA biogenesis, likely originated via gene duplication before the diversification of the grasses. This curated database of phylogenetically diverse monocot miRNAs, siRNAs, and phasiRNAs represents a large collection of data that should facilitate continued exploration of sRNA diversification in flowering plants. 
    more » « less
  2. Abstract

    Small RNAs (sRNAs) associate with ARGONAUTE (AGO) proteins forming effector complexes with key roles in gene regulation and defense responses against molecular parasites. In multicellular eukaryotes, extensive duplication and diversification of RNA interference (RNAi) components have resulted in intricate pathways for epigenetic control of gene expression. The unicellular alga Chlamydomonas reinhardtii also has a complex RNAi machinery, including 3 AGOs and 3 DICER-like proteins. However, little is known about the biogenesis and function of most endogenous sRNAs. We demonstrate here that Chlamydomonas contains uncommonly long (>26 nt) sRNAs that associate preferentially with AGO1. Somewhat reminiscent of animal PIWI-interacting RNAs, these >26 nt sRNAs are derived from moderately repetitive genomic clusters and their biogenesis is DICER-independent. Interestingly, the sequences generating these >26-nt sRNAs have been conserved and amplified in several Chlamydomonas species. Moreover, expression of these longer sRNAs increases substantially under nitrogen or sulfur deprivation, concurrently with the downregulation of predicted target transcripts. We hypothesize that the transposon-like sequences from which >26-nt sRNAs are produced might have been ancestrally targeted for silencing by the RNAi machinery but, during evolution, certain sRNAs might have fortuitously acquired endogenous target genes and become integrated into gene regulatory networks.

    more » « less
  3. Summary

    In plants, 24 nucleotide long heterochromatic siRNAs (het‐siRNAs) transcriptionally regulate gene expression byRNA‐directedDNAmethylation (RdDM). The biogenesis of most het‐siRNAs depends on the plant‐specificRNApolymeraseIV(PolIV), andARGONAUTE4 (AGO4) is a major het‐siRNAeffector protein. Through genome‐wide analysis ofsRNA‐seq data sets, we found thatAGO4is required for the accumulation of a small subset of het‐siRNAs. The accumulation ofAGO4‐dependent het‐siRNAs also requires several factors known to participate in the effector portion of the RdDMpathway, includingRNA POLYMERASEV (POLV),DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) andSAWADEE HOMEODOMAIN HOMOLOGUE1 (SHH1). Like manyAGOproteins,AGO4 is an endonuclease that can ‘slice’RNAs. We found that a slicing‐defectiveAGO4 was unable to fully recoverAGO4‐dependent het‐siRNAaccumulation fromago4mutant plants. Collectively, our data suggest thatAGO4‐dependent siRNAs are secondary siRNAs dependent on the prior activity of the RdDMpathway at certain loci.

    more » « less
  4. Summary

    Plant smallRNAs (sRNAs) modulate key physiological mechanisms through post‐transcriptional and transcriptional silencing of gene expression. SmallRNAs fall into two major categories: those are reliant onRNA‐dependentRNApolymerases (RDRs) for biogenesis and those that are not. KnownRDR1/2/6‐dependentsRNAs include phased and repeat‐associated short interferingRNAs, while knownRDR1/2/6‐independentsRNAs are primarily microRNAs (miRNA) and other hairpin‐derivedsRNAs. In this study we produced and analyzedsRNA‐seq libraries fromrdr1/rdr2/rdr6triple mutant plants. We found 58 previously annotated miRNAloci that were reliant onRDR1, ‐2, or ‐6function, casting doubt on their classification. We also found 38RDR1/2/6‐independentsRNAloci that are notMIRNAs or otherwise hairpin‐derived, and did not fit into other known paradigms forsRNAbiogenesis. These 38sRNA‐producing loci have as‐yet‐undescribed biogenesis mechanisms, and are frequently located in the vicinity of protein‐coding genes. Altogether, our analysis suggests that these 38 loci represent one or more undescribed types ofsRNAinArabidopsis thaliana.

    more » « less
  5. Summary

    Phased secondary siRNAs (phasiRNAs) are broadly present in the reproductive tissues of flowering plants, with spatial–temporal specificity. However, the ARGONAUTE (AGO) proteins associated with phasiRNAs and their miRNA triggers remain elusive.

    Here, through histological and high‐throughput sequencing analyses, we show that rice AGO1d, which is specifically expressed in anther wall cells before and during meiosis, associates with both miR2118 and miR2275 to mediate phasiRNA biogenesis.

    AGO1d preferentially binds to miR2118‐triggered 21‐nucleotide (nt) phasiRNAs with a 5′‐terminal uridine, suggesting a dual role in phasiRNA biogenesis and function. Depletion of AGO1d causes a reduction of 21‐ and 24‐nt phasiRNAs and temperature‐sensitive male sterility. At lower temperatures, anthers of theago1dmutant predominantly show excessive tapetal cells with little starch accumulation during pollen formation, possibly caused by the dysregulation of cell metabolism.

    These results uncover an essential role of AGO1d in rice anther development at lower temperatures and demonstrate coordinative roles of AGO proteins during reproductive phasiRNA biogenesis and function.

    more » « less