Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants

Plant Cell. 2012 Sep;24(9):3613-29. doi: 10.1105/tpc.112.099945. Epub 2012 Sep 28.


In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Argonaute Proteins / genetics
  • Argonaute Proteins / metabolism*
  • Catalytic Domain
  • Genome-Wide Association Study
  • High-Throughput Nucleotide Sequencing
  • Mutation
  • Phenotype
  • Plant Diseases / virology
  • Plant Leaves / genetics
  • Plant Leaves / metabolism
  • Plants, Genetically Modified
  • Potyvirus / physiology
  • Protein Stability
  • RNA Interference
  • RNA, Plant / genetics
  • RNA, Plant / metabolism
  • RNA, Small Interfering / genetics*
  • RNA, Small Interfering / metabolism
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Sequence Analysis, RNA
  • Transgenes


  • AGO1 protein, Arabidopsis
  • Arabidopsis Proteins
  • Argonaute Proteins
  • RNA, Plant
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • ZIPPY protein, Arabidopsis
  • argonaute 2 protein, Arabidopsis

Associated data

  • GEO/GSE40259