Global and local perturbation of the tomato microRNA pathway by a trans-activated DICER-LIKE 1 mutant

J Exp Bot. 2014 Feb;65(2):725-39. doi: 10.1093/jxb/ert428. Epub 2013 Dec 27.

Abstract

DICER-like 1 (DCL1) is a major player in microRNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested development. Consequently, generation of dcl1 mutants by reverse genetics and functional analysis of DCL1 in late-developing organs are challenging. Here, these challenges were resolved through the unique use of trans-activated RNA interference. Global, as well as organ-specific tomato DCL1 (SlDCL1) silencing was induced by crossing the generated responder line (OP:SlDCL1IR) with the appropriate driver line. Constitutive trans-activation knocked down SlDCL1 levels by ~95%, resulting in severe abnormalities including post-germination growth arrest accompanied by decreased miRNA and 21-nucleotide small RNA levels, but prominently elevated levels of 22-nucleotide small RNAs. The increase in the 22-nucleotide small RNAs was correlated with specific up-regulation of SlDCL2b and SlDCL2d, which are probably involved in their biogenesis. Leaf- and flower-specific OP:SlDCL1IR trans-activation inhibited blade outgrowth, induced premature bud senescence and produced pale petals, respectively, emphasizing the importance of SlDCL1-dependent small RNAs in these processes. Together, these results establish OP:SlDCL1IR as an efficient tool for analysing processes regulated by SlDCL1-mediated gene regulation in tomato.

Keywords: Carotenoid; DCL1; leaf; miRNA; mutant; petal; tomato..

Publication types

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

MeSH terms

  • Base Sequence
  • Carotenoids / metabolism
  • Flowers / metabolism
  • Gene Expression Regulation, Plant
  • Gene Silencing
  • Genes, Reporter
  • Lycopersicon esculentum / genetics*
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Molecular Sequence Data
  • Mutation / genetics*
  • Phenotype
  • Plant Leaves / anatomy & histology
  • Plant Leaves / genetics
  • Plant Proteins / metabolism*
  • RNA, Plant / genetics
  • RNA, Plant / metabolism
  • RNA, Small Interfering / metabolism
  • Ribonuclease III / metabolism*
  • Seedlings / genetics
  • Sequence Homology, Amino Acid
  • Transcriptional Activation / genetics*
  • Up-Regulation / genetics

Substances

  • MicroRNAs
  • Plant Proteins
  • RNA, Plant
  • RNA, Small Interfering
  • Carotenoids
  • Ribonuclease III