title: Generation and characterization of a tomato DCL3-silencing mutant.
abstract: DICER-like 3 (DCL3) is a major player in heterochromatic 24-nucleotide (nt) small RNA (sRNA) and long microRNA (lmiRNA) biogenesis, and higher plant DCL3 mutants have been characterized from Arabidopsis thaliana and rice. Here, a tomato DCL3 (SlDCL3) mutant was generated through the use of trans-activated artificial miRNA and characterized. Constitutive trans-activation knocked down SlDCL3 levels by ∼64%, resulting in dramatically decreased 24-nt sRNA levels and a significant increase in 21- and 22-nt sRNAs. The latter was correlated with specific upregulation of SlDCL4 and SlDCL2b, which function in the biogenesis of 21- and 22-nt sRNAs, respectively. Moreover, at the majority of sRNA-generating genomic loci, an almost complete overlap between small RNA signatures of control and silenced seedlings was observed, suggesting that the reductions in 24-nt sRNAs at these loci were compensated for by biogenesis of 21- and 22-nt sRNAs from the same double-stranded RNA substrates. In addition, bioinformatic analysis and reduced expression in SlDCL3-silenced seedlings identified four novel tomato lmiRNAs, two of which were found to be developmentally regulated. Taken together, these results establish the requirement of SlDCL3 for the biogenesis of 24-nt sRNAs and lmiRNAs in tomato and suggest SlDCL4 and SlDCL2b as surrogates for SlDCL3.
authors.affiliation: Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel.
authors.forename: Michael
authors.initials: M
authors.lastname: Kravchik
authors.affiliation: Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel.
authors.forename: Subha
authors.initials: S
authors.lastname: Damodharan
authors.affiliation: Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel.
authors.forename: Ran
authors.initials: R
authors.lastname: Stav
authors.affiliation: Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel. Electronic address: tarazi@agri.gov.il.
authors.forename: Tzahi
authors.initials: T
authors.lastname: Arazi
mesh_terms: D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D018551:Solanum lycopersicum / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism; D036226:Seedlings / Q000235:genetics; D020539:Sequence Analysis, Protein
chemical_list: D035683:MicroRNAs; D010940:Plant Proteins; D043244:Ribonuclease III
keywords: DCL3; Long miRNA; Mutant; Silencing; Tomato