| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2017091 | Plant Science | 2014 | 9 Pages |
•Cloning the tomato DCL3 homolog and its silencing by artificial miRNA.•SlDCL3 silencing decreases 24 nt small RNA numbers and diversity.•SlDCL3 silencing increases 21–22 nt small RNA numbers and diversity.•SlDCL2b and SlDCL4 are upregulated upon SlDCL3 silencing.•Identification of novel SlDCL3-dependent 24 nt tomato long miRNAs.
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.
