Generation and characterization of a tomato DCL3-silencing mutant

DICER-like 3 (DCL3) is a major player in heterochromatic 24-nucleotide (nt) small RNA (sRNA) and long microRNA (ImiRNA) biogenesis, and higher plant DCL3 mutants have been characterized from Arabidopsis thaliana and rice. Here, a tomato DCL3 (SIDCL3) mutant was generated through the use of trans-activated artificial miRNA and characterized. Constitutive trans-activation knocked down SIDCL3 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 S1DCL4 and S1DCL2b, 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 21and 22-nt sRNAs from the same double-stranded RNA substrates. In addition, bioinformatic analysis and reduced expression in S1DCL3-silenced seedlings identified four novel tomato lmiRNAs, two of which were found to be developmentally regulated. Taken together, these results establish the requirement of SIDCL3 for the biogenesis of 24-nt sRNAs and lmiRNAs in tomato and suggest SIDCL4 and S1DCL2b as surrogates for S1DCL3. (C) 2014 Elsevier Ireland Ltd. All rights reserved.