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Kravchik, M., Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel
Damodharan, S., Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel
Stav, R., Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel
Arazi, T., Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel
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. © 2014 Elsevier Ireland Ltd.
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Generation and characterization of a tomato DCL3-silencing mutant
221-222
Kravchik, M., Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel
Damodharan, S., Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel
Stav, R., Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel
Arazi, T., Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO BOX 6, Bet Dagan 50250, Israel
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 (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. © 2014 Elsevier Ireland Ltd.
Scientific Publication
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