GSE51959
Global and local perturbation of the tomato microRNA pathway by a trans-activated DICER-LIKE 1 mutant
DICER-like 1 (DCL1) is a major player in micro RNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested seedling 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 elevation in the 22- nucleotide small RNAs was correlated with specific upregulation of SlDCL2b and SlDCL2d, which are likely 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, our results establish OP:SlDCL1IR as an efficient tool for analyzing processes regulated by SlDCL1-mediated gene regulation in tomato. Overall design: Examination of small RNA populations in control (35S:LhG4) and SlDCL1 silenced seedlings (35S>>SlDCL1IR) 2 biological replicates each