Higher plants encode four or five DICER-LIKE (DCL) enzymes responsible for the production of small non-coding RNAs which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense, but in other settings, DCL2 has the opposite function of DCL4, at least in formal genetic terms. For example, knockout of DCL4 causes growth defects that are suppressed by inactivation of DCL2. Current models maintain that the biochemical basis of both of these effects is RNAi via DCL2-dependent small interfering RNAs (siRNAs). Here, we report that neither DCL2-mediated antiviral resistance nor growth defects can be explained by silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either through specific point mutations in DCL2 or simply by reducing DCL2 dosage in plants heterozygous for dcl2 knockout alleles. Intriguingly, however, all functions of DCL2 depend on it having some level of catalytic activity. We discuss this requirement for catalytic activity, but not for the resulting siRNAs, in the light of recent findings that reveal a function of DCL2 in activation of innate immunity in response to cytoplasmic double-stranded RNA.

The proteotypic peptide YVAGNNSGLQNQTR of DCL2 was quantified in rosette tissue lysate of arabidopsis thalian (21-day old plants). 10 reference peptides covering 10 endogenous proteins were included.

Rosette tissue of 21-day old plants germinated on soil and grown under long day conditions were harvested and ground to a fine powder in liquid nitrogen. 100 mg ground tissue were then homogenized in 400 µl lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM MgCl2, 0.1% NP40, freshly supplemented with 4 mM DTT and 1x Roche complete protease inhibitor), incubated for 5 mins on a rotator at 4ºC and cleared by centrifugation at max. speed for 10 min at 4ºC. The supernatant was transferred to a new tube and the procedure was repeated twice. Four volumes of acetone (pre-chilled at -20ºC) were then added to the supernatant and proteins were precipitated at -80ºC overnight.