Incomplete DRB4-dependence of the DCL4-mediated antiviral defense

The double-stranded RNA-binding protein DRB4 of Arabidopsis was shown previously to contribute to the DICER-LIKE 4 (DCL4)-mediated biogenesis of viral small interfering RNAs (vsiRNAs) of 21 nucleotides (nt) in size. However, it is unclear whether all 21-nt vsiRNAs are dependent on this DRB4-DCL4 partnership. To resolve this question, we generated dcl2drb4 and dcl4drb4 double knockout mutants, and subjected them to infections with CPB-CC-PDS, a turnip crinkle virus mutant capable of inducing silencing of the PHYTOENE DESATURASE gene. The dcl2drb4 double knockouts caused a far smaller loss of antiviral silencing than dcl2dcl4. In addition, although both drb4 and dcl4 single mutants permitted a consistent (but small) increase in viral RNA levels, the drb4 mutant correlated with a less pronounced reduction of 21-nt vsiRNAs. Therefore, a substantial subset of DCL4 antiviral activity is DRB4-independent, and may involve other DRB proteins that compensate for loss of DRB4.