DICER-LIKE (DCL) enzymes process double-stranded RNA into small RNAs that act as regulators of gene expression. Arabidopsis DCL4 and DCL2 each allow the post-transcriptional gene silencing (PTGS) of viruses and transgenes, but primary PTGS-prone DCL4 outcompetes transitive PTGS-prone DCL2 in wild-type plants. This hierarchy likely prevents DCL2 having any detrimental effects on endogenous genes. Indeed, dcl4 mutants exhibit developmental defects and increased sensitivity to genotoxic stress. In this study, the mechanism underlying dcl4 defects was investigated using genetic, biochemical and high-throughput sequencing approaches. We show that the purple phenotype of dcl4 leaves correlates with carbohydrate over-accumulation and defective phloem transport, and depends on the activity of SUPPRESSOR OF GENE SILENCING 3, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DCL2. This phenotype correlates with the downregulation of two genes expressed in the apex and the vasculature, SMAX1-LIKE 4 (SMXL4) and SMXL5, and the accumulation of DCL2- and RDR6-dependent small interfering RNAs derived from these two genes. Supporting a causal effect, smxl4 smxl5 double mutants exhibit leaf pigmentation, enhanced starch accumulation and defective phloem transport, similar to dcl4 plants. Overall, this study elucidates the detrimental action of DCL2 when DCL4 is absent, and indicates that DCL4 outcompeting DCL2 in wild-type plants is crucial to prevent the degradation of endogenous transcripts by DCL2- and RDR6-dependent transitive PTGS.

Plant microRNAs (miRNAs) are predominantly 21 nucleotides (nt) long but non-canonical lengths of 22 and 20 nt are commonly observed in diverse plant species. While miRNAs longer than 21 nt can be attributed to the neglect of unpaired bases within asymmetric bulges by the ruler function of DICER-LIKE 1 (DCL1), how 20-nt miRNA is generated remains obscure. Analysis of small RNA data revealed that 20-nt miRNA can be divided into 3 main groups featured by atypical 3 0 overhangs or shorter duplex regions. Asymmetric bulges or mismatches at specific positions are commonly observed within each group and were shown to be crucial for 20-nt miRNA formation. Analysis of DCL1 cleavage sites on 20-nt miRNA precursors suggests that these determinants might alter precursor structure or trigger 3 0 -end decay of mature miRNA. The results herein advance our understanding of miRNA biogenesis and demonstrate that the effect of asymmetric bulges on miRNA length could be position-dependent.