Processing of microRNA (miRNA) precursors results in the release of a double-stranded miRNA/miRNA* duplex. The miRNA or guide strand, is loaded onto the Argonaute (AGO) effector, and the miRNA* or passenger strand is typically degraded. The loaded AGO-containing RNA-induced silencing complex specifically recognizes a target mRNA, leading to its degradation or translational inhibition. In plants, miRNA-mediated cleavage of a target triggers in some cases the production of secondary small interfering RNAs (siRNAs), which in turn can silence other genes in trans . This alternative pathway depends on the length of the miRNA and the specific AGO in the effector complex. However, 22-nt miRNAs are sufficient, but not essential for this pathway. Using a combination of computational and experimental approaches, we show that transitivity can be triggered when the small RNA that is not retained in AGO is 22-nt long. Moreover, we demonstrate that asymmetrically positioned bulged bases in the miRNA:miRNA* duplex, regardless of miRNA or miRNA* length, are sufficient for the initiation of transitivity. We propose that the RNA-induced silencing complex reprogramming occurs during the early steps of miRNA loading, before the miRNA duplex is disassembled and the guide strand is selected.

• MicroRNAs (miRNAs) are small 21‐nucleotide RNAs that post‐transcriptionally regulate gene expression. MiR396 controls leaf development by targeting GRF and bHLH transcription factors in Arabidopsis. WRKY transcription factors, unique to plants, have been identified as mediating varied stress responses. The sunflower (Helianthus annuus) HaWRKY6 is a particularly divergent WRKY gene exhibiting a putative target site for the miR396. A possible post‐transcriptional regulation of HaWRKY6 by miR396 was investigated. • Here, we used expression analyses, performed by quantitative reverse transcription polymerase chain reaction (qRT‐PCR) and northern blots together with computational approaches to establish the regulatory interaction between HaWRKY6 and the identified sunflower miR396. Arabidopsis plants expressing a mi396‐resistant version of HaWRKY6 confirmed the miRNA‐dependency of the HaWRKY6 silencing. • Sunflower plants exposed to high temperatures or salicylic acid presented opposite expression of HaWRKY6 and miR396. Experiments using the wildtype and miRNA‐resistant versions of HaWRKY6 showed altered stress responses. Our results showed a role of the recently evolved miR396 regulation of HaWRKY6 during early responses to high temperature. • Our study reveals how a miRNA that normally regulates development has been recruited for high‐temperature protection in sunflower, a plant particularly well adapted to this type of stress.