In flowering plants, small RNA pathways are the key components of TE silencing and control. At the transcription level, TEs are silenced by a mechanism called RNA-directed DNA methylation (RdDM), where 24-nucleotide-long small interfering RNAs (siRNAs) facilitate methylation of TE sequences leading to heterochromatin formation. Another mode of TE regulation is posttranscriptional gene silencing (PTGS): expressed TEs induce the production of 21 and 22nt siRNAs targeting AGO proteins toward these transcripts. This pathway is also a part of plants' antiviral immunity. 21nt siRNAs mostly lead to mRNA cleavage and degradation, whilst 22nt siRNAs were shown to induce translational repression and, most importantly, recruit RNA-dependent RNA polymerase to the mRNA to produce more double-stranded RNA (dsRNA) leading to amplification of the PTGS.

Duckweeds, or Lemnoideae, small fast-growing aquatic flowering plants, possess a somewhat simplified RdDM lacking many components of this pathway as well as PTGS, missing some elements considered antiviral factors such as AGO2. DICER-LIKE PROTEIN 2 (DCL2), responsible for producing 22nt siRNAs in plants, is also missing in duckweeds, yet we observe 22nt siRNA production in all three species studied in our lab. For instance, transgenic silencing in Lemna minor is characterized by the presence of transgene-derived 21/22-nt siRNAs. Transient expression of inverted repeat (IR) in Spirodela polyrhiza leads to the production of equal amounts of 21/22-nt siRNAs from the resulting dsRNA. On the other hand, 22-nt siRNAs, together with 21-nt, also come from transposons expressed in Spirodela, similarly to Wolffia brasiliensis.

The aim of this project is to understand 22-nt siRNA biogenesis in the absence of DCL2 and its biological function in Duckweeds through a combination of genetic and biochemical approaches. Complementation assays in Arabidopsis, knockouts in Lemna's transgenic line and in vitro analysis will be the next steps to investigate this problem.