A genetics screen uncovers the role of RNA-binding protein RBP45D in flowering time under heat and DCL3-independent RNA-directed DNA methylation (WGBS)

RNA-directed DNA methylation (RdDM) is a prominent pathway in plants to defend against invasive nucleic acids. In the canonical RdDM, 24-nt small interfering RNAs (siRNAs) are produced through DICER-LIKE 3 (DCL3). Here, we describe the Arabidopsis thaliana prors1 (LUC) transgenic system in which transcriptional gene silencing (TGS) is independent of DLC3. In a forward genetics screen performed with this system, already known components of RdDM and RNA-binding protein RBP45D were identified. RBP45D promotes DNA methylation, and a lack of RBP45D causes late flowering especially under heat, presumably mediated by elevated FLC levels. RBP45D is localized to the nucleus where it is associated with snRNAs and snoRNAs. RBP45D maintains siRNA production originating from the transgene, but does not promote mRNA levels or influence pre-mRNA splicing of known RdDM genes. We hypothesize that RBPD45 facilitates DCL3-independent siRNA production through stabilisation of either the precursor RNA or the – not yet identified – slicer protein. Overall design: Examination of differential methalytion in the Arabidopsis thaliana rbp-ko-1 (KO27) mutant in comparison to Col-0