The role of RNA-dependent RNA polymerases RDR1 and RDR6 in shaping the small RNA landscape of tobacco BY-2 cells
(2025)
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- Plant cell suspensions, such as Nicotiana tabacum BY-2 cells, are an emerging expression system to produce recombinant proteins. However, the production yields are usually lower than those obtained with other expression systems. RNA silencing is a general issue for reaching high recombinant protein yields in plant molecular farming. In this process, small interfering RNAs (siRNAs) mediate degradation of transgenic transcripts via post-transcriptional gene silencing (PTGS) and promote RNA-directed DNA methylation of promoter sequences, leading to transcriptional gene silencing (TGS). RNA-dependent RNA polymerase RDR1 and RDR6 are the first enzymes involved in siRNA production during PTGS. Our research aimed at generating BY-2 cell lines inactivated for RDR1 and RDR6 activities, via a multiplex CRISPR/Cas9 strategy. The knock-out of RDR1 and RDR6 genes was confirmed by sequencing of the edited regions. Before studying the impact on transgenic siRNA production, we investigated changes in the endogenous small RNA (sRNA) landscape. Despite growing interest in plant molecular farming and fundamental research, sRNA studies in BY-2 cells were almost missing. We identified novel microRNAs in BY-2 cells and developed a robust validation pipeline to assess their reliability. As RDR6 plays a central role in the biogenesis of phasiRNAs, their synthesis was severely impaired in the RDR1/6KO cell lines. This enabled us to validate novel PHAS loci predicted using a phasing-score algorithm. Finally, we showed that inactivation of RDR1/6 genes effectively impeded the production of transgenic siRNAs involved in PTGS. PTGS deficiency led to an approximate 10-fold increase in GusPlus or GUS-Venus (GV) reporter gene expression, upon transient transformation of RDR1/6KO cell lines with Agrobacterium tumefaciens. However, no increase in GV expression was observed when stably expressed in BY-2 RDR1/6KO cells. Potential reasons for this difference are discussed, and strategies to further enhance recombinant protein expression in BY-2 cell cultures are proposed.
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Bouhon, L. (2025). The role of RNA-dependent RNA polymerases RDR1 and RDR6 in shaping the small RNA landscape of tobacco BY-2 cells. https://hdl.handle.net/2078.5/245641