Polymerase IV Plays a Crucial Role in Pollen Development in Capsella

In Arabidopsis thaliana, DNA-dependent RNA polymerase IV (Pol IV) is required for the formation of transposable element (TE)-derived small RNA (sRNA) transcripts. These transcripts are processed by DICER-LIKE3 into 24-nt small interfering RNAs (siRNAs) that guide RNA-directed DNA methylation. In the pollen grain, Pol IV is also required for the accumulation of 21/22-nt epigenetically activated siRNAs (easiRNAs), which likely silence TEs via post-transcriptional mechanisms. Despite this proposed role of Pol IV, its loss of function in Arabidopsis does not cause a discernable pollen defect. Here, we show that the knockout of NRPD1, encoding the largest subunit of Pol IV in the Brassicaceae species Capsella rubella, caused post-meiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were 2 depleted in Capsella nrpd1 microspores. In the wild-type background, the same TEs produced 21/22-nt and 24-nt siRNAs; these processes required Pol IV activity. Arrest of Capsella nrpd1 microspores was accompanied by the deregulation of genes targeted by Pol IV-dependent siRNAs. TEs were much closer to genes in Capsella rubella compared to Arabidopsis thaliana, perhaps explaining the essential role of Pol IV in pollen development in Capsella. Our discovery that Pol IV is functionally required in Capsella microspores emphasizes the relevance of investigating different plant models. 12 small RNA sequencing libraries, 8 RNA-seq libraries, 4 genome-wide cytosine methylation libraries and 6 whole genome sequencing libraries. 4 small RNA libraries were generated from Arabidopsis microspore of Col-0 wt and nrpd1a mutants. 4 small RNA libraries were generated from Capsella microspore of wt and nrpd1a mutants. 4 small RNA libraries were generated from Capsella leaves of wt and nrpd1a mutants. 4 RNA libraries were generated from Arabidopsis microspore of Col-0 wt and nrpd1a mutants. 4 RNA libraries were generated from Capsella microspore of wt and nrpd1a mutants. 4 genome-wide cytosine methylation libraries were generated from Capsella leaves of wt and nrpd1a mutants. 6 whole genome sequencing libraries were generated from Capsella leaves of wt and nrpd1a mutants.