Published December 12, 2019In eukaryotes, RNA and chromatin-based pathways control transposable elements (TE) to minimize the deleterious consequences of genetic invasion, transposition, mutation and chromosome instability (Matzke and Mosher, 2014). In higher plants, the multi-subunit nuclear RNA polymerase IV (Pol IV) specializes in transcribing the 24 nucleotide class of small RNAs that target TE’s for DNA cytosine methylation and silencing in the RNA-directed DNA methylation (RdDM) pathway (Herr et al., 2005; Onodera et al., 2005). In Arabidopsis, the Pol IV has two alternative subunits encoded by the NRPD1a and NRPD1b and a common subunit encoded by NRPD2A (Herr et al., 2005; Kanno et al., 2005; Onodera et al., 2005; Pontier et al., 2005). The null mutant for NRPD1a is defective in the RdDM pathway and also displays a late flowering phenotype under short day conditions (Pontier et al., 2005; Eamens et al., 2008).
Interestingly, we observed a root hair defective phenotype in the nrpd1a-3 mutant allele which has not been previously reported (Fig. 1A). When grown on vertically oriented agar medium, nrpd1a-3 seedlings lacked root hairs or defective root hair elongation when compared to the Col-0 wild type control which showed normal root hair distribution and length in the root maturation zone (Fig. 1A). Further investigation of other NRPD1A mutant alleles, nrpd1a-1, nrpd1a-2, in the C-24 accession, and nrpd1a-4 in the Col-0 accession revealed that these alleles had a similar root hair number to the wild type controls (Fig. 1B). To investigate if the root hair defective phenotype in nrpd1a-3 was a spurious event in our laboratory’s seed stock or an earlier event, we re-ordered the same mutant from the ABRC stock Centre and tested the root hair phenotype. We observed the same root hair defective phenotype in both nrpd1a-3 seed stocks. We next crossed the nrpd1a-3 mutant (Col-0) to wild type accession C-24, self-fertilized the F1to create a F2mapping population and mapped polymorphic DNA markers across the 5 chromosomes revealed the gene for the root hair defective phenotype was located between DNA markers ciw3 and F26B6 on chromosome 1 (Berendzen et al., 2005). Next we whole-genome sequenced DNA from both Col-0 and nrpd1a-3 using Illumina short read technology, and after GATK (McKenna et al., 2010) and DELLY (Rausch et al., 2012) analysis of the annotated gene models in the genetic window, we identified only one nucleotide mutation, a 1,310 bp deletion in Root Hair Defective 6 (RHD6), in nrpd1a-3 (Fig. 1C). RHD6 is a bHLH transcription factor that positively regulates root hair initiation (Masucci and Schiefelbein, 1994) and loss of function mutations cause a root hair defective phenotype. Semi-quantitative RT-PCR showed RHD6 mRNA was undetectable in the nrpd1a-3 roots when compared to Col-0 wild-type and nrpd1a-4 suggesting that the loss of RHD6 was the likely candidate for the root hair defective phenotype observed in the nrpd1a-3 mutant (Fig. 1D). We confirmed the 1,310 bp deletion that deleted part of exon 2, all of exons 3-5 and part of the 3’ UTR by PCR and Sanger sequencing. Together the genetic mapping and the undetectable RHD6 mRNA transcript strongly suggests that the root hair defective phenotype only observed in the nrpd1a-3 mutant background is caused by the deletion in RHD6. In the Arabidopsis research community, sometimes phenotypes caused by an unlinked mutation to a gene of interest have been incorrectly associated in the research field for many years (Enders et al., 2015; Habets and Offringa, 2015), and so our discovery of a deletion in RHD6 in nrpd1a-3 will allow the community to not incorrectly associate the defective root hair phenotype with POLIV function.Rakesh David, R. Daniel Kortschak, and Iain Searl
