Low dose ribosomal DNA P-loop mutation affects development and enforces autophagy in Arabidopsis

To investigate the ribosomal RNA subpopulation types in a mutant created by CRISPR-Cas9 editing of rDNA copies in Arabidopsis thaliana / Original Abstract: Arabidopsis contains hundreds of ribosomal DNA copies organised within the nucleolar organising regions (NORs) in chromosomes 2 and 4. There are four major types of variants of rDNA, VAR1-4, based on the polymorphisms of 3' external transcribed sequences. The variants are differentially expressed during plant development. We created a mutant by the CRISPR-Cas9-mediated excision of ~25 nt from only the copies of NOR4 ribosomal DNA, obtaining mosaic mutational events on ~5% of all rDNA copies. The excised region consists of P-loop and Helix-82 segments of 25S rRNA. The mutation led to allelic, dosage-dependent defects marked by lateral root inhibition, reduced size, and pointy leaves, all previously observed for defective ribosomal function. The mutation in NOR4 led to dosage compensation from the NOR2 copies, marked by its associated single-nucleotide variants and, thus, resulted in an altered rRNA sub-population. Furthermore, it caused rRNA maturation defects specifically in the minor pathway characterised by 32S pre-rRNA accumulation. The mutated rRNAs were not incorporated into the translating ribosomes, while it induced an elevated autophagic flux, likely related to ribophagy. Comparative rRNA expression profiling of BG wildtype and homozygous ploop KD -/- mutant.