Establishment of a Heterologous RNA Editing Event in Chloroplasts

Abstract

ORCID IDs: 0000-0001-8210-1216 (F.V.L.); 0000-0003-3210-0260 (M.A.S.); 0000-0001-7502-6940 (R.B.).In chloroplasts and plant mitochondria, specific cytidines in mRNAs are posttranscriptionally converted to uridines by RNA editing. Editing sites are recognized by nucleus-encoded RNA-binding proteins of the pentatricopeptide repeat (PPR) family, which bind upstream of the editing site in a sequence-specific manner and direct the editing activity to the target position. Editing sites have been lost many times during evolution by C-to-T mutations. Loss of an editing site is thought to be accompanied by loss or degeneration of its cognate PPR protein. Consequently, foreign editing sites are usually not recognized when introduced into species lacking the site. Previously, the spinach (Spinacia oleracea) psbF-26 editing site was introduced into the tobacco (Nicotiana tabacum) plastid genome. Tobacco lacks the psbF-26 site and cannot edit it. Expression of the "unedited" PsbF protein resulted in impaired PSII function. In Arabidopsis (Arabidopsis thaliana), the PPR protein LPA66 is required for editing at psbF-26. Here, we show that introduction of the Arabidopsis LPA66 reconstitutes editing of the spinach psbF-26 site in tobacco and restores a wild-type-like phenotype. Our findings define the minimum requirements for establishing new RNA editing sites and suggest that the evolutionary dynamics of editing patterns is largely explained by coevolution of editing sites and PPR proteins.

Advancing organelle genome transformation and editing for crop improvement

“…For example, when the spinach psbF- 26 editing site was introduced into the psbF gene of tobacco, which naturally lacks a corresponding editing site, tobacco plastids were incapable of editing the spinach site, resulting in strongly impaired PSII function ( Bock et al., 1994 ). Interestingly, introduction of the Arabidopsis DYW-type PPR editing factor LPA66 was sufficient to reconstitute editing of the spinach psbF-26 site in tobacco chloroplasts and rescued the PSII-deficient phenotype caused by the unedited psbF -26 site ( Loiacono et al., 2019 ). These results demonstrate that although additional non-PPR trans -acting factors also contribute to editing reactions ( Bentolila et al., 2012 ; Hackett et al., 2017 ), new RNA editing sites can be established through introduction of the co-evolving editing site and its corresponding PPR protein.…”

Neither the availability of D2 nor CP43 limits the biogenesis of PSII in tobacco

“…These mutants are referred to as "antisense" (as)-mutants. Because insertion of the selectable marker in antisense orientation may negatively affect the expression of neighbouring genes transcribed in the opposite direction (Loiacono et al, 2019), transcript accumulation of psbD could be strongly reduced in all as-mutants. Additionally, to alter translation initiation efficiency, we introduced point mutations into the translation initiation codon.…”

Genetic and Molecular Genetic Basis of Nuclear-Plastid Incompatibilities

“…Thus, the identified mutation did not change the amino acid sequence of the AtpA protein but abolished the need for RNA editing. RNA-editing factors are known to be encoded in the nucleus [84,85], and apparently belladonna genome lacks the factor which specifically edits the cytosine in the 264-th codon of the atpA gene, resulting in the amino acid replacement of leucine by proline L264P [82].…”