Engineered PPR proteins as inducible switches to activate the expression of chloroplast transgenes

Rojas, Margarita; Yu, Qiguo; Williams‐Carrier, Rosalind; Maliga, Pál; Barkan, Alice

doi:10.1038/s41477-019-0412-1

Cited by 60 publications

(62 citation statements)

References 54 publications

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“…Given that phylogenetically close crosses (HORT × FRUT, HORT × ZONA, HORT × ACET) require the one, two, or three gene model with the assumption of lethality to explain the phenotypic ratios for both the seedling and seed phase we evaluated, we may just be viewing the tip of the iceberg for the phylogenetically more distant crosses. Generally, genes thought to be involved in chloroplast management and expression are Whirly genes (Maréchal et al, 2009;Isemer et al, 2012;Krupinska et al, 2014Krupinska et al, , 2019, involved in importing proteins into chloroplasts (Krausea et al, 2005;Chateigner-Boutin et al, 2008;Mackenzie and Kundariya, 2019), and PPR genes, acting at the level of RNA editing (Takenaka et al, 2013;Wang et al, 2016a,b;Rojas et al, 2019;Small et al, 2020). These genes are good candidates to study in Pelargonium and a closer study of the proteins they encode for as well as the type of RNA editing taking place, may explain both biparental inheritance as well as early stage processes of speciation.…”

Section: Model Of > 3 Interacting Nuclear Genesmentioning

confidence: 99%

Interspecific Hybrids Between Pelargonium × hortorum and Species From P. Section Ciconium Reveal Biparental Plastid Inheritance and Multi-Locus Cyto-Nuclear Incompatibility

et al. 2020

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The genetics underlying Cyto-Nuclear Incompatibility (CNI) was studied in Pelargonium interspecific hybrids. We created hybrids of 12 closely related crop wild relatives (CWR) with the ornamental P. × hortorum. Ten of the resulting 12 (F1) interspecific hybrids segregate for chlorosis suggesting biparental plastid inheritance. The segregation ratios of the interspecific F2 populations show nuclear interactions of one, two, or three nuclear genes regulating plastid function dependent on the parents. We further validated that biparental inheritance of plastids is common in section Ciconium, using diagnostic PCR primers. Our results pave the way for using the diverse species from section Ciconium, each with its own set of characteristics, as novel sources of desired breeding traits for P. × hortorum cultivars.

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Section: Model Of > 3 Interacting Nuclear Genesmentioning

confidence: 99%

Interspecific Hybrids Between Pelargonium × hortorum and Species From P. Section Ciconium Reveal Biparental Plastid Inheritance and Multi-Locus Cyto-Nuclear Incompatibility

et al. 2020

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“…DNA/RNA engineering tools based on TALEs or PUFs, such as artificial nucleases, have already been developed [39,40]. In a recent study, Rojas et al [41] successfully engineered a P-class PPR10 to activate the expression of chloroplast transgene. In the same way, PLS-class PPR proteins could be engineered and modified to edit any cytidine in any transcript.…”

Section: Plos Onementioning

confidence: 99%

The L motifs of two moss pentatricopeptide repeat proteins are involved in RNA editing but predominantly not in RNA recognition

2020

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Pentatricopeptide repeat (PPR) proteins, composed of PPR motifs repeated in tandem, are sequence-specific RNA binding proteins. Recent bioinformatic studies have shown that the combination of polar amino acids at positions 5 and last in each PPR motif recognizes RNA bases, and an RNA recognition code for PPR proteins has been proposed. Subsequent studies confirmed that the P (canonical length) and S (short) motifs bind to specific nucleotides according to this code. However, the contribution of L (long) motifs to RNA recognition is mostly controversial, owing to the presence of a nonpolar amino acid at position 5. The PLS-class PPR protein PpPPR_56 is a mitochondrial RNA editing factor in the moss Physcomitrella patens. Here, we performed in vitro RNA binding and in vivo complementation assays with PpPPR_56 and its variants containing mutated L motifs to investigate their contributions to RNA recognition. In vitro RNA binding assay showed that the original combination of amino acids at positions 5 and last in the L motifs of PpPPR_56 is not required for RNA recognition. In addition, an in vivo complementation assay with RNA editing factors PpPPR_56 and PpPPR_78 revealed the importance of nonpolar amino acids at position 5 of C-terminal L motifs for efficient RNA editing. Our findings suggest that L motifs function as non-binding spacers, not as RNA-binding motifs, to facilitate the formation of a complex between PLS-class PPR protein and RNA. As a result, the DYW domain, a putative catalytic deaminase responsible for C-to-U RNA editing, is correctly placed in proximity to C, which is to be edited.

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“…The PPR code has been used to recode several natural PPR proteins to bind nonnative RNA ligands in vitro (Barkan et al, 2012) and in vivo (Kindgren et al, 2015;Colas des Francs-Small et al, 2018;Rojas et al, 2019). However, the engineering of native PPR proteins is complicated by irregularities in their PPR tracts, which result in variable and unpredictable contributions of their PPR motifs to RNA affinity and specificity (Fujii et al, 2013;Okuda et al, 2014;Miranda et al, 2017;Rojas et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Ribonucleoprotein Capture by in Vivo Expression of a Designer Pentatricopeptide Repeat Protein in Arabidopsis

et al. 2019

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Pentatricopeptide repeat (PPR) proteins bind RNA via a mechanism that facilitates the customization of sequence specificity. However, natural PPR proteins have irregular features that limit the degree to which their specificity can be predicted and customized. We demonstrate here that artificial PPR proteins built from consensus PPR motifs selectively bind the intended RNA in vivo, and we use this property to develop a new tool for ribonucleoprotein characterization. We show by RNA coimmunoprecipitation sequencing (RIP-seq) that artificial PPR proteins designed to bind the Arabidopsis (Arabidopsis thaliana) chloroplast psbA mRNA bind with high specificity to psbA mRNA in vivo. Analysis of coimmunoprecipitating proteins by mass spectrometry showed the psbA translational activator HCF173 and two RNA binding proteins of unknown function (CP33C and SRRP1) to be highly enriched. RIP-seq revealed that these proteins are bound primarily to psbA RNA in vivo, and precise mapping of the HCF173 and CP33C binding sites placed them in different locations on psbA mRNA. These results demonstrate that artificial PPR proteins can be tailored to bind specific endogenous RNAs in vivo, add to the toolkit for characterizing native ribonucleoproteins, and open the door to other applications that rely on the ability to target a protein to a specified RNA sequence.

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Engineered PPR proteins as inducible switches to activate the expression of chloroplast transgenes

Cited by 60 publications

References 54 publications

Interspecific Hybrids Between Pelargonium × hortorum and Species From P. Section Ciconium Reveal Biparental Plastid Inheritance and Multi-Locus Cyto-Nuclear Incompatibility

Interspecific Hybrids Between Pelargonium × hortorum and Species From P. Section Ciconium Reveal Biparental Plastid Inheritance and Multi-Locus Cyto-Nuclear Incompatibility

The L motifs of two moss pentatricopeptide repeat proteins are involved in RNA editing but predominantly not in RNA recognition

Ribonucleoprotein Capture by in Vivo Expression of a Designer Pentatricopeptide Repeat Protein in Arabidopsis

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