DNA base editing in nuclear and organellar genomes.

Genome editing continues to revolutionize biological research. Due to its simplicity and flexibility, CRISPR/Cas-based editing has become the preferred technology in most systems. Cas nucleases tolerate fusion to large protein domains, thus allowing combination of their DNA recognition properties with new enzymatic activities. Fusion to nucleoside deaminase or reverse transcriptase domains has produced base editors and prime editors that, instead of generating double-strand breaks in the target sequence, induce site-specific alterations of single (or a few adjacent) nucleotides. The availability of protein-only genome editing reagents based on transcription activator-like effectors has enabled the extension of base editing to the genomes of chloroplasts and mitochondria. In this review, we summarize currently available base editing methods for nuclear and organellar genomes. We highlight recent advances with improving precision, specificity, and efficiency and discuss current limitations and future challenges. We also provide a brief overview of applications in agricultural biotechnology and gene therapy.

Mitochondrial ferredoxin-like is essential for forming complex I-containing supercomplexes in Arabidopsis.

In eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of 5 multiprotein complexes (complexes I-V). Analyses of the OXPHOS system by native gel electrophoresis have revealed an organization of OXPHOS complexes into supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mitochondrial ferredoxin-like, mFDX-like). This protein was previously found to be part of the bridge domain linking the matrix and membrane arms of the complex I. Phylogenetic analysis suggested that the Arabidopsis (Arabidopsis thaliana) mFDX-like evolved from classical mitochondrial ferredoxins (mFDXs) but lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster, supposedly essential for the electron transfer function of FDXs. Accordingly, our biochemical study showed that AtmFDX-like does not bind an Fe-S cluster and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9-based strategy. These lines did not show any abnormal phenotype under standard growth conditions. However, the characterization of the OXPHOS system demonstrated that mFDX-like is important for the assembly of complex I and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III2 to form supercomplexes.

Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis.

Shoot regeneration can be achieved in vitro through a two-step process involving the acquisition of pluripotency on callus-induction media (CIM) and the formation of shoots on shoot-induction media. Although the induction of root-meristem genes in callus has been noted recently, the mechanisms underlying their induction and their roles in de novo shoot regeneration remain unanswered. Here, we show that the histone acetyltransferase HAG1/AtGCN5 is essential for de novo shoot regeneration. In developing callus, it catalyzes histone acetylation at several root-meristem gene loci including WOX5, WOX14, SCR, PLT1, and PLT2, providing an epigenetic platform for their transcriptional activation. In turn, we demonstrate that the transcription factors encoded by these loci act as key potency factors conferring regeneration potential to callus and establishing competence for de novo shoot regeneration. Thus, our study uncovers key epigenetic and potency factors regulating plant-cell pluripotency. These factors might be useful in reprogramming lineage-specified plant cells to pluripotency.

A Comprehensive Toolkit for Inducible, Cell Type-Specific Gene Expression in Arabidopsis.

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell type-specific transactivation in Arabidopsis (Arabidopsis thaliana) based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic pOp promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of transgenic lines termed GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems. When we combined these transgenic lines with effectors under the control of the pOp promoter, we observed tight temporal and spatial control of gene expression. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows for rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible method is suitable for overcoming the limitations of ubiquitous genetic approaches, the outputs of which often are difficult to interpret due to the widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number.

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.

RNA PROCESSING FACTOR2 is required for 5' end processing of nad9 and cox3 mRNAs in mitochondria of Arabidopsis thaliana.

In mitochondria of higher plants, the majority of 5' termini of mature mRNAs are generated posttranscriptionally. To gain insight into this process, we analyzed a natural 5' end polymorphism in the species Arabidopsis thaliana. This genetic approach identified the nuclear gene At1g62670, encoding a pentatricopeptide repeat protein. The functional importance of this mitochondrial restorer of fertility-like protein, designated RNA PROCESSING FACTOR2 (RPF2), is confirmed by the analysis of a respective T-DNA knockout mutant and its functional restoration by in vivo complementation. RPF2 fulfills two functions: it is required for the generation of a distinct 5' terminus of transcripts of subunit 9 of the NADH DEHYDROGENASE complex (nad9) and it determines the efficiency of 5' end formation of the mRNAs for subunit 3 of the CYTOCHROME C OXIDASE (cox3), the latter also being influenced by mitochondrial DNA sequences. Accordingly, recombinant RPF2 protein directly binds to a nad9 mRNA fragment in vitro. Two-dimensional gel electrophoresis and immunodetection analyses reveal that altered 5' processing does not influence accumulation of the nad9 and cox3 polypeptides. In accessions C24, Oystese-1, and Yosemite-0, different inactive RPF2 alleles exist, demonstrating the variability of this gene in Arabidopsis. The identification of RPF2 is a major step toward the characterization of 5' mRNA processing in mitochondria of higher plants.

Targeted knockout of a conserved plant mitochondrial gene by genome editing.

Fusion proteins derived from transcription activator-like effectors (TALEs) have emerged as genome editing tools for mitochondria. TALE nucleases (TALENs) have been applied to delete chimaeric reading frames and duplicated (redundant) genes but produced complex genomic rearrangements due to the absence of non-homologous end-joining. Here we report the targeted deletion of a conserved mitochondrial gene, nad9, encoding a subunit of respiratory complex I. By generating a large number of TALEN-mediated mitochondrial deletion lines, we isolated, in addition to mutants with rearranged genomes, homochondriomic mutants harbouring clean nad9 deletions. Characterization of the knockout plants revealed impaired complex I biogenesis, male sterility and defects in leaf and flower development. We show that these defects can be restored by expressing a functional Nad9 protein from the nuclear genome, thus creating a synthetic cytoplasmic male sterility system. Our data (1) demonstrate the feasibility of using genome editing to study mitochondrial gene functions by reverse genetics, (2) highlight the role of complex I in plant development and (3) provide proof-of-concept for the construction of synthetic cytoplasmic male sterility systems for hybrid breeding by genome editing.

A RESTORER OF FERTILITY-like PPR gene is required for 5'-end processing of the nad4 mRNA in mitochondria of Arabidopsis thaliana.

Processing of 5'-ends is a frequently observed step during maturation of plant mitochondrial mRNAs. Up to now, very little is known about the biochemistry of this process and the proteins involved in the removal of 5' leader sequences. Based on natural genetic variation we have used linkage mapping and complementation studies to identify a nuclear gene required for the efficient generation of a 5'-end 228 nucleotides upstream of the mitochondrial nad4 gene encoding subunit 4 of the NADH dehydrogenase complex. This nuclear gene, At1g12700, that we designate RNA PROCESSING FACTOR 1 (RPF1), encodes a pentatricopeptide repeat (PPR) protein of the P-class containing canonical PPR-repeats. RPF1 belongs to a subgroup of PPR proteins, which includes the RESTORER OF FERTILITY (RF) gene products restoring cytoplasmic male sterility (CMS) in various plant species. CMS is a mitochondrially inherited trait caused by the expression of aberrant, chimeric genes, which has not been observed in the predominantly inbreeding species Arabidopsis thaliana. The here reported results are a further step towards the characterization of the plant mitochondrial RNA processing machinery and provide additional insights into the function of RF-like PPR proteins.

RNA PROCESSING FACTOR3 is crucial for the accumulation of mature ccmC transcripts in mitochondria of Arabidopsis accession Columbia.

RNA PROCESSING FACTOR1 (RPF1) and RPF2 are pentatricopeptide repeat (PPR) proteins involved in 5' processing of different mitochondrial mRNAs in Arabidopsis (Arabidopsis thaliana). Both factors are highly similar to RESTORERS OF FERTILITY (RF), which are part of cytoplasmic male sterility/restoration systems in various plant species. These findings suggest a predominant role of RF-like PPR proteins in posttranscriptional 5' processing. To further explore the functions of this group of proteins, we examined a number of T-DNA lines carrying insertions in the corresponding PPR genes. This screening identified a nearly complete absence of mature ccmC transcripts in an At1g62930 T-DNA insertion line, a phenotype that could be restored by the introduction of the intact At1g62930 gene into the mutant. The insertion in this nuclear gene, which we now call RPF3, also leads to a severe reduction of the CcmC protein in mitochondria. The analysis of C24/rpf3-1 F2 hybrids lacking functional RPF3 genes revealed that this gene has less influence on the generation of the mature ccmC 5' transcript end derived from a distinct ccmC 5' upstream configuration found in mitochondrial DNAs from C24 and other accessions. These data show that a particular function of an RF-like protein is required only in connection with a distinct mtDNA configuration. Our new results further substantiate the fundamental role of RF-like PPR proteins in the posttranscriptional generation of plant mitochondrial 5' transcript termini.

Targeted introduction of heritable point mutations into the plant mitochondrial genome.

The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.

Expression strategies for the efficient synthesis of antimicrobial peptides in plastids.

Antimicrobial peptides (AMPs) kill microbes or inhibit their growth and are promising next-generation antibiotics. Harnessing their full potential as antimicrobial agents will require methods for cost-effective large-scale production and purification. Here, we explore the possibility to exploit the high protein synthesis capacity of the chloroplast to produce AMPs in plants. Generating a large series of 29 sets of transplastomic tobacco plants expressing nine different AMPs as fusion proteins, we show that high-level constitutive AMP expression results in deleterious plant phenotypes. However, by utilizing inducible expression and fusions to the cleavable carrier protein SUMO, the cytotoxic effects of AMPs and fused AMPs are alleviated and plants with wild-type-like phenotypes are obtained. Importantly, purified AMP fusion proteins display antimicrobial activity independently of proteolytic removal of the carrier. Our work provides expression strategies for the synthesis of toxic polypeptides in chloroplasts, and establishes transplastomic plants as efficient production platform for antimicrobial peptides.

Germline-Transmitted Genome Editing Methodology in Arabidopsis thaliana Using TAL Effector Nucleases.

TAL effector nucleases (TALENs) are powerful tools to create specific knockout mutants in plants. The use of an optimized TALEN backbone and the choice of promoters that are strongly active in the stem cells of the shoot apical meristem are key to a high rate of heritable targeted mutations. Recommendations for construct design and screening for mutants are given in this chapter.

WUSCHEL triggers innate antiviral immunity in plant stem cells.

Stem cells in plants constantly supply daughter cells to form new organs and are expected to safeguard the integrity of the cells from biological invasion. Here, we show how stem cells of the Arabidopsis shoot apical meristem and their nascent daughter cells suppress infection by cucumber mosaic virus (CMV). The stem cell regulator WUSCHEL responds to CMV infection and represses virus accumulation in the meristem central and peripheral zones. WUSCHEL inhibits viral protein synthesis by repressing the expression of plant S-adenosyl-l-methionine-dependent methyltransferases, which are involved in ribosomal RNA processing and ribosome stability. Our results reveal a conserved strategy in plants to protect stem cells against viral intrusion and provide a molecular basis for WUSCHEL-mediated broad-spectrum innate antiviral immunity in plants.

Mapping of mitochondrial mRNA termini in Arabidopsis thaliana: t-elements contribute to 5' and 3' end formation.

With CR-RT-PCR as primary approach we mapped the 5' and 3' transcript ends of all mitochondrial protein-coding genes in Arabidopsis thaliana. Almost all transcripts analyzed have single major 3' termini, while multiple 5' ends were found for several genes. Some of the identified 5' ends map within promoter motifs suggesting these ends to be derived from transcription initiation while the majority of the 5' termini seems to be generated post-transcriptionally. Assignment of the extremities of 5' leader RNAs revealed clear evidence for an endonucleolytic generation of the major cox1 and atp9 5' mRNA ends. tRNA-like structures, so-called t-elements, are associated either with 5' or with 3' termini of several mRNAs. These secondary structures most likely act as cis-signals for endonucleolytic cleavages by RNase Z and/or RNase P. Since no conserved sequence motif is evident at post-transcriptionally derived ends, we suggest t-elements, stem-loops and probably complex higher order structures as cis-elements for processing. This analysis provides novel insights into 5' and 3' end formation of mRNAs. In addition, the complete transcript map is a substantial and important basis for future studies of gene expression in mitochondria of higher plants.

High-efficiency generation of fertile transplastomic Arabidopsis plants.

The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here, we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as a source tissue for biolistic transformation. The method produces fertile transplastomic plants at high frequency when combined with a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-generated knockout allele of a nuclear locus that enhances sensitivity to the selection agent used for isolation of transplastomic events. Our work makes the model organism of plant biology amenable to routine engineering of the plastid genome, facilitates the combination of plastid engineering with the power of Arabidopsis nuclear genetics, and informs the future development of plastid transformation protocols for other recalcitrant species.

WUSCHEL acts as an auxin response rheostat to maintain apical stem cells in Arabidopsis.

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.

The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants.

BACKGROUND: Intrinsically fluorescent proteins have revolutionized studies in molecular cell biology. The parallel application of these proteins in dual- or multilabeling experiments such as subcellular localization studies requires non-overlapping emission spectra for unambiguous detection of each label. In the red spectral range, almost exclusively DsRed and derivatives thereof are used today. To test the suitability of the red fluorescent protein eqFP611 as an alternative in higher plants, the behavior of this protein was analyzed in terms of expression, subcellular targeting and compatibility with GFP in tobacco. RESULTS: When expressed transiently in tobacco protoplasts, eqFP611 accumulated over night to levels easily detectable by fluorescence microscopy. The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed. When fused to N-terminal mitochondrial and peroxisomal targeting sequences, the red fluorescence was located exclusively in the corresponding organelles in transfected protoplasts. Upon co-expression with GFP in the same cells, fluorescence of both eqFP611 and GFP could be easily distinguished, demonstrating the potential of eqFP611 in dual-labeling experiments with GFP. A series of plasmids was constructed for expression of eqFP611 in plants and for simultaneous expression of this fluorescent protein together with GFP. Transgenic tobacco plants constitutively expressing mitochondrially targeted eqFP611 were generated. The red fluorescence was stably transmitted to the following generations, making these plants a convenient source for protoplasts containing an internal marker for mitochondria. CONCLUSION: In plants, eqFP611 is a suitable fluorescent reporter protein. The unmodified protein can be expressed to levels easily detectable by epifluorescence microscopy without adverse affect on the viability of plant cells. Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

Transcript end mapping and analysis of RNA editing in plant mitochondria.

Mitochondria are genetic compartments with their own enzymatic equipment for maintenance and expression of their genetic information. As in all genetic systems, gene expression has to be regulated, and in mitochondria this also has to be coordinated with the expression of nuclear-encoded mitochondrial proteins. Presently, there is virtually no information available about the mechanistic details and the enzymes involved in these processes. There is still much to be learned about how plant mitochondrial gene expression is managed and to what extent the contribution of transcription initiation and posttranscriptional processes, respectively, contribute to this control. As one prerequisite for better understanding of the mechanisms and regulatory controls, more fundamental data on mitochondrial transcription initiation and posttranscriptional RNA processing are necessary. As part of the essential methodology, we present methods for the analysis of the 5' and 3' extremities of mitochondrial transcripts and the identification of transcription initiation sites. An in organello system is described for the functional investigation of ribonucleic acid editing in plant mitochondria.

Distant sequences determine 5' end formation of cox3 transcripts in Arabidopsis thaliana ecotype C24.

The genomic environments and the transcripts of the mitochondrial cox3 gene are investigated in three Arabidopsis thaliana ecotypes. While the proximate 5' sequences up to nucleotide position -584, the coding regions and the 3' flanking regions are identical in Columbia (Col), C24 and Landsberg erecta (Ler), genomic variation is detected in regions further upstream. In the mitochondrial DNA of Col, a 1790 bp fragment flanked by a nonanucleotide direct repeat is present beyond position -584 with respect to the ATG. While in Ler only part of this insertion is conserved, this sequence is completely absent in C24, except for a single copy of the nonanucleotide direct repeat. Northern hybridization reveals identical major transcripts in the three ecotypes, but identifies an additional abundant 60 nt larger mRNA species in C24. The extremities of the most abundant mRNA species are identical in the three ecotypes. In C24, an extra major 5' end is abundant. This terminus and the other major 5' ends are located in identical sequence regions. Inspection of Atcox3 transcripts in C24/Col hybrids revealed a female inheritance of the mRNA species with the extra 5' terminus. Thus, a mitochondrially encoded factor determines the generation of an extra 5' mRNA end.

Control of plant cell fate transitions by transcriptional and hormonal signals.

Plant meristems carry pools of continuously active stem cells, whose activity is controlled by developmental and environmental signals. After stem cell division, daughter cells that exit the stem cell domain acquire transit amplifying cell identity before they are incorporated into organs and differentiate. In this study, we used an integrated approach to elucidate the role of HECATE (HEC) genes in regulating developmental trajectories of shoot stem cells in Arabidopsis thaliana. Our work reveals that HEC function stabilizes cell fate in distinct zones of the shoot meristem thereby controlling the spatio-temporal dynamics of stem cell differentiation. Importantly, this activity is concomitant with the local modulation of cellular responses to cytokinin and auxin, two key phytohormones regulating cell behaviour. Mechanistically, we show that HEC factors transcriptionally control and physically interact with MONOPTEROS (MP), a key regulator of auxin signalling, and modulate the autocatalytic stabilization of auxin signalling output.