Editing of ORF138 restores fertility of Ogura cytoplasmic male sterile broccoli via mitoTALENs.

Cytoplasmic male sterility (CMS), encoded by the mitochondrial open reading frames (ORFs), has long been used to economically produce crop hybrids. However, the utilization of CMS also hinders the exploitation of sterility and fertility variation in the absence of a restorer line, which in turn narrows the genetic background and reduces biodiversity. Here, we used a mitochondrial targeted transcription activator-like effector nuclease (mitoTALENs) to knock out ORF138 from the Ogura CMS broccoli hybrid. The knockout was confirmed by the amplification and re-sequencing read mapping to the mitochondrial genome. As a result, knockout of ORF138 restored the fertility of the CMS hybrid, and simultaneously manifested a cold-sensitive male sterility. ORF138 depletion is stably inherited to the next generation, allowing for direct use in the breeding process. In addition, we proposed a highly reliable and cost-effective toolkit to accelerate the life cycle of fertile lines from CMS-derived broccoli hybrids. By applying the k-mean clustering and interaction network analysis, we identified the central gene networks involved in the fertility restoration and cold-sensitive male sterility. Our study enables mitochondrial genome editing via mitoTALENs in Brassicaceae vegetable crops and provides evidence that the sex production machinery and its temperature-responsive ability are regulated by the mitochondria.

Mitochondrial DNA editing in potato through mitoTALEN and mitoTALECD: molecular characterization and stability of editing events.

BACKGROUND: The aim of this study was to evaluate and characterize the mutations induced by two TALE-based approaches, double-strand break (DSB) induction by the FokI nuclease (mitoTALEN) and targeted base editing by the DddA cytidine deaminase (mitoTALECD), to edit, for the first time, the mitochondrial genome of potato, a vegetatively propagated crop. The two methods were used to knock out the same mitochondrial target sequence (orf125). RESULTS: Targeted chondriome deletions of different sizes (236-1066 bp) were induced by mitoTALEN due to DSB repair through ectopic homologous recombination of short direct repeats (11-12 bp) present in the target region. Furthermore, in one case, the induced DSB and subsequent repair resulted in the amplification of an already present substoichiometric molecule showing a 4288 bp deletion spanning the target sequence. With the mitoTALECD approach, both nonsense and missense mutations could be induced by base substitution. The deletions and single nucleotide mutations were either homoplasmic or heteroplasmic. The former were stably inherited in vegetative offspring. CONCLUSIONS: Both editing approaches allowed us to obtain plants with precisely modified mitochondrial genomes at high frequency. The use of the same plant genotype and mtDNA region allowed us to compare the two methods for efficiency, accuracy, type of modifications induced and stability after vegetative propagation.

Genome Editing of Plant Mitochondrial and Chloroplast Genomes.

Plastids (including chloroplasts) and mitochondria are remnants of endosymbiotic bacteria, yet they maintain their own genomes, which encode vital components for photosynthesis and respiration, respectively. Organellar genomes have distinctive features, such as being present as multicopies, being mostly inherited maternally, having characteristic genomic structures and undergoing frequent homologous recombination. To date, it has proven to be challenging to modify these genomes. For example, while CRISPR/Cas9 is a widely used system for editing nuclear genes, it has not yet been successfully applied to organellar genomes. Recently, however, precise gene-editing technologies have been successfully applied to organellar genomes. Protein-based enzymes, especially transcription activator-like effector nucleases (TALENs) and artificial enzymes utilizing DNA-binding domains of TALENs (TALEs), have been successfully used to modify these genomes by harnessing organellar-targeting signals. This short review introduces and discusses the use of targeted nucleases and base editors in organellar genomes, their effects and their potential applications in plant science and breeding.

Targeted A-to-G base editing in the organellar genomes of Arabidopsis with monomeric programmable deaminases.

Plastids and mitochondria are 2 intracellular organelles containing DNA-encoding partial but essential components for their roles, photosynthesis, and respiration. Precise base editing in both plastid and mitochondrial genomes would benefit their gene functional analysis and crop breeding. Targeted base editing in organellar genomes relies on a protein-based genome-editing system that uses the TALE-DNA recognition motif with deaminases. This is because the efficient delivery of guide RNA for clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems into organelles is currently impossible. Since TALE-based base editors used in organellar genomes are usually dimeric types, in this study, we used targeted A-to-G base editing in Arabidopsis (Arabidopsis thaliana) plastid and mitochondrial genomes with monomeric TALE-based deaminase for easier assembling of vectors. As a result, inheritable targeted A-to-G base editing of adenosine triphosphatase subunit 6-2 (atp6-2) in plant mitochondrial genomes and of 16S ribosomal RNA (16S rRNA) in plastid genomes of Arabidopsis was successfully induced by monomeric TALE-based adenine deaminase (AD) without off-target mutations. The monomeric TALE-based adenine deaminases also demonstrated a preference for editing the 8th T on the same strand from the recognition end. Phenotypic analysis showed that A-to-G conversion at 1139A of plastid 16S rRNA conferred substantial spectinomycin resistance in Arabidopsis, but not the other 2 potential-resistant mutations at 1131T and 1137T, predicted from the previous bacterial data. Our study demonstrated the feasibility of monomeric TALE-based ADs in plant organelles and their potential contribution to the functional analyses of plant organelles with easier assembling.

Characterization and development of a plastid genome base editor, ptpTALECD.

The modification of photosynthesis-related genes in plastid genomes may improve crop yields. Recently, we reported that a plastid-targeting base editor named ptpTALECD, in which a cytidine deaminase DddA functions as the catalytic domain, can homoplasmically substitute a targeted C to T in plastid genomes of Arabidopsis thaliana. However, some target Cs were not substituted. In addition, although ptpTALECD could substitute Cs on the 3' side of T and A, it was unclear whether it could also substitute Cs on the 3' side of G and C. In this study, we identified the preferential positions of the substituted Cs in ptpTALECD-targeting sequences in the Arabidopsis plastid genome. We also found that ptpTALECD could substitute Cs on the 3' side of all four bases in plastid genomes of Arabidopsis. More recently, a base editor containing an improved version of DddA (DddA11) was reported to substitute Cs more efficiently, and to substitute Cs on the 3' side of more varieties of bases in human mitochondrial genomes than a base editor containing DddA. Here, we also show that ptpTALECD_v2, in which a modified version of DddA11 functions as the catalytic domain, more frequently substituted Cs than ptpTALECD in the Arabidopsis plastid genome. We also found that ptpTALECD_v2 tended to substitute Cs at more positions than ptpTALECD. Our results reveal that ptpTALECD can cause a greater variety of codon changes and amino acid substitutions than previously thought, and that ptpTALECD and ptpTALECD_v2 are useful tools for the targeted base editing of plastid genomes.

A Method for Precisely Identifying Modifications to Plant Mitochondrial Genomes by mitoTALENs.

The ability to transform plant mitochondrial genomes has many benefits. Although delivery of foreign DNA to mitochondria is presently very difficult, it is now possible to knock out mitochondrial genes using mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs). Such knockouts have been achieved by a genetic transformation of mitoTALENs encoding genes into the nuclear genome. Previous studies have shown that double-strand breaks (DSBs) induced by mitoTALENs are repaired by ectopic homologous recombination. As a result of DNA repair by homologous recombination, a portion of the genome containing the mitoTALEN target site is deleted. The deletion and repair process cause the mitochondrial genome to become more complex. Here, we describe a method for identifying the ectopic homologous recombination events that occur following the repair of double-strand breaks induced by mitoTALENs.

Mitochondrial gene defects in Arabidopsis can broadly affect mitochondrial gene expression through copy number.

How mitochondria regulate the expression of their genes is poorly understood, partly because methods have not been developed for stably transforming mitochondrial genomes. In recent years, the disruption of mitochondrial genes has been achieved in several plant species using mitochondria-localized TALEN (mitoTALEN). In this study, we attempted to disrupt the NADH dehydrogenase subunit7 (NAD7) gene, a subunit of respiratory chain complex I, in Arabidopsis (Arabidopsis thaliana) using the mitoTALEN method. In some of the transformants, disruption of NAD7 was accompanied by severe growth inhibition and lethality, suggesting that NAD7 has an essential function in Arabidopsis. In addition, the mitochondrial genome copy number and overall expression of genes encoding mitochondrial proteins were generally increased by nad7 knockout. Similar increases were also observed in mutants with decreased NAD7 transcripts and with dysfunctions of other mitochondrial respiratory complexes. In these mutants, the expression of nuclear genes involved in mitochondrial translation or protein transport was induced in sync with mitochondrial genes. Mitochondrial genome copy number was also partly regulated by the nuclear stress-responsive factors NAC domain containing protein 17 and Radical cell death 1. These findings suggest the existence of overall gene-expression control through mitochondrial genome copy number in Arabidopsis and that disruption of single mitochondrial genes can have additional broad consequences in both the nuclear and mitochondrial genomes.

The mitochondrial and plastid genomes of Oryza sativa L. cv. Taichung 65.

A highly contiguous mitochondrial and plastid genome sequences of a japonica rice cultivar, Taichung 65, were determined by a hybrid approach with long- and short-read sequences. The assembled mitochondrial genome was 465,453 bases in length with an overall GC content of 43.8%. It was predicted to harbor 62 protein-encoding genes, 16 kinds (33 copies) of transfer RNA, and three kinds (six copies) of ribosomal RNA genes. The mitochondrial genome structure in Taichung 65 is largely the same as that of Nipponbare, but the first ∼9.5 kb sequence in Nipponbare (DQ167400) is replaced with a ∼27 kb sequence duplicated from other parts of the mitochondrial genome. Phylogenetic and sequence polymorphism analysis indicated that Taichung 65 is classified as typical japonica. The assembled plastid genome sequence was 134,551 bases in length and completely identical to the previously reported Nipponbare sequence. These near-complete organelle genome sequences will serve as fundamental resources for investigating alloplasmic cytoplasmic male sterile lines and other organelle-controlled phenomena in rice.

A Wolbachia factor for male killing in lepidopteran insects.

Bacterial symbionts, such as Wolbachia species, can manipulate the sexual development and reproduction of their insect hosts. For example, Wolbachia infection induces male-specific death in the Asian corn borer Ostrinia furnacalis by targeting the host factor Masculinizer (Masc), an essential protein for masculinization and dosage compensation in lepidopteran insects. Here we identify a Wolbachia protein, designated Oscar, which interacts with Masc via its ankyrin repeats. Embryonic expression of Oscar inhibits Masc-induced masculinization and leads to male killing in two lepidopteran insects, O. furnacalis and the silkworm Bombyx mori. Our study identifies a mechanism by which Wolbachia induce male killing of host progeny.

Targeted base editing in the mitochondrial genome of Arabidopsis thaliana.

Beyond their well-known role in respiration, mitochondria of land plants contain biologically essential and/or agriculturally important genes whose function and regulation are not fully understood. Until recently, it has been difficult to analyze these genes or, in the case of crops, to improve their functions, due to a lack of methods for stably modifying plant mitochondrial genomes. In rice, rapeseed, and Arabidopsis thaliana, mitochondria-targeting transcription activator-like effector nucleases (mitoTALENs) have recently been used to disrupt targeted genes in an inheritable and stable manner. However, this technique can also induce large deletions around the targeted sites, as well as cause ectopic homologous recombinations, which can change the sequences and gene order of mitochondrial genomes. Here, we used mitochondria-targeting TALEN-based cytidine deaminase to successfully substitute targeted C:G pairs with T:A pairs in the mitochondrial genomes of plantlets of A. thaliana without causing deletions or changes in genome structure. Expression vectors of the base editor genes were stably introduced into the nuclear genome by the easy-to-use floral dipping method. Some T1 plants had apparent homoplasmic substitutions that were stably inherited by seed progenies, independently of the inheritance of nuclear-introduced genes. As a demonstration of the method, we used it to restore the growth of an organelle transcript processing 87 (otp87) mutant that is defective in the editing of RNA transcripts of the mitochondrial atp1 gene and to identify bases in atp1 that affect the efficiency of RNA editing by OTP87.

TALEN-mediated depletion of the mitochondrial gene orf312 proves that it is a Tadukan-type cytoplasmic male sterility-causative gene in rice.

Cytoplasmic male sterility (CMS) is a trait that causes pollen or anther dysfunctions, resulting in the lack of seed setting. CMS is considered to be caused by the expression of a unique mitochondrial open reading frame referred to as CMS-associated gene. orf312 has been reported as a CMS-associated gene of Tadukan-type CMS (TAA) in rice (Oryza sativa L.), which exhibits impaired anther dehiscence; however, evidence thereof has not yet been reported. Here, we took a loss-of-function approach, using a mitochondria-targeted transcription activator-like effector nuclease (mitoTALEN) designed to knock out orf312 in TAA, to prove that orf312 indeed is a CMS-causative gene. Out of 28 transgenic TAA plants harboring the mitoTALEN expression vector, deletion of orf312 was detected in 24 plants by PCR, Southern blot, and sequencing analyses. The 24 plants were grouped into three groups based on the deleted regions. All orf312-depleted TAA plants exhibited recovery of anther dehiscence and seed setting. The depletion of orf312 and fertility restoration was maintained in the next generation, even in mitoTALEN expression cassette null segregants. In contrast, orf312-retaining plants were sterile. These results provide robust evidence that orf312 is a Tadukan-type CMS-causative gene.

Existence of giant mitochondria-containing sheet structures lacking cristae and matrix in the etiolated cotyledon of Arabidopsis thaliana.

Mitochondria are essential organelles involved in the production and supply of energy in eukaryotic cells. Recently, the use of serial section scanning electron microscopy (S3EM) has allowed accurate three-dimensional (3D) reconstructed images of even complex organelle structures. Using this method, ultrathin sections of etiolated cotyledons were observed 4 days after germination of Arabidopsis thaliana in the dark, and giant mitochondria were found. To exclude the possibility of chemical fixation artifacts, this study confirmed the presence of giant mitochondria in high-pressure frozen samples. The 3D reconstructed giant mitochondria had a complex structure that included not only the elongated region but also the flattened shape of a disk. It contained the characteristic sheet structure, and the sheet lacked cristae and matrix but consisted of outer and inner membranes. Whether this phenomenon could be observed in living cells was investigated using the transformant with mitochondrial matrix expressing green fluorescent protein. Small globular mitochondria observed in light-treated samples were also represented in etiolated cotyledons. Although no giant mitochondria were observed in light-treated samples, they were found in the dark 3 days after germination and rapidly increased in number on the fourth day. Therefore, giant mitochondria were observed only in dark samples. These findings were supported by electron microscopy results.

A glossary of plant cell structures: Current insights and future questions.

In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.

MitoTALENs: A Method for Targeted Gene Disruption in Plant Mitochondrial Genomes.

Modification of plant mitochondrial genomes is still a difficult task, especially in multicellular plants. Transcription activator-like effector nucleases with a mitochondrial localization signal (mitoTALENs) can cut out a desired sequence from the mitochondrial genome in plants. Although vector construction of mitoTALENs is complicated, the modification efficiency is high enough to achieve homoplasmy of multicopy mitochondrial genomes. Here I describe how to design mitoTALENs to select a target, construct a vector, and select the mitochondrial transformants.

Plant genome engineering from lab to field-a Keystone Symposia report.

Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. Enhancing crop resiliency and yield via genome engineering will undoubtedly be a key part of the solution. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering. However, several serious challenges remain in achieving this goal. Among them are efficient transformation and plant regeneration for most crop species, low frequency of some editing applications, and high attrition rates. On March 8 and 9, 2021, experts in plant genome engineering and breeding from academia and industry met virtually for the Keystone eSymposium "Plant Genome Engineering: From Lab to Field" to discuss advances in genome editing tools, plant transformation, plant breeding, and crop trait development, all vital for transferring the benefits of novel technologies to the field.

Targeted base editing in the plastid genome of Arabidopsis thaliana.

Bacterial cytidine deaminase fused to the DNA binding domains of transcription activator-like effector nucleases was recently reported to transiently substitute a targeted C to a T in mitochondrial DNA of mammalian cultured cells1. We applied this system to targeted base editing in the Arabidopsis thaliana plastid genome. The targeted Cs were homoplasmically substituted to Ts in some plantlets of the T1 generation and the mutations were inherited by their offspring independently of their nuclear-introduced vectors.

Investigating the mitochondrial genomic landscape of Arabidopsis thaliana by long-read sequencing.

Plant mitochondrial genomes have distinctive features compared to those of animals; namely, they are large and divergent, with sizes ranging from hundreds of thousands of to a few million bases. Recombination among repetitive regions is thought to produce similar structures that differ slightly, known as "multipartite structures," which contribute to different phenotypes. Although many reference plant mitochondrial genomes represent almost all the genes in mitochondria, the full spectrum of their structures remains largely unknown. The emergence of long-read sequencing technology is expected to yield this landscape; however, many studies aimed to assemble only one representative circular genome, because properly understanding multipartite structures using existing assemblers is not feasible. To elucidate multipartite structures, we leveraged the information in existing reference genomes and classified long reads according to their corresponding structures. We developed a method that exploits two classic algorithms, partial order alignment (POA) and the hidden Markov model (HMM) to construct a sensitive read classifier. This method enables us to represent a set of reads as a POA graph and analyze it using the HMM. We can then calculate the likelihood of a read occurring in a given cluster, resulting in an iterative clustering algorithm. For synthetic data, our proposed method reliably detected one variation site out of 9,000-bp synthetic long reads with a 15% sequencing-error rate and produced accurate clustering. It was also capable of clustering long reads from six very similar sequences containing only slight differences. For real data, we assembled putative multipartite structures of mitochondrial genomes of Arabidopsis thaliana from nine accessions sequenced using PacBio Sequel. The results indicated that there are recurrent and strain-specific structures in A. thaliana mitochondrial genomes.