Witnessing Genome Evolution: Experimental Reconstruction of Endosymbiotic and Horizontal Gene Transfer.

Present day mitochondria and plastids (chloroplasts) evolved from formerly free-living bacteria that were acquired through endosymbiosis more than a billion years ago. Conversion of the bacterial endosymbionts into cell organelles involved the massive translocation of genetic material from the organellar genomes to the nucleus. The development of transformation technologies for organellar genomes has made it possible to reconstruct this endosymbiotic gene transfer in laboratory experiments and study the mechanisms involved. Recently, the horizontal transfer of genetic information between organisms has also become amenable to experimental investigation. It led to the discovery of horizontal genome transfer as an asexual process generating new species and new combinations of nuclear and organellar genomes. This review describes experimental approaches towards studying endosymbiotic and horizontal gene transfer processes, discusses the new knowledge gained from these approaches about both the evolutionary significance of gene transfer and the underlying molecular mechanisms, and highlights exciting possibilities to exploit gene and genome transfer in biotechnology and synthetic biology.

Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops.

Addressing nutritional deficiencies in food crops through biofortification is a sustainable approach to tackling malnutrition. Biofortification is continuously being attempted through conventional breeding as well as through various plant biotechnological interventions, ranging from molecular breeding to genetic engineering and genome editing for enriching crops with various health-promoting metabolites. Genetic engineering is used for the rational incorporation of desired nutritional traits in food crops and predominantly operates through nuclear and chloroplast genome engineering. In the recent past, chloroplast engineering has been deployed as a strategic tool to develop model plants with enhanced nutritional traits due to the various advantages it offers over nuclear genome engineering. However, this approach needs to be extended for the nutritional enhancement of major food crops. Further, this platform could be combined with strategies, such as synthetic biology, chloroplast editing, nanoparticle-mediated rapid chloroplast transformation, and horizontal gene transfer through grafting for targeting endogenous metabolic pathways for overproducing native nutraceuticals, production of biopharmaceuticals, and biosynthesis of designer nutritional compounds. This review focuses on exploring various features of chloroplast genome engineering for nutritional enhancement of food crops by enhancing the levels of existing metabolites, restoring the metabolites lost during crop domestication, and introducing novel metabolites and phytonutrients needed for a healthy daily diet.

'Yes' to mitochondrial replacement techniques and lesbian motherhood: a reply to Françoise Baylis.

In a recent paper - Lesbian motherhood and mitochondrial replacement techniques: reproductive freedom and genetic kinship - we argued that lesbian couples who wish to have children who are genetically related to both of them should be allowed access to mitochondrial replacement techniques (MRTs). Françoise Baylis wrote a reply to our paper -'No' to lesbian motherhood using human nuclear genome transfer- where she challenges our arguments on the use of MRTs by lesbian couples, and on MRTs more generally. In this reply we respond to her claims and further clarify our position.

Peeking at a plant through the holes in the wall - exploring the roles of plasmodesmata.

Plasmodesmata (PD) are membrane-lined pores that connect neighbouring plant cells and allow molecular exchange via the symplast. Past studies have revealed the basic structure of PD, some of the transport mechanisms for molecules through PD, and a variety of physiological processes in which they function. Recently, with the help of newly developed technologies, several exciting new features of PD have been revealed. New PD structures were observed during early formation of PD and between phloem sieve elements and phloem pole pericycle cells in roots. Both observations challenge our current understanding of PD structure and function. Research into novel physiological responses, which are regulated by PD, indicates that we have not yet fully explored the potential contribution of PD to overall plant function. In this Viewpoint article, we summarize some of the recent advances in understanding the structure and function of PD and propose the challenges ahead for the community.

Human Nuclear Genome Transfer (So-Called Mitochondrial Replacement): Clearing the Underbrush.

In this article, I argue that there is no compelling therapeutic 'need' for human nuclear genome transfer (so-called mitochondrial replacement) to prevent mitochondrial diseases caused by mtDNA mutations. At most there is a strong interest in (i.e. 'want' for) this technology on the part of some women and couples at risk of having children with mitochondrial disease, and perhaps also a 'want' on the part of some researchers who see the technology as a useful precedent - one that provides them with 'a quiet way station' in which to refine the micromanipulations techniques essential for other human germline interventions and human cloning. In advance of this argument, I review basic information about mitochondrial disease and novel genetic strategies to prevent the transmission of mutated mitochondria. Next, I address common features of contemporary debates and discussions about so-called mitochondrial replacement. First, I contest the cliché that science-and-(bio)technology is fast outpacing ethics. Second, I dispute the accuracy of the term 'mitochondrial replacement'. Third, I provide a sustained critique of the purported 'need' for genetically-related children. In closing, I call into question the mainly liberal defense of human nuclear genome transfer. I suggest an alternative frame of reference that pays particular attention to issues of social justice. I conclude that our limited resources (time, talent, human eggs, and money) should be carefully expended in pursuit of the common good, which does not include pandering to acquired desires (i.e., wants).

Preventing the transmission of mitochondrial DNA disorders: selecting the good guys or kicking out the bad guys.

Mitochondrial disorders represent the most common group of inborn errors of metabolism. Clinical manifestations can be extremely variable, ranging from single affected tissues to multisystemic syndromes. Maternally inherited mitochondrial DNA (mtDNA) mutations are a frequent cause, affecting about one in 5000 individuals. The expression of mtDNA mutations differs from nuclear gene defects. Mutations are either homoplasmic or heteroplasmic, and in the latter case disease becomes manifest when the mutation load exceeds a tissue-specific threshold. Mutation load can vary between tissues and in time, and often an exact correlation between mutation load and clinical manifestations is lacking. Because of the possible clinical severity, the lack of treatment and the high recurrence risk of affected offspring for female carriers, couples request prevention of transmission of mtDNA mutations. Previously, choices have been limited due to a segregational bottleneck, which makes the mtDNA mutation load in embryos highly variable and the consequences largely unpredictable. However, recently it was shown that preimplantation genetic diagnosis offers a fair chance of unaffected offspring to carriers of heteroplasmic mtDNA mutations. Technically and ethically challenging possibilities, such maternal spindle transfer and pronuclear transfer, are emerging and providing carriers additional prospects of giving birth to a healthy child.

Cross-species microbial genome transfer: a Review.

Synthetic biology combines the disciplines of biology, chemistry, information science, and engineering, and has multiple applications in biomedicine, bioenergy, environmental studies, and other fields. Synthetic genomics is an important area of synthetic biology, and mainly includes genome design, synthesis, assembly, and transfer. Genome transfer technology has played an enormous role in the development of synthetic genomics, allowing the transfer of natural or synthetic genomes into cellular environments where the genome can be easily modified. A more comprehensive understanding of genome transfer technology can help to extend its applications to other microorganisms. Here, we summarize the three host platforms for microbial genome transfer, review the recent advances that have been made in genome transfer technology, and discuss the obstacles and prospects for the development of genome transfer.

Combining Fusion of Cells with CRISPR-Cas9 Editing for the Cloning of Large DNA Fragments or Complete Bacterial Genomes in Yeast.

The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy.

Genome transfer for the prevention of female infertility caused by maternal gene mutation.

Poor oocyte quality is associated with early embryo developmental arrest and infertility. Maternal gene plays crucial roles in the regulation of oocyte maturation, and its mutation is a common cause of female infertility. However, how to improve oocyte quality and develop effective therapy for maternal gene mutation remains elusive. Here, we use Zar1 as an example to assess the feasibility of genome transfer to cure maternal gene mutation-caused female infertility. We first discover that cytoplasmic deficiency primarily leads to Zar1-null embryo developmental arrest by disturbing maternal transcript degradation and minor zygotic genome activation (ZGA) during the maternal-zygotic transition. We next perform genome transfer at the oocyte (spindle transfer or polar body transfer) and zygote (early pronuclear transfer or late pronuclear transfer) stages to validate the feasibility of preventing Zar1 mutation-caused infertility. We finally demonstrate that genome transfer either at the oocyte or at the early pronuclear stage can support normal preimplantation embryo development and produce live offspring. Moreover, those pups grow to adulthood and show normal fertility. Therefore, our findings provide an effective basis of therapies for the treatment of female infertility caused by maternal gene mutation.

Rewriting the human genome, rewriting human rights law? Human rights, human dignity, and human germline modification in the CRISPR era.

In most legal orders, human germline modification is either prohibited or severely restricted. A recurring thought in these legal frameworks is that heritable genome editing would result in practices that are at odds with principles of human rights, such as dignity, justice, and equality. However, now that CRISPR is bringing heritable genome editing within human reach, the question has risen as to whether these human rights bans still make sense. The call is growing louder to lift the ban on heritable genome editing for therapeutic purposes as soon as the technology is safe for introduction in the clinic. This article critically examines these recent proposals from a human rights perspective. First, it examines the question as to how realistic the proposed distinction between the therapeutic and the nontherapeutic uses of human germline modification is in the CRISPR era. Second, it argues that these proposals rely on a one-dimensional understanding of the meaning of human rights for this issue. Finally, it suggests that this one-dimensional understanding paves the way for a regime of self-regulation by the scientific community that leaves little room for public debate on the question as to whether or how human germline modification fits in the long-term aspirations of society.

Direct Transfer of a Mycoplasma mycoides Genome to Yeast Is Enhanced by Removal of the Mycoides Glycerol Uptake Factor Gene glpF.

We previously discovered that intact bacterial chromosomes can be directly transferred to a yeast host cell where they can propagate as centromeric plasmids by fusing bacterial cells with S accharomyces cerevisiae spheroplasts. Inside the host any desired number of genetic changes can be introduced into the yeast centromeric plasmid to produce designer genomes that can be brought to life using a genome transplantation protocol. Earlier research demonstrated that the removal of restriction-systems from donor bacteria, such as Mycoplasma mycoides, Mycoplasma capricolum, or Haemophilus influenzae increased successful genome transfers. These findings suggested that other genetic factors might also impact the bacteria-to-yeast genome transfer process. In this study, we demonstrated that the removal of a particular genetic factor, the glycerol uptake facilitator protein gene glpF from M. mycoides, significantly increased direct genome transfer by up to 21-fold. Additionally, we showed that intact bacterial cells were endocytosed by yeast spheroplasts producing organelle-like structures within these yeast cells. These might lead to the possibility of creating novel synthetic organelles.

Horizontal Transfer of a Synthetic Metabolic Pathway between Plant Species.

Transgene expression from the plastid (chloroplast) genome provides unique advantages, including high levels of foreign protein accumulation, convenient transgene stacking in operons, and increased biosafety due to exclusion of plastids from pollen transmission [1, 2]. However, applications in biotechnology and synthetic biology are severely restricted by the very small number of plant species whose plastid genomes currently can be transformed [3, 4]. Here we report a simple method for the introduction of useful plastid transgenes into non-transformable species. The transgenes tested comprised a synthetic operon encoding three components of a biosynthetic pathway for producing the high-value ketocarotenoid astaxanthin in the plastids of the cigarette tobacco, Nicotiana tabacum. Transplastomic N. tabacum plants accumulated astaxanthin to up to 1% of the plants' dry weight. We then used grafting, a procedure recently shown to facilitate horizontal genome transfer between plants [5-7], to let the transgenic chloroplast genome move across the graft junction from N. tabacum plants into plants of the nicotine-free tree species Nicotiana glauca. Transplastomic N. glauca trees expressing the synthetic pathway were recovered at high frequency, thus providing a straightforward method for extension of the transplastomic technology to new species.

Plant grafting: new mechanisms, evolutionary implications.

Grafting, an old plant propagation practice, is still widely used with fruit trees and in recent decades also with vegetables. Taxonomic proximity is a general prerequisite for successful graft-take and long-term survival of the grafted, composite plant. However, the mechanisms underlying interspecific graft incompatibility are as yet insufficiently understood. Hormonal signals, auxin in particular, are believed to play an important role in the wound healing and vascular regeneration within the graft union zone. Incomplete and convoluted vascular connections impede the vital upward and downward whole plant transfer routes. Long-distance protein, mRNA and small RNA graft-transmissible signals currently emerge as novel mechanisms which regulate nutritional and developmental root/top relations and may play a pivotal role in grafting physiology. Grafting also has significant pathogenic projections. On one hand, stock to scion mechanical contact enables the spread of diseases, even without a complete graft union. But, on the other hand, grafting onto resistant rootstocks serves as a principal tool in the management of fruit tree plagues and vegetable soil-borne diseases. The 'graft hybrid' historic controversy has not yet been resolved. Recent evidence suggests that epigenetic modification of DNA-methylation patterns may account for certain graft-transformation phenomena. Root grafting is a wide spread natural phenomenon; both intraspecific and interspecific root grafts have been recorded. Root grafts have an evolutionary role in the survival of storm-hit forest stands as well as in the spread of devastating diseases. A more fundamental evolutionary role is hinted by recent findings that demonstrate plastid and nuclear genome transfer between distinct Nicotiana species in the graft union zone, within a tissue culture system. This has led to the formation of alloploid cells that, under laboratory conditions, gave rise to a novel, alloploid Nicotiana species, indicating that natural grafts may play a role in plant speciation, under certain circumstances.

Why did eukaryotes evolve only once? Genetic and energetic aspects of conflict and conflict mediation.

According to multi-level theory, evolutionary transitions require mediating conflicts between lower-level units in favour of the higher-level unit. By this view, the origin of eukaryotes and the origin of multicellularity would seem largely equivalent. Yet, eukaryotes evolved only once in the history of life, whereas multicellular eukaryotes have evolved many times. Examining conflicts between evolutionary units and mechanisms that mediate these conflicts can illuminate these differences. Energy-converting endosymbionts that allow eukaryotes to transcend surface-to-volume constraints also can allocate energy into their own selfish replication. This principal conflict in the origin of eukaryotes can be mediated by genetic or energetic mechanisms. Genome transfer diminishes the heritable variation of the symbiont, but requires the de novo evolution of the protein-import apparatus and was opposed by selection for selfish symbionts. By contrast, metabolic signalling is a shared primitive feature of all cells. Redox state of the cytosol is an emergent feature that cannot be subverted by an individual symbiont. Hypothetical scenarios illustrate how metabolic regulation may have mediated the conflicts inherent at different stages in the origin of eukaryotes. Aspects of metabolic regulation may have subsequently been coopted from within-cell to between-cell pathways, allowing multicellularity to emerge repeatedly.