Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

CDKG1 Is Required for Meiotic and Somatic Recombination Intermediate Processing in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) cyclin-dependent kinase G1 (CDKG1) is necessary for recombination and synapsis during male meiosis at high ambient temperature. In the cdkg1-1 mutant, synapsis is impaired and there is a dramatic reduction in the number of class I crossovers, resulting in univalents at metaphase I and pollen sterility. Here, we demonstrate that CDKG1 is necessary for the processing of recombination intermediates in the canonical ZMM recombination pathway and that loss of CDKG1 results in increased class II crossovers. While synapsis and events associated with class I crossovers are severely compromised in a cdkg1-1 mutant, they can be restored by increasing the number of recombination intermediates in the double cdkg1-1 fancm-1 mutant. Despite this, recombination intermediates are not correctly resolved, leading to the formation of chromosome aggregates at metaphase I. Our results show that CDKG1 acts early in the recombination process and is necessary to stabilize recombination intermediates. Finally, we show that the effect on recombination is not restricted to meiosis and that CDKG1 is also required for normal levels of DNA damage-induced homologous recombination in somatic tissues.

Methyl-CpG-binding (SmMBD2/3) and chromobox (SmCBX) proteins are required for neoblast proliferation and oviposition in the parasitic blood fluke Schistosoma mansoni.

While schistosomiasis remains a significant health problem in low to middle income countries, it also represents a recently recognised threat to more economically-developed regions. Until a vaccine is developed, this neglected infectious disease is primarily controlled by praziquantel, a drug with a currently unknown mechanism of action. By further elucidating how Schistosoma molecular components cooperate to regulate parasite developmental processes, next generation targets will be identified. Here, we continue our studies on schistosome epigenetic participants and characterise the function of a DNA methylation reader, the Schistosoma mansoni methyl-CpG-binding domain protein (SmMBD2/3). Firstly, we demonstrate that SmMBD2/3 contains amino acid features essential for 5-methyl cytosine (5mC) binding and illustrate that adult schistosome nuclear extracts (females > males) contain this activity. We subsequently show that SmMBD2/3 translocates into nuclear compartments of transfected murine NIH-3T3 fibroblasts and recombinant SmMBD2/3 exhibits 5mC binding activity. Secondly, using a yeast-two hybrid (Y2H) screen, we show that SmMBD2/3 interacts with the chromo shadow domain (CSD) of an epigenetic adaptor, S. mansoni chromobox protein (SmCBX). Moreover, fluorescent in situ hybridisation (FISH) mediated co-localisation of Smmbd2/3 and Smcbx to mesenchymal cells as well as somatic- and reproductive- stem cells confirms the Y2H results and demonstrates that these interacting partners are ubiquitously expressed and found within both differentiated as well as proliferating cells. Finally, using RNA interference, we reveal that depletion of Smmbd2/3 or Smcbx in adult females leads to significant reductions (46-58%) in the number of proliferating somatic stem cells (PSCs or neoblasts) as well as in the quantity of in vitro laid eggs. Collectively, these results further expand upon the schistosome components involved in epigenetic processes and suggest that pharmacological inhibition of SmMBD2/3 and/or SmCBX biology could prove useful in the development of future schistosomiasis control strategies.

CDKG1 protein kinase is essential for synapsis and male meiosis at high ambient temperature in Arabidopsis thaliana.

The Arabidopsis cyclin-dependent kinase G (CDKG) gene defines a clade of cyclin-dependent protein kinases related to CDK10 and CDK11, as well as to the enigmatic Ph1-related kinases that are implicated in controlling homeologous chromosome pairing in wheat. Here we demonstrate that the CDKG1/CYCLINL complex is essential for synapsis and recombination during male meiosis. A transfer-DNA insertional mutation in the cdkg1 gene leads to a temperature-sensitive failure of meiosis in late Zygotene/Pachytene that is associated with defective formation of the synaptonemal complex, reduced bivalent formation and crossing over, and aneuploid gametes. An aphenotypic insertion in the cyclin L gene, a cognate cyclin for CDKG, strongly enhances the phenotype of cdkg1-1 mutants, indicating that this cdk-cyclin complex is essential for male meiosis. Since CYCLINL, CDKG, and their mammalian homologs have been previously shown to affect mRNA processing, particularly alternative splicing, our observations also suggest a mechanism to explain the widespread phenomenon of thermal sensitivity in male meiosis.

Natural and artificial sources of genetic variation used in crop breeding: A baseline comparator for genome editing.

Traditional breeding has successfully selected beneficial traits for food, feed, and fibre crops over the last several thousand years. The last century has seen significant technological advancements particularly in marker assisted selection and the generation of induced genetic variation, including over the last few decades, through mutation breeding, genetic modification, and genome editing. While regulatory frameworks for traditional varietal development and for genetic modification with transgenes are broadly established, those for genome editing are lacking or are still evolving in many regions. In particular, the lack of "foreign" recombinant DNA in genome edited plants and that the resulting SNPs or INDELs are indistinguishable from those seen in traditional breeding has challenged development of new legislation. Where products of genome editing and other novel breeding technologies possess no transgenes and could have been generated via traditional methods, we argue that it is logical and proportionate to apply equivalent legislative oversight that already exists for traditional breeding and novel foods. This review analyses the types and the scale of spontaneous and induced genetic variation that can be selected during traditional plant breeding activities. It provides a base line from which to judge whether genetic changes brought about by techniques of genome editing or other reverse genetic methods are indeed comparable to those routinely found using traditional methods of plant breeding.

Quantification of Synapsis Using Immunolocalization in Embedded Nuclei of Lolium.

The establishment, formation and disassembly of the synaptonemal complex (SC) is intimately associated with other essential processes that occur during prophase I of meiosis, including recombination. Labeling the SC using primary antibodies raised against key proteins, detected using secondary antibodies conjugated to fluorescent dyes, differentiate between synapsed and unsynapsed regions, revealing the dynamics of the process. Embedding meiotic nuclei in acrylamide pads preserves the three-dimensional (3D) organization of the chromosomes, which can be optically sectioned using confocal laser scanning microscopy to produce a faithful representation of the SC at the point of fixation. Deconvolution, and processing using Imaris allows the axes to be isolated from the nucleus and their features measured. Here, I describe a robust protocol to quantify the SC using immunofluorescence in Lolium perenne and L. temulentum.

Genetic Transformation of Protoplasts Isolated from Leaves of Lolium temulentum and Lolium perenne.

Transient expression of inserted recombinant DNA in plant protoplasts is a widely used tool for functional genomics research. Recently it has been utilized to screen potential sgRNA guides for CRISPR-mediated genome editing. However, little research has been conducted into the use of transient expression of protoplasts in Lolium perenne (a globally important pasture, hay, and amenity grass), and no studies have been conducted into Lolium temulentum (a weed in cereal crops but a potentially useful model species for Lolium research). In this chapter, we describe a methodology of protoplast extraction and transformation from 14-day-old leaf mesophyll cells from L. perenne and L. temulentum. We believe this is the first report of a procedure for obtaining high density, viable protoplasts from L. temulentum. The method of polyethylene glycol (PEG)-mediated transformation is also described to achieve genetic transformation of protoplasts.

Anthropogenic Impacts on Meiosis in Plants.

As the human population grows and continues to encroach on the natural environment, organisms that form part of such ecosystems are becoming increasingly exposed to exogenous anthropogenic factors capable of changing their meiotic landscape. Meiotic recombination generates much of the genetic variation in sexually reproducing species and is known to be a highly conserved pathway. Environmental stresses, such as variations in temperature, have long been known to change the pattern of recombination in both model and crop plants, but there are other factors capable of causing genome damage, infertility and meiotic abnormalities. Our agrarian expansion and our increasing usage of agrochemicals unintentionally affect plants via groundwater contamination or spray drift; our industrial developments release heavy metals into the environment; pathogens are spread by climate change and a globally mobile population; imperfect waste treatment plants are unable to remove chemical and pharmaceutical residues from sewage leading to the release of xenobiotics, all with potentially deleterious meiotic effects. In this review, we discuss the major classes of exogenous anthropogenic factors known to affect meiosis in plants, namely environmental stresses, agricultural inputs, heavy metals, pharmaceuticals and pathogens. The possible evolutionary fate of plants thrust into their new anthropogenically imposed environments are also considered.

Potential impact of genome editing in world agriculture.

Changeable biotic and abiotic stress factors that affect crop growth and productivity, alongside a drive to reduce the unintended consequences of plant protection products, will demand highly adaptive farm management practices as well as access to continually improved seed varieties. The former is limited mainly by cost and, in theory, could be implemented in relatively short time frames. The latter is fundamentally a longer-term activity where genome editing can play a major role. The first targets for genome editing will inevitably be loss-of-function alleles, because these are straightforward to generate. In addition, they are likely to focus on traits under simple genetic control and where the results of modification are already well understood from null alleles in existing gene pools or other knockout or silencing approaches such as induced mutations or RNA interference. In the longer term, genome editing will underpin more fundamental changes in agricultural performance and food quality, and ultimately will merge with the tools and philosophies of synthetic biology to underpin and enable new cellular systems, processes and organisms completely. The genetic changes required for simple allele edits or knockout phenotypes are synonymous with those found naturally in conventional breeding material and should be regulated as such. The more radical possibilities in the longer term will need societal engagement along with appropriate safety and ethical oversight.