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.

Homoeologous crossovers are distally biased and underlie genomic instability in first-generation neo-allopolyploid Arabidopsis suecica.

First-generation polyploids often suffer from more meiotic errors and lower fertility than established wild polyploid populations. One such example is the allopolyploid model species Arabidopsis suecica which originated c. 16 000 generations ago. We present here a comparison of meiosis and its outcomes in naturally evolved and first-generation 'synthetic' A. suecica using a combination of cytological and genomic approaches. We show that while meiosis in natural lines is largely diploid-like, synthetic lines have high levels of meiotic errors including incomplete synapsis and nonhomologous crossover formation. Whole-genome re-sequencing of progeny revealed 20-fold higher levels of homoeologous exchange and eightfold higher aneuploidy originating from synthetic parents. Homoeologous exchanges showed a strong distal bias and occurred predominantly in genes, regularly generating novel protein variants. We also observed that homoeologous exchanges can generate megabase scale INDELs when occurring in regions of inverted synteny. Finally, we observed evidence of sex-specific differences in adaptation to polyploidy with higher success in reciprocal crosses to natural lines when synthetic plants were used as the female parent. Our results directly link cytological phenotypes in A. suecica with their genomic outcomes, demonstrating that homoeologous crossovers underlie genomic instability in neo-allopolyploids and are more distally biased than homologous crossovers.

Modulation of physio-biochemical and photosynthesis parameters by overexpressing SbPIP2 gene improved abiotic stress tolerance of transgenic tobacco.

The present study aims to explore the potential of a plasma-membrane localized PIP2-type aquaporin protein sourced from the halophyte Salicornia brachiata to alleviate salinity and water deficit stress tolerance in a model plant through transgenic intervention. Transgenic plants overexpressing SbPIP2 gene showed improved physio-biochemical parameters like increased osmolytes (proline, total sugar, and amino acids), antioxidants (polyphenols), pigments and membrane stability under salinity and drought stresses compared to control plants [wild type (WT) and vector control (VC) plants]. Multivariate statistical analysis showed that, under water and salinity stresses, osmolytes, antioxidants and pigments were correlated with SbPIP2-overexpressing (SbPIP2-OE) plants treated with salinity and water deficit stress, suggesting their involvement in stress tolerance. As aquaporins are also involved in CO2 transport, SbPIP2-OE plants showed enhanced photosynthesis performance than wild type upon salinity and drought stresses. Photosynthetic gas exchange (net CO2 assimilation rate, PSII efficiency, ETR, and non-photochemical quenching) were significantly higher in SbPIP2-OE plants compared to control plants (wild type and vector control plants) under both unstressed and stressed conditions. The higher quantum yield for reduction of end electron acceptors at the PSI acceptor side [Φ( R0 )] in SbPIP2-OE plants compared to control plants under abiotic stresses indicates a continued PSI functioning, leading to retained electron transport rate, higher carbon assimilation, and less ROS-mediated injuries. In conclusion, the SbPIP2 gene functionally validated in the present study could be a potential candidate for engineering abiotic stress resilience in important crops.

Meiosis in allopolyploid Arabidopsis suecica.

Polyploidy is a major force shaping eukaryote evolution but poses challenges for meiotic chromosome segregation. As a result, first-generation polyploids often suffer from more meiotic errors and lower fertility than established wild polyploid populations. How established polyploids adapt their meiotic behaviour to ensure genome stability and accurate chromosome segregation remains an active research question. We present here a cytological description of meiosis in the model allopolyploid species Arabidopsis suecica (2n = 4x = 26). In large part meiosis in A. suecica is diploid-like, with normal synaptic progression and no evidence of synaptic partner exchanges. Some abnormalities were seen at low frequency, including univalents at metaphase I, anaphase bridges and aneuploidy at metaphase II; however, we saw no evidence of crossover formation occurring between non-homologous chromosomes. The crossover number in A. suecica is similar to the combined number reported from its diploid parents Arabidopsis thaliana (2n = 2x = 10) and Arabidopsis arenosa (2n = 2x = 16), with an average of approximately 1.75 crossovers per chromosome pair. This contrasts with naturally evolved autotetraploid A. arenosa, where accurate chromosome segregation is achieved by restricting crossovers to approximately 1 per chromosome pair. Although an autotetraploid donor is hypothesized to have contributed the A. arenosa subgenome to A. suecica, A. suecica harbours diploid A. arenosa variants of key meiotic genes. These multiple lines of evidence suggest that meiosis in the recently evolved allopolyploid A. suecica is essentially diploid like, with meiotic adaptation following a very different trajectory to that described for autotetraploid A. arenosa.

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.

A cytological approach to studying meiotic recombination and chromosome dynamics in Arabidopsis thaliana male meiocytes in three dimensions.

During meiotic prophase I chromosomes undergo dramatic conformational changes that accompany chromosome condensation, pairing and recombination between homologs. These changes include the anchoring of telomeres to the nuclear envelope and their clustering to form a bouquet. In plants, these events have been studied and illustrated in intact meiocytes of species with large genomes. Arabidopsis thaliana is an excellent genetic model in which major molecular pathways that control synapsis and recombination between homologs have been uncovered. Yet the study of chromosome dynamics is hampered by current cytological methods that disrupt the three-dimensional (3D) architecture of the nucleus. Here we set up a protocol to preserve the 3D configuration of A. thaliana meiocytes. We showed that this technique is compatible with the use of a variety of antibodies that label structural and recombination proteins and were able to highlight the presence of clustered synapsis initiation centers at the nuclear periphery. By using fluorescence in situ hybridization we also studied the behavior of chromosomes during pre-meiotic G2 and prophase I, revealing the existence of a telomere bouquet during A. thaliana male meiosis. In addition we showed that the number of telomeres in a bouquet and its volume vary greatly, thus revealing the complexity of telomere behavior during meiotic prophase I. Finally, by using probes that label subtelomeric regions of individual chromosomes, we revealed differential localization behaviors of chromosome ends. Our protocol opens new areas of research for investigating chromosome dynamics in A. thaliana meiocytes.

Instability of Alien Chromosome Introgressions in Wheat Associated with Improper Positioning in the Nucleus.

Alien introgressions introduce beneficial alleles into existing crops and hence, are widely used in plant breeding. Generally, introgressed alien chromosomes show reduced meiotic pairing relative to the host genome, and may be eliminated over generations. Reduced pairing appears to result from a failure of some telomeres of alien chromosomes to incorporate into the leptotene bouquet at the onset of meiosis, thereby preventing chiasmate pairing. In this study, we analysed somatic nuclei of rye introgressions in wheat using 3D-FISH and found that while introgressed rye chromosomes or chromosome arms occupied discrete positions in the Rabl's orientation similar to chromosomes of the wheat host, their telomeres frequently occupied positions away from the nuclear periphery. The frequencies of such abnormal telomere positioning were similar to the frequencies of out-of-bouquet telomere positioning at leptotene, and of pairing failure at metaphase I. This study indicates that improper positioning of alien chromosomes that leads to reduced pairing is not a strictly meiotic event but rather a consequence of a more systemic problem. Improper positioning in the nuclei probably impacts the ability of introgressed chromosomes to migrate into the telomere bouquet at the onset of meiosis, preventing synapsis and chiasma establishment, and leading to their gradual elimination over generations.

Nuclear Disposition of Alien Chromosome Introgressions into Wheat and Rye Using 3D-FISH.

During interphase, the chromosomes of eukaryotes decondense and they occupy distinct regions of the nucleus, called chromosome domains or chromosome territories (CTs). In plants, the Rabl's configuration, with telomeres at one pole of nucleus and centromeres at the other, appears to be common, at least in plants with large genomes. It is unclear whether individual chromosomes of plants adopt defined, genetically determined addresses within the nucleus, as is the case in mammals. In this study, the nuclear disposition of alien rye and barley chromosomes and chromosome arm introgressions into wheat while using 3D-FISH in various somatic tissues was analyzed. All of the introgressed chromosomes showed Rabl's orientation, but their relative positions in the nuclei were less clear. While in most cases pairs of introgressed chromosomes occupied discrete positions, their association (proximity) along their entire lengths was rare, and partial association only marginally more frequent. This arrangement is relatively stable in various tissues and during various stages of the cell cycle. On the other hand, the length of a chromosome arm appears to play a role in its positioning in a nucleus: shorter chromosomes or chromosome arms tend to be located closer to the centre of the nucleus, while longer arms are more often positioned at the nuclear periphery.

B chromosomes are associated with redistribution of genetic recombination towards lower recombination chromosomal regions in perennial ryegrass.

Supernumerary 'B' chromosomes are non-essential components of the genome present in a range of plant and animal species-including many grasses. Within diploid and polyploid ryegrass and fescue species, including the forage grass perennial ryegrass (Lolium perenne L.), the presence of B chromosomes has been reported as influencing both chromosome pairing and chiasma frequencies. In this study, the effects of the presence/absence of B chromosomes on genetic recombination has been investigated through generating DArT (Diversity Arrays Technology) marker genetic maps for six perennial ryegrass diploid populations, the pollen parents of which contained either two B or zero B chromosomes. Through genetic and cytological analyses of these progeny and their parents, we have identified that, while overall cytological estimates of chiasma frequencies were significantly lower in pollen mother cells with two B chromosomes as compared with zero B chromosomes, the recombination frequencies within some marker intervals were actually increased, particularly for marker intervals in lower recombination regions of chromosomes, namely pericentromeric regions. Thus, in perennial ryegrass, the presence of two B chromosomes redistributed patterns of meiotic recombination in pollen mother cells in ways which could increase the range of allelic variation available to plant breeders.

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.

A spontaneous mutation in MutL-Homolog 3 (HvMLH3) affects synapsis and crossover resolution in the barley desynaptic mutant des10.

Although meiosis is evolutionarily conserved, many of the underlying mechanisms show species-specific differences. These are poorly understood in large genome plant species such as barley (Hordeum vulgare) where meiotic recombination is very heavily skewed to the ends of chromosomes. The characterization of mutant lines can help elucidate how recombination is controlled. We used a combination of genetic segregation analysis, cytogenetics, immunocytology and 3D imaging to genetically map and characterize the barley meiotic mutant DESYNAPTIC 10 (des10). We identified a spontaneous exonic deletion in the orthologue of MutL-Homolog 3 (HvMlh3) as the causal lesion. Compared with wild-type, des10 mutants exhibit reduced recombination and fewer chiasmata, resulting in the loss of obligate crossovers and leading to chromosome mis-segregation. Using 3D structured illumination microscopy (3D-SIM), we observed that normal synapsis progression was also disrupted in des10, a phenotype that was not evident with standard confocal microscopy and that has not been reported with Mlh3 knockout mutants in Arabidopsis. Our data provide new insights on the interplay between synapsis and recombination in barley and highlight the need for detailed studies of meiosis in nonmodel species. This study also confirms the importance of early stages of prophase I for the control of recombination in large genome cereals.

The effect of temperature on the male and female recombination landscape of barley.

Barley (Hordeum vulgare) is a crop of global significance. However, a third of the genes of barley are largely inaccessible to conventional breeding programmes as crossovers are localised to the ends of the chromosomes. This work examines whether crossovers can be shifted to more proximal regions simply by elevating growth temperature. We utilised a genome-wide marker set for linkage analysis combined with cytological mapping of crossover events to examine the recombination landscape of plants grown at different temperatures. We found that barley shows heterochiasmy, that is, differences between female and male recombination frequencies. In addition, we found that elevated temperature significantly changes patterns of recombination in male meiosis only, with a repositioning of Class I crossovers determined by cytological mapping of HvMLH3 foci. We show that the length of synaptonemal complexes in male meiocytes increases in response to temperature. The results demonstrate that the distribution of crossover events are malleable and can be shifted to proximal regions by altering the growth temperature. The shift in recombination is the result of altering the distribution of Class I crossovers, but the higher recombination at elevated temperatures is potentially not the result of an increase in Class I events.

Quantitative high resolution mapping of HvMLH3 foci in barley pachytene nuclei reveals a strong distal bias and weak interference.

In barley (Hordeum vulgare L.), chiasmata (the physical sites of genetic crossovers) are skewed towards the distal ends of chromosomes, effectively consigning a large proportion of genes to recombination coldspots. This has the effect of limiting potential genetic variability, and of reducing the efficiency of map-based cloning and breeding approaches for this crop. Shifting the sites of recombination to more proximal chromosome regions by forward and reverse genetic means may be profitable in terms of realizing the genetic potential of the species, but is predicated upon a better understanding of the mechanisms governing the sites of these events, and upon the ability to recognize real changes in recombination patterns. The barley MutL Homologue (HvMLH3), a marker for class I interfering crossovers, has been isolated and a specific antibody has been raised. Immunolocalization of HvMLH3 along with the synaptonemal complex transverse filament protein ZYP1, used in conjunction with fluorescence in situ hybridization (FISH) tagging of specific barley chromosomes, has enabled access to the physical recombination landscape of the barley cultivars Morex and Bowman. Consistent distal localization of HvMLH3 foci throughout the genome, and similar patterns of HvMLH3 foci within bivalents 2H and 3H have been observed. A difference in total numbers of HvMLH3 foci between these two cultivars has been quantified, which is interpreted as representing genotypic variation in class I crossover frequency. Discrepancies between the frequencies of HvMLH3 foci and crossover frequencies derived from linkage analysis point to the existence of at least two crossover pathways in barley. It is also shown that interference of HvMLH3 foci is relatively weak compared with other plant species.

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.

Schistosoma mansoni venom allergen-like protein 6 (SmVAL6) maintains tegumental barrier function.

The Schistosoma mansoni venom allergen-like protein (SmVAL) superfamily is a collection of at least 29 molecules that have been classified into two distinctive groups (Group 1 and Group 2 SmVALs). The fundamental basis for SmVAL segregation relates to signal peptide and conserved cysteine retention (present in all Group 1 SmVALs, but absent in all Group 2 SmVALs). These structural differences have led to the hypothesis that most Group 1 SmVALs, found as components of schistosome excretory/secretory (E/S) products, predominantly interact with their environment (intermediate or definitive hosts) whereas the Group 2 SmVALs are retained within the schistosome to fulfil parasite-related functions. While experimental evidence to support Group 1 SmVAL/host interactions is growing, similar support for identification of parasite-related Group 2 SmVAL functions is currently lacking. By applying a combination of approaches to the study of SmVAL6, we provide the first known evidence for an essential function of a Group 2 SmVAL in schistosome biology. After whole mount in situ hybridisation (WISH) localised Smval6 to the anterior region of the oesophageal gland (AOG) and cells scattered through the mesenchyme in adult schistosomes, short interfering RNA (siRNA)-mediated silencing of Smval6 was employed to assess loss of function phenotypes. Here, siSmval6-mediated knockdown of transcript and protein levels led to an increase in tegumental permeability as assessed by the quantification of TAMRA-labelled dextran throughout sub-tegumental cells/tissues. Yeast two hybrid screening using SmVAL6 as a bait revealed Sm14 (a fatty acid binding protein) and a dynein light chain (DLC) as directly interacting partners. Interrogation of single-cell RNA-seq (scRNA-seq) data supported these protein interactions by demonstrating the spatial co-expression of Smval6/dlc/Sm14 in a small proportion of adult cell types (e.g. neurons, tegumental cells and neoblasts). In silico modelling of SmVAL6 with Sm14 and DLC provided evidence that opposing faces of SmVAL6 were likely responsible for these protein/protein interactions. Our results suggest that SmVAL6 participates in oesophageal biology, formation of higher order protein complexes and maintenance of tegumental barrier function. Further studies of other Group 2 SmVALs may reveal additional functions of this enigmatic superfamily.

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.

Cupid, a cell permeable peptide derived from amoeba, capable of delivering GFP into a diverse range of species.

Cell permeating peptides (CPPs) are attracting great interest for use as molecular delivery vehicles for the transport of biologically active cargo across the cell membrane. The sequence of a novel CPP sequence, termed 'Cupid', was identified from the genome of Dictyostelium discoideum. A Cupid-Green Fluorescent Protein (Cupid-GFP) fusion protein was tested on mammalian, whole plant cells, plant leaf protoplast and fungal cell cultures and observed using confocal microscopy. GFP fluorescence builds up within the cell cytosol in 60 min, demonstrating Cupid-GFP has permeated them and folded correctly into its fluorescent form. Our combined data suggest Cupid can act as a molecular vehicle capable of delivering proteins, such as GFP, into the cytosol of a variety of cells.