Search Results (76)

Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease with few options for treatment and no available vaccine. Deletion mutants for live attenuated vaccines, particularly deletions of proteins related to the cytoskeleton, have been widely tested in related parasites but candidates have not been tested in T. cruzi. Kharon is one such protein, identified as being associated with the cytoskeleton in Leishmania and essential for amastigote replication. Here we investigated the T. cruzi Kharon ortholog (TcKharon) to test if it has orthologous function and thus potential in generating a live attenuated vaccine. In silico analysis predicted TcKharon to be an intrinsically disordered protein, consistent with its ortholog feature, and GFP fusion protein revealed that TcKharon is associated with the cytoskeleton of epimastigotes. CRISPR-Cas9-mediated gene disruption impaired epimastigote proliferation and cytokinesis, resulting in altered nucleus-to-kinetoplast ratios and pronounced morphological defects, particularly in the posterior cell region. Despite these abnormalities, TcKharon^−/− mutants retained the ability to differentiate into metacyclic trypomastigotes and exhibited in vitro infection rates comparable to wild-type parasites. Our data show that TcKharon is crucial for cell morphology. However, in contrast to close related parasites, TcKharon is not essential for in vitro infectivity. Full article

The syndrome “bassess richesses” is a vector-borne disease of sugar beet in Germany. The gammaproteobacterium ‘Candidatus Arsenophonus phytopathogenicus’ causes reduced sugar content and biomass, growth abnormalities, and yellowing. Co-infection with the 16SrXII-P stolbur phytoplasmas often leads to more severe symptoms and a risk of complete economic loss. This yellowing agent of the Mollicutes class had not been described before, so its differences from other stolbur phytoplasmas remained unanswered. The genome of strain GOE was sequenced, providing a resource to analyze its characteristics. Phylogenetic position was revised, genome organization was compared, and functional reconstructions of metabolic and virulence factors were performed. Average nucleotide identity analysis indicates that GOE represents a new ‘Ca. Phytoplasma’ species. Our results show that GOE is also distinct from other stolbur phytoplasmas in terms of smaller genome size and G+C content. Its reductive evolution is reflected in conserved membrane protein repertoire and minimal metabolism. The encoding of a riboflavin kinase indicates a lost pathway of phytoplasmas outside the groups 16SrXII and 16SrXIII. GOE shows a complete tra5 transposon harboring orthologs of SAP11, SAP54, and SAP05 effectors indicating an original phytoplasma pathogenicity island. Our results deepen the understanding of phytoplasma evolution and reaffirm the heterogeneity of stolbur phytoplasmas. Full article

Arabidopsis thaliana LEAFY COTYLEDON1 (LEC1) gene is shown to have numerous diverse functions in plant development, including the regulation of embryo morphogenesis and maturation, hypocotyl elongation, flowering transition, etc. However, the functions of LEC1 orthologs in different plant species have not been extensively studied. In this study, we obtained a line of Medicago truncatula, a model leguminous plant, carrying the loss-of-function mutation in the MtLEC1 (MtNF-YB10) gene, orthologous to LEC1, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas9) genome editing system. Edited plants with loss of MtNF-YB10 function did not demonstrate any severe abnormalities during their normal growth and gave viable seeds, but their capability for somatic embryogenesis in vitro was dramatically reduced. The T1 progeny of unedited plants with a Cas9-gRNA cassette insertion was also analyzed based on the suggestion that editing could occur during seed formation. However, no edited plants were found in the T1 generation. These results suggest divergent functions of LEC1 orthologs and make it possible to investigate potential specific MtNF-YB10 functions. Full article

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. Previously cysteine proteases have been found to be highly expressed during leaf senescence in different plant species. Using biochemical and immunoblotting approaches, we characterized developmental senescence of barley (Hordeum vulgare L. var. ‘GemCraft’) leaves collected from 0 to 6 weeks after the onset of flowering. A decrease in total protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunits occurred in parallel with an increase in proteolytic activity measured using the fluorogenic substrates Z-RR-AMC, Z-FR-AMC, and casein labeled with fluorescein isothiocyanate (casein-FITC). Aminopeptidase activity detected with R-AMC peaked at week 3 and then decreased, reaching a low level by week 6. Maximal proteolytic activity with Z-FR-AMC and Z-RR-AMC was detected from pH 4.0 to pH 5.5 and pH 6.5 to pH 7.4, respectively, while two pH optima (pH 3.6 to pH 4.5 and pH 6.5 to pH 7.4) were found for casein-FITC. Compound E-64, an irreversible cysteine protease inhibitor, and CAA0225, a selective cathepsin L inhibitor, effectively inhibited proteolytic activity with IC₅₀ values in the nanomolar range. CA-074, a selective cathepsin B inhibitor, was less potent under the same experimental conditions, with IC₅₀ in the micromolar range. Inhibition by leupeptin and phenylmethylsulfonyl fluoride (PMSF) was weak, and pepstatin A, an inhibitor of aspartic acid proteases, had no effect at the concentrations studied (up to 0.2 mM). Maximal proteolytic activity with the aminopeptidase substrate R-AMC was detected from pH 7.0 to pH 8.0. The pH profile of DCG-04 (a biotinylated activity probe derived from E-64) binding corresponded to that found with Z-FR-AMC, suggesting that the major active proteases are related to cathepsins B and L. Moreover, immunoblotting detected increased levels of barley SAG12 orthologs and aleurain, confirming a possible role of these enzymes in senescing leaves. Full article

The plant-specific IDD transcription factors (TFs) are vital for regulating plant growth and developmental processes. However, the characteristics and biological roles of the IDD gene family in tomato (Solanum lycopersicum) are still largely unexplored. In this study, 17 SlIDD genes were identified in the tomato genome and classified into seven subgroups according to the evolutionary relationships of IDD proteins. Analysis of exon–intron structures and conserved motifs reflected the evolutionary conservation of SlIDDs in tomato. Collinearity analysis revealed that segmental duplication promoted the expansion of the SlIDD family. Ka/Ks analysis indicated that SlIDD gene orthologs experienced predominantly purifying selection throughout evolution. The analysis of cis-acting elements revealed that the promoters of SlIDD genes contain numerous elements associated with light, plant hormones, and abiotic stresses. The RNA-seq data and qRT-PCR experimental results showed that the SlIDD genes exhibited tissue-specific expression. Additionally, Group A members from Arabidopsis thaliana and rice are known to play a role in regulating plant shoot gravitropism. QRT-PCR analysis confirmed that the expression level of SlIDD15 in Group A was high in the hypocotyls and stems. Subcellular localization demonstrated that the SlIDD15 protein was localized in the nucleus. Surprisingly, the loss-of-function of SlIDD15 by CRISPR/Cas9 gene editing technology did not display obvious gravitropic response defects, implying the existence of functional redundant factors within SlIDD15. Taken together, this study offers foundational insights into the tomato IDD gene family and serves as a valuable guide for exploring their molecular mechanisms in greater detail. Full article

The human GBA1 gene encodes lysosomal acid β-glucocerebrosidase, whose activity is deficient in Gaucher disease (GD). In Drosophila, there are two GBA1 orthologs, Gba1a and Gba1b, and Gba1b is the bona fide GCase encoding gene. Several fly lines with different deletions in the Gba1b were studied in the past. However, since most GD-associated GBA1 mutations are point mutations, we created missense mutations homologous to the two most common GD mutations: the mild N370S mutation (D415S in Drosophila) and the severe L444P mutation (L494P in Drosophila), using the CRISPR-Cas9 technology. Flies homozygous for the D415S mutation (dubbed D370S hereafter) presented low GCase activity and substrate accumulation, which led to lysosomal defects, activation of the Unfolded Protein Response (UPR), inflammation/neuroinflammation, and neurodegeneration along with earlier death compared to control flies. Surprisingly, the L494P (called L444P hereafter) flies presented higher GCase activity with fewer lysosomal defects and milder disease in comparison to that presented by the D370S homozygous flies. Treatment with ambroxol had a limited effect on all homozygous fly lines tested. Overall, our results underscore the differences between the fly and human GCase enzymes, as evidenced by the distinct phenotypic outcomes of mutations in flies compared to those observed in human GD patients. Full article

The advent of Clustered Regularly Interspaced Palindromic Repeat (CRISPR)/CRISPR-associated (Cas) proteins as a revolutionary innovation in genome editing has greatly promoted targeted modification and trait improvement in most plant species. For grapevine (Vitis vinifera L.), a perennial woody plant species, CRISPR/Cas genome editing is an extremely promising technique for genetic improvement in a short period. Advances in grapevine genome editing have been achieved by using CRISPR technology in recent years, which promises to accelerate trait improvement in grapevine. In this review, we describe the development and advances in CRISPR/Cas9 and its orthologs and variants. We summarize the applications of genome editing in grapevine and discuss the challenges facing grapevine genome editing as well as the possible strategies that could be used to improve genome editing in grapevine. In addition, we outline future perspectives for grapevine genome editing in a model system, precise genome editing, accelerated trait improvement, and transgene-free genome editing. We believe that CRISPR/Cas will play a more important role in grapevine genome editing, and an exciting and bright future is expected in this economically significant species. Full article

Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3, -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses in the hypotonic female oviduct. In contrast, the ejaculated spermatozoa of marine teleosts, such as the gilthead seabream (Sparus aurata), experience a high hypertonic shock in seawater, initially resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. Further regulatory recovery of cell volume in post-activated spermatozoa is mediated by Aqp4a in cooperation with the Trpv4 Ca²⁺ channel and other ion channels and transporters. Using a paralog-specific antibody, here, we show that seabream spermatozoa also express the aquaglyceroporin AQP3 ortholog Aqp3a, which is highly accumulated in the mid posterior region of the spermatozoon flagella, in a similar pattern to that described in mouse and human sperm. To investigate the role of Aqp3a in seabream sperm motility, we used a recently developed AQP3 antagonist (DFP00173), as well as the seabream Aqp3a-specific antibody (α-SaAqp3a), both of which specifically inhibit Aqp3a-mediated water conductance when the channel was heterologously expressed in Xenopus laevis oocytes. Inhibition with either DFP00173 or α-SaAqp3a did not affect sperm motility activation but did impair the spermatozoon motion kinetics at 30 s post activation in a dose-dependent manner. Interestingly, in close resemblance to the phenotypes of AQP3-deficient murine sperm, electron microscopy image analysis revealed that both Aqp3a inhibitors induce abnormal sperm tail morphologies, including swelling and angulation of the tail, with complete coiling of the flagella in some cases. These findings suggest a conserved role of Aqp3a as an osmosensor that regulates cell volume in fish spermatozoa under a high hypertonic stress, thereby controlling the efflux of water and/or solutes in the post-activated spermatozoon. Full article

Variants in membrane trafficking proteins are known to cause rare disorders with severe symptoms. The highly conserved transport protein particle (TRAPP) complexes are key membrane trafficking regulators that are also involved in autophagy. Pathogenic genetic variants in specific TRAPP subunits are linked to neurological disorders, muscular dystrophies, and skeletal dysplasias. Characterizing these variants and their phenotypes is important for understanding the general and specialized roles of TRAPP subunits as well as for patient diagnosis. Patient-derived cells are not always available, which poses a limitation for the study of these diseases. Therefore, other systems, like the yeast Saccharomyces cerevisiae, can be used to dissect the mechanisms at the intracellular level underlying these disorders. The development of CRISPR/Cas9 technology in yeast has enabled a scar-less editing method that creates an efficient humanized yeast model. In this study, core yeast subunits were humanized by replacing them with their human orthologs, and TRAPPC1, TRAPPC2, TRAPPC2L, TRAPPC6A, and TRAPPC6B were found to successfully replace their yeast counterparts. This system was used for studying the first reported individual with an autosomal recessive disorder caused by biallelic TRAPPC1 variants, a girl with a severe neurodevelopmental disorder and myopathy. We show that the maternal variant (TRAPPC1 p.(Val121Alafs*3)) is non-functional while the paternal variant (TRAPPC1 p.(His22_Lys24del)) is conditional-lethal and affects secretion and non-selective autophagy in yeast. This parallels defects seen in fibroblasts derived from this individual which also showed membrane trafficking defects and altered Golgi morphology, all of which were rescued in the human system by wild-type TRAPPC1. This study suggests that humanized yeast can be an efficient means to study TRAPP subunit variants in the absence of human cells and can assign significance to variants of unknown significance (VUS). This study lays the foundation for characterizing further TRAPP variants through this system, rapidly contributing to disease diagnosis. Full article

The pollen wall protects pollen during dispersal and is critical for pollination recognition. In the Poaceae family, the pollen exine stereostructure exhibits a high degree of conservation with similar patterns across species. However, there remains controversy regarding the conservation of key factors involved in its formation among various Poaceae species. EPAD1, as a gene specific to the Poaceae family, and its orthologous genes play a conserved role in pollen wall formation in wheat and rice. However, they do not appear to have significant functions in maize. To further confirm the conserved function of EPAD1 in Poaceae, we performed an analysis on four EPAD1 orthologs from two distinct sub-clades within the Poaceae family. The two functional redundant barley EPAD1 genes (HvEPAD1 and HvEPAD2) from the BOP clade, along with the single copy of sorghum (SbEPAD1) and millet (SiEPAD1) from the PACMAD clade were examined. The CRISPR-Cas9-generated mutants all exhibited defects in pollen wall formation, consistent with previous findings on EPAD1 in rice and wheat. Interestingly, in barley, hvepad2 single mutant also showed apical spikelets abortion, aligning with a decreased expression level of HvEPAD1 and HvEPAD2 from the apical to the bottom of the spike. Our finding provides evidence that EPAD1 orthologs contribute to Poaceae specific pollen exine pattern formation via maintaining primexine integrity despite potential variations in copy numbers across different species. Full article

The human louse (Pediculus humanus) is an obligatory blood feeding ectoparasite with two ecotypes: the human body louse (Pediculus humanus humanus), a competent vector of several bacterial pathogens, and the human head louse (Pediculus humanus capitis), responsible for pediculosis and affecting millions of people around the globe. GABA (γ-aminobutyric acid) receptors, members of the cys-loop ligand gated ion channel superfamily, are among the main pharmacological targets for insecticides. In insects, there are four subunits of GABA receptors: resistant-to-dieldrin (RDL), glycin-like receptor of drosophila (GRD), ligand-gated chloride channel homologue3 (LCCH3), and 8916 are well described and form distinct phylogenetic clades revealing orthologous relationships. Our previous studies in the human body louse confirmed that subunits Phh-RDL, Phh-GRD, and Phh-LCCH3 are well clustered in their corresponding clades. In the present work, we cloned and characterized a putative new GABA receptor subunit in the human body louse that we named HoCas, for Homologous to Cys-loop α like subunit. Extending our analysis to arthropods, HoCas was found to be conserved and clustered in a new (fifth) phylogenetic clade. Interestingly, the gene encoding this subunit is ancestral and has been lost in some insect orders. Compared to the other studied GABA receptor subunits, HoCas exhibited a relatively higher expression level in all development stages and in different tissues of human body louse. These findings improved our understanding of the complex nature of GABA receptors in Pediculus humanus and more generally in arthropods. Full article

Due to their immobility and possession of underground parts, plants have evolved various mechanisms to endure and adapt to abiotic stresses such as extreme temperatures, drought, and salinity. However, the contribution of long noncoding RNAs (lncRNAs) to different abiotic stresses and distinct rice seedling parts remains largely uncharacterized beyond the protein-coding gene (PCG) layer. Using transcriptomics and bioinformatics methods, we systematically identified lncRNAs and characterized their expression patterns in the roots and shoots of wild type (WT) and ososca1.1 (reduced hyperosmolality-induced [Ca²⁺]_i increase in rice) seedlings under hyperosmolarity and salt stresses. Here, 2937 candidate lncRNAs were identified in rice seedlings, with intergenic lncRNAs representing the largest category. Although the detectable sequence conservation of lncRNAs was low, we observed that lncRNAs had more orthologs within the Oryza. By comparing WT and ososca1.1, the transcription level of OsOSCA1.1-related lncRNAs in roots was greatly enhanced in the face of hyperosmolality stress. Regarding regulation mode, the co-expression network revealed connections between trans-regulated lncRNAs and their target PCGs related to OsOSCA1.1 and its mediation of hyperosmolality stress sensing. Interestingly, compared to PCGs, the expression of lncRNAs in roots was more sensitive to hyperosmolarity stress than to salt stress. Furthermore, OsOSCA1.1-related hyperosmolarity stress-responsive lncRNAs were enriched in roots, and their potential cis-regulated genes were associated with transcriptional regulation and signaling transduction. Not to be ignored, we identified a motif-conserved and hyperosmolarity stress-activated lncRNA gene (OSlncRNA), speculating on its origin and evolutionary history in Oryza. In summary, we provide a global perspective and a lncRNA resource to understand hyperosmolality stress sensing in rice roots, which helps to decode the complex molecular networks involved in plant sensing and adaptation to stressful environments. Full article

Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD’s ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo’s histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, we utilized CRISPR/Cas9 technology to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions. Full article

Ermp1 is a putative metalloprotease from Schizosaccharomyces pombe and a member of the Fxna peptidases. Although their function is unknown, orthologous proteins from rats and humans have been associated with the maturation of ovarian follicles and increased ER stress. This study focuses on proposing the first prediction of PPI by comparison of the interologues between humans and yeasts, as well as the molecular docking and dynamics of the M28 domain of Ermp1 with possible target proteins. As results, 45 proteins are proposed that could interact with the metalloprotease. Most of these proteins are related to the transport of Ca²⁺ and the metabolism of amino acids and proteins. Docking and molecular dynamics suggest that the M28 domain of Ermp1 could hydrolyze leucine and methionine residues of Amk2, Ypt5 and Pex12. These results could support future experimental investigations of other Fxna peptidases, such as human ERMP1. Full article

Originally developed as a chemotherapeutic agent, miltefosine (hexadecylphosphocholine) is an inhibitor of phosphatidylcholine synthesis with proven antiparasitic effects. It is the only oral drug approved for the treatment of Leishmaniasis and American Trypanosomiasis (Chagas disease). Although its precise mechanisms are not yet fully understood, miltefosine exhibits broad-spectrum anti-parasitic effects primarily by disrupting the intracellular Ca²⁺ homeostasis of the parasites while sparing the human hosts. In addition to its inhibitory effects on phosphatidylcholine synthesis and cytochrome c oxidase, miltefosine has been found to affect the unique giant mitochondria and the acidocalcisomes of parasites. Both of these crucial organelles are involved in Ca²⁺ regulation. Furthermore, miltefosine has the ability to activate a specific parasite Ca²⁺ channel that responds to sphingosine, which is different to its L-type VGCC human ortholog. Here, we aimed to provide an overview of recent advancements of the anti-parasitic mechanisms of miltefosine. We also explored its multiple molecular targets and investigated how its pleiotropic effects translate into a rational therapeutic approach for patients afflicted by Leishmaniasis and American Trypanosomiasis. Notably, miltefosine’s therapeutic effect extends beyond its impact on the parasite to also positively affect the host’s immune system. These findings enhance our understanding on its multi-targeted mechanism of action. Overall, this review sheds light on the intricate molecular actions of miltefosine, highlighting its potential as a promising therapeutic option against these debilitating parasitic diseases. Full article