RESUMO
Morphogenesis of many protozoans depends on a polarized establishment of cytoskeletal structures. In malaria-causing parasites, this can be observed when a round zygote develops into an elongated motile ookinete within the mosquito stomach. This morphogenesis is mediated by the pellicle cytoskeletal structures, including the inner membrane complex (IMC) and the underlying subpellicular microtubules (SPMs). How the parasite maintains the IMC-SPM connection and establishes a dome-like structure of SPM to support cell elongation is unclear. Here, we show that palmitoylation of N-terminal cysteines of two IMC proteins (ISP1/ISP3) regulates the IMC localization of ISP1/ISP3 and zygote-to-ookinete differentiation. Palmitoylation of ISP1/ISP3 is catalyzed by an IMC-residing palmitoyl-S-acyl-transferase (PAT) DHHC2. Surprisingly, DHHC2 undergoes self-palmitoylation at C-terminal cysteines via its PAT activity, which controls DHHC2 localization in IMC after zygote formation. IMC-anchored ISP1 and ISP3 interact with microtubule component ß-tubulin, serving as tethers to maintain the proper structure of SPM during zygote elongation. This study identifies the first PAT-substrate pair in malaria parasites and uncovers a protein palmitoylation cascade regulating microtubule cytoskeleton.
Assuntos
Lipoilação , Microtúbulos/metabolismo , Plasmodium yoelii/metabolismo , Proteínas de Protozoários/metabolismo , Zigoto/metabolismo , Animais , Anopheles/parasitologia , Camundongos , Camundongos Endogâmicos ICRRESUMO
Male and female gametocytes are sexual precursor cells essential for mosquito transmission of malaria parasite. Differentiation of gametocytes into fertile gametes (known as gametogenesis) relies on the gender-specific transcription program. How the parasites establish distinct repertoires of transcription in the male and female gametocytes remains largely unknown. Here, we report that an Apetala2 family transcription factor AP2-O3 operates as a transcription repressor in the female gametocytes. AP2-O3 is specifically expressed in the female gametocytes. AP2-O3-deficient parasites produce apparently normal female gametocytes. Nevertheless, these gametocytes fail to differentiate into fully fertile female gametes, leading to developmental arrest in fertilization and early development post-fertilization. AP2-O3 disruption causes massive upregulation of transcriptionally dormant male genes and simultaneously downregulation of highly transcribed female genes in the female gametocytes. AP2-O3 targets a substantial proportion of the male genes by recognizing an 8-base DNA motif. In addition, the maternal AP2-O3 is removed after fertilization, which is required for the zygote to ookinete development. Therefore, the global transcriptional repression of the male genes in the female gametocytes is required for safeguarding female-specific transcriptome and essential for the mosquito transmission of Plasmodium.
Assuntos
Plasmodium berghei , Plasmodium falciparum , Animais , Feminino , Gametogênese/genética , Masculino , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Fatores de Transcrição/genética , TranscriptomaRESUMO
The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a transcription factor family specific to the land plants, have been implicated in multiple biological processes including organ development, pathogen response and the uptake of inorganic nitrogen. The study focused on LBDs in legume forage Alfalfa. The genome-wide analysis revealed that in Alfalfa 178 loci across 31 allelic chromosomes encoded 48 unique LBDs (MsLBDs), and the genome of its diploid progenitor M. sativa spp. Caerulea encoded 46 LBDs. Synteny analysis indicated that the expansion of AlfalfaLBDs was attributed to the whole genome duplication event. The MsLBDs were divided into two major phylogenetic classes, and the LOB domain of the Class I members was highly conserved relative to that of the Class II. The transcriptomic data demonstrated that 87.5% of MsLBDs were expressed in at least one of the six test tissues, and Class II members were preferentially expressed in nodules. Moreover, the expression of Class II LBDs in roots was upregulated by the treatment of inorganic nitrogen such as KNO3 and NH4Cl (0.3 mM). The overexpression of MsLBD48, a Class II member, in Arabidopsis resulted in growth retardance with significantly declined biomass compared with the non-transgenic plants, and the transcription level of the genes involved in nitrogen uptake or assimilation, including NRT1.1, NRT2.1, NIA1 and NIA2 was repressed. Therefore, the LBDs in Alfalfa are highly conserved with their orthologs in embryophytes. Our observations that ectopic expression of MsLBD48 inhibited Arabidopsis growth by repressing nitrogen adaption suggest the negative role of the transcription factor in plant uptake of inorganic nitrogen. The findings imply the potential application of MsLBD48 in Alfalfa yield improvement via gene editing.
Assuntos
Proteínas de Transporte de Ânions , Medicago sativa , Fixação de Nitrogênio , Proteínas de Plantas , Fatores de Transcrição , Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Medicago sativa/genética , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fixação de Nitrogênio/genéticaRESUMO
Bioactive gibberellic acids (GAs) are diterpenoid plant hormones that are biosynthesized through complex pathways and control various aspects of growth and development. Although GA biosynthesis has been intensively studied, the downstream metabolic pathways regulated by GAs have remained largely unexplored. We investigated Tnt1 retrotransposon insertion mutant lines of Medicago truncatula with a dwarf phenotype by forward and reverse genetics screening and phylogenetic, molecular, biochemical, proteomic and metabolomic analyses. Three Tnt1 retrotransposon insertion mutant lines of the gibberellin 3-beta-dioxygenase 1 gene (GA3ox1) with a dwarf phenotype were identified, in which the synthesis of GAs (GA3 and GA4) was inhibited. Phenotypic analysis revealed that plant height, root and petiole length of ga3ox1 mutants were shorter than those of the wild type (Medicago truncatula ecotype R108). Leaf size was also much smaller in ga3ox1 mutants than that in wild-type R108, which is probably due to cell-size diminution instead of a decrease in cell number. Proteomic and metabolomic analyses of ga3ox1/R108 leaves revealed that in the ga3ox1 mutant, flavonoid isoflavonoid biosynthesis was significantly up-regulated, while nitrogen metabolism was down-regulated. Additionally, we further demonstrated that flavonoid and isoflavonoid biosynthesis was induced by prohexadione calcium, an inhibitor of GA3ox enzyme, and inhibited by exogenous GA3. In contrast, nitrogen metabolism was promoted by exogenous GA3 but inhibited by prohexadione calcium. The results of this study further demonstrated that GAs play critical roles in positively regulating nitrogen metabolism and transport and negatively regulating flavonoid biosynthesis through GA-mediated signaling pathways in leaves.
Assuntos
Flavonoides/antagonistas & inibidores , Giberelinas/farmacologia , Medicago truncatula/metabolismo , Metaboloma/efeitos dos fármacos , Nitrogênio/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/efeitos dos fármacos , Flavonoides/biossíntese , Regulação da Expressão Gênica de Plantas , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/genética , Medicago truncatula/crescimento & desenvolvimento , Mutação , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Proteoma/análise , Proteoma/metabolismoRESUMO
Soil salinization is one of most crucial environmental problems around the world and negatively affects plant growth and production. Carex rigescens is a turfgrass with favorable stress tolerance and great application prospect in salinity soil remediation and utilization; however, the molecular mechanisms behind its salt stress response are unknown. We performed a time-course transcriptome analysis between salt tolerant 'Huanghua' (HH) and salt sensitive 'Beijing' (BJ) genotypes. Physiological changes within 24 h were observed, with the HH genotype exhibiting increased salt tolerance compared to BJ. 5764 and 10752 differentially expressed genes were approved by transcriptome in BJ and HH genotype, respectively, and dynamic analysis showed a discrepant profile between two genotypes. In the BJ genotype, genes related to carbohydrate metabolism and stress response were more active and ABA signal transduction pathway might play a more important role in salt stress tolerance than in HH genotype. In the HH genotype, unique increases in the regulatory network of transcription factors, hormone signal transduction, and oxidation-reduction processes were observed. Moreover, trehalose and pectin biosynthesis and chitin catabolic related genes were specifically involved in the HH genotype, which may have contributed to salt tolerance. Moreover, some candidate genes like mannan endo-1,4-beta-mannosidase and EG45-like domain-containing protein are highlighted for future research about salt stress resistance in C. rigescens and other plant species. Our study revealed unique salt adaptation and resistance characteristics of two C. rigescens genotypes and these findings could help to enrich the currently available knowledge and clarify the detailed salt stress regulatory mechanisms in C. rigescens and other plants.
Assuntos
Carex (Planta)/genética , Transcriptoma , Pequim , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genótipo , Salinidade , Tolerância ao Sal , Estresse Fisiológico/genética , Fatores de Transcrição/genéticaRESUMO
KEY MESSAGE: CrCOMT, a COMT gene in Carex rigescens, was verified to enhance salt stress tolerance in transgenic Arabidopsis. High salinity severely restricts plant growth and development while melatonin can alleviate salt damage. Caffeic acid O-methyltransferase (COMT) plays an important role in regulating plant growth, development, and stress responses. COMT could also participate in melatonin biosynthesis. The objective of this study was to identify CrCOMT from Carex rigescens (Franch.) V. Krecz, a stress-tolerant grass species with a widespread distribution in north China, and to determine its physiological functions and regulatory mechanisms that impart tolerance to salt stress. The results showed that the transcription of CrCOMT exhibited different expression patterns under salt, drought, and ABA treatments. Transgenic Arabidopsis with the overexpression of CrCOMT exhibited improved growth and physiological performance under salt stress, such as higher lateral root numbers, proline level, and chlorophyll content, than in the wild type (WT). Overexpression of CrCOMT also increased dehydration tolerance in Arabidopsis. The transcription of salt response genes was more highly activated in transgenic plants than in the WT under salt stress conditions. In addition, the melatonin content in transgenic plants was higher than that in the WT after stress treatment. Taken together, our results indicated that CrCOMT may positively regulate stress responses and melatonin synthesis under salt stress.
Assuntos
Arabidopsis/metabolismo , Carex (Planta)/metabolismo , Melatonina/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Carex (Planta)/genética , Regulação da Expressão Gênica de Plantas , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tolerância ao SalRESUMO
Carex rigescens is an ornamental turfgrass in northern China which has a relatively low maintenance cost and robust tolerance to many adverse environmental conditions, so it could be considered a new material for researching into plant stress resistance. However, suitable reference genes are vacant for obtaining reliable results in quantitative real-time PCR (qRT-PCR) analysis of C. rigescens in adversity research. In this study, we tested the expression stability of nine potential reference genes in leaves and roots under five different abiotic stress conditions, including cold, salt, heat, osmotic and cadmium (Cd). We then selected the best reference genes according to the analysis results calculated by three algorithmic programs (geNorm, NormFinder and BestKeeper) and used the RankAggreg package to merge the outputted data. The results showed that combinations of at least two reference genes should be used for reliable normalization except in heat-treated root samples, which require three reference genes. eIF-4α, GADPH, SAND and PEPKR1 and their combination were found to be the most stably expressed reference genes, while SAM, TUA-α and UPL7 were the three least stable reference genes among most of experimental samples. In addition, five stress-induced genes (Cu-Zn SOD, P5CS, LEA, GST, and APX) were chosen to verify the stability of the selected reference genes in various tissues and under various stress conditions. The results of this study will provide an important fundamental basis both for gene expression verification for transcriptomic and proteomic analyses and for gene expression analysis for future gene function research in C. rigescens.
Assuntos
Cyperaceae/genética , Genes de Plantas , Folhas de Planta/genética , Raízes de Plantas/genética , Estresse Fisiológico/genética , Regulação da Expressão Gênica de Plantas , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Seleção Genética , TranscriptomaRESUMO
Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.
Assuntos
Axonema , Íntrons , Proteínas de Protozoários , Splicing de RNA , Proteínas de Ligação a RNA , Íntrons/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Masculino , Axonema/metabolismo , Feminino , Gametogênese/genética , Spliceossomos/metabolismo , Spliceossomos/genética , Plasmodium berghei/genética , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Malária/parasitologia , Plasmodium/genética , Plasmodium/metabolismoRESUMO
Bispecific antibodies (biAbs) that mediate cytotoxicity by recruiting and activating endogenous immune cells are an emerging class of next-generation antibody therapeutics. Of particular interest are biAbs of relatively small size (â¼50 kDa) that can redirect cytotoxic T cells through simultaneous binding of tumor cells. Here we describe a conceptually unique class of biAbs in which the tumor cell specificity of a humanized antibody fragment that recognizes CD3 on T cells is chemically programmed through a C-terminal selenocysteine (Sec) residue. We demonstrate that through chemically programmed specificity for integrin α(4)ß(1) or folate receptor 1 (FOLR1), and common specificity for CD3, these hybrid molecules exert potent and specific in vitro and ex vivo cytotoxicity toward tumor cell lines and primary tumor cells in the presence of primary T cells. Importantly, the generic nature of chemical programming allows one to apply our approach to virtually any specificity, promising a broad utility of chemically programmed biAbs in cancer therapy.
Assuntos
Anticorpos Biespecíficos/imunologia , Especificidade de Anticorpos , Complexo CD3/imunologia , Ativação Linfocitária , Selenocisteína/imunologia , Linfócitos T/imunologia , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Complexo CD3/genética , Receptor 1 de Folato/genética , Receptor 1 de Folato/imunologia , Células HeLa , Humanos , Integrina alfa4beta1/genética , Integrina alfa4beta1/imunologia , Células Jurkat , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/imunologia , Selenocisteína/genéticaRESUMO
Faithful chromosome segregation of 8 duplicated haploid genomes into 8 daughter gametes is essential for male gametogenesis and mosquito transmission of Plasmodium. Plasmodium undergoes endomitosis in this multinucleated cell division, which is highly reliant on proper spindle-kinetochore attachment. However, the mechanisms underlying the spindle-kinetochore attachment remain elusive. End-binding proteins (EBs) are conserved microtubule (MT) plus-end binding proteins and play an important role in regulating MT plus-end dynamics. Here, we report that the Plasmodium EB1 is an orthologue distinct from the canonical eukaryotic EB1. Both in vitro and in vivo assays reveal that the Plasmodium EB1 losses MT plus-end tracking but possesses MT-lattice affinity. This MT-binding feature of Plasmodium EB1 is contributed by both CH domain and linker region. EB1-deficient parasites produce male gametocytes that develop to the anucleated male gametes, leading to defective mosquito transmission. EB1 is localized at the nucleoplasm of male gametocytes. During the gametogenesis, EB1 decorates the full-length of spindle MTs and regulates spindle structure. The kinetochores attach to spindle MTs laterally throughout endomitosis and this attachment is EB1-dependent. Consequently, impaired spindle-kinetochore attachment is observed in EB1-deficient parasites. These results indicate that a parasite-specific EB1 with MT-lattice binding affinity fulfills the spindle-kinetochore lateral attachment in male gametogenesis.
Assuntos
Proteínas Associadas aos Microtúbulos , Plasmodium , Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Plasmodium/genética , Fuso Acromático/metabolismoRESUMO
We have previously described an approach whereby antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec) are directed against a variety of targets by changing the peptide or small molecule to which they are conjugated. In the present work, we describe methodology for improving the efficacy of these Fc-Sec conjugates by incorporating cytotoxic drugs. The Fc-Sec protein is first programmed to target specific tumor cell types by attachment of a bifunctional linker that contains a "clickable" handle (e.g., cyclobutane or cyclooctyne) in addition to a tumor cell-binding peptide or small molecule. Following Fc-Sec conjugation, a cytotoxic warhead is then attached by cycloaddition reactions of tetrazine or azide-containing linker. To validate this approach, we used a model system in which folic acid (FA) is the targeting moiety and a disulfide-linked biotin moiety serves as a cytotoxic drug surrogate. We demonstrated successful targeting of Fc-Sec proteins to folate-receptor expressing tumor cells. Tetrazine ligation was found to be an efficient method for biotin "arming" of the folate-targeted Fc-Sec proteins. We also report novel bioconjugation methodologies that use [4 + 2] cycloaddition reactions between tetrazines and cyclooctynes.
Assuntos
Alcinos/química , Antineoplásicos/química , Azidas/química , Reação de Cicloadição , Fragmentos Fc das Imunoglobulinas/química , Tetrazóis/química , Antineoplásicos/farmacologia , Ciclobutanos/química , Células HeLa , Humanos , Selenocisteína/químicaRESUMO
Melatonin is an important, multifunctional protective agent against a variety of abiotic and biotic stressors in plants. Caffeic acid O-methyltransferase (COMT) catalyzes the last step of melatonin synthesis in plants and reportedly participates in the regulation of stress response and tolerance. However, few studies have reported its function in melatonin-mediated drought resistance. In this study, CrCOMT was identified and was strongly induced by drought stress in Carex rigescens. CrCOMT overexpression in transgenic tobacco increased tolerance to drought stress with high levels of seed germination, relative water content, and survival rates. CrCOMT overexpression in tobacco improved membrane stability, and plants exhibited lower relative electrolytic leakage and malondialdehyde content, as well as higher photochemical efficiency than the wildtype (WT) under drought stress. The transgenic plants also had higher levels of proline accumulation and antioxidant enzyme activity, which decreased oxidative stress damage due to reactive oxygen species (ROS) hyperaccumulation under drought stress. The transcription of drought stress response and ROS scavenging genes was significantly higher in the CrCOMT overexpression plants than in the WT plants. In addition, CrCOMT transgenic tobacco plants exhibited higher melatonin content under drought stress conditions. Exogenous melatonin was applied to C. rigescens under drought stress to confirm the function of melatonin in mediating drought tolerance; the relative water content and proline content were higher, and the relative electrolytic leakage was lower in melatonin-treated C. rigescens than in the untreated plants. In summary, these results show that CrCOMT plays a positive role in plant drought stress tolerance by regulating endogenous melatonin content.
RESUMO
Malaria is caused by infection of the erythrocytes by the parasites Plasmodium. Inside the erythrocytes, the parasites multiply via schizogony, an unconventional cell division mode. The inner membrane complex (IMC), an organelle located beneath the parasite plasma membrane, serving as the platform for protein anchorage, is essential for schizogony. So far, the complete repertoire of IMC proteins and their localization determinants remain unclear. Here we used biotin ligase (TurboID)-based proximity labeling to compile the proteome of the schizont IMC of the rodent malaria parasite Plasmodium yoelii. In total, 300 TurboID-interacting proteins were identified. 18 of 21 selected candidates were confirmed to localize in the IMC, indicating good reliability. In light of the existing palmitome of Plasmodium falciparum, 83 proteins of the P. yoelii IMC proteome are potentially palmitoylated. We further identified DHHC2 as the major resident palmitoyl-acyl-transferase of the IMC. Depletion of DHHC2 led to defective schizont segmentation and growth arrest both in vitro and in vivo. DHHC2 was found to palmitoylate two critical IMC proteins CDPK1 and GAP45 for their IMC localization. In summary, this study reports an inventory of new IMC proteins and demonstrates a central role of DHHC2 in governing the IMC localization of proteins during the schizont development.
Assuntos
Malária , Parasitos , Animais , Eritrócitos/parasitologia , Lipoilação , Malária/parasitologia , Parasitos/metabolismo , Plasmodium falciparum/fisiologia , Proteoma/metabolismo , Proteômica , Proteínas de Protozoários/metabolismo , Reprodutibilidade dos Testes , EsquizontesRESUMO
Morphogenesis of many protozoans depends on a polarized establishment of cortical cytoskeleton containing the subpellicular microtubules (SPMTs), which are apically nucleated and anchored by the apical polar ring (APR). In malaria parasite Plasmodium, APR emerges in the host-invading stages, including the ookinete for mosquito infection. So far, the fine structure and molecular components of APR as well as the underlying mechanism of APR-mediated apical positioning of SPMTs are largely unknown. Here, we resolve an unprecedented APR structure composed of a top ring plus approximate 60 radiating spines. We report an APR-localizing and SPMT-binding protein APR2. APR2 disruption impairs ookinete morphogenesis and gliding motility, leading to Plasmodium transmission failure in mosquitoes. The APR2-deficient ookinetes display defective apical anchorage of APR and SPMT due to the impaired integrity of APR. Using protein proximity labeling, we obtain a Plasmodium ookinete APR proteome and validate ten undescribed APR proteins. Among them, APRp2 and APRp4 directly interact with APR2 and also mediate the apical anchorage of SPMTs. This study sheds light on the molecular basis of APR in the organization of Plasmodium ookinete SPMTs.
Assuntos
Culicidae , Malária , Animais , Citoesqueleto , MicrotúbulosRESUMO
The evolutionary origins of mammalian immunoglobulin H chain isotypes (IgM, IgD, IgG, IgE, and IgA) are still incompletely understood as these isotypes differ considerably in structure and number from their counterparts in nonmammalian tetrapods. We report in this study that the platypus (Ornithorhynchus anatinus) Ig H chain constant region gene locus contains eight Ig encoding genes, which are arranged in an mu-delta-omicron-gamma2-gamma1-alpha1-epsilon-alpha2 order, spanning a total of approximately 200 kb DNA, encoding six distinct isotypes. The omicron (omicron for Ornithorhynchus) gene encodes a novel Ig H chain isotype that consists of four constant region domains and a hinge, and is structurally different from any of the five known mammalian Ig classes. This gene is phylogenetically related to upsilon (epsilon) and gamma, and thus appears to be a structural intermediate between these two genes. The platypus delta gene encodes ten heavy chain constant region domains, lacks a hinge region and is similar to IgD in amphibians and fish, but strikingly different from that in eutherian mammals. The platypus Ig H chain isotype repertoire thus shows a unique combination of genes that share similarity both to those of nonmammalian tetrapods and eutherian animals and demonstrates how phylogenetically informative species can be used to reconstruct the evolutionary history of functionally important genes.
Assuntos
Evolução Molecular , Imunoglobulina D/genética , Isotipos de Imunoglobulinas/genética , Imunoglobulinas/genética , Mamíferos/genética , Mamíferos/imunologia , Ornitorrinco/genética , Ornitorrinco/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Humanos , Regiões Constantes de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias delta de Imunoglobulina/genética , Masculino , Dados de Sequência Molecular , FilogeniaRESUMO
Mosquito midgut epithelium traversal is essential for malaria parasite transmission. Phospholipid flippases are eukaryotic type 4 P-type adenosine triphosphatases (P4-ATPases), which, in association with CDC50, translocate phospholipids across the membrane lipid bilayers. In this study, we investigated the function of a putative P4-ATPase, ATP7, from the rodent malaria parasite Plasmodium yoelii Disruption of ATP7 blocks the parasite infection of mosquitoes. ATP7 is localized on the ookinete plasma membrane. While ATP7-depleted ookinetes are capable of invading the midgut, they are eliminated within the epithelial cells by a process independent from the mosquito complement-like immunity. ATP7 colocalizes and interacts with the flippase cofactor CDC50C. Depletion of CDC50C phenocopies ATP7 deficiency. ATP7-depleted ookinetes fail to uptake phosphatidylcholine across the plasma membrane. Ookinete microinjection into the mosquito hemocoel reverses the ATP7 deficiency phenotype. Our study identifies Plasmodium flippase as a mechanism of parasite survival in the midgut epithelium that is required for mosquito transmission.
RESUMO
Acetic acid priming may mitigate salt stress to plants by modulating lipid metabolism. Carex rigescens is a stress-tolerant turfgrass species with a widespread distribution in north China. The objective of this study was to figure out whether modification of lipid profiles, including the contents, compositions and saturation levels of leaf lipids, may contribute to acetic acid modulated salt tolerance in C. rigescens. Plants of C. rigescens were primed with or without acetic acid (30 mM) and subsequently exposed to salt stress (300 mM NaCl) for 15 days. Salt stress affected the physiological performance of C. rigescens, while acetic acid-primed plants showed significantly lower malondialdehyde content, proline content, and electrolyte leakage than non-primed plants under salt stress. Acetic acid priming enhanced the contents of phospholipids and glycolipids involved in membrane stabilization and stress signaling (phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, digalactosyl diacylglycerol, monogalactosyl diacylglycerol, and sulfoquinovosyldiacylglycerol), reduced the content of toxic lipid intermediates (free fatty acids) during subsequent exposure to salt stress. Furthermore, expression levels of genes involved in lipid metabolism such as CK and PLDα changed due to acetic acid priming. These results demonstrated that acetic acid priming could enhance salt tolerance of C. rigescens by regulating lipid metabolism. The lipids could be used as biomarkers to select for salt-tolerant grass germplasm.
Assuntos
Carex (Planta) , Tolerância ao Sal , Ácido Acético , Lipidômica , Estresse Salino , Estresse FisiológicoRESUMO
The auxin-inducible degron (AID) system is a robust chemical-genetic method for manipulating endogenous protein level by conditional proteasomal degradation via a small molecule. So far, this system has not been adapted in the P. yoelii, an important and widely used Plasmodium rodent parasite model for malaria biology. Here, using the CRISPR/Cas9 genome editing method, we generated two marker-free transgenic P. yoelii parasite lines (eef1a-Tir1 and soap-Tir1) stably expressing the Oryza sativa gene tir1 under the promoters of eef1a and soap respectively. These two lines develop normally during the parasite life cycle. In these backgrounds, we used the CRISPR/Cas9 method to tag two genes (cdc50c and fbxo1) with the AID motif and interrogate the expression of these two proteins with auxin. The eef1a-Tir1 line allows efficient degradation of the AID-tagged endogenous protein in the asexual schizont and sexual gametocyte stages, while the soap-Tir1 line allows protein degradation in the ookinetes. These two lines will be a useful resource for studying the Plasmodium parasite biology based on the P. yoelii.
Assuntos
Animais Geneticamente Modificados , Engenharia Genética , Plasmodium yoelii/genética , Plasmodium yoelii/metabolismo , Animais , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Feminino , Imunofluorescência , Edição de Genes , Expressão Gênica , Marcação de Genes , Malária/parasitologia , Malária/transmissão , Camundongos , Proteólise , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Arabidopsis thaliana MUT9-like kinases (MLKs), a family of the plant-specific casein kinase 1 (CK1), have been implicated collectively in multiple biological processes including flowering. Three of the four MLKs (MLK1/2/4) have been characterized, however, little is known about MLK3, the most divergent member of MLKs. Here, we demonstrated that disruption of MLK3 transcript in mlk3 caused early flowering with retarded leaf growth under long-day conditions. In vitro kinase assay showed the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3) and mutation of a conserved residue (K146R) abolished the catalytic activity. Ectopic expression of MLK3 but not MLK3(K146R) rescued the morphological defects of mlk3, indicating that an intact MLK3 is critical for maintaining proper flowering time. Transcriptomic analysis revealed that the floral repressor FLOWERINGLOCUS C (FLC) was down-regulated significantly in mlk3, suggesting that MLK3 negatively regulates flowering. Hence, MLK3 plays a role in repressing the transition from vegetative to reproductive phase in A. thaliana. This study sheds light on the delicate control of flowering time by A. thaliana CK1 specific to the plant kingdom.