RESUMEN
In female mammals, the oviduct and uterus are essential sites for female and male gamete transport, fertilization, implantation, and maintenance of a successful pregnancy. To delineate the reproductive function of Mothers against decapentaplegic homolog 4 (Smad4), we specifically inactivated Smad4 in ovarian granulosa cells and, oviduct and uterine mesenchymal cells using the Amhr2-cre mouse line. Deletion of exon 8 of Smad4 results in the production of an MH2-truncated SMAD4 protein. These mutant mice are infertile due to the development of oviductal diverticula and defects during the implantation process. The ovaries are fully functional as demonstrated in an ovary transfer experiment. The development of oviductal diverticula occurs shortly after puberty and is dependent on estradiol. The diverticula interfere with sperm migration and embryo transit to the uterus, reducing the number of implantation sites. Analysis of the uterus shows that, even if implantation occurs, decidualization and vascularization are defective resulting in embryo resorption as early as the seventh day of pregnancy. Thus, Smad4 plays an important function in female reproduction by controlling the structural and functional integrity of the oviduct and uterus.
Asunto(s)
Estradiol , Proteína Smad4 , Animales , Femenino , Humanos , Masculino , Ratones , Embarazo , Implantación del Embrión , Estradiol/metabolismo , Mamíferos/metabolismo , Oviductos/metabolismo , Semen/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Útero/metabolismoRESUMEN
EXOSC10 is a catalytic subunit of the nuclear RNA exosome, and possesses a 3'-5' exoribonuclease activity. The enzyme processes and degrades different classes of RNAs. To delineate the role of EXOSC10 during oocyte growth, specific Exosc10 inactivation was performed in oocytes from the primordial follicle stage onward using the Gdf9-iCre; Exosc10 f/- mouse model (Exosc10 cKO(Gdf9)). Exosc10 cKO(Gdf9) female mice are infertile. The onset of puberty and the estrus cycle in mutants are initially normal and ovaries contain all follicle classes. By the age of eight weeks, vaginal smears reveal irregular estrus cycles and mutant ovaries are completely depleted of follicles. Mutant oocytes retrieved from the oviduct are degenerated, and occasionally show an enlarged polar body, which may reflect a defective first meiotic division. Under fertilization conditions, the mutant oocytes do not enter into an embryonic development process. Furthermore, we conducted a comparative proteome analysis of wild type and Exosc10 knockout mouse ovaries, and identified EXOSC10-dependent proteins involved in many biological processes, such as meiotic cell cycle progression and oocyte maturation. Our results unambiguously demonstrate an essential role for EXOSC10 in oogenesis and may serve as a model for primary ovarian insufficiency in humans. Data are available via ProteomeXchange with identifier PXD039417.
Asunto(s)
Fenómenos Biológicos , Reserva Ovárica , Animales , Femenino , Humanos , Lactante , Ratones , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Oocitos/metabolismo , Oogénesis/genéticaRESUMEN
During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function, and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82 to 92%) during differentiation, presumably due to the shift from α4 to α4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between α4 and α4s. We propose that these differences induced by α4s incorporation result in significant changes in the way the s20S interacts with its partners and dictate its role in germ cell differentiation.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Espermatogénesis , Espermatogonias , Humanos , Masculino , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Espermatogonias/enzimologíaRESUMEN
Chlordecone (CD) is an insecticide that was used in the French West Indies for several years to control the banana root borer pest. Given its nonsignificant degradation, it persists in the environment. CD is a carcinogenic compound with reproductive and developmental toxicity and is a recognized endocrine-disrupting chemical. In this study, we examined the effects of CD on female reproductive system of mice with the focus on epigenetic features in ovary. Our data show that gestational exposure to low dose of CD affects meiotic double-strand breaks repair in female embryos. In adult mice derived from CD-treated pregnant females, we observed delayed puberty, decreased number of primordial and increased number of atretic follicles. Gene expression analysis revealed that Rcbtb2 and Rbpms genes were not expressed in embryonic gonads. Estrogen signaling- and oocyte maturation-associated genes were downregulated in adult ovaries. The morphological changes were associated with altered epigenetic features: increased H2Aub and increased H3K27me3 and decreased H4ac and H3K4me3 in embryonic oocytes. The DNA damage-associated, γH2AX marks were detected in the follicles of treated but not control adult ovaries. We also found reduced H3K4me3 and H4ac in fully grown oocytes of the treated ovaries. The ChIP-seq analysis of H3K4me3 in adult ovaries showed that target genes of ZFP57 and TRIM28, which regulate pluripotency and imprinting, were significantly enriched in altered regions. Our study clearly demonstrates that gestational exposure to a low dose of CD impairs the function of female reproductive system and the changes are associated with altered epigenetic features.
Asunto(s)
Clordecona/efectos adversos , Epigénesis Genética/efectos de los fármacos , Ovario/efectos de los fármacos , Animales , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Epigénesis Genética/genética , Epigenómica/métodos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genitales Femeninos/efectos de los fármacos , Insecticidas/efectos adversos , Ratones , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oogénesis/efectos de los fármacos , Ovario/metabolismo , Embarazo , Efectos Tardíos de la Exposición PrenatalRESUMEN
Glyphosate is the most widely used herbicide in the world. Several studies have investigated the effects of glyphosate and glyphosate-based herbicides (GBHs) on male reproduction, but there is still little and conflicting evidence for its toxicity. In this study, we analyzed the effects of glyphosate, alone or in formula, on the male reproductive system. Pregnant mice were treated from E10.5 to 20 days postpartum by adding glyphosate or a GBH (Roundup 3 Plus) to their drinking water at 0.5 (the acceptable daily intake, ADI dose), 5 and 50 mg/kg/day. Male offspring derived from treated mice were sacrificed at 5, 20, and 35 days old (d.o.) and 8 months old (m.o.) for analysis. Our result showed that exposure to glyphosate, but not GBH, affects testis morphology in 20 d.o. and decrease serum testosterone concentrations in 35 d.o. males. We identified that the spermatozoa number decreased by 89% and 84% in 0.5 and 5 mg/kg/day of GBH and glyphosate groups, respectively. Moreover, the undifferentiated spermatogonia numbers were decreased by 60% in 5 mg/kg/day glyphosate group, which could be due to the alterations in the expression of genes involved in germ cell differentiation such as Sall4 and Nano3 and apoptosis as Bax and Bcl2. In 8 m.o. animals, a decreased testosterone level was observed in GBH groups. Our data demonstrate that glyphosate and GBHs could cause endocrine-disrupting effects on male reproduction at low doses. As glyphosate has effects at the ADI level, our data suggest that the current ADI for glyphosate could be overestimated.
Asunto(s)
Disruptores Endocrinos/toxicidad , Glicina/análogos & derivados , Herbicidas/toxicidad , Infertilidad Masculina/inducido químicamente , Espermatogénesis/efectos de los fármacos , Espermatogonias/efectos de los fármacos , Testículo/efectos de los fármacos , Factores de Edad , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Glicina/toxicidad , Infertilidad Masculina/sangre , Infertilidad Masculina/patología , Infertilidad Masculina/fisiopatología , Masculino , Ratones , Nivel sin Efectos Adversos Observados , Embarazo , Efectos Tardíos de la Exposición Prenatal , Medición de Riesgo , Recuento de Espermatozoides , Espermatogonias/metabolismo , Espermatogonias/patología , Testículo/metabolismo , Testículo/patología , Testículo/fisiopatología , Testosterona/sangre , GlifosatoRESUMEN
Environmental factors can affect epigenetic events during germline reprogramming and impose distinctive transgenerational consequences onto the offspring. In this study, we examined the transgenerational effects of chlordecone (CD), an organochlorine insecticide with well-known estrogenic properties. We exposed pregnant mice to CD from embryonic day 6.5 to 15.5 and observed a reduction in spermatogonia (SG) numbers in F3, meiotic defects in spermatocytes and decrease in spermatozoa number in the first and third generation of male progeny. The RNA qRT-PCR expression analysis in F1 and transcriptomics analysis in F3 males using the whole testes revealed changes in the expression of genes associated with chromosome segregation, cell division and DNA repair. The expression of the master regulator of pluripotency, Pou5f1, decreased in foetal and increased in adult F1, but not in F3 adult testes. Analysis of histone H3K4me3 distribution revealed widespread changes in its occupancy in the genome of F1 and F3 generations. We established that 7.1% of altered epigenetic marks were conserved between F1 and F3 generations. The overlapping changes common to F1 and F3 include genes implicated in cell adhesion and transcription factor activities functions. Differential peaks observed in F1 males are significantly enriched in predicted ESR1 binding sites, some of which we confirmed to be functional. Our data demonstrate that CD-mediated impairment of reproductive functions could be transmitted to subsequent generations.
Asunto(s)
Clordecona/toxicidad , Epigénesis Genética , Regulación de la Expresión Génica/efectos de los fármacos , Insecticidas/toxicidad , Exposición Materna/efectos adversos , Efectos Tardíos de la Exposición Prenatal/patología , Espermatogonias/patología , Animales , Metilación de ADN , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Histonas/metabolismo , Masculino , Ratones , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Recuento de Espermatozoides , Espermatogonias/efectos de los fármacosRESUMEN
EXOSC10 is a catalytic subunit of the exosome that processes biologically active transcripts, degrades aberrant mRNAs and targets certain long non-coding RNAs (lncRNAs). The yeast orthologue Rrp6 is required for efficient growth and gametogenesis, and becomes unstable during meiosis. However, nothing is known about the localization, stability and function of EXOSC10 in the rodent male germline. We detect the protein in nucleoli and the cytoplasm of mitotic and meiotic germ cells, and find that it transiently associates with the XY body, a structure targeted by meiotic sex chromosome inactivation (MSCI). Finally, EXOSC10 becomes unstable at later stages of gamete development. To determine Exosc10's meiotic function, we inactivated the gene specifically in male germ cells using cre recombinase controlled by Stra8 or Ddx4/Vasa promoters. Mutant mice have small testes, show impaired germ cell differentiation and are subfertile. Our results demonstrate that EXOSC10 is post-translationally regulated in germ cells, associate the protein with epigenetic chromosome silencing, and reveal its essential role in germ cell growth and development.
Asunto(s)
Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Espermatogénesis/genética , Animales , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Femenino , Fertilidad/genética , Masculino , Meiosis/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas Sprague-Dawley , Testículo/metabolismoRESUMEN
The widely-used herbicide atrazine (ATZ) is detected in ground and surface water in many countries. Several studies in animals have demonstrated that ATZ has endocrine-disrupting effects on male and female reproduction in many vertebrate species. In this study, we investigated the effects of ATZ exposure on meiosis, a key step in gametogenesis in mammals. The treatment was initiated before oocyte entry into meiosis, which occurs during the embryonic period in females. We found that embryonic exposure to ATZ increases the level of 8-oxo-guanine in the nucleus of meiotic cells, reflecting oxidative stress and affecting meiotic double-strand break repair, chromosome synapsis and crossover numbers. Finally, embryonic exposure to ATZ reduces the number of primordial follicles and increases the incidence of multi-oocyte follicles in adult mice. Our data demonstrate that embryonic exposure to ATZ disrupts prophase I of meiosis and affects normal follicle formation in female mice.
Asunto(s)
Atrazina/toxicidad , Herbicidas/toxicidad , Meiosis/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/embriología , Animales , Femenino , Incidencia , RatonesRESUMEN
The epigenetic events imposed during germline reprogramming and affected by harmful exposure can be inherited and transferred to subsequent generations via gametes inheritance. In this study, we examine the transgenerational effects promoted by widely used herbicide atrazine (ATZ). We exposed pregnant outbred CD1 female mice and the male progeny was crossed for three generations with untreated females. We demonstrate here that exposure to ATZ affects meiosis, spermiogenesis and reduces the spermatozoa number in the third generation (F3) male mice. We suggest that changes in testis cell types originate from modified transcriptional network in undifferentiated spermatogonia. Importantly, exposure to ATZ dramatically increases the number of transcripts with novel transcription initiation sites, spliced variants and alternative polyadenylation sites. We found the global decrease in H3K4me3 occupancy in the third generation males. The regions with altered H3K4me3 occupancy in F3 ATZ-derived males correspond to altered H3K4me3 occupancy of F1 generation and 74% of changed peaks in F3 generation are associated with enhancers. The regions with altered H3K4me3 occupancy are enriched in SP family and WT1 transcription factor binding sites. Our data suggest that the embryonic exposure to ATZ affects the development and the changes induced by ATZ are transferred up to three generations.
Asunto(s)
Atrazina/efectos adversos , Exposición a Riesgos Ambientales/efectos adversos , Epigénesis Genética/efectos de los fármacos , Herbicidas/efectos adversos , Histonas/metabolismo , Efectos Tardíos de la Exposición Prenatal , Transcripción Genética/efectos de los fármacos , Animales , Sitios de Unión , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Exposición Materna , Meiosis/efectos de los fármacos , Metilación/efectos de los fármacos , Ratones , Motivos de Nucleótidos , Especificidad de Órganos/genética , Posición Específica de Matrices de Puntuación , Embarazo , Unión Proteica , ARN Largo no Codificante/genética , Espermatogénesis/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Testículo/efectos de los fármacos , Testículo/metabolismo , Testículo/patologíaRESUMEN
BACKGROUND: Environmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. We studied the effects of the widely used herbicide atrazine (ATZ) on meiosis, a key step of gametogenesis, in male mice. METHODS: Gene expression pattern was analysed by Gene-Chip array. Genome-wide mapping of H3K4me3 marks distribution was done by ChIP-sequencing of testis tissue using Illumina technologies. RT-qPCR was used to validate differentially expressed genes or differential peaks. RESULTS: We demonstrate that exposure to ATZ reduces testosterone levels and the number of spermatozoa in the epididymis and delays meiosis. Using Gene-Chip and ChIP-Seq analysis of H3K4me3 marks, we found that a broad range of cellular functions, including GTPase activity, mitochondrial function and steroid-hormone metabolism, are affected by ATZ. Furthermore, treated mice display enriched histone H3K4me3 marks in regions of strong recombination (double-strand break sites), within very large genes and reduced marks in the pseudoautosomal region of X chromosome. CONCLUSIONS: Our data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production.
Asunto(s)
Atrazina/farmacología , Epigénesis Genética/efectos de los fármacos , Herbicidas/farmacología , Meiosis/efectos de los fármacos , Meiosis/genética , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Supervivencia Celular , Inmunoprecipitación de Cromatina , Biología Computacional/métodos , Roturas del ADN de Doble Cadena/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Hormonas Esteroides Gonadales/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/metabolismo , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Motivos de Nucleótidos , Posición Específica de Matrices de Puntuación , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Recuento de Espermatozoides , Testículo/efectos de los fármacos , Testículo/metabolismo , Testosterona/sangreRESUMEN
Genome-wide RNA profiling studies have identified hundreds of transcripts that are highly expressed in mammalian male germ cells, including many that are undetectable in somatic control tissues. Among them, genes important for spermatogenesis are significantly enriched. Information about mRNAs and their cognate proteins facilitates the identification of novel conserved target genes for functional studies in the mouse. By inspecting genome-wide RNA profiling data, we manually selected 81 genes for which RNA is detected almost exclusively in the human male germline and, in most cases, in rodent testicular germ cells. We observed corresponding mRNA/protein patterns in 43 cases using immunohistochemical data from the Human Protein Atlas and large-scale human protein profiling data obtained via mass spectroscopy. Protein network information enabled us to establish an interaction map of 38 proteins that points to potentially important testicular roles for some of them. We further characterized six candidate genes at the protein level in the mouse. We conclude that conserved genes induced in testis tend to show similar mRNA/protein expression patterns across species. Specifically, our results suggest roles during embryogenesis and adult spermatogenesis for Foxr1 and Sox30 and during spermiogenesis and fertility for Fam71b, 1700019N19Rik, Hmgb4, and Zfp597.
Asunto(s)
Perfilación de la Expresión Génica , Redes Reguladoras de Genes/genética , Estudio de Asociación del Genoma Completo , Análisis por Matrices de Proteínas , ARN Mensajero/genética , Espermatogénesis/genética , Secuencia de Aminoácidos , Animales , Fertilidad/genética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de la Especie , Factores de Transcripción/genéticaRESUMEN
It was recently reported that the sizes of many mRNAs change when budding yeast cells exit mitosis and enter the meiotic differentiation pathway. These differences were attributed to length variations of their untranslated regions. The function of UTRs in protein translation is well established. However, the mechanism controlling the expression of distinct transcript isoforms during mitotic growth and meiotic development is unknown. In this study, we order developmentally regulated transcript isoforms according to their expression at specific stages during meiosis and gametogenesis, as compared to vegetative growth and starvation. We employ regulatory motif prediction, in vivo protein-DNA binding assays, genetic analyses and monitoring of epigenetic amino acid modification patterns to identify a novel role for Rpd3 and Ume6, two components of a histone deacetylase complex already known to repress early meiosis-specific genes in dividing cells, in mitotic repression of meiosis-specific transcript isoforms. Our findings classify developmental stage-specific early, middle and late meiotic transcript isoforms, and they point to a novel HDAC-dependent control mechanism for flexible transcript architecture during cell growth and differentiation. Since Rpd3 is highly conserved and ubiquitously expressed in many tissues, our results are likely relevant for development and disease in higher eukaryotes.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Histona Desacetilasas/metabolismo , Meiosis/genética , Mitosis/genética , Isoformas de ARN/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Mutación , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Subunidades de Proteína/metabolismo , Isoformas de ARN/genética , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sitio de Iniciación de la Transcripción , Regiones no Traducidas , Proteínas de Transporte Vesicular/genética , Factores de Escisión y Poliadenilación de ARNm/genética , ARNt MetiltransferasasRESUMEN
In a recent proteomic study of rat spermatogenesis, we identified CLPH (for Casein-Like PHosphoprotein), a new testis-specific protein expressed exclusively in postmeiotic germ cells. In situ hybridization showed that the CLPH transcript was mainly present in round spermatids, whereas the protein was specifically detected by immunohistochemistry in elongated spermatids and in residual bodies. Electron microscopy showed the protein to be mostly cytoplasmic, but also frequently associated with the mitochondrial inner membrane during the last steps of spermatid differentiation. The Clph gene was found to be present solely in mammalian genomes, in a chromosomal region syntenic to the mammalian cluster of secretory calcium-binding phosphoprotein (SCPP) genes. CLPH has several distinctive properties in common with SCPPs: calcium overlay experiments showed that CLPH was a calcium-binding protein, whereas trypsin digestion assay, circular dichroism and fluorescence experiments demonstrated its intrinsically disordered structure. We also showed that CLPH was phosphorylated in vitro and in vivo by casein kinase 2, an enzyme critical for spermatid elongation. Given the specific and strong production of CLPH during rat spermiogenesis, together with the particular biochemical properties of this protein, we suggest that CLPH is involved in the extremely complex structural rearrangements occurring in haploid germ cells during spermiogenesis.
Asunto(s)
Caseínas/metabolismo , Espermátides/metabolismo , Espermatogénesis , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína II/metabolismo , Caseínas/química , Caseínas/genética , Simulación por Computador , Electroforesis en Gel Bidimensional , Humanos , Hibridación in Situ , Masculino , Espectrometría de Masas , Meiosis , Datos de Secuencia Molecular , Fosforilación , Pliegue de Proteína , Proteómica , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/citología , Testículo/metabolismoRESUMEN
One of the major factors determining the incidence of Barley yellow dwarf virus (BYDV) on autumn-sown cereals is the viruliferous state of immigrant winged aphids. This variable is assessed routinely using the enzyme-linked immunosorbant assay (ELISA). However, the threshold for virus detection by ELISA can lead to false negative results for aphids carrying less than 10(6) particles. Although molecular detection techniques enabling the detection of lower virus quantities in samples are available, the relatively laborious sample preparation and data analysis have restricted their use in routine applications. A gel-free real-time one-step reverse transcription polymerase chain reaction (RT-PCR) protocol is described for specific detection and quantitation of BYDV-PAV, the most widespread BYDV species in Western Europe. This new assay, based on TaqMan technology, detects and quantifies from 10(2) to 10(8) BYDV-PAV RNA copies. This test is 10 and 10(3) times more sensitive than the standard RT-PCR and ELISA assays published previously for BYDV-PAV detection and significantly improves virus detection in single aphids. Extraction of nucleic acids from aphids using either phenol/chloroform or chelatin resin-based protocols allow the use of pooled samples or of a small part (up to 1/1600th) of a single aphid extract for efficient BYDV-PAV detection.
Asunto(s)
Áfidos/virología , Hordeum/virología , Luteovirus/aislamiento & purificación , Enfermedades de las Plantas/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Polimerasa Taq/metabolismo , Animales , Secuencia de Bases , Ensayo de Inmunoadsorción Enzimática , Colorantes Fluorescentes , Luteovirus/genética , Datos de Secuencia Molecular , Virus de Plantas/aislamiento & purificación , ARN Viral/genética , ARN Viral/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
Successful mechanical inoculation of plant with viruses requires an efficient method to introduce the viral pathogen into the appropriate cells of the plant. Barley yellow dwarf virus-PAV (BYDV-PAV, Luteovirus), transmitted naturally by aphids, must be inoculated into the phloem tissue to infect systemically inoculated hosts. The particle bombardment method used widely for nucleic acid transfer into plant tissues was adapted to inoculate immature embryos of winter and spring wheat cultivars with either BYDV-PAV particles or viral full-length RNAs. DAS-ELISA and RT-PCR were carried out on extracts of developed leaves at 7 weeks post-bombardment and revealed that up to 14% of bombarded embryos produced BYDV-infected wheat plants. This is the first report of an aphid-free inoculation method for BYDV.