RESUMO
Control of fanleaf disease induced by the Grapevine Fanleaf Nepovirus (GFLV) today is based on sanitary selection and soil disinfection with nematicides. This way of control is not always efficient and nematicides can be dangerous pollutants. Coat protein (CP) mediated protection could be an attractive alternative. We have transferred a chimeric CP gene of GFLV-F13 via Agrobacterium tumefaciens LBA4404 into two rootstock varieties: Vitis rupestris and 110 Richter (V. rupestris X V. Berlandieri). Transformation was performed on embryogenic callus obtained from anthers and on hypocotyl fragments from mature embryos. Success of the transformation was assessed by polymerase chain reaction and Southern analyses. Transformants with a number of copies of the CP gene varying from one to five were obtained. Enzyme-linked immunosorbent assay with virus-specific antibodies revealed various levels of expression of the coat protein in the different transformants.
RESUMO
The presence of a genome-linked protein (VPg) at the RNA 5'-end of the genome is a characteristic of different groups of animal and plant positive-sense single-stranded RNA viruses. These viruses express their structural and functional proteins from polyproteins that are sequentially processed by at least one viral proteinase. The grapevine fanleaf nepovirus 24K chymotrypsin-like cysteine proteinase, located between the VPg and the RNA polymerase in the RNA-1 encoded polyprotein P1, is active in its free form and in various precursors forms. The VPg proteinase precursor (VPg-Pro) constitutes a stable protein and its maturation in the reticulocyte lysate system occurs at a very low rate. Differences on cleavage activity were observed between the proteinase and its VPg-Pro precursor forms, depending upon the cleavage site considered. The proteinase alone has a greater cleavage efficiency than VPg-Pro at the Arg605/Gly606 and Cys257/Ala258 sites of polyprotein P2. On the other hand, the presumed Cys415/Ala416 site, present at the amino terminus of polyprotein P1, was preferentially cleaved by the VPg-Pro precursor. During their in vitro maturation, proteins containing the VPg proteinase-polymerase coding region or the proteinase-polymerase region were similar in their ability to cleave in cis between the proteinase and the RNA polymerase.
Assuntos
Cisteína Endopeptidases/metabolismo , Nepovirus/enzimologia , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas do Core Viral/metabolismo , Sequência de Bases , Cisteína Endopeptidases/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Dados de Sequência Molecular , Nepovirus/genética , Precursores de Proteínas/genética , Proteínas do Core Viral/genéticaRESUMO
Grapevine fanleaf nepovirus (GFLV) is responsible for the economically significant "court-noué" disease in vineyards. Its genome is made up of two single-stranded RNA molecules (RNA1 and RNA2) which direct the synthesis of polyproteins P1 and P2 respectively. A chimeric coat protein gene derived from the C-terminal part of P2 was constructed and subsequently introduced into a binary transformation vector. Transgenic Nicotiana benthamiana plants expressing the coat protein under the control of the CaMV 35S promoter were engineered by Agrobacterium tumefaciens-mediated transformation. Protection against infection with virions or viral RNA was tested in coat protein-expressing plants. A significant delay of systemic invasion was observed in transgenic plants inoculated with virus compared to control plants. This effect was also observed when plants were inoculated with viral RNA. No coat protein-mediated cross-protection was observed when transgenic plants were infected with arabis mosaic virus (ArMV), a closely related nepovirus also responsible for a "court-noué" disease.
RESUMO
We have constructed a series of promoterprobe cassettes that provides powerful tools for insertion mutagenesis, transcription fusions and genetic constructions. These cassettes contain the Tn9 chloramphenicol (CmR) and the Tn903 kanamycin (KmR) resistance genes which are expressed in a large variety of microorganisms; these antibiotic-resistance markers were associated with the uidA promoterless gene. This beta-glucuronidase-encoding gene of Escherichia coli K-12 has been successfully used as reporter gene for various organisms including prokaryotes and eukaryotes. The resulting 'uidA-KmR and 'uidA-CmR cassettes (truncated at the ') can be excized with most of the commonly used restriction enzymes. Furthermore, they are borne by ApR or CmR plasmids which facilitate their utilization. These promoter-probe cassettes allow transcriptional signal localization and regulation studies.
Assuntos
Elementos de DNA Transponíveis/genética , Resistência Microbiana a Medicamentos/genética , Vetores Genéticos/genética , Mutagênese Insercional/métodos , Plasmídeos/genética , Resistência ao Cloranfenicol/genética , DNA Recombinante/genética , Endodesoxirribonucleases , Glucuronidase/genética , Resistência a Canamicina/genética , Regiões Promotoras Genéticas/genéticaRESUMO
New promoter-probe and terminator-probe shuttle vectors for Escherichia coli and corynebacteria were constructed. These vectors, working with the uidA gene of E. coli as reporter gene, are very useful for the cloning and subsequent analysis of transcriptional regulatory signals. The beta-glucuronidase encoding activity of uidA can be easily detected on agar plates containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-gluc). In the terminator-probe vector pUT2, uidA is expressed from a promoter of the Brevibacterium lactofermentum cryptic plasmid pBL1. Multiple cloning sites (MCS) located immediately upstream of uidA allow introduction and selection of terminators or regulatory signals. In the promoter-probe vector pUT3, transcription readthrough from vector promoters is prevented by a terminator of B. lactofermentum isolated using pUT2. We have successfully used pUT2 and pUT3 to isolate several terminators and promoter regions active in B. lactofermentum and shown that the E. coli strong terminator cartridge omega appears less efficient in corynebacteria.
Assuntos
Corynebacterium/genética , Vetores Genéticos , Plasmídeos , Transcrição Gênica , Sequência de Bases , Brevibacterium/genética , Clonagem Molecular , DNA Bacteriano , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA BacterianoRESUMO
We have constructed promoter-probe plasmids, pNB4 and pNB5, based on the promoterless gene for beta-glucuronidase (uidA) of Escherichia coli. Unique restriction sites for EcoRI, SacI, KpnI, SmaI, XmaI, XbaI, SalI, SphI and HindIII in pNB4 and for HindIII, PstI and BglII in pNB5 were included upstream of the uidA structural gene. The usefulness of these plasmids was demonstrated by cloning the promoter-operator region of the E. coli uxaB gene. We observed that expression of the uxaB-uidA operon fusion followed the transcription-regulating properties of the uxaB promoter. Another construct, pNB2, can be used to detect operator and terminator signals. Recipient cells transformed with such recombinant plasmids can be revealed by growth on medium containing a chromogenic beta-glucuronidase substrate.
