A novel strategy for the expression of foreign genes from plant virus vectors.

Potato virus X (PVX)-based vector constructs were generated to investigate the use of an internal ribosome entry site (IRES) sequence to direct translation of a viral gene. The 148-nucleotide IREScp sequence from a crucifer-infecting strain of tobacco mosaic virus was used to direct expression of the PVX coat protein (CP). The IRES was inserted downstream of the gene encoding green fluorescent protein (GFP) and upstream of the PVX CP, in either sense or antisense orientation, such that CP expression depended on ribosome recruitment to the IRES. Stem-loop structures were inserted at either the 3'- or 5'-end of the IRES sequence to investigate its mode of action. In vitro RNA transcripts were inoculated to Nicotiana benthamiana plants and protoplasts: levels of GFP and CP expression were analysed by enzyme-linked immunosorbent assay and the rate of virus cell-to-cell movement was determined by confocal laser scanning microscope imaging of GFP expression. PVX CP was expressed, allowing cell-to-cell movement of virus, from constructs containing the IRES sequence in either orientation, and from the construct containing a stem-loop structure at the 5'-end of the IRES sequence. No CP was expressed from a construct containing a stem-loop at the 3'-end of the IRES sequence. Our results suggest that the IRES sequence is acting in vivo to direct expression of the 3'-proximal open reading frame in a bicistronic mRNA thereby demonstrating the potential of employing IRES sequences for the expression of foreign proteins from plant virus-based vectors.

[Vagal chemodectoma with atypical cells. Case report]. / Quemodectoma vagal con atipias celulares. Presentación de un caso.

We report a clinical case of vagal chemodectoma with cell atypies, which was presented as slowly growing neck mass without any other symptoms. At the beginning and due to the histologic report of the needle biopsy, which morphologic study based on cellular atypia was misinterpreted as a malignant tumor and with the images obtained by computed tomography (CT) suggesting a malignant neoplasia, we proceeded to approach the case as a metastatic tumor of unknown origin. We made a revision of the epidemiologic factors, and diagnostic and therapeutic management of this type of growths.

Bax-induced cell death in tobacco is similar to the hypersensitive response.

Bax, a death-promoting member of the Bcl-2 family of proteins, triggered cell death when expressed in plants from a tobacco mosaic virus vector. Analysis of Bax deletion mutants demonstrated a requirement for the BH1 and BH3 domains in promoting rapid cell death, whereas deletion of the carboxyl-terminal transmembrane domain completely abolished the lethality of Bax in plants. The phenotype of cell death induced by Bax closely resembled the hypersensitive response induced by wild-type tobacco mosaic virus in tobacco plants carrying the N gene. The cell death-promoting function of Bax in plants correlated with accumulation of the defense-related protein PR1, suggesting Bax activated an endogenous cell-death program in plants. In support of this view, both N gene- and Bax-mediated cell death was blocked by okadaic acid, an inhibitor of protein phosphatase activity. The ability of Bax to induce cell death and a defense reaction in plants suggests that some features of animal and plant cell death processes may be shared.

Perspective: phloem transport of viruses and macromolecules - what goes in must come out.

Phloem transport of endogenous macromolecules and plant viruses remains poorly understood. Selective movement into and out of the phloem is tightly regulated, yet the mechanisms governing this selectivity have not been elucidated. Recent advances in identifying nonviral proteins and nucleic acids with the capacity for phloem transport will hopefully shed new light on the regulation of phloem loading and unloading.

Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves.

Leaves undergo a sink-source transition during which a physiological change occurs from carbon import to export. In sink leaves, biolistic bombardment of plasmids encoding GFP-fusion proteins demonstrated that proteins with an Mr up to 50 kDa could move freely through plasmodesmata. During the sink-source transition, the capacity to traffic proteins decreased substantially and was accompanied by a developmental switch from simple to branched forms of plasmodesmata. Inoculation of sink leaves with a movement protein-defective virus showed that virally expressed GFP, but not viral RNA, was capable of trafficking between sink cells during infection. Contrary to dogma that plasmodesmata have a size exclusion limit below 1 kDa, the data demonstrate that nonspecific "macromolecular trafficking" is a general feature of simple plasmodesmata in sink leaves.

Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector.

We have used a plant virus episomal vector, based on potato virus X (PVX) to transiently express a single-chain Fv (scFv) and its diabody derivative in plants. The scFv was directed against a continuous epitope (cryptotope) on the coat protein of potato virus V. A cloned, full-length PVX vector sequence, containing the scFv gene, was used to direct in vitro transcription and the resulting RNA was used to inoculate Nicotiana clevelandii plants. Within a few days, plants developed characteristic symptoms and immunoblot analysis showed that accumulation of scFv protein coincided with accumulation of PVX. Targeting of the scFv to the apoplast greatly increased protein accumulation compared with cytosolic scFv and produced more severe symptoms on infected plants. ELISA demonstrated that the scFv and diabody extracted from infected plants showed the same antigen-binding specificity as that of the parental monoclonal antibody. The PVX vector is a convenient, rapid, low-cost in planta expression system that can also be used for assessment of scFv production and function prior to stable plant transformation.

Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network.

We have visualized the relationship between the endoplasmic reticulum (ER) and Golgi in leaf cells of Nicotiana clevelandii by expression of two Golgi proteins fused to green fluorescent protein (GFP). A fusion of the transmembrane domain (signal anchor sequence) of a rat sialyl transferase to GFP was targeted to the Golgi stacks. A second construct that expressed the Arabidopsis H/KDEL receptor homologue aERD2, fused to GFP, was targeted to both the Golgi apparatus and ER, allowing the relationship between these two organelles to be studied in living cells for the first time. The Golgi stacks were shown to move rapidly and extensively along the polygonal cortical ER network of leaf epidermal cells, without departing from the ER tubules. Co-localization of F-actin in the GFP-expressing cells revealed an underlying actin cytoskeleton that matched precisely the architecture of the ER network, while treatment of cells with the inhibitors cytochalasin D and N-ethylmaleimide revealed the dependency of Golgi movement on actin cables. These observations suggest that the leaf Golgi complex functions as a motile system of actin-directed stacks whose function is to pick up products from a relatively stationary ER system. Also, we demonstrate for the first time in vivo brefeldin A-induced retrograde transport of Golgi membrane protein to the ER.

HLA-DR2 gene and Spanish patients with ulcerative colitis.

BACKGROUND: An association with class II MHC genes has been described in ulcerative colitis, as in other diseases with immunological pathogenesis. Heterogeneous results have been reported, depending on the studied population. OBJECTIVE: To study the importance of these genes in our population, mainly the alleles of group HLA DR2 (gene HLA-DRB1). PATIENTS AND METHODS: We studied a total of 107 patients diagnosed of ulcerative colitis and 200 unaffected controls. Complete information about sex, age, family antecedent, age of onset, localization, complications, surgery and treatment was obtained from these patients. DNA was extracted from peripheral blood leukocytes and all the individuals were HLA-DRB1 genotyping. RESULTS AND CONCLUSIONS: We conclude that a positive association exists between DR15 and ulcerative colitis (p < 0.05). This positive association was characterized and various clinical parameters were analyzed, being concluded that DR1501 is more frequent in this disease (p < 0.05) and in benign manifestations; the frequency of the allele DR1502 was also found to be elevated in severe manifestations.

Intracellular location of two groundnut rosette umbravirus proteins delivered by PVX and TMV vectors.

The proteins encoded by open reading frames (ORF) 3 and 4 of groundnut rosette umbravirus (GRV) were expressed in Nicotiana benthamiana as fusions with green fluorescent protein (GFP) from modified potato virus X (PVX) and tobacco mosaic virus (TMV) vectors. Regardless of which plant virus vector was used, GFP fused to the ORF3 protein accumulated in large cytoplasmic inclusion bodies and in nucleoli, whereas GFP fused to the ORF4 protein was found in cell walls close to plasmodesmata. Cell-to-cell movement of PVX requires three proteins encoded by the triple gene block (TGB) and also the coat protein (CP). However, when GRV ORF4 was substituted for the PVX CP gene, the hybrid virus was able to move normally in inoculated leaves but not into noninoculated leaves. In contrast, when GRV ORF4 was substituted for the TGB, or for both the TGB and the CP gene, movement of the hybrid viruses was limited to a few epidermal cells neighboring the infection site. Thus, the GRV ORF4 protein can replace the movement proteins of PVX for some of their functions.

Production of a functional single chain antibody attached to the surface of a plant virus.

A potato virus X (PVX) vector was used to express a single chain antibody fragment (scFv) against the herbicide diuron, as a fusion to the viral coat protein. The modified virus accumulated in inoculated Nicotiana clevelandii plants and assembled to give virus particles carrying the antibody fragment. Electron microscopy was used to show that virus particles from infected leaf sap were specifically trapped on grids coated with a diuron-BSA conjugate. The results demonstrate that the PVX vector can be used as a presentation system for functional scFv.

Positive and negative associations of distinct HLA-DR2 subtypes with ulcerative colitis (UC).

In UC, as in many other diseases with a suspected autoimmune etiology or pathogenesis, an association with certain HLA class II specificities has been investigated. In the Japanese, several studies have shown a positive association with DR2, but in Caucasian populations the results are conflicting. Therefore we undertook a study of HLA class II gene association with UC in a large group of white Madrid patients and ethnically matched controls, using molecular biology techniques to investigate whether any allelic subspecificity within the HLA-DR2 group is associated with susceptibility to or protection against UC. Patients with ulcerative colitis (n = 107) and 200 controls were typed using molecular, DNA-based techniques for HLA-DRB1, DQA1 and DQB1 alleles. Those HLA-DR2+ were then specifically typed for the individual alleles within the HLA-DR2 group. We observed a positive association with HLA-DR15 (P=0.021) and its subtype DRB1*1501 (P=0.018). HLA-DRB1*1502 was also increased, although its frequency both in patients and controls was very low. When the HLA-DR2+ population was studied, HLA-DRB1*1601 was significantly decreased in patients (P=0.026). Both HLA-DR3 (P=0.002) and HLA-DQB1*02 (P=0.001) were also negatively associated with the disease, the latter especially with pancolitis. Therefore, HLA class II association with UC is complex, and separate alleles confer either susceptibility or resistance. Conflicting results with HLA-DR2 appear to be due to the presence in this group of both positively associated (HLA-DRB1*1501 and DRB1*1502) and negatively associated (HLA-DRB1*1601) subspecificities. Moreover, HLA-DR3 and HLA-DQB1*02 are associated negatively.

Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV).

Plasmodesmatal gating in epidermal cells of Nicotiana tabacum was examined in expanding infection sites of tobacco mosaic virus (TMV) expressing a fusion between the viral movement protein and the green fluorescent protein (MP-GFP). The infection sites were circular in profile and within 3 days post-inoculation had developed a brightly fluorescent leading edge, giving them a characteristic 'halo' shape. Co-localization of MP-GFP with callose demonstrated that nearly all epidermal cell plasmodesmata were targeted with MP-GFP. The fusion protein was located in the centre of the plasmodesmal pore, between paired callose platelets. Increase in plasmodesmatal size exclusion limit, as determined by the passage of microinjected 10 kDa Texas Red dextran, was restricted predominantly to cells within the fluorescent halo, and was virtually absent from cells in the centre of the expanding infection site. The plasmodesmata of these cells, however, remained fluorescently labelled with MP-GFP. Injections outside the fluorescent infection site failed to show movement of dextran, while dextran injected into cells at the leading edge moved inwards towards the centre of the lesion but not outwards into cells lacking GFP. Leaf incisions through cells ahead of the infection front halted the advance of the virus, indicating that virus replication was absent in non-fluorescent cells outside the infection site. The data provide the first demonstration that within an expanding infection site plasmodesmatal gating is under temporal control.

[Kukuchi's disease of the neck: report of two cases]. / Enfermedad de Kikuchi de localización cervical. Presentación de dos casos.

Kikuchi's disease is an unusual disease, described in 1972, that is characterized by lymph node enlargement and fever. Cervical lymph nodes are affected so often that the ear, nose and throat specialist should be aware of this entity during differential diagnosis. Two cases treated in our service are reported and the literature is reviewed.

[Bilateral Warthin tumors. Case report and review of literature]. / Bilateralidad en los tumores de Warthin. Presentación de un caso y revisión de la literatura.

Tumors of the salivary glands are fairly frequent among the tumors of the head and neck. Cystoadenolymphoma is a benign salivary tumor. A case of bilateral Warthin tumor was diagnosed eight years after the appearance of the first homolateral lesion. The diagnosis, literature review, and treatment are discussed.

Analysis of potato virus X coat protein genes in relation to resistance conferred by the genes Nx, Nb and Rx1 of potato.

The coat protein gene nucleotide sequences from eight previously uncharacterized strains of potato virus X (PVX) were determined. Comparison of the deduced amino acid sequences showed that two classes of PVX coat protein, designated types X and B, could be distinguished based on protein length and overall amino acid identities. In all there were 14 amino acid positions where all of the type X proteins differed from all of the type B proteins. The PVX coat protein is the principal viral determinant of the outcome of interactions between the virus and potatoes carrying either the Nx or Rx1 resistance genes. The different strains of PVX were tested for their ability to overcome resistance conferred by three potato resistance genes: Nx, Nb and Rx1. All of the strains that were avirulent on potato cultivars carrying the Nx resistance gene were found to have type X coat proteins whereas strains capable of overcoming the Nx resistance had type B coat proteins.

Jellyfish green fluorescent protein as a reporter for virus infections.

The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.

A feature of the coat protein of potato virus X affects both induced virus resistance in potato and viral fitness.

The coat protein of PVX determines whether isolates of PVX are affected by Rx-mediated resistance in potato. Isolates with the coat protein of PVXHB are not affected by the resistance; those with the coat protein of PVXUK3 elicit an extreme resistance in the Rx potato that prevents virus accumulation, even on the inoculated leaf. In this paper we describe the analysis of a series of hybrid and mutant isolates of PVXHB and PVXCP4 which were inoculated to plants and protoplasts of Rx and rx cultivars of potato. From the virulence phenotypes of these isolates we conclude that elicitation of the resistance is affected by amino acids 121 and 127 of the viral coat protein, with codon 121 being the major determinant. PVXHB and hybrid or mutant isolates with lysine and arginine at positions 121 and 127 were able to overcome the resistance of Rx, whereas those with threonine and arginine were resistance sensitive both on plants and in protoplasts. The viral isolates with single mutations at either codon 121 or 127 were less infectious than the wild-type or double mutant isolates although, in protoplasts of the susceptible cultivar of potato, they accumulated as well as the wild-type virus. Taken together these data suggest that amino acids 121 and 127 affect a feature of the viral coat protein which may interact with cellular components involved in the spread of PVX and with the product of the Rx resistance gene.

Molecular analysis of a resistance-breaking strain of potato virus X.

Full-length cDNA clones of potato virus X (PVX) strains PVXUK3 and PVXHB have been constructed in plasmid vectors to allow in vitro transcription of infectious PVX RNA. In both instances the transcript-derived virus infected tobacco and potato identically to the respective progenitor strains: in tobacco and susceptible potato cultivars both strains infected systemically, producing symptomless or mild mosaic symptoms. In potato carrying the Rx or Nx resistance genes, the virus derived from the PVXHB cDNA infected systemically, whereas the virus derived from the PVXUK3 cDNA failed to infect the Rx plants or induced apical necrosis, characteristic of a hypersensitive response of the Nx gene. Three hybrid viral genomes were constructed at the cDNA level to localize the resistance breaking determinants of PVXHB. Transcripts of all three hybrids were infectious on tobacco. On potato cultivars with either the Rx or Nx resistance genes, the hybrid viruses infected in the same way as PVXHB, rather than PVXUK3. The common feature of these hybrid viruses, the coat protein gene, is therefore the determinant of Nx and Rx resistance breaking of PVXHB.