RESUMEN
Intercellular spread of plant viruses involves passage of the viral genome or virion through a plasmodesma (PD). Some viruses severely modify the PD structure, as they assemble a virion carrying tubule composed of the viral movement protein (MP) inside the PD channel. Successful modulation of the host plant to allow infection requires an intimate interaction between viral proteins and both structural and regulatory host proteins. To date, however, very few host proteins are known to promote virus spread. Plasmodesmata-located proteins (PDLPs) localised in the PD have been shown to contribute to tubule formation in cauliflower mosaic virus and grapevine fanleaf virus infections. In this study, we have investigated the role of PDLPs in intercellular transport of another tubule-forming virus, cowpea mosaic virus. The MP of this virus was found to interact with PDLPs in the PD, as was shown for other tubule-forming viruses. Expression of PDLPs and MPs in protoplasts in the absence of a PD revealed that these proteins do not co-localise at the site of tubule initiation. Furthermore, we show that tubule assembly in protoplasts does not require an interaction with PDLPs at the base of the tubule, as has been observed in planta. These results suggest that a physical interaction between MPs and PDLPs is not required for assembly of the movement tubule and that the beneficial role of PDLPs in virus movement is confined to the structural context of the PD.
Asunto(s)
Comovirus/fisiología , Nicotiana/virología , Proteínas de Plantas/metabolismo , Proteínas de Movimiento Viral en Plantas , Plasmodesmos/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Hojas de la Planta/fisiología , Hojas de la Planta/virología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Transporte de Proteínas , Nicotiana/genética , Nicotiana/fisiologíaRESUMEN
The movement protein (MP) of Cowpea mosaic virus forms tubules in plasmodesmata to enable the transport of mature virions. Here it is shown that the MP is capable of specifically binding riboguanosine triphosphate and that mutational analysis suggests that GTP binding plays a role in the targeted transport of the MP. Furthermore, the MP is capable of binding both single-stranded RNA and single-stranded DNA in a non-sequence-specific manner, and the GTP- and RNA-binding sites do not overlap.
Asunto(s)
Comovirus/metabolismo , ADN de Cadena Simple/metabolismo , ADN Viral/metabolismo , Guanosina Trifosfato/metabolismo , ARN Viral/metabolismo , Proteínas Virales/metabolismo , Animales , Células Cultivadas , Comovirus/fisiología , Proteínas de Movimiento Viral en Plantas , Protoplastos/virología , Spodoptera/virologíaRESUMEN
Cowpea mosaic virus (CPMV) moves from cell to cell as virus particles which are translocated through a plasmodesmata-penetrating transport tubule made up of viral movement protein (MP) copies. To gain further insight into the roles of the viral MP and capsid proteins (CP) in virus movement, full-length and truncated forms of the MP were expressed in insect cells using the baculovirus expression system. Using ELISA and blot overlay assays, affinity purified MP was shown to bind specifically to intact CPMV virions and to the large CP, but not to the small CP. This binding was not observed with a C-terminal deletion mutant of the MP, although this mutant retained the capacity to bind to other MP molecules and to form tubules. These results suggest that the C-terminal 48 amino acids constitute the virion-binding domain of the MP.
Asunto(s)
Proteínas de la Cápside/metabolismo , Comovirus/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Animales , Western Blotting , Células Cultivadas , Comovirus/fisiología , Ensayo de Inmunoadsorción Enzimática , Eliminación de Gen , Proteínas de Movimiento Viral en Plantas , Spodoptera , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Virión/metabolismoRESUMEN
The route of tomato spotted wilt virus (TSWV) in the body of its vectors, Frankliniella occidentalis and Thrips tabaci (Thysanoptera: Thripidae) was studied during their development. First instar larvae were allowed, immediately upon hatching, to acquire virus from mechanically infected Datura stramonium plants for 24 h. The rate of transmission by adults was determined in inoculation access feeding test on Emilia sonchifolia leaf disks. Thrips tissues were analysed for infection at 24 h intervals after the acquisition-access feeding period, and assayed by the whole-mount immuno-fluorescent staining technique. The virus was initially detected in the proximal midgut region in larvae of both species, and then in the second and third midgut regions, foregut, and salivary glands. Occasionally the first infections of the salivary glands were already detected in one-day-old second stage larvae. The intensity of the infection in the various organs of the thrips of each species was positively related to the transmission efficiency. In both thrips populations good agreement was found between the percentage of second instar larvae and adults with at least one infected salivary gland lobe and the percentage of transmitting adults. These results support the contention that the virus must reach the salivary glands before thrips pupation in order to be transmitted by old second instar larvae and adults.
Asunto(s)
Insectos Vectores/virología , Insectos/virología , Tospovirus/fisiología , Animales , Ensayo de Inmunoadsorción Enzimática , Intestinos/virología , Larva/virología , Glándulas Salivales/virología , Latencia del VirusRESUMEN
Tomato spotted wilt virus (TSWV) is able to infect both its botanical hosts and its insect vector (thrips). In plant tissue the NS(M) protein of TSWV functions as viral movement protein (MP), aggregating into plasmodesma-penetrating tubules to establish cell-to-cell movement. As upon heterologous expression NS(M) was able to form similar tubules on the surface of insect (Spodoptera frugiperda) cells, we have now investigated the expression and cellular manifestation of this protein in infected thrips tissue. It is shown that NS(M), though detectably expressed in both the L2 larval and adult thrips stages, does not aggregate into tubules, indicating that this requirement is associated to its function as MP in plants, and raising the question if NS(M) has a function at all during the insect life cycle of TSWV.
Asunto(s)
Insectos Vectores/virología , Insectos/virología , Tospovirus/metabolismo , Proteínas Virales/metabolismo , Animales , Inmunohistoquímica , Larva/virología , Estadios del Ciclo de Vida , Proteínas de Movimiento Viral en Plantas , Proteínas Virales/análisisRESUMEN
Within their host plants, viruses spread from the initially infected cell through plasmodesmata to neighbouring cells (cell-to-cell movement), until reaching the phloem for rapid invasion of the younger plant parts (long-distance or vascular movement). Cowpea mosaic virus (CPMV) moves from cell-to-cell as mature virions via tubules constructed of the viral movement protein (MP). The mechanism of vascular movement, however, is not well understood. The characteristics of vascular movement of CPMV in Vigna unguiculata (cowpea) were examined using GFP-expressing recombinant viruses. It was established that CPMV was loaded into both major and minor veins of the inoculated primary leaf, but was unloaded exclusively from major veins, preferably class III, in cowpea trifoliate leaves. Phloem loading and unloading of CPMV was scrutinized at the cellular level in sections of loading and unloading veins. At both loading and unloading sites it was shown that the virus established infection in all vascular cell types with the exception of companion cells (CC) and sieve elements (SE). Furthermore tubular structures, indicative of virion movement, were never found in plasmodesmata connecting phloem parenchyma cells and CC or CC and SE. In cowpea, SE are symplasmically connected only to the CC and these results therefore suggest that CPMV employs a mechanism for phloem loading and unloading that is different from the typical tubule-guided cell-to-cell movement in other cell types.
Asunto(s)
Comovirus/metabolismo , Fabaceae/virología , Transporte Biológico , Comovirus/genética , Comovirus/aislamiento & purificación , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Microscopía Electrónica , Hojas de la Planta/virología , Recombinación GenéticaRESUMEN
The role of polyhedrin in the occlusion of virions was studied by substituting two heterologous polyhedrin-coding sequences, one from a multiple-nucleocapsid (M) nucleopolyhedrovirus (NPV) of Spodoptera exigua (Se) and one from a single-nucleocapsid (S) NPV of Buzura suppressaria (BusuNPV), into the genome of Autographa californica (Ac) MNPV. Both heterologous polyhedrin genes were highly expressed and polyhedra were produced in the nuclei of cells infected with the respective recombinant AcMNPVs. Polyhedra produced by the recombinant with BusuNPV polyhedrin showed normal occlusion of multiple-nucleocapsid virions and were equally as infectious to S. exigua larvae as were wild-type AcMNPV polyhedra. This indicates that virion occlusion is not specific with respect to whether the virions or polyhedrin are from an SNPV or MNPV. Polyhedra produced by the recombinant containing the SeMNPV polyhedrin had an altered morphology, being pyramidal rather than polyhedral in shape, and many fewer virions were occluded. These occlusion bodies were less infectious to S. exigua larvae than were those of wild-type AcMNPV. These results indicate that virion occlusion is a finely controlled process that is to some extent specific to the polyhedrin involved and may also require other viral or host factors for optimal morphogenesis.
Asunto(s)
Nucleopoliedrovirus/fisiología , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Microscopía Electrónica , Microscopía de Contraste de Fase , Datos de Secuencia Molecular , Nucleopoliedrovirus/ultraestructura , Proteínas de la Matriz de Cuerpos de Oclusión , Proteínas Recombinantes/biosíntesis , Spodoptera , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Estructurales ViralesRESUMEN
The p10 gene of Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus (BusuNPV) was identified by virtue of its localization downstream from the Autographa californica (Ac) MNPV p26 homologue. The BusuNPV p10 gene encodes a protein of 94 amino acids. The amino acid sequence contains domains characteristic of baculovirus P10 proteins, e.g. a coiled-coil domain, a proline-rich motif and a positively charged C terminus. The highest amino acid homologies were found with the Spodoptera littoralis (Spli) NPV and Spodoptera exigua (Se) MNPV P10 proteins. An AcMNPV recombinant expressing the BusuNPV P10 formed fibrillar structures in the cytoplasm of Spodoptera frugiperda cells. BusuNPV P10 could not fully replace AcMNPV P10 in its nuclear disintegration function, since polyhedra were not efficiently liberated from infected cells late in infection. The BusuNPV p26 gene encodes a protein of 263 amino acid residues with 70% amino acid similarity with SeMNPV P26. Downstream of the BusuNPV p10 gene, the gene for the occlusion-derived virus protein ODVP-6e is located. This is unlike the situation in many other NPVs, including SeMNPV, where the p10 gene neighbours the p74 gene. The data presented here suggest that although the p10 gene is not conserved in sequence, evolutionary pressure preserves the structure of P10 and hence its function. These data also indicate that all NPVs, MNPVs as well as SNPVs, contain this gene.
Asunto(s)
Mariposas Nocturnas/virología , Nucleopoliedrovirus/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Viral , Genes Virales , Datos de Secuencia Molecular , Nucleocápside , Homología de Secuencia de Aminoácido , Proteínas Virales/fisiologíaRESUMEN
In systemically infected tissues of Nicotiana benthamiana, alfalfa mosaic virus (AMV) coat protein (CP) and movement protein (MP) are detected in plasmodesmata in a layer of three to four cells at the progressing front of infection. Besides the presence of these viral proteins, the plasmodesmata are structurally modified in that the desmotubule is absent and the diameter has increased drastically (almost twofold) when compared to plasmodesmata in uninfected cells or cells in which AMV infection had been fully established. Previously reported observations on virion-containing tubule formation at the surface of AMV-infected protoplasts suggest that AMV employs a tubule-guided mechanism for intercellular movement. Although CP and MP localization to plasmodesmata is consistent with such a mechanism, no tubules were found in plasmodesmata of AMV-infected tissues. The significance of these observations is discussed.
Asunto(s)
Virus del Mosaico de la Alfalfa/metabolismo , Virus del Mosaico de la Alfalfa/patogenicidad , Proteínas de la Cápside , Cápside/metabolismo , Proteínas Virales/metabolismo , Uniones Intercelulares/ultraestructura , Uniones Intercelulares/virología , Microscopía Inmunoelectrónica , Movimiento/fisiología , Enfermedades de las Plantas/virología , Hojas de la Planta/ultraestructura , Hojas de la Planta/virología , Proteínas de Movimiento Viral en Plantas , Plantas Tóxicas , Nicotiana/ultraestructura , Nicotiana/virología , VirulenciaRESUMEN
Tubular structures involved in the cell-to-cell movement of cowpea mosaic virus (CPMV) were partially purified from infected cowpea protoplasts to identify the structural components. A relatively pure fraction could be obtained by differential centrifugation and this was analysed by PAGE and immunoblotting. Besides the movement protein (MP) and capsid proteins (CP) of CPMV, no other major infection-specific proteins could be detected, suggesting that host proteins are not a major structural component of the movement tubule.
Asunto(s)
Comovirus/ultraestructura , Plantas/virología , Comovirus/química , Proteínas Estructurales Virales/ultraestructuraRESUMEN
The structural phenotype of the movement proteins (MPs) of two representatives of the Bromoviridae, alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), was studied in protoplasts. Immunofluorescence microscopy showed that the MPs of these viruses, for which there has been no evidence of a tubule-guided mechanism, assemble into long tubular structures at the surface of the infected protoplast. Electron microscopy and immunogold analysis confirmed the presence of both MP and virus particles in the tubules induced by AMV and BMV. The significance of the tubule-forming properties of these viral MPs is discussed.
Asunto(s)
Alfamovirus/fisiología , Bromovirus/fisiología , Proteínas Virales/genética , Alfamovirus/ultraestructura , Bromovirus/ultraestructura , Fabaceae/ultraestructura , Fabaceae/virología , Técnica del Anticuerpo Fluorescente , Microscopía Electrónica , Microscopía Inmunoelectrónica , Proteínas de Movimiento Viral en Plantas , Plantas Medicinales , Protoplastos/ultraestructura , Protoplastos/virología , Proteínas Virales/metabolismoRESUMEN
The movement proteins (MP) of cowpea mosaic virus and cauliflower mosaic virus (CaMV) are associated with tubular structures in vivo which participate in the transmission of virus particles from cell to cell. Both proteins have been expressed in plant protoplasts and insect cells. In all cases, immunofluorescent histochemistry showed that the MPs accumulate intracellularly as tubular extensions projecting from the cell surface. Additionally, electron microscopy revealed intracellular MP aggregates in CaMV MP-expressing cells. The data presented establish common features for the tubule-forming MPs: no other virus gene products are required for tubule formation and unique plant components (e.g. plasmodesmata) are not essential for tubule synthesis.
Asunto(s)
Caulimovirus/fisiología , Comovirus/fisiología , Plantas/virología , Spodoptera/virología , Proteínas Virales/fisiología , Animales , Microscopía Fluorescente , Proteínas de Movimiento Viral en Plantas , Plantas/ultraestructura , Conejos , Proteínas Recombinantes/biosíntesis , Spodoptera/ultraestructura , Proteínas Virales/análisis , Proteínas Virales/genéticaRESUMEN
Phytase from Aspergillus niger increases the availability of phosphorus from feed for monogastric animals by releasing phosphate from the substrate phytic acid. A phytase cDNA was constitutively expressed in transgenic tobacco (Nicotiana tabacum) plants. Secretion of the protein to the extracellular fluid was established by use of the signal sequence from the tobacco pathogen-related protein S. The specific phytase activity in isolated extracellular fluid was found to be approximately 90-fold higher than in total leaf extract, showing that the enzyme was secreted. This was confirmed by use of immunolocalization. Despite differences in glycosylation, specific activities of tobacco and Aspergillus phytase were identical. Phytase was found to be biologically active and to accumulate in leaves up to 14.4% of total soluble protein during plant maturation. Comparison of phytase accumulation and relative mRNA levels showed that phytase stably accumulated in transgenic leaves during plant growth.
Asunto(s)
6-Fitasa/biosíntesis , Aspergillus niger/enzimología , 6-Fitasa/análisis , 6-Fitasa/genética , Secuencia de Aminoácidos , Aspergillus niger/genética , Western Blotting , Espacio Extracelular/enzimología , Expresión Génica , Glicósido Hidrolasas , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Hojas de la Planta , Plantas Modificadas Genéticamente , Plantas Tóxicas , Plásmidos , Señales de Clasificación de Proteína/química , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , NicotianaRESUMEN
The expression and subcellular location of the 33.6-kDa nonstructural protein NSm of tomato spotted wilt virus (TSWV) was analyzed in Nicotiana rustica plants and protoplasts as a function of time. Immunofluorescent studies in protoplasts isolated from TSWV-infected N. rustica leaves showed that this protein could first be detected close to the periphery of the cell, near the plasmamembrane, and later in tubular structures emerging from the cell surface. In situ, these tubules appeared specifically in the plasmodesmata, suggesting their involvement in cell-to-cell movement of the virus during systemic infection. In protoplasts transfected with an expression vector containing the NSm gene, similar tubules were formed, indicating that NSm has the ability to form these structures in the absence of other virus-specific components. To test whether plant-specific components were involved in tubule formation, the NSm gene was also expressed in a heterologous expression system, i.e., insect cells. Spodoptera frugiperda and Trichoplusia ni cells were infected with a recombinant baculovirus expressing the NSm-gene (AcNPV/NSm). The efficient formation of NSm-containing tubules emerging from the surface of both cell types indicate that no plant-specific cell structures or proteins are involved in their development.
Asunto(s)
Tospovirus/fisiología , Proteínas no Estructurales Virales/fisiología , Proteínas Virales/fisiología , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Vectores Genéticos , Solanum lycopersicum/virología , Microscopía Fluorescente , Datos de Secuencia Molecular , Mariposas Nocturnas , Nucleopoliedrovirus/genética , Hojas de la Planta/virología , Proteínas de Movimiento Viral en Plantas , Plantas Tóxicas , Protoplastos/virología , Nicotiana/virología , Tospovirus/genética , Tospovirus/ultraestructura , Transfección , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
A special recombinant of Autographa californica multicapsid nuclear polyhedrosis virus (AcNPV) was designed to study the early histopathological events of baculovirus infection in Spodoptera exigua larvae. This recombinant contained a Drosophila melanogaster heat shock 70 promoter driving an Escherichia coli beta-galactosidase (Lac-Z) reporter gene to monitor the presence of early viral gene expression and a second reporter gene, the E. coli beta-glucuronidase (GUS) gene, under control of the very late AcNPV p10 promoter to monitor viral replication. In S. exigua larvae, permissive Spodoptera spp. cultured cells, and nonpermissive D. melanogaster cultured cells early viral gene expression was indicated by the appearance of Lac-Z as early as 3 hr p.i. Late viral gene expression was indicated by the appearance of GUS and occurred only in the permissive cultured cells and larvae. Early and late viral gene expression could be detected simultaneously using differential enzyme histochemistry. Analysis of infected S. exigua larvae revealed that midgut columnar cells and, at a low frequency, midgut regenerative cells were the primary sites of infection. Parental nucleocapsids were apparently transported through columnar cells to underlaying regenerative cells before virus replication and progeny production. Infection of tissues beside the midgut epithelium was not detected prior to viral replication within the midgut, suggesting that infection of the midgut is an important prelude to systemic infection.
Asunto(s)
Nucleopoliedrovirus/fisiología , Virus Reordenados/fisiología , Spodoptera/virología , Animales , Cápside/genética , Sistema Digestivo/virología , Epitelio/virología , Genes Reporteros , Larva/virología , Nucleopoliedrovirus/patogenicidad , Replicación ViralRESUMEN
We have assessed the functional importance of the NTP-binding motif (NTBM) in the cowpea mosaic virus (CPMV) B-RNA-encoded 58K domain by changing two conserved amino acids within the consensus A and B sites (GKSRTGK500S and MDD545, respectively). Both Lys-500 to Thr and Asp-545 to Pro substitutions are lethal as mutant B-RNAs were no longer replicated in cowpea protoplasts. Transiently produced mutant proteins were not able to support trans-replication of CPMV M-RNA in cowpea protoplasts in contrast to transiently produced wild-type B proteins. Therefore loss of viral RNA synthesis was a result of a protein defect rather than an RNA template defect. Mutant B polyproteins were correctly processed in vitro and in vivo and the regulatory function of the 32K protein on processing of B proteins was not affected by these mutations. Since regulation of processing by the 32K protein depends on interaction with the 58K domain, the mutations in the NTBM apparently do not interfere with this interaction. The Asp-545 to Pro substitution left intact the binding properties of the 84K precursor of the 58K protein, with respect to ATP-agarose, whereas the Lys-500 to Thr substitution decreased the binding capacity of the 84K protein, suggesting that the Lys-500 residue is directly involved in ATP binding. The Lys-500 to Thr substitution in the 58K domain resulted in an altered distribution of viral proteins, which failed to aggregate into large cytopathic structures as observed in protoplasts infected with wild-type B-RNA. However viral proteins containing the Asp-545 to Pro substitution showed a normal distribution in protoplasts.
Asunto(s)
Comovirus/fisiología , Proteínas de Unión al GTP/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Northern Blotting , Cromatografía de Afinidad , Comovirus/metabolismo , Secuencia de Consenso , Fabaceae/virología , Proteínas de Unión al GTP/biosíntesis , Expresión Génica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Plantas Medicinales , Protoplastos/virología , ARN Viral/metabolismo , Transfección , Proteínas Virales/biosíntesisRESUMEN
Chimeric proteins consisting of the VP2 capsid protein of human parvovirus B19 and defined linear epitopes from human herpes simplex virus type 1 and mouse hepatitis virus A59 inserted at the N-terminus and at a predicted surface region were expressed by recombinant baculoviruses. The chimeric proteins expressed the inserted epitopes and assembled into empty capsids. Immunoelectron microscopy indicated that the epitopes inserted in the loop were exposed on the surface of the chimeric particles. The chimeric capsids were immunogenic in mice and antibodies specific for the inserted sequences were induced. In the case of MHV, antibodies were produced that recognized the epitope in the context of native virus. Mice immunized with the chimeric capsids were partially protected against a lethal challenge infection with either MHV or HSV.
Asunto(s)
Presentación de Antígeno , Cápside/inmunología , Epítopos/inmunología , Parvovirus B19 Humano/inmunología , Proteínas Recombinantes de Fusión/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/biosíntesis , Secuencia de Bases , Cápside/genética , Células Cultivadas , Femenino , Herpes Simple/inmunología , Herpes Simple/prevención & control , Herpesvirus Humano 1/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mariposas Nocturnas/citología , Virus de la Hepatitis Murina/inmunología , Nucleopoliedrovirus/genética , Parvovirus B19 Humano/genética , Vacunas Sintéticas/uso terapéuticoRESUMEN
Mechanisms of resistance to beet necrotic yellow vein virus (BNYVV) were studied by comparing the multiplication and distribution of BNYVV in root tissue of some beet accessions. Seedlings were infected either by soil containing resting spores of Polymyxa betae with BNYVV, or by a viruliferous zoospore suspension. With both inoculation methods high virus concentrations were obtained in rootlets of the susceptible cultivar 'Regina'. Using infested soil, low virus concentrations were found in the partially resistant cultivar 'Rima' and in the resistant accessions Holly and WB42. When a zoospore suspension was used, similar virus concentrations occurred in 'Rima' and Holly as in 'Regina', while a low virus concentration was found in WB42. By in situ localisation studies, using immunogold-silver labelling, virus was detected in 'Regina' after infection by soil or a zoospore suspension, but it could only be detected in the resistant accessions after infection by a zoospore suspension. In rootlets of 'Regina', 'Rima' and Holly, virus was found in the epidermis, cortex parenchyma, endodermis, and interstitial parenchyma, but in general not inside the vascular tissue. In WB42 the virus, occurring in small aggregates, seemed to be restricted to the epidermis and some cortex parenchyma cells. Comparing both the multiplication and distribution of BNYVV in rootlets of the accessions studied, it is concluded that the virus resistance mechanism in 'Rima' and Holly is different from that in WB42.
Asunto(s)
Virus de Plantas/aislamiento & purificación , Virus ARN/aislamiento & purificación , Verduras/microbiología , Inmunohistoquímica , Hibridación in SituRESUMEN
During an infection with cowpea mosaic virus (CPMV) both virion assembly and formation of tubules associated with plasmodesmata are required for cell-to-cell movement. These functions are encoded by the M-RNA of CPMV. To study the mechanism of CPMV movement, mutant N123 was used in complementation studies with sunn-hemp mosaic virus (SHMV), a legume-infecting tobamovirus. Previous studies have shown that N123 fails to spread in cowpea plants because of mutation(s) in its M-RNA. However, the mutant was efficiently replicated in cowpea protoplasts, in which virions were formed and tubular transport structures were induced. After high-dose inoculation of cowpeas with N123, only a few infected protoplasts could be isolated, indicating that cell-to-cell transport of N123 was greatly impaired, if not completely abolished. Upon coinoculation with SHMV, mutant N123 infected cowpea plants systemically and accumulated to levels which were comparable to those of wild-type CPMV. In contrast, separate B-RNA of CPMV and a CPMV deletion mutant lacking the tubule-inducing function, were complemented by SHMV to only low levels. It is concluded that SHMV-facilitated spread of CPMV in the non-virion tobamovirus mode is inefficient and that spread of mutant N123 is probably in the CPMV mode, SHMV providing an as yet unidentified helper function.
Asunto(s)
Brassica/microbiología , Virus Defectuosos/fisiología , Virus del Mosaico/fisiología , Cápside/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Prueba de Complementación Genética , Cinética , Virus del Mosaico/genética , Mutagénesis , Protoplastos/microbiología , ARN Viral/genética , ARN Viral/metabolismo , Factores de TiempoRESUMEN
Tubular structures extending from plasmodesmata in cowpea mosaic virus (CPMV)-infected tissue have been implicated to play an important role in cell-to-cell movement of this virus. Using a cauliflower mosaic virus 35S promoter-based transient expression vector, we show that expression of only the CPMV M RNA-encoded 48-kDa protein (48K protein) in cowpea protoplasts is sufficient to induce these structures. Strikingly, expression of the 48K protein in protoplasts from a number of nonhost plant species, such as barley, Arabidopsis thaliana, and carrot, also resulted in tubular structure formation. Thus, it is not likely that the viral 48K protein, though playing a key role in cell-to-cell movement of CPMV, has a role in determining the host range of CPMV.