RESUMEN
BACKGROUND: The non conventional RTM (Restricted Tobacco etch virus Movement) resistance which restricts long distance movement of some plant viruses in Arabidopsis thaliana is still poorly understood. Though at least three RTM genes have been identified, their precise role(s) in the process as well as whether other genes are involved needs to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the natural variation of the RTM genes was analysed at the amino acid level in relation with their functionality to restrict the long distance movement of Lettuce mosaic potyvirus (LMV). We identified non-functional RTM alleles in LMV-susceptible Arabidopsis accessions as well as some of the mutations leading to the non-functionality of the RTM proteins. Our data also indicate that more than 40% of the resistant accessions to LMV are controlled by the RTM genes. In addition, two new RTM loci were genetically identified. CONCLUSIONS/SIGNIFICANCE: Our results show that the RTM resistance seems to be a complex biological process which would involves at least five different proteins. The next challenges will be to understand how the different RTM protein domains are involved in the resistance mechanism and to characterise the new RTM genes for a better understanding of the blocking of the long distance transport of plant viruses.
Asunto(s)
Arabidopsis/genética , Arabidopsis/virología , Variación Genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Lectinas de Plantas/genética , Potyvirus/fisiología , Alelos , Sustitución de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Mapeo Cromosómico , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Predisposición Genética a la Enfermedad , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Lectinas de Plantas/químicaRESUMEN
In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro. The analysis of mutated forms of the α(5) subunit demonstrated that mutation of a glutamic acid at position 110 affects RNase activity. Furthermore, it was demonstrated, using a bimolecular fluorescence complement assay, that the multifunctional helper component proteinase (HcPro) of LMV, already known to interfere with the 20S proteasome RNase activity in vitro, can interact in vivo with the recombinant α(5) subunit. Further experiments demonstrated that, in LMV-infected lettuce cells, α(5) is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α(5) , showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thaliana α(5) the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cisteína Endopeptidasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/metabolismo , Ribonucleasas/metabolismo , Proteínas Virales/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Escherichia coli , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Ácido Glutámico/genética , Proteínas Fluorescentes Verdes/metabolismo , Lactuca , Mutación/genética , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Subunidades de Proteína/genética , ARN Viral/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleasas/genética , Fracciones Subcelulares/metabolismoRESUMEN
Restriction of long-distance movement of several potyviruses in Arabidopsis (Arabidopsis thaliana) is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2, and RTM3. RTM1 encodes a protein belonging to the jacalin family, and RTM2 encodes a protein that has similarities to small heat shock proteins. In this article, we describe the positional cloning of RTM3, which encodes a protein belonging to an undescribed protein family of 29 members that has a meprin and TRAF homology (MATH) domain in its amino-terminal region and a coiled-coil domain at its carboxy-terminal end. Involvement in the RTM resistance system is the first biological function experimentally identified for a member of this new gene family in plants. Our analyses showed that the coiled-coil domain is not only highly conserved between RTM3-homologous MATH-containing proteins but also in proteins lacking a MATH domain. The cluster organization of the RTM3 homologs in the Arabidopsis genome suggests the role of duplication events in shaping the evolutionary history of this gene family, including the possibility of deletion or duplication of one or the other domain. Protein-protein interaction experiments revealed RTM3 self-interaction as well as an RTM1-RTM3 interaction. However, no interaction has been detected involving RTM2 or the potyviral coat protein previously shown to be the determinant necessary to overcome the RTM resistance. Taken together, these observations strongly suggest the RTM proteins might form a multiprotein complex in the resistance mechanism to block the long-distance movement of potyviruses.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Genes de Plantas/genética , Familia de Multigenes/genética , Potyvirus/metabolismo , Tiopronina/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/virología , Proteínas de Arabidopsis/química , Transporte Biológico , Proteínas de la Cápside/metabolismo , Genotipo , Datos de Secuencia Molecular , Lectinas de Plantas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus, Fabavirus and Nepovirus) and Sequiviridae (genera Sequivirus and Waikavirus) and into the unassigned genera Cheravirus and Sadwavirus. These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed "Secoviridae" to include the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus and Waikavirus. Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae.
Asunto(s)
Filogenia , Virus de Plantas/clasificación , Virus ARN/clasificación , Genoma Viral , Virus de Plantas/genética , Virus ARN/genética , ARN Viral/genética , Secoviridae/clasificación , Secoviridae/genética , Análisis de Secuencia de ARN , Sequiviridae/clasificación , Sequiviridae/genéticaRESUMEN
The capacity of Lettuce mosaic virus to overcome the lettuce resistance conferred by the mo1(1) and mo1(2) alleles of the gene for eukaryotic translation initiation factor 4E (eIF4E) was analysed using reverse genetics. Mutations in the virus genome-linked protein (VPg) allowed mo1(1) only to be overcome, but mutations in the C-terminal portion of the cylindrical inclusion (CI) protein allowed both alleles to be overcome. Site-directed mutagenesis pinpointed a key role of the amino acid at position 621 in the virulence. This is the first example of the involvement of a potyviral CI protein in the breaking of an eIF4E-mediated resistance.
Asunto(s)
Factor 4E Eucariótico de Iniciación/fisiología , Lactuca/virología , Potyvirus/metabolismo , Proteínas Virales/fisiología , Secuencia de Bases , Cartilla de ADN , Mutagénesis Sitio-Dirigida , Potyvirus/patogenicidad , Proteínas Virales/genéticaRESUMEN
TAXONOMY: Lettuce mosaic virus (LMV) belongs to the genus Potyvirus (type species Potato virus Y) in the family Potyviridae. PHYSICAL PROPERTIES: The virion is filamentous, flexuous with a length of 750 nm and a width of 15 nm. The particles are made of a genomic RNA of 10 080 nucleotides, covalently linked to a viral-encoded protein (the VPg) at the 5' end and with a 3' poly A tail, and encapsidated in a single type of capsid protein. The molecular weight of the capsid protein subunit has been estimated electrophoretically to be 34 kDa and estimated from the amino acid sequence to be 31 kDa. GENOME ORGANIZATION: The genome is expressed as a polyprotein of 3255 amino-acid residues, processed by three virus-specific proteinases into ten mature proteins. HOSTS: LMV has a worldwide distribution and a relatively broad host range among several families. Weeds and ornamentals can act as local reservoirs for lettuce crops. In particular, many species within the family Asteraceae are susceptible to LMV, including cultivated and ornamental species such as common (Lactuca sativa), prickly (L. serriola) or wild (L. virosa) lettuce, endive/escarole (Cichorium endiva), safflower (Carthamus tinctorius), starthistle (Centaurea solstitialis), Cape daisy (Osteospermum spp.) and gazania (Gazania rigens). In addition, several species within the families Brassicaceae, Cucurbitaceae, Fabaceae, Solanaceae and Chenopodiaceae are natural or experimental hosts of LMV. Genetic control of resistance to LMV: The only resistance genes currently used to protect lettuce crops worldwide are the recessive genes mo1(1) and mo1(2) corresponding to mutant alleles of the gene encoding the translation initiation factor eIF4E in lettuce. It is believed that at least one intact copy of eIF4E must be present to ensure virus accumulation. TRANSMISSION: LMV is transmitted in a non-persistent manner by a high number of aphid species. Myzus persicae and Macrosiphum euphorbiae are particularly active in disseminating this virus in the fields. LMV is also seedborne in lettuce. The effectiveness of LMV transmission depends on the cultivar and the age of the seed carrier at the inoculation time. SYMPTOMS: The characteristic symptoms on susceptible lettuce cultivars are dwarfism, mosaic, distortion and yellowing of the leaves with sometimes a much reduced heart of lettuce (failure to form heads). The differences in virus strains, cultivars and the physiological stage of the host at the moment of the attack cause different symptom severity: from a very slight discoloration of the veins to severe necrosis leading to the death of the plant.
Asunto(s)
Lactuca/virología , Enfermedades de las Plantas/virología , Virus de Plantas/fisiología , Variación Genética , Interacciones Huésped-Patógeno , Inmunidad Innata/genética , Lactuca/genética , Lactuca/crecimiento & desarrollo , Virus del Mosaico/clasificación , Virus del Mosaico/genética , Virus del Mosaico/fisiología , Filogenia , Enfermedades de las Plantas/genética , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/virología , Virus de Plantas/clasificación , Virus de Plantas/genéticaRESUMEN
The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo1(1) and mo1(2) against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo1(1) or mo1(2) varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Inmunidad Innata , Lactuca/fisiología , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/metabolismo , Potyvirus/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Análisis Mutacional de ADN , Factor 4E Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Prueba de Complementación Genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Proteínas de Plantas/genética , Mutación Puntual , Unión Proteica , Conformación Proteica , Caperuzas de ARN/metabolismo , Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
Despite the apparent natural grouping of "picorna-like" viruses, the taxonomical significance of this putative "supergroup" was never addressed adequately. We recently proposed to the ICTV that an order should be created and named Picornavirales, to include viruses infecting eukaryotes that share similar properties: (i) a positive-sense RNA genome, usually with a 5'-bound VPg and 3'-polyadenylated, (ii) genome translation into autoproteolytically processed polyprotein(s), (iii) capsid proteins organized in a module containing three related jelly-roll domains which form small icosahedral, non-enveloped particles with a pseudo-T = 3 symmetry, and (iv) a three-domain module containing a superfamily III helicase, a (cysteine) proteinase with a chymotrypsin-like fold and an RNA-dependent RNA polymerase. According to the above criteria, the order Picornavirales includes the families Picornaviridae, Comoviridae, Dicistroviridae, Marnaviridae, Sequiviridae and the unassigned genera Cheravirus, Iflavirus and Sadwavirus. Other taxa of "picorna-like" viruses, e.g. Potyviridae, Caliciviridae, Hypoviridae, do not conform to several of the above criteria and are more remotely related: therefore they are not being proposed as members of the new order. Newly described viruses, not yet assigned to an existing taxon by ICTV, may belong to the proposed order.
Asunto(s)
Virus ARN/clasificación , Virión , Proteínas de la Cápside/química , Cisteína Endopeptidasas , Genoma Viral/genética , Picornaviridae/clasificación , Picornaviridae/genética , Poliproteínas/química , Virus ARN/química , Virus ARN/genética , Virus ARN/ultraestructura , ARN Viral/química , ARN Viral/ultraestructura , ARN Polimerasa Dependiente del ARN , Virión/química , Virión/ultraestructura , Virología/métodosRESUMEN
In compatible interactions between plants and viruses that result in systemic infection, symptom development is a major phenotypic trait. However, host determinants governing this trait are mostly unknown, and the mechanisms underlying it are still poorly understood. In a previous study on the Arabidopsis thaliana-Plum pox virus (PPV) pathosystem, we showed a large degree of variation in symptom development among susceptible accessions. In particular, Cvi-1 (Cape Verde islands) accumulates viral particules but remains symptomless, Col-0 (Columbia) sometimes shows weak symptoms compared with Ler (Landsberg erecta), which always shows severe symptoms. Genetic analyses of Col x Ler and Cvi x Ler F2 and recombinant inbred line (RIL) populations suggested that symptom development as well as viral accumulation traits are polygenic and quantitative. Three of the symptom quantitative trait loci (QTL) identified could be confirmed in near-isogenic lines, including PSI1 (PPV symptom induction 1), which was identified on the distal part of chromosome 1 in both RIL populations. With respect to viral accumulation, several factors have been detected and, interestingly, in the Col x Ler population, two out of three viral accumulation QTL colocalized with loci controlling symptom development, although correlation analysis showed weak linearity between symptom severity and virus accumulation. In addition, in the Cvi x Ler RIL population, a digenic recessive determinant controlling PPV infection was identified.
Asunto(s)
Arabidopsis/genética , Arabidopsis/virología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Virus de Plantas/fisiología , Sitios de Carácter Cuantitativo/genética , Mapeo Cromosómico , Cromosomas de las Plantas , Inmunidad Innata/inmunología , Endogamia , Patrón de Herencia , Fenotipo , Enfermedades de las Plantas/inmunologíaRESUMEN
A panel of 19 monoclonal antibodies (mAbs) was used to study the immunological variability of Lettuce mosaic virus (LMV), a member of the genus Potyvirus, and to perform a first epitope characterization of this virus. Based on their specificity of recognition against a panel of 15 LMV isolates, the mAbs could be clustered in seven reactivity groups. Surface plasmon resonance analysis indicated the presence, on the LMV particles, of at least five independent recognition/binding regions, correlating with the seven mAbs reactivity groups. The results demonstrate that LMV shows significant serological variability and shed light on the LMV epitope structure. The various mAbs should prove a new and efficient tool for LMV diagnostic and field epidemiology studies.
Asunto(s)
Anticuerpos Monoclonales , Variación Genética , Lactuca/virología , Virus del Mosaico/genética , Virus de Plantas/genética , Geografía , Virus del Mosaico/clasificación , Virus del Mosaico/aislamiento & purificación , Filogenia , Virus de Plantas/clasificación , Virus de Plantas/aislamiento & purificación , Resonancia por Plasmón de Superficie/métodosRESUMEN
The translation initiation factors eIF4E and eIF(iso)4E play a key role during virus infection in plants. During mRNA translation, eIF4E provides the cap-binding function and is associated with the protein eIF4G to form the eIF4F complex. Susceptibility analyses of Arabidopsis mutants knocked-out for At-eIF4G genes showed that eIF4G factors are indispensable for potyvirus infection. The colonization pattern by a viral recombinant carrying GFP indicated that eIF4G is involved at a very early infection step. Like eIF4E, eIF4G isoforms are selectively recruited for infection. Moreover, the eIF4G selective involvement parallels eIF4E recruitment. This is the first report of a coordinated and selective recruitment of eIF4E and eIF4G factors, suggesting the whole eIF4F recruitment.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/virología , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Potyvirus/patogenicidad , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , ADN Viral/genética , Factor 4E Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/genética , Genes de Plantas , Prueba de Complementación Genética , Mutación , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Potyvirus/genética , Biosíntesis de ProteínasRESUMEN
We investigated the changes in the expression profiles of the partially resistant apricot (Prunus armeniaca L.) cultivar Goldrich following inoculation with Plum pox virus (PPV) using cDNA-amplification fragment length polymorphism (AFLP). Altered expression patterns were detected and twenty-one differentially expressed cDNA had homologies with genes in databases coding for proteins involved in metabolism, signal transduction, defense, stress and intra/intercellular connections. Seven of the modified expressed patterns were further investigated by semi-quantitative RT-PCR or Northern blotting. The expression patterns of five of these genes were confirmed in the partially resistant P. armeniaca cv. 'Goldrich' and assessed in a susceptible genotype. One of these cDNAs, coding for a putative class III chitinase, appeared to be repressed in infected plants of the partially resistant genotype and expressed in the susceptible one which could be related to the partially resistant phenotype. On the contrary, the expression patterns of the genes coding for a transketolase, a kinesin-like and an ankyrin-like protein, were clearly linked to the susceptible interaction. These candidate genes could play a role either in the compatible interaction leading to virus invasion or to the quantitative resistance of apricot to PPV.
Asunto(s)
Expresión Génica/fisiología , Genes de Plantas , Enfermedades de las Plantas/virología , Virus Eruptivo de la Ciruela/fisiología , Prunus/virología , Frutas/virología , Perfilación de la Expresión Génica , Virus Eruptivo de la Ciruela/genética , Virus Eruptivo de la Ciruela/patogenicidad , Prunus/genéticaRESUMEN
The virus protein linked to the genome (VPg) of plant potyviruses is a 25-kDa protein covalently attached to the genomic RNA 5' end. It was previously reported that VPg binds specifically to eIF4E, the mRNAcap-binding protein of the eukaryotic translation initiation complex. We performed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV). The cap analogue m7GDP and VPg bind to eIF4E at two distinct sites with similar affinity (K(d) = 0.3 microm). A deeper examination of the interaction pathway showed that the binding of one ligand induces a decrease in the affinity for the other by a factor of 15. GST pull-down experiments from plant extracts revealed that VPg can specifically trap eIF4G, the central component of the complex required for the initiation of protein translation. Our data suggest that eIF4G recruitment by VPg is indirectly mediated through VPg-eIF4E association. The strength of interaction between eIF4E and pep4G, the eIF4E-binding domain on eIF4G, was increased significantly by VPg. Taken together these quantitative data show that VPg is an efficient modulator of eIF4E biochemical functions.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Proteínas de Plantas/metabolismo , Análogos de Caperuza de ARN/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Sitios de Unión/genética , Glutatión Transferasa/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza , Unión Proteica , ARN Mensajero/metabolismo , Proteínas de Unión al ARNRESUMEN
The interactions between Plum pox virus (PPV), a member of the Potyvirus genus, and Prunus host plants are, up to now, poorly understood. In the current paper, fluorescence stereomicroscopy, in situ hybridisation and immunogold detection were performed in order to evaluate the virus transport and cellular distribution. The behavior of PPV in several susceptible (cv. "Moniqui" and "Screara") and resistant apricot genotypes (cv. "Harlayne", "Henderson", "Harcot", "Goldrich", "Stella" and "Stark Early Orange") were compared. Viral RNA was detected by in situ hybridisation in stem tissues close to the inoculation point, irrespective of the resistance status of the variety. Systemic infection was evidenced by virus immunodetection and by fluorescence detection of a GFP-tagged PPV in distant leaf sections. The signal obtained by in situ hybridisation colocalised with the fluorescence produced by GFP-tagged PPV in the same plant material but did not colocalise with the signal obtained by immunostaining. Intensity of the PPV infection in susceptible apricot cultivars varied depending on genotypes. The behavior of PPV in systemic leaves was clearly distinct between susceptible and resistant cultivars. While PPV was spreading widely around the major and minor veins in susceptible leaves, in the resistant apricot genotypes it was restricted to isolated spots consisting of few cells embedded in the mesophyll tissue. In summary, differences in the ability of PPV to systemically infect susceptible and resistant apricot cultivars were evident but nevertheless, long-distance transport of PPV occured in resistant apricot scions.
Asunto(s)
Virus Eruptivo de la Ciruela/fisiología , Prunus/virología , Susceptibilidad a Enfermedades , Locomoción , Enfermedades de las Plantas/virología , Hojas de la Planta/virología , Prunus/genética , Especificidad de la EspecieRESUMEN
Positive-sense single-stranded RNA viruses have developed strategies to exploit cellular resources at the expense of host mRNAs. The genomes of these viruses display a variety of structures at their 5' and 3' ends that differentiate them from cellular mRNAs. Despite this structural diversity, viral RNAs are still circularized by juxtaposition of their 5' and 3' ends, similar to the process used by cellular mRNAs. Also reminiscent of the mechanisms used by host mRNAs, translation of viral RNAs involves the recruitment of translation initiation factors. However, the roles played by these factors likely differ from those played by cellular mRNAs. In keeping with the general parsimony typical of RNA viruses, these host factors also participate in viral RNA replication. However, the dual use of host factors requires that viral RNA template utilization be regulated to avoid conflict between replication and translation. The molecular composition of the large ribonucleoprotein complexes that form the viral RNA replication and translation machineries likely evolves over the course of infection to allow for switching template use from translation to replication.
Asunto(s)
Virus de Plantas/fisiología , Biosíntesis de Proteínas , ARN Viral/metabolismo , Secuencia de Bases , Regulación Viral de la Expresión Génica , Genoma Viral , Virus ARN/fisiología , ARN Viral/química , Replicación ViralRESUMEN
The isolate AF199 of Lettuce mosaic virus (LMV, genus Potyvirus) causes local lesions followed by systemic wilting and plant death in the lettuce cultivars Ithaca and Vanguard 75. Analysis of the phenotype of virus chimeras revealed that a region within the P1 protein coding region (nucleotides 112-386 in the viral genome) and/or another one within the CI protein coding region (nucleotides 5496-5855) are sufficient together to cause the lethal wilting in Ithaca, but not in Vanguard 75. This indicates that the determinants of this particular symptom are different in these two lettuce cultivars. The wilting phenotype was not directly correlated with differences in the deduced amino acid sequence of these two regions. Furthermore, transient expression of the LMV-AF199 proteins, separately or in combination, did not induce local necrosis or any other visible reaction in the plants. Together, these results suggest that the systemic wilting reaction might be due to RNA rather than protein sequences.
Asunto(s)
Genes Virales , Lactuca/virología , Enfermedades de las Plantas/virología , Potyvirus/genética , Potyvirus/patogenicidad , ARN Helicasas/genética , ARN Helicasas/fisiología , ARN Viral/fisiología , Proteínas Virales/genética , Proteínas Virales/fisiología , Virulencia/genéticaRESUMEN
Lettuce mosaic virus (LMV)-Most isolates can infect and are seed-borne in cultivars containing the mo1 gene. A reverse transcription and polymerase chain reaction (RT-PCR)-based test was developed for the specific detection of LMV-Most isolates. Based on the complete genome sequences of three LMV isolates belonging respectively to the Most type, the Common type and neither of these two types, three different assays were compared: (i) presence of a diagnostic restriction site in the region of the genome encoding the variable N-terminus of the capsid protein, in the 3' end of the genome, (ii) RT-PCR using primers designed to amplify a cDNA corresponding to a portion of the P1 coding region, in the 5' end of the genome and (iii) RT-PCR using primers designed to amplify a central region of the genome. The assays were performed against a collection of 21 isolates from different geographical origins and representing the molecular variability of LMV. RT-PCR of the central region of the genome was preferred because its results are expected to be less affected by natural recombination between LMV isolates, and it allows sensitive detection of LMV-Most in situations of single as well as mixed contamination.
Asunto(s)
Lactuca/virología , Enfermedades de las Plantas/virología , Potyvirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Potyvirus/clasificación , Potyvirus/genética , ARN Viral/análisis , ARN Viral/genéticaRESUMEN
The potential of a new in vitro inoculation and propagation method developed for Lettuce mosaic virus (LMV) on lettuce (Lactuca sativa L.) was evaluated by studying LMV infection on in vitro cultivated seedlings or on newly regenerated plantlets. Lettuce cultivars differing by their LMV-resistance status were inoculated with various natural LMV isolates as well as with Green Fluorescent Protein (GFP)-tagged recombinant virus isolates. A good correlation was observed between the known resistance status of the cultivars and the results obtained by in vitro screening. The results show that this resistance assay can be greatly improved by the use of GFP-tagged virus isolates. The main advantages of this method are reduced space requirements and an improved environmental safety, especially for the handling of recombinant virus, of quarantine virus or of transgenic plants.
Asunto(s)
Lactuca/virología , Potyvirus/crecimiento & desarrollo , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/análisis , Enfermedades de las Plantas/virología , Potyvirus/aislamiento & purificación , Potyvirus/patogenicidad , Proteínas Recombinantes/análisis , Transfección , Virología/métodosRESUMEN
Lettuce mosaic virus (LMV) isolates LMV-E and LMV-0 differ in their virulence on lettuce varieties carrying the mo1(2) resistance gene, which reduces viral accumulation and blocks the expression of symptoms after infection with avirulent isolates such as LMV-0. Previous work had indicated that reporter genes such as GUS or GFP affect the biological properties of recombinant LMV isolates in both susceptible and resistant lettuce varieties when fused to the N-terminus of the viral protein HC-Pro. The impact of the addition of a cleavage site for the NIa proteinase between the reporter gene and HC-Pro was evaluated, in an effort to recover the full spectrum of the biological properties of parental isolates. Symptoms, accumulation, cell-to-cell and long distance movement of the recombinant viruses containing the NIa cleavage site were studied in susceptible and mo1(2) lettuce varieties. Both LMV-0 and LMV-E recombinant viruses recovered the behaviour of their wild-type parent in susceptible plants upon addition of the NIa cleavage site. While the recombinant LMV-E modified in this way recovered the breaking properties of its wild-type counterpart in mo1(2) plants, similar modification of the LMV-0 derived recombinants failed to rescue a severe inhibition in systemic accumulation in mo1(2) plants, despite the fact that neither cell-to-cell movement nor phloem loading or unloading seemed to be severely affected.
Asunto(s)
Lactuca/virología , Virus del Mosaico/fisiología , Proteínas Virales/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Endopeptidasas , Genes Reporteros , Proteínas Fluorescentes Verdes , Lactuca/metabolismo , Proteínas Luminiscentes , Virus del Mosaico/genética , Virus del Mosaico/aislamiento & purificación , Virus del Mosaico/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/genéticaRESUMEN
With the aim to characterize plant and viral factors involved in the molecular interactions between plants and potyviruses, a Lettuce mosaic virus (LMV)-Arabidopsis thaliana pathosystem was developed. Screening of Arabidopsis accessions with LMV isolates indicated the existence of a large variability in the outcome of the interaction, allowing the classification of Arabidopsis accessions into seven susceptibility groups. Using a reverse genetic approach, the genome-linked protein of LMV, a multifunctional protein shown to be involved in viral genome amplification and movement of potyviruses, was established as the viral determinant responsible for the ability to overcome the resistance of the Niederzenz accession to LMV-0. Preliminary genetic analyses from F2 and recombinant inbred lines available between susceptible and resistant Arabidopsis accessions revealed the existence of at least three resistance phenotypes to LMV with different genetic bases. One dominant resistance gene, designated LLM1, involved in blocking the replication or cell-to-cell movement of the LMV-0 isolate in the Columbia accession, was mapped to chromosome I and shown to be linked to the marker nga280. At the same time, genetic analyses of segregating F2 populations were consistent with the restriction of the systemic movement of the LMV-AF199 isolate in Columbia being controlled by two dominant genes and with the complete resistance to all tested LMV isolates of the Cape Verde islands (Cvi) accession being conferred by a single recessive resistance gene. Sequencing of the eukaryotic translation initiation factor 4E genes from the different LMV-resistant Arabidopsis accessions showed that these factors are not directly involved in the characterized resistance phenotypes.