The Experts below are selected from a list of 768 Experts worldwide ranked by ideXlab platform
Sylvie Germanretana - One of the best experts on this subject based on the ideXlab platform.
-
gibson assembly an easy way to clone potyviral full length infectious cdna clones expressing an ectopic vpg
Virology Journal, 2015Co-Authors: Mariechristine Houvenaghel, Sylvie Germanretana, Amandine BordatAbstract:Approaches to simplify and accelerate the construction of full-length infectious cDNA clones for plant potyViruses have been described, based on cloning strategies involving in vitro ligation or homologous recombination in yeast. In the present study, we developed a faster and more efficient in vitro recombination system using Gibson assembly (GA), to engineer a Lettuce Mosaic Virus (LMV) infectious clone expressing an ectopic mcherry-tagged VPg (Viral protein genome-linked) for in planta subcellular localization of the viral protein in an infection context. Three overlapping long distance PCR fragments were amplified and assembled in a single-step process based on in vitro recombination (Gibson assembly). The resulting 17.5 kbp recombinant plasmids (LMVmchVPg_Ec) were inoculated by biolistic on Lettuce plants and then propagated mechanically on Nicotiana benthamiana. Confocal microscopy was used to analyze the subcellular localization of the ectopically expressed mcherry-VPg fusion protein. The Gibson assembly allowed the cloning of the expected plasmids without any deletion. All the inoculated plants displayed symptoms characteristic of LMV infection. The majority of the mcherry fluorescent signal observed using confocal microscopy was located in the nucleus and nucleolus as expected for a potyviral VPg. This is the first report of the use of the Gibson assembly method to construct full-length infectious cDNA clones of a potyVirus genome. This is also the first description of the ectopic expression of a tagged version of a potyviral VPg without affecting the viability of the recombinant potyVirus.
-
key mutations in the cylindrical inclusion involved in Lettuce Mosaic Virus adaptation to eif4e mediated resistance in Lettuce
Molecular Plant-microbe Interactions, 2014Co-Authors: Maud Sorel, Thierry Candresse, Mariechristine Houvenaghel, L Svanelladumas, G Acelin, A Pitarch, Sylvie GermanretanaAbstract:We previously showed that allelic genes mo11 and mo12 used to protect Lettuce crops against Lettuce Mosaic Virus (LMV) correspond to mutant alleles of the gene encoding the eukaryotic translation initiation factor 4E. LMV resistance-breaking determinants map not only to the main potyVirus virulence determinant, a genome-linked viral protein, but also to the C-terminal region of the cylindrical inclusion (CI), with a key role of amino acid at position 621. Here, we show that the propagation of several non-Lettuce isolates of LMV in mo11 plants is accompanied by a gain of virulence correlated with the presence in the CI C terminus of a serine at position 617 and the accumulation of mutations at positions 602 or 627. Whole-genome sequencing of native and evolved isolates showed that no other mutation could be associated with adaptation to mo1 resistance. Site-directed mutagenesis pinpointed the key role in the virulence of the combination of mutations at positions 602 and 617, in addition to position 621. Th...
-
adaptation of Lettuce Mosaic Virus to catharanthus roseus involves mutations in the central domain of the vpg
Molecular Plant-microbe Interactions, 2014Co-Authors: Laurence Svanelladumas, Sylvie Germanretana, Eric Verdin, Chantal Faure, Patrick Gognalons, Jean Luc Danet, Armelle Marais, Thierry CandresseAbstract:An isolate of Lettuce Mosaic Virus (LMV, a PotyVirus) infecting Madagascar periwinckle (Catharanthus roseus) was identified and characterized by Illumina deep sequencing. LMV-Cr has no close affinities to previously sequenced LMV isolates and represents a novel, divergent LMV clade. Inoculation experiments with other representative LMV isolates showed that they are unable to infect C. roseus, which was not known to be a host for LMV. However, three C. roseus variants of one of these isolates, LMV-AF199, could be selected and partially or completely sequenced. These variants are characterized by the accumulation of mutations affecting the C-terminal part of the cylindrical inclusion (CI) helicase and the central part of the VPg. In particular, a serine to proline mutation at amino acid 143 of the VPg was observed in all three independently selected variants and is also present in the LMV-Cr isolate, making it a prime candidate as a host-range determinant. Other mutations at VPg positions 65 and 144 could a...
-
involvement of the cylindrical inclusion ci protein in the overcoming of an eif4e mediated resistance against Lettuce Mosaic potyVirus
Molecular Plant Pathology, 2009Co-Authors: Anas Abdulrazzak, Martine Peypelut, Thierry Candresse, Mariechristine Houvenaghel, Olivier Le Gall, Thomas Guiraud, Jocel Ne Y Walter, Sylvie GermanretanaAbstract:SUMMARY 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.
-
mutational analysis of plant cap binding protein eif4e reveals key amino acids involved in biochemical functions and potyVirus infection
Journal of Virology, 2008Co-Authors: Sylvie Germanretana, Mariechristine Houvenaghel, Jocelyne Walter, Benedicte Doublet, Genevieve Roudettavert, Valerie Nicaise, Cecile Lecampion, Christophe Robaglia, Thierry Michon, Olivier Le GallAbstract: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 mo11 and mo12 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 mo11 or mo12 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.
Olivier Le Gall - One of the best experts on this subject based on the ideXlab platform.
-
The C terminus of Lettuce Mosaic potyVirus cylindrical inclusion helicase interacts with the viral VPg and with Lettuce translation eukaryotic initiation factor 4E
Journal of General Virology, 2012Co-Authors: Genevieve Tavert-roudet, Jocelyne Walter, Benedicte Doublet, Thierry Michon, Anas Abdul Razzak, Thierry Delaunay, Olivier Le GallAbstract:Recessive resistance to Lettuce Mosaic Virus (LMV) is conferred in Lettuce by the mo1 gene, encoding the eukaryotic translation initiation factor 4E (eIF4E). The C terminus of the viral cylindrical inclusion helicase (CI-Cter), together with the VPg, is involved directly in overcoming mo1 resistance. In this study, recombinant LMV VPg and CI-Cter proteins from wild-type or resistance-breaking isolates were expressed and purified from Escherichia coli. The allelic forms of eIF4E from susceptible or resistant Lettuce cultivars were produced similarly and these proteins were used in ELISA-based assays to demonstrate the in vitro binding of the various forms of LMV CI-Cter to both Lettuce eIF4E and LMV VPg proteins. All combinations tested displayed significant and specific interactions, and the interaction between the C-terminal part of the LMV CI and eIF4E was confirmed in vivo in bimolecular fluorescence complementation assays. Higher interaction signals for both CI–eIF4E and CI–VPg were observed for LMV-E, indicating that the eIF4E interaction network involving CI and VPg appears to be stronger in the case of this resistance-breaking isolate. This could suggest the need for a minimal interaction threshold for infection success in resistant Lettuce, but more precise measurement of the interaction parameters linking eIF4E, VPg and CI is needed in order to reinforce such a hypothesis.
-
The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the Lettuce Mosaic potyVirus HcPro protein
Molecular Plant Pathology, 2011Co-Authors: Anne-sophie Dielen, Jocelyne Walter, Thierry Michon, Renate Krause-sakate, Flavio Tetsuo Sassaki, Guillaume Menard, Gaelle Pagny, Ivan De Godoy Maia, Saloua Badaoui, Olivier Le GallAbstract: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 α5 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 α5 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 α5 subunit. Further experiments demonstrated that, in LMV-infected Lettuce cells, α5 is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α5, showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thalianaα5 the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.
-
Quantitative control of Lettuce Mosaic Virus fitness and host defence inhibition by P1-HcPro
Summa Phytopathologica, 2007Co-Authors: Renate Krause-sakate, Thierry Candresse, Elise Redondo, Marcelo Agenor Pavan, Florence Richard-forget, Francisco Murilo Zerbini, Olivier Le GallAbstract:Two Lettuce Mosaic Virus isolates capable of overcoming the resistance afforded by the resistance gene mo12 in Lettuce, LMV-AF199 from Brazil, and LMV-E, an European isolate, were evaluated for the rapidity and severity of symptoms induced on the Lettuce variety Salinas 88 (mo12). The Mosaic symptoms on Salinas 88 plants inoculated with LMV-AF199 appeared 7 days post-inoculation (dpi) and 15 dpi for LMV-E. The symptoms induced by LMV-AF199 in this cultivar were also more severe than those induced by LMV-E. In order to identify the region of the viral genome responsible for this phenotype, recombinant Viruses were constructed between these isolates and the phenotype of each recombinant was analysed. The region encoding proteins P1 and HcPro from LMV-AF199 was associated with the increased virulence in Salinas 88.
-
The potyviral Virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue
FEBS Journal, 2006Co-Authors: Thierry Michon, Jocelyne Walter, Sylvie German-retana, Yannick Estevez, Olivier Le GallAbstract: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.
-
A naturally occurring recombinant isolate of Lettuce Mosaic Virus
Archives of Virology, 2004Co-Authors: R. Krause-sakate, H. Fakhfakh, Martine Peypelut, M.a. Pavan, F.m. Zerbini, M. Marrakchi, Thierry Candresse, Olivier Le GallAbstract:LMV-Common and LMV-Most are two seed-borne types of Lettuce Mosaic Virus (LMV), genus PotyVirus. LMV-Most, but not LMV-Common, overcomes the resistance afforded to Lettuce by two recessive genes, mo1 1 and mo1 2. An RT-PCR-based assay thought to be specific for LMV-Most also amplified LMV-Tn2, previously typified as LMV-Common. The sequence of selected regions along the genome indicated that LMV-Tn2 is a natural recombinant between LMV-Most and LMV-Common isolates, with a putative recombination site located within the P3 coding region. This is the first evidence of a naturally occurring LMV recombinant isolate.
Thierry Michon - One of the best experts on this subject based on the ideXlab platform.
-
the potyVirus particle recruits the plant translation initiation factor eif4e by means of the vpg covalently linked to the viral rna
Molecular Plant-microbe Interactions, 2017Co-Authors: Genevieve Tavertroudet, Agnes Anne, Amandine Barra, Arnaud Chovin, Christophe Demaille, Thierry MichonAbstract:The viral protein genome-linked (VPg) of potyViruses is a protein covalently linked to the 5′ end of viral RNA. It interacts with eIF4E, a component of the cellular translation initiation complex. It has been suggested that the 5′ RNA-linked VPg could mimic the cellular mRNA cap, promoting synthesis of viral proteins. Here, we report evidence for recruitment of the plant eIF4E by Lettuce Mosaic Virus (LMV, potyVirus) particles via the 5′ RNA-linked VPg. Analysis of the viral population was performed by enzyme-linked immunosorbent assay–based tests, either with crude extracts of LMV-infected tissues or purified viral particles. In both cases, LMV-VPg and LMV-eIF4E subpopulations could be detected. After reaching a maximum within the first 2 weeks postinoculation, these populations decreased and very few labeled particles were found later than 3 weeks postinoculation. The central domain of VPg (CD-VPg) was found to be exposed at the surface of the particles. Using a purified recombinant Lettuce eIF4E and CD...
-
The potyVirus particle recruits the plant translation initiation factor eIF4E by means of the VPg covalently linked to the viral RNA.
Molecular Plant-Microbe Interactions, 2017Co-Authors: Genevieve Roudet, Agnes Anne, Amandine Barra, Arnaud Chovin, Christophe Demaille, Thierry MichonAbstract:The VPg of potyViruses is a protein covalently linked to the 5' end of viral RNA. It interacts with eIF4E, a component of the cellular translation initiation complex. It has been suggested that the 5' RNA linked VPg could mimic the cellular mRNA cap, promoting synthesis of viral proteins. We report here evidences for evidence for recruitment of the plant eIF4E by Lettuce Mosaic Virus (LMV, potyVirus) particles via the 5' RNA linked VPg. Analysis of the viral population was performed by ELISA-based tests, either with crude extracts of LMV-infected tissues or purified viral particles. In both cases, LMV-VPg and LMV-eIF4E subpopulations could be detected. After reaching a maximum within the first two weeks post inoculation, these populations decreased and very few labeled particles were found after 3 weeks post-inoculation. The central domain of VPg (CD-VPg) was found to be exposed at the surface of the particles. Using a purified recombinant Lettuce eIF4E and CD-VPg specific antibodies, we demonstrate that the plant factor binds to the VPg via its central domain. Moreover, the plant eIF4E factor could be imaged at one end of the particles purified from LMV plant extracts, by immunoredox atomic force microscopy coupled to scanning electrochemical microscopy (AFM-SECM). We discuss the biological significance of these results.
-
The C terminus of Lettuce Mosaic potyVirus cylindrical inclusion helicase interacts with the viral VPg and with Lettuce translation eukaryotic initiation factor 4E
Journal of General Virology, 2012Co-Authors: Genevieve Tavert-roudet, Jocelyne Walter, Benedicte Doublet, Thierry Michon, Anas Abdul Razzak, Thierry Delaunay, Olivier Le GallAbstract:Recessive resistance to Lettuce Mosaic Virus (LMV) is conferred in Lettuce by the mo1 gene, encoding the eukaryotic translation initiation factor 4E (eIF4E). The C terminus of the viral cylindrical inclusion helicase (CI-Cter), together with the VPg, is involved directly in overcoming mo1 resistance. In this study, recombinant LMV VPg and CI-Cter proteins from wild-type or resistance-breaking isolates were expressed and purified from Escherichia coli. The allelic forms of eIF4E from susceptible or resistant Lettuce cultivars were produced similarly and these proteins were used in ELISA-based assays to demonstrate the in vitro binding of the various forms of LMV CI-Cter to both Lettuce eIF4E and LMV VPg proteins. All combinations tested displayed significant and specific interactions, and the interaction between the C-terminal part of the LMV CI and eIF4E was confirmed in vivo in bimolecular fluorescence complementation assays. Higher interaction signals for both CI–eIF4E and CI–VPg were observed for LMV-E, indicating that the eIF4E interaction network involving CI and VPg appears to be stronger in the case of this resistance-breaking isolate. This could suggest the need for a minimal interaction threshold for infection success in resistant Lettuce, but more precise measurement of the interaction parameters linking eIF4E, VPg and CI is needed in order to reinforce such a hypothesis.
-
The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the Lettuce Mosaic potyVirus HcPro protein
Molecular Plant Pathology, 2011Co-Authors: Anne-sophie Dielen, Jocelyne Walter, Thierry Michon, Renate Krause-sakate, Flavio Tetsuo Sassaki, Guillaume Menard, Gaelle Pagny, Ivan De Godoy Maia, Saloua Badaoui, Olivier Le GallAbstract: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 α5 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 α5 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 α5 subunit. Further experiments demonstrated that, in LMV-infected Lettuce cells, α5 is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α5, showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thalianaα5 the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.
-
Intrinsic disorder in Viral Proteins Genome-Linked: experimental and predictive analyses
Virology Journal, 2009Co-Authors: Eugénie Hébrard, Vladimir N Uversky, Jocelyne Walter, Thierry Michon, Sonia Longhi, Yannick Bessin, François Delalande, Alain Van Dorsselaer, Pedro Romero, Nathalie DeclerckAbstract:Background VPgs are viral proteins linked to the 5' end of some viral genomes. Interactions between several VPgs and eukaryotic translation initiation factors eIF4Es are critical for plant infection. However, VPgs are not restricted to phytoViruses, being also involved in genome replication and protein translation of several animal Viruses. To date, structural data are still limited to small picornaviral VPgs. Recently three phytoviral VPgs were shown to be natively unfolded proteins. Results In this paper, we report the bacterial expression, purification and biochemical characterization of two phytoviral VPgs, namely the VPgs of Rice yellow mottle Virus (RYMV, genus SobemoVirus ) and Lettuce Mosaic Virus (LMV, genus PotyVirus ). Using far-UV circular dichroism and size exclusion chromatography, we show that RYMV and LMV VPgs are predominantly or partly unstructured in solution, respectively. Using several disorder predictors, we show that both proteins are predicted to possess disordered regions. We next extend theses results to 14 VPgs representative of the viral diversity. Disordered regions were predicted in all VPg sequences whatever the genus and the family. Conclusion Based on these results, we propose that intrinsic disorder is a common feature of VPgs. The functional role of intrinsic disorder is discussed in light of the biological roles of VPgs.
Thierry Candresse - One of the best experts on this subject based on the ideXlab platform.
-
key mutations in the cylindrical inclusion involved in Lettuce Mosaic Virus adaptation to eif4e mediated resistance in Lettuce
Molecular Plant-microbe Interactions, 2014Co-Authors: Maud Sorel, Thierry Candresse, Mariechristine Houvenaghel, L Svanelladumas, G Acelin, A Pitarch, Sylvie GermanretanaAbstract:We previously showed that allelic genes mo11 and mo12 used to protect Lettuce crops against Lettuce Mosaic Virus (LMV) correspond to mutant alleles of the gene encoding the eukaryotic translation initiation factor 4E. LMV resistance-breaking determinants map not only to the main potyVirus virulence determinant, a genome-linked viral protein, but also to the C-terminal region of the cylindrical inclusion (CI), with a key role of amino acid at position 621. Here, we show that the propagation of several non-Lettuce isolates of LMV in mo11 plants is accompanied by a gain of virulence correlated with the presence in the CI C terminus of a serine at position 617 and the accumulation of mutations at positions 602 or 627. Whole-genome sequencing of native and evolved isolates showed that no other mutation could be associated with adaptation to mo1 resistance. Site-directed mutagenesis pinpointed the key role in the virulence of the combination of mutations at positions 602 and 617, in addition to position 621. Th...
-
adaptation of Lettuce Mosaic Virus to catharanthus roseus involves mutations in the central domain of the vpg
Molecular Plant-microbe Interactions, 2014Co-Authors: Laurence Svanelladumas, Sylvie Germanretana, Eric Verdin, Chantal Faure, Patrick Gognalons, Jean Luc Danet, Armelle Marais, Thierry CandresseAbstract:An isolate of Lettuce Mosaic Virus (LMV, a PotyVirus) infecting Madagascar periwinckle (Catharanthus roseus) was identified and characterized by Illumina deep sequencing. LMV-Cr has no close affinities to previously sequenced LMV isolates and represents a novel, divergent LMV clade. Inoculation experiments with other representative LMV isolates showed that they are unable to infect C. roseus, which was not known to be a host for LMV. However, three C. roseus variants of one of these isolates, LMV-AF199, could be selected and partially or completely sequenced. These variants are characterized by the accumulation of mutations affecting the C-terminal part of the cylindrical inclusion (CI) helicase and the central part of the VPg. In particular, a serine to proline mutation at amino acid 143 of the VPg was observed in all three independently selected variants and is also present in the LMV-Cr isolate, making it a prime candidate as a host-range determinant. Other mutations at VPg positions 65 and 144 could a...
-
Adaptation of Lettuce Mosaic Virus to Catharanthus roseus involves mutations in the central domain of the VPg.
Molecular Plant-Microbe Interactions, 2014Co-Authors: Laurence Svanella-dumas, Eric Verdin, Chantal Faure, Patrick Gognalons, Jean Luc Danet, Armelle Marais, Sylvie German-retana, Thierry CandresseAbstract:An isolate of Lettuce Mosaic Virus (LMV, a PotyVirus) infecting Madagascar periwinckle (Catharanthus roseus) was identified and characterized by Illumina deep sequencing. LMV-Cr has no close affinities to previously sequenced LMV isolates and represents a novel, divergent LMV clade. Inoculation experiments with other representative LMV isolates showed that they are unable to infect C. roseus, which was not known to be a host for LMV. However, three C. roseus variants of one of these isolates, LMV-AF199, could be selected and partially or completely sequenced. These variants are characterized by the accumulation of mutations affecting the C-terminal part of the CI helicase and the central part of the VPg. In particular, a Serine to Proline mutation at amino acid 143 of the VPg was observed in all three independently selected variants and is also present in the LMV-Cr isolate, making it a prime candidate as a host range determinant. Other mutations at VPg positions 65 and 144 could also contribute to the ability to infect C. roseus. Inoculation experiments involving a recombinant LMV expressing a permissive Lettuce translation initiation factor 4E (eIF4E) suggest that eIF4E does not contribute to the interaction of most LMV isolates with C. roseus.
-
Key Mutations in the Cylindrical Inclusion Involved in Lettuce Mosaic Virus Adaptation to eIF4E-Mediated Resistance in Lettuce.
Molecular Plant-Microbe Interactions, 2014Co-Authors: Maud Sorel, Thierry Candresse, Mariechristine Houvenaghel, G Acelin, A Pitarch, Laurence Svanella-dumas, Sylvie German-retanaAbstract:We previously showed that allelic genes mo1(1) and mo1(2) used to protect Lettuce crops against Lettuce Mosaic Virus (LMV) correspond to mutant alleles of the gene encoding the eukaryotic translation initiation factor 4E. LMV resistance-breaking determinants map not only to the main potyVirus virulence determinant, a genome-linked viral protein, but also to the C-terminal region of the cylindrical inclusion (CI), with a key role of amino acid at position 621. Here, we show that the propagation of several non-Lettuce isolates of LMV in mo1(1) plants is accompanied by a gain of virulence correlated with the presence in the CI C terminus of a serine at position 617 and the accumulation of mutations at positions 602 or 627. Whole-genome sequencing of native and evolved isolates showed that no other mutation could be associated with adaptation to mo1 resistance. Site-directed mutagenesis pinpointed the key role in the virulence of the combination of mutations at positions 602 and 617, in addition to position 621. The impact of these mutations on the fitness of the Virus was evaluated, suggesting that the durability of mo1 resistance in the field relies on the fitness cost associated with the resistance-breaking mutations, the nature of the mutations, and their potential antagonistic effects.
-
the determinant of potyVirus ability to overcome the rtm resistance of arabidopsis thaliana maps to the n terminal region of the coat protein
Molecular Plant-microbe Interactions, 2009Co-Authors: Veronique Decroocq, Frederic Revers, L Svanelladumas, Beatriz Salvador, Ophelie Sicard, M Glasa, Patrick Cosson, Juan Antonio Garcia, Thierry CandresseAbstract:In Arabidopsis thaliana Columbia (Col-0) plants, the restriction of Tobacco etch Virus (TEV) long-distance movement involves at least three dominant RTM (restricted TEV movement) genes named RTM1, RTM2, and RTM3. Previous work has established that, while the RTM-mediated resistance is also effective against other potyViruses, such as Plum pox Virus (PPV) and Lettuce Mosaic Virus (LMV), some isolates of these Viruses are able to overcome the RTM mechanism. In order to identify the viral determinant of this RTM-resistance breaking, the biological properties of recombinants between PPV-R, which systemically infects Col-0, and PPV-PSes, restricted by the RTM resistance, were evaluated. Recombinants that contain the PPV-R coat protein (CP) sequence in an RTM-restricted background are able to systemically infect Col-0. The use of recombinants carrying chimeric CP genes indicated that one or more PPV resistance-breaking determinants map to the 5′ half of the CP gene. In the case of LMV, sequencing of independent...
Olivier Legall - One of the best experts on this subject based on the ideXlab platform.
-
the eukaryotic translation initiation factor 4e controls Lettuce susceptibility to the potyVirus Lettuce Mosaic Virus
Plant Physiology, 2003Co-Authors: Valerie Nicaise, Carole Caranta, Raquel Sanjuan, Mariepierre Dubrana, Marianne Mazier, Brigitte Maisonneuve, Olivier LegallAbstract:The eIF4E and eIF(iso)4E cDNAs from several genotypes of Lettuce ( Lactuca sativa ) that are susceptible, tolerant, or resistant to infection by Lettuce Mosaic Virus (LMV; genus PotyVirus ) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E 1 and Ls-eIF4E 2 were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 Lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1 1 and mo1 2 . Ls-eIF4E 1 and mo1 1 cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1 1 genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1 2 plants and a recombinant LMV expressing Ls-eIF4E° from its genome, but not Ls-eIF4E 1 or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1 1 or mo1 2 genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in Lettuce and that mo1 1 and mo1 2 are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyViruses in plants, probably through a general mechanism yet to be clarified.
-
the eukaryotic translation initiation factor 4e controls Lettuce susceptibility to the potyVirus Lettuce Mosaic Virus
Plant Physiology, 2003Co-Authors: Valerie Nicaise, Carole Caranta, Raquel Sanjuan, Mariepierre Dubrana, Marianne Mazier, Brigitte Maisonneuve, Olivier LegallAbstract:The eIF4E and eIF(iso)4E cDNAs from several genotypes of Lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce Mosaic Virus (LMV; genus PotyVirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 Lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in Lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyViruses in plants, probably through a general mechanism yet to be clarified.