Frederic Aparicio - Academia.edu (original) (raw)

Papers by Frederic Aparicio

Molecular Plant Pathology, 2016

During virus infection, the specific viral components-host factors interaction elicits transcript... more During virus infection, the specific viral components-host factors interaction elicits transcriptional reprogramming of diverse cellular pathways. Alfalfa mosaic virus (AMV) establishes a compatible interaction in tobacco and Arabidopsis hosts. We show that the coat protein (CP) of AMV directly interacted with transcription factor (TF) ILR3 of both species. ILR3 is a basic helix-loop-helix (bHLH) family member of TFs, previously proposed to participate in diverse metabolic pathways. ILR3 has been shown to regulate NEET in Arabidopsis, a critical protein in plant development, senescence, iron metabolism and reactive oxygen species (ROS) homeostasis. We observed that the AMV CP-ILR3 interaction caused a fraction of this TF to relocate from the nucleus to the nucleolus. ROS, PR1 mRNAs, SA and JA contents were increased in healthy Arabidopsis loss-of function ILR3 mutant (ilr3.2 plants), which suggests the implication of ILR3 in the regulation of plant defense responses. In AMVinfected wild-type (wt) plants, NEET expression slightly reduced, but was significantly induced in ilr3.2 mutant plants. Furthermore, SA and JA accumulation was induced in Arabidopsis wt-infected plants. AMV infection in ilr3.2 plants increased JA over 10-fold, and SA significantly reduced, which indicates an antagonist crosstalk effect. The accumulation levels of viral RNAs significantly lowered in ilr3.2 mutants, but the virus could still systemically invade the plant. The AMV CP-TF ILR3 interactions might down-regulate a host factor, NEET, which would lead to plant hormone responses being activated to obtain a hormonal equilibrium state, where infection would remain at a level that would not affect plant viability. This article is protected by copyright. All rights reserved.

Virus genes, 2002

The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRS... more The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRSV) varying in the symptomatology they cause in six different Prunus spp. were determined. Analysis of the molecular variability has allowed, in addition to study the phylogenetic relationships among them, to evaluate the minimal requirements for the synthesis of the subgenomic RNA in Ilarvirus genus and their comparison to other members of the Bromoviridae family. Computer assisted comparisons led recently to Jaspars (Virus Genes 17, 233-242, 1998) to propose that a hairpin structure in viral minus strand RNA is required for subgenomic promoter activity of viruses from at least two, and possibly all five, genera in the family of Bromoviridae. For PNRSV and Apple mosaic virus two stable hairpins were proposed whereas for the rest of Ilarviruses and the other four genera of the Bromoviridae family only one stable hairpin was predicted. Comparative analysis of this region among the fifteen ...

Virology, 2003

Binding of coat protein (CP) to the 3Ј nontranslated region (3Ј-NTR) of viral RNAs is a crucial r... more Binding of coat protein (CP) to the 3Ј nontranslated region (3Ј-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo-and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3Ј-NTR of its genomic RNA using purified E. coli-expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3Ј-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3Ј-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg 2ϩ , lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3Ј-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3Ј-NTR level in Alfamo-and Ilarvirus genera.

Molecular Plant-Microbe Interactions, 2012

In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved ... more In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation.

Journal of General Virology, Jun 1, 2006

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent com... more Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N-and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

European Journal of Plant Pathology, 2015

Methods in molecular biology (Clifton, N.J.), 2009

Multiplex Polymerase Chain Reaction (PCR) can be used for the simultaneous detection of plant vir... more Multiplex Polymerase Chain Reaction (PCR) can be used for the simultaneous detection of plant viruses. Multiple primer pairs or polyvalent primer pairs can be used to detect and identify several viruses in a single PCR.

The Journal of general virology, 2001

Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera ALFAMO... more Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera ALFAMOVIRUS: and ILARVIRUS:, respectively, of the family BROMOVIRIDAE: Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3' termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. CIS:-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV-PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.

Virus Research, 2014

The movement protein (MP) of parietaria mottle virus (PMoV) is required for virus cell-to-cell mo... more The movement protein (MP) of parietaria mottle virus (PMoV) is required for virus cell-to-cell movement. Bioinformatics analysis identified two hydrophilic non-contiguous regions (R1 and R2) rich in the basic amino acids lysine and arginine and with the predicted secondary structure of an ␣-helix. Different approaches were used to determine the implication of the R1 and R2 regions in RNA binding, plasmodesmata (PD) targeting and cell-to-cell movement. EMSA (Electrophoretic Mobility Shift Assay) showed that both regions have RNA-binding activity whereas that mutational analysis reported that either deletion of any of these regions, or loss of the basic amino acids, interfered with the viral intercellular movement. Subcellular localization studies showed that PMoV MP locates at PD. Mutants designed to impeded cellto-cell movement failed to accumulate at PD indicating that basic residues in both R1 and R2 are critical for binding the MP at PD.

Advances in Virus Research, 2013

The e-Xtra logo stands for "electronic extra" and indicates that the online version contains one ... more The e-Xtra logo stands for "electronic extra" and indicates that the online version contains one supplemental figure.

THE PLANT CELL ONLINE, 2009

In common with a range of environmental and biological stresses, heat shock results in the accumu... more In common with a range of environmental and biological stresses, heat shock results in the accumulation of misfolded proteins and a collection of downstream consequences for cellular homeostasis and growth. Within this complex array of responses, the sensing of and responses to misfolded proteins in specific subcellular compartments involves specific chaperones, transcriptional regulators, and expression profiles. Using biological (ectopic protein expression and virus infection) and chemical triggers for misfolded protein accumulation, we have profiled the transcriptional features of the response to misfolded protein accumulation in the cytosol (i.e., the cytoplasmic protein response [CPR]) and identified the effects as a subcomponent of the wider effects induced by heat shock. The CPR in Arabidopsis thaliana is associated with the heat shock promoter element and the involvement of specific heat shock factors (HSFs), notably HSFA2, which appears to be regulated by alternative splicing and non-sense-mediated decay. Characterization of Arabidopsis HSFA2 knockout and overexpression lines showed that HSFA2 is one of the regulatory components of the CPR.

PLoS ONE, 2011

Virus life cycle heavily depends on their ability to command the host machinery in order to trans... more Virus life cycle heavily depends on their ability to command the host machinery in order to translate their genomes. Animal viruses have been shown to interfere with host translation machinery by expressing viral proteins that either maintain or inhibit eIF2a function by phosphorylation. However, this interference mechanism has not been described for any plant virus yet. Prunnus necrotic ringspot virus (PNRSV) is a serious pathogen of cultivated stone fruit trees. The movement protein (MP) of PNRSV is necessary for the cell-to-cell movement of the virus. By using a yeast-based approach we have found that overexpression of the PNRSV MP caused a severe growth defect in yeast cells. cDNA microarrays analysis carried out to characterise at the molecular level the growth interference phenotype reported the induction of genes related to amino acid deprivation suggesting that expression of MP activates the GCN pathway in yeast cells. Accordingly, PNRSV MP triggered activation of the Gcn2p kinase, as judged by increased eIF2a phosphorylation. Activation of Gcn2p by MP expression required a functional Tor1p kinase, since rapamycin treatment alleviated the yeast cell growth defect and blocked eIF2a phosphorylation triggered by MP expression. Overall, these findings uncover a previously uncharacterised function for PNRSV MP viral protein, and point out at Tor1p and Gcn2p kinases as candidate susceptibility factors for plant viral infections.

PLANT PHYSIOLOGY, 2005

Different cytoplasmically replicating RNA viruses were shown to induce a specific subset of heat-... more Different cytoplasmically replicating RNA viruses were shown to induce a specific subset of heat-inducible heat shock protein 70 (HSP70) genes in Arabidopsis (Arabidopsis thaliana). To identify the inducing principle, a promoterreporter system was developed for the facile analysis of differentially responding Arabidopsis HSP70 genes, by infiltration into Nicotiana benthamiana leaves. Through transient expression of individual viral cistrons or through deletion analysis of a viral replicon, we were unable to identify a unique inducer of HSP70. However, there was a positive correlation between the translatability of the test construct and the differential induction of HSP70. Since these data implied a lack of specificity in the induction process, we also expressed a random series of cytosolically targeted Arabidopsis genes and showed that these also differentially induced HSP70. Through a comparison of different promoterreporter constructs and through measurements of the steady-state levels of the individual proteins, it appeared that the HSP70 response reflected the ability of the cytosol to sense individual properties of particular proteins when expressed at high levels. This phenomenon is reminiscent of the unfolded protein response observed when the induced accumulation of proteins in the endoplasmic reticulum also induces a specific suite of chaperones.

Plant Pathology, 1998

Three methods were compared for the detection of prunus necrotic ringspot virus in herbaceous and... more Three methods were compared for the detection of prunus necrotic ringspot virus in herbaceous and woody plants: DAS-ELISA, nonisotopic dot-blot hybridization and reverse transcriptional polymerase chain reaction (RT-PCR). When purified virus preparations were used, the detection limit of the RT-PCR technique was 1·28 pg mL ¹1 whereas nonisotopic molecular hybridization and DAS-ELISA allowed detection of 0·8 ng mL ¹1 and 4 ng mL ¹1 , respectively. Several sample processing procedures were evaluated for virus detection by the nonisotopic molecular hybridization technique. When a very short and simple sample processing method was used, the detection limit of the nonisotopic molecular hybridization technique was 25 times higher than that of DAS-ELISA and 625 times lower than that of RT-PCR. A comparison of the level of virus accumulation in mature fruits and in leaf tissue showed that, on average, 125 times more virus was found in the fruits.

Plant Disease, 1999

The apple scar skin disease was first re- ported 60 years ago in China (Manchuria) with the name ... more The apple scar skin disease was first re- ported 60 years ago in China (Manchuria) with the name “Manshu-sabika-byo” (19). Twenty years later, two apple disorders were described simultaneously in the United States: the apple scar skin disease on cv. Red Delicious in Missouri (18), ...

Phytopathology, 2000

ABSTRACT The three most economically damaging ilarviruses affecting stone fruit trees on a worldw... more ABSTRACT The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

Phytopathology, 1999

ABSTRACT Viral sequences amplified by polymerase chain reaction from 25 isolates of Prunus necrot... more ABSTRACT Viral sequences amplified by polymerase chain reaction from 25 isolates of Prunus necrotic ringspot virus (PNRSV), varying in the symptomatology they cause in six different Prunus spp., were analyzed for restriction fragment polymorphisms. Most of the isolates could be discriminated by using a combination of three different restriction enzymes. The nucleotide sequences of the RNA 4 of 15 of these isolates were determined. Sequence comparisons and phylogenetic analyses of the RNA 4 and coat proteins (CPs) revealed that all of the isolates clustered into three different groups, represented by three previously sequenced PNRSV isolates: PV32, PE5, and PV96. The PE5-type group was characterized by a 5' untranslated region that was clearly different from that of the other two groups. The PV32-type group was characterized by an extra hexanucleotide consisting of a duplication of the six immediately preceding nucleotides. Although most of the variability was observed in the first third of the CP, the amino acid residues in this region, which were previously thought to be functionally important in the replication cycle of the virus, were strictly conserved. No clear correlation with the type of symptom or host specificity could be observed. The validity of this grouping was confirmed when other isolates recently characterized by other authors were included in these analyses.

Molecular Plant Pathology, 2014

Movement proteins (MPs) encoded by plant viruses interact with host proteins to facilitate or int... more Movement proteins (MPs) encoded by plant viruses interact with host proteins to facilitate or interfere with intra- and/or intercellular viral movement. Using yeast two-hybrid and bimolecular fluorescence complementation assays, we herein present in vivo evidence for the interaction between Alfalfa mosaic virus (AMV) MP and Arabidopsis Patellin 3 (atPATL3) and Patellin 6 (atPATL6), two proteins containing a Sec14 domain. Proteins with Sec14 domains are implicated in membrane trafficking, cytoskeleton dynamics, lipid metabolism and lipid-mediated regulatory functions. Interestingly, the overexpression of atPATL3 and/or atPATL6 interfered with the plasmodesmata targeting of AMV MP and correlated with reduced infection foci size. Consistently, the viral RNA levels increased in the single and double Arabidopsis knockout mutants for atPATL3 and atPATL6. Our results indicate that, in general, MP-PATL interactions interfere with the correct subcellular targeting of MP, thus rendering the intracellular transport of viral MP-containing complexes less efficient and diminishing cell-to-cell movement.

Journal of Virological Methods, 2005

A new strategy for the simultaneous detection of plant viruses by molecular hybridization has bee... more A new strategy for the simultaneous detection of plant viruses by molecular hybridization has been developed. Two, four or six viral sequences were fused in tandem and transcribed to render unique riboprobes and designated as 'polyprobes'. The 'polyprobe four' (poly 4) covered the four ilarviruses affecting stone fruit trees including apple mosaic virus (ApMV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), and American plum line pattern virus (APLPV) whereas the 'polyprobe two' (poly 2) was designed to detect simultaneously, plum pox virus (PPV) and apple chlorotic leaf spot virus (ACLSV), the two more important viruses affecting these trees. Finally, a 'polyprobe six' (poly 6) was generated to detect any of the six viruses. The three polyprobes were comparable to the individual riboprobes in terms of end-point dilution limit and specificity. The validation of the new simultaneous detection strategy was confirmed by the analysis of 46 field samples from up to seven different hosts collected from 10 different geographical areas.

Molecular Plant Pathology, 2016

During virus infection, the specific viral components-host factors interaction elicits transcript... more During virus infection, the specific viral components-host factors interaction elicits transcriptional reprogramming of diverse cellular pathways. Alfalfa mosaic virus (AMV) establishes a compatible interaction in tobacco and Arabidopsis hosts. We show that the coat protein (CP) of AMV directly interacted with transcription factor (TF) ILR3 of both species. ILR3 is a basic helix-loop-helix (bHLH) family member of TFs, previously proposed to participate in diverse metabolic pathways. ILR3 has been shown to regulate NEET in Arabidopsis, a critical protein in plant development, senescence, iron metabolism and reactive oxygen species (ROS) homeostasis. We observed that the AMV CP-ILR3 interaction caused a fraction of this TF to relocate from the nucleus to the nucleolus. ROS, PR1 mRNAs, SA and JA contents were increased in healthy Arabidopsis loss-of function ILR3 mutant (ilr3.2 plants), which suggests the implication of ILR3 in the regulation of plant defense responses. In AMVinfected wild-type (wt) plants, NEET expression slightly reduced, but was significantly induced in ilr3.2 mutant plants. Furthermore, SA and JA accumulation was induced in Arabidopsis wt-infected plants. AMV infection in ilr3.2 plants increased JA over 10-fold, and SA significantly reduced, which indicates an antagonist crosstalk effect. The accumulation levels of viral RNAs significantly lowered in ilr3.2 mutants, but the virus could still systemically invade the plant. The AMV CP-TF ILR3 interactions might down-regulate a host factor, NEET, which would lead to plant hormone responses being activated to obtain a hormonal equilibrium state, where infection would remain at a level that would not affect plant viability. This article is protected by copyright. All rights reserved.

Virus genes, 2002

The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRS... more The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRSV) varying in the symptomatology they cause in six different Prunus spp. were determined. Analysis of the molecular variability has allowed, in addition to study the phylogenetic relationships among them, to evaluate the minimal requirements for the synthesis of the subgenomic RNA in Ilarvirus genus and their comparison to other members of the Bromoviridae family. Computer assisted comparisons led recently to Jaspars (Virus Genes 17, 233-242, 1998) to propose that a hairpin structure in viral minus strand RNA is required for subgenomic promoter activity of viruses from at least two, and possibly all five, genera in the family of Bromoviridae. For PNRSV and Apple mosaic virus two stable hairpins were proposed whereas for the rest of Ilarviruses and the other four genera of the Bromoviridae family only one stable hairpin was predicted. Comparative analysis of this region among the fifteen ...

Virology, 2003

Binding of coat protein (CP) to the 3Ј nontranslated region (3Ј-NTR) of viral RNAs is a crucial r... more Binding of coat protein (CP) to the 3Ј nontranslated region (3Ј-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo-and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3Ј-NTR of its genomic RNA using purified E. coli-expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3Ј-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3Ј-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg 2ϩ , lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3Ј-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3Ј-NTR level in Alfamo-and Ilarvirus genera.

Molecular Plant-Microbe Interactions, 2012

In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved ... more In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation.

Journal of General Virology, Jun 1, 2006

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent com... more Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N-and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

European Journal of Plant Pathology, 2015

Methods in molecular biology (Clifton, N.J.), 2009

Multiplex Polymerase Chain Reaction (PCR) can be used for the simultaneous detection of plant vir... more Multiplex Polymerase Chain Reaction (PCR) can be used for the simultaneous detection of plant viruses. Multiple primer pairs or polyvalent primer pairs can be used to detect and identify several viruses in a single PCR.

The Journal of general virology, 2001

Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera ALFAMO... more Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera ALFAMOVIRUS: and ILARVIRUS:, respectively, of the family BROMOVIRIDAE: Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3' termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. CIS:-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV-PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.

Virus Research, 2014

The movement protein (MP) of parietaria mottle virus (PMoV) is required for virus cell-to-cell mo... more The movement protein (MP) of parietaria mottle virus (PMoV) is required for virus cell-to-cell movement. Bioinformatics analysis identified two hydrophilic non-contiguous regions (R1 and R2) rich in the basic amino acids lysine and arginine and with the predicted secondary structure of an ␣-helix. Different approaches were used to determine the implication of the R1 and R2 regions in RNA binding, plasmodesmata (PD) targeting and cell-to-cell movement. EMSA (Electrophoretic Mobility Shift Assay) showed that both regions have RNA-binding activity whereas that mutational analysis reported that either deletion of any of these regions, or loss of the basic amino acids, interfered with the viral intercellular movement. Subcellular localization studies showed that PMoV MP locates at PD. Mutants designed to impeded cellto-cell movement failed to accumulate at PD indicating that basic residues in both R1 and R2 are critical for binding the MP at PD.

Advances in Virus Research, 2013

The e-Xtra logo stands for "electronic extra" and indicates that the online version contains one ... more The e-Xtra logo stands for "electronic extra" and indicates that the online version contains one supplemental figure.

THE PLANT CELL ONLINE, 2009

In common with a range of environmental and biological stresses, heat shock results in the accumu... more In common with a range of environmental and biological stresses, heat shock results in the accumulation of misfolded proteins and a collection of downstream consequences for cellular homeostasis and growth. Within this complex array of responses, the sensing of and responses to misfolded proteins in specific subcellular compartments involves specific chaperones, transcriptional regulators, and expression profiles. Using biological (ectopic protein expression and virus infection) and chemical triggers for misfolded protein accumulation, we have profiled the transcriptional features of the response to misfolded protein accumulation in the cytosol (i.e., the cytoplasmic protein response [CPR]) and identified the effects as a subcomponent of the wider effects induced by heat shock. The CPR in Arabidopsis thaliana is associated with the heat shock promoter element and the involvement of specific heat shock factors (HSFs), notably HSFA2, which appears to be regulated by alternative splicing and non-sense-mediated decay. Characterization of Arabidopsis HSFA2 knockout and overexpression lines showed that HSFA2 is one of the regulatory components of the CPR.

PLoS ONE, 2011

Virus life cycle heavily depends on their ability to command the host machinery in order to trans... more Virus life cycle heavily depends on their ability to command the host machinery in order to translate their genomes. Animal viruses have been shown to interfere with host translation machinery by expressing viral proteins that either maintain or inhibit eIF2a function by phosphorylation. However, this interference mechanism has not been described for any plant virus yet. Prunnus necrotic ringspot virus (PNRSV) is a serious pathogen of cultivated stone fruit trees. The movement protein (MP) of PNRSV is necessary for the cell-to-cell movement of the virus. By using a yeast-based approach we have found that overexpression of the PNRSV MP caused a severe growth defect in yeast cells. cDNA microarrays analysis carried out to characterise at the molecular level the growth interference phenotype reported the induction of genes related to amino acid deprivation suggesting that expression of MP activates the GCN pathway in yeast cells. Accordingly, PNRSV MP triggered activation of the Gcn2p kinase, as judged by increased eIF2a phosphorylation. Activation of Gcn2p by MP expression required a functional Tor1p kinase, since rapamycin treatment alleviated the yeast cell growth defect and blocked eIF2a phosphorylation triggered by MP expression. Overall, these findings uncover a previously uncharacterised function for PNRSV MP viral protein, and point out at Tor1p and Gcn2p kinases as candidate susceptibility factors for plant viral infections.

PLANT PHYSIOLOGY, 2005

Different cytoplasmically replicating RNA viruses were shown to induce a specific subset of heat-... more Different cytoplasmically replicating RNA viruses were shown to induce a specific subset of heat-inducible heat shock protein 70 (HSP70) genes in Arabidopsis (Arabidopsis thaliana). To identify the inducing principle, a promoterreporter system was developed for the facile analysis of differentially responding Arabidopsis HSP70 genes, by infiltration into Nicotiana benthamiana leaves. Through transient expression of individual viral cistrons or through deletion analysis of a viral replicon, we were unable to identify a unique inducer of HSP70. However, there was a positive correlation between the translatability of the test construct and the differential induction of HSP70. Since these data implied a lack of specificity in the induction process, we also expressed a random series of cytosolically targeted Arabidopsis genes and showed that these also differentially induced HSP70. Through a comparison of different promoterreporter constructs and through measurements of the steady-state levels of the individual proteins, it appeared that the HSP70 response reflected the ability of the cytosol to sense individual properties of particular proteins when expressed at high levels. This phenomenon is reminiscent of the unfolded protein response observed when the induced accumulation of proteins in the endoplasmic reticulum also induces a specific suite of chaperones.

Plant Pathology, 1998

Three methods were compared for the detection of prunus necrotic ringspot virus in herbaceous and... more Three methods were compared for the detection of prunus necrotic ringspot virus in herbaceous and woody plants: DAS-ELISA, nonisotopic dot-blot hybridization and reverse transcriptional polymerase chain reaction (RT-PCR). When purified virus preparations were used, the detection limit of the RT-PCR technique was 1·28 pg mL ¹1 whereas nonisotopic molecular hybridization and DAS-ELISA allowed detection of 0·8 ng mL ¹1 and 4 ng mL ¹1 , respectively. Several sample processing procedures were evaluated for virus detection by the nonisotopic molecular hybridization technique. When a very short and simple sample processing method was used, the detection limit of the nonisotopic molecular hybridization technique was 25 times higher than that of DAS-ELISA and 625 times lower than that of RT-PCR. A comparison of the level of virus accumulation in mature fruits and in leaf tissue showed that, on average, 125 times more virus was found in the fruits.

Plant Disease, 1999

The apple scar skin disease was first re- ported 60 years ago in China (Manchuria) with the name ... more The apple scar skin disease was first re- ported 60 years ago in China (Manchuria) with the name “Manshu-sabika-byo” (19). Twenty years later, two apple disorders were described simultaneously in the United States: the apple scar skin disease on cv. Red Delicious in Missouri (18), ...

Phytopathology, 2000

ABSTRACT The three most economically damaging ilarviruses affecting stone fruit trees on a worldw... more ABSTRACT The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

Phytopathology, 1999

ABSTRACT Viral sequences amplified by polymerase chain reaction from 25 isolates of Prunus necrot... more ABSTRACT Viral sequences amplified by polymerase chain reaction from 25 isolates of Prunus necrotic ringspot virus (PNRSV), varying in the symptomatology they cause in six different Prunus spp., were analyzed for restriction fragment polymorphisms. Most of the isolates could be discriminated by using a combination of three different restriction enzymes. The nucleotide sequences of the RNA 4 of 15 of these isolates were determined. Sequence comparisons and phylogenetic analyses of the RNA 4 and coat proteins (CPs) revealed that all of the isolates clustered into three different groups, represented by three previously sequenced PNRSV isolates: PV32, PE5, and PV96. The PE5-type group was characterized by a 5' untranslated region that was clearly different from that of the other two groups. The PV32-type group was characterized by an extra hexanucleotide consisting of a duplication of the six immediately preceding nucleotides. Although most of the variability was observed in the first third of the CP, the amino acid residues in this region, which were previously thought to be functionally important in the replication cycle of the virus, were strictly conserved. No clear correlation with the type of symptom or host specificity could be observed. The validity of this grouping was confirmed when other isolates recently characterized by other authors were included in these analyses.

Molecular Plant Pathology, 2014

Movement proteins (MPs) encoded by plant viruses interact with host proteins to facilitate or int... more Movement proteins (MPs) encoded by plant viruses interact with host proteins to facilitate or interfere with intra- and/or intercellular viral movement. Using yeast two-hybrid and bimolecular fluorescence complementation assays, we herein present in vivo evidence for the interaction between Alfalfa mosaic virus (AMV) MP and Arabidopsis Patellin 3 (atPATL3) and Patellin 6 (atPATL6), two proteins containing a Sec14 domain. Proteins with Sec14 domains are implicated in membrane trafficking, cytoskeleton dynamics, lipid metabolism and lipid-mediated regulatory functions. Interestingly, the overexpression of atPATL3 and/or atPATL6 interfered with the plasmodesmata targeting of AMV MP and correlated with reduced infection foci size. Consistently, the viral RNA levels increased in the single and double Arabidopsis knockout mutants for atPATL3 and atPATL6. Our results indicate that, in general, MP-PATL interactions interfere with the correct subcellular targeting of MP, thus rendering the intracellular transport of viral MP-containing complexes less efficient and diminishing cell-to-cell movement.

Journal of Virological Methods, 2005

A new strategy for the simultaneous detection of plant viruses by molecular hybridization has bee... more A new strategy for the simultaneous detection of plant viruses by molecular hybridization has been developed. Two, four or six viral sequences were fused in tandem and transcribed to render unique riboprobes and designated as 'polyprobes'. The 'polyprobe four' (poly 4) covered the four ilarviruses affecting stone fruit trees including apple mosaic virus (ApMV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), and American plum line pattern virus (APLPV) whereas the 'polyprobe two' (poly 2) was designed to detect simultaneously, plum pox virus (PPV) and apple chlorotic leaf spot virus (ACLSV), the two more important viruses affecting these trees. Finally, a 'polyprobe six' (poly 6) was generated to detect any of the six viruses. The three polyprobes were comparable to the individual riboprobes in terms of end-point dilution limit and specificity. The validation of the new simultaneous detection strategy was confirmed by the analysis of 46 field samples from up to seven different hosts collected from 10 different geographical areas.