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1.
J Virol Methods ; 329: 114997, 2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-39059502

RESUMO

The extraction of double stranded (ds) RNA is a common enrichment method for the study, characterization, and detection of RNA viruses. In addition to RNA viruses, viroids, and some DNA viruses, can also be detected from dsRNA enriched extracts which makes it an attractive method for detecting a wide range of viruses when coupled with HTS. Several dsRNA enrichment strategies have been developed. The oldest utilizes the selective binding properties of dsRNA to cellulose. More recent methods are based on the application of anti-dsRNA antibodies and viral proteins with a specific affinity for dsRNA. All three methods have been used together with HTS for plant virus detection and study. To our knowledge, this is the first comparative study of three alternative dsRNA enrichment methods for virus and viroid detection through HTS using virus-infected, and healthy grapevine test plants. Extracts were performed in triplicate using methods based on, the anti-dsRNA antibody mAb rJ2 (Millipore Sigma Canada Ltd, Oakville, ON, Canada), the B2 dsRNA binding protein, and ReliaPrep™ Resin (Promega Corporation, Madison, WI, USA). The results show that the workflows for all three methods are effectively comparable, apart from purification steps related to antibody and binding protein construct. Both the cellulose resin and dsRNA binding protein construct methods provide highly enriched dsRNA extracts suitable for HTS with the B2 method providing a 36× and the ReliaPrep™ Resin a 163× increase in dsRNA enrichment compared to the mAb rJ2 antibody. The overall consistency and cost effectiveness of the ReliaPrep™ cellulose resin-based method and the potentially simpler adaptation to robotics made it the method of choice for future transfer to a semi-automated workflow.

2.
Plant Cell ; 33(11): 3402-3420, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34436604

RESUMO

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Interações Hospedeiro-Patógeno , Defesa das Plantas contra Herbivoria/genética , Vírus de Plantas/fisiologia , RNA de Cadeia Dupla/genética , RNA de Plantas/genética , Proteínas de Ligação a RNA/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral
3.
Viruses ; 12(10)2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33023227

RESUMO

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant-microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant-virus interactions in complement to Saccharomyces cerevisiae.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Ribonuclease III/metabolismo , Tombusvirus/isolamento & purificação , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica de Plantas , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas , Interferência de RNA , RNA de Cadeia Dupla , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Saccharomyces cerevisiae/genética , Nicotiana/virologia , Replicação Viral
4.
Methods Mol Biol ; 2166: 307-327, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32710417

RESUMO

Plant RNA viruses are obligate intracellular parasites that hijack specific cellular membranes to replicate their genomes in what are commonly known as viral replication complexes (VRC). These contain host- and virus-encoded proteins and viral RNA. Double-stranded RNA (dsRNA) is a mandatory intermediate of RNA replication and a hallmark feature of VRCs. We have recently developed a method to isolate viral dsRNA and its associated proteins through pull-down of an ectopically expressed dsRNA-binding protein (B2:GFP) from infected Arabidopsis thaliana plants. After mass spectrometry analysis to identify the dsRNA-associated proteins, resulting candidate proteins of interest are tagged with a red fluorescent protein and their subcellular localization in relation to VRCs is assessed by transient expression within leaves of B2:GFP-transgenic Nicotiana benthamiana plants. In this chapter we describe in detail these experimental procedures to allow investigators to characterize the replication complexes of their plant RNA virus of interest.


Assuntos
Imunoprecipitação/métodos , Microscopia Confocal/métodos , Vírus de Plantas/metabolismo , Plantas/metabolismo , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/isolamento & purificação , Replicação Viral/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas Luminescentes , Espectrometria de Massas , Microscopia Confocal/instrumentação , Folhas de Planta/metabolismo , Vírus de Plantas/genética , Plantas/virologia , Plantas Geneticamente Modificadas , RNA de Cadeia Dupla/metabolismo , Nicotiana/metabolismo , Nicotiana/virologia
5.
Methods Mol Biol ; 2149: 443-462, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32617950

RESUMO

The purification of plant cell walls is challenging because they constitute an open compartment which is not limited by a membrane like the cell organelles. Different strategies have been established to limit the contamination by proteins of other compartments in cell wall proteomics studies. Non-destructive methods rely on washing intact cells with various types of solutions without disrupting the plasma membrane in order to elute cell wall proteins. In contrast, destructive protocols involve the purification of cell walls prior to the extraction of proteins with salt solutions. In both cases, proteins known to be intracellular have been identified by mass spectrometry in cell wall proteomes. The aim of this chapter is to provide tools to assess the subcellular localization of the proteins identified in cell wall proteomics studies, including: (1) bioinformatic predictions, (2) immunocytolocalization of proteins of interest on tissue sections and (3) in muro observation of proteins of interest fused to reporter fluorescent proteins by confocal microscopy. Finally, a qualitative assessment of the work can be performed and the strategy used to prepare the samples can be optimized if necessary.


Assuntos
Parede Celular/química , Biologia Computacional/métodos , Imuno-Histoquímica/métodos , Células Vegetais/metabolismo , Proteínas de Plantas/análise , Proteoma/metabolismo , Proteômica/métodos , Parede Celular/metabolismo , Técnicas de Transferência de Genes , Proteínas Luminescentes/metabolismo , Espectrometria de Massas , Microscopia Confocal , Folhas de Planta/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Inclusão do Tecido/métodos
6.
Proc Natl Acad Sci U S A ; 117(20): 10848-10855, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32371486

RESUMO

Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV-Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.


Assuntos
Nepovirus/efeitos dos fármacos , Doenças das Plantas/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacologia , Animais , Anticorpos Antivirais/imunologia , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/efeitos dos fármacos , Microscopia Crioeletrônica , Epitopos/química , Modelos Moleculares , Nematoides/virologia , Nepovirus/ultraestrutura , Doenças das Plantas/virologia , Folhas de Planta/virologia , Vírus de Plantas/imunologia , Vírus de Plantas/fisiologia , Conformação Proteica , Vitis
7.
Viruses ; 11(12)2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31835698

RESUMO

Grapevine fanleaf virus (GFLV) and arabis mosaic virus (ArMV) are nepoviruses responsible for grapevine degeneration. They are specifically transmitted from grapevine to grapevine by two distinct ectoparasitic dagger nematodes of the genus Xiphinema. GFLV and ArMV move from cell to cell as virions through tubules formed into plasmodesmata by the self-assembly of the viral movement protein. Five surface-exposed regions in the coat protein called R1 to R5, which differ between the two viruses, were previously defined and exchanged to test their involvement in virus transmission, leading to the identification of region R2 as a transmission determinant. Region R4 (amino acids 258 to 264) could not be tested in transmission due to its requirement for plant systemic infection. Here, we present a fine-tuning mutagenesis of the GFLV coat protein in and around region R4 that restored the virus movement and allowed its evaluation in transmission. We show that residues T258, M260, D261, and R301 play a crucial role in virus transmission, thus representing a new viral determinant of nematode transmission.


Assuntos
Vetores de Doenças , Nematoides/virologia , Nepovirus/classificação , Nepovirus/fisiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/virologia , Sequência de Aminoácidos , Animais , Genes Reporter , Modelos Moleculares , Nepovirus/ultraestrutura , Conformação Proteica , RNA Viral , Recombinação Genética , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/genética
8.
Plasmid ; 105: 102436, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31449836

RESUMO

Transient expression of proteins based on agro-infiltration techniques has proven very efficient and straightforward to study the intrinsic properties of proteins. The level of protein expression has been enhanced by the use of vector plasmids containing virus-derived sequences and the cloning step has been facilitated by recombination technologies. The pEAQ-HT-DEST series of vectors fulfilling these improvements are vectors of choice. However, they lack the possibility to directly and easily fuse the protein of interest to a fluorescent tag or to address it to the secretion pathway. In the present work we describe the production of 15 pEAQ-HT-DEST1-based plasmids designed to use the Gateway® cloning technology and to generate high levels of fluorescent fusion protein by agro-infiltration, in planta. This collection of plasmids includes binary vectors allowing N-terminal or C-terminal fusion to the bright tags EGFP or TagRFP for cytoplasmic accumulation or secretion and represents therefore a valuable tool for subcellular localization or biochemical studies. A viral protein, the blue fluorescent protein TagBFP, the green fluorescent protein variant T-Sapphire and an Arabidopsis protein were transiently expressed in N. benthamiana to demonstrate the potential of these vectors.


Assuntos
Vetores Genéticos/genética , Proteínas de Plantas/genética , Plasmídeos/genética , Arabidopsis/genética , Clonagem Molecular , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Fluorescência Verde/genética , Plantas Geneticamente Modificadas/genética
9.
Front Plant Sci ; 9: 70, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29449856

RESUMO

Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.

10.
Front Plant Sci ; 9: 135, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29479364

RESUMO

Peroxisomes are organelles that play key roles in eukaryotic metabolism. Their protein complement is entirely imported from the cytoplasm thanks to a unique pathway that is able to translocate folded proteins and protein complexes across the peroxisomal membrane. The import of molecules bound to a protein targeted to peroxisomes is an active process known as 'piggybacking' and we have recently shown that P15, a virus-encoded protein possessing a peroxisomal targeting sequence, is able to piggyback siRNAs into peroxisomes. Here, we extend this observation by analyzing the small RNA repertoire found in peroxisomes of P15-expressing plants. A direct comparison with the P15-associated small RNA retrieved during immunoprecipitation (IP) experiments, revealed that in vivo piggybacking coupled to peroxisome isolation could be a more sensitive means to determine the various small RNA species bound by a given protein. This increased sensitivity of peroxisome isolation as opposed to IP experiments was also striking when we analyzed the small RNA population bound by the Tomato bushy stunt virus-encoded P19, one of the best characterized viral suppressors of RNA silencing (VSR), artificially targeted to peroxisomes. These results support that peroxisomal targeting should be considered as a novel/alternative experimental approach to assess in vivo interactions that allows detection of labile binding events. The advantages and limitations of this approach are discussed.

11.
Plant Biotechnol J ; 16(2): 660-671, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28796912

RESUMO

Since their discovery, single-domain antigen-binding fragments of camelid-derived heavy-chain-only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode-transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell-to-cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs.


Assuntos
Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Nepovirus/patogenicidade , Vírus de Plantas/genética , Vírus de Plantas/fisiologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/fisiologia
12.
Methods Mol Biol ; 1701: 169-187, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29116505

RESUMO

Single-domain antibodies libraries of heavy-chain only immunoglobulins from camelids or shark are enriched for high-affinity antigen-specific binders by a short in vivo immunization. Thus, potent binders are readily retrieved from relatively small-sized libraries of 107-108 individual transformants, mostly after phage display and panning on a purified target. However, the remaining drawback of this strategy arises from the need to generate a dedicated library, for nearly every envisaged target. Therefore, all the procedures that shorten and facilitate the construction of an immune library of best possible quality are definitely a step forward. In this chapter, we provide the protocol to generate a high-quality immune VHH library using the Golden Gate Cloning strategy employing an adapted phage display vector where a lethal ccdB gene has to be substituted by the VHH gene. With this procedure, the construction of the library can be shortened to less than a week starting from bleeding the animal. Our libraries exceed 108 individual transformants and close to 100% of the clones harbor a phage display vector having an insert with the length of a VHH gene. These libraries are also more economic to make than previous standard approaches using classical restriction enzymes and ligations. The quality of the Nanobodies that are retrieved from immune libraries obtained by Golden Gate Cloning is identical to those from immune libraries made according to the classical procedure.


Assuntos
Camelus/genética , Clonagem Molecular/métodos , Biblioteca Gênica , Vetores Genéticos , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/genética , Animais , Camelus/imunologia , Anticorpos de Cadeia Única/imunologia
14.
Mol Plant Pathol ; 18(5): 708-719, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27216084

RESUMO

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis-related (PR) proteins. Endo-ß-1,3-glucanases (EGases) belong to the PR-2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti-P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR-2 family from Vitis.


Assuntos
Anti-Infecciosos/farmacologia , Oomicetos/patogenicidade , Proteínas de Plantas/farmacologia , Vitis/enzimologia , Anti-Infecciosos/metabolismo , Resistência à Doença/genética , Resistência à Doença/fisiologia , Regulação da Expressão Gênica de Plantas , Oomicetos/efeitos dos fármacos , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia
15.
Plant Biotechnol J ; 14(12): 2288-2299, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27178344

RESUMO

Virus-like particles (VLPs) derived from nonenveloped viruses result from the self-assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self-assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N- and C-terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant-based production of nucleic acid-free VLPs. Remarkably, expression of N- or C-terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.


Assuntos
Nepovirus/fisiologia , Proteínas Recombinantes/metabolismo , Vitis/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Nanopartículas , Nepovirus/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética
16.
PLoS One ; 9(2): e90072, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24587212

RESUMO

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Teste de Complementação Genética , Mutação , Saccharomyces cerevisiae/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Temperatura
17.
J Gen Virol ; 94(Pt 12): 2803-2813, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24088345

RESUMO

Factors involved in symptom expression of viruses from the genus Nepovirus in the family Secoviridae such as grapevine fanleaf virus (GFLV) are poorly characterized. To identify symptom determinants encoded by GFLV, infectious cDNA clones of RNA1 and RNA2 of strain GHu were developed and used alongside existing infectious cDNA clones of strain F13 in a reverse genetics approach. In vitro transcripts of homologous combinations of RNA1 and RNA2 induced systemic infection in Nicotiana benthamiana and Nicotiana clevelandii with identical phenotypes to WT virus strains, i.e. vein clearing and chlorotic spots on N. benthamiana and N. clevelandii for GHu, respectively, and lack of symptoms on both hosts for F13. The use of assorted transcripts mapped symptom determinants on RNA1 of GFLV strain GHu, in particular within the distal 408 nt of the RNA-dependent RNA polymerase (1E(Pol)), as shown by RNA1 transcripts for which coding regions or fragments derived thereof were swapped. Semi-quantitative analyses indicated no significant differences in virus titre between symptomatic and asymptomatic plants infected with various recombinants. Also, unlike the nepovirus tomato ringspot virus, no apparent proteolytic cleavage of GFLV protein 1E(Pol) was detected upon virus infection or transient expression in N. benthamiana. In addition, GFLV protein 1E(Pol) failed to suppress silencing of EGFP in transgenic N. benthamiana expressing EGFP or to enhance GFP expression in patch assays in WT N. benthamiana. Together, our results suggest the existence of strain-specific functional domains, including a symptom determinant module, on the RNA-dependent RNA polymerase of GFLV.


Assuntos
Nepovirus/genética , Nepovirus/patogenicidade , Nicotiana/virologia , Doenças das Plantas/virologia , RNA Polimerase Dependente de RNA/genética , Vitis/virologia , Sequência de Aminoácidos , Dados de Sequência Molecular , Nepovirus/isolamento & purificação , Filogenia , RNA Viral/genética , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Análise de Sequência de DNA , Especificidade da Espécie , Proteínas Virais/genética , Proteínas Virais/metabolismo
18.
J Struct Biol ; 182(1): 1-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23376736

RESUMO

Arabis mosaic virus (ArMV) and Grapevine fanleaf virus (GFLV) are two picorna-like viruses from the genus Nepovirus, consisting in a bipartite RNA genome encapsidated into a 30 nm icosahedral viral particle formed by 60 copies of a single capsid protein (CP). They are responsible for a severe degeneration of grapevines that occurs in most vineyards worldwide. Although sharing a high level of sequence identity between their CP, ArMV is transmitted exclusively by the ectoparasitic nematode Xiphinema diversicaudatum whereas GFLV is specifically transmitted by the nematode X. index. The structural determinants involved in the transmission specificity of both viruses map solely to their respective CP. Recently, reverse genetic and crystallographic studies on GFLV revealed that a positively charged pocket in the CP B domain located at the virus surface may be responsible for vector specificity. To go further into delineating the coat protein determinants involved in transmission specificity, we determined the 6.5 Å resolution cryo-electron microscopy structure of ArMV and used homology modeling and flexible fitting approaches to build its pseudo-atomic structure. This study allowed us to resolve ArMV CP architecture and delineate connections between ArMV capsid shell and its RNA. Comparison of ArMV and GFLV CPs reveals structural differences in the B domain pocket, thus strengthening the hypothesis of a key role of this region in the viral transmission specificity and identifies new potential functional domains of Nepovirus capsid.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/ultraestrutura , Nepovirus/fisiologia , Nepovirus/ultraestrutura , RNA Viral/metabolismo , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Enoplídios/virologia , Modelos Moleculares , Vírus do Mosaico/química , Vírus do Mosaico/fisiologia , Vírus do Mosaico/ultraestrutura , Nepovirus/química , Doenças das Plantas/virologia , Estrutura Terciária de Proteína
19.
PLoS Pathog ; 7(10): e1002327, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22046131

RESUMO

Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD), organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP) modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and provide a conduit for virion translocation. The MP of tubule-forming viruses including Grapevine fanleaf virus (GFLV) recruit the plant PD receptors called Plasmodesmata Located Proteins (PDLP) to mediate tubule assembly and virus movement. Here we show that PDLP1 is transported to PD through a specific route within the secretory pathway in a myosin-dependent manner. This transport relies primarily on the class XI myosins XI-K and XI-2. Inactivation of these myosins using dominant negative inhibition results in mislocalization of PDLP and MP and suppression of GFLV movement. We also found that the proper targeting of specific markers of the Golgi apparatus, the plasma membrane, PD, lipid raft subdomains within the plasma membrane, and the tonoplast was not affected by myosin XI-K inhibition. However, the normal tonoplast dynamics required myosin XI-K activity. These results reveal a new pathway of the myosin-dependent protein trafficking to PD that is hijacked by GFLV to promote tubule-guided transport of this virus between plant cells.


Assuntos
Miosinas/metabolismo , Nepovirus/fisiologia , Proteínas do Movimento Viral em Plantas/fisiologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/fisiologia , Complexo de Golgi/virologia , Interações Hospedeiro-Patógeno , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/virologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Microtúbulos/virologia , Miosinas/antagonistas & inibidores , Nepovirus/efeitos dos fármacos , Nepovirus/patogenicidade , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Tiazolidinas/farmacologia , Proteínas não Estruturais Virais
20.
PLoS Pathog ; 7(5): e1002034, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21625570

RESUMO

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.


Assuntos
Proteínas do Capsídeo/genética , Nematoides/virologia , Nepovirus , Estrutura Quaternária de Proteína , Substituição de Aminoácidos , Animais , Capsídeo , Mutação , Nepovirus/genética , Nepovirus/metabolismo , Nepovirus/ultraestrutura , Doenças das Plantas/genética , Doenças das Plantas/virologia , Vírus de Plantas/genética , RNA Viral/genética , Alinhamento de Sequência , Análise de Sequência de Proteína , Eletricidade Estática , Difração de Raios X
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