RESUMEN
The movement of potyviruses, the largest genus of single-stranded, positive-sense RNA viruses responsible for serious diseases in crops, is very complex. As potyviruses developed strategies to hijack the host secretory pathway and plasmodesmata (PD) for their transport, the goal of this study was to identify membrane and/or PD-proteins that interact with the 6K2 protein, a potyviral protein involved in replication and cell-to-cell movement of turnip mosaic virus (TuMV). Using split-ubiquitin membrane yeast two-hybrid assays, we screened an Arabidopsis cDNA library for interactors of TuMV6K2. We isolated AtHVA22a (Hordeum vulgare abscisic acid responsive gene 22), which belongs to a multigenic family of transmembrane proteins, homologous to Receptor expression-enhancing protein (Reep)/Deleted in polyposis (DP1)/Yop1 family proteins in animal and yeast. HVA22/DP1/Yop1 family genes are widely distributed in eukaryotes, but the role of HVA22 proteins in plants is still not well known, although proteomics analysis of PD fractions purified from Arabidopsis suspension cells showed that AtHVA22a is highly enriched in a PD proteome. We confirmed the interaction between TuMV6K2 and AtHVA22a in yeast, as well as in planta by using bimolecular fluorescence complementation and showed that TuMV6K2/AtHVA22a interaction occurs at the level of the viral replication compartment during TuMV infection. Finally, we showed that the propagation of TuMV is increased when AtHVA22a is overexpressed in planta but slowed down upon mutagenesis of AtHVA22a by CRISPR-Cas9. Altogether, our results indicate that AtHVA22a plays an agonistic effect on TuMV propagation and that the C-terminal tail of the protein is important in this process.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Potyvirus , Potyvirus/patogenicidad , Potyvirus/fisiología , Arabidopsis/virología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Enfermedades de las Plantas/virología , Proteínas Virales/metabolismo , Proteínas Virales/genética , Replicación Viral , Nicotiana/virología , Nicotiana/genéticaRESUMEN
The genus Potyvirus is considered as the largest among plant single-stranded (positive-sense) RNA viruses, causing considerable economic damage to vegetable and fruit crops worldwide. Through the coordinated action of four viral proteins and a few identified host factors, potyviruses exploit the endomembrane system of infected cells for their replication and for their intra- and intercellular movement to and through plasmodesmata (PDs). Although a significant amount of data concerning potyvirus movement has been published, no synthetic review compiling and integrating all information relevant to our current understanding of potyvirus transport is available. In this review, we highlight the complexity of potyvirus movement pathways and present three potential nonexclusive mechanisms based on (1) the use of the host endomembrane system to produce membranous replication vesicles that are targeted to PDs and move from cell to cell, (2) the movement of extracellular viral vesicles in the apoplasm, and (3) the transport of virion particles or ribonucleoprotein complexes through PDs. We also present and discuss experimental data supporting these different models as well as the aspects that still remain mostly speculative.
Asunto(s)
Potyvirus , Potyvirus/genética , Proteínas Virales/metabolismo , Enfermedades de las Plantas , NicotianaRESUMEN
Resistance to viruses is an important aspect of plant breeding. One way to achieve it is to select genetic resistances based on the susceptibility factors hijacked by the virus to infect the plants. Here, we recount work done on genes encoding translation initiation factors eIF4E, some of the most successful targets for obtaining resistance to potyviruses, starting from their characterization 20 years ago. With examples from different plant species, pepper, tomato, tobacco and arabidopsis, we present the basis of this type of resistances and their characteristics, highlighting the role of gene redundancy among 4E factors, their specificity for the virus and the need for the plant of a trade-off between resistance and development. Finally, we show how the new genome editing techniques could be used in plant breeding to develop eIF4E-based resistances in crops, mimicking the functional alleles that have been selected during evolution in many crops.
Asunto(s)
Resistencia a la Enfermedad , Factor 4E Eucariótico de Iniciación , Enfermedades de las Plantas , Plantas , Potyvirus , Alelos , Factor 4E Eucariótico de Iniciación/genética , Fitomejoramiento , Potyvirus/genética , Plantas/virología , Enfermedades de las Plantas/virologíaRESUMEN
REMORIN proteins belong to a plant-specific multigene family that localise in plasma membrane nanodomains and in plasmodesmata. We previously showed that in Nicotiana benthamiana, group 1 StREM1.3 limits the cell-to-cell spread of a potexvirus without affecting viral replication. This prompted us to check whether an effect on viral propagation could apply to potyvirus species Turnip mosaic virus (TuMV) and Potato virus A (PVA). Our results show that StREM1.3 transient or stable overexpression in transgenic lines increases potyvirus propagation, while it is slowed down in transgenic lines underexpressing endogenous NbREMs, without affecting viral replication. TuMV and PVA infection do not alter the membranous localisation of StREM1.3. Furthermore, StREM1.3-membrane anchoring is necessary for its agonist effect on potyvirus propagation. StREM1.3 phosphocode seems to lead to distinct plant responses against potexvirus and potyvirus. We also showed that StREM1.3 interacts in yeast and in planta with the key potyviral movement protein CI (cylindrical inclusion) at the level of the plasma membrane but only partially at plasmodesmata pit fields. TuMV infection also counteracts StREM1.3-induced plasmodesmata callose accumulation at plasmodesmata. Altogether, these results showed that StREM1.3 plays an agonistic role in potyvirus cell-to-cell movement in N. benthamiana.
Asunto(s)
Potexvirus , Potyvirus , Movimiento Celular , Enfermedades de las Plantas , Proteínas de Plantas , Potexvirus/genética , Potyvirus/fisiología , Nicotiana , Proteínas Virales/metabolismoRESUMEN
REMORINs (REMs) are a plant-specific protein family, proposed regulators of membrane-associated molecular assemblies and well-established markers of plasma membrane nanodomains. REMs play a diverse set of functions in plant interactions with pathogens and symbionts, responses to abiotic stresses, hormone signaling and cell-to-cell communication. In this review, we highlight the established and more putative roles of REMs throughout the literature. We discuss the physiological functions of REMs, the mechanisms underlying their nanodomain-organization and their putative role as regulators of nanodomain-associated molecular assemblies. Furthermore, we discuss how REM phosphorylation may regulate their functional versatility. Overall, through data-mining and comparative analysis of the literature, we suggest how to further study the molecular mechanisms underpinning the functions of REMs.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Many potyvirus species are among the most economically-significant plant viruses as they cause substantial yield losses to crop plants globally [...].
Asunto(s)
Enfermedades de las Plantas/virología , Potyvirus/genética , Potyvirus/metabolismo , Nicotiana/virología , Proteínas Virales/genéticaRESUMEN
Many recessive resistances against potyviruses are mediated by eukaryotic translation initiation factor 4E (eIF4E). In tobacco, the va resistance gene commonly used to control Potato virus Y (PVY) corresponds to a large deletion affecting the eIF4E-1 gene on chromosome 21. Here, we compared the resistance durability conferred by various types of mutations affecting eIF4E-1 (deletions of various sizes, frameshift or nonsense mutations). The 'large deletion' genotypes displayed the broadest and most durable resistance, whereas frameshift and nonsense mutants displayed a less durable resistance, with rapid and frequent apparition of resistance-breaking variants. In addition, genetic and transcriptomic analyses revealed that resistance durability is strongly impacted by a complex genetic locus on chromosome 14, which contains three other eIF4E genes. One of these, eIF4E-3, is rearranged as a hybrid gene between eIF4E-2 and eIF4E-3 (eIF4E-2-3 ) in the genotypes showing the most durable resistance, while eIF4E-2 is differentially expressed between the tested varieties. RNA-seq and quantitative reverse transcriptase-polymerase chain reaction experiments demonstrated that eIF4E-2 expression level is positively correlated with resistance durability. These results suggest that besides the nature of the mutation affecting eIF4E-1, three factors linked with a complex locus may potentially impact va durability: loss of an integral eIF4E-3, presence of eIF4E-2-3 and overexpression of eIF4E-2. This latter gene might act as a decoy in a non-productive virus-plant interaction, limiting the ability of PVY to evolve towards resistance breaking. Taken together, these results show that va resistance durability can in large part be explained by complex redundancy effects in the eIF4E gene family.
Asunto(s)
Resistencia a la Enfermedad , Factor 4E Eucariótico de Iniciación/genética , Genes de Plantas , Sitios Genéticos , Nicotiana/inmunología , Nicotiana/virología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Potyvirus/fisiología , Sustitución de Aminoácidos/genética , Cromosomas de las Plantas/genética , Ecotipo , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas , Genotipo , Modelos Biológicos , Mutación/genética , Fenotipo , Filogenia , Enfermedades de las Plantas/genética , Eliminación de Secuencia , Nicotiana/genéticaRESUMEN
Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3's phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/inmunología , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Potexvirus/patogenicidad , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de la Cápside/fisiología , Membrana Celular/metabolismo , Movimiento Celular , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Plantas Modificadas Genéticamente/virología , Plasmodesmos/metabolismo , Proteínas Quinasas/metabolismoRESUMEN
In view of major economic problems caused by viruses, the development of genetically resistant crops is critical for breeders but remains limited by the evolution of resistance-breaking virus mutants. During the plant breeding process, the introgression of traits from Crop Wild Relatives results in a dramatic change of the genetic background that can alter the resistance efficiency or durability. Here, we conducted a meta-analysis on 19 Quantitative Trait Locus (QTL) studies of resistance to viruses in plants. Frequent epistatic effects between resistance genes indicate that a large part of the resistance phenotype, conferred by a given QTL, depends on the genetic background. We next reviewed the different resistance mechanisms in plants to survey at which stage the genetic background could impact resistance or durability. We propose that the genetic background may impair effector-triggered dominant resistances at several stages by tinkering the NB-LRR (Nucleotide Binding-Leucine-Rich Repeats) response pathway. In contrast, effects on recessive resistances by loss-of-susceptibility-such as eIF4E-based resistances-are more likely to rely on gene redundancy among the multigene family of host susceptibility factors. Finally, we show how the genetic background is likely to shape the evolution of resistance-breaking isolates and propose how to take this into account in order to breed plants with increased resistance durability to viruses.
Asunto(s)
Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/genética , Virus de Plantas , Plantas/genética , Epistasis Genética , Plantas/inmunología , Plantas/virología , Plantas Modificadas Genéticamente , Sitios de Carácter CuantitativoRESUMEN
Potato virus Y (PVY) is one of the most damaging viruses of tobacco. In particular, aggressive necrotic strains (PVYN ) lead to considerable losses in yield. The main source of resistance against PVY is linked to the va locus. However, va-overcoming PVY isolates inducing necrotic symptoms were observed in several countries. In this context, it is important to find va-independent protection strategies. In a previous study, the phenotyping of 162 tobacco varieties revealed 10 accessions that do not carry the va allele and do not exhibit typical PVYN -induced veinal necrosis. Despite the absence of necrotic symptoms, normal viral accumulation in these plants suggests a va-independent mechanism of tolerance to PVYN -induced systemic veinal necrosis. Fine mapping of the genetic determinant(s) was performed in a segregating F2 population. The tolerance trait is inherited as a single recessive gene, and allelism tests demonstrated that eight of the 10 tolerant varieties carry the same determinant. Anchoring the linkage map to the tobacco genome physical map allowed the identification of a RPP8-like R gene, called NtTPN1 (for Nicotiana tabacum Tolerance to PVY-induced Necrosis1), with the same single-nucleotide polymorphism in the eight tolerant accessions. Functional assays using homozygous NtTPN1 EMS mutants confirmed the role of NtTPN1 in the tolerance phenotype. PVYN -induced systemic veinal necrosis in tobacco likely represents an inefficient defense response with hypersensitive response-like characteristics. The identification of NtTPN1 opens breeding options to minimize the impact of emerging and so far uncontrolled va-breaking necrotic PVY isolates.
RESUMEN
To infect plants, viruses rely heavily on their host's machinery. Plant genetic resistances based on host factor modifications can be found among existing natural variability and are widely used for some but not all crops. While biotechnology can supply for the lack of natural resistance alleles, new strategies need to be developed to increase resistance spectra and durability without impairing plant development. Here, we assess how the targeted allele modification of the Arabidopsis thaliana translation initiation factor eIF4E1 can lead to broad and efficient resistance to the major group of potyviruses. A synthetic Arabidopsis thaliana eIF4E1 allele was designed by introducing multiple amino acid changes associated with resistance to potyvirus in naturally occurring Pisum sativum alleles. This new allele encodes a functional protein while maintaining plant resistance to a potyvirus isolate that usually hijacks eIF4E1. Due to its biological functionality, this synthetic allele allows, at no developmental cost, the pyramiding of resistances to potyviruses that selectively use the two major translation initiation factors, eIF4E1 or its isoform eIFiso4E. Moreover, this combination extends the resistance spectrum to potyvirus isolates for which no efficient resistance has so far been found, including resistance-breaking isolates and an unrelated virus belonging to the Luteoviridae family. This study is a proof-of-concept for the efficiency of gene engineering combined with knowledge of natural variation to generate trans-species virus resistance at no developmental cost to the plant. This has implications for breeding of crops with broad-spectrum and high durability resistance using recent genome editing techniques.
RESUMEN
Allele mining on susceptibility factors offers opportunities to find new sources of resistance among crop wild relatives for breeding purposes. As a proof of concept, we used available RNAseq data to investigate polymorphisms among the four tomato genes encoding translation initiation factors [eIF4E1 and eIF4E2, eIFiso4E and the related gene new cap-binding protein(nCBP)] to look for new potential resistance alleles to potyviruses. By analysing polymorphism among RNAseq data obtained for 20 tomato accessions, 10 belonging to the cultivated type Solanum lycopersicum and 10 belonging to the closest related wild species Solanum pimpinellifolium, we isolated one new eIF4E1 allele, in the S. pimpinellifolium LA0411 accession, which encodes a potential new resistance allele, mainly due to a polymorphism associated with an amino acid change within eIF4E1 region II. We confirmed that this new allele, pot12, is indeed associated with resistance to potato virus Y, although with a restricted resistance spectrum and a very low durability potential. This suggests that mutations occurring in eIF4E region II only may not be sufficient to provide efficient and durable resistance in plants. However, our study emphasizes the opportunity brought by RNAseq data to mine for new resistance alleles. Moreover, this approach could be extended to seek for putative new resistance alleles by screening for variant forms of susceptibility genes encoding plant host proteins known to interact with viral proteins.
Asunto(s)
Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/inmunología , Enfermedades de las Plantas/virología , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Potyvirus/fisiología , Solanum lycopersicum/genética , Alelos , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/clasificación , Solanum lycopersicum/inmunología , Solanum lycopersicum/virología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Potyvirus/inmunologíaRESUMEN
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Asunto(s)
Clonación Molecular/métodos , ADN Complementario , Expresión Génica , Genoma Viral , Potyvirus/genética , Proteínas Recombinantes de Fusión , Orden Génico , Vectores Genéticos/genética , Lactuca , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The Triple Gene Block 1 (TGBp1) protein encoded by the Potato virus X is a multifunctional protein that acts as a suppressor of RNA silencing or facilitates the passage of virus from cell to cell by promoting the plasmodesmata opening. We previously showed that the membrane raft protein StRemorin1.3 is able to impair PVX infection. Here, we show that overexpressed StRemorin1.3 does not impair the silencing suppressor activity of TGBp1, but affects its ability to increase plasmodesmata permeability. A similar effect on plasmodesmata permeability was observed with other movement proteins, suggesting that REM is a general regulator of plasmodesmal size exclusion limit. These results add to our knowledge of the mechanisms underlying the StREM1.3 role in virus infection.
Asunto(s)
Proteínas Portadoras/fisiología , Fosfoproteínas/fisiología , Proteínas de Plantas/fisiología , Plasmodesmos/metabolismo , Potexvirus/fisiología , Solanum tuberosum/virología , Proteínas Virales/fisiología , Agrobacterium/genética , Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno , Microscopía Fluorescente , Permeabilidad , Plasmodesmos/virología , Isoformas de Proteínas/fisiología , Transporte de Proteínas , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Solanum tuberosum/metabolismo , Nicotiana/metabolismoRESUMEN
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 also contribute to the ability to infect C. roseus. Inoculation experiments involving a recombinant LMV expressing a permissive lettuce eukaryotic translation initiation factor 4E (eIF4E) suggest that eIF4E does not contribute to the interaction of most LMV isolates with C. roseus.
Asunto(s)
Catharanthus/virología , Genoma Viral/genética , Lactuca/virología , Enfermedades de las Plantas/virología , Potyvirus/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , ADN Helicasas/genética , ADN Helicasas/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Especificidad del Huésped , Datos de Secuencia Molecular , Mutación , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Potyvirus/fisiología , Análisis de Secuencia de ADN , Proteínas Virales/metabolismoRESUMEN
The formation of plasma membrane (PM) microdomains plays a crucial role in the regulation of membrane signaling and trafficking. Remorins are a plant-specific family of proteins organized in six phylogenetic groups, and Remorins of group 1 are among the few plant proteins known to specifically associate with membrane rafts. As such, they are valuable to understand the molecular bases for PM lateral organization in plants. However, little is known about the structural determinants underlying the specific association of group 1 Remorins with membrane rafts. We used a structure-function approach to identify a short C-terminal anchor (RemCA) indispensable and sufficient for tight direct binding of potato (Solanum tuberosum) REMORIN 1.3 (StREM1.3) to the PM. RemCA switches from unordered to α-helical structure in a nonpolar environment. Protein structure modeling indicates that RemCA folds into a tight hairpin of amphipathic helices. Consistently, mutations reducing RemCA amphipathy abolished StREM1.3 PM localization. Furthermore, RemCA directly binds to biological membranes in vitro, shows higher affinity for Detergent-Insoluble Membranes lipids, and targets yellow fluorescent protein to Detergent-Insoluble Membranes in vivo. Mutations in RemCA resulting in cytoplasmic StREM1.3 localization abolish StREM1.3 function in restricting potato virus X movement. The mechanisms described here provide new insights on the control and function of lateral segregation of plant PM.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Potexvirus/metabolismo , Solanum tuberosum/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas Portadoras/genética , Membrana Celular/genética , Membrana Celular/virología , Dicroismo Circular , Clonación Molecular , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/metabolismo , Microdominios de Membrana/genética , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Mutación , Fosfoproteínas/genética , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Proteínas de Plantas/genética , Potexvirus/patogenicidad , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas , Solanum tuberosum/genética , Solanum tuberosum/virología , Relación Estructura-ActividadRESUMEN
In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro. The analysis of mutated forms of the α(5) subunit demonstrated that mutation of a glutamic acid at position 110 affects RNase activity. Furthermore, it was demonstrated, using a bimolecular fluorescence complement assay, that the multifunctional helper component proteinase (HcPro) of LMV, already known to interfere with the 20S proteasome RNase activity in vitro, can interact in vivo with the recombinant α(5) subunit. Further experiments demonstrated that, in LMV-infected lettuce cells, α(5) is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α(5) , showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thaliana α(5) the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cisteína Endopeptidasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/metabolismo , Ribonucleasas/metabolismo , Proteínas Virales/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Escherichia coli , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Ácido Glutámico/genética , Proteínas Fluorescentes Verdes/metabolismo , Lactuca , Mutación/genética , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Subunidades de Proteína/genética , ARN Viral/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleasas/genética , Fracciones Subcelulares/metabolismoRESUMEN
The ubiquitin/26S proteasome system (UPS) plays a central role in plant protein degradation. Over the past few years, the importance of this pathway in plant-pathogen interactions has been increasingly highlighted. UPS is involved in almost every step of the defence mechanisms in plants, regardless of the type of pathogen. In addition to its proteolytic activities, UPS, through its 20S RNase activity, may be part of a still unknown antiviral defence pathway. Strikingly, UPS is not only a weapon used by plants to defend themselves, but also a target for some pathogens that have evolved mechanisms to inhibit and/or use this system for their own purposes. This article attempts to summarize the current knowledge on UPS involvement in plant-microbe interactions, a complex scheme that illustrates the never-ending arms race between hosts and microbes.
Asunto(s)
Interacciones Huésped-Patógeno , Enfermedades de las Plantas/microbiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Biocatálisis , Enfermedades de las Plantas/virología , Estabilidad ProteicaRESUMEN
Previous resistance analyses of Arabidopsis thaliana mutants knocked out for eukaryotic translation initiation factors showed that disruption of the At-eIF(iso)4E or both the At-eIF(iso)4G1 and At-eIF(iso)4G2 genes resulted in resistance against turnip mosaic virus (TuMV). This study selected TuMV virulent variants that overcame this resistance and showed that two independent mutations in the region coding for the viral genome-linked protein (VPg) were sufficient to restore TuMV virulence in At-eIF(iso)4E and At-eIF(iso)4G1xAt-eIF(iso)4G2 knockout plants. As a VPg-eIF(iso)4E interaction has been shown previously to be critical for TuMV infection, a systematic analysis of the interactions between A. thaliana eIF4Es and VPgs of virulent and avirulent TuMVs was performed. The results suggest that virulent TuMV variants may use an eIF4F-independent pathway.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/virología , Factor 4G Eucariótico de Iniciación/genética , Factores Eucarióticos de Iniciación/genética , Mutación Missense , Enfermedades de las Plantas/virología , Potyvirus/patogenicidad , Proteínas no Estructurales Virales/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Arabidopsis/genética , Técnicas de Inactivación de Genes , Interacciones Huésped-Patógeno , Datos de Secuencia Molecular , Potyvirus/genética , Mapeo de Interacción de Proteínas , Supresión Genética , Proteínas no Estructurales Virales/genética , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/fisiologíaRESUMEN
Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.