RESUMO
Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.
Assuntos
Potexvirus/genética , Potexvirus/metabolismo , Proteínas do Complexo da Replicase Viral/genética , Sequência de Aminoácidos/genética , Proteínas de Membrana/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Doenças das Plantas/virologia , Prolina/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Replicon/genética , Nicotiana/virologia , Proteínas Virais/metabolismo , Proteínas do Complexo da Replicase Viral/metabolismo , Replicação Viral/genéticaRESUMO
Pleioblastus mosaic virus (PleMV) is a tentative member of the genus Potyvirus in the family Potyviridae and was discovered in bamboo with mosaic symptoms in Tokyo, Japan. Since no information on the genome sequence of PleMV has been reported, its taxonomic position has long been uncertain. Here, we report the first complete genome sequences of two distinct PleMV isolates. Excluding the 3'-terminal poly(A) tail, their genomic RNA sequences consist of 9,634 and 9,643 nucleotides (nt); both contain a large open reading frame (ORF) encoding a polyprotein and a small ORF termed PIPO. The large ORFs of the two isolates share 79.2% and 87.6% sequence identity at the nucleotide (nt) and amino acid (aa) level, respectively, and were found to have the highest nt and aa sequence identity (69.0% and 69.9%) to the potyvirus johnsongrass mosaic virus (JGMV). Phylogenetic analysis showed that PleMV is most closely related to JGMV but forms its own clade. These results suggest that PleMV is a distinct member of the genus Potyvirus.
Assuntos
Genoma Viral/genética , Potyvirus/genética , Sequência de Aminoácidos , Sequência de Bases , Genômica/métodos , Japão , Fases de Leitura Aberta/genética , Filogenia , Poliproteínas/genética , RNA Viral/genética , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma/métodosRESUMO
Since the propagation of plant viruses depends on various host susceptibility factors, deficiency in them can prevent viral infection in cultivated and model plants. Recently, we identified the susceptibility factor Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana, and revealed that EXA1-mediated resistance was effective against three potexviruses. Although EXA1 homolog genes are found in tomato and rice, little is known about which viruses depend on EXA1 for their infection capability and whether the function of EXA1 homologs in viral infection is conserved across multiple plant species, including crops. To address these questions, we generated knockdown mutants using virus-induced gene silencing in two Solanaceae species, Nicotiana benthamiana and tomato. In N. benthamiana, silencing of an EXA1 homolog significantly compromised the accumulation of potexviruses and a lolavirus, a close relative of potexviruses, whereas transient expression of EXA1 homologs from tomato and rice complemented viral infection. EXA1 dependency for potexviral infection was also conserved in tomato. These results indicate that EXA1 is necessary for effective accumulation of potexviruses and a lolavirus, and that the function of EXA1 in viral infection is conserved among diverse plant species.
Assuntos
Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Nicotiana/virologia , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Potexvirus/fisiologia , Solanum lycopersicum/virologia , Doenças das Plantas/virologia , Proteínas de Plantas/genéticaRESUMO
Understanding the innate immune mechanisms of plants is necessary for the breeding of disease-resistant lines. Previously, we identified the antiviral resistance gene JAX1 from Arabidopsis thaliana, which inhibits infection by potexviruses. JAX1 encodes a unique jacalin-type lectin protein. In this study, we analyzed the molecular mechanisms of JAX1-mediated resistance. JAX1 restricted the multiplication of a potexviral replicon lacking movement-associated proteins, suggesting inhibition of viral replication. Therefore, we developed an in vitro potato virus X (PVX) translation/replication system using vacuole- and nucleus-free lysates from tobacco protoplasts, and we revealed that JAX1 inhibits viral RNA synthesis but not the translation of the viral RNA-dependent RNA polymerase (RdRp). JAX1 did not affect the replication of a resistance-breaking mutant of PVX. Blue native polyacrylamide gel electrophoresis of fractions separated by sucrose gradient sedimentation showed that PVX RdRp constituted the high-molecular-weight complex that seems to be crucial for viral replication. JAX1 was detected in this complex of the wild-type PVX replicon but not in that of the resistance-breaking mutant. In addition, JAX1 interacted with the RdRp of the wild-type virus but not with that of a virus with a point mutation at the resistance-breaking residue. These results suggest that JAX1 targets RdRp to inhibit potexviral replication.IMPORTANCE Resistance genes play a crucial role in plant antiviral innate immunity. The roles of conventional nucleotide-binding leucine-rich repeat (NLR) proteins and the associated defense pathways have long been studied. In contrast, recently discovered resistance genes that do not encode NLR proteins (non-NLR resistance genes) have not been investigated extensively. Here we report that the non-NLR resistance factor JAX1, a unique jacalin-type lectin protein, inhibits de novo potexviral RNA synthesis by targeting the huge complex of viral replicase. This is unlike other known antiviral resistance mechanisms. Molecular elucidation of the target in lectin-type protein-mediated antiviral immunity will enhance our understanding of the non-NLR-mediated plant resistance system.
Assuntos
Farmacorresistência Viral , Nicotiana/enzimologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Replicação Viral , Antivirais/metabolismo , Regulação Enzimológica da Expressão Gênica , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/virologia , Potexvirus/fisiologia , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Nicotiana/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Plant viruses in the genus Carlavirus include more than 65 members. Plants infected with carlaviruses exhibit various symptoms, including leaf malformation and plant stunting. Cysteine-rich protein (CRP) encoded by carlaviruses has been reported to be a pathogenicity determinant. Carlavirus CRPs contain two motifs in their central part: a nuclear localization signal (NLS) and a zinc finger motif (ZF). In addition to these two conserved motifs, carlavirus CRPs possess highly divergent, N-terminal, 34 amino acid residues with unknown function. In this study, to analyse the role of these distinct domains, we tested six carlavirus CRPs for their RNA silencing suppressor activity, ability to enhance the pathogenicity of a heterologous virus and effects on virus accumulation levels. Although all six tested carlavirus CRPs showed RNA silencing suppressor activity at similar levels, symptoms induced by the Potato virus X (PVX) heterogeneous system exhibited two different patterns: leaf malformation and whole-plant stunting. The expression of each carlavirus CRP enhanced PVX accumulation levels, which were not correlated with symptom patterns. PVX-expressing CRP with mutations in either NLS or ZF did not induce symptoms, suggesting that both motifs play critical roles in symptom expression. Further analysis using chimeric CRPs, in which the N-terminal region was replaced with the corresponding region of another CRP, suggested that the N-terminal region of carlavirus CRPs determined the exhibited symptom types. The up-regulation of a plant gene upp-L, which has been reported in a previous study, was also observed in this study; however, the expression level was not responsible for symptom types.
Assuntos
Carlavirus/metabolismo , Doenças das Plantas/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Carlavirus/patogenicidade , Sinais de Localização Nuclear/metabolismo , Folhas de Planta/virologia , Potexvirus/metabolismo , Interferência de RNA , RNA Viral/metabolismo , Especificidade da Espécie , Nicotiana/virologia , Proteínas Virais/químicaRESUMO
Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MPFMV) using live cell imaging, focusing on its ER-directing signal peptide (SPFMV). Transiently expressed MPFMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SPFMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MPFMV mutant lacking SPFMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MPFMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with Arabidopsis synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SPFMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MPFMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MPFMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MPFMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MPFMV into PD.
Assuntos
Arabidopsis/virologia , Membrana Celular/virologia , Retículo Endoplasmático/metabolismo , Proteínas do Movimento Viral em Plantas/metabolismo , Plasmodesmos/virologia , Vírus do Mosaico do Tabaco/isolamento & purificação , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/virologia , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/virologia , Microtúbulos/metabolismo , Microtúbulos/virologia , Plasmodesmos/metabolismo , Transporte Proteico/fisiologia , Nicotiana/virologia , Vírus do Mosaico do Tabaco/metabolismoRESUMO
Members of the SEPALLATA (SEP) gene sub-family encode class E floral homeotic MADS-domain transcription factors (MADS TFs) that specify the identity of floral organs. The Arabidopsis thaliana genome contains 4 ancestrally duplicated and functionally redundant SEP genes, SEP1-4. Recently, a gene family of unique effectors, phyllogens, was identified as an inducer of leaf-like floral organs in phytoplasmas (plant pathogenic bacteria). While it was shown that phyllogens target some MADS TFs, including SEP3 for degradation, it is unknown whether the other SEPs (SEP1, SEP2, and SEP4) of Arabidopsis are also degraded by them. In this study, we found that all 4 SEP proteins of Arabidopsis are degraded by a phyllogen using a transient co-expression assay in Nicotiana benthamiana. This finding indicates that phyllogens may broadly target class E MADS TFs of plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas de Bactérias , Flores/crescimento & desenvolvimento , Proteínas de Domínio MADS/metabolismo , Phytoplasma/metabolismo , Doenças das Plantas/microbiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/farmacologia , Regulação da Expressão Gênica de Plantas , Família Multigênica , Folhas de Planta/crescimento & desenvolvimento , Proteólise/efeitos dos fármacos , Nicotiana/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Systemic necrosis is one of the most severe symptoms caused by plant RNA viruses. Recently, systemic necrosis has been suggested to have similar features to a defense response referred to as the hypersensitive response (HR), a form of programmed cell death. In virus-infected plant cells, host intracellular membrane structures are changed dramatically for more efficient viral replication. However, little is known about whether this replication-associated membrane modification is the cause of the symptoms. In this study, we identified an amino-terminal amphipathic helix of the helicase encoded by Radish mosaic virus (RaMV) (genus Comovirus) as an elicitor of cell death in RaMV-infected plants. Cell death caused by the amphipathic helix had features similar to HR, such as SGT1-dependence. Mutational analyses and inhibitor assays using cerulenin demonstrated that the amphipathic helix-induced cell death was tightly correlated with dramatic alterations in endoplasmic reticulum (ER) membrane structures. Furthermore, the cell death-inducing activity of the amphipathic helix was conserved in Cowpea mosaic virus (genus Comovirus) and Tobacco ringspot virus (genus Nepovirus), both of which are classified in the family Secoviridae. Together, these results indicate that ER membrane modification associated with viral intracellular replication may be recognized to prime defense responses against plant viruses.
Assuntos
Comovirus/enzimologia , Nicotiana/virologia , Doenças das Plantas/virologia , Raphanus/virologia , Sequência de Aminoácidos , Morte Celular , Cerulenina/farmacologia , Comovirus/genética , Comovirus/fisiologia , DNA Helicases/genética , DNA Helicases/metabolismo , Retículo Endoplasmático/metabolismo , Genes Reporter , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Necrose , Folhas de Planta/citologia , Folhas de Planta/fisiologia , Folhas de Planta/virologia , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Nicotiana/citologia , Nicotiana/efeitos dos fármacos , Nicotiana/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
UNLABELLED: Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE: Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.
Assuntos
Citoplasma/virologia , Retículo Endoplasmático/virologia , Proteínas do Nucleocapsídeo/metabolismo , Vírus de Plantas/fisiologia , Multimerização Proteica , Vírus de RNA/fisiologia , Ficus , Microscopia Imunoeletrônica , Transporte Proteico , NicotianaRESUMO
Despite plants infected by pathogens are often unable to produce offspring, it remains unclear how sterility is induced in host plants. In this study, we demonstrate that TENGU, a phytoplasmal virulence peptide known as a dwarfism inducer, acts as an inducer of sterility. Transgenic expression of TENGU induced both male and female sterility in Arabidopsis thaliana flowers similar to those observed in double knockout mutants of auxin response factor 6 (ARF6) and ARF8, which are known to regulate floral development in a jasmonic acid (JA)-dependent manner. Transcripts of ARF6 and ARF8 were significantly decreased in both tengu-transgenic and phytoplasma-infected plants. Furthermore, JA and auxin levels were actually decreased in tengu-transgenic buds, suggesting that TENGU reduces the endogenous levels of phytohormones by repressing ARF6 and ARF8, resulting in impaired flower maturation. TENGU is the first virulence factor with the effects on plant reproduction by perturbation of phytohormone signaling.
Assuntos
Arabidopsis , Proteínas de Bactérias , Ciclopentanos/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Ácidos Indolacéticos/metabolismo , Oxilipinas/metabolismo , Peptídeos , Phytoplasma , Infertilidade das Plantas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Flores/genética , Flores/metabolismo , Flores/microbiologia , Peptídeos/genética , Peptídeos/metabolismo , Phytoplasma/genética , Phytoplasma/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Plant virus expression vectors provide a powerful tool for basic research as well as for practical applications. Here, we report the construction of an expression vector based on plantago asiatica mosaic virus (PlAMV), a member of the genus Potexvirus. Modification of a vector to enhance the expression of a foreign gene, combined with the use of the foot-and-mouth disease virus 2A peptide, allowed efficient expression of the foreign gene in two model plant species, Arabidopsis thaliana and Nicotiana benthamiana. Comparison with the widely used potato virus X (PVX) vector demonstrated that the PlAMV vector retains an inserted foreign gene for a longer period than PVX. Moreover, our results showed that the GFP expression construct PlAMV-GFP exhibits stronger RNA silencing suppression activity than PVX-GFP, which is likely to contribute to the stability of the PlAMV vector.
Assuntos
Arabidopsis/virologia , Regulação Viral da Expressão Gênica/fisiologia , Nicotiana/virologia , Potexvirus/metabolismo , Proteínas Virais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Doenças das Plantas/virologia , Potexvirus/genética , Interferência de RNARESUMO
Lectin-mediated resistance (LMR) has been suggested to comprise an uncharacterized branch of antiviral plant innate immunity. To unveil the feature of resistance conferred by jacalin-type lectin required for potexvirus resistance 1 (JAX1), a recently isolated LMR gene against potexviruses, we analyzed the resistance-breaking variants to find the viral component involved in resistance. We employed grafting-mediated inoculation, a high-pressure virus inoculation method, to obtain Potato virus X (PVX) variants that can overcome JAX1-mediated resistance. Whole-genome sequencing of the variants suggested that a single amino acid in the methyl transferase domain of the replicase encoded by PVX is responsible for this resistance-breaking property. Reintroduction of the amino-acid substitution to avirulent wild-type PVX was sufficient to overcome the JAX1-mediated resistance. These results suggest that viral replicase is involved in JAX1-mediated resistance. The residue that determines the resistance-breaking properties was highly conserved among potexviruses, suggesting a general role of the residue in potexvirus-JAX1 interactions.
Assuntos
Nicotiana/virologia , Doenças das Plantas/virologia , Imunidade Vegetal , Potexvirus/enzimologia , Potexvirus/patogenicidade , RNA Polimerase Dependente de RNA/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Dados de Sequência Molecular , Doenças das Plantas/imunologia , Lectinas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Potexvirus/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Nicotiana/imunologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Phytoplasmas are insect-borne plant pathogenic bacteria that alter host morphology. TENGU, a small peptide of 38 residues, is a virulence factor secreted by phytoplasmas that induces dwarfism and witches' broom in the host plant. In this study, we demonstrate that plants process TENGU in order to generate small functional peptides. First, virus vector-mediated transient expression demonstrated that the amino-terminal 11 amino acids of TENGU are capable of causing symptom development in Nicotiana benthamiana plants. The deletion of the 11th residue significantly diminished the symptom-inducing activity of TENGU, suggesting that these 11 amino acids constitute a functional domain. Second, we found that TENGU undergoes proteolytic processing in vitro, generating peptides of 19 and 21 residues including the functional domain. Third, we observed similar processing of TENGU in planta, and an alanine substitution mutant of TENGU, for which processing was compromised, showed reduced symptom induction activity. All TENGU homologs from several phytoplasma strains possessed similar symptom induction activity and went through processing, which suggests that the processing of TENGU might be related to its function.
Assuntos
Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Nicotiana/microbiologia , Phytoplasma/patogenicidade , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/metabolismo , Filogenia , Phytoplasma/metabolismo , Doenças das Plantas/microbiologia , Extratos Vegetais/metabolismo , Estrutura Terciária de Proteína , RNA Ribossômico 16S , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Nicotiana/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Radish mosaic virus (RaMV) is a crucifer-infecting comovirus that has been detected worldwide. Here, we report the successful construction of a full-length infectious cDNA clone of RaMV. The full-length cDNA clones corresponding to RNA1 and RNA2 of a Japanese isolate of RaMV were cloned into the pBlueScript plasmid or the binary vector pCAMBIA1301 downstream of the cauliflower mosaic virus 35S promoter. Mechanical inoculation or agroinoculation of Nicotiana benthamiana with these vectors resulted in systemic RaMV infections causing symptoms similar to those caused by the wild-type parental virus. The presence of progeny virus was verified by western blot analysis and electron microscopy.
Assuntos
Comovirus/genética , Comovirus/patogenicidade , DNA Complementar/genética , RNA Viral/genética , Raphanus/virologia , Western Blotting , Comovirus/isolamento & purificação , Microscopia Eletrônica , Doenças das Plantas/virologia , Nicotiana/virologia , Replicação ViralRESUMO
Fig mosaic virus (FMV), a member of the newly formed genus Emaravirus, is a segmented negative-strand RNA virus. Each of the six genomic FMV segments contains a single ORF: that of RNA4 encodes the protein p4. FMV-p4 is presumed to be the movement protein (MP) of the virus; however, direct experimental evidence for this is lacking. We assessed the intercellular distribution of FMV-p4 in plant cells by confocal laser scanning microscopy and we found that FMV-p4 was localized to plasmodesmata and to the plasma membrane accompanied by tubule-like structures. A series of experiments designed to examine the movement functions revealed that FMV-p4 has the capacity to complement viral cell-to-cell movement, prompt GFP diffusion between cells, and spread by itself to neighbouring cells. Altogether, our findings demonstrated that FMV-p4 shares several properties with other viral MPs and plays an important role in cell-to-cell movement.
Assuntos
Proteínas do Movimento Viral em Plantas/fisiologia , Vírus de Plantas/fisiologia , Agrobacterium tumefaciens , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica de Plantas , Regulação Viral da Expressão Gênica , Genes Virais , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Proteínas do Movimento Viral em Plantas/genética , Proteínas do Movimento Viral em Plantas/metabolismo , Plantas Geneticamente Modificadas , Plasmodesmos/virologia , RNA Viral/genética , Nicotiana/virologiaRESUMO
BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily ancient mechanism of signal transduction found in eukaryotic cells. In plants, MAPK cascades are associated with responses to various abiotic and biotic stresses such as plant pathogens. MAPK cascades function through sequential phosphorylation: MAPK kinase kinases (MAPKKKs) phosphorylate MAPK kinases (MAPKKs), and phosphorylated MAPKKs phosphorylate MAPKs. Of these three types of kinase, the MAPKKKs exhibit the most divergence in the plant genome. Their great diversity is assumed to allow MAPKKKs to regulate many specific signaling pathways in plants despite the relatively limited number of MAPKKs and MAPKs. Although some plant MAPKKKs, including the MAPKKKα of Nicotiana benthamiana (NbMAPKKKα), are known to play crucial roles in plant defense responses, the functional relationship among MAPKKK genes is poorly understood. Here, we performed a comparative functional analysis of MAPKKKs to investigate the signaling pathway leading to the defense response. RESULTS: We cloned three novel MAPKKK genes from N. benthamiana: NbMAPKKKß, NbMAPKKKγ, and NbMAPKKKε2. Transient overexpression of full-length NbMAPKKKß or NbMAPKKKγ or their kinase domains in N. benthamiana leaves induced hypersensitive response (HR)-like cell death associated with hydrogen peroxide production. This activity was dependent on the kinase activity of the overexpressed MAPKKK. In addition, virus-induced silencing of NbMAPKKKß or NbMAPKKKγ expression significantly suppressed the induction of programmed cell death (PCD) by viral infection. Furthermore, in epistasis analysis of the functional relationships among NbMAPKKKß, NbMAPKKKγ, and NbMAPKKKα (previously shown to be involved in plant defense responses) conducted by combining transient overexpression analysis and virus-induced gene silencing, silencing of NbMAPKKKα suppressed cell death induced by the overexpression of the NbMAPKKKß kinase domain or of NbMAPKKKγ, but silencing of NbMAPKKKß failed to suppress cell death induced by the overexpression of NbMAPKKKα or NbMAPKKKγ. Silencing of NbMAPKKKγ suppressed cell death induced by the NbMAPKKKß kinase domain but not that induced by NbMAPKKKα. CONCLUSIONS: These results demonstrate that in addition to NbMAPKKKα, NbMAPKKKß and NbMAPKKKγ also function as positive regulators of PCD. Furthermore, these three MAPKKKs form a linear signaling pathway leading to PCD; this pathway proceeds from NbMAPKKKß to NbMAPKKKγ to NbMAPKKKα.
Assuntos
Apoptose , MAP Quinase Quinase Quinases/metabolismo , Nicotiana/citologia , Nicotiana/enzimologia , Transdução de Sinais , Apoptose/genética , Biocatálise , Clonagem Molecular , Epistasia Genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas/genética , MAP Quinase Quinase Quinases/química , MAP Quinase Quinase Quinases/genética , Vírus do Mosaico/fisiologia , Filogenia , Folhas de Planta/citologia , Folhas de Planta/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estrutura Terciária de Proteína , Transdução de Sinais/genética , Nicotiana/genéticaRESUMO
The first open-reading frame (ORF) of apple stem grooving virus (ASGV), of the genus Capillovirus, encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP). However, our previous study revealed that ASGV mutants with distinct and discontinuous Rep- and CP-coding regions successfully infect plants, indicating that CP expressed via a subgenomic RNA (sgRNA) is sufficient for viability of the virus. Here we identified a transcription start site of the CP sgRNA and revealed that CP translated from the sgRNA is essential for ASGV infection. We mapped the transcription start sites of both the CP and the movement protein (MP) sgRNAs of ASGV and found a hexanucleotide motif, UUAGGU, conserved upstream from both sgRNA transcription start sites. Mutational analysis of the putative CP initiation codon and of the UUAGGU sequence upstream from the transcription start site of CP sgRNA demonstrated their importance for ASGV accumulation. Our results also demonstrated that potato virus T (PVT), an unassigned species closely related to ASGV, produces two sgRNAs putatively deployed for the CP and MP expression and that the same hexanucleotide motif as found in ASGV is located upstream from the transcription start sites of both sgRNAs. This motif, which constituted putative core elements of the sgRNA promoter, is broadly conserved among viruses in the families Alphaflexiviridae and Betaflexiviridae, suggesting that the gene expression strategy of the viruses in both families has been conserved throughout evolution.
Assuntos
Flexiviridae/genética , Flexiviridae/fisiologia , Regulação Viral da Expressão Gênica , Regiões Promotoras Genéticas , RNA Viral/genética , Transcrição Gênica , Sequência de Bases , Chenopodium quinoa/virologia , Sequência Conservada , Flexiviridae/química , Flexiviridae/isolamento & purificação , Genoma Viral , Dados de Sequência Molecular , Doenças das Plantas/virologia , RNA Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. Lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. This study analyzed the resistance of certain Arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (PlAMV). Map-based positional cloning revealed that the lectin gene JACALIN-TYPE LECTIN REQUIRED FOR POTEXVIRUS RESISTANCE1 (JAX1) is responsible for the resistance. JAX1-mediated resistance did not show the properties of conventional resistance (R) protein-mediated resistance and was independent of plant defense hormone signaling. Heterologous expression of JAX1 in Nicotiana benthamiana showed that JAX1 interferes with infection by other tested potexviruses but not with plant viruses from different genera, indicating the broad but specific resistance to potexviruses conferred by JAX1. In contrast with the lectin gene RESTRICTED TEV MOVEMENT1, which inhibits the systemic movement of potyviruses, which are distantly related to potexviruses, JAX1 impairs the accumulation of PlAMV RNA at the cellular level. The existence of lectin genes that show a variety of levels of virus resistance, their targets, and their properties, which are distinct from those of known R genes, suggests the generality of lectin-mediated resistance in plant innate immunity.
Assuntos
Arabidopsis/imunologia , Lectinas/imunologia , Doenças das Plantas/virologia , Imunidade Vegetal , Potexvirus/patogenicidade , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , Resistência à Doença , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/virologia , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/virologiaRESUMO
Viruses encode RNA silencing suppressors to counteract host antiviral silencing. In this study, we analyzed the suppressors encoded by potato virus M (PVM), a member of the genus Carlavirus. In the conventional green fluorescent protein transient coexpression assay, the cysteine-rich protein (CRP) of PVM inhibited both local and systemic silencing, whereas the triple gene block protein 1 (TGBp1) showed suppressor activity only on systemic silencing. Furthermore, to elucidate the roles of these two suppressors during an active viral infection, we performed PVX vector-based assays and viral movement complementation assays. CRP increased the accumulation of viral RNA at the single-cell level and also enhanced viral cell-to-cell movement by inhibiting RNA silencing. However, TGBp1 facilitated viral movement but did not affect viral accumulation in protoplasts. These data suggest that CRP inhibits RNA silencing primarily at the viral replication step, whereas TGBp1 is a suppressor that acts at the viral movement step. Thus, our findings demonstrate a sophisticated viral infection strategy that suppresses host antiviral silencing at two different steps via two mechanistically distinct suppressors. This study is also the first report of the RNA silencing suppressor in the genus Carlavirus.
Assuntos
Carlavirus/imunologia , Carlavirus/patogenicidade , Inativação Gênica , Interações Hospedeiro-Patógeno , Doenças das Plantas/virologia , Proteínas Virais/metabolismo , Nicotiana/virologiaRESUMO
Systemic necrosis is the most destructive symptom induced by plant pathogens. We previously identified amino acid 1154, in the polymerase domain (POL) of RNA-dependent RNA polymerase (RdRp) of Plantago asiatica mosaic virus (PlAMV), which affects PlAMV-induced systemic necrosis in Nicotiana benthamiana. By point-mutation analysis, we show that amino acid 1,154 alone is not sufficient for induction of necrotic symptoms. However, PlAMV replicons that can express only RdRp, derived from a necrosis-inducing PlAMV isolate, retain their ability to induce necrosis, and transient expression of PlAMV-encoded proteins indicated that the necrosis-eliciting activity resides in RdRp. Moreover, inducible-overexpression analysis demonstrated that the necrosis was induced in an RdRp dose-dependent manner. In addition, during PlAMV infection, necrotic symptoms are associated with high levels of RdRp accumulation. Surprisingly, necrosis-eliciting activity resides in the helicase domain (HEL), not in the amino acid 1,154-containing POL, of RdRp, and this activity was observed even in HELs of PlAMV isolates of which infection does not cause necrosis. Moreover, HEL-induced necrosis had characteristics similar to those induced by PlAMV infection. Overall, our data suggest that necrotic symptoms induced by PlAMV infection depend on the accumulation of a non-isolate specific elicitor HEL (even from nonnecrosis isolates), whose expression is indirectly regulated by amino acid 1,154 that controls replication.