RESUMEN

The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b's nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b's C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b's nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b's efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm.

Asunto(s)

Transporte Activo de Núcleo Celular/fisiología , Cucumovirus/fisiología , Inmunidad de la Planta/fisiología , Interferencia de ARN/fisiología , Proteínas Virales/metabolismo , Carioferinas/metabolismo

RESUMEN

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus in the family Bromoviridae. We cloned the coat protein (CP) and the 2b protein (2b) genes of AV-2 isolates from asparagus plants from various regions and found that the sequence for CP and for 2b was highly conserved among the isolates, suggesting that AV-2 from around the world is almost identical. We then made an AV-2 infectious clone by simultaneous inoculation with in vitro transcripts of RNAs 1-3 of AV-2 and in vitro-synthesized CP, which is necessary for initial infection. Because 2b of cucumoviruses in Bromoviridae can suppress systemic silencing as well as local silencing, we analyzed whether there is functional synteny of 2b between AV-2 and cucumovirus. Using the AV-2 infectious clone, we here provided first evidence that Ilarvirus 2b functions as an RNA silencing suppressor; AV-2 2b has suppressor activity against systemic silencing but not local silencing.

Asunto(s)

Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno , Ilarvirus/crecimiento & desarrollo , Ilarvirus/inmunología , Interferencia de ARN , Proteínas Virales/metabolismo , Replicación Viral , Asparagus/inmunología , Asparagus/virología , Clonación Molecular , Secuencia Conservada , Ilarvirus/genética , Ilarvirus/aislamiento & purificación , Datos de Secuencia Molecular , ARN Viral/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Virulencia/genética , Factores de Virulencia/metabolismo

RESUMEN

In plants, jasmonic acid (JA) and its derivatives are thought to be involved in mobile forms of defense against biotic and abiotic stresses. In this study, the distal transport of JA-isoleucine (JA-Ile) that is synthesized de novo in response to leaf wounding in tomato (Solanum lycopersicum) plants was investigated. JA-[¹³C6]Ile was recovered in distal untreated leaves after wounded leaves were treated with [¹³C6]Ile. However, as [¹³C6]Ile was also recovered in the distal untreated leaves, whether JA-Ile was synthesized in the wounded or in the untreated leaves was unclear. Hence, stem exudates were analyzed to obtain more detailed information. When [¹³C6]Ile and [²H6]JA were applied separately into the wounds on two different leaves, JA-[¹³C6]Ile and [²H6]JA-Ile were detected in the stem exudates but [²H6]JA-[¹³C6]Ile was not, indicating that JA was conjugated with Ile in the wounded leaf and that the resulting JA-Ile was then transported into systemic tissues. The [²H3]JA-Ile that was applied exogenously to the wounded tissues reached distal untreated leaves within 10 min. Additionally, applying [²H3]JA-Ile to the wounded leaves at concentrations of 10 and 60 nmol/two leaves induced the accumulation of PIN II, LAP A, and JAZ3 mRNA in the distal untreated leaves of the spr2 mutant S. lycopersicum plants. These results demonstrate the transportation of de novo synthesized JA-Ile and suggest that JA-Ile may be a mobile signal.

Asunto(s)

Ciclopentanos/metabolismo , Isoleucina/análogos & derivados , Solanum lycopersicum/metabolismo , Ciclopentanos/química , Isoleucina/biosíntesis , Isoleucina/química , Isoleucina/metabolismo , Cinética , Conformación Molecular , Hojas de la Planta/química , Hojas de la Planta/metabolismo

RESUMEN

RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.

Asunto(s)

Silenciador del Gen , Nicotiana/metabolismo , Virus ARN/patogenicidad , ARN Viral/genética , Autofagia , Hidrólisis , Unión Proteica , Interferencia de ARN , Virus ARN/genética

RESUMEN

Mixed infection of Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV) induced more severe symptoms on Nicotiana benthamiana than single infection. To dissect the relationships between spatial infection patterns and the 2b protein (2b) of CMV in single or mixed infections, the CMV vectors expressing enhanced green fluorescent or Discosoma sp. red fluorescent proteins (EGFP [EG] or DsRed2 [Ds], respectively were constructed from the same wild-type CMV-Y and used for inoculation onto N. benthamiana. CMV2-A1 vector (C2-A1 [A1]) has a functional 2b while CMV-H1 vector (C2-H1 [H1]) is 2b deficient. As we expected from the 2b function as an RNA silencing suppressor (RSS), in a single infection, A1Ds retained a high level of accumulation at initial infection sites and showed extensive fluorescence in upper, noninoculated leaves, whereas H1Ds disappeared rapidly at initial infection sites and could not spread efficiently in upper, noninoculated leaf tissues. In various mixed infections, we found two phenomena providing novel insights into the relationships among RSS, viral synergism, and interference. First, H1Ds could not spread efficiently from vasculature into nonvascular tissues with or without TuMV, suggesting that RNA silencing was not involved in CMV unloading from vasculature. These results indicated that 2b could promote CMV to unload from vasculature into nonvascular tissues, and that this 2b function might be independent of its RSS activity. Second, we detected spatial interference (local interference) between A1Ds and A1EG in mixed infection with TuMV, between A1Ds (or H1Ds) and TuMV, and between H1Ds and H1EG. This observation suggested that local interference between two viruses was established even in the synergism between CMV and TuMV and, again, RNA silencing did not seem to contribute greatly to this phenomenon.

Asunto(s)

Cucumovirus/patogenicidad , Nicotiana/virología , Enfermedades de las Plantas/virología , Potyvirus/patogenicidad , ARN Viral/genética , Proteínas Virales/metabolismo , Coinfección , Cucumovirus/genética , Cucumovirus/fisiología , ADN Complementario/genética , Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Interacciones Microbianas , Hojas de la Planta/virología , Plantas Modificadas Genéticamente , Potyvirus/genética , Potyvirus/fisiología , Protoplastos , Interferencia de ARN , ARN Mensajero/genética , Análisis de Secuencia de ADN , Temperatura , Nicotiana/fisiología , Proteínas Virales/genética , Proteína Fluorescente Roja

RESUMEN

Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5'-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene.

Asunto(s)

Clorofila/biosíntesis , Satélite del Virus del Mosaico del Pepino/genética , Nicotiana/virología , Enfermedades de las Plantas/virología , Interferencia de ARN , ARN Viral/genética , Arabidopsis/genética , Arabidopsis/virología , Secuencia de Bases , Capsicum/genética , Capsicum/virología , Clorofila/genética , Satélite del Virus del Mosaico del Pepino/metabolismo , Cucumovirus/metabolismo , Cucumovirus/patogenicidad , Regulación hacia Abajo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Interacciones Huésped-Patógeno , Liasas/genética , Liasas/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/virología , Datos de Secuencia Molecular , Fenotipo , Enfermedades de las Plantas/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/virología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/metabolismo , Nicotiana/enzimología , Nicotiana/genética

RESUMEN

The RNA silencing suppressor 2b protein of Cucumber mosaic virus (CMV) is difficult to produce in Escherichia coli. We compared two CMV 2b proteins that differ in their toxicity against E. coli and found that the acidic amino acid residues in the C-terminal significantly affected the toxicity and expression level of the protein in E. coli. In addition, in a DNA-binding assay, 2b had the ability to bind to DNA, and this ability was affected by the charge on the C-terminal residues of 2b. We concluded that the C-terminal residues were important for 2b's DNA-binding ability, which may partly explain the toxicity of the protein.

Asunto(s)

ADN/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Unión Proteica/genética , Unión Proteica/fisiología , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Proteínas Virales/genética