RESUMEN
Potato virus X (PVX) belongs to genus Potexvirus. This study characterizes the cellular transcriptome responses to PVX infection in Russet potato at 2 and 3 days post infection (dpi). Among the 1242 differentially expressed genes (DEGs), 268 genes were upregulated, and 37 genes were downregulated at 2 dpi while 677 genes were upregulated, and 265 genes were downregulated at 3 dpi. DEGs related to signal transduction, stress response, and redox processes. Key stress related transcription factors were identified. Twenty-five pathogen resistance gene analogs linked to effector triggered immunity or pathogen-associated molecular pattern (PAMP)-triggered immunity were identified. Comparative analysis with Arabidopsis unfolded protein response (UPR) induced DEGs revealed genes associated with UPR and plasmodesmata transport that are likely needed to establish infection. In conclusion, this study provides an insight on major transcriptional regulatory networked involved in early response to PVX infection and establishment.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/genética , Inmunidad de la Planta/genética , Potexvirus/genética , Solanum tuberosum/genética , Factores de Transcripción/genética , Transcriptoma , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/virología , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Potexvirus/crecimiento & desarrollo , Potexvirus/patogenicidad , Transducción de Señal , Solanum tuberosum/inmunología , Solanum tuberosum/virología , Factores de Transcripción/clasificación , Factores de Transcripción/metabolismo , Transcripción Genética , Respuesta de Proteína DesplegadaRESUMEN
The propensity of viruses to acquire genetic material from relatives and possibly from infected hosts makes them excellent candidates as vectors for horizontal gene transfer. However, virus-mediated acquisition of host genetic material, as deduced from historical events, appears to be rare. Here, we report spontaneous and surprisingly efficient generation of hybrid virus/host DNA molecules in the form of minicircles during infection of Beta vulgaris by Beet curly top Iran virus (BCTIV), a single-stranded DNA virus. The hybrid minicircles replicate, become encapsidated into viral particles, and spread systemically throughout infected plants in parallel with the viral infection. Importantly, when co-infected with BCTIV, B. vulgaris DNA captured in minicircles replicates and is transcribed in other plant species that are sensitive to BCTIV infection. Thus, we have likely documented in real time the initial steps of a possible path of virus-mediated horizontal transfer of chromosomal DNA between plant species.
Asunto(s)
Beta vulgaris/genética , Beta vulgaris/virología , ADN Circular/genética , ADN de Plantas/genética , ADN Viral/genética , Geminiviridae/genética , Transferencia de Gen Horizontal/genética , Arabidopsis/virología , ADN de Cadena Simple/genética , Enfermedades de las Plantas/virología , Nicotiana/virologíaRESUMEN
Ribosomal protein S6 (RPS6) is an indispensable plant protein regulated, in part, by ribosomal protein S6 kinase (S6K) which, in turn, is a key regulator of plant responses to stresses and developmental cues. Increased expression of RPS6 was detected in Nicotiana benthamiana during infection by diverse plant viruses. Silencing of the RPS6 and S6K genes in N. benthamiana affected accumulation of Cucumber mosaic virus, Turnip mosaic virus (TuMV), and Potato virus A (PVA) in contrast to Turnip crinkle virus and Tobacco mosaic virus. In addition, the viral genome-linked protein (VPg) of TuMV and PVA interacted with S6K in plant cells, as detected by bimolecular fluorescence complementation assay. The VPg-S6K interaction was detected in cytoplasm, nucleus, and nucleolus, whereas the green fluorescent protein-tagged S6K alone showed cytoplasmic localization only. These results demonstrate that the requirement for RPS6 and S6K differs for diverse plant viruses with different translation initiation strategies and suggest that potyviral VPg-S6K interaction may affect S6K functions in both the cytoplasm and the nucleus.
Asunto(s)
Nicotiana/metabolismo , Nicotiana/virología , Potyvirus/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteína S6 Ribosómica/metabolismo , Proteínas Virales/metabolismo , Arabidopsis/virología , Proteínas de Arabidopsis/metabolismo , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Silenciador del Gen , Genoma Viral , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Huésped-Patógeno , Fenotipo , Epidermis de la Planta/citología , Potyvirus/genética , Unión Proteica , Solanum tuberosum/virología , Fracciones Subcelulares/metabolismoRESUMEN
The unfolded protein response (UPR) signaling network encompasses two pathways in plants, one mediated by inositol-requiring protein-1 (IRE1)-bZIP60 mRNA and the other by site-1/site-2 proteases (S1P/S2P)-bZIP17/bZIP28. As the major sensor of UPR in eukaryotes, IRE1, in response to endoplasmic reticulum (ER) stress, catalyzes the unconventional splicing of HAC1 in yeast, bZIP60 in plants and XBP1 in metazoans. Recent studies suggest that IRE1p and HAC1 mRNA, the only UPR pathway found in yeast, evolves as a cognate system responsible for the robust UPR induction. However, the functional connectivity of IRE1 and its splicing target in multicellular eukaryotes as well as the degree of conservation of IRE1 downstream signaling effectors across eukaryotes remains to be established. Here, we report that IRE1 and its substrate bZIP60 function as a strictly cognate enzyme-substrate pair to control viral pathogenesis in plants. Moreover, we show that the S1P/S2P-bZIP17/bZIP28 pathway, the other known branch of UPR in plants, does not play a detectable role in virus infection, demonstrating the distinct function of the IRE1-bZIP60 pathway in plants. Furthermore, we provide evidence that bZIP60 and HAC1, products of the enzyme-substrate duet, rather than IRE1, are functionally replaceable to cope with ER stress in yeast. Taken together, we conclude that the downstream signaling of the IRE1-mediated splicing is evolutionarily conserved in yeast and plants, and that the IRE1-bZIP60 UPR pathway not only confers overlapping functions with the other UPR branch in fundamental biology but also may exert a unique role in certain biological processes such as virus-plant interactions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Interacciones Huésped-Patógeno , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Tymovirus/patogenicidad , Respuesta de Proteína Desplegada , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/virología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arseniato Reductasas/genética , Arseniato Reductasas/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Datos de Secuencia Molecular , Proteínas Quinasas/química , Proteínas Quinasas/genética , Empalme del ARN , Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
Viral genome-linked protein (VPg) of potyviruses is involved in multiple steps of the potyvirus infection cycle, including viral multiplication and movement in plants. Recently, we showed that VPg of Potato virus A (PVA; genus Potyvirus) suppresses sense-mediated RNA silencing, which is linked to one or both nuclear or nucleolar localization. Here, we studied interactions between VPg and components of the plant RNA silencing pathway. Results showed that VPg interacts with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana, as shown by yeast two-hybrid analysis and bimolecular fluorescence complementation assays. VPg-SGS3 interactions co-localized with small cytoplasmic bodies that contained plant RNA-dependent RNA polymerase 6 (RDR6) (likely SGS3/RDR6 bodies). The N-terminal zinc finger (ZF) domain of SGS3 was the main determinant of the VPg interaction. Our data also suggest that the ZF domain controls SGS3 localization. SGS3 homodimerization was controlled by multiple protein regions. The VPg-SGS3 interaction appeared beneficial for PVA, as viral RNA levels correlated positively with sgs3 mRNA levels in the SGS3-silenced and SGS3-overexpressing leaves of Nicotiana benthamiana. The data support the idea that VPg acts as a suppressor of RNA silencing and suggest that an interaction with SGS3 may be important, especially in suppression of sense-mediated RNA silencing.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Enfermedades de las Plantas/virología , Potyvirus/genética , Solanum tuberosum/genética , Proteínas no Estructurales Virales/metabolismo , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Secuencia de Bases , Datos de Secuencia Molecular , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/virología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Potyvirus/metabolismo , Interferencia de ARN , ARN de Planta/genética , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Recombinantes de Fusión , Análisis de Secuencia de ADN , Eliminación de Secuencia , Solanum tuberosum/virología , Nicotiana/citología , Nicotiana/genética , Nicotiana/virología , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/genética , Dedos de ZincRESUMEN
Plant infection by poleroviruses is restricted to phloem tissues, preventing any classical leaf rub inoculation with viral RNA or virions. Efficient virus inoculation to plants is achieved by viruliferous aphids that acquire the virus by feeding on infected plants. The use of promoter-driven infectious cDNA is an alternative means to infect plants and allows reverse genetic studies to be performed. Using Beet mild yellowing virus isolate 2ITB (BMYV-2ITB), we produced a full-length infectious cDNA clone of the virus (named BMYV-EK) placed under the control of the T7 RNA polymerase and the Cauliflower mosaic virus 35S promoters. Infectivity of the engineered BMYV-EK virus was assayed in different plant species and compared with that of the original virus. We showed that in vitro- or in planta-derived transcripts were infectious in protoplasts and in whole plants. Importantly, the natural aphid vector Myzus persicae efficiently transmitted the viral progeny produced in infected plants. By comparing agroinoculation and aphid infection in a host range assay, we showed that the engineered BMYV-EK virus displayed a similar host range to BMYV-2ITB, except for Nicotiana benthamiana, which proved to be resistant to systemic infection with BMYV-EK. Finally, both the BMYV-EK P0 and the full-length clone were able to strongly interfere with post-transcriptional gene silencing.
Asunto(s)
Beta vulgaris/virología , ADN Complementario/genética , Especificidad del Huésped , Enfermedades de las Plantas/virología , Virus de Plantas/genética , Virus de Plantas/aislamiento & purificación , Animales , Áfidos/virología , Arabidopsis/virología , Secuencia de Bases , Células Clonales , Silenciador del Gen , Genoma Viral/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Protoplastos/virología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Virión/metabolismoRESUMEN
Arabidopsis thaliana infected with Beet severe curly top virus (BSCTV) exhibits systemic symptoms such as stunting of plant growth, callus induction on shoot tips, and curling of leaves and shoot tips. The regulation of sucrose metabolism is essential for obtaining the energy required for viral replication and the development of symptoms in BSCTV-infected A. thaliana. We evaluated the changed transcript level and enzyme activity of invertases in the inflorescence stems of BSCTV-infected A. thaliana. These results were consistent with the increased pattern of ribulose-1,5-bisphosphate carboxylase/oxygenase activity and photosynthetic pigment concentration in virus-infected plants to supply more energy for BSCTV multiplication. The altered gene expression of invertases during symptom development was functionally correlated with the differential expression patterns of D-type cyclins, E2F isoforms, and invertase-related genes. Taken together, our results indicate that sucrose sensing by BSCTV infection may regulate the expression of sucrose metabolism and result in the subsequent development of viral symptoms in relation with activation of cell cycle regulation.
Asunto(s)
Arabidopsis/enzimología , Geminiviridae/fisiología , Regulación Enzimológica de la Expresión Génica , Enfermedades de las Plantas/virología , beta-Fructofuranosidasa/genética , Arabidopsis/genética , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Beta vulgaris/virología , Carotenoides/metabolismo , Proteínas de Ciclo Celular/genética , Clorofila/metabolismo , Ciclinas/genética , Factores de Transcripción E2F/genética , Regulación de la Expresión Génica de las Plantas , Glucosiltransferasas/metabolismo , Inflorescencia/enzimología , Inflorescencia/genética , Inflorescencia/virología , Datos de Secuencia Molecular , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/virología , Tallos de la Planta/enzimología , Tallos de la Planta/genética , Tallos de la Planta/virología , Plantas Modificadas Genéticamente , Ribulosa-Bifosfato Carboxilasa/metabolismo , Sacarosa/metabolismo , beta-Fructofuranosidasa/metabolismoRESUMEN
Microtubules (MT) outline and maintain the overall shape of cells and can reorganize cellular membranes to serve as sites of RNA virus replication. Here, we provide data on involvement of an MT-associated protein in infection of plants with a potyvirus, Potato virus A (PVA), representing the largest family of plant-infecting RNA viruses. Our results showed that helper-component proteinase (HCpro)-interacting protein 2 (HIP2) of potato (Solanum tuberosum) is an MT-associated protein similar to Arabidopsis SPR2. Virus-induced silencing of HIP2 in Nicotiana benthamiana resulted in a spiral-like growth phenotype, similar to the Arabidopsis spr2 mutant, and the spr2 phenotype in Arabidopsis was complemented with potato HIP2. HCpro of PVA interacted with HIP2 of potato and tobacco (Nicotiana tabacum). The interaction was detected by bimolecular fluorescence complementation in PVA-infected leaves on MT and MT intersections at the cell cortex. HIP2-HCpro interaction was determined by the C-proximal α-helix-rich domain of HIP2, whereas the N-proximal putative TOG domain and the central coiled-coil domain of HIP2 controlled HIP2 dimerization and binding to MT. Accumulation of PVA was significantly reduced in the HIP2-silenced leaves of N. benthamiana, which indicates that HIP2-HCpro interactions are important for virus infection.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Interacciones Huésped-Patógeno , Proteínas Asociadas a Microtúbulos/metabolismo , Nicotiana/metabolismo , Potyvirus/fisiología , Proteínas Virales/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Cisteína Endopeptidasas/genética , Técnicas de Inactivación de Genes , Silenciador del Gen , Prueba de Complementación Genética , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Fenotipo , Filogenia , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Potyvirus/enzimología , Potyvirus/patogenicidad , Mapeo de Interacción de Proteínas , Análisis de Secuencia de ADN , Solanum tuberosum/genética , Solanum tuberosum/inmunología , Solanum tuberosum/metabolismo , Solanum tuberosum/virología , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/virología , Proteínas Virales/genéticaRESUMEN
Beet necrotic yellow vein virus (BNYVV) is a multipartite RNA virus. BNYVV RNA3 does not accumulate in non-host transgenic Arabidopsis thaliana plants when expressed using a 35S promoter. However, a 3'-derivative species has been detected in transgenic plants and in transient expression assays conducted in Nicotiana benthamiana and Beta macrocarpa. The 3'-derivative species is similar to the previously reported subgenomic RNA3 produced during virus infection. 5' RACE revealed that the truncated forms had identical 5' ends. The 5' termini carried the coremin motif also present on BNYVV RNA5, beet soil-borne mosaic virus RNA3 and 4, and cucumber mosaic virus group 2 RNAs. This RNA3 species lacks a m(7)Gppp at the 5' end of the cleavage products, whether expressed transiently or virally. Mutagenesis revealed the importance of the coremin sequence for both long-distance movement and stabilization of the cleavage product in vivo and in vitro. The isolation of various RNA3 5'-end products suggests the existence of a cleavage between nt 212 and 1234 and subsequent exonucleolytic degradation, leading to the accumulation of a non-coding RNA. When RNA3 was incubated in wheatgerm extracts, truncated forms appeared rapidly and their appearance was protein- and divalent ion-dependent.
Asunto(s)
Genoma Viral , Virus de Plantas/patogenicidad , ARN no Traducido/metabolismo , ARN Viral/metabolismo , Arabidopsis/virología , Beta vulgaris/virología , Virus de Plantas/genética , Estabilidad del ARN , ARN Viral/genética , Nicotiana/virologíaRESUMEN
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/genéticaRESUMEN
Salicylic acid (SA) is required for plant systemic acquired resistance (SAR) to viruses. However, SA-deficient plants adapt to RNA virus infections better, which show a lighter symptom and have less reactive oxygen species (ROS) accumulation. The virus replication levels are higher in the SA-deficient plants during the first 10 days, but lower than the wild-type seedlings after 20 dpi. The higher level of glutathione and ascorbic acid (AsA) in SA-deficient plants may contribute to their alleviated symptoms. Solo virus-control method for mortal viruses results in necrosis and chlorosis, no matter what level of virus RNAs would accumulate. Contrastingly, early and high-dose AsA treatment alleviates the symptom, and eventually inhibits virus replication after 20 days. ROS eliminators could not imitate the effect of AsA, and could neither alleviate symptom nor inhibit virus replication. It suggests that both symptom alleviation and virus replication control should be considered for plant virus cures.
Asunto(s)
Arabidopsis/virología , Ácido Ascórbico/farmacología , Glutatión/farmacología , Enfermedades de las Plantas/virología , Virus de Plantas/fisiología , Virus ARN/fisiología , Ácido Salicílico/metabolismo , Inmunidad Innata , Enfermedades de las Plantas/terapia , Replicación Viral/efectos de los fármacosRESUMEN
Potato spindle tuber viroid (PSTVd) contains an element of tertiary structure -loop E- also present in eukaryotic 5S rRNA. The ribosomal protein L5 and transcription factor IIIA (TFIIIA) from Arabidopsis thaliana bind 5S rRNA in vitro and in vivo, mediating different functions that include nucleocytoplasmic transport and transcription activation, respectively. We show that A. thaliana L5 and TFIIIA also bind PSTVd (+) RNA in vitro with the same affinity as they bind 5S rRNA, whereas the affinity for a chloroplastic viroid is significantly lower. These two proteins might participate in the synthesis and delivery of PSTVd RNA in vivo.
Asunto(s)
Arabidopsis/virología , Proteínas de Plantas/metabolismo , ARN Viral/metabolismo , Proteínas Ribosómicas/metabolismo , Solanum tuberosum/virología , Factor de Transcripción TFIIIA/metabolismo , Viroides/genética , Unión Proteica , ARN Ribosómico 5S/metabolismoRESUMEN
The RNA-3-encoded p25 protein was previously characterized as one of the major symptom determinants of the Beet necrotic yellow vein virus. Previous analyses reported the influence of the p25 protein in root proliferation phenotype observed in rhizomania disease on infected sugar beets (Beta vulgaris). A transgenic approach was developed, in which the p25 protein was constitutively expressed in Arabidopsis thaliana Columbia (Col-0) ecotype in order to provide new clues as to how the p25 protein might promote alone disease development and symptom expression. Transgenic plants were characterized by Southern blot and independent lines carrying single and multiple copies of the transgene were selected. Mapping of the T-DNA insertion was performed on the monocopy homozygote lines. P25 protein was localized both in the nucleus and in the cytoplasm of epidermal and root cells of transgenic plants. Although A. thaliana was not described as a susceptible host for BNYVV infection, abnormal root branching was observed on p25 protein-expressing A. thaliana plants. Moreover, these transgenic plants were more susceptible than wild-type plants to auxin analog treatment (2,4-D) but more resistant to methyl jasmonate (MeJA), abscisic acid (ABA) and to lesser extend to salicylic acid (SA). Hormonal content assays measuring plant levels of auxin (IAA), jasmonate (JA) and ethylene precursor (ACC) revealed major hormonal changes. Global transcript profiling analyses on roots displayed differential gene expressions that could corroborate root branching phenotype and stress signaling modifications.
Asunto(s)
Arabidopsis/metabolismo , Beta vulgaris/virología , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/fisiología , Plantas Modificadas Genéticamente/metabolismo , Virus ARN/metabolismo , Proteínas Virales/metabolismo , Arabidopsis/genética , Arabidopsis/virología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Ciclopentanos/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , ADN Bacteriano/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ácidos Indolacéticos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxilipinas/metabolismo , Fenotipo , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Raíces de Plantas/metabolismo , Raíces de Plantas/virología , Virus de Plantas/genética , Virus de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/virología , Virus ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Virales/genéticaRESUMEN
The promoter of an Indian isolate of the pararetrovirus Rice tungro bacilliform virus (RTBV-WB) contains a negative element downstream of the transcription start site (TSS), between nucleotide residues +58 and +195 (Mathur and Dasgupta, 2007). To further characterize the element, we show, by using transient gus reporter gene assays in the cells of onion peel, rice calli and Arabidopsis leaves, that it down-regulates heterologous promoters CaMV35S and Maize ubiquitin. Quantitative measurements of transient GUS activity indicated more than 90% inhibition of reporter gene expression by the negative element. We also show, by reversing the orientation of the element downstream and by placing it in a position upstream to a constitutively expressing RTBV promoter, that the negative element is orientation- and position-independent, pointing towards its activity at the transcriptional and not post-transcriptional level.
Asunto(s)
Regulación Viral de la Expresión Génica , Elementos Reguladores de la Transcripción , Tungrovirus/fisiología , Arabidopsis/genética , Arabidopsis/virología , Genes Reporteros , Glucuronidasa/genética , Glucuronidasa/metabolismo , India , Cebollas/genética , Cebollas/virología , Oryza/genética , Oryza/virología , Hojas de la Planta/genética , Hojas de la Planta/virología , Proteínas de Plantas/genética , Tungrovirus/genética , Tungrovirus/aislamiento & purificación , Ubiquitina/genética , Zea mays/genéticaRESUMEN
AtMPB2C is the Arabidopsis (Arabidopsis thaliana) homolog of MPB2C, a microtubule-associated host factor of tobacco mosaic virus movement protein that was been previously identified in Nicotiana tabacum. To analyze the endogenous function of AtMPB2C and its role in viral infections, transgenic Arabidopsis plant lines stably overexpressing green fluorescent protein (GFP)-AtMPB2C were established. The GFP-AtMPB2C fusion protein was detectable in various cell types and organs and localized at microtubules in a punctuate pattern or in filaments. To determine whether overexpression impacted on the cortical microtubular cytoskeleton, GFP-AtMPB2C-overexpressing plants were compared to known microtubular marker lines. In rapidly elongated cell types such as vein cells and root cells, GFP-AtMPB2C overexpression caused highly unordered assemblies of cortical microtubules, a disturbed, snake-like microtubular shape, and star-like crossing points of microtubules. Phenotypically, GFP-AtMPB2C transgenic plants showed retarded growth but were viable and fertile. Seedlings of GFP-AtMPB2C transgenic plants were characterized by clockwise twisted leaves, clustered stomata, and enhanced drought tolerance. GFP-AtMPB2C-overexpressing plants showed increased resistance against oilseed rape mosaic virus, a close relative of tobacco mosaic virus, but not against cucumber mosaic virus when compared to Arabidopsis wild-type plants. These results suggest that AtMPB2C is involved in the alignment of cortical microtubules, the patterning of stomata, and restricting tobamoviral infections.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/virología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Estomas de Plantas/metabolismo , Estomas de Plantas/virología , Tobamovirus/patogenicidad , Secuencia de Aminoácidos , Arabidopsis/citología , Proteínas de Arabidopsis/química , Sequías , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Asociadas a Microtúbulos/química , Microtúbulos/virología , Datos de Secuencia Molecular , Extractos Vegetales/metabolismo , Estomas de Plantas/citología , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Alineación de SecuenciaRESUMEN
Phytic acid (myo-inositol hexakisphosphate, InsP6) is an important phosphate store and signal molecule in plants. However, low-phytate plants are being developed to minimize the negative health effects of dietary InsP6 and pollution caused by undigested InsP6 in animal waste. InsP6 levels were diminished in transgenic potato plants constitutively expressing an antisense gene sequence for myo-inositol 3-phosphate synthase (IPS, catalysing the first step in InsP6 biosynthesis) or Escherichia coli polyphosphate kinase. These plants were less resistant to the avirulent pathogen potato virus Y and the virulent pathogen tobacco mosaic virus (TMV). In Arabidopsis thaliana, mutation of the gene for the enzyme catalysing the final step of InsP6 biosynthesis (InsP5 2-kinase) also diminished InsP6 levels and enhanced susceptibility to TMV and to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae. Arabidopsis thaliana has three IPS genes (AtIPS1-3). Mutant atips2 plants were depleted in InsP6 and were hypersusceptible to TMV, turnip mosaic virus, cucumber mosaic virus and cauliflower mosaic virus as well as to the fungus Botrytis cinerea and to P. syringae. Mutant atips2 and atipk1 plants were as hypersusceptible to infection as plants unable to accumulate salicylic acid (SA) but their increased susceptibility was not due to reduced levels of SA. In contrast, mutant atips1 plants, which were also depleted in InsP6, were not compromised in resistance to pathogens, suggesting that a specific pool of InsP6 regulates defence against phytopathogens.
Asunto(s)
Arabidopsis/metabolismo , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Ácido Fítico/biosíntesis , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Arabidopsis/genética , Arabidopsis/microbiología , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Botrytis/patogenicidad , Caulimovirus/patogenicidad , Cucumovirus/patogenicidad , ADN Bacteriano/genética , Susceptibilidad a Enfermedades/microbiología , Susceptibilidad a Enfermedades/virología , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Inmunidad Innata/genética , Mutagénesis Insercional , Mutación , Mio-Inositol-1-Fosfato Sintasa/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/microbiología , Plantas Modificadas Genéticamente/virología , Potyvirus/patogenicidad , Pseudomonas syringae/patogenicidad , ARN de Planta/genética , Ácido Salicílico/metabolismo , Transducción de Señal , Solanum tuberosum/genética , Solanum tuberosum/virología , Virus del Mosaico del Tabaco/patogenicidad , Tymovirus/patogenicidadRESUMEN
Spinach curly top virus (SCTV), the fifth characterized Curtovirus species belonging to the family Geminiviridae, is an agriculturally significant plant pathogen representing an emerging disease threat in the southern United States. The SCTV genome comprises a single DNA chromosome of approximately 3.0 kb, with the potential to code for seven proteins larger than 10 kDa but which relies extensively on the host for replication and transcription of its genome. In this study, we have identified viral and complementary sense transcripts in SCTV-infected plants, confirming a bidirectional transcription strategy for SCTV. The most abundant RNA maps to the virion sense (1.1-kb transcript) and is comparable in size and location to that observed in Beet curly top virus (BCTV). Two complementary sense transcripts (1.7 and 0.7 kb) were identified in SCTV-infected plants. The large, 1.7-kb transcript is comparable in size and position to that identified in BCTV and several begomoviruses and most likely encodes the C1 protein. Both complementary sense RNAs could potentially direct expression of C2 and C3 from polycistronic mRNAs. A mutation in the C2 gene of SCTV results in expression of a truncated protein of 38 amino acids that is capable of interacting with two cellular kinases, AKIN11 and ADK2, and the resulting mutant virus remains highly infectious. A second mutant virus can only express the first three amino acids of the C2 protein and is unable to interact with the same kinases. However, this mutant virus still remains infectious, although a reduction in infectivity and symptom severity was seen in both Arabidopsis and spinach. A possible relationship between the interaction of C2 with AKIN11 and ADK2 and disease severity is presented.
Asunto(s)
Geminiviridae/genética , Genoma Viral/genética , Virión/genética , Arabidopsis/virología , Secuencia de Bases , Beta vulgaris/virología , Geminiviridae/crecimiento & desarrollo , Regulación Viral de la Expresión Génica , Modelos Genéticos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Hojas de la Planta/virología , Spinacia oleracea/virología , Nicotiana/virología , Transcripción Genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virión/crecimiento & desarrolloRESUMEN
Thermal stress of PSTVd-infected Nicotiana benthamiana led to appearance of a broad PSTVd sequence distribution, where most of mutations accumulated in the left half of the viroid's secondary structure including the "pathogenicity" domain. A similar effect had been reported for hop latent viroid [Virology 287 (2001) 349]. The pool of viroid "thermomutants" progenies was transcribed into cDNA and used for biolistic inoculation of Raphanus sativa, where the PSTVd infection was detectable by reverse transcription and polymerase chain reaction (RT-PCR). Newly generated inoculum from R. sativa was used for biolistic transfer to Arabidopsis thaliana wild-type and silencing-deficient mutants bearing one of sde1, sde2, and sde3 locuses. Irrespective to A. thaliana silencing mutants, viroid levels in Brasicaceae species infected with mutated PSTVd variants were of approximately 300 times lower than it is expected for tomato. At the same time, no systemic infection of A. thaliana was achieved with the wild-type PSTVd. In Arabidopsis, a population of PSTVd, consisting of frequent and minor variants, was present and the sequence distribution differed from that of the original viroid "thermomutants"; that is, mutations were not predominantly restricted to the left half of viroid's secondary structure. At least 65% of viroid sequences from Arabidopsis library accumulated mutations in the upper conserved central region (UCCR). In addition, mutants having changes in "hairpin II" domain (C-->A transition at position 229) and in the conserved internal loop element in the left part of viroid structure (single insertion of G at position 39) were detected. All those mutants were inoculated biolistically to tomato and promoted infection especially after prolonged period of plant cultivation (50-80 days pi) when infection reached 70-90%. However, the sequence variants were unstable and reverted to the wild type and to other sequence variants stable in tomato. Our results demonstrate that heat stress-mediated production of viroid quasi-species could be of significance for viroid adaptations.
Asunto(s)
Arabidopsis/virología , Biolística , Variación Genética , Calor , Raphanus/virología , Viroides/genética , Secuencia de Bases , Respuesta al Choque Térmico , Datos de Secuencia Molecular , Mutagénesis , Mutación , Enfermedades de las Plantas/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Solanum tuberosum/virología , Nicotiana/virología , Viroides/clasificación , Viroides/crecimiento & desarrollo , Viroides/patogenicidadRESUMEN
Little is known about the mechanisms that couple regulation of secondary metabolic pathways to the synthesis of primary metabolic precursors. Camalexin, an indolic secondary metabolite, appears to be the major phytoalexin in Arabidopsis. It was previously shown that camalexin accumulation is caused by infection with plant pathogens, by abiotic elicitors, and in spontaneous lesions in the accelerated cell death mutant acd2. We demonstrate that the accumulation of this phytoalexin is accompanied by the induction of the mRNAs and proteins for all of the tryptophan biosynthetic enzymes tested. A strong correlation was observed between the magnitude of camalexin accumulation and the induction of tryptophan biosynthetic proteins, indicating coordinate regulation of these processes. Production of disease symptoms is not sufficient for the response because systemic infection with cauliflower mosaic virus or cucumber mosaic virus did not induce the tryptophan pathway enzymes or camalexin accumulation. Salicylic acid appears to be required, but unlike other documented pathogenesis-related proteins, it is not sufficient for the coordinate induction. Results with trp mutants suggest that the tryptophan pathway is not rate limiting for camalexin accumulation. Taken together, these results are consistent with the hypothesis that the regulation of the tryptophan pathway in plants responds to needs for biosynthesis of secondary metabolites.