RESUMEN
To identify positive regulators of cell death in plants, we performed a high-throughput screening, employing potato virus X-based overexpression in planta of a cDNA library derived from paraquat-treated Nicotiana benthamiana leaves. The screening of 30,000 cDNA clones enabled the identification of an ADP-ribosylation factor 1 (ARF1) that induces cell death when overexpressed in N. benthamiana. Overexpression of the guanosine diphosphate (GDP)-locked mutant of ARF1 did not trigger cell death, suggesting that ARF1 guanosine triphosphatase (GTPase) activity is necessary for the observed cell death-inducing activity. The ARF1 transcript level increased strongly following treatment with Phytophthora infestans elicitor INF1, as well as inoculation with a non-host pathogen Pseudomonas cichorii in N. benthamiana. In addition, ARF1 was induced in the interaction between the N gene and tobacco mosaic virus (TMV) in Nicotiana tabacum. By contrast, inoculation with the virulent pathogen Pseudomonas syringae pv. tabaci did not affect ARF1 expression in N. benthamiana. Virus-induced gene silencing of ARF1 in N. benthamiana resulted in a stunted phenotype, and severely hampered non-host resistance towards P. cichorii. In addition, ARF1 silencing partially compromised resistance towards TMV in N. benthamiana containing the N resistance gene. By contrast, and in accordance with the ARF1 gene expression profile, silencing of ARF1 transcription did not alter the susceptibility of N. benthamiana towards the pathogen P. syringae pv. tabaci. These results strongly implicate ARF1 in the non-host resistance to bacteria and N gene-mediated resistance in N. benthamiana.
Asunto(s)
Factor 1 de Ribosilacion-ADP/genética , Genes de Plantas , Nicotiana/genética , Muerte Celular , ADN Complementario/genética , Expresión Génica , Silenciador del Gen , Inmunidad Innata , Datos de Secuencia Molecular , Mutación , Fenotipo , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Pseudomonas syringae/patogenicidad , ARN de Planta/genética , Nicotiana/inmunología , Nicotiana/microbiología , Nicotiana/virología , Virus del Mosaico del Tabaco/patogenicidadRESUMEN
There is no question that protein- and RNA-based measurements are complementary, but which approach has the highest return in the case of a non-model crop and what is the correlation between mRNA and proteins? We describe and evaluate in detail the advantages and pitfalls of both a proteomics and a transcriptomics approach. The information on the abundance of transcripts was obtained by serial analysis of gene expression (SAGE), while information on the abundance of proteins was obtained via two-dimensional gel electrophoresis.
Asunto(s)
Productos Agrícolas/química , Productos Agrícolas/genética , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Musa/química , Musa/genética , Proteómica/métodos , Electroforesis en Gel Bidimensional , Etiquetas de Secuencia Expresada , Regulación de la Expresión Génica de las Plantas , Punto Isoeléctrico , Modelos Biológicos , Hojas de la Planta/química , Proteínas de Plantas/análisis , Proteínas de Plantas/aislamiento & purificación , Proteoma/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Especificidad de la EspecieRESUMEN
Super-serial analysis of gene expression (SuperSAGE) was used to characterize, for the first time, the global gene expression pattern in banana (Musa acuminata). A total of 10,196 tags were generated from leaf tissue, representing 5,292 expressed genes. Forty-nine tags of the top 100 most abundantly expressed transcripts were annotated by homology to cDNA or EST sequences. Typically for leaf tissue, analysis of the transcript profiles showed that the majority of the abundant transcripts are involved in energy production, mainly photosynthesis. However, the most abundant tag was derived from a type 3 metallothionein transcript, which accounted for nearly 3% of total transcripts analysed. Furthermore, the 26-bp long SuperSAGE tags were applied in 3'-rapid amplification of cDNA ends (3'RACE) for the identification of unknown tags. In combination with thermal asymmetric interlaced PCR (TAIL-PCR), this allowed the recovery of a full gene sequence of a novel NADPH:protochlorophyllide oxidoreductase, the key enzyme in chlorophyll biosynthesis. SuperSAGE in conjunction with 3'RACE and TAIL-PCR will be a powerful tool for transcriptomics of non-model, but otherwise important organisms.
Asunto(s)
Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Biblioteca de Genes , Musa/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Biología Computacional , Cartilla de ADN , ADN Complementario/genética , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico , Análisis de Secuencia de ADNRESUMEN
Transferred DNA (T-DNA) tagging is a powerful tool for tagging and in planta characterization of plant genes on a genome-wide scale. An improved promoter tagging vector is described here, which contains the codon-optimized luciferase (luc+) reporter gene 31 bp from the right border of the T-DNA. Compared to the wild-type luciferase gene, this construct provides significantly increased reporter gene expression and a 40 times higher tagging frequency. The utility of the construct is demonstrated in banana, a tropical monocot species, by screening embryogenic cell colonies and regenerated plants with an ultrasensitive charged-coupled device (CCD) camera. The improved vector resulted in a luciferase activation frequency of 2.5% in 19,000 cell colonies screened. Detailed molecular analysis of flanking DNA sequences in a tagged line revealed insertion of the luciferase tag in a novel gene with near-constitutive expression.
