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Background    Nicotiana benthamiana  has been widely used for transient gene expression assays and as a model plant in the study of plant-microbe interactions, lipid engineering and RNA silencing pathways. Assembling the sequence of its transcriptome provides information that, in conjunction with the genome sequence, will facilitate gaining insight into the plant’s capacity for high-level transient transgene expression, generation of mobile gene silencing signals, and hyper-susceptibility to viral infection.    Methodology/Results  RNA-seq libraries from 9 different tissues were deep sequenced and assembled, de novo, into a representation of the transcriptome. The assembly, of16GB of sequence, yielded 237,340 contigs, clustering into 119,014 transcripts (unigenes). Between 80 and 85% of reads from all tissues could be mapped back to the full transcriptome. Approximately 63% of the unigenes exhibited a match to the Solgenomics tomato predicted proteins database. Approximately 94% of the Solgenomics  N. benthamiana  unigene set (16,024 sequences) matched our unigene set (119,014 sequences). Using homology searches we identified 31 homologues that are involved in RNAi-associated pathways in  Arabidopsis thaliana , and show that they possess the domains characteristic of these proteins. Of these genes, the RNA dependent RNA polymerase gene, Rdr1, is transcribed but has a 72 nt insertion in exon1 that would cause premature termination of translation. Dicer-like 3 (DCL3) appears to lack both the DEAD helicase motif and second dsRNA binding motif, and DCL2 and AGO4b have unexpectedly high levels of transcription.    Conclusions  The assembled and annotated representation of the transcriptome and list of RNAi-associated sequences are accessible at  www.benthgenome.com  alongside a draft genome assembly. These genomic resources will be very useful for further study of the developmental, metabolic and defense pathways of  N. benthamiana  and in understanding the mechanisms behind the features which have made it such a well-used model plant.

De Novo Transcriptome Sequence Assembly and Analysis of RNA Silencing Genes of Nicotiana benthamiana