Nicotiana benthamiana Transcriptome or Gene expression. Nicotiana benthamiana

We developed a novel platform for genome-wide gene expression analysis in any eukaryotic organism, that we coin SuperSAGE Array. The SuperSAGE Array is a microarray onto which oligonucleotides of 26 nucleotides corresponding to SuperSAGE tag sequences are directly synthesized. A SuperSAGE Array combines the advantages of the highly quantitative SuperSAGE expression analysis with the high-throughput microarray technology. This technology was also applied to the detailed study of expressed genes identified by SuperSAGE in Nicotiana benthamiana, an organism without available DNA database. Keywords: transgene overexpression, transient expression Overall design: Using SuperSAGE, we first explored N. benthamiana genes that were up- or down-regulated by NbCD1- or NbCD3-overexpression, respectively. N. benthamiana leaves were infiltrated with Agrobacterium tumefaciens carrying plasmids harboring genes for NbCD1, NbCD3 and GFP (control), respectively, in the glucocorticoid-inducible gene expression cassette GVG9. Two days after agroinfiltration, the leaves were treated with dexamethasone (DEX) to induce expression of the transgenes. Four hours after DEX treatment, leaves were harvested, and RNA was isolated for SuperSAGE analysis. For the identification of genes whose expression is altered by NbCD1-overexpression, 10,269 and 10,884 tags (6,659 and 6,796 genes) were compared between GFP- and NbCD1-overexpressing leaves. Similarly, 10,063 tags (5,564 genes) from GFP-overexpressing leaves and 10,677 tags (5,858 genes) from NbCD3-overexpressing leaves were compared. We selected those tags that show more than four-fold representational differences between NbCD1- or NbCD3-, and GFP-overexpressing leaves, respectively . These tags were altogether used to establish a SuperSAGE Array. In total, 154 tags corresponding to the genes up- or down-regulated by either NbCD1- or NbCD3-overexpression were selected for an oligonucleotide array .