Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-Type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco

Using mRNA differential display analysis, we isolated a salt-induced transc ript that showed a significant sequence homology with an EREBP/AP2 DNA bind ing motif from oilseed rape plants. With this cDNA fragment as a probe, cDN A clone Tsi1 (for Tobacco stress-induced gene1) was isolated from a tobacco cDNA library. RNA gel blot analysis indicated that transcripts homologous with Tsi1 were induced not only in NaCl-treated leaves but also in leaves t reated with ethephon or salicylic acid. Transient expression analysis using a Tsi1::smGFP fusion gene in BY-2 cells indicated that the Tsi1 protein wa s targeted to the nucleus. Fusion protein of Tsi1 with GAL4 DNA binding dom ain strongly activated transcription in yeast, and the transactivating acti vity was localized to the 13 C-terminal amino acids of Tsi1. Electrophoreti c mobility shift assays revealed that Tsi1 could bind specifically to the G CC and the DRE/CRT sequences, although the binding activity to the former w as stronger than that to the latter. Furthermore, Agrobacterium-mediated tr ansient expression and transgenic plants expressing Tsi1 demonstrated that overexpression of the Tsi1 gene induced expression of several pathogenesis- related genes under normal conditions, resulting in improved tolerance to s alt and pathogens. These results suggest that Tsi1 might be involved as a p ositive trans-acting factor in two separate signal transduction pathways un der abiotic and biotic stress.