Subtractive expressed sequence tag analysis and screening of cDNA libraries derived from Brassica napus leaves subjected to mechanical wounding, flea beetle feeding or cold temperatures revealed eight genes encoding NAC-domain transcription factors. The genes were found to be differentially regulated in response to biotic and abiotic stresses including wounding, insect feeding, Sclerotinia sclerotiorum infection, cold shock and dehydration. Five BnNAC proteins were orthologous to Arabidopsis thaliana ATAF1 or ATAF2 and gave rise to developmental abnormalities similar to the A. thaliana nam and cuc mutants when expressed ectopically in A. thaliana. Transgenic lines expressing BnNAC14, exhibited large leaves, thickened stems and hyper-developed lateral root systems similar to that observed with A. thaliana NAC1, but also were delayed in bolting and lacked an apical dominant tap root. Several of the BnNAC proteins were capable of activating gene expression in yeast and recognized an element within the CaMV35S promoter. A yeast two-hybrid screen revealed that BnNAC14 interacted with other select BnNAC proteins in vitro and identified an additional BnNAC gene, BnNAC485. The protein interaction and transcriptional activation domains were mapped by deletion analysis.