PR-1a is a salicylic acid-inducible defense gene of tobacco ( Nicotiana tabacum ). One-hybrid screens identified a novel tobacco WRKY transcription factor (NtWRKY12) with specific binding sites in the PR-1a promoter at positions −564 (box WK 1 ) and −859 (box WK 2 ). NtWRKY12 belongs to the class of transcription factors in which the WRKY sequence is followed by a GKK rather than a GQK sequence. The binding sequence of NtWRKY12 (WK box TTTTCCAC) deviated significantly from the consensus sequence (W box TTGAC(C/T)) shown to be recognized by WRKY factors with the GQK sequence. Mutation of the GKK sequence in NtWRKY12 into GQK or GEK abolished binding to the WK box. The WK 1 box is in close proximity to binding sites in the PR-1a promoter for transcription factors TGA1a ( as-1 box) and Myb1 (MBSII box). Expression studies with PR-1a promoter∷ β-glucuronidase ( GUS ) genes in stably and transiently transformed tobacco indicated that NtWRKY12 and TGA1a act synergistically in PR-1a expression induced by salicylic acid and bacterial elicitors. Cotransfection of Arabidopsis thaliana protoplasts with 35S ∷ NtWRKY12 and PR-1a ∷ GUS promoter fusions showed that overexpression of NtWRKY12 resulted in a strong increase in GUS expression, which required functional WK boxes in the PR-1a promoter.
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