Tobacco pathogenesis‐related protein 1a (PR‐1a) is induced in plants during the hypersensitive response (HR) after exposure of plants to salicylic acid (SA) and by developmental cues. Gene activation by these diverse stimuli is mediated via an as‐1‐like element in the PR‐1a upstream region. To further analyze the significance of this cis‐acting sequence, an authentic as‐1 element from the cauliflower mosaic virus 35S RNA promoter was inserted into the PR‐1a promoter in place of the as‐1‐like motif. Reporter gene analysis in transgenic tobacco plants demonstrated that as‐1 can functionally replace the as‐1‐like element in the PR‐1a promoter in response to all stimuli. However, reporter gene induction from the as‐1 carrying promoter was enhanced in response to SA compared to the wild‐type promoter, and the ratio of reporter gene activities in SA treated leaf tissue to tissue exhibiting the HR increased with the as‐1 promoter construct. Our findings support a model where PR‐1a gene expression relies on at least two distinct signal transduction pathways initiated by SA and by a yet unknown signal produced during the HR, that promote different, albeit related, transcription complexes on the PR‐1a as‐1‐like element. Analysis of PR‐1 proteins in plants expressing salicylate hydroxylase yielded additional evidence that an HR dependent pathway leads to high level PR‐1 gene induction in tobacco.

Pathogenesis-related proteins (PR proteins) are a heterogeneous group of proteins which are induced in plants by diverse stimuli, e.g. PR proteins are elicited by pathogen attack in the course of the hypersensitive defense reaction of the plant. To examine the regulation of these genes, the 5'-flanking region of the tobacco (Nicotiana tabacum cv. Wisconsin 38) PR-1 a gene up to position -1533 was isolated from genomic DNA by the polymerase chain reaction. Two chimeric gene constructs containing 1533 bp and 906 bp, respectively, of the PR-1 a upstream region fused to the GUS reporter gene were stably integrated into the tobacco genome. All primary transformants exhibited induced expression of the reporter gene after infection of the plants with tobacco mosaic virus or treatment with acetylsalicylic acid. In addition, similar expression of the reporter gene was observed in leaves of adult transgenic plants-without any prior inductive treatments. To study this phenomenon in more detail, the F1 progeny of independent transgenic lines were monitored during the ontogeny of the plants. In normally developing tobacco plants, strong GUS activities were typically detected approximately 12 weeks after germination in the lowest leaves of vegetative plants. When successive leaves of individual plants were tested during the following weeks, a clear gradient of reporter gene activity had developed in the green leaves including the sepals from the bottom to the top of the plants. In all cases analyzed, this gradient of reporter gene expression was strictly parallelled by the expression of the endogenous PR-1 proteins. These results suggest that the acidic PR-1 proteins from tobacco fulfiIl a role during the later stages of plant development and that the PR-1 a upstream region -906 to -335 contains positive regulatory elements for both environmental and developmental signals.Infection of plants with pathogens usually results in a distinct host response depending on the genetic constitution of the plant and the pathogen. In an incompatible reaction, the plant succeeds in localizing the pathogen at the primary infection sites which often become visible as necrotic local lesions on the leaves (hypersensitive response). This local defense reaction is accompanied by a general resistance of the whole plant against further attack by pathogens, a phenomenon called systemic acquired resistance. During the defense reaction of the plant, several host-encoded proteins are induced which are known as pathogenesis-related proteins (PR proteins) and which are believed to play a role in restricting the pathogen. PR proteins were first described in 1970 in tobacco plants exhibiting the hypersensitive response after infection with toCorrespondence to U. M. Pfitzner, Botanisches Institut, MenFax: +49 89 1782 274.

Pathogenesis-related proteins of group 1 (PR-1) are strongly induced in plants by pathogen attack, exposure of the plants to (acetyl)salicylic acid (ASA, SA), and by developmental cues. Functional analysis of the PR-1a promoter identified a region of 139 bp (from -691 to -553) mediating expression of the GUS reporter gene in response to ASA. Inspection of this region revealed two TGACG elements reminiscent of activation sequence-1 (as-1). Recently, as-1 has been reported to be responsive to SA in the context of the CaMV 35S RNA promoter. To address the question of whether the as-1-like sequence may be of functional significance for the expression of the PR-1a gene, gel shift assays were performed with TGA1a, a protein been shown to interact with as-1 in vitro. TGA1a was found to bind to the PR-1a as-1-like sequence with similar specificity and affinity as to as-1. Furthermore, mutations were introduced in the as-1-like sequence in the context of the inducible 906 bp PR-1a promoter which are impaired in binding TGA1a in vitro. Significantly reduced levels of GUS reporter gene activity were obtained with the mutant promoter regions as compared to the wild-type PR-1a promoter in response to all stimuli in transgenic tobacco plants. Yet, mutation of the as-1-like sequence did not abolish induction of reporter gene expression. Taken together, these results suggest that the level of expression of the tobacco PR-1a gene is controlled by an as-1-like sequence motif in the PR-1a upstream region, possibly interacting with a factor related to TGA1a.

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