Tobacco genes encoding the PR-la protein and a glycine-rich protein are expressed after treatment of plants with salicylate or infection with tobacco mosaic virus. Upstream sequences of these genes were fused to reporter genes, and these constructs were used to transform tobacco. Upstream sequences of the PR-la gene of 689 base pairs or longer were sufficient for induction of the reporter gene in tobacco mosaic virus-inoculated leaves, systemically induced leaves from infected plants, and leaves treated with salicylate. N o such induction was found with upstream sequences of 643 base pairs or shorter of the PR-la gene. When the PR-la upstream sequence from nucleotides -625 to -902 was fused to the cauliflower mosaic virus 35s core promoter, a construct was obtained that conferred tobacco mosaic virus and salicylate inducibility to the reporter gene in transgenic plants. This confirmed the localization of tobacco mosaic vírus-and salicylate-responsive elements between positions -643 and -689 in the PR-la promoter. With the glycine-rich protein gene, an upstream sequence of 645 base pairs was sufficient for tobacco mosaic virus and salicylate inducibility of the reporter gene, whereas constructs containing 400 base pairs or fewer of the glycine-rich protein promoter were largely inactive.

Application of biotechnology to the cultivated mushroom, Agaricus bisporus, has been hampered thus far by the lack of a transformation system. Here, transformation of both a homo- and a heterokaryotic strain of A. bisporus to hygromycin B resistance is described. Transforming DNA was integrated into the A. bisporus genome and stably maintained throughout vegetative growth. Transformants of the heterokaryotic strain formed transgenic fruiting bodies. Promoters derived from the unrelated ascomycete Aspergillus nidulans and from A. bisporus itself, were able to drive expression of the hygromycin B resistance gene. Expression controlled by a fragment of 265 bp from the A. bisporus GPD promoter was sufficient to generate transformants. However, transformation efficiency was not enhanced by using this homologous promoter.

Infection of tobacco by tobacco mosaic virus (TMV) induces coordinate expression of genes encoding acidic and basic beta-1,3-glucanase isoforms. These genes are differentially expressed in response to other treatments. Salicylate treatment induces acidic glucanase mRNA to a higher level than basic glucanase mRNA. Ethylene treatment and wounding strongly induce the basic glucanase genes but have little effect on genes encoding the acidic isoforms. Furthermore, the basic glucanase genes are constitutively expressed in roots and lower leaves of healthy plants, whereas the acidic glucanase genes are not. In order to investigate how these expression patterns are established, we fused promoter regions of an acidic and a basic glucanase gene to the beta-glucuronidase (GUS) reporter gene and examined expression of these constructs in transgenic tobacco plants. A fragment of 1750 bp and two 5'-truncated fragments of 650 bp and 300 bp of the acidic glucanase promoter were tested for induction of GUS gene expression after salicylate treatment and TMV infection. Upstream sequences of 1750 bp and 650 bp were sufficient for induction of the reporter gene by salicylate treatment and TMV infection, but the activity of the 300 bp fragment was strongly reduced. The results suggest that the 1750 bp upstream sequence of the acidic glucanase gene contains multiple regulatory elements. For the basic glucanase promoter it is shown that 1476 bp of upstream sequences were able to drive expression in response to TMV infection and ethylene treatment, but no response was found to incision wounding. Furthermore, high GUS activity was found in lower leaves and roots of healthy transgenic plants, carrying the 1476 bp basic glucanase promoter/GUS construct. When the promoter was truncated up to position -446 all activity was lost, indicating that the region between -1476 and -446 of the basic glucanase promoter is necessary for organ-specific and developmentally regulated expression as well as for induced expression in response to infection and other stress treatments.

SummaryExpression of the tobacco PR-la gene is induced by treatment of plants with salicylate or infection with tobacco mosaic virus (TMV). Cis-acting sequences involved in regulation of the PR-la gene by salicylate and TMV were identified by investigating fusion constructs of PR-la upstream sequences with a heterologous minimal promoter and the GUSreporter gene in transformed tobacco plants. The PR-la promoter was found to contain a number of interacting elements which function in a context-dependent way. A minimum of four regulatory elements was identified, located between nucleotides -902 to -691 (element l ) , -689 to -643 (element 2), -643 to -287 (element 3) and -287 to +29 (element 4). Alone, these elements have no promoter activity and they are all four required for maximum induction of the reporter gene by salicylate or TMV. The P R -l a promoter is positively regulated by elements 1 , 2, and 3 ; constructs containing two of these three elements had about half of the maximum activity. Replacement of the minimal heterologous promoter by element 4 differentially affected the activity of these various constructs, suggesting that this element may be important for a correct spacing between elements 1 to 3 and the transcription start site. The observation that all PR-la promoter constructs respond similarly to salicylate treatment and TMV infection supports a role of salicylate in the signal transduction pathway leading to P R -l a gene expression.

Two tobacco genes encoding thaumatin-like proteins were cloned and sequenced. Both genes are expressed after infection of tobacco with tobacco mosaic virus (TMV). Comparison of the upstream sequences of these genes with those of other TMV-inducible tobacco genes revealed limited regions of homology.