NIMIN-1, NIMIN-2 and NIMIN-3, members of a novel family of proteins from Arabidopsis that interact with NPR1/NIM1, a key regulator of systemic acquired resistance in plants.

NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR) in Arabidopsis. Using the yeast two-hybrid system, we have identified three novel genes, NIMIN-1, NIMIN-2 and NIMIN-3 (NIMIN for NIM1-interacting) that encode structurally related proteins interacting physically with NPR1/NIM1. NIMIN-1 and NIMIN-2 both bind strongly to NPR1/NIM1 via a common binding motif interacting with the C-terminal moiety of NPR1/NIM1, whereas NIMIN-3 interacts with NPR1/NIM1 via the N-terminal part of NPR1/NIM1. In addition, NIMIN-1, NIMIN-2, and NIMIN-3 are able to interact via NPR1/NIM1 with basic leucine zipper transcription factors of the TGA family in a yeast tri-hybrid system. A mutant protein of NPR1/NIM1, npr1-2, which has been shown to be severely impaired in induction of SAR gene expression, failed to bind the NIMIN proteins. The NIMIN genes are expressed in Arabidopsis plants in response to SAR-inducing treatments, and the NIMIN proteins, like NPR1/NIM1, carry functional nuclear localization signals as revealed by expression of fusion proteins in yeast and in transgenic plants. Taken together, these data indicate that the NIMIN proteins, via physical interaction with NPR1/NIM1, are part of the signal transduction pathway leading to SAR gene expression in Arabidopsis.

An as-1-like motif controls the level of expression of the gene for the pathogenesis-related protein 1a from tobacco.

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.

Characterization and expression of a heptaubiquitin gene from tomato.

Ubiquitin is highly conserved 76 amino acid protein involved, among other functions, in the selective degradation of proteins in the cell. From a tomato (Lycopersicon esculentum Mill. cv. Craigella) genomic library, we have isolated a clone encoding a polyubiquitin gene, designated ubq1-1 comprising seven repeats of ubiquitin and two C-terminal extension amino acids. The ubq1-1 gene contains an intron of 1128bp immediately upstream of the translation start codon. DNA sequence comparison revealed that the 5' and 3' non-coding regions of the tomato ubq1-1 gene are nearly identical to the sequence of a polyubiquitin cDNA clone isolated from potato (Garbarino et al., 1992; Plant Mol. Biol. 20, 235-244). The ubq1-1 gene is expressed in leaves to rather low levels in tomato, and the abundance of ubq1-1 transcripts is increased under heat shock conditions. For functional analyses, a chimeric gene construct containing the intron and 1.6kb of ubq1-1 sequence 5' to the intron fused to the gus reporter gene was introduced into the tobacco genome. In leaves of transgenic tobacco plants, reporter gene expression was generally lower from the ubq1-1 promoter than from the cauliflower mosaic virus 35S RNA promoter. In addition, the tomato ubq1-1 promoter was not found to respond to heat shock in transgenic tobacco plants. Histochemical analysis of the plants demonstrated localization of gus reporter gene activity in the vascular systems of the leaves and the roots. Deletion of the intron from the reporter gene construct markedly reduced reporter gene expression in transformed tobacco plants, thus suggesting that the intron may influence transcript levels deriving from the ubq1-1 promoter.

The upstream region of the gene for the pathogenesis-related protein 1a from tobacco responds to environmental as well as to developmental signals in transgenic plants.

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-1a 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-1a 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 fulfill a role during the later stages of plant development and that the PR-1a upstream region -906 to -335 contains positive regulatory elements for both environmental and developmental signals.

Expression of a viral avirulence gene in transgenic plants is sufficient to induce the hypersensitive defense reaction.

Tobacco plants containing the N' resistance gene exhibit a hypersensitive defense reaction when infected with tomato mosaic virus (ToMV); infection results in necrotic lesions at the primary infection sites. In an attempt to investigate the molecular mechanism(s) underlying this plant-pathogen interaction, the ToMV coat protein gene was joined by a transcriptional fusion to the strong constitutive 35S RNA promoter from cauliflower mosaic virus. This chimeric gene was introduced via Agrobacterium-mediated transformation into isogenic tobacco cultivars differing only with respect to the N' gene. Strong necrotic reactions were observed on most emerging calli of the N' genotype, but never on calli lacking the N' resistance gene. These data indicate that the coat protein of ToMV is, on its own, sufficient to induce a hypersensitive reaction in tobacco. Thus, recognition of a single viral gene product may be the only prerequisite for the induction of a specific defense reaction in higher plants.

Activation of a truncated PR-1 promoter by endogenous enhancers in transgenic plants.

PR-1 genes are induced by various environmental stimuli such as pathogen attack or exposure of the plants to certain chemicals. To examine the regulation of these genes, the 5' flanking regions of the PR-la gene and of two PR-1 pseudogenes were joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene. These constructs were stably integrated into the tobacco genome and independent primary transformants were monitored for the expression of the reporter gene. Unexpectedly, out of 55 transformants analysed, four plants exhibited considerable GUS activities without any inductive treatment of the plants. Expression of the endogenous PR-1 genes, however, could not be detected in these plants. Primer extension analyses revealed correct initiation of the PR1/GUS hybrid transcripts from the PR-1a TATA box. When the plants were analysed at the cellular level, clear differences regarding the tissue specificity of expression of the reporter gene were observed. These results strongly suggest that the PR1/GUS hybrid promoter expression cassettes may be activated when integrated in the vicinity of heterologous enhancer elements dispersed in the tobacco genome. In order to support this hypothesis, domain B of the enhancer of the 35S RNA promoter from cauliflower mosaic virus (CaMV) was fused to various PR1/GUS hybrid genes upstream as well as downstream from the RNA start site. These constructs were stably introduced into the tobacco genome. In any primary transformant analysed, strong GUS activities were observed with the PR1/GUS hybrid RNAs originating from the normal transcription start site of the PR-1a gene. The tissue specificity of gene expression was identical to that described previously for the CaMV 35S domain B enhancer element. Thus, modulations of the transcriptional activity of the PR-1 promoter can be achieved by heterologous enhancers in transgenic plants and may be encountered upon random integration of PR-1 promoter constructs into the tobacco genome.

Functional analysis of the pathogenesis-related 1a protein gene minimal promoter region. Comparison of reporter gene expression in transient and in stable transfections.

Pathogenesis-related (PR) proteins are a heterogeneous group of host encoded, low-molecular-mass proteins that are induced in plants by various external stimuli, such as pathogen attack or exposure of the plants to certain chemicals. To examine the regulation of these genes, the 5'-flanking region of the tobacco PR-1a gene [Pfitzner U.M., Pfitzner, A.J.P. & Goodman, H.M. (1988) Mol. Gen. Genet. 211, 290-295] was joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene. Expression of the reporter gene was monitored in transient expression assays as well as in stable transformants. The PR-1a 5'-flanking sequences from -335, -149 or -71 to +28 are functional promoter elements in tobacco and carrot protoplasts, as determined by transient expression. These constructs direct correct initiation at the normal transcription-start site of the PR-1a gene. The level of gene expression was about twofold less than that obtained with the cauliflower mosaic virus 35S RNA promoter. Regulation of gene expression by acetylsalicylic acid, however, could not be detected in the transient assays. When the same constructs were stably integrated into the tobacco genome, neither constitutive nor induced beta-glucuronidase activity was observed. A comparison of the results from the transient and the stable transfection experiments suggests that expression of the reporter gene may be due to a constitutive transcriptional activity of the PR-1a 5'-flanking regions under the conditions of the transient assays and that the PR-1a promoter may contain at least two functional domains.

Molecular analysis of two PR-1 pseudogenes from tobacco.

Two independent PR-1 lambda genomic clones (W38/1 and W38/3) were isolated and characterized from a tobacco (Nicotiana tabacum cv. Wisconsin 38) library. Neither clone is identical to the previously described PR-1 cDNA clones, and both clones carry mutations within the highly conserved PR-1 protein coding region. For example, clone W38/1 has a GAA Glu codon instead of the translation stop codon thus harbouring an open reading frame extended by 16 additional amino acids. Furthermore, both clones display considerable variations in the genomic flanking sequences when compared to the PR-1a gene. In order to test whether the encoded genes are active, their upstream sequences were fused to the E. coli beta-glucuronidase (GUS) reporter gene. While significant GUS activities as compared to the 35S RNA promoter from cauliflower mosaic virus (CaMV) were obtained with the W38/1 and W38/3 sequences in transient gene expression assays, no transcriptional activities could be observed upon stable transformation of the same constructs. In addition, the protein coding region of W38/1 was joined to the CaMV 35S RNA promoter and transgenic tobacco plants were generated. However, neither transcripts nor a protein could be detected deriving from the W38/1 structural gene with this chimaeric construct in the transformants. Taken together, these data indicate that the genes contained in lambda clones W38/1 and W38/3 are not active in planta.

Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants.

Infection of the tobacco cultivar Samsun NN by tobacco mosaic virus (TMV) results in a hypersensitive response. During this defense reaction several host encoded proteins, known as pathogenesis-related proteins (PR-proteins), are induced. Poly(A)+ RNA from TMV infected tobacco plants was used to construct a cDNA library. Thirty two cDNA clones were isolated and after digestion with different restriction endonucleases, twenty clones were found to code for PR-1a, six clones for PR-1b, and four clones for PR-1c. Two independent cDNA clones of each class were further characterized by DNA sequence analysis. All clones analyzed contained the 138 amino acid coding regions of their respective mature proteins, but only partial sequences of the signal peptides. Minor differences between the nucleotide sequences for clones belonging to the same class were detected. Comparison of the amino acid sequence for PR-1a deduced from its nucleotide sequence with published data obtained by Edman degradation of the protein showed four differences. Analysis of the 3' ends of the cDNA clones indicates that various alternate poly(dA) addition sites are used. Southern blot analysis using these cDNAs as probes suggests the presence of multiple PR-protein genes in the genomes of tobacco and tomato plants.