By utilizing a homologous transient expression system, we have demonstrated that the Opaque-2 (O2) gene product O2 confers positive trans-regulation on a 22-kD zein promoter. This trans-acting function of the O2 protein is mediated by its sequence-specific binding to a cis element (the O2 target site) present in the 22-kD zein promoter. A multimer of a 32-bp promoter fragment containing this O2 target site confers transactivation by O2. A single nucleotide substitution in the O2 target sequence not only abolishes O2 binding in vitro, but also its response to transactivation by O2 in vivo. We have also demonstrated that an amino acid domain including the contiguous basic region and the heptameric leucine repeat is essential for the trans-acting function of the O2 protein. Similar but not identical O2 target sequence motifs can be found in the promoters of zein genes of different molecular weight classes. Conversion of such a motif in the 27-kD zein promoter to an exact O2 target sequence by site-directed mutagenesis was sufficient to increase the binding affinity of the O2 protein in vitro and to confer transactivation by O2 in vivo.
Polyclonal antibodies to human (3-interferon reacted specifically with two plant proteins (gp22 and gp35) by Western blot analysis of crude protein extracts from tobacco leaves infected with tobacco mosaic virus. Immunoaffinity chromatography of these extracts on a column of immobilized monoclonal antibodies to human f-interferon and then reversed-phase HPLC yielded gp22 and gp35 in a pure state. Both proteins reacted with the Schiff reagent and concanavalin A (indicating their glycoprotein nature) and exhibited antiviral activity (inhibiting tobacco mosaic virus replication in tobaccoleaf discs at concentrations of ng/ml). Each protein was cleaved by cyanogen bromide and the resultant peptides, separated by HPLC, were sequenced as far as the Edman degradation allowed, giving a total of 61 amino acid residues for gp22 and 105 residues for gp35, which represent 30-50% of their expected length. Computer analyses of the sequenced segments revealed no significant homology to human (-interferon In this study we report the purification to homogeneity and the partial sequencing of two virus-stimulated antiviral tobacco proteins (AVFs, gp22 and gp35). Immunoaffinity chromatography on an immobilized monoclonal antibody to human IFN-/ was a key step in this purification. The analogy of these plant proteins to the human IFN system is discussed. MATERIALS AND METHODSPlants. The antiviral plant proteins were isolated from the tobacco (Nicotiana tabacum) cultivar "Samsun NN," carrying the N gene, which determines the localization of TMV infection.Preparation of Crude Plant Proteins. Tobacco leaves were inoculated with TMV and, after 48 hr, leaves were homogenized in 10 mM sodium phosphate (pH 7.6) and treated with hydrated calcium phosphate, as described by Sela (4). This treatment removed about 85% of the proteins as well as TMV, leaving a noninfectious antiviral fraction. Prior to large-scale immunoaffinity purification, this fraction was dialyzed against water and freeze-dried. In Western blottings of the crude preparation, the calcium phosphate step was omitted.Immunoaffinity tTo whom reprint requests should be addressed. 588The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Two antivirally active, TMV-inducible, tobacco proteins (gp22 and gp35) were recently isolated by immunoaffinity chromatography using monoclonal antibodies to human interferon-fl [2]. The proteins were characterized by determining the partial amino acid sequence of the amino termini of the proteins and of several cyanogen bromidederived peptides. At the time of characterization, these proteins were found to be unique and not homologous to known protein sequences on available databases. However, in a recent search of protein sequence information from our own database on TMV-inducible proteins (J. Ryals et al., unpublished database) and from the GenBank and EMBL databases, we have found that gp35 corresponds to the basic, vacuolar form of fl-l,3-glucanase from tobacco [6] and gp22 is closely related to both the acidic and basic forms ofpathogenesis-related protein 5 (i.e. PR-5), from tobacco [1,3,4,7].The alignment of the gp35 sequences with the amino acid sequence of the basic form of glucanase is shown in Fig. 1A. The four cyanogen bromide-derived peptide sequences match the glucanase sequence at 105 of 108 residues. Further, the molecular mass of the mature, basic glucanase is about 35000 [6] which is in close agreement to the estimated molecular mass of gp35. Therefore, we conclude that the gp35 protein is equivalent to the basic form of ~l,3-glucanase.Two of the three amino acid sequences derived from gp22 can be aligned with the amino acid sequences of the acidic and basic forms of PR-5 (Fig. 1B). The amino terminal sequence from gp22 matches the acidic form of PR-5 at 17 of 32 residues (53~o) and marches the basic form of PR-5 (e.g. osmotin) at 20 of 32 residues (63 ~o). The amino acid sequence of the peptide in Fraction 20 is identical to the acidic form of PR-5 at 5 of 19 residues (26 ~o) and identical to the basic form of PR-5 at either 8 or 9 of 18 residues (44 or 50~o). Since the gp22 protein was not reduced and alkylated prior to Edman degradation, cysteine residues would not be detected [2]. Assuming that the cysteines were missed in sequencing, the identity for the amino terminal sequence would increase to 18 of 32 (56~o) for the acidic form of PR-5 and 21 of 32 (65~o) for the basic form of PR-5. The Fraction 20 peptides would match the acidic form at 7 of 18 (39~o) and match the basic form at 10 (55~/o) or 11 (61~o) of 18
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