The 6b gene in the T-DNA from Agrobacterium has oncogenic activity in plant cells, inducing tumor formation, the phytohormone-independent division of cells, and alterations in leaf morphology. The product of the 6b gene appears to promote some aspects of the proliferation of plant cells, but the molecular mechanism of its action remains unknown. We report here that the 6b protein associates with a nuclear protein in tobacco that we have designated NtSIP1 (for Nicotiana tabacum 6b-interacting protein 1). NtSIP1 appears to be a transcription factor because its predicted amino acid sequence includes two regions that resemble a nuclear localization signal and a putative DNA binding motif, which is similar in terms of amino acid sequence to the triple helix motif of rice transcription factor GT-2. Expression in tobacco cells of a fusion protein composed of the DNA binding domain of the yeast GAL4 protein and the 6b protein activated the transcription of a reporter gene that was under the control of a chimeric promoter that included the GAL4 upstream activating sequence and the 35S minimal promoter of Cauliflower mosaic virus . Furthermore, nuclear localization of green fluorescent protein-fused 6b protein was enhanced by NtSIP1. A cluster of acidic residues in the 6b protein appeared to be essential for nuclear localization and for transactivation as well as for the hormone-independent growth of tobacco cells. Thus, it seems possible that the 6b protein might function in the proliferation of plant cells, at least in part, through an association with NtSIP1. INTRODUCTIONAgrobacterium cells that harbor a Ti plasmid induce the formation of crown gall tumors on dicotyledonous plants. Upon infection of a plant by Agrobacterium, a specific region of the Ti plasmid, known as T-DNA, is transferred to the plant cell and integrated into the chromosomal DNA in the nucleus. Plant cells that have been transformed with T-DNA can proliferate autonomously to generate a tumor, which is a consequence, for the most part, of the expression of genes that are responsible for the biosynthesis of auxin; and cytokinin ( iaaM [ tms1 ] or iaaH [ tms2 ] for auxin; ipt [ tmr ] for cytokinin) in the T-DNA (Weiler and Spanier, 1981; Akiyoshi et al., 1984).In addition to these genes, gene 6b , which is localized at the tml locus (Garfinkel et al., 1981) and has been found in the T-DNA of all strains of Agrobacterium (Willmitzer et al., 1983;Otten and De Ruffray, 1994), also is expressed in tumor cells (Willmitzer et al., 1983) and appears to play a role in the proliferation of plant cells. Various phenotypic effects associated with the expression of 6b have been reported, as follows: (1) formation of tumors on certain plants (Hooykaas et al., 1988;Spanier et al., 1989;Tinland et al., 1989Tinland et al., , 1992; (2) stimulation of the ipt -induced and iaaM / iaaH -induced division of cells (Tinland et al., 1989(Tinland et al., , 1990Wabiko and Minemura, 1996); (3) reduction in the formation of shoots on leaf discs that is normally induced by appr...

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Protein 6b, encoded by T-DNA from the pathogen Agrobacterium tumefaciens, stimulates the plant hormone–independent division of cells in culture in vitro and induces aberrant cell growth and the ectopic expression of various genes, including genes related to cell division and meristem-related class 1 KNOX homeobox genes, in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b is found in nuclei and binds to several plant nuclear proteins. Here, we report that 6b binds specifically to histone H3 in vitro but not to other core histones. Analysis by bimolecular fluorescence complementation revealed an interaction in vivo between 6b and histone H3. We recovered 6b from a chromatin fraction from 6b-expressing plant cells. A supercoiling assay and digestion with micrococcal nuclease indicated that 6b acts as a histone chaperone with the ability to mediate formation of nucleosomes in vitro. Mutant 6b, lacking the C-terminal region that is required for cell division–stimulating activity and interaction with histone H3, was deficient in histone chaperone activity. Our results suggest a relationship between alterations in nucleosome structure and the expression of growth-regulating genes on the one hand and the induction of aberrant cell proliferation on the other.

A 3778-bp DNA sequence of the insecticidal protoxin gene coding sequence and flanking regions from Bacillus thuringiensis subspecies berliner 1715 has been determined. The protoxin is composed of 1155 amino acids, deduced from the nucleotide sequence, and has a calculated molecular mass of 130,615 daltons. To determine the DNA portion that encodes toxicity, sequential deletions were constructed from the 3' end of the coding region using nuclease Bal-31. Using these mutants in an insect bioassay, we found that an amino-terminal 612-amino-acid peptide is toxic, whereas, a 603-amino-acid peptide is not toxic to insects. Ninety percent of the amino acid residues were homologous to the protoxins from closely related subspecies kurstaki HD-1-Dipel and sotto. The differences occurred both in the amino-terminal half, or toxic portion, and in the carboxy-terminal half. These differences were clustered in several regions. From comparative analysis of subspecies berliner and kurstaki, we propose a model whereby the protoxin molecule is divided into distinct structural and functional domains. These domains may be responsible for the differences in specific toxicities and spectra of insect host range among these subspecies.

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