Protein encoded by oncogene 6b from Agrobacterium tumefaciens has a reprogramming potential and histone chaperone-like activity.

Crown gall tumors are formed mainly by actions of a group of genes in the T-DNA that is transferred from Agrobacterium tumefaciens and integrated into the nuclear DNA of host plants. These genes encode enzymes for biosynthesis of auxin and cytokinin in plant cells. Gene 6b in the T-DNA affects tumor morphology and this gene alone is able to induce small tumors on certain plant species. In addition, unorganized calli are induced from leaf disks of tobacco that are incubated on phytohormone-free media; shooty teratomas, and morphologically abnormal plants, which might be due to enhanced competence of cell division and meristematic states, are regenerated from the calli. Thus, the 6b gene appears to stimulate a reprogramming process in plants. To uncover mechanisms behind this process, various approaches including the yeast-two-hybrid system have been exploited and histone H3 was identified as one of the proteins that interact with 6b. It has been also demonstrated that 6b acts as a histone H3 chaperon in vitro and affects the expression of various genes related to cell division competence and the maintenance of meristematic states. We discuss current views on a role of 6b protein in tumorigenesis and reprogramming in plants.

Interaction between Agrobacterium tumefaciens oncoprotein 6b and a tobacco nucleolar protein that is homologous to TNP1 encoded by a transposable element of Antirrhinum majus.

When gene 6b on the T-DNA of Agrobacterium tumefaciens is transferred to plant cells, its expression causes plant hormone-independent division of cells in in vitro culture and abnormal cell growth, which induces various morphological defects in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b localizes to the nuclei, a requirement for the abnormal cell growth, and binds to a tobacco nuclear protein called NtSIP1 and histone H3. In addition, 6b has histone chaperone-like activity in vitro and affects the expression of various plant genes, including cell division-related genes and meristem-related class 1 KNOX homeobox genes, in transgenic Arabidopsis. Here, we report that 6b binds to a newly identified protein NtSIP2, whose amino acid sequence is predicted to be 30% identical and 51% similar to that of the TNP1 protein encoded by the transposon Tam1 of Antirrhinum majus. Immunolocalization analysis using anti-T7 antibodies showed nucleolar localization of most of the T7 epitope-tagged NtSIP2 proteins. A similar analysis with the T7-tagged 6b protein also showed subnucleolar as well as nuclear localization of the 6b protein. These results suggest the involvement of 6b along with NtSIP2 in certain molecular processes in the nucleolus as well as the nucleoplasm.

An oncoprotein from the plant pathogen agrobacterium has histone chaperone-like activity.

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.

Histone deacetylases and ASYMMETRIC LEAVES2 are involved in the establishment of polarity in leaves of Arabidopsis.

We show that two Arabidopsis thaliana genes for histone deacetylases (HDACs), HDT1/HD2A and HDT2/HD2B, are required to establish leaf polarity in the presence of mutant ASYMMETRIC LEAVES2 (AS2) or AS1. Treatment of as1 or as2 plants with inhibitors of HDACs resulted in abaxialized filamentous leaves and aberrant distribution of microRNA165 and/or microRNA166 (miR165/166) in leaves. Knockdown mutations of these two HDACs by RNA interference resulted in phenotypes like those observed in the as2 background. Nuclear localization of overproduced AS2 resulted in decreased levels of mature miR165/166 in leaves. This abnormality was abolished by HDAC inhibitors, suggesting that HDACs are required for AS2 action. A loss-of-function mutation in HASTY, encoding a positive regulator of miRNA levels, and a gain-of-function mutation in PHABULOSA, encoding a determinant of adaxialization, suppressed the generation of abaxialized filamentous leaves by inhibition of HDACs in the as1 or as2 background. AS2 and AS1 were colocalized in subnuclear bodies adjacent to the nucleolus where HDT1/HD2A and HDT2/HD2B were also found. Our results suggest that these HDACs and both AS2 and AS1 act independently to control levels and/or patterns of miR165/166 distribution and the development of adaxial-abaxial leaf polarity and that there may be interactions between HDACs and AS2 (AS1) in the generation of those miRNAs.

Oncogene 6b from Agrobacterium tumefaciens induces abaxial cell division at late stages of leaf development and modifies vascular development in petioles.

The 6b gene in the T-DNA region of the Ti plasmids of Agrobacterium tumefaciens and A. vitis is able to generate shooty calli in phytohormone-free culture of leaf sections of tobacco transformed with 6b. In the present study, we report characteristic morphological abnormalities of the leaves of transgenic tobacco and Arabidopsis that express 6b from pTiAKE10 (AK-6b), and altered expression of genes related to cell division and meristem formation in the transgenic plants. Cotyledons and leaves of both transgenic tobacco and Arabidopsis exhibited various abnormalities including upward curling of leaf blades, and transgenic tobacco leaves produced leaf-like outgrowths from the abaxial side. Transcripts of some class 1 KNOX homeobox genes, which are thought to be related to meristem functions, and cell cycle regulating genes were ectopically accumulated in mature leaves. M phase-specific genes were also ectopically expressed at the abaxial sides of mature leaves. These results suggest that the AK-6b gene stimulates the cellular potential for division and meristematic functions preferentially in the abaxial side of leaves and that the leaf phenotypes generated by AK-6b are at least in part due to such biased cell division during polar development of leaves. The results of the present experiments with a fusion gene between the AK-6b gene and the glucocorticoid receptor gene showed that nuclear import of the AK-6b protein was essential for upward curling of leaves and hormone-free callus formation, suggesting a role for AK-6b in nuclear events.

The protein encoded by oncogene 6b from Agrobacterium tumefaciens interacts with a nuclear protein of tobacco.

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.