Hiroetsu Wabiko

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.

Exogenous Phytohormone-Independent Growth and Regeneration of Tobacco Plants Transgenic for the 6b Gene of Agrobacterium tumefaciens AKE10

The 6b gene of Agrobacterium tumefaciens AKE10 (AK-6b) induces crown gall tumors on certain plants but so far there have been no reports of the gene being able to induce tumors on culture medium, We cloned T-DNA segments containing the 6b gene but lacking the auxin and cytokinin biosynthesis genes from A. tumefaciens AKE10. Tobacco (Nicotiana tabacum) leaf discs infected with A. tumefaciens LBA4404 carrying the clones produced shooty calli on hormone-free Murashige-Skoog medium. The relevant T-DNA segment was integrated into plant DNA as determined by Southern hybridization. Some of these immature shoots spontaneously developed into mature shoots, of which several leaves displayed morphological abnormalities. When leaf discs of these mature plants were placed onto the same medium numerous shoots developed from the wounding sites, indicating that the transgenic plants possessed a high regenerative potential. Northern blot and reverse transcriptase-polymerase chain reaction analyses showed a large accumulation of the AK-6b transcripts in the shooty calli, but only a limited degree in mature plants, demonstrating that AK-6b expression is regulated in plants and essential for the early stages of regeneration. Cytokinin levels in the shooty calli were comparable to those in normal shoots, suggesting that shoot regeneration is not mediated by the modulation of cytokinin content.

Analysis of core genes supports the reclassification of strains Agrobacterium radiobacter K84 and Agrobacterium tumefaciens AKE10 into the species Rhizobium rhizogenes

Some strains of the former genus Agrobacterium have high biotechnological interest and are currently misclassified. Consequently, in this study, the taxonomic status of the non-pathogenic strain Agrobacterium radiobacter K84, used in biological control, and the tumourigenic strain Agrobacterium tumefaciens AKE10, able to regenerate tobacco transgenic plants, was revised. The phylogenetic analysis of the chromosomal genes rrs, atpD and recA showed that they should be reclassified into Rhizobium rhizogenes. The analysis of virulence genes located in the Ti plasmid (pTi) outside T-DNA showed a common phylogenetic origin among strains AKE10, R. rhizogenes 163C and A. tumefaciens (currently R. radiobacter) C58. However, the genes located inside the T-DNA, mainly the 6b gene, of strain AKE10 were phylogenetically close to those of strain 163C but divergent from those of strain C58. Furthermore, the T-DNA of tumourigenic strains from R. rhizogenes conferred on them the ability to regenerate tumour tissue resembling fasciation in tobacco plants. These results showed the existence of a highly mosaic genetic organization in tumourigenic strains of the genus Rhizobium and provided evidence of the involvement of T-DNA from tumourigenic strains of R. rhizogenes in fasciation of Nicotiana leaves. The data further suggested that pathogenic strains of Rhizobium could be good models to analyse bacterial evolution.

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.

Reduction of polar auxin transport in tobacco by the tumorigenic Agrobacterium tumefaciens AK-6b gene

The plant-tumorigenic 6b (AK-6b) gene of Agrobacterium tumefaciens strain AKE10 induces morphological alterations to tobacco plants, Nicotiana tabacum. To investigate the molecular mechanisms underlying these processes, we generated transgenic tobacco harboring the AK-6b gene under the control of a dexamethazone-inducible promoter. Upon induction, transgenic tobacco seedlings exhibited distinct classes of aberrant morphologies, most notably adventitious outgrowths and stunted epicotyls. Histological analysis revealed massive proliferation and altered venation in the newly established outgrowths. Prominent vascular development suggested that auxin metabolism or signaling had been altered. Indeed, basipetal auxin transport in the hypocotyls of the transgenic seedlings was reduced by 50–80%, whereas intracellular auxin contents were only slightly reduced. Analysis of cell extracts by HPLC revealed a large accumulation of phenolic compounds, including the flavonoid kaempferol-3-rutinoside, in transgenic plants compared with wild-type seedlings. As some naturally occurring flavonoids have been shown to affect auxin transport, we suggest that the AK-6b gene expression impairs auxin transport via modulation of phenylpropanoid metabolism, and ultimately results in the observed morphological alterations.

The Agrobacterium tumefaciens C58-6b gene confers resistance to N 6 -benzyladenine without modifying cytokinin metabolism in tobacco seedlings

Abstract. The 6b gene of Agrobacterium tumefaciens has been demonstrated to modify the activity of the plant growth regulators, auxin and cytokinin. To study the possible mode of action of the gene, tobacco (Nicotiana tabacum L. cv. Samsun) plants were transformed with the A. tumefaciens C58-6b gene. Seeds obtained from morphologically normal transgenic as well as wild-type plants were germinated on media supplemented with growth-inhibitory levels of cytokinin, N6-benzyladenine (BA). The transgenic seedlings showed increased resistance to cytokinins, as reflected by continuous shoot development, whereas further growth of the wild-type plants beyond the cotyledonary stage was inhibited. Concurrently, the levels of 6b gene transcripts in transgenic seedlings increased greatly upon BA treatment. Since glucosylation of BA represents the main inactivation mechanism of the hormone, we analyzed BA glucoside formation during the early stages of seedling growth. Intracellular levels of the major BA metabolite, N6-benzyladenine-7-glucoside (80–92%), as well as other BA-derived components were found to be comparable in transgenic and wild-type seedlings. Therefore, increased resistance of the C58-6b transgenic seedlings to cytokinins could not be directly attributed to enhanced BA glucosylation and subsequent hormone inactivation.

Collapsed abnormal pollen1 gene encoding the Arabinokinase-like protein is involved in pollen development in rice.

Pollen-expressed arabinokinase-like protein gene CAP1 is important for rice pollen development, and its related genes are conserved in both monocotyledonous and dicotyledonous plants. We isolated a pollen-defective mutant, collapsed abnormal pollen1 (cap1), from Tos17 insertional mutant lines of rice (Oryza sativa). The cap1 heterozygous plant produced equal numbers of normal and collapsed abnormal grains. The abnormal pollen grains lacked almost all cytoplasmic materials, nuclei, and intine cell walls and did not germinate. Genetic analysis of crosses revealed that the cap1 mutation did not affect female reproduction or vegetative growth. CAP1 encodes a protein consisting of 996 amino acids that showed high similarity to Arabidopsis (Arabidopsis thaliana) l-arabinokinase, which catalyzes the conversion of l-arabinose to l-arabinose 1-phosphate. A wild-type genomic DNA segment containing CAP1 restored mutants to normal pollen grains. During rice pollen development, CAP1 was preferentially expressed in anthers at the bicellular pollen stage, and the effects of the cap1 mutation were mainly detected at this stage. Based on the metabolic pathway of l-arabinose, cap1 pollen phenotype may have been caused by toxic accumulation of l-arabinose or by inhibition of cell wall metabolism due to the lack of UDP-l-arabinose derived from l-arabinose 1-phosphate. The expression pattern of CAP1 was very similar to that of another Arabidopsis homolog that showed 71% amino acid identity with CAP1. Our results suggested that CAP1 and related genes are critical for pollen development in both monocotyledonous and dicotyledonous plants.

Agrobacterium tumefaciens AKE10-mediated transformation of an Asian pea pear, Pyrus betulaefolia Bunge: host specificity of bacterial strains

Abstract. The Asian pea pear, Pyrusbetulaefolia Bunge, is tolerant to several disorders in the fruit bodies caused by high humidity and dryness and is hence widely used as a rootstock for many pear plants suitable for food sources. We have now successfully transformed P. betulaefolia Bunge by an Agrobacterium-mediated gene transfer system. Among several wild-type A. tumefaciens strains examined, only AKE10 induced shoot-forming tumors at a high frequency on excised cotyledons of P. betulaefolia Bunge cultured on phytohormone-free medium. Both the nptII (kanamycin resistance) and GUS (β-glucuronidase) genes were introduced into the cotyledons by infection with AKE10 harboring a binary vector, and regenerated plants were obtained. Southern hybridization and polymerase chain reaction analyses and histochemical GUS assay indicated that morphologically normal transformed plants faithfully contained genes from the vector but not from wild-type oncogenic T-DNA. However, morphologically abnormal plants additionally possessed the 6b gene (AK-6b) of AKE10. These results show that non-disarmed A. tumefaciens is adequate to transfer genes to the Asian pea pear, P. betulaefolia Bunge.

Ectopic localization of auxin and cytokinin in tobacco seedlings by the plant-oncogenic AK-6b gene of Agrobacterium tumefaciens AKE10.

Abstract The oncogenic 6b gene of Agrobacterium tumefaciens induces a number of morphological and metabolic alterations in plants. Although molecular functions associated with the 6b genes have been proposed, including auxin transport, sugar transport, transcriptional regulation, and miRNA metabolism, so far an unequivocal conclusion has not been obtained. We investigated the association between auxin accumulation and tumor development of the tobacco seedlings expressing the AK-6b gene under the control of the dexamethasone-inducible promoter. Indole-3-acetic acid (IAA) localization was examined by immunochemical staining with monoclonal antibody against IAA and by histochemical analysis using the IAA-specific induced construct, DR5::GUS (β-glucuronidase). Both procedures indicated that IAA preferentially accumulated in the tumorous protrusions as well as in newly developing vascular bundles in the tumors. Furthermore, true leaves also showed abaxial IAA localization, leading to altered leaves in which the adaxial and abaxial identities were no longer evident. Co-localization of cytokinin and auxin in the abaxial tumors was verified by immunochemical staining with an antibody against cytokinin. Treatment of AK-6b-seedlings with N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport, promoted the morphological severity of phenotypes, whereas 1-naphthoxyacetic acid, a specific auxin influx carrier inhibitor, induced tumor regression on cotyledons and new tumorous proliferations on hypocotyls. Prominent accumulation of both auxin and cytokinin was observed in both regressed and newly developing tumors. We suggest from these results that modulation of auxin/cytokinin localization as a result of AK-6b gene expression is responsible for the tumorous proliferation.

Modulation of the venation pattern of cotyledons of transgenic tobacco for the tumorigenic 6b gene of Agrobacterium tumefaciens AKE10

Neoplastic plant-tissue formation, termed crown gall disease, is induced on infection with Agrobacterium tumefaciens. The tumorous tissues develop an extensive vascular system, with a venation pattern distinct from that of native host plants. We report here that the plant-tumorigenic 6b gene of the A. tumefaciens strain AKE10 is capable of inducing extensive vein formation in transgenic tobacco seedlings with distinct pattern formation. Unlike the wild-type cotyledons, transgenic cotyledons had wavy and striate veins depending on the extent of severity of leaf morphology. Graph analysis of the transgenic cotyledonous vein patterns revealed an increase in the number of branch points of veins, end-points of veins, and areas surrounded by the veins. Histological analysis showed abnormal tissue growth on the abaxial side of the cotyledon blades and continual formation of adventitious veins. These adventitiously formed veins included inverted dorso-ventrality and formation of a radial axis.