Léon Otten

Agrobacterium tumefaciens 6bgenes are strain-specific and affect the activity of auxin as well as cytokinin genes

SummaryThe T-region located 6b gene of Agrobacterium tumefaciens has been found to interfere with cytokinin effects produced by the cotransferred ipt gene. We have compared the biological activity of three different 6b genes: A-6b from Ach5 (octopine, biotype 1), C-6b from C58 (nopaline, biotype 1) and T-6b from Tm4 (octopine, biotype III) by using different biological assays. Each 6b gene was inserted into a disarmed vector and tested on tobacco stems in coinfection experiments with the Ach5 cytokinin (ipt) gene (A-ipt). A-ipt/C-6b coinfections produced tumours with shoots, A-ipt/A-6b coinfections green tumours and A-ipt/T-6b coinfections tumours with a necrotic surface. The tumour phenotypes obtained were independent of the 6b/A-ipt coinfection ratios, indicating that the strain-specific 6b effects result from the activity of a non-diffusible 6b encoded product. Studies with ipt-less Tm4 mutants showed that 6b genes affect other tumour genes besides the ipt gene and pointed to an influence of T-6b on auxin effects resulting from the Tm4 iaa system. T-iaa/T-6b coinfection experiments showed that T-6b did indeed strongly increase tumour formation by the Tm4 iaa genes. The three 6b genes also have effects which do not require other T-region genes. The complex role of the 6b gene in crown gall induction and Agrobacterium host range will be discussed.

Agrobacterium tumefaciens T-DNA gene 6b stimulates rol-induced root formation, permits growth at high auxin concentrations and increases root size

SummaryAll Agrobacterium tumefaciens strains studied up to now transfer an active 6b gene to plant cells. However, the role of this gene in natural tumour induction is unknown. Various effects of 6b on plant cell growth have been described, but the precise mechanism by which 6b causes these effects has not been elucidated. Earlier experiments indicated that the 6b gene might increase auxin sensitivity as do the A. rhizogenes rol genes. The 6b gene from Tm4 (T-6b) was therefore compared with the rolB and rolABC genes. Although T-6b was unable to induce root formation, it strongly interfered with root induction and root elongation. In rolABC/T-6b coinfection experiments on carrots, T-6b-transformed cells stimulated root outgrowth of rolABC-transformed cells, indicating that the biologically active T-6b product is diffusible. Carrot rolABC roots containing the T6b gene rapidly developed into unorganized calli. Nicotiana rustica roots with rolABC and T-6b continued their development, but became very large. Fragments of such roots formed callus at α-naphthaleneacetic acid concentrations which inhibited growth of rolABC and normal root fragments, suggesting that the role of 6b genes in natural tumour induction may be to reduce the inhibitory effects of high auxin levels and to keep cells in an undifferentiated state.

Expression of a chimaeric heat-shock-inducible Agrobacterium 6b oncogene in Nicotiana rustica

The T-6b gene of Agrobacterium tumefaciens strain Tm4 induces tumours on Nicotiana rustica by an as yet unknown mechanism. These tumours cannot be regenerated into normal plants. To study the effect of the T-6b gene product on normal plant cells, the T-6b gene was placed under control of the Drosophila melanogaster hsp70 heat-shock promoter and introduced into N. rustica. Progeny of an hsp70-T-6b transformant developed into normal plants. The inducibility of the hsp70-T-6b construct was shown by northern analysis and by heat-shock-dependent growth alterations on the level of whole seedlings. Upon wounding at normal temperature conditions hsp70-T-6b plants formed small tumours on leaves and stems. Grafts between transformed plants and normal plants led to a wound callus which remained limited to transformed tissues, indicating that the T-6b gene product does not diffuse. Protoplasts of hsp70-T-6b plants divided in the same way as control protoplasts under standard culture conditions. However, when protoplast cultures were started in the absence of hormones, normal cells rapidly lost their sensitivity towards hormones, whereas hsp70-T-6b cells remained sensitive for a significantly longer period. Thus, the T-6b gene product alters hormone sensitivity during the initial phases of protoplast culture.

Nucleotide sequence, evolutionary origin and biological role of a rearranged cytokinin gene isolated from a wide host range biotype III Agrobacterium strain

SummaryA DNA fragment with homology to the cytokinin (ipt) gene from biotype I Agrobacterium tumefaciens strain Ach5 was cloned from the Ti plasmid of the wide host range biotype III Agrobacterium strain Tm-4 and sequenced. The fragment contains an intact ipt coding sequence. However, the 3′ non-coding region of this ipt gene is rearranged due to a 0.9 kb deletion fusing it to the 3′ coding region of the neighbouring gene 6a, most of which was found to be deleted. The Tm-4 ipt gene is strongly related to the partially deleted ipt gene of the limited host range biotype III strain Ag162. To test its biological activity, the Tm-4 ipt gene was inserted into a specially constructed, disarmed Ti vector lacking tzs and tested on tobacco, where the rearranged ipt gene induced shoot formation. The cloned Tm-4 ipt gene was mutated with Tn5 and the intact gene on the wild-type Tm-4 Ti plasmid was replaced by the mutated gene. The resulting strain was avirulent on tobacco but normally virulent on the natural host of the wild-type strain Tm-4, grapevine. As the biotype 1 6b gene diminishes the effect of a corresponding ipt gene, a larger Tm-4 fragment carrying both the ipt gene and an adjacent 6b-like gene was also tested on tobacco and compared with the Tm-4 ipt fragment alone and with an ipt and 6b/ipt fragment derived from Ach5. The Tm-4 6b gene diminishes the effect of the Tm-4 ipt gene, showing the Tm-4 6b gene to be active as well. The Tm-4 6b/ipt combination is less effective than the Ach5 combination. These results provide further insight into the molecular basis of the host range differences between limited host range and wide host range biotype III Agrobacterium strains and show that the WHR cytokinin gene, although active, does not significantly contribute to tumour formation on the natural host of the WHR biotype III strains, grapevine.

Organization and functional analysis of three T-DNAs from the vitopine Ti plasmid pTiS4.

SummaryThe vitopine Ti plasmid pTiS4 of Agrobacterium vitis has an unusual T-DNA organization. The pTiS4 oncogenes, localized by screening selected pTiS4 clones for growth-inducing activity, are localized on three T-DNAs, whereas in all other characterized Ti plasmids one or two T-DNAs are found. The nucleotide sequences and predicted amino acid sequences of the pTiS4 oncogenes set them apart from the corresponding genes from other Ti or Ri plasmids. The oncogenes induce the same type of reaction on various test plants as the well-known pTiAch5 oncogenes but the pTiS4 ipt gene induces considerably more shoots than its Ach5 homologue. We have also identified the gene coding for vitopine synthase as well as a vitopine synthase pseudogene. Both sequences show homology to the octopine synthase gene. In terms of both nucleotide sequence and overall organization, the pTiS4 T-DNAs appear to be only distantly related to previously characterized T-DNAs.

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.

Distribution of two Agrobacterium tumefaciens insertion elements in natural isolates: Evidence for stable association between Ti plasmids and their bacterial hosts

SummaryA large number of Agrobacterium tumefaciens strains of the biotype III group carry two to ten copies of two related IS elements, IS866 and IS867. A study of the distribution and localization of these elements in 54 strains showed that one IS866 and two IS867 copies are always found at characteristic sites on the octopine/cucumopine and vitopine Ti plasmids, whereas varying amounts of IS866 and IS867 copies occur at different positions on the chromosome. By comparison of the IS patterns, an evolutionary tree could be deduced which shows the phylogenetic relationships between 23 different types of Agrobacterium strains. The structures of the T-regions of the different strains were also compared. Within the octopine/cucumopine group, eight T-region patterns could be defined. These patterns were found to be correlated with the chromosomal IS patterns. This strongly suggests that the IS866 and IS867 containing Ti plasmids are stably associated with their bacterial hosts. The possible role of the IS866 and IS867 elements in Ti plasmid evolution is discussed.

Biological Activity of the Agrobacterium rhizogenes–Derived trolC Gene of Nicotiana tabacum and Its Functional Relation to Other plast Genes

Agrobacterium rhizogenes induces hairy roots through the activity of three essential T-DNA genes, rolA, rolB, and rolC, whereas the orf13 gene acts as an accessory root-inducing gene. rolB, rolC, and orf13 belong to the highly diverged plast gene family with remotely related representatives in the endomycorrhizal basidiomycete Laccaria bicolor. Nicotiana glauca and N. tabacum contain A. rhizogenes-derived T-DNAs with active plast genes. Here, we report on the properties of a rolC homolog in N. tabacum, trolC. Dexamethasone-inducible trolC and A4-rolC genes from A. rhizogenes A4 induce comparable, strong growth effects affecting all parts of the plants. Several have not been described earlier and were found to be very similar to the effects of the distantly related plast gene 6b. They include leaf chlorosis and starch accumulation, enations, increase of sucrose-dependent leaf disk expansion, growth of isolated roots on low-sucrose media, and stimulation of sucrose uptake by small root fragments. Collectively, our findings indicate that enhancement of sucrose uptake plays an important role in generating the complex 6b and rolC phenotypes and might be an ancestral property of the plast genes.

The Agrobacterium oncogene AB-6b causes a graft-transmissible enation syndrome in tobacco.

Agrobacterium 6b oncogenes induce tumours and modify plant growth in various ways. Here we show that the AB-6b gene from strain AB4 placed under 2x35S promoter control (2x35S-AB-6b) induces a complex enation syndrome in transgenic Nicotiana tabacum plants, that also occurs in a few rare cases of genetic enations. In Arabidopsis thaliana, 2x35S-AB-6b induced radially symmetrical tubes on the abaxial side of the leaves, which must therefore be considered as the Arabidopsis equivalents of enations on other plant species. Tobacco and Arabidopsis 2x35S-AB-6b leaves contained small, supernumerary densely packed cells between the spongy mesophyll and the abaxial epidermis, close to vascular strands arising at an early stage of leaf development. On tobacco, the 2x35S-AB-6b enation syndrome could be transmitted across graft junctions to growing tissues of untransformed plants, both acropetally and basipetally. We propose that the AB-6b gene encodes the synthesis of one or more enation factor(s) that are transported by the phloem and modify the growth of developing tissues.

Sequence and distribution of IS866, a novel T region-associated insertion sequence from Agrobacterium tumefaciens

We have identified a new insertion sequence, IS866, located in the auxin synthesis gene TA iaaH of Tm4, a wide host range biotype III octopine/cucumopine type Agrobacterium tumefaciens strain with two T regions on its tumor-inducing (Ti) plasmid, TA, and TB. IS866 is 2716 bp long, has inverted repeats of 27 bp with three mismatches, and generates 8-bp direct repeats upon integration. In addition to IS866, pTiTm4 carries two copies of a related element, IS867, associated with TA and TB, respectively. A systematic study of 92 virulent Agrobacterium strains has shown that among the three biotypes all octopine/cucumopine and vitopine biotype III isolates contain IS866-like elements. The various octopine/cucumopine Ti plasmids always carry IS866 and IS867 at the same position as in pTiTm4. The chromosomes of the bacteria which contain these Ti plasmids also carry IS866 and IS867 copies but in varying numbers and locations.