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

Summary The 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-mediated hairy root production from seedlings of Chinese cabbage

Cruciferous hairy roots are often used for improving drought adaptability, peroxidase production, andin vitro subculturing ofPlasmodiophora brassicae. For metabolic engineering,Agrobacterium tumefaciens-mediated systems have previously been developed for hairy root production in other plant species. Here, we used therolABC gene binary construct inA. tumefaciens strain GV3101 to establish cultures of Chinese cabbage hairy roots. On both solid and liquid media, therolABC hairy root lines exhibited a wild-type hairy root syndrome in terms of their growth and morphology. This demonstrates that those three genes are sufficient to induce high-quality hairy roots in Chinese cabbage. Such a system could be useful for the stable production of secondary metabolites in that species.     

Developmental and pathogen-induced activation of an msr gene, str246C, from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5 flanking DNA sequence from the str246C gene fused to the -glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5 deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.Joint first authors     

Expression of transferred genes during hairy root development in pea

Root border cell development and expression of reporter genes were evaluated in transgenic pea hairy roots. Successful induction of hairy roots in pea is conditioned by bacterial strain and plant genotype, as well as by developmental and environmental factors. Morphological changes sometimes occur when hairy roots are transferred from infected plants to tissue culture media, but such changes are confined to specific clones. Expression of reporter genes under the control of promoters from bean (Phaseolus vulgaris L.) stress genes encoding phenylalanine ammonia lyase and chalcone synthase were evaluated. Expression patterns vary between hairy roots taken directly from infected plants, and those grown in culture; most hairy roots taken from infected plants exhibit expression throughout all tissues, whereas expression in cultured hairy roots is most often localized to specific tissues. Patterns of expression that occur during different stages of hairy root development are very similar to those observed in transgenic plants expressing the same fusion genes. Border cell separation and release in hairy roots is normal, and expression of glucuronidase in border cells of some transgenic roots resulted in development of bright blue single cells. Cultured hairy roots should provide a very useful model for studying the effect of defined changes in root border cells on microbial associations with roots of this important legume.Abbreviations YEM yeast extract-mannitol - GUS glucuronidase - PAL phenylalanine ammonium lyase - CHS chalcone syntase     

Transgenic white clover. Studies with the auxin-responsive promoter,GH3, in root gravitropism and lateral root development

We report an improved method for white clover (Trifolium repens) transformation usingAgrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. Thebar gene and phosphinothricin selection was shown to be a more effective selection system thannptII (kanamycin selection) oraadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.     

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.     

Influence of exogenous hormones on the growth and secondary metabolite formation in transformed root cultures

Transformed organ cultures formed following transformation of plant tissues with Agrobacterium species owe their phenotypes to alterations in hormone metabolism. Exogenously supplied hormones have been used to probe the relationship between the growth and morphology of transformed root cultures of a number of species and their ability to accumulate secondary products. Auxins in the presence of low levels of kinetin induce the rapid disorganisation of transformed roots of Nicotiana rustica ultimately toform suspension cultures of transformed cells and this process is associated with a decrease in nicotine content of the cells. This is related to cells in the culture losing competence in alkaloid biosynthesis. In contrast, exogenously supplied GA₃ enhanced branching in two transformed root clones of the tropane-alkaloid producing species, Brugmansia candida and so enhanced their typical hairy root phenotype. This growth substance had the effect of reducing the overall alkaloid accumulation but in one case significantly altered the relative concentrations of different tropine esters.In transformed roots of Cucumis sativus, the phenotype of the roots is influenced by the expression of auxin synthesis genes on T_R-DNA resulting in roots with two distinct morphologies. The pattern of expression of the enzyme ascorbate oxidase in populations of control roots of different morphologies is described. The significance of these phenotypic variations on the utility of transformed root cultures for the study of secondary metabolic pathways will be discussed.Abbreviations AO ascorbate oxidase - DW dry weight - FW fresh weight - GA₃ gibberellic acid     

Agrobacterium rhizogenes mediated induction of apparently untransformed roots and callus in chrysanthemum

The agropine type Agrobacterium rhizogenes strain LBA9402 induced callus and roots on stems of greenhouse grown plants and on leaf disks of in vitro grown plantlets of chrysanthemum (Dendranthema grandiflora Tzvel.). In this callus and roots no opines were detected, nor were any of the other features of the hairy root syndrome observed. Experiments aimed to identify the nature of the tumour-like growth revealed that induction was correlated with the presence of the TR-DNA on the Ri-plasmid. Root induction was probably the result of auxin synthesis following transient expression of iaaM and iaaH genes, present on the TR-DNA. The chrysanthemum cultivar used, cv. Parliament, showed a high auxin sensitivity compared to tobacco. Analysis of early transformation events using the GUSintron reporter gene revealed that low efficiency gene transfer and transient gene expression took place, but most probably without stable integration of the T-DNA in the plant genome. The results presented here stress the fact that callus formation or root induction as measures for transformation efficiency should be used with caution.     

Callus,shoot and hairy root formation in vitro as affected by the sensitivity to auxin and ethylene in tomato mutants

We analyzed the impact of ethylene and auxin disturbances on callus, shoots and Agrobacterium rhizogenes-induced hairy root formation in tomato (Solanum lycopersicum L.). The auxin low-sensitivity dgt mutation showed little hairy root initiation, whereas the ethylene low-sensitivity Nr mutation did not differ from the control Micro-Tom cultivar. Micro-Tom and dgt hairy roots containing auxin sensitivity/biosynthesis rol and aux genes formed prominent callus onto media supplemented with cytokinin. Under the same conditions, Nr hairy roots did not form callus. Double mutants combining Rg1, a mutation conferring elevated shoot formation capacity, with either dgt or Nr produced explants that formed shoots with little callus proliferation. The presence of rol + aux genes in Rg1 hairy roots prevented shoot formation. Taken together, the results suggest that although ethylene does not affect hairy root induction, as auxin does, it may be necessary for auxin-induced callus formation in tomato. Moreover, excess auxin prevents shoot formation in Rg1.     

Molecular basis for novel root phenotypes induced by Agrobacterium rhizogenes A4 on cucumber

We have used the wild-type Agrobacterium rhizogenes strain A4 to induce roots on cucumber stem explants. Cultures of transformed roots obtained that were capable of hormone-autonomous growth could be grouped in three phenotypic classes. Of particular interest were extremely thick roots of a type not previously described. Characterization of the transferred DNA and of the expression of the corresponding genes allowed us to determine that the genes rolABC of the TL region of the Ri plasmid are sufficient to induce thin roots similar to those observed in other species, while the aux genes of the TR region are sufficient to induce thick roots. Among clones bearing the aux genes, there was a correlation between level of expression of aux2 and root phenotype.     

In planta analysis of the Agrobacterium tumefaciens T-cyt gene promoter: identification of an upstream region essential for promoter activity in leaf,stem and root cells of transgenic tobacco

The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a -glucuronidase (gusA) reporter gene and introduced into tobacco plants. Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants. Developmental stage-dependent promoter activity was observed in seedlings. Analysis of 5-deleted promoter fragments showed that sequences located between positions–185 and –139 with respect to the T-cyt translational start codon are essential for T-cyt promoter activity in transfected tobacco protoplasts as well as in transformed tobacco plants.     

Effect of auxins on <Emphasis Type="Italic">Karwinskia humboldtiana</Emphasis> root cultures

Effects of auxins (IAA, IBA and NAA) on K. humboldtiana root culture cultivated in 16-h photoperiod or in dark have been observed. Light affected positively the production of biomass when cultivated on medium supplemented with NAA in 10 and 25 mol ^–1 concentrations. In the presence of IAA and IBA these values were significantly lower. The growth dynamics of root cultures depended on the auxin used. The best adventitious roots elongation and lateral roots induction on media supplemented with IBA has been ascertained. Morphological and anatomical differences in dependence on auxin used were observed. NAA supported the formation of huge callus-like mass besides mostly very short roots, especially under the light. Similarly IAA induced short roots, and IBA seems to be the most effective substance for the root elongation in this model system. NAA induced roots with larger diameter under the light compared with the other two auxins used. The reason is in the different anatomical structure of roots which was characterized by higher number of cell layers and large intercellulars in the cortex. The shape of cortical cells in the presence of IBA depended on the light conditions. Isodiametric cortical cells were present in roots cultivated in 16-h photoperiod, irregularly-shaped cells in the dark. The effect of light conditions was the smallest in the case of roots grown on IAA enriched media.     

Rhizobium nod genes are involved in the induction of two early nodulin genes in Vicia sativa root nodules

Nodulin gene expresison was studied in Vicia sativa (common vetch) root nodules induced by several Rhizobium and Agrobacterium strains. An Agrobacterium transconjugant containing a R. leguminosarum symplasmid instead of its Ti-plasmid, that was previously shown to form empty nodules on pea, induced nodules on Vicia roots in which nodule cells were infected with bacteria. In the Vicia nodules induced by this transconjugant, two so-called early nodulin genes were found to be expressed, whereas in the nodules formed on pea the expression of only one early nodulin gene was detected. In both cases the majority of the nodulin genes was not expressed.Apparently, an intracellular location of the bacteria is not sufficient for the induction of the majority of the nodulin genes. All nodulin genes were expressed in nodules induced by cured Rhizobium strains containing cosmid clones that have a 10 kb nod region of the sym-plasmid in common. Since in tumours no nodulin gene expression was found at all, the Agrobacterium chromosome does not contribute to the induction of nodulin genes. Therefore it is concluded that the signal for the induction of the expression of the two Vicia early nodulin genes is encoded by the nod-region, and the signal involved in the induction of all other nodulin genes has to be located outside the sym-plasmid, on the Rhizobium chromosome. The apparent difference in early nodulin gene expression between pea and Vicia is discussed in the light of the usefulness of Agrobacterium transconjugants in the study of nodulin gene expression.     

Upstream non-coding region which confers polar expression to Ri plasmid root inducing gene rolB

Summary Root differentiation could be elicited on carrot discs by transformation with the agropine Ri plasmid rolB gene cloned in the binary vector Bin19, provided two conditions were met. Firstly, an adequate auxin supply had to be provided. This was achieved by co-inoculation with a strain carrying only the auxin synthetic genes of the T_R-DNA. Most of the resulting roots were then shown to harbour only rolB and no aux genes. Secondly, an extended non-coding region (1200 bp) at the 5 end of rolB had to be included in the construction. A shorter (300 bp) 5 region, including TATA and CCAAT boxes, was not sufficient to trigger root differentiation. Both the extended (B1185) and reduced (B310) 5 regions of rolB were then cloned upstream of the -glucuronidase (GUS) reporter gene and infections carried out both on the apical and on the basal side of carrot discs. Strong expression of GUS, visualized histochemically as an intense blue colouring of transformed cells was observed with B1185-GUS constructions on the apical side of the discs. Only occasionally could coloured cells be observed on the basal side of the discs with B1185-GUS and on both apical and basal sides with B310-GUS constructions. Strong GUS expression was, on the contrary, achieved on cells of both auxin-rich (apical) and auxin-depleted (basal) sides of the discs with the strong constitutive viral promoter, CaMV35S. These results indicate the presence of an upstream regulatory region which confers polar expression to the rolB gene and suggest a role for auxin in its activation.     

Regulation of Agrobacterium tumefaciens T-cyt gene expression in leaves of transgenic potato (Solanum tuberosum L. cv. Désirée) is strongly influenced by plant culture conditions

The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for -glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3 flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11 000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10–15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5 expression control sequences.     

Functional analysis of a complex oncogene arrangement in biotype III Agrobacterium tumefaciens strains

The ubiquitous grapevine-associated octopine/cucumopine Ti plasmids of biotype III Agrobacterium tumefaciens strains carry two T regions, TA and TB, with a complex oncogene arrangement. Within the octopine/cucumopine group, two main strain types were identified: large TA strains with a TA region resembling the TL region of the biotype I octopine strain Ach5 and small TA strains with a similar T region organization as the large TA strains but with a large internal TA deletion. Structural and functional studies of the representative large TA strain Tm4 revealed six oncogenes. Each oncogene was inserted in a disarmed vector and tested for biological activity using the corresponding oncogenes of Ach5 as standards. Five Tm4 oncogenes, TA-iaaM, T-ipt, T-6b, TB-iaaH and TB-iaaM, were shown to be active, the IS-interrupted TA-iaaH gene was inactive. To study the role of each gene in the pTiTm4 context, several single and multiple pTiTm4 mutations were constructed. It was shown that whereas TA-iaaM and TB-iaaH are essential for tumour formation on grapevine, T-ipt, T-6b and TB-iaaM are not. The avirulence of the TA-iaaM ^- mutant was shown to be due to an inhibitory effect of the T-ipt gene, since a TA-iaaM ^- /T-ipt ^- double mutant was fully virulent. We conclude that the TA-iaaM gene of large TA strains is specifically required to counteract the tumour growth inhibiting activity of the T-ipt gene. Both TA-iaaM and T-ipt are absent from the small TA strains. A model on the roles and interactions of the different oncogenes in large TA and small TA strains is presented.     

Residual phosphorus and long-term management strategies for an Ultisol

Due to their extensive growth potential, transgenic root systems arising from inoculation withAgrobacterium rhizogenes became popular in the last decade as model systems in domains as diverse as production of secondary metabolites, interactions with pathogens and symbionts, examination of gene importance in control of root development or in regulation of gene expression in roots. Wild-type bacterial strains have also been considered as useful tools to stimulate rooting on recalcitrant cuttings or microcuttings as they cause abundant root initiation at the site of inoculation.Root initiation and the in vitro growth characteristics of transformed roots result from the transfer of genes located on the root-inducing plasmid (Ri) to plant cells and their expression therein. Two sets of pRi genes are involved in the root induction process: therol (rootloci) genes located in the TL region and theaux genes of the TR region. Some of these genes being able to interact, the system appears also as a new tool to study the role of auxin in the process of root initiation. The distinctive phenotype of the transformed roots which are capable of hormone autonomous growth seems to be controlled mainly by therol genes. Theserol genes, i.e. the geneticloci rol A, rol B, rol C androl D correspond to open reading frames ORFs 10, 11, 12 and 15. In vitro experiments determined the functions of the Rol B and Rol C proteins but the functions of Rol A and Rol D are still unknown. Altered metabolism of developmental regulators or modified sensitivity to auxin have been suspected to mediate root induction and morphological abnormalities of transformed roots and plants.The target cells for transformation and the cells which are competent for root initiation will be characterized as well as the subsequent development of transgenic roots provided with various constructs from the whole T-DNA to singlerol genes. Results dealing with auxin contents in relation with root growth kinetics, phenotype and structure, will also be presented and discussed with the potential use of therol genes to control root biomass. F J de Bruijn Section editor     

The <Emphasis Type="Italic">rolB</Emphasis> gene promotes rooting <Emphasis Type="Italic">in vitro </Emphasis>and increases fresh root weight <Emphasis Type="Italic">in vivo</Emphasis> of transformed apple scion cultivar ‘Florina’

Transgenic apple (Malus × domestica Borkh.) Florina plants were obtained by Agrobacterium-mediated transformation. The efficiency of gene transfer was 7.9%, calculated as a number of explants producing at least one transgenic shoot, after co-cultivation of leaf explants from in vitro-grown shoots in a thin layer of the A. tumefaciens C58C1 strain with the binary vector pCMB-B:GUS. Polymerase chain reaction revealed that all the clones contained the nptII and rolB genes, while four of them did not contain the gus gene. Southern blot analysis confirmed the integration of the nptII and rolB genes, with one to three copies per genome being present. All independent rolB-transgenic lines were able to produce roots in vitro on the hormone free medium, while the plants, transformed with the vector pIB16.1, or untransformed control plants did not root, and only half of shoots of MM106 rootstock rooted on this medium. The average root number in the rolB-transgenic clones ranged from 4 to 7.7. Pretreatment with indole-3-butyric acid caused root formation in all transgenic and control plants and significantly increased root number in the rolB-transgenic lines, compared to untransformed plants. RolB-transgenic plants, grown in vivo in greenhouse for 2 years, did not differ phenotypically from the wild type line with the exception of root parts. All rolB-transformed plants produced altered root systems containing more fine roots leading to significantly increased fresh root weight in five plant lines.     

Expression of Agrobacterium rhizogenes auxin biosynthesis genes in transgenic tobacco plants

Plant oncogenes aux1 and aux2 carried by the TR-DNA of Agrobacterium rhizogenes strain A4 encode two enzymes involved in the auxin biosynthesis pathway in transformed plant cells. The short divergent promoter region between the two aux-coding sequences contains the main regulatory elements. This region was fused to the uidA reporter gene and introduced into Nicotiana tabacum in order to investigate the regulation and the tissue specificity of these genes. Neither wound nor hormone induction could be detected on transgenic leaf discs. However, phytohormone concentration and auxin/cytokinin balance controlled the expression of the chimaeric genes in transgenic protoplasts. The expression was localised in apical meristems, root tip meristems, lateral root primordia, in cells derived from transgenic protoplasts and in transgenic calli. Histological analysis showed that the expression was located in cells reactivated by in vitro culture. Experiments using cell-cycle inhibitors such as hydroxyurea or aphidicolin on transgenic protoplast cultures highly decreased the -glucuronidase activity of the chimaeric genes. These results as well as the histological approach suggest a correlation between expression of the aux1 and aux2 genes and cell division.