The non-conserved region of cucumopine-type Agrobacterium rhizogenes T-DNA is responsible for hairy root induction

The T-DNA regions of three strains of Ri plasmids 1855, 8196, 2659 (agropine, mannopine and cucumopine type respectively) share two highly conserved regions flanking a non-homologous central part [1,2]. We have cloned segments of the cucumopine Ri plasmid 2659 T-DNA in the binary vector system Bin 19 and infected carrot discs with recombinant Agrobacterium strains. We show here that the central non-conserved region is crucial in hairy root induction as it is sufficient to induce rooting on the apical (auxin-rich) surface of carrot discs; in order to observe rooting on the basal (auxin-depleted) side of the discs, a longer T-DNA fragment, also encompassing part of the right conserved region, had to be utilized in conjunction with a Agrobacterium strain carrying aux genes. Differences of growth properties in culture are exhibited by roots transformed with different fragments of pRi 2659 T-DNA, although all transformed roots show the plagiotropic behaviour typical of hairy roots.     

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.     

A reporter gene under the control of tms or aux promoters is differentially expressed in tobacco and barley protoplasts

Summary Agrobacterium tumefaciens and some Agrobacterium rhizogenes strains possess auxin biosynthesis genes (tms and aux genes respectively), responsible for a de novo auxin biosynthetic pathway in transformed plant cells. A comparison is presented of the potential expression of these genes in a monocotyledonous (barley) and a dicotyledonous plant (tobacco). The promoters of the genes were translationally fused to the -glucuronidase reporter gene and analysed in transient expression experiments. The tms and aux fusions were highly expressed in tobacco, but not in barley. However, the aux enhancer active in tobacco, conferred low -glucuronidase expression in barley when fused to a truncated cauliflower mosaic virus 35S promoter. The results are discussed in relation to the differential responses to Agrobacterium infection in monocots and dicots.Abbreviations CaMV cauliflower mosaic virus - PCR polymerase chain reaction - PEG polyethylene glycol - Mu 4-methyl umbelliferone     

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     

Low level expression of prokaryotic tzs gene enhances growth performance of transgenic poplars

We modulated the level of a hormone gene expression in poplars using either 35S promoter (p35S) of cauliflower mosaic virus (CaMV) or aux promoter (pAUX) of A. rhizogenes. The transgenic poplars (Populus alba × P. tremula var. glandulosa), in which the bacterial trans-zeatin secretion (tzs) gene was attached either to the 35S promoter or to the aux promoter, were compared for their performance in tissue culture as well as in nursery. Northern blot analysis of total RNA probed with tzs coding region showed that the total tzs mRNA expression by p35S was approximately 200–300-fold higher than that driven by pAUX. In contrast, the cellular zeatin content of p35S-tzs transgenic poplars was merely 13-fold of those found in pAUX-tzs plants. Due to different levels of cellular zeatin levels, the two types of transgenic poplars showed different morphogenetic as well as growth responses. The p35S-tzs transgenic plants showed morphological characteristics typical of those treated with cytokinin in culture. These include multiple axillary shoot formation, thick stems, narrow leaves and absence of roots. In contrast, the pAUX-tzs plants had slightly higher cellular cytokinin levels than did control plants and showed a lower degree of cytokinin-related phenotypes, including a few axillary shoots in root-inducing media. Since p35S-tzs did not develop roots, only pAUX-tzs transgenic poplars could be transplanted to the nursery where they resumed a close-to-normal growth. Nevertheless, pAUX-tzs plants transferred to the nursery developed cytokinin-related phenotypes, including greater number of shoots, smaller leaves and slightly retarded growth in height, but with a high total biomass.     

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.     

Genetic and functional analysis of the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI

Thirty Tn5- or Tn1831-induced nodulation (nod) mutants of Rhizobium leguminosarum were examined for their genetic and symbiotic properties. Thirteen mutants contained a deletion in Sym plasmid pRL1JI. These deletions cover the whole nod region and are 50 kb in size. All remaining seventeen mutations are located in a 6.6 kb EcoRI nod fragment of the Sym plasmid. Mutations in a 3.5 kb part on the right hand side of this 6.6 kb fragment completely prevent nodulation on Vicia sativa. All mutants in this 3.5 kb area are unable to induce marked root hair curling and thick and short roots.Mutations in a 1.5 kb area on the left hand side of the 6.6 kb nod fragment generate other symbiotic defects in that nodules are only rarely formed and only so after a delay of several days. Moreover, infection thread formation is delayed and root hair curling is more excessive than that caused by the parental strain. Their ability to induce thick and short roots is unaltered.Mutations in this 1.5 kb region are not complemented by pRmSL26, which carries nod genes of R. meliloti, whereas mutations in the 3.5 kb region are all complemented by pRmSL26.Abbreviations Rps repression of production of small bacteriocin - Mep medium bacteriocin production - Nod nodulation - Fix fixation - Tsr thick and short roots - Flac root hair curling - Hsp host specificity - Flad root hair deformation - Tc tetracycline - Km kanamycin - Cm chloramphenicol - Sp spectinomycin - Sm streptomycin - R resistant     

Independent induction of transformed roots by the TL and TR regions of the Ri plasmid of agropine type Agrobacterium rhizogenes

Summary To analyse the respective role of TL- and TR-DNA in root induction by agropine-type Agrobacterium rhizogenes Ri plasmids, deletions covering the TL- or the TR-regions were constructed in vitro and introduced into pRiA4 by marker exchange. Each T-region of pRiHRI was also cloned separately on an independent replicon and used in a binary system with the virulence functions of either an Ri or a Ti plasmid provided in trans. Transformed roots were induced on tobacco and tomato explants by TL-DNA as well as by TR-DNA, suggesting that agropine type Ri plasmids from strains A4 and HRI can induce root proliferation by two independent transformation mechanisms. The root induction by the TR-DNA is probably due to auxin biosynthesis by gene products of aux loci homologous to the tms genes of Ti plasmid T-DNA. The molecular mechanism of root proliferation induced by the TL-DNA is probably equivalent to that of mannopine type Ri plasmid T-DNA.     

The role of Rhizobium conserved and host specific nodulation genes in the infection of the non-legume Parasponia andersonii

Summary Rhizobium and Bradyrhizobium bacteria gain intercellular entry into roots of the non-legume Parasponia andersonii by stimulating localized sites of cell division which disrupt the epidermis. Infection threads are then initiated from intercellular colonies within the cortex. Infection via the information of infection threads within curled root hairs, which commonly occurs in legumes, was not observed in Parasponia. The conserved nodulation genes nodABC, necded for the curling of legume root hairs, were not essential for the initiation of infection, however, these genes were required for Parasponia prenodule development. In contrast, the nodD gene of Rhizobium strain NGR234 was essential for the initiation of infection. In addition, successful infection required not only nodD but a region of the NGR234 symbiotic plasmid which is not needed for the nodulation of legumes. Agrobacterium tumefaciens carrying this Parasponia specific region, as well as legume nod genes, was able to form nodules on Parasponia which reached an advanced stage of development.     

Isolation and functional analysis of a set of auxin genes with low root-inducing activity from an Agrobacterium tumefaciens biotype III strain

A new type of root-inducing iaa gene set was cloned from the Ti plasmid of the biotype III Agrobacterium tumefaciens strain Tm-4. These iaa genes are characterized by a very low DNA homology with the well-characterized iaa gene set, iaaM and iaaH, of the common DNA region of the biotype I strain Ach5 and by a low root-inducing activity.The biological activities of both iaa gene sets were compared by transferring each into a disarmed Ti vector and by testing the resulting strains on Nicotiana rustica leaf discs, decapitated Datura stramonium stems, tomato plants and Kalanchoë daigremontiana. Tm-4 iaa genes have a reproducibly weaker root-inducing ability on Nicotiana rustica, induce very little tumour growth on decapitated Datura plants or on tomato plants and do not induce roots on Kalanchoë daigremontiana. The Tm-4 iaa region was mapped by :: Tn5 transposon mutagenesis and tested on Nicotiana rustica. These tests combined with complementation experiments map the iaa genes to a 4.5-kb region.The Tm-4 iaa genes were able to complement the corresponding Ach5 iaa genes on Nicotiana rustica, indicating that the differences between these genes are quantitative rather than qualitative. Complementation experiments on Kalanchoë showed the iaaM gene of Tm-4 responsible for the overall weak auxin activity of the intact iaa set. In view of the observed structural and functional differences we propose to call the Tm-4 iaa genes TB-iaaM and TB-iaaH and the Ach5 iaa genes A-iaaM and A-iaaH.     

Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal,P<Subscript>i</Subscript>-fertilised and phytohormone-treated <Emphasis Type="Italic">Medicago truncatula</Emphasis> roots

A microarray carrying 5,648 probes of Medicago truncatula root-expressed genes was screened in order to identify those that are specifically regulated by the arbuscular mycorrhizal (AM) fungus Gigaspora rosea, by P_i fertilisation or by the phytohormones abscisic acid and jasmonic acid. Amongst the identified genes, 21% showed a common induction and 31% a common repression between roots fertilised with P_i or inoculated with the AM fungus G. rosea, while there was no obvious overlap in the expression patterns between mycorrhizal and phytohormone-treated roots. Expression patterns were further studied by comparing the results with published data obtained from roots colonised by the AM fungi Glomus mosseae and Glomus intraradices, but only very few genes were identified as being commonly regulated by all three AM fungi. Analysis of P_i concentrations in plants colonised by either of the three AM fungi revealed that this could be due to the higher P_i levels in plants inoculated by G. rosea compared with the other two fungi, explaining that numerous genes are commonly regulated by the interaction with G. rosea and by phosphate. Differential gene expression in roots inoculated with the three AM fungi was further studied by expression analyses of six genes from the phosphate transporter gene family in M. truncatula. While MtPT4 was induced by all three fungi, the other five genes showed different degrees of repression mirroring the functional differences in phosphate nutrition by G. rosea, G. mosseae and G. intraradices. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Long-distance signals positively regulate the expression of iron uptake genes in tobacco roots

Long-distance signals generated in shoots are thought to be associated with the regulation of iron uptake from roots; however, the signaling mechanism is still unknown. To elucidate whether the signal regulates iron uptake genes in roots positively or negatively, we analyzed the expressions of two representative iron uptake genes: NtIRT1 and NtFRO1 in tobacco (Nicotiana tabacum L.) roots, after shoots were manipulated in vitro. When iron-deficient leaves were treated with Fe(II)-EDTA, the expressions of both genes were significantly reduced; nevertheless iron concentration in the roots maintained a similar level to that in roots grown under iron-deficient conditions. Next, all leaves from tobacco plants grown under the iron-deficient condition were excised. The expression of two genes were quickly reduced below half within 2 h after the leaf excision and gradually disappeared by the end of a 24-h period. The NtIRT1 expression was compared among the plants whose leaves were cut off in various patterns. The expression increased in proportion to the dry weight of iron-deficient leaves, although no relation was observed between the gene expression and the position of excised leaves. Interestingly, the NtIRT1 expression in hairy roots increased under the iron-deficient condition, suggesting that roots also have the signaling mechanism of iron status as well as shoots. Taken together, these results indicate that the long-distance signal generated in iron-deficient tissues including roots is a major factor in positive regulation of the expression of NtIRT1 and NtFRO1 in roots, and that the strength of the signal depends on the size of plants.     

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.     

The <Emphasis Type="Italic">Medicago truncatula SUNN</Emphasis> Gene Encodes a <Emphasis Type="Italic">CLV1</Emphasis>-like Leucine-rich Repeat Receptor Kinase that Regulates Nodule Number and Root Length

Four Medicago truncatula sunn mutants displayed shortened roots and hypernodulation under all conditions examined. The mutants, recovered in three independent genetic screens, all contained lesions in a leucine-rich repeat (LRR) receptor kinase. Although the molecular defects among alleles varied, root length and the extent of nodulation were not significantly different between the mutants. SUNN is expressed in shoots, flowers and roots. Although previously reported grafting experiments showed that the presence of the mutated SUNN gene in roots does not confer an obvious phenotype, expression levels of SUNN mRNA were reduced in sunn-1 roots. SUNN and the previously identified genes HAR1 (Lotus japonicus) and NARK (Glycine max) are orthologs based on gene sequence and synteny between flanking sequences. Comparison of related LRR receptor kinases determined that all nodulation autoregulation genes identified to date are the closest legume relatives of AtCLV1 by sequence, yet sunn, har and nark mutants do not display the fasciated clv phenotype. The M. truncatula region is syntenic with duplicated regions of Arabidopsis chromosomes 2 and 4, none of which harbor CLV1 or any other LRR receptor kinase genes. A novel truncated copy of the SUNN gene lacking a kinase domain, RLP1, is found immediately upstream of SUNN and like SUNN is expressed at a reduced level in sunn-1 roots.     

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.     

Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: Nucleotide sequence and plant-inducible expression

Summary Azorhizobium caulinodans strain ORS571 induces nitrogen-fixing nodules on roots and stem-located root primordia of Sesbania rostrata. Two essential Nod loci have been previously identified in the bacterial genome, one of which (Nod locus 1) shows weak homology with the common nodC gene of Rhizobium mehloti. Here we present the nucleotide sequence of this region and show that it contains three contiguous open reading frames (ORFA, ORFB and ORFC) that are related to the nodABC genes of Rhizobium and Bradyrhizobium species. ORFC is followed by a fourth (ORF4) and probably a fifth (ORF5) open reading frame. ORF4 may be analogous to the nod[ gene of R. leguminosarum, whereas ORF5 could be similar to the rhizobial nodF genes. Coordinated expression of this set of five genes seems likely from the sequence organization. There is no typical nod promoter consensus sequence (nod box) in the region upstream of the first gene (ORFA) and there is no nodD-like gene. LacZ fusions constructed with ORFA, ORFB, ORFC, and ORF4 showed inducible -galactosidase expression in the presence of S. rostrata seedlings as well as around stem-located root primordia. Among a series of phenolic compounds tested, the flavanone naringenin was the most efficient inducer of the expression of this ORS571 nod gene cluster.     

The Agrobacterium tumefaciens T-DNA gene 6b is an onc gene

In this article it is shown that the T-DNA of Agrobacterium tumefaciens contains besides the well-known cyt and aux genes another gene with an oncogenic effect in plants. The gene in question is called 6^b and causes the formation of small tumors in plant species such as Nicotiana glauca and Kalanchoe tubiflora.     

Activation and analysis of crypticcrt genes for carotenoid biosynthesis fromStreptomyces griseus

Genes encoding enzymes with sequence similarity to carotenoid biosynthetic enzymes of other organisms were cloned fromStreptomyces griseus JA3933 and transformed into the colourless (non-daunorubicin producing) mutantStreptomyces griseus IMET JA3933/956/2. Cells harbouring these genes showed an orange-red pigmentation, caused by the strongly hydrophobic, membrane-bound lycopene. The cloned fragment (9 kb) contained seven genes, four transcribed in one direction (crtEIBV) and three (crtYTU) transcribed convergently to them. Three of these genes encode polypeptides that resemble geranylgeranyl-pyrophosphate (GGPP) synthases (CrtE), phytoene synthases (PS) (CrtB) and phytoene dehydrogenases (PDH) (CrtI), respectively, of various bacteria. These enzymes are sufficient for the formation of lycopene.crtE alone was sufficient to induce zeaxanthin formation in anEscherichia coli clone containing thecrt gene cluster fromErwinia herbicola deleted forcrtE. The combination ofcrtE andcrtB led to formation of phytoene inS. griseus. The putativecrtEp promoter region was cloned and mapped by primer extension analysis. In a gel retardation experiment, this fragment was specifically shifted by an unknown protein. CrtY shows similarity to lycopene cyclases that convert lycopene into-carotene, CrtT resembles various methyltransferases and CrtU a dehydrogenase. We conclude that these genes are functionally intact, but not expressed (cryptic) in the wild-typeS. griseus strain.     

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.