Variation in Binding and Virulence of Agrobacterium tumefaciens Chromosomal Virulence (chv) Mutant Bacteria on Different Plant Species

Chromosomal virulence (chv) mutants of Agrobacterium tumefaciens have been reported to be deficient in binding to cells of zinnia, tobacco, and bamboo. The mutants are nonpathogenic on stems of Kalanchoë, sunflower, tomato, Jerusalem artichoke, and tobacco, but they cause tumors on tubers of Solanum tuberosum. We used a root cap cell binding assay to test ability of cells from individual plants of 13 different plant species to bind parent or chv mutant bacteria. The same plants were then inoculated to test for disease response. Cells from nine of the plant species were grossly deficient in their abilities to bind mutant bacteria, and the plants inoculated with mutant bacteria failed to form tumors. In contrast, root cap cells as well as root hairs and root surfaces of S. tuberosum, S. okadae, and S. hougasii bound chv mutant bacteria as well as wild type. Nevertheless, S. tuberosum roots inoculated with mutant bacteria did not develop tumors. Although S. okadae plants inoculated with mutant bacteria formed a few tumors, and S. hougasii developed as many tumors in response to chv mutants as in response to the parent strain, the tumors induced by mutant bacteria were smaller.     

Crown gall disease and hairy root disease : a sledgehammer and a tackhammer

The neoplastic diseases crown gall and hairy root are incited by the phytopathogenic bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes, respectively. Although the molecular mechanism of T-DNA transfer to the plant most likely is the same for both species, the physiological basis of tumorigenesis is fundamentally different. Crown gall tumors result from the over-production of the phytohormones auxin and cytokinin specified by A. tumefaciens T-DNA genes. Although the T-DNA of some Riplasmids of A. rhizogenes contains auxin biosynthetic genes, these loci are not always necessary for hairy root formation. Recent experiments suggest that hairy root tumors result from the increased sensitivity of transformed cells to endogenous auxin levels. An understanding of hairy root tumorigenesis will likely result in an increased knowledge of plant developmental processes.     

Agrobacterium tumefaciens Interaction with Suspension-Cultured Tomato Cells

Adherence of Agrobacterium tumefaciens to suspension-cultured tomato cells has been characterized using a quantitative binding assay. Saturable binding of radiolabeled A. tumefaciens to plant cells resulted in 100 to 300 bacteria bound per cell. Specificity of A. tumefaciens binding was also inferred from two additional results: (a) an initial incubation of plant cells with A. tumefaciens reduced subsequent binding of radiolabeled A. tumefaciens by 60% to 75%; (b) tomato cells bound less than three E. coli per cell. Protease treatment of plant cells had no effect on subsequent bacterial binding, but prior treatment of plant cells with pectinolytic enzymes increased binding 2- to 3-fold. Pectin-enriched and neutral polymer-enriched fractions were obtained from tomato cell walls. The soluble pectin-enriched fraction inhibited binding of bacteria to plant cells by 85% to 95%, whereas the neutral polymer fraction only partially inhibited binding. Preliminary characterization of the activity showed it is heat stable, partially inactivated by protease treatment, and substantially inactivated by acid hydrolysis.     

A morphometric analysis of cellular differentiation in root caps ofCucurbita peop

In order to quantify the ultrastructural changes that occur during cellular differentiation in an “open” type of root cap, we have performed a morphometric analysis of the ultrastructures of calyptrogen, columella, and peripheral cells of the root cap ofCucurbita pepo. The relative volumes of nuclei, nucleoli, and mitochondria decrease as cells move (i.e., differentiate) through the root cap. Before cells are sloughed from the cap, the relative volume of the vacuole increases by 250%. The relative volumes of plastids and plastid starch increase as calyptrogen cells differentiate into columella cells, but decrease as columella cells differentiate into peripheral cells. Dictyosomal volumes increase only as columella cells differentiate into peripheral cells. These results indicate that the five cell types comprising the root cap ofC.pepo are each characterized by a unique structure, and that the ultrastructural changes associated with cellular differentiation in root caps are organelle specific. These results are discussed relative to the functions of the various cell types of the root cap.     

Potential of Agrobacterium tumefaciens and Octopine-Utilizing Fluorescent Pseudomonas Strains To Attach to Susceptible Potato Tissues

The binding characteristics of two octopine-catabolizing pseudomonads, Pseudomonas fluorescens B99A and E175D, which were isolated from crown galls, have been examined. The binding of strain B99A to potato disks was very weak, followed a Freundlich isotherm, and was temperature and pH independent. Strain E175D displayed strong attachment and followed a Langmuir isotherm. Despite these fundamental differences in binding characteristics, when each strain was placed in competitive binding assays with either Agrobacterium tumefaciens B6 or A. tumefaciens ATCC 15955, the number of bound pseudomonad cells decreased compared with those obtained in independent trials. Furthermore, the binding of A. tumefaciens cells was increased. In prebinding experiments, in which the potato disks were bound with the pseudomonads before exposure to the agrobacteria, the number of bound pseudomonad cells again decreased. This implies that increased desorption was occurring. In these prebinding studies, the numbers of bound A. tumefaciens ATCC 15955 increased, but the number of bound A. tumefaciens B6 remained the same. The mechanism for this observed synergism on the binding of agrobacterial cells and the depression in bound pseudomonad cells is believed to be alterations in the electrostatic or ionic charges on the plant and bacterial cell surfaces. The synergistic effect on A. tumefaciens undermines the use of these pseudomonads as potential biocontrol agents for crown gall.     

Effect of Plasmid pSa and of Auxin on Attachment of Agrobacterium tumefaciens to Carrot Cells

When the plasmid pSa is introduced into Agrobacterium tumefaciens, its presence results in the suppression of bacterial virulence. A. tumefaciens(pSa) cells are virulent on Bryophyllum diagremontiana only when inoculated with auxin. A. tumefaciens(pSa) cells also bind to plant cells only in the presence of auxin. The effect of auxin is on the bacteria rather than on the plant cells, since the bacteria require auxin to bind to heat-killed carrot cells. Bacteria containing pSa and grown in the absence of auxin showed a lag in binding to carrot cells in auxin-containing medium. This lag was not seen during the binding of wild-type strains. Tetracycline inhibited the binding of A. tumefaciens(pSa) in auxin-containing medium, suggesting that bacterial protein synthesis is required for the auxin effect. No difference was seen in the size or ability to inhibit bacterial binding of lipopolysaccharides from bacteria containing or lacking pSa and grown with or without auxin. A. tumefaciens(pSa) cells grown in the absence of auxin lacked surface polypeptide(s) found in bacteria grown in the presence of auxin and in the wild-type bacteria, which do not contain pSa. Thus, the presence of certain polypeptides appears to be associated with the ability of the bacteria to bind to plant cells.     

Observation by SEM of the attachment ofAgrobacterium tumefaciens to the surface of vinca,asparagus and rice cells

Transformation of vinca cells was performed by the co-cultivation of cell-wall regenerated vinca protoplasts withAgrobacterium tumefaciens. Using thisin vitro and single cell system, attachment of the bacteria to the surface of vinca cells was observed by scanning electron microscopy (SEM). Figures of the bacteria polarly binding to the plant cell wall were often observed. AsEscherichia coli does not attach to the plant cells at all, the observed attachment ofA. tumefaciens is suggested as a characteristic feature in crown gall induction. Even though no evidence of transformation was obtained by the co-cultivation methods, a similar attachment was observed in the cell-wall regenerated protoplasts of rice. The bacteria also attached to the surface of isolated mesophyll cells of asparagus and root hairs of rice. From these observation, we concluded that the attachment is not the limiting step of crown gall induction byA. tumefaciens in monocotyledonous plants. Extracellular fibrils like pili were observed with a few strains of A.tumefaciens for the first time. These fibrils were observed regardless of their ability of attachment and infectivity.     

Genetic transformation of 9 in vitro clones of Alnus and Betula by Agrobacterium tumefaciens

Crown gall tumorigenesis, integration and expression of T-DNA encoded genes from Agrobacterium tumefaciens were investigated in 9 clones of Alnus glutinosa, A. incana and Betula papyrifera. Tumor formation on in vitro shoots was frequent in all clones with strain Ach5 and present in 8 clones with strain C58. Tumors excised from shoots were selected for autotrophic growth in vitro and axenic cultures were established. Octopine or nopaline, respective of the strain type used for inoculation, was detected in tumorous cultures. Southern blot analyses demonstrated T-DNA integration by hybridization of DNA from tumors with tmr and nos gene probes. One clone of B. papyrifera produced tumors with a morphogenic character, unusual in calli of this species, generating viable shoots which did not synthesize opine.Abbreviations Cb Carbenicillin - Cf Cefotaxime - 2,4-D 2,4-Dichloro-phenoxyacetic acid     

Reduced rhizosphere colonization ability ofAgrobacterium tumefaciens chromosomal virulence (chv) mutants

Mutations in the chromosomal virulence (chv) region ofA. tumefaciens strain A723 reduce virulence, motility, and ability of the bacteria to bind to plant cells. We conducted experiments to assess the ability ofchv mutants to colonize the rhizosphere ofPisum sativum. The mutation had no effect on ability of bacteria to grow with a defined number of root cap cells as the sole carbon and nitrogen source. Ten days after inoculation, there were up to 103-fold more wild type thanchv mutant bacteria present in the rhizosphere of inoculated plants.     

Association of Azospirillum with Grass Roots

The association between grass roots and Azospirillum brasilense Sp 7 was investigated by the Fahraeus slide technique, using nitrogen-free medium. Young inoculated roots of pearl millet and guinea grass produced more mucilaginous sheath (mucigel), root hairs, and lateral roots than did uninoculated sterile controls. The bacteria were found within the mucigel that accumulated on the root cap and along the root axes. Adherent bacteria were associated with granular material on root hairs and fibrillar material on undifferentiated epidermal cells. Significantly fewer numbers of azospirilla attached to millet root hairs when the roots were grown in culture medium supplemented with 5 mM potassium nitrate. Under these growth conditions, bacterial attachment to undifferentiated epidermal cells was unaffected. Aseptically collected root exudate from pearl millet contained substances which bound to azospirilla and promoted their adsorption to the root hairs. This activity was associated with nondialyzable and proteasesensitive substances in root exudate. Millet root hairs adsorbed azospirilla in significantly higher numbers than cells of Rhizobium, Pseudomonas, Azotobacter, Klebsiella, or Escherichia. Pectolytic activities, including pectin transeliminase and endopolygalacturonase, were detected in pure cultures of A. brasilense when this species was grown in a medium containing pectin. These studies describe colonization of grass root surfaces by A. brasilense and provide a possible explanation for the limited colonization of intercellular spaces of the outer root cortex.     

Transformation of white spruce and other conifer species byAgrobacterium tumefaciens

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0–80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced gall formation on more than 50% of the inoculated trees and at greater than 10% of the inoculated sites. One strain, B2/74 gave rise to galls at 28% of the inoculated sites on white spruce and induced the highest overall frequency of gall formation on all the conifer species tested. Relative frequency of gall formation was consistent among species, although the overall frequency was much higher on Douglas-fir. Of the well characterized strains for which disarmed derivatives are available only A281 (carrying the supervirulent tumor inducing plasmid, pTiBo542) gave efficient transformation. Stable integration of T-DNA encoded genes has been confirmed by the expression of opine synthesis and hormone autonomous growth. The transfer and long-term stable expression of kanamycin resistance and firefly luciferase activity using binary vector systems was also achieved.     

Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

Inhibitory Effects of a Pectin-Enriched Tomato Cell Wall Fraction on Agrobacterium tumefaciens Binding and Tumor Formation

A pectin-enriched soluble cell wall fraction (CWF) prepared from suspension cultured tomato cells inhibits binding of Agrobacterium tumefaciens to these cells. It was hypothesized that the CWF contains the plant surface binding site for A. tumefaciens (NT Neff, AN Binns 1985 Plant Physiol 77: 35-42). Experiments described here demonstrate that tomato CWF inhibited tumor formation on potato slices and Agrobacterium binding to intact tomato cells in a dose-dependent fashion. Boiling the fraction reduced both its binding and tumor inhibitory activities. Tumor inhibitory activity was titrated out by increased concentrations of bacterial inocula with no inhibition apparent at 1 × 10⁸ bacteria per milliliter. These results indicate that a tomato CWF is enriched for a putative A. tumefaciens binding site which may also be involved in tumor formation in potato.     

Gene transfer into peanut (Arachis hypogaea L.) by Agrobacterium tumefaciens

Introduction of foreign genes into plant tissues via Agrobacterium tumefaciens based vectors requires specific knowledge of Agrobacterium-host compatibility. Therefore, to develop a transformation protocol for peanut (Arachis hypogaea L.), five Brazilian cultivars were screened with four wild-type A.tumefaciens strains. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions and strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain. Tumors induced on seed and seedling explants by A281 (pTD02) also expressed the reporter genes gus and npt-II contained in the binary vector. These results show that peanut is a permissive host for the acceptance of genes from specific A.tumefaciens gene vectors.Abbreviations GUS ß-glucuronidase (EC 3.2.1.31) - NPT-II neomycin phosphotransferase II (EC 2.7.1.95) - EDTA ethylene-diamine-tetracetic acid     

Involvement of Carrot Cell Surface Proteins in Attachment of Agrobacterium tumefaciens

The initial step in tumor formation by Agrobacterium tumefaciens is the site-specific attachment of the bacteria to plant cells. A similar attachment to plant tissue culture cells has been observed. Binding to carrot suspension culture cells was not dependent on the presence of divalent cations and was not inhibited by the addition of mannose, α-methyl mannoside, galactose, arabinose, glucosamine, 2-deoxyglucose, or 0.25 molar NaCl to the culture medium. The ability of the carrot cells to bind A. tumefaciens was markedly reduced by elution of the cells with dilute detergent or CaCl₂ or by incubation of the cells with proteolytic enzymes. The carrot cells were not killed by these treatments and recovered the ability to bind A. tumefaciens within 3 to 6 hours. A. tumefaciens did not bind to carrot cells which had been induced to form embryos (AG Matthysse, RHG Gurlitz 1982 Physiol Plant Pathol 21: 381-387). A comparison of the peptides eluted from embryos and from uninduced cells using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that there were several changes in extractable polypeptides after embryo induction. One or more of the polypeptides present before embryo induction and absent from embryos may be involved in the binding of A. tumefaciens to the carrot cell surface.     

Fusion of Agrobacterium and E. coli spheroplasts with Nicotiana tabacum protoplasts — Direct gene transfer from microorganism to higher plant

Spheroplasts of Agrobacterium tumefaciens strains and E. coli were fused with protoplasts of Nicotiana tabacum. Fusion products were cultured in the presence of antibiotics to eliminate remaining bacterial spheroplasts. On hormone free medium, tobacco protoplasts treated with wild type Agrobacterium-strains formed colonies with an average frequency of 10^–4. Opine synthesis was detected in the tissues. Some calli derived from protoplasts treated with A. tumefaciens C58C1pRi15834 formed typical hairy roots. Kanamycin resistant calli were obtained after fusion with A. tumefaciens containing pLGVTi23 neo (frequency=10^–3). Fusion of E. coli spheroplasts containing a virulent pTiB6S3::RP4 co-integrate with tobacco protoplasts yielded two hormone independent growing calli producing octopine out of 10⁵ microcalli.Abbreviations PEG Polyethylene glycol - PVA Polyvinyl alcohol     

Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector

The disamed Ti-binary vector pBIN 6 in Agrobacterium tumefaciens has been used in leaf disc transfomations to produce transgenic apple (Malus pumila Mill.) plants with a nomal phenotype except for a somewhat reduced capacity to root. The presence of the genes for nopaline synthase and neomycin phosphotrans ferase (conferring kanamycin resistance), inserted into the host genome by the vector, was confirmed by Southern blot analysis, the detection of nopaline synthase activity and rooting in the presence of the antibiotic.The nopaline synthase gene continued to be expressed in glasshouse-grown plants several months after removal from in vitro growth conditions.     

Transformation of Solanum integrifolium poir via Agrobacterium tumefaciens: Plant regeneration and progeny analysis

Summary The wild species Solanum integrifolium represents a source of pest and disease resistance genes for breeding strategies of the cultivated species Solanum melongena. Somatic hybridization via protoplast fusion between the two species may provide a valuable tool for transferring polygenic traits into the cultivated species. The availability of S.integrifolium cells carrying dominant selectable markers would facilitate the heterokaryon rescue. An appropriate methodology for in vitro culture and plant regeneration from leaf explants of S.integrifolium is reported. Efficient leaf-disk transformation via co-cultivation with Agrobacterium tumefaciens led to the regeneration of transformed plants carrying the reporter genes GUS and NPT-II. Transformed individuals were obtained through selection on kanamycin-containing medium. Stable genetic transformation was assessed by histochemical and enzymatic assays for GUS and NPT-II activity, by the ability of leaf disks to initiate callus on Km-containing medium, Southern blot analyses of the regenerated plants, and genetic analysis of their progenies. Selfed-seed progeny of individual transformed plants segregated seedlings capable to root and grow in selective condition, while untransformed progeny did not. Genetic analyses of progeny behaviour showed that the reporter gene NPT-II segregated as single as well as two independent Mendelian factors. In two cases an excess of kanamycin-sensitive seedlings was obtained, not fitting into any genetic hypothesis.Abbreviations MS Murashige and Skoog (1962) medium - NOS nopaline synthase - NPT-II neomycin phosphotransferase - GUS beta-glucuronidase - LB Luria and Bertani medium - KIN 6-furfurylaminopurine - BAP 6-benzylaminopurine - 2iP N⁶-(2-isopentyl)adenine - ZEA zeatin - TDZ Thidiazuron     

Restoration of attachment, virulence and nodulation of Agrobacterium tumefaciens chvB mutants by rhicadhesin

In contrast to wild-type Agrobacterium tumefaciens strains, β-1,2-glucan-deficient chvB mutants were found to be unable to attach to pea root hair tips. The mutants appeared to produce rhicadhesin, the protein that mediates the first step in attachment of Rhizobiaceae cells to plant root hairs, but the protein was inactive. Both attachment to root hairs and virulence of the ChvB mutants could be restored by treatment of the plants with active rhicadhesin, whereas treatment of plants with β-1,2-glucan had no effect on attachment or virulence. Moreover, nodulation ability of a chvB mutant carrying a Sym plasmid could be restored by pretreatment of the host plant with rhicadhesin. Apparently the attachment-minus and avirulence phenotype of chvB mutants is caused by lack of active rhicadhesin, rather than directly being caused by a deficiency in β-1,2-glucan synthesis. The results strongly suggest that rhicadhesin is essential for attachment and virulence of A. tumefaciens cells. They also indicate that the mechanisms of binding of Agrobacterium and Rhizobium bacteria to plant target cells are similar, despite differences between these target cells.     

Conditioned medium promotes the attachment ofAgrobacterium tumefaciens strain NT 1 to carrot cells

Summary Wild-typeAgrobacterium tumefaciens bind to carrot suspension culture cells. Avirulent strain NT 1 did not bind to carrot cells when they were incubated together in Murashige and Skoog medium. Conditioned medium was prepared by incubatingA. tumefaciens virulent strain C 58 with carrot cells and removing the bacteria and carrot cells using filter sterilization. This conditioned medium promoted the binding of NT 1 to carrot cells. Conditioned medium did not promote the nonspecific attachment ofEscherichia coli to carrot cells. These results suggest that when wild-typeA. tumefaciens are incubated with plant host cells, some substance(s) involved in bacterial attachment are released into the medium. Filter-sterilized medium from the incubation of the nonattachingchvB mutant A 1045 with carrot cells promoted the attachment of strain NT 1 even though A 1045 bacteria did not bind to the carrot cells. However, filter-sterilized medium from the incubation of the non-attachingatt mutant Att-B 123 with carrot cells was unable to promote the binding of strain NT 1. This suggests that nonattaching mutants ofA. tumefaciens can be divided into two groups on the basis of the properties of the substances released into the medium when the bacteria are incubated with carrot cells.Abbreviations MS Murashige and Skoog tissue culture medium Dedicated to the memory of Professor John G. Torrey