Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells.

The attachment of virulent strains of Agrobacterium tumefaciens to plant cells is the first step in the bacterial induction of tumors. Binding of A. tumefaciens to carrot tissue culture cells occurred as a two-step process. The initial step was the attachment of the bacteria to the plant cell wall. Living plant cells were not required. Bacterial attachment to heat-killed or glutaraldehyde-fixed carrot cells proceeded with only slightly altered kinetics and unaltered bacterial strain specificity. After the bacteria bound to the carrot cell surface, scanning electron microscopy showed that fibrils developed, surrounded the bacteria, and anchored them to the plant cell surface. These fibrils were synthesized by the bacteria and not by the plant cell since they were also made after the attachment of A. tumefaciens to dead carrot cells and since under some conditions the bacteria synthesized fibrils in the absence of plant cells. Calcofluor staining, acid hydrolysis, enzymatic digestion studies, and infrared spectroscopy showed that the fibrils were composed of cellulose. The formation of these cellulose fibrils occurred during the attachment of virulent strains of A. tumefaciens to plant cells in vitro. The fibrils anchored the bacteria to the plant cell surface and entrapped additional bacteria. The multiplication of entrapped and attached bacteria resulted in the formation of large clusters of bacteria held close to the plant cell wall and plasma membrane by cellulose fibrils. This high concentration of bacteria may facilitate transfer of Ti plasmid deoxyribonucleic acid to the plant cell resulting in the formation of tumors.     

Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells.

Infections of dicotyledonous plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. Attachment of the bacteria to plant host cells is required for tumor formation. Human vitronectin and antivitronectin antibodies both inhibited the binding of A. tumefaciens to carrot cells. Wild-type bacteria are able to bind radioactive vitronectin; nonattaching mutants showed a reduction in the ability to bind vitronectin. The binding of biotype 1 A. tumefaciens to carrot cells or to radioactive vitronectin was not affected by high ionic strength. Detergent extraction of carrot cells removed the receptor to which the bacteria bind. The extract was found to contain a vitronectin-like protein. These results suggest that A. tumefaciens utilizes a vitronectin-like protein on the plant cell surface as the receptor for its initial attachment to host cells.     

Structural determination of the O-chain polysaccharide from Agrobacterium tumefaciens, strain DSM 30205.

Agrobacterium tumefaciens is a Gram-negative, phytopathogenic bacterium and is characterized by an unique mode of action on dicotyledonous plants: it is able to genetically modify the host, and because of this feature, it is used as a tool for transgenic plants. Many experiments have demonstrated that lipopolysaccharides (LPSs) play an important role for the disease development, as they are involved in the adhesion process of the bacterium on the plant cell wall. Despite the wealth of information on the role of LPS on phytopathogenesis, the present paper appears as the first report on the molecular primary structure of the O-chain produced from Agrobacterium. Its repeating unit was determined by means of chemical and spectroscopical analysis, and has the following structure: (3)-alpha-D-Araf-(1-->3)-alpha-l-Fucp-(1-->.     

Attachment to roots and virulence of a chvB mutant of Agrobacterium tumefaciens are temperature sensitive

Agrobacterium tumefaciens chvB mutants are unable to produce beta-1,2 glucan. They are nonattaching and avirulent and show reduced motility at room temperature. At lower temperatures (16 degrees C), chvB mutants became virulent on Bryophyllum daigremontiana and Lycopersicon esculentum and were able to attach to L. esculentum, Arabidopsis thaliana, Daucus carota, and Tagetes erecta roots. The mutant bacteria also recovered wild-type motility at lower temperatures. Two other nonattaching mutants of A. tumefaciens, AttR and AtrA, were unaffected by the lowered temperature, remaining nonattaching and avirulent.     

Attachment of Agrobacterium tumefaciens B6 and A. radiobacter K84 to Tomato Root Tips

Agrobacterium tumefaciens B6 and the avirulent Agrobacterium radiobacter strain K84 attached to in vitro-cultured tomato root tips, but the binding of strain B6 to root tips was greater than the binding of strain K84. Strain K84 was not able to block the attachment of A. tumefaciens B6 to in vitro-cultured tomato root tips.     

Altered development of Arabidopsis thaliana carrying the Agrobacterium tumefaciens ipt gene is partially due to ethylene effects

Transgenic Arabidopsis thaliana plants containingthe Agrobacterium tumefaciens cytokinin-biosynthesis geneipt were produced to study the effect of increasedcytokinin (CK) levels on the development of this rosette plant species. Inthreeindependently transformed lines (ipt-156, 158 and 161),Arabidopsis plants had smaller leaves, an underdevelopedroot system and decreased apical dominance in inflorescence stems. The smallertransgenic leaves were highly serrated along the margins, pale green and hadpointed leaf tips. In cross section, transgenic leaves had smaller cells andirregularly shaped epidermal cells. In the ipt-161 line,leaves and hypocotyls frequently exhibited purple color due to anthocyaninproduction. The most severe phenotype was observed in tissue cultureconditions,while growth in soil reduced or eliminated some phenotypic effects. Compared toC24 wild type plants, ipt-161 plants accumulated zeatinandzeatin riboside with an approximate 10-fold increase in the total pool of CKs.Astudy of the progeny resulting from crosses between theipt-161 transgenic line and the ethylene insensitivemutants ein1, ein2 andeti5 suggested that part of the altered developmentexhibited by the ipt transgenic plants was caused byincreased ethylene levels.     

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.     

Specific attachment of Agrobacterium tumefaciens to bamboo cells in suspension cultures.

Agrobacterium tumefaciens was tested for its ability to attach to tissue culture cells of bamboo, a monocotyledonous plant. Phase-contrast microscopy and kinetic experiments with radiolabeled bacteria showed that attachment to bamboo cells was indistinguishable from attachment to cells of dicotyledonous plants. Bacterial mutants defective in attachment to dicotyledonous plants showed similar behavior with bamboo, and extensive washing of the bamboo cells had no effect on the number of bacteria which attached.     

Adherence of Agrobacterium tumefaciens to the cells of immature wheat embryos

Agrobacterium attached to wheat embryos in vitro. This attachment was plasmid independent, and occurred on both wounded and unwounded cell surfaces. The pattern of attachment clearly demonstrated that bacterial attachment to cereal cells follows the same trends observed for dicotyledonous plants. During the inoculation period the bacterial cells attach to the plant cell walls either with lateral or polar orientation. Wounding (mechanical or enzymatic) preferentially promoted adherence of the bacteria at the wound site, however, attachment was not wound dependent.     

Agrobacterium tumefaciens virulence locus pscA is related to the Rhizobium meliloti exoC locus.

Agrobacterium tumefaciens and Rhizobium meliloti carry related genetic loci which have important roles in virulence and symbiosis. Previously, it was shown that two virulence loci of A. tumefaciens, chvA and chvB, are related to two R. meliloti symbiosis loci, ndvA and ndvB, respectively (T. Dylan, L. Ielpi, S. Stanfield, L. Kashyap, C. Douglas, M. Yanofsky, E. Nester, D. R. Helinski, and G. Ditta, Proc. Natl. Acad. Sci. USA 83:4403-4407, 1986). Here we show that these two phytobacteria possess additional related virulence/symbiosis genes. Results of genetic complementation and DNA hybridization experiments indicate that the pscA virulence locus of A. tumefaciens is structurally and functionally related to the exoC symbiosis locus of R. meliloti. Thus, A. tumefaciens and R. meliloti bear at least three related genetic loci that have crucial roles in establishing the interactions that each bacterium has with its respective host plants.     

Multiple copies of virG enhance the transient transformation of celery,carrot and rice tissues by Agrobacterium tumefaciens

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical -glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.Current address: Department of Agronomy, Purdue University     

A Ti-plasmid determined function is responsible for chemotaxis of Agrobacterium tumefaciens towards the plant wound product acetosyringone

Agrobacterium tumefaciens C58C¹[pTiB6S3] harbouring the octopine Ti-plasmid pTiB6S3, showed positive chemotaxis towards the phenolic plant wound exudate acetosyringone (AS). Maximal attraction was observed at 10⁻⁷ M. In contrast, A. tumefaciens C58C¹ lacking a Ti-plasmid, exhibited no chemotactic response to AS. However, chemotaxis did occur towards the plant phenolic vanillyl alcohol, but at higher concentrations (10⁻² M) and in both Ti-plasmid-containing and cured A. tumefaciens.These results indicate that at least one Ti-plasmid function is involved in the specific chemotactic response to AS, although chemotaxis per se is not Ti-plasmid-encoded. This correlates well with the specific induction of vir-operons mediated by this plant wound product [1].     

Attachment, Chemotaxis, and Multiplication of Agrobacterium tumefaciens Biovar 1 and Biovar 3 on Grapevine and Pea

Tumorigenic (CG49) and nontumorigenic (CG484) strains of Agrobacterium tumefaciens bv. 3 attached to grape roots at a higher level than did a nonpectinolytic mutant of CG49 (CG50) or a tumorigenic strain of A. tumefaciens bv. 1 (CG628). Strains attached equally well to wounded and unwounded grape roots. Strains responded differently to pea plants in that biovar 3 strains consistently attached to unwounded roots at a lower level than they did to wounded roots, whereas CG628 attached equally well regardless of wounding. The lowest levels of attachment to pea roots were consistently observed for CG50. Population curves were calculated for the strains inoculated into wound sites on grape and pea roots. A. tumefaciens bv. 3 wild-type strains developed greater populations at wound sites on grape roots after 100 h (resulting in root decay) than did CG50 or CG628. Population curves for strains at wound sites on pea roots were different from those on grape roots. There were no significant differences in populations after 100 h, and no strains caused root decay. No differences in the chemotaxis of wild-type and mutant A. tumefaciens bv. 3 strains towards grape roots, crown pieces, or root extracts were observed, but the biovar 1 strain, CG628, always migrated the greatest distance towards all substrates. Polygalacturonase production may affect attachment to grape roots and multiplication of A. tumefaciens bv. 3 at wound sites and thus be associated with the specificity of the bacterium for grape.     

Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment.

We have identified a new virulence locus in Agrobacterium tumefaciens. Strains carrying Tn5 inserts at this locus could not incite tumors on Kalanchoe daigremontiana, Nicotiana rustica, tobacco, or sunflower and had severely attenuated virulence on carrot disks. We termed the locus pscA, because the mutants that defined the locus were initially isolated as having an altered polysaccharide composition; they were nonfluorescent on media containing Leucophor or Calcofluor, indicating a defect in the production of cellulose fibrils. Further analysis showed that the pscA mutants produced little, if any, of the four species of exopolysaccharide synthesized by the wild-type strain. DNA hybridization analysis and genetic complementation experiments indicated that the pscA locus is not encoded by the Ti plasmid and that it is distinct from the previously described chromosomal virulence loci chvA and chvB. However, like chvA and chvB mutants, the inability of the pscA mutants to form tumors is apparently due to a defect in plant cell attachment. Whereas we could demonstrate binding of the wild-type strain to tobacco suspension cells, attachment of the pscA mutants was drastically reduced or completely absent.     

picA, a novel plant-inducible locus on the Agrobacterium tumefaciens chromosome.

We used the transposon Mu dI1681 to identify genes on the Agrobacterium tumefaciens chromosome that are inducible by extracts from carrot roots. One such locus (picA, for plant inducible chromosomal), harbored by A. tumefaciens At156, was inducible 10- to 50-fold by these extracts. Mutation of picA had no detectable effect upon bacterial growth or virulence under laboratory assay conditions. However, A. tumefaciens cells harboring a mutated picA locus aggregated into long "ropes" when incubated with pea root tip cells. Such aggregation was not displayed by the parental strain A. tumefaciens A136. A preliminary characterization of the inducing compound in the carrot root extract suggests that the active substance is an acidic polysaccharide that is most likely derived from the pectic portion of the plant cell wall.     

Serological studies with Agrobacterium radiobacter,A. tumefaciens,and Rhizobium strains

Summary Antisera were prepared against cell material from 3 strains of A. radiobacter and 6 of A. tumefaciens. Agar diffusion and immune absorption techniques revealed 3 antigens common to each strain of these organisms. However, 5 different lipopolysaccharide antigens occurred in the 9 test strains. There was no obvious species differences in the distribution of these antigens. Mannose, and possibly glucuronic acid were immunologically active in one of the lipopolysaccharides.The agrobacterial antisera were further crosstested with antigenic material for 34 strains of Rhizobium. Fast-growing rhizobia showed extensive cross-reaction, but only one of 7 R. lupini strains tested reacted with any antiserum.     

The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA.

Agrobacterium tumefaciens transfers T-DNA into the plant genome by a process mediated by Ti plasmid-encoded vir genes. Cleavage at T-DNA border sequences by the VirD endonuclease generates linear, single-stranded T-DNA molecules. In the work described in this report, we used electrophoretic mobility shift assays to show that the purified virE2 gene product binds to single-stranded DNA. VirE2 protein associates with T-DNA as shown by immunoprecipitation studies with VirE2-specific antiserum. The VirE2 protein was detected primarily in the cytoplasm, but also in the inner and outer membrane and periplasmic fractions. Virulence of a virE2 mutant was restored by mixed infection with strains carrying an intact vir region, but not with virA, virB, virD, virE, or virG mutants or chvA, chvB, or exoC mutants. We propose that the VirE2 protein is involved in the processing of T-DNA and in T-strand protection during transfer to the plant cell.     

VirB7 lipoprotein is exocellular and associates with the Agrobacterium tumefaciens T pilus

Agrobacterium tumefaciens transfers oncogenic T-DNA and effector proteins to plant cells via a type IV secretion pathway. This transfer system, assembled from the products of the virB operon, is thought to consist of a transenvelope mating channel and the T pilus. When screened for the presence of VirB and VirE proteins, material sheared from the cell surface of octopine strain A348 was seen to possess detectable levels of VirB2 pilin, VirB5, and the VirB7 outer membrane lipoprotein. Material sheared from the cell surface of most virB gene deletion mutants also possessed VirB7, but not VirB2 or VirB5. During purification of the T pilus from wild-type cells, VirB2, VirB5, and VirB7 cofractionated through successive steps of gel filtration chromatography and sucrose density gradient centrifugation. A complex containing VirB2 and VirB7 was precipitated from a gel filtration fraction enriched for T pilus with both anti-VirB2 and anti-VirB7 antiserum. Both the exocellular and cellular forms of VirB7 migrated as disulfide-cross-linked dimers and monomers when samples were electrophoresed under nonreducing conditions. A mutant synthesizing VirB7 with a Ser substitution of the lipid-modified Cys15 residue failed to elaborate the T pilus, whereas a mutant synthesizing VirB7 with a Ser substitution for the disulfide-reactive Cys24 residue produced very low levels of T pilus. Together, these findings establish that the VirB7 lipoprotein localizes exocellularly, it associates with the T pilus, and both VirB7 lipid modification and disulfide cross-linking are important for T-pilus assembly. T-pilus-associated VirB2 migrated in nonreducing gels as a monomer and a disulfide-cross-linked homodimer, whereas cellular VirB2 migrated as a monomer. A strain synthesizing a VirB2 mutant with a Ser substitution for the reactive Cys64 residue elaborated T pilus but exhibited an attenuated virulence phenotype. Dithiothreitol-treated T pilus composed of native VirB2 pilin and untreated T pilus composed of the VirB2C64S mutant pilin distributed in sucrose gradients more predominantly in regions of lower sucrose density than untreated, native T pili. These findings indicate that intermolecular cross-linking of pilin monomers is not required for T-pilus production, but cross-linking does contribute to T-pilus stabilization.