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.     

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.     

Agrobacterium tumefaciens Site Attachment as a Necessary Prerequisite for Crown Gall Tumor Formation on Potato Discs

The infectivity of Agrobacterium tumefaciens strain B6 was inhibited about 50% when these bacteria were inoculated on potato discs with equal viable cell counts of a weakly virulent strain of A. tumefaciens (B-48) or autoclaved strains of B6 or B-48. Inhibition by B-48 or autoclaved B6 could still be obtained when these cells were added up to a maximum of 10 minutes after the addition of viable B6. Maximum inhibition occurred when these cells were added 10 minutes prior to the addition of B6. There was no inhibition observed when equal cell counts of B6 were added along with a Gram-positive bacterium or yeast cell, while inhibition was observed when these B6 cells were added simultaneously with other Gram-negative cells. These results suggest that a physical, specific bacterial attachment that occurs within 10 minutes is necessary for tumor formation on potato discs.     

Correlation between Binding Affinity and Necrosis-Inducing Activity of Mutant AVR9 Peptide Elicitors

The race-specific peptide elicitor AVR9 of the fungus Cladosporium fulvum induces a hypersensitive response only in tomato (Lycopersicon esculentum) plants carrying the complementary resistance gene Cf-9 (MoneyMaker-Cf9). A binding site for AVR9 is present on the plasma membranes of both resistant and susceptible tomato genotypes. We used mutant AVR9 peptides to determine the relationship between elicitor activity of these peptides and their affinity to the binding site in the membranes of tomato. Mutant AVR9 peptides were purified from tobacco (Nicotiana clevelandii) inoculated with recombinant potato virus X expressing the corresponding avirulence gene Avr9. In addition, several AVR9 peptides were synthesized chemically. Physicochemical techniques revealed that the peptides were correctly folded. Most mutant AVR9 peptides purified from potato virus X::Avr9-infected tobacco contain a single N-acetylglucosamine. These glycosylated AVR9 peptides showed a lower affinity to the binding site than the nonglycosylated AVR9 peptides, whereas their necrosis-inducing activity was hardly changed. For both the nonglycosylated and the glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membrane-localized binding site and their necrosis-inducing activity in MoneyMaker-Cf9 tomato was found. The perception of AVR9 in resistant and susceptible plants is discussed.     

Differential accumulation of proteinase inhibitor I in normal and crown gall tissue of tobacco, tomato, and potato

A proteinase inhibitor (inhibitor I) is induced in crown gall tumors of tobacco (Nicotiana tabacum) initiated through infection with the tumorinducing bacterium, Agrobacterium tumefaciens, strains B6 or CG-14. Uninfected tissues do not contain immunologically detectable quantities of inhibitor I. Inhibitor I synthesis in tobacco crown gall tumors paralleled tumor growth at the average rate of about 4.5 μg of inhibitor I per 200 mg of fresh tissue per day. Infection of variegated tobacco mutant Dp-I with A. tumefaciens strain CG-14 produced tumors with 25% more inhibitor than tumors induced with strain B6. Unlike tobacco, tumors induced by either bacterial strain on potato (Solanum tuberosum) and on tomato (Lycopersicum esculentum) did not accumulate inhibitor I. Consequently, inhibitor I accumulation is modulated by the type of plant host used in spite of familial relatedness (Solanaceae) and the strain of A. tumefaciens used for infection.     

Crown-gall tumors possess tumor-specific antigenic sites on their cell walls

Rabbits were injected with cell walls obtained from crown-gall tumor tissue or the corresponding cell walls from normal potato tissue. The serum obtained from rabbits 53 days after they were injected with tumor cell walls contained immunoglobins that reacted with both tumor and normal cell walls as well as with the cells from the inciting strain of Agrobacterium tumefaciens. When this serum was repeatedly absorbed against normal cell walls and the cells of the inciting strain of Agrobacterium tumefaciens, only tumor-specific immunoglobins remained. These immunoglobins did not react with cell walls obtained from meristematic (nontumorous) potato tissue. Yet this same serum reacted with crown-gall tumor cell walls obtained from turnip and carrot discs.     

A simple method for in planta tomato transformation by inoculating floral buds with a sticky Agrobacterium tumefaciens suspension

Tomato transformation is conventionally performed using Agrobacterium tumefaciens-infected cotyledons. Here, we propose a simple procedure for tomato transformation, by which A. tumefaciens cells were smeared onto floral buds of a tomato plant using a paintbrush. Sufficient numbers of fruits were obtained from them, although the smearing of an excess number of A. tumefaciens cells led to an adverse effect on the plant growth. Progeny plants were screened by growth on a kanamycin-containing selection medium plate. The nptII gene was detected in 10 plants among 1,599 progenies. These transformants were derived from fruits other than those obtained from the smeared buds. This suggested that A. tumefaciens cells moved to the buds located near the smeared buds and caused the transformation event. Our findings suggest that this procedure can be used for the introduction of a foreign gene into plant cells.     

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.     

A Method for Eliminating the Contribution of Agrobacterium tumefaciens to Studies of Nucleic Acid Biosynthesis in Crown Gall Tumors

A procedure is described that permits the use of radiotracer methods for examining crown gall tumors in the presence of the initiating agent Agrobacterium tumefaciens. On the basis of sensitivity testing using the disc diffusion technique and the minimum inhibitory concentration procedure and incorporation of radioactive phosphorus into nucleic acids, it was determined that A. tumefaciens was most sensitive to tetracycline, oxytetracycline, and methacycline. It was further demonstrated that nucleic acid synthesis in tumor tissue from four representative plants including tomato (Lycopersicum esculentum L.), Bryophyllum daigremontiana (Hamet and Perrier), pinto beans (Phaseolus vulgaris L.), and carrot (Daucus carota L.) was unaffected by a concentration of tetracycline (50 μg/ml) which completely inhibited nucleic acid synthesis in the bacterium. On this basis a procedure is described which first eliminates surface contamination and then uses tetracycline to nullify the contribution of A. tumefaciens in radioactive studies using tumors initiated by this bacterium.     

Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan? assay

A Serratia plymuthica-specific TaqMan? assay was designed based on the consensus nucleotide sequence from the 3??- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various species that can potentially coexist with S. plymuthica in the same environment. Positive reactions in the TaqMan? assay were observed only for S. plymuthica isolates and not for other bacteria. The TaqMan? assay could detect down to 1.95 ng of S. plymuthica DNA, down to 5 bacterial cells per reaction (100?cfu ml^?1) in vitro, down to 50 bacterial cells per reaction (1,000?cfu ml^?1) in spiked potato root extracts and down to 5 bacterial cells per reaction (100?cfu ml^?1) in spiked potato haulm extracts. We used this assay to quantify S. plymuthica A30 cells in potato and tomato haulms and roots grown from S. plymuthica A30-inoculated potato seed tubers and tomato seeds. The results were comparable with the spread-plating of plant extracts on a newly developed S. plymuthica A30 selective medium (CVTR2Arif). The TaqMan? assay can be used to quantify S. plymuthica isolates in different ecosystems and in complex substrates.     

CROWN GALL TUMORIGENESIS IN POTATO TUBER TISSUE

Potato tuber discs were examined as a possible quantitative bioassay for studying tumor induction by Agrobacterium tumefaciens. Discs from two potato varieties, Pontiac and Russett Burbank, were inoculated and cultured on water agar plates. Tumors appeared within 10 days. Both the number and weight of tumors per disc increased linearly for inoculum concentration between 1 × 10⁷–1 × 10⁹ cells per ml. Polarity, position of the disc relative to tuber epidermis, potato variety and the light conditions did not influence the tumor formation. The simplicity of the procedure and the homogeneity of the tissue, together with the fact that it is a quantitative bioassay, makes the potato tuber disc an ideal system for the investigation of biochemical step(s) associated with the transformation process.     

Role of bacterial lipopolysaccharide in attachment of agrobacterium to moss

Gametophore induction in moss by Agrobacterium tumefaciens was inhibited by addition of lipopolysaccharide (LPS) from A. tumefaciens. The LPS did not affect bacterial viability or appear to bind to bacterial cells. LPS from nonbinding Agrobacterium radiobacter was not effective in reducing gametophore formation. A. tumefaciens LPS, if added 24 hours after addition of viable bacterial cells, had no effect in reducing gametophore formation. The polysaccharide portion of the LPS was identified as the binding component necessary for attachment of agrobacteria for induction of gametophores in moss and tumors in higher plants.     

Use of phosphomannose isomerase-based selection system for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of tomato and potato

Two selection systems for Agrobacterium tumefaciens mediated transformation of tomato and potato were compared. In the tomato (Lycopersicon esculentum cv. Moneymaker), the highest transformation rate, 4.2 %, of cotyledon explants on mannose-selection medium was obtained when mannose/sucrose concentration in the regeneration medium was 5/15 g dm⁻³. The best transformation efficacy with the commonly used concentration of 100 mg dm⁻³ kanamycin as a selection agent was 9 %. In the potato (Solanum tuberosum cv. Bintje), the highest transformation frequency was 53.3 % when mannose concentration in the regeneration medium was 5 g dm⁻³ during the first 3 weeks after transformation and 10 g dm⁻³ afterwards. The optimum concentration of sucrose was 20 g dm⁻³. The transformation efficiency using kanamycin as a selection agent at a concentration 100 mg dm⁻³ was 33.3 % with potato. Our results demonstrate that the transformation efficiency using mannose selection is 1.6-fold higher for potato and about 2 times lower for tomato comparing with the ordinary protocol using kanamycin.     

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.     

A Conserved Mechanism of GABA Binding and Antagonism Is Revealed by Structure-Function Analysis of the Periplasmic Binding Protein Atu2422 in Agrobacterium tumefaciens

Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus flytrap modules) of G-protein-coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422, which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution x-ray structures of Atu2422 liganded to GABA, Pro, Ala, and Val and of point mutant Atu2422-F77A liganded to Leu were determined. Structural analysis of the ligand-binding site revealed two essential residues, Phe⁷⁷ and Tyr²⁷⁵, the implication of which in GABA signaling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe⁷⁷ restricts ligand specificity to α-amino acids with a short lateral chain, which act as antagonists of GABA signaling in A. tumefaciens. Tyr²⁷⁵ specifically interacts with the GABA γ-amino group. Conservation of these two key residues in proteins phylogenetically related to Atu2422 brought to light a subfamily of PBPs in which all members could bind GABA and short α-amino acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors and revealed their occurrence among host-interacting proteobacteria.     

The Conjugal Intermediate of Plasmid RSF1010 Inhibits Agrobacterium tumefaciens Virulence and VirB-Dependent Export of VirE2

Agrobacterium tumefaciens causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from A. tumefaciens to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 between Agrobacterium strains or from Agrobacterium to plants also requires the virB-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of Agrobacterium inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with virE mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.     

Transformation of Tobacco, Tomato, Potato, and Arabidopsis thaliana Using a Binary Ti Vector System

Using a binary tumor-inducing (Ti) plasmid vector system, several plant species were transformed with a kanamycin resistance marker (neomycin phosphotransferase gene). Four Nicotiana species, seven tomato cultivars, two potato cultivars, and Arabidopsis thaliana were transformed by the binary vector transformation method. In this method, various plant organ pieces were co-cultivated with Agrobacterium tumefaciens cells carrying the binary vector, pGA472, and a helper Ti plasmid. We have also demonstrated that a wild type Ti plasmid can be used as a helper to obtain a transformed plant.     

Role of the mitochondria in the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene in plant tissues

Intact plant mitochondria, isolated from climacteric (Lycopersicon esculentum, Mill., tomato) or non-climacteric (Solanum tuberosum, L., potato) tissues, and purified on Percoll density gradients, were unable to convert 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene. Energization or sonication did not enhance ethylene production. For both tissues, the low activity of ACC conversion found in crude mitochondrial fractions from both tissues was increased by sonication. After mitochondrial purification, this activity was located on top of the gradient together with the microsomal membrane fraction containing a high lipoxygenase activity. Addition of exogenous lipoxygenase and linoleic acid to isolated tomato or potato mitochondria greatly enhanced ACC conversion (to approx. 300 pmol h⁻¹ mg⁻¹ protein). Direct measurements of ACC uptake by mitochondria indicated that ACC uptake is not dependent on energization.     

Root Colonization by Agrobacterium tumefaciens Is Reduced in cel, attB, attD, and attR Mutants

Root colonization by Agrobacterium tumefaciens was measured by using tomato and Arabidopsis thaliana roots dipped in a bacterial suspension and planted in soil. Wild-type bacteria showed extensive growth on tomato roots; the number of bacteria increased from 10³ bacteria/cm of root length at the time of inoculation to more than 10⁷ bacteria/cm after 10 days. The numbers of cellulose-minus and nonattaching attB, attD, and attR mutant bacteria were less than 1/10,000th the number of wild-type bacteria recovered from tomato roots. On roots of A. thaliana ecotype Landsberg erecta, the numbers of wild-type bacteria increased from about 30 to 8,000 bacteria/cm of root length after 8 days. The numbers of cellulose-minus and nonattaching mutant bacteria were 1/100th to 1/10th the number of wild-type bacteria recovered after 8 days. The attachment of A. tumefaciens to cut A. thaliana roots incubated in 0.4% sucrose and observed with a light microscope was also reduced with cel and att mutants. These results suggest that cellulose synthesis and attachment genes play a role in the ability of the bacteria to colonize roots, as well as in bacterial pathogenesis.     

Spezifität beim „Attachment” von Agrobacterium an Wundrandzellen von Kalanchoe?

Specificity of the Attachment of Agrobacterium to Wound Cells of Kalanchoe? Tumor induction by A. tumefaciens on leaves of Kalanchoe is severely inhibited by cell wall preparations from young and mature tissucs of monocotyledons and dicoryledons and from tumor cells and also by filter paper homogenates and living or dead Pseudomonas cells. The inhibition a demonstrable if the cell wall preparations or Pseudomonas cells are inoculated into the plant wounds at the same time with A tumefaciens or before A. tumefaciens a. Postinoculation does affect tumor These results demonstrate site binding as an important step of tumor induction without any doubt but at the same time they question the specificity of this process.