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.     

DEVELOPMENT AND GAMETOPHORE INITIATION IN THE MOSS PYLAISIELLA SELWYNII AS INFLUENCED BY AGROBACTERIUM TUMEFACIENS

Spores of the heterotrichous moss Pylaisiella selwynii Kindb. were sown in a defined inorganic liquid culture medium and incubated at 27 C with a 16-hr photoperiod. They germinated at 7–10 days, and formed a few caulonemal buds at 27–30 days which developed into gametophores by 40 days. Bud formation and gametophore development followed a pattern common to many mosses. Addition of a virulent strain of Agrobacterium tumefaciens (B6) to the moss cultures increased bud formation and hastened the time of their appearance by 5–6 days. With 10⁹ or more bacteria per ml of moss culture medium the percentage of plants with gametophores at day 35 after the spores were sown was 96 % or greater, as opposed to 0–24 % in the controls. The mean number of gametophores per responding plant was also increased from one per plant in controls to 4–6 per plant in inoculated cultures. Addition of the bacterium at day 17–18 of culture was as effective as early additions of the bacterium, suggesting that the moss must become ready to bud before the bacterium can influence its development. The promotion of gametophore formation was directly related to the number of bacteria added and depended upon the presence of viable bacteria. The supernatant from bacterial cultures did not promote gametophore formation. The changes induced by A. tumefaciens were similar to those reported for cytokinins.     

ADHERENCE OF AGROBACTERIA TO MOSS PROTONEMA AND GAMETOPHORES VIEWED BY SCANNING ELECTRON MICROSCOPY

The attachment of certain Agrobacterium species to moss protonema was examined by scanning electron microscopy. The bacteria cultured with the moss adhered to the protonemal filaments of Pylaisiella selwynii and Funaria hygrometrica throughout the fixation and washing procedure prior to examination. The number of bacteria adhering per unit length of filament did not correlate with the ability of four species (A. tumefaciens, A. rubi, A. rhizogenes, A. radiobacter) to induce gametophore formation in P. selwynii. Little difference was found between the number of A. tumefaciens on P. selwynii or on F. hygrometrica, and although this bacterial species induced gametophores on P. selwynii, it had no effect on F. hygrometrica. A. rhizogenes, which is capable of inducing rhizoid formation on both P. selwynii and F. hygrometrica without physical attachment, did associate with filaments of both mosses. Larger numbers of A. tumefaciens and A. rubi, which induce gametophores or callus, were associated with the spore and germ tube of P. selwynii than were A. rhizogenes or A. radiobacter which do not initiate these changes. These results suggest that the germ tube may constitute the site where effective attachment occurs. The number of A. tumefaciens adhering to P. selwynii was greatest at 2–6 h after addition and then decreased by 26 h, raising the possibility that some bacteria lyse following attachment. None of the bacteria adhered to the gametophore walls of either moss, indicating that a change in cell wall surface may occur. The four species of Agrobacterium differed in their orientation when adhering to the moss. These results suggest that the process of bacterial adherence to higher plant cells in Agrobacterium infection is similar to that which we observe in mosses and which leads to developmental changes.     

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.     

Inhibition of cell division in a moss protonema by some cytokinin isomers

Summary The cytokinin isomers, 1- and 9-isopentenyladenine, are very active inhibitors of cell division in the protonema of the moss Ceratodon purpureus. They also abolish the N⁶-isopentenyladenine-induced stimulation of cell divisions in this material whereas they are inactive on the induction of gametophore buds in moss protonemata by cytokinins.This work was supported by the Polish Academy of Sciences within the project 09.3.1.     

Ti plasmid containing Rhizobium meliloti are non-tumorigenic on plants,despite proper virulence gene induction and T-strand formation

We examined the expression of the vir genes of the Agrobacterium tumefaciens Ti plasmid in Rhizobium meliloti, which remains non-tumorigenic on plants after introduction of a Ti- or Ri-plasmid. Both the levels of virulence (vir) gene expression, induced by the plant phenolic compound acetosyringone, and of subsequent T-strand formation were comparable to what is observed in Agrobacterium. In contrast to the situation in Agrobacterium, though, vir induction in R. meliloti did not require a low pH (5.3) of the induction medium and the optimum temperature for induction in R. meliloti was significantly lower than in Agrobacterium. At 37°C no induction of the vir genes was found both in Agrobacterium and R. meliloti. We postulate that the lack of tumorigenicity of Ti carrying R. meliloti strains is due either to a lack of proper attachment of the bacteria to plant cells, or to an improper assembly of a virB-determined essential structure in the cell wall of R. meliloti.     

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.     

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.     

Acclimation and endogenous abscisic acid in the moss Physcomitrella patens during acquisition of desiccation tolerance

The moss Physcomitrella patens has been used as a model organism to study the induction of desiccation tolerance (DT), but links between dehydration rate, the accumulation of endogenous abscisic acid (ABA) and DT remain unclear. In this study, we show that prolonged acclimation of P. patens at 89% relative humidity (RH) [?16 MPa] can induce tolerance of desiccation at 33% RH (?153 MPa) in both protonema and gametophore stages. During acclimation, significant endogenous ABA accumulation occurred after 1 day in gametophores and after 2 days in protonemata. Physcomitrella patens expressing the ABA‐inducible EARLY METHIONINE promoter fused to a cyan fluorescent protein (CFP) reporter gene revealed a mostly uniform distribution of the CFP increasing throughout the tissues during acclimation. DT was measured by day 6 of acclimation in gametophores, but not until 9 days of acclimation for protonemata. These results suggest that endogenous ABA accumulating when moss cells experience moderate water loss requires sufficient time to induce the changes that permit cells to survive more severe desiccation. These results provide insight for ongoing studies of how acclimation induces metabolic changes to enable DT in P. patens.     

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.     

Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division

Development of Physcomitrella patens (Hedw.) B.S.G. starts with a filamentous protonema growing by apical cell division. As a developmental switch, some subapical cells produce three-faced apical cells, the so-called buds, which grow to form leafy shoots, the gametophores. Application of cytokinins enhances bud formation but no subsequent gametophore development in several mosses. We used the ipt gene of Agrobacterium tumefaciens, encoding a protein which catalyzes the rate-limiting step in cytokinin biosynthesis, to transform two developmental Physcomitrella mutants. One mutant (P24) was defective in budding (bud) and thus did not produce three-faced cells, while the other one (PC22) was a double mutant, defective in plastid division (pdi), thus possessing at the most one giant chloroplast per cell, and in gametophore development (gad), resulting in malformed buds which could not differentiate into leafy gametophores. Expression of the ipt gene rescued the mutations in budding and in plastid division but not the one in gametophore development. By mutant rescue we provide evidence for a distinct physiological difference between externally applied and internally produced cytokinins. Levels of immunoreactive cytokinins and indole-3-acetic acid were determined in tissues and in culture media of the wild-type moss, both mutants and four of their stable ipt transformants. Isopentenyl-type cytokinins were the most abundant cytokinins in Physcomitrella, whereas zeatin-type cytokinins, the major native cytokinins of higher plants, were not detectable. Cytokinin as well as auxin levels were enhanced in ipt transgenics, demonstrating a cross-talk between both metabolic pathways. In all genotypes, most of the cytokinin and auxin was found extracellularly. These extracellular pools may be involved in hormone transport in the non-vascular mosses. We suggest that both mutants are defective in signal-transduction rather than in cytokinin metabolism. Received: 24 October 1997 / Accepted: 20 March 1998     

EFFECT OF HORMONES AND VITAMIN B12 ON GAMETOPHORE DEVELOPMENT IN THE MOSS PYLAISIELLA SELWYNII

Bud formation in the moss Pylaisiella selwynii is greatly enhanced by cytokinins at concentrations as low as 10⁻¹²m , yet these buds usually fail to develop into normal gametophores. Various ratios at different concentrations of the cytokinin N-6-γ,γ-dimethylallylaminopurine to indoleacetic acid failed to enhance bud initiation over that obtained with cytokinin alone or to permit normal gametophore development. Deletion of the cobaltous ions from the culture medium prevented the appearance of the few gametophores usually formed in the complete medium, but different amounts of cobaltous ion did not significantly enhance initiation of gametophore development. Bud initiation was enhanced 3- to 20-fold by vitamin B₁₂ at 10⁻⁵m or by B₁₂ coenzyme at 10⁻⁴m , and the time of appearance of these buds was advanced by 6–12 days compared to control plants. At these concentrations of the B₁₂ compounds the buds formed normal gametophores, but at 10⁻⁴m vitamin B₁₂ they grew into callus-like masses similar to those obtained with cytokinins. Although the effects of B₁₂ on bud initiation and development mimicked those of cytokinins, except in permitting normal development, no additive or synergistic effects were observed when they were tested together. It is suggested that B₁₂ may play a regulatory role in the control of gametophore initiation and development in mosses.     

Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts

Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the delivery method. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an Agrobacterium-mediated standard binary vector system to target maize immature embryos. Eleven maize inbred lines were pre-screened for transformation frequency using N6 salts. A subset of three maize inbred lines was then systematically evaluated for frequency of post-infection embryogenic callus induction and transformation on four media regimes: N6 or MS salts in each of two distinct media backgrounds. Transgenic plants recovered from inbred lines B104, B114, and Ky21 were analyzed for transgene integration, expression, and transmission. Average transformation frequencies of 6.4% (for B104), 2.8% (for B114), and 8% (for Ky21) were achieved using MS salts. Availability of Agrobacterium-mediated maize inbred line transformation will improve future opportunities for maize genetic and functional genomic studies.     

Centrifugation Assisted Agrobacterium tumefaciens-mediated Transformation (CAAT) of embryogenic cell suspensions of banana (Musa spp. Cavendish AAA and Lady finger AAB)

Centrifugation-assisted Agrobacterium-mediated transformation (CAAT) protocol, developed using banana cultivars from two economically important genomic groups (AAA and AAB) of cultivated Musa, is described. This protocol resulted in 25-65 plants/50mg of settled cell volume of embryogenic suspension cells, depending upon the Agrobacterium strain used, and gave rise to hundreds of morphologically normal, transgenic plants in two banana cultivars from the two genomic groups. Development of a highly efficient Agrobacterium-mediated transformation protocol for a recalcitrant species like banana, especially the Cavendish group (AAA) cultivars, required the identification and optimisation of the factors affecting T-DNA delivery and subsequent plant regeneration. We used male-flower-derived embryogenic cell suspensions of two banana cultivars (Cavendish and Lady Finger) and Agrobacterium strains AGL1 and LBA4404, harbouring binary vectors carrying hpt (hygromycin phosphotransferase) and gusA (-glucuronidase) or nptII (neomycin phosphotransferase) and a modified gfp (green fluorescent protein) gene in the T-DNA, to investigate and optimise T-DNA delivery and tissue culture variables. Factors evaluated included pre-induction of Agrobacterium, conditions and media used for inoculation and co-cultivation, and the presence of acetosyringone and Pluronic F68 in the co-cultivation media. One factor that led to a significant enhancement in transformation frequency was the introduction of a centrifugation step during co-cultivation. Post co-cultivation liquid-media wash and recovery step helped avoid Agrobacterium overgrowth on filters supporting suspension culture cells. Marker-gene expression and molecular analysis demonstrated that transgenes integrated stably into the banana genome. T-DNA:banana DNA boundary sequences were amplified and sequenced in order to study the integration profile.     

Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium

Summary T-DNA circularization is one of the molecular events specifically induced in agrobacterial cells upon their infection of dicotyledonous plant cells. We developed a seedling co-cultivation procedure to determine whether or not monocotyledonous plants have the ability to induce T-DNA circularization and vir gene expression. Co-cultivation of Agrobacterium tumefaciens with seedlings of dicotyledonous plants showed that the circularization event takes place efficiently. The exudates and extracts of the seedlings also effectively induced T-DNA circularization and vir gene expression, indicating that dicotyledonous seedlings contain diffusible factors capable of inducing these molecular events. In contrast, neither T-DNA circularization nor vir gene expression was detectable when Agrobacterium was incubated with seedlings of monocotyledonous plants. Supplementing with acetosyringone, a known inducer of vir gene expression and T-DNA circularization, resulted in the induction of circularization during co-cultivation with monocotyledonous seedlings. These results indicate that the seedlings of monocotyledonous plants have no detectable amounts of diffusible inducers, unlike dicotyledonous seedlings. Therefore, it is unlikely that the vir genes are expressed in Agrobacterium inoculated in monocotyledonous plants. This may be one of the blocks in tumorigenesis of monocotyledonous plants by Agrobacterium.     

Rapid,in situ detection of Agrobacterium tumefaciens attachment to leaf tissue

Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging‐based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum‐infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post‐infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Tissue specific response of Agrobacterium tumefaciens attachment to Sorghum bicolor (L) Moench

Agrobacterium mediated genetic transformation of plants have advantages over other methods, especially for making single copy transgenic plants with reduced chances of gene silencing and instability. However, monocotyledonous plant species could not utilize the full potential of this system because of possible limitations in Agrobacterium interaction with monocot plant cells. Agrobacterium attachment as a factor in genetic transformation was studied in the leaf, shoot apex, and leaf derived callus of sorghum (Sorghum bicolor (L) Moench). Pre-induction of Agrobacterium with acetosyringone was found necessary for Agrobacterium attachment to sorghum tissues. All the explants responded positively, with preferential Agrobacterium attachment and colonization around the tissues having actively dividing cells. Callus proved to be the best explant for Agrobacterium attachment as observed in scanning electron microscopy and transient GUS expression. Loss of Agrobacterium attachment was observed with an increase in the degree of tissue differentiation.Key words: Genetic transformation, Acetosyringone, Scanning electron microscopy, Transient gene expression, GUS assays, qRT-PCR     

Lifeact-mEGFP Reveals a Dynamic Apical F-Actin Network in Tip Growing Plant Cells

Background

Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments.

Methodology/Principal Findings

In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore.

Conclusions/Significance

Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin''s role in tip growing plant cells.