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PubMed1990

vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens.

Mutation of the genes virA, virB, virC, and virG of the Agrobacterium tumefaciens octopine-type Ti plasmid pTiR10 was found to cause a 100- to 10,000-fold decrease in the frequency of conjugal transfer of this plasmid between Agrobacterium cells. This effect was not absolute, however, in that it occurred only during early times (18 to 24 h) of induction of the conjugal transfer apparatus by octopine. Induction of these mutant Agrobacterium strains by octopine for longer periods (48 to 72 h) resulted in a normal conjugal transfer frequency. The effect of these vir gene mutations upon conjugation could be restored by the introduction of cosmids harboring wild-type copies of the corresponding disrupted vir genes into the mutant Agrobacterium strains. In addition, transfer of the self-mobilizable plasmid pPH1JI was not impaired in any of the mutant Agrobacterium strains tested. The effect of vir gene function on the conjugal transfer of the Ti plasmid suggests that a relationship may exist between the processes that control the transfer of the T-DNA from Agrobacterium to plant cells and the conjugal transfer of the Ti plasmid between bacterial cells.

Bacterial Proteins↗

PubMed1992

Two-way chemical signaling in Agrobacterium-plant interactions.

The discovery in 1977 that Agrobacterium species can transfer a discrete segment of oncogenic DNA (T-DNA) to the genome of host plant cells has stimulated an intense interest in the molecular biology underlying these plant-microbe associations. This attention in turn has resulted in a series of insights about the biology of these organisms that continue to accumulate at an ever-increasing rate. This excitement was due in part to the notion that this unprecedented interkingdom DNA transfer could be exploited to create transgenic plants containing foreign genes of scientific or commercial importance. In the course of these discoveries, Agrobacterium became one of the best available models for studying the molecular interactions between bacteria and higher organisms. One extensively studied aspect of this association concerns the exchange of chemical signals between Agrobacterium spp. and host plants. Agrobacterium spp. can recognize no fewer than five classes of low-molecular-weight compounds released from plants, and other classes probably await discovery. The most widely studied of these are phenolic compounds, which stimulate the transcription of the genes needed for infection. Other compounds include specific monosaccharides and acidic environments which potentiate vir gene induction, acidic polysaccharides which induce one or more chromosomal genes, and a family of compounds called opines which are released from tumorous plant cells to the bacteria as nutrient sources. Agrobacterium spp. in return release a variety of chemical compounds to plants. The best understood is the transferred DNA itself, which contains genes that in various ways upset the balance of phytohormones, ultimately causing neoplastic cell proliferation. In addition to transferring DNA, some Agrobacterium strains directly secrete phytohormones. Finally, at least some strains release a pectinase, which degrades a component of plant cell walls.

Cell Communication↗

PubMed1990

Agrobacterium in plant disease, biological disease control and plant genetic engineering.

Plant pathogenic strains of Agrobacterium cause crown gall and hairy-root diseases. The abnormal cell proliferation of diseased tissues results from elevated plant hormonal levels within them. These levels are consequent upon the transfer of DNA portions (T-DNA) from Agrobacterium plasmids to host nuclei where they are integrated and where genes for hormone synthesis, borne on the T-DNA, are expressed in transformed host cells. This process has been exploited in plant genetic engineering by producing Agrobacterium mutants with deleterious genes removed from T-DNA, and with selected beneficial genes inserted in their place. Such mutants are able to infect and transform plants so that the beneficial genes are transferred to, and expressed in, the host plants, endowing them with desirable characteristics, including insect and disease resistance and herbicide tolerance. Biological control of Agrobacterium disease has been achieved using non-pathogenic Agrobacterium strains which produce an antibiotic specifically inhibitory to Agrobacterium pathogens. Use of one of these biocontrol strains is the first instance of a genetically engineered micro-organism being released commercially.

DNA Mutational Analysis↗

PubMed1998

A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation.

The introduction of binary plasmids into Agrobacterium hosts for Agrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular combinations of Agrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized. We have observed that there is significant variation between transformed Agrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of three Argobacterium tumefaciens (EHA101, LBA4404, C58) and two Agrobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin(res), kanamycin(res), and gentamycin(res)).

Agrobacterium tumefaciens↗

PubMed1999

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda).

Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

Acetophenones↗

PubMed2002

Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation.

Transformation of plant cells by Agrobacterium tumefaciens involves both bacterial virulence proteins and host proteins. We have previously shown that the Arabidopsis thaliana gene H2A-1 (RAT5), which encodes histone H2A-1, is involved in T-DNA integration into the plant genome. Mutation of RAT5 results in a severely decreased frequency of transformation, whereas overexpression of RAT5 enhances the transformation frequency (Mysore et al., 2000b). We show here that the expression pattern of RAT5 correlates with plant root cells most susceptible to transformation. As opposed to a cyclin-GUS fusion gene whose expression is limited to meristematic tissues, the H2A-1 gene is expressed in many non-dividing cells. Under normal circumstances, the H2A-1 gene is expressed in the elongation zone of the root, the region that is most susceptible to Agrobacterium transformation. In addition, when roots are incubated on medium containing phytohormones, a concomitant shift in H2A-1 expression and transformation susceptibility to the root base is observed. Inoculation of root segments with a transfer-competent, but not a transformation-deficient Agrobacterium strain induces H2A-1 expression. Furthermore, pre-treatment of Arabidopsis root segments with phytohormones both induces H2A-1 expression and increases the frequency of Agrobacterium transformation. Our results suggest that the expression of the H2A-1 gene is both a marker for, and a predictor of, plant cells most susceptible to Agrobacterium transformation.

Agrobacterium tumefaciens↗

PubMed2003

Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression.

Agrobacterium tumefaciens is a plant pathogen that incites crown gall tumors by transferring to and expressing a portion of a resident plasmid in plant cells. Currently, little is known about the host response to Agrobacterium infection. Using suppressive subtractive hybridization and DNA macroarrays, we identified numerous plant genes that are differentially expressed during early stages of Agrobacterium-mediated transformation. Expression profiling indicates that Agrobacterium infection induces plant genes necessary for the transformation process while simultaneously repressing host defense response genes, thus indicating successful utilization of existing host cellular machinery for genetic transformation purposes. A comparison of plant responses to different strains of Agrobacterium indicates that transfer of both T-DNA and Vir proteins modulates the expression of host genes during the transformation process.

Agrobacterium tumefaciens↗

PubMed2001

Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue.

A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated. The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here. The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of sonication treatments, and Agrobacterium cell density. Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated. Increasing Agrobacterium cell density, the duration of inoculation/co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression. The investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue.

Agrobacterium tumefaciens↗

PubMed2006

Integration of genes into the chromosome of Agrobacterium tumefaciens C58.

Agrobacterium tumefaciens has been widely used to transform numerous plant species. Frequently, investigators want to place more than one gene into Agrobacterium in order to manipulate various bacterial functions during plant genetic transformation. These genes are frequently brought into the bacterium by multiple plasmids. It is difficult to maintain several plasmids in the same bacterium without the use of a variety of antibiotics. However, the use of some antibiotics is not feasible for certain Agrobacterium strains. Also, too many different antibiotics in the culture medium generally results in slow growth of Agrobacterium. Therefore, it is to one's advantage to place genes of interest onto the bacterial chromosome to eliminate the use of multiple antibiotics during bacterial growth. This chapter describes in detail a method to integrate a gene of interest into the pgl/picA locus of the Agrobacterium tumefaciens C58 chromosome. The integrated gene is stably maintained as single copy per cell without the need for selection.

Agrobacterium tumefaciens↗

PubMed2003

Agrobacterium-mediated transient GUS gene expression in buffel grass (Cenchrus ciliaris L.).

The study was conducted to standardize a protocol for Agrobacterium-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.). Embryogenic calli, produced from one-year-old mature seeds of buffel grass, were used as target cells for Agrobacterium-mediated transformation. A. tumefaciens strain LBA4404, harbouring pCAMBIA-1301 or pCAMBIA-2301, was used for co-cultivation with embryogenic calli from three genotypes (IG-3108, IG-9757 and IG-97101). Co-culturing of calli with Agrobacterium for 30 minutes, followed by co-cultivation with 0.1 mM acetosyringone for 3 days was found to be optimum for maximum transformation efficiency. Presence of acetosyringone during co-cultivation was found to be necessary for transformation. Transient GUS (beta-glucuronidase) gene expression was used to monitor T-DNA delivery into the target cells. Significant genotypic variations in response to transformation were observed among the tested genotypes. A very high frequency (63.3%) of GUS gene expression was obtained following Agrobacterium-mediated gene transfer into embryogenic calli. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of buffel grass with genes of agronomic importance.

Agrobacterium tumefaciens↗

PubMed2026

Development of an Efficient Regeneration and Agrobacterium-Mediated Transformation Protocol for Hosta 'Light Star' Using the RUBY Reporter Gene.

Hosta plantaginea is a perennial shade-tolerant herb of the Liliaceae family, with high ornamental and urban greening value. Hosta 'Light Star' is a newly developed ornamental cultivar with yellow-margined leaves and lilac flowers, but no efficient in vitro regeneration or genetic transformation system has been established for this cultivar to date. In this study, we established a highly efficient in vitro regeneration system for Hosta 'Light Star,' and developed an Agrobacterium-mediated genetic transformation protocol using the RUBY visual reporter gene for non-invasive screening of positive transformants. The optimal callus induction medium was MS + 2 mg/L 6-BA + 0.3 mg/L NAA + 0.05 mg/L 2, 4-D, with a callus induction rate of 53.33% for leaf explants (the optimal explant for sterile seedlings). The optimal adventitious bud proliferation medium was MS + 2 mg/L 6-BA + 0.1 mg/L NAA, with a proliferation coefficient of 5.87. The optimal rooting medium was 1/2 MS + 0.5 mg/L NAA + 0.5 mg/L IBA, with a 100% rooting rate. The optimal transplant substrate was perlite:vermiculite = 2:1, with a 100% transplant survival rate after acclimatization. For Agrobacterium-mediated transformation, the optimal infection parameters were as follows: Agrobacterium suspension OD600 = 0.6, infection time of 10 min, and 200 μM acetosyringone; the optimal selection conditions were 300 mg/L cefotaxime for bacteriostasis and 30 mg/L hygromycin for transformant screening. The final stable transformation efficiency was 2.50% (95% CI 1.23-3.77%), with an escape rate of 16.13%. Transgenic plants showed distinct purplish-red coloration in roots, stems, and leaves, with significantly higher betacyanin accumulation than wild-type plants (p < 0.05). Stable integration and expression of the RUBY gene were confirmed by PCR, RT-PCR, and RT-qPCR. This study establishes the first efficient regeneration and Agrobacterium-mediated transformation system for Hosta 'Light Star,' and validates the feasibility of the RUBY reporter gene as a visual marker for Hosta transformation. This system provides a solid technical platform for functional genomic studies, CRISPR/Cas9-mediated gene editing, and molecular breeding of ornamental traits in Hosta.

Transformation, Genetic↗

PubMed1990

Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation.

Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to overcome this obstacle a modified prokaryotic indicator gene was constructed. The expression of this indicator gene and its use in analysing early events in Agrobacterium-mediated plant transformation are described. A portable intron, derived from a plant intron, was introduced into the beta-glucuronidase (GUS) gene. In transgenic plants containing this chimaeric gene the intron is spliced efficiently, giving rise to GUS enzymatic activity. Mapping of the splice junction indicates the exact removal of the intron. No GUS activity is detected in agrobacteria containing this construct due to the lack of a eukaryotic splicing apparatus in prokaryotes. Early phases after transformation of Arabidopsis cotyledon explants were analysed using this GUS-intron chimaeric gene showing that as early as 36 h after Agrobacterium infection significant GUS activity is detected. In vivo GUS staining of transformed cells clearly shows that quickly proliferating calli expressing GUS activity are formed, mainly at the cut surface. Minor transformation events occur however throughout the whole cotyledon. These data indicate that Agrobacterium-mediated T-DNA transfer to plants is much more efficient than has been judged from experiments where selection is applied immediately. The intron-containing GUS gene can be used as an optimised marker gene in transient and stable transformation experiments.

Base Sequence↗

PubMed2003

Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection.

An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study-selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50-80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of theAROA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.

Biolistics↗

PubMed2004

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques.

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.

Agriculture↗

PubMed1998

Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting.

To investigate early events of Agrobacterium-mediated transformation of apple cultivars, a synthetic green fluorescent protein gene (SGFP) was used as a highly sensitive, vital reporter gene. Leaf explants from four apple cultivars ('Delicious', 'Golden Delicious', 'Royal Gala' and 'Greensleeves') were infected with Agrobacterium EHA101 harboring plasmid pDM96.0501. Fluorescence microscopy indicated that SGFP expression was first detected 48 h after infection and quantitative analysis revealed a high T-DNA transfer rate. Plant cells with stably incorporated T-DNA exhibited cell division and developed transgenic calli, followed by formation of transgenic shoots at low frequencies. The detection of SGFP expression with an epifluorescence stereomicroscope confirmed the effectiveness of SGFP as a reporter gene for detection of very early transformation events and for screening of putative transformants. The efficiency of the transformation and regeneration process decreased ca. 10,000-fold from Agrobacterium infection to transgenic shoot regeneration, suggesting that factors other than Agrobacterium interaction and T-DNA transfer are rate-limiting steps in Agrobacterium-mediated transformation of apple.

Blotting, Southern↗

PubMed2007

Biological systems of the host cell involved in Agrobacterium infection.

Genetic transformation of plants by Agrobacterium, which in nature causes neoplastic growths, represents the only known case of trans-kingdom DNA transfer. Furthermore, under laboratory conditions, Agrobacterium can also transform a wide range of other eukaryotic species, from fungi to sea urchins to human cells. How can the Agrobacterium virulence machinery function in such a variety of evolutionarily distant and diverse species? The answer to this question lies in the ability of Agrobacterium to hijack fundamental cellular processes which are shared by most eukaryotic organisms. Our knowledge of these host cellular functions is critical for understanding the molecular mechanisms that underlie genetic transformation of eukaryotic cells. This review outlines the bacterial virulence machinery and provides a detailed discussion of seven major biological systems of the host cell-cell surface receptor arrays, cellular motors, nuclear import, chromatin targeting, targeted proteolysis, DNA repair, and plant immunity--thought to participate in the Agrobacterium-mediated genetic transformation.

Active Transport, Cell Nucleus↗

PubMed1987

Complementary methodologies to identify specific agrobacterium strains.

Serological techniques and restriction enzyme cleavage patterns of total DNA were used to differentiate strains of Agrobacterium spp. Forty-five wild-type and plasmid-cured Agrobacterium strains were tested by immunodiffusion and immunofluorescence against polyclonal antisera to a crude ribosome preparation from Agrobacterium strains K84, U11, B6, A323, NT1, and C58. In immunodiffusion gels, these antisera reacted only with water-phenol extracts of the homologous strain, producing a single, strain-specific precipitin line. In contrast, when the same antisera were used in immunofluorescence staining, cross-reactions occurred with a limited number of heterologous Agrobacterium strains. However, the cross-reacting heterologous cells fluoresced generally less brightly than the homologous cells. When the EcoRI-digested DNA profiles from the same Agrobacterium strains were compared, 34 distinct cleavage patterns were observed. The DNA profiles were the same for all strains sharing a common chromosomal background and correlated with the strain-specific serological reaction. The presence or absence of plasmid DNA did not alter the strain-specific serological reaction or the DNA cleavage patterns. Both the serological reaction and the restriction enzyme digestion of total DNA were complementary to each other. These methods were used successfully to identify A. radiobacter K84 strains which were recovered 6 months after being inoculated to young trees in the field.

Journal Article↗

PubMed1987

Isolation of different agrobacterium biovars from a natural oak savanna and tallgrass prairie.

Populations of agrobacteria in excess of 10 CFU/g were recovered from 12 soil and root samples obtained from the Allison Savanna, Minn., a natural oak savanna and tallgrass prairie which has never been disturbed agriculturally. Of 126 strains picked randomly from selective media, 54 were identified as Agrobacterium spp. Biovar 2 strains predominated (35 of 54), but these strains were distributed into three phenotypically distinct subgroups. Of the remaining Agrobacterium strains, four were biovar 1-2, one was biovar 1, and none were biovar 3. The last 14 Agrobacterium strains formed a homogeneous group which differed biochemically from the hitherto reported biovars. Opine utilization (coded for by genes on the tumor-inducing plasmid in pathogenic Agrobacterium spp.) by these agrobacteria was limited to two biovar 2 strains. In contrast, 10 nonfluorescent gram-negative strains utilized either nopaline or octopine as the sole carbon and nitrogen source. There may be a need to reexamine the source and role of opines in the terrestrial environment because (i) all of these opine utilizers were isolated from an environment free of crown gall, the only known terrestrial source of opines, and (ii) 83% of the opine utilizers were not Agrobacterium spp.

Journal Article↗