Three Methods for the Introduction of Foreign DNA into &lt;i&gt;Agrobacterium&lt;/i&gt;

Wise, Arlene A.; Liu, Zhenying; Binns, Andrew N.

doi:10.1385/1-59745-130-4:43

Cited by 175 publications

(135 citation statements)

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“…Bacterial DNA transformation was conducted by using chemically competent E. coli DH5a (Invitrogen), electroporation of E. coli Rosetta (DE3), or through freeze/thaw transformation of CaCl 2 -competent A. tumefaciens (29). N. tabacum (variety Turk), and A. thaliana plants were grown in a controlled growth chamber at 24°C at 16-h light/8-h dark photoperiod before infiltrations and switched to 24-h light after infiltrations.…”

Section: Methodsmentioning

confidence: 99%

Hyaloperonospora arabidopsidis ATR1 effector is a repeat protein with distributed recognition surfaces

Chou¹,

Krasileva

Holton

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The in planta association of the Hyaloperonospora arabidopsidis effector ATR1 with the cognate Arabidopsis thaliana RPP1 immune receptor activates a disease-resistance signaling pathway that inhibits pathogen growth. To define the molecular events specifying effector recognition by RPP1, we determined the crystal structure of ATR1 and assayed in planta the effects of surface polymorphisms that are critical to activating plant immunity. ATR1 adopts an elongated, all-helical, two-domain, seahorse-like structure with an overall architecture unlike any previously described fold. Structural comparisons highlight a tandemly duplicated, five-helix motif in the C-terminal domain that creates a structural framework for rapid diversification. Identification and mapping of critical recognition sites suggest that ATR1 detection by the RPP1 resistance protein is mediated by several distinct protein surfaces that allow the effectors to escape recognition through diverse surface polymorphisms. ATR1 gain-of-recognition mutants demonstrate that multiple amino acid substitutions are necessary for recognition and that surface polymorphisms exert additive effects. These results suggest that ATR1 is a modular repeat protein belonging to an ancient family of oomycete effectors that rapidly evolves to escape host detection and adopt diverse virulence functions.plant innate immunity | obligate biotroph

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Section: Methodsmentioning

confidence: 99%

Hyaloperonospora arabidopsidis ATR1 effector is a repeat protein with distributed recognition surfaces

Chou¹,

Krasileva

Holton

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…After incubation at 37°C for 1 hour, 50-μl aliquots were plated onto LB agar supplemented with rifampicin and other antibiotics that were selective for recipient cells that had acquired incoming plasmid. Electroporation was performed as described by Wise et al (2006) using a Cell-Porator (BRL, Gaithersburg U.S.A) with Bio-Rad Pulser cubettes ( 1 mm electrode gap ) under the following conditions: voltage, 300 V; capacitance, 330 μF; impedance, low ohms; charge rate, fast; voltage boost resistance, 4 K. After electroporation the cells were immediately re-suspended in 1 ml of SOC medium, and incubated at 28°C with gentle shaking for 1 hour.…”

Section: Methodsmentioning

confidence: 99%

Identification of pTi-SAKURA DNA region conferring enhancement of plasmid incompatibility and stability

et al. 2007

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In Agrobacterium tumefaciens, the stability of Ti plasmids differs depending on the strain. So far, little is known about genes that cause the difference in stability. The repABC operon is responsible for replication and incompatibility of Ti plasmids. We constructed recombinant plasmids carrying the repABC operon and different portions of pTi-SAKURA. Cells having the recombinant plasmids that harbored a 2.6-kbp NheI fragment of pTi-SAKURA were found to be transformed via conjugation 100-fold less frequently with a small incompatible repABC plasmid than cells having the recombinant plasmids lacking the 2.6-kbp NheI fragment. Since the phenomenon occurred only when the resident and incoming plasmids belonged to the same incompatibility group, it was suggested that the 2.6-kbp NheI fragment bears the potential enhancing incompatibility. The fragment contained an operon consisting of two open reading frames, tiorf24 and tiorf25. tiorf24 is an orphan gene, whereas tiorf25 is a homologue of a group of plasmid stability genes. Removal of the 2.6-kbp fragment from the resident pTi-SAKURA increased the resident plasmid ejection ratio by the incoming repABC plasmid, whereas addition of the fragment to pTiC58 decreased the ejection ratio, and the loss ratio during growth at 37°C. These data suggest that tiorf24 and tiorf25 are responsible for the stability of pTi-SAKURA, and reduce, in the host bacterium, the frequency of ejection of the resident plasmid, presumably through an incompatibility mechanism.Key words: Agrobacterium, incompatibility, stability, Ti plasmid INTRODUCTIONAgrobacterium tumefaciens causes crown gall disease in dicot plants. The tumor-inducing plasmid (Ti) is indispensable for the phytopathogenicity of this bacterium, because it harbors oncogenic genes in its T-DNA region and many virulence genes for infection and T-DNA transport functions. The plasmids are stably maintained at a low copy number in the bacterial cells (at an equimolar ratio with the bacterial linear and circular chromosomes) . The stability of Ti plasmids still differs depending on the strain (Holsters et al., 1982). The stability difference is likely to exert a serious effect on Ti plasmid evolution. The repABC locus is sufficient for autonomous replication of the plasmids (Tabata et al., 1989;Li and Farrand, 2000). To date, the nucleotide sequence of the repABC region of Ti has been determined in an octopine-type plasmid, pTiB6S3 (Tabata et al., 1989), and two nopaline-type plasmids, pTi-SAKURA and pTiC58 (Suzuki et al., 1998;Li and Farrand, 2000).Rhizobiaceae bacteria commonly possess repABC plasmids (Rigottier-Gois et al., 1998). The repABC region consists of three clustered genes (repA, repB and repC) and a replication origin (oriV) required for replication and partitioning. The RepA and RepB proteins are involved in plasmid partitioning and copy number control, which are important for stable inheritance. RepC is an indispensable protein for replication initiation. These genes are organized as an operon (Ramírez-Romero et al., 20...

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“…Following a recovery, this protocol yielded a surprisingly high transformation efficiency of~10 4 cfu. Low-temperature transformations in bacteria have been previously reported, originally by Dityatkin et al in 1972 [18]; however, they have only found widespread use in Agrobacterium tumefaciens transformations [19][20][21], and examples in E. coli are rare. This is most likely due to relatively low transformation efficiencies compared to other methods and the overall prevalence of both electroporation and heat shock protocols.…”

Section: E V E L O P M E N T Of M I C Row a V E -M E D I A T E D Bamentioning

confidence: 99%

Development of rapid microwave-mediated and low-temperature bacterial transformations

2013

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The introduction of exogenous DNA into Escherichia coli is a cornerstone of molecular biology. Herein, we investigate two new mechanisms for bacterial transformation involving either the use of microwave irradiation or a freeze-thaw protocol in liquid nitrogen. Ultimately, both methods afforded successful transfer of plasmid DNA into bacterial cells, with the freeze-thaw technique yielding efficiencies of~10 5 . More importantly, both techniques effectively eliminated the need for the preparation of competent cells.

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Three Methods for the Introduction of Foreign DNA into <i>Agrobacterium</i>

Cited by 175 publications

References 11 publications

Hyaloperonospora arabidopsidis ATR1 effector is a repeat protein with distributed recognition surfaces

Hyaloperonospora arabidopsidis ATR1 effector is a repeat protein with distributed recognition surfaces

Identification of pTi-SAKURA DNA region conferring enhancement of plasmid incompatibility and stability

Development of rapid microwave-mediated and low-temperature bacterial transformations

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