Gene. 90 (1990) 149-151

Elsevier

149

GENE 03570

Efficient transformation o f A g r o b a c t e r i u m t u m e f a c i e n s by e l e c t r o p o r a t i o n (Recombinant DNA; soil bacteria; methylation; restriction system; phytopathogen; Ti plasmid)

Mark Mersereau, Gregory J. Pazour and Anath Das Department of Biochemistry and Plant Molecular GeneticsInstitute. Universityof Minnesotu. St. Paul. MN 55108 (U.S.A.) Tel. (612)624-2236 Received by J.L. Slightom: 15 December 1989 Accepted: 25 February 1990

SUMMARY

High-voltage electroporation was used to transform Agrobacte6um tumefaciens strains A136 and A348, reaching the efficiency of 1-3 × l0 s transformants/#g DNA. Transformation frequency was dependent on the electrical field strength and the pulse length. No significant reduction in transformation efficiency was observed when the transforming DNA contained sites sensitive to endonuclease AtuCI ofA. tumefaciens.

INTRODUCTION

Agrobacterium tumefaciens, a phytopathogenic soil bacterium, has been widely used for transformation of plant cells (Rogers et al., 1986). The most efficient way to introduce plasmids to A. tumefaciens is by conjugal transfer using Escherichia coli as an intermediate (Ditta et al., 1980). Direet DNA transfer to A. tumefaciens by freeze-thaw procedure and by electroporation has been described (Holsters et al., 1978; Wirth et al., 1989). However, a low frequency of transformation (102-104/ttg DNA) was usually obtained in these studies. Recent studies of Wen-jun and Forde (1989) and of Mattanovich et al. (1989) demonstrate that electroporation can be used for A. tumefaciens transformation to a frequency of 8-15 x l0 s transformants//~g DNA. Similar procedures have been used successfully to

transform E. coil and other bacteria to a high frequency (Dower et al., 1988; Miller et ai., 1988). The aim of this study was to use electroporation to achieve high frequency of transformation ofA. tumefaciens. Such procedure would allow the recovery oftransformants in a single day as compared to a week normally required for DNA transfer by the conjugal transfer method (Ditta et ai., 1980). A. tumefaciens C58 contains a restriction endonuclease AtuCl, an isoschizomer of Bcll (Kessler and HOltke, 1986). We investigated if the presence of AtuCl sites in most commonly used wide-host range" plasmids leads to a reduction in frequency of transformation. To test this we constructed plasmids pGP213 and pGP239 which contain 0 and 4 AtuCI sites, respectively.

EXPERIMENTAL AND DISCUSSION Correspondence to: Dr. A. Das, Dept. of Biochemistry, 1479Gortner Ave.,

University of Minnesota, St. Paul, Tel. (612)624-3239; Fax (612)625-5780.

MN

55108 (U.S.A.),

Abbreviations: A, absorbance; bp, base pair(s); Cb, carbenicillin; kb, kilobase(s) or 1000 bp; Kin, kanamycin; kV, kilovolts; LB, Luria-Bertani (medium); MTase, methyltransferase; nt, nucleotide(s); R resistance/resistant; TB, terrific broth (Tartoff and Hobbs, 1987); Tc, tetracycline. 0378-1119/90/S03.50 © 1990ElsevierSciencePublishersB.V.(BiomedicalDivision)

(a) Plasmids Plasmid pGP213 was constructed by cloning pTJ $75 Kn (Klee et aL, 1985) DNA linearized with Bcll into the BamHl site ofplasmid pUC19 (Vieira and Messing, 1982). The conjugant pGP213 is a 9.5-kb plasmid resistant to Km and Cb, and contains no BclI sites. Plasmid pGP239 was constructed by cloning pUC 119 DNA into the unique Xbal

150

site of plasmid pAD1052. This produced a 15-kb plasmid containing fourBclI sites and genes for Cb R, T c R and Km R (low level of Km). Plasmid pADI052 is a derivative of pTJS75 (Schmidhauser and Helinski, 1985) containing sequences from the cauliflower mosaic virus 35S promoter region, a chimeric KmR gene and A. tumefaciens Ti plasmid sequences. E. cell strains DH5=F' and GM48 (dam-, dcm- ) were used for plasmid amplification. Plasmid DNA was isolated by Triton X-100 lysis procedure (Clewell and Helinski, 1970) and purified by banding twice in CsCIethidium bromide gradients. (b) Bacterial growth and transformation A. tumefaciens strains were grown at 30 ° C in TB medium (Tartoff and Hobbs, 1987) to an A6oo = 1.5-2.0. The cells were collected by centrifugation, washed five to seven times with cold (4°C) sterile water, resuspended in 10% glycerol at a cell density of 4-6 x 101~ ceUs/mi, and stored frozen at -70°C. A. tumefaciens A136 was mixed with 2 ng pGP213 DNA and subjected to 1, 5 or 10 ms pulses at a series of field strengths (Fig. 1). No transformants were recovered when cells were exposed for 1 ms. Increasing field strength

VOLTAGE APPLIED 400 I

500 I

600 I

700

800

I

I

o

yielded a linear increase in transformation frequency when a 5 ms pulse was applied. At the highest field strength a transformation frequency of 1-3 × 10a transformants//~g DNA was routinely observed. Due to limitation of the instrument, no higher field strengths could be applied to the cells. Frequency of transformation was higher at lower field strengths for a 10 ms pulse than for a 5 ms pulse; however, at higher field strengths transformation frequency from a 10 ms pulse was lower than that from a 5 ms pulse. This decrease was not due to cell death (data not shown). Restriction analysis of plasmid DNA isolated from several independent transformants showed patterns identical to that of the transforming plasmid (data not shown). To study if the presence of a Ti plasmid had any effect on transformation frequency we compared A. tumefaciens A136 to its isogenic derivative A348 that harbors an octopine-type Ti plasmid, pTiA6. Under optimal conditions both A. tumefaciens A136 and A348 showed equally high efficiency of transformation (data not shown). Similar studies were performed at varying DNA concentration (0.05-1.25 #g DNA/ml) and with cells grown to logarithmic or stationary phase. No effectof these variables on transformation frequency was observed (data not shown). (e) Effect of the presence of restriction endonuclease AtuCl sites in the transforming DNA on transformation A. tumefaciens C58, the parent of A. tumefaciens A136, contains a restriction endonuclease AtuCI. To study if this restriction system is a barrier for transformation we compared transformation frequency ofplasmid pGP239, a plas-

10 6 TABLE ! Effect of the presence of A:uCi sites on transformation frequency

I=. it. 0 m LI.I m

107

Plasmid a

Number of AtuCl sites

Transformation frequency b

106

pGP213 pGP213 pGP239 pGP239

0 0 4 4

2.2 × 1.7 x 1.1 x 1.7 x

10 5

z I

I

7.2

9.0

I

10.8

!

12,6

I

14.4

F I E L D STRENGTH, kVIcm Fig. 1. Effect of field strength on transformation frequency: 2 ng pGP213 was introduced into A. tumefactens A136 by electroporation using a pulse length of 5ms (O O) or 10ms (0---------0) at indicated field strengths. Number oftransformants on ABCo plates were counted and are represented as transformants per/~g input DNA. Values presented are an average of at least four independent experiments.

(methylated) (unmethylated) (methylated) (unmethylated)

10s 107 l0 s i0 T

" Constructions ofthe plasmids are described in the text. Methylated and unmethylated DNAs were isolated from E. colt DHS=F' and GM48 (dam-, dcm- ), respectively, harboring the appropriate plasmid. b Transformation frequency is defined as the number ofCb R colonies per /Jg ofplasmid DNA. The values presented are an average of four indepen. dent experiments. For electroporation frozen cells were thawed in ice and diluted with sterile water to a density of I x 101°cells/ml prior to use. The 40 pl of cells were mixed with plasmid DNA, transferred to a disposable chamber with a 0.58 mm gap and alectroporated in a BTX T100 electroporator (BTX, Inc., San Diego, CA). After electroporation the chamber was rinsed with 400 #1 LB medium and the cells were collected into a sterile tube. Cells were incubated without agitation for 1 h at room temperature and plated on minimal AB plates (Chilton et al., 1974) supplemented with I00 Pg Cb/ml. Electroporation was performed under optimal conditions: 5 ms pulse length, 14.4 kV/cm field strength.

151

mid containing four AmCI sites, and pGP213, which contains no A tuCI sites. AmCI sites (recognition sequence: 5'-TGATCA) contain the sequence GATC which is methylated by the E. coil dam MTase. Plasmid DNA isolated from A. tumefaciens A136 is not cleaved by the methylation sensitive enzyme BclI, an isoschizomer of AtuCl. Both methylated (dam, dcm MTases of E. coil) and nonmethylated DNA were used as transforming DNA in an electroporation experiment (Table I). Methylated pGP213 DNA and pGP239 DNA showed a transformation frequency of 2.2 x 108 and 1.1 x l0 s, respectively (lines 1, 3). Unmethylated plasmids showed a transformation frequency of 1.3' x i07 (lines 2, 4). Since pGP239 DNA and pGP213 DNA showed a similar frequency of transformarion, the presence of an AtuCI site in the transforming plasmid is not a major barrier for transformation. The nonmethylated DNA consistently exhibited a five- to tenfold lower efficiency of transformation. The reason for this is not apparent; however, at least five other restriction endonucleases (AtuAI, AtuIAMI, AtuBI, AtuII and AtuBVI) are known to exist in different strains OfA. tumefaciens (Kessler and HOltke, 1986). The role of these enzymes in transformation efficiencycould not be assessed in this study.

(d) Conclusions Our study demonstrates that high-voltage electroporation can be effectively used to transform A. tumefaciens. Even in the presence of a restriction endonuclease(s) a reasonably high frequency of transformation could be achieved in these studies. This procedure will facilitate studies on plant gene transformation and understanding of the biology of A. tumefaciens.

ACKNOWLEDGEMENTS

We thank Neil Olszewski for the use ofthe electroporator in this study and Bonnie Allen for typing of the manuscript. This work was supported by a grant (GM 37555) from the National Institutes of Health. A.D. is a recipient of a Junior Faculty Research Award (JFRA-170) of the American Cancer Society and a McKnight Land-Grant Professorship of the University of Minnesota.

REFERENCES

Chilton, M.D., Currier, T., Farrand, S., Bendich, A., Gordon, M. and Nester, E.: ,4[~,robacterfmnDNA and PS8 bacteriophage DNA not detected in crown gall tumors, proc. Nail. Acad. Sci. USA 71 (1974) 3672-3676. Clewell, D.B. and Helinski, D.R.: Properties of a deoxyribonucleic acidprotein relaxation complex and strand specificity of the relaxation event. Biochemistry 9 (1970) 4 4 2 8 - ~ 0 . Ditta, G., Stanfield, S., Corbin, D. and Helinski, D.P~: Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank ofRhlzobium melilotti. Proc. Natl. Acad. Sci. USA 77 (1980) 7347-7351. Dower, J.W., Miller, FJ. and Ragsdale, W.C.: High efficiency transformarion of£. coliby high voltage electroporation. Nucleic Acids Res. 16 (1988) 6127-6145. Holsters, M., De Waele, D., Depicker, A., Messens, E., Van Montagu, M. and Schell, J.: Transfection and transformation of Agrobacterium tumefaciens. Mol. Gen. Genet. 163 (1978) 181-187. Kessler, C. and Htltke, H.-J.: Specificityof restrictionendonucleases and methylases - a review (Edition 2~ Gene 47 (1986) 1-153. Klee, H., Yanofsky, M.F. and Nester, E.W.: Vectors for transformation of higher plants. Bin/Technology 3 (1985) 637-642. Mattanovich, D., Ruker, F., da Camera Machado, A., Laimer, M., Regner, F., Steinkellner, H., Himmler, G. and Katinger, H.: Efficient transformation of Agrobacterium spp. by electroporation. Nucleic Acids Res. 17 (1989) 6747. Miller, J., Dower, WJ. and Tompkins, L.S.: High-voltage electroporation of bacteria: genetic transformation of Campylobacterjejuniwith pissmid DNA. Proc. Natl. Acad. SCI.USA 85 (1988) 856-860. Rogers, S., Horsch, S.B. and Fraley, R.T.: Gene transfer in plants: production of transformed plants using Ti plasmid vectors. Methods Enzymol. 118 (1986) 627-640. Schmidhanser, T. and Helinski, D.R.: Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J. BacterioL 164 (1985) 446-455. Tartoff, K. and Hobbs, C.: Improved media for growing plasmid and cosmid clones. BRL Focus 9 (2) (1987) 12. Vieira, J. and Messing, J.: The pUC plasmids, an M I3mp7-derived system for insertion mntagenesis and sequencing with synthetic universal primers. Gene 19 (1982) 259-268. Wen-jun, S. and Forde, B.: Efficienttransformation of Agrobacterlumapp. by high voltage electroporarion. Nucleic Acids Res. 17 (1989) 8385. Wirth, R., Friesenegger, A. and Fiedler, S.: Transformation of various species ofgram-negarive bacteria belonging to I ! different genera by electroporatinn. Mol. Gun, Genet. 216 (1989) 175-177.

Efficient transformation of Agrobacterium tumefaciens by electroporation.

High-voltage electroporation was used to transform Agrobacterium tumefaciens strains A136 and A348, reaching the efficiency of 1-3 x 10(8) transforman...
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