Gene. 104 (1991) 247-252 rc: 1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

247

0378-l I19/91/$03.50

05065

Geminivirus-based shuttle vectors capable of replication in Escherichia coli and monocotyledonous cells (Genetic engineering; suspension culture cells; ~r~ticu~?2~z~~~ococcu~ protoplasts; DNA; prokaryotic and eukaryotic vectors; transposon trap)

Matthias Kammann”, Volker Matzeit a, Bodo Schmidt a, Jeff Schell”,

plant virus;

transfection;

plant

reconlbinant

Richard Walden * and Bruno Gronenborn a,b

” Map-Planck-lnstitutf~r ~ii~htu~gs~ors&hung,WSOOO~oiog~e-30 (F. R. G.j, and& I~~titut des Sciences Vt?g&ales, Centre ~~t~on~~ de /a ~echerche Scientifique, 91198 Gif-sur-Yvette Cedex (France) Tel. (33) 149823833 Received by 5. Messing: 9 November 1990 Revised/Accepted: 21 March/30 April 1991 Received at publishers: 21 May 1991

SUMMARY

Shuttle vectors have been constructed that are able to replicate in either ~sc~eric~~u cofi or plant cells. They contain the ColEl origin of replication and parts of the wheat dwarf virus genome, a geminivirus infecting a variety of species of monocotyledonous plants. Such plasmids are able to replicate in E. coli and wheat cells. The plasmids can be rescued in E. coli and show no changes during their passage through plant cells. Such an E. co/i/plant cell shuttle vector system could be used for the amplification of foreign genes in plant cells, for studies on DNA rearrangement or the isolation of plant transposons.

Shuttle vectors are able to replicate in prokaryotic as well as in eukaryotic cells. Therefore, such plasmids are amenable to standard recombinant DNA technology in E. coli prior to introduction into a eukaryotic host cell. in the eukaryotic host, replication of the plasmid allows accumulation of the DNA to a high copy number with the potential of achieving high levels of gene expression. Subsequently, Corre.~p#fl~en~e

to:

Z~chtungsforschul~g,

Dr.

Tel. (49)221-5062284; Abbreviations: circular;

Kammann,

ARS, autonomous

BPV, bovine

DSV. Digitaria

Max-Planck-Institut

fXir

10, WSOOO K&X-30 (F.R.G.)

Fax (49)221-5062213.

AC, activator;

bp, base pair(s);

M.

Carl-van-Li~ln~-Weg

papilloma

virus;

replicating

streak virus; EBV, Epstein-Barr

base(s) or 1000 bp; lin. linear; nt, nucleotide(s)~

sequence(s);

ccc, covalently

closed

virus; kb, kilo-

oc, open circuiar;

ORF,

open reading frame(s); on‘, origin of DNA replication; Polfk, Kienow (1arge)fragment ofE. c&DNA polymerase I; ss, single strand(ed); SV40, simian

virus 40; T., Triticum; WDV, wheat dwarf virus; wt, wild type.

such vectors can be rescued in E. coli by transforming bacteria with DNA extracted from the eukaryotic host. Shuttle vectors were used first in yeast where the bacterial vector pBR322 was engineered to contain either the 2 pm yeast plasmid or yeastARS sequences (Broach, 1983; Stinchcomb et al., 1980). In higher eukaryotes, only viruses replicate to a high copy number as extrachromosomal eiements. Hence, mammalian shuttle vectors have employed pBR322 fused to sequences derived from SV40 (Lusky and Botchan, 1981). BPV (Sarver et al., 1982) or EBV (Margolskee et al., 1988). In plants, a large variety of T-DNA vectors have been constructed based upon the Agrobacteriurtl-mediated transformation system (reviewed in Walden and Schell, 1990). This system aims at the stable integration of the introduced DNA rather than extrachromosomal replication (Zambryski, 1988). The main groups of plant DNA viruses that have been used for vector construction are the caulimoviruses and the geminiviruses (see Gronenborn and

248 Matzeit, 1989; Davies and Stanley, 1989; Shepherd, 1989, for reviews). The shuttle vectors described here consist of derivatives of pBR322 and WDV, a geminivirus plants (Lindsten et al., 1980).

of monocotyledonous

is identical to that produced by Sau3A (lanes 4, 8) indicating that the viral DNA has been replicated. It also shows that ORFI replication.

and ORFII

of WDV are dispensable

(b) The WDV genome replicates plant cells EXPERIMENTAL

AND

DISCUSSION

(a) A WDV deletion mutant replicates in plant cells Cloned WDV genomes are able to replicate in suspension culture cells of wheat, rice, maize and rye grass (Matzeit et al., 1991). For developing a shuttle vector, a bacterial replicon has to be inserted which does not interfere with replication in plant cells when combined with the viral genome or parts thereof. The interruption of ORFIII and ORFIV of WDV inhibits viral DNA replication, and the bacterial plasmid inserted into these genes has to be removed prior to introduction into plant cells to allow viral replication (Schalk et al., 1989). Alternatively, clones containing redundant viral sequences lead to the release of a full-length viral genome after transfection by either recombination or a replicative mechanism (Woolston et al., 1989). A variety of deletions encompassing ORFI and ORFII of WDV were generated and tested for their ability to replicate in protoplast derived cells of T. nmzococcurt~. A 982-bp deletion mutant (WDVSXS), in which ORFI and ORFII were almost completely deleted (Fig. 1) still replicates. A comparison of the DNAs accumulating in protoplasts transfected with pWH5X5 or the wt clone pWH8 1 is shown in Fig. 2. Prior to transfection, the WDV DNAs were excised from the cloning vector by HirldIII. Total DNA was isolated two and seven days after transfection and analysed by Southern blotting. At day 2 after transfection the major hybridisation signal is caused by the linearised input DNAs. The full-length viral DNA and the pUC8 vector DNA, 2749 bp and 2686 bp, respectively, comigrate (‘lin’, lane 1). The DNA of the deletion mutant WDVSX5 (1775 bp) migrates ahead (‘lin’, lane 5) of the pUC8 moiety (‘i’. lane 5). The hybridisation intensity of the oc and ccc forms of both wt and mutant genome increases from days 2 to 7 (lanes 1, 2 and 5, 6) suggesting an accumulation of WDV DNA by replication. To discriminate between residual input DNA and newly replicated DNA, DNA from day 7 was digested with either MhoI or Suu3A prior to the Southern analysis (Fig. 2, lanes 3, 4, 7, 8). Sau3A is not inhibited by methylation of the adenine in the recognition sequence (GATC) and hence digests DNA methylated in E. co/i; Mb01 cleaves only the unmethylated sequence. While linear input DNA remains uncleaved, the de novo synthesized oc and ccc DNAs are cleaved byMho1 (lanes 3, 7). Apart from the residual input DNA, the pattern obtained

a bacterial

for viral

plasmid in

The SV40-based shuttle vectors can only replicate to high copy numbers in mammalian cells, if the so-called ‘poisonous sequences’ of pBR322 are deleted (Lusky and Botchan, 1981). Accordingly, we used pXf3 (Hanahan, 1983), a deletion variant of pBR322, to test whether it could be replicated by the WDV genome in plant cells. A monomeric unit of the deletion mutant WDV5X5 was excised from the dimeric clone pWH5XSdi by XhoI and inserted into the &r/I site of pXf3 to yield pXf5X5 (Fig. 1). The replication behaviour of pXf5X5 in T. tmmococcwn cells is illustrated in Fig. 3. The input DNA linearised with BuII~HI and a sample of the undigested vector DNA are shown in lanes 1 and 13, respectively. Circular DNA appears two days after transfection and increases in amount by day 7 (lanes 4, 5). The de novo synthesis of this DNA was confirmed by differential digestion with MhoI and Suu3A. MhoI does not cut the input DNA (lane 2) whereas SLIU~A digests it to completion (lane 3). The oc and ccc forms (lanes 4, 5) were digested by MhoI (lanes 6, 8) to produce a pattern expected from digestion of pXf5X5 by Sau3A (lanes 7,9) while a low amount ofthe input DNA is still detectable (lanes 6, 8). The plasmid DNA had been linearised with BLIIIIHI (lane 1) to distinguish easily between potentially replicating molecules (ccc, oc) and input DNA (lin). The same level of replication was observed, if supcrcoiled DNA was used for transfection, but the differential A4hoI and Su113A digestion was still required to identify the de novo synthesized molccults (data not shown). These results indicate that the WDV genome is able to replicate an entire bacterial plasmid. We next tested the ability of the pXfSX5 vector DNA, which had been replicated in plant cells, to be recover-cd in E. co/i. To be sure that the molecules transforming E. coli did not represent residual input DNA, the isolated plant DNA was digested with QnI. Contrary toMho1, Q117I only cleaves the methylated GATC recognition sequence (Fig. 3, lane lo), hence leaving de novo synthesized DNA intact. To check whether DpnI is active under the conditions used, 1.3 ng of pXf5X5 DNA wcrc mixed with 12-15 pg of DNA isolated from nontransfected cells and digested with Dpn I, Transformation of E. coli with this treated DNA did not yield any transformants. In a second mixing experiment 1.3 ng of pXf5X5 DNA were added to 15 pg total plant DNA isolated seven days after transfection with pXf5X5 and digested with Dprrl. An

249 12345678

oc -

lin -

I

m

OC lin BY

ccc

Fig. I.

Fig. 2.

Fig. 1. Map of the WDV genome numbered

5X5 (A). DNA of a full-length

with BAL 31 nuclease, set of deletions.

subsequently

All manipulations

et al. (1989) are deleted] pBY5X51

WDV sequences

I through IV. ORFIII in pWH81 and pWH5X5 is interrupted

(982 bp) in mutant

was cloned

and the shuttle plasmids.

cleaved

and B.C., unpublished).

clone of the WDV genome in the Hind111 site ofpUC8 in Maniatis

B, BclI; BE, BstEII; of DNA isolated

BH, Ban2HI;

ofpUC18

Fig. 2. Southern

analysis

of the T. rno~rococcum cells were done as described

on a 1.1 “I agarose

(D) ([S/X]:

of the oc, lin, and ccc forms of each of the DNAs are indicated. (lanes 5-8) two days (lanes 1,5) and seven days after transfection of the linear pUCX DNA.

recombinant

transfected

(Matzeit

(Hybond-N,

pWH5X5

SalI/XhoI

[nt 185-1167

and hybridised digested

according

by XhoI a monomeric

to Schalk unit, which

site) or into the XhoI site of pBY to yield S. Schaefer

Sp, SphI; X, XhoI.

with wt WDV and the deletion

from protoplasts

(lanes 2,3,4,6,7,8);

aliquot of the digestion assay was subjected to Southern blot analysis (lane 11). @VII, while leaving the DNA synthesized in plant cells intact, digested to completion the added plasmid DNA, hence also any residual input DNA. This treated DNA was used to transform E. coliBMH7 l- 18 (Messing et al., 1977). Transformation of bacteria was carried out basically as outlined in Maniatis et al. (1982). A transformation frequency of about 1 x lo5 colonies per pg rescued plasmid was obtained. This frequency is about ten times lower when compared to that obtained with shuttle vectors used in mammalian cell/E. ccrfi systems (2-5 x 10” colonies per pg plasmid DNA) (Lusky and Botchan, 198 1; Sarver et al., 1982). This may be due to the excess of plant

and

with BstEIl, treated

(EcoRI~SstI/KpnIiXhoI/~~~I~~~hI/HindIII;

et al., 1991). Total

Amersham)

DNA isolated

by arrows

the extent of the deletion

(B) was linearised

served to release

M. MluI; N, NcoI; Sm, SmaI;

from T. m~nococcunz protoplasts

gel, blotted to nylon membrane

(pWH81)

plasmid

(pWH5XSdi)

with a modified polylinker

H, HindIII;

and transfection fractionated

et al. (1982). The mutant

1983) to yield pXf5X5

(F). Plasmid pBY is a derivative

arc delineates

with PolIk. XhoI linkers were ligated to the ends of the thus created

in this way (C). A cloned dimer of this mutant

into the Sal1 site of pXf3 (Hanahan, (E) and pBY5X52

areas. ORFs of WDV are represented

by cloning in pUC8. An arrowhead-less

by MluI, followed by a fill-in reaction

were done as described

was obtained

are shown as shaded

DNA

was isolated

with a “P-labeled

transfected

with pWHRI

mutant

WH5X5.

Maintenance

from transfected

protoplasts.

probe of pWS6. The positions (wt) (lanes 1-4) or pWH5X5

with Mb01 (lanes 3,7), or Sau3A

(lanes 4,8). i, position

over plasmid DNA as explained by Hanahan (1983). After transformation of E. coli with the DpnI-digested plant DNA, all of the bacterial colonies obtained harboured a plasmid whose restriction pattern was identical to the original pXf5X5 (data not shown). Plasmid DNA was prepared from two bacterial clones and used again to transfect T. monococcum protoplasts. No difference in the ability to replicate in the plant cell, rescue frequency in E. cd’ and restriction pattern compared to the original vector molecule was found. We conclude that passage between E. coli and plant cells results in no obvious change of the pXf5X5 shuttle plasmid. The plasmids of the pUC series are versatile as cloning

250 23456789

1

10

2

1

11 1213

3

4

5

6

7

8

9

10 11

12

13 1415

-

Itn

-

ccc

_

_

Fig. 3. Southern with a deletion

analysis derivative

nation and blotting was hybridised digested gested

of DNA isolated

from protoplasts

of the WDV genome

transfected

linked to pXl3. Fractio-

of the DNA were done as in Fig. 2. The blotted filter

to a “P-labeled

with BamHI

probe

of pXf5X5.

Input

(lane l), MhoI (lane 2) Sau3A

(lane 13). DNA isolated

from transfected

vector

DNA

(lane 3) and undi-

T. ~IOI~CO~CUMproto-

plasts two days (lanes 4,12) or seven days (lane 5) post transfection, and digested with M/x/I (lanes 6, 8) or Sau3A (lanes 7, 9). Vector DNA digested

with Dpnl (lane 10). Digestion

with Dpnl of 1.3 ng of vector (lane 11).

DNA mixed with 15 pg of DNA from transfected

protoplasts

The bars on the left margin

of the MhoI, Sau3A,

and Dpnl fragments

indicate

of pXf5X5.

802,665,4X3,341,283/272,258/253. represent

partially

cleaved

the positions

From top to bottom Larger fragments

(sizes in bp): 862, in lanes 10 and 11

DprlI fragments.

Fig. 4. Southern

analysis

with the mutant

WDV5X5

blotting

of DNA isolated genome

from protoplasts

of the DNA were done as in Fig. 2. The blotted membrane

hybridised molecule DNA

to a “P-labeled pBY5X51

isolated

(lanes 2, 4,s) digested

from transfected

of pBY5X52. T. monococc’u,n

of the vector

with SphI (lane I).

protoplasts

two days and either

(lanes 5, 7). Analysis

(lanes 8-15): undigested

with SphI prior to transfection T. ,,ronococcum

(lanes 12, 14) or Sau3A the positions top to bottom

doublet).

and was

or seven days (lanes 3, 6,7) post transfection,

protoplasts

of the Mb01 and Sau3A

(lanes 10. 12, 13) or

and digested

(lanes 13. 15). The bars

with MboI

on the left margin

fragments

of pBY5X5 I.

(sizes in bp): 1051, X22, 585, 483, 341, 258:‘253

The bars on the right margin indicate

and Srru3A fragments

of the

vector DNA (lane 8)

(lane 9). DNA isolated from

two days

seven days (lanes 11, 14,lS) post transfection, indicate

Analysis

input DNA digested

with Mb01 (lanes 4, 6) or Suu3A

and digested transfcctcd

probe

(lanes l-7):

vector molecule pBY5X52

From

transfected

linked to pBY. Fractionation

of pBY5X52.

the positions

From top to bottom

of the Mhol (sizes in bp):

1619, 585, 483, 341. 25X/2541253 triplet.

vectors. Therefore, we also tested their suitability to serve as the bacterial replicon in combination with the WDV genome. Into theXho1 site ofpBY, a pUC1S derivative with a modified polylinker, the deletion mutant WDV5X5 was inserted to yield the pBY5X51 and pBY5X52 (Fig. 1). DNA of these plasmids linearised by SphI was used to transfect protoplasts of T. monococcum (Fig. 4). From days 2 to 7 replicative forms (ccc, oc) of both hybrid plasmids accumulate (lanes 2, 3, 10, 11). They are sensitive to digestion by Mb01 (lanes 4, 6, 12, 14), except the residual input DNA. After seven days of passage through the plant cells, the shuttle plasmids can be recovered by transformation of E. cd’ in the same way as with pXf5X5.

(c) Conclusions (1) The shuttle vectors described here are the first example of the combination of a ColEl derived rcplicon with a geminivirus genome. The geminivirus replicon directs the amplification of the hybrid molecule in the plant cell while the bacterial plasmid sequences drive the replication in E. coli. Plasmids of the pUC series can serve as part of a shuttle vector without further modification; their versatility may be improved by the insertion of the viral replicon outside the multiple cloning sequence to restore the ability for chromogenic selection or adding an origin for ss DNA production in E. loli to facilitate site-directed mutagenesis.

251 (2) Shuttle vectors offer some additional features over the vectors currently available in plant research. Plasmid

REFERENCES

vectors routinely used in transient assays cannot replicate inside the plant cell. The replicating vectors described offer the possibility to amplify the level of expression of foreign genes. Moreover, the WDV replicon aids the maintenance of the introduced DNA in suspension cells cultured for periods more than three weeks (Matzeit et al., 1991) a much longer period than obtained in standard transient expression assays. The general utility of WDV-based vectors for replication and expression of foreign DNA in plant cells has been demonstrated (Matzeit et al., 1991). WDVbased vectors replicate in cell lines derived from wheat, maize, rice and rye grass. However, replication of WDV is

Broach,

greatly reduced in protoplasts

of tobacco

mesophyll

(V.M.,

unpublished results). Also, replication of DSV, a related geminivirus of monocotyledonous plants, was not observed in transgenic tobacco plants (Mullineaux et al., 1990). This restriction probably reflects the splicing of the WDV and DSV transcripts specific for monocotyledonous plants. (3) The easy rescue of the shuttle plasmids in E. coli offers the opportunity to simplify the study of DNA rearrangement in plant cells. Recently, it has been demonstrated that the maize-transposable element AC is able to transpose from replicating WDV genomes (Laufs et al., 1990). If a shuttle vector contains a genetic marker screenable in E. co/i, it may be used as a ‘transposon trap’ to identify as yet undetected mobile elements in a variety of monocotyledonous plant species, simply by isolating a population of shuttle vector molecules, transforming into bacteria and screening for loss of the marker function. Similarly, shuttle vectors could be used to study recombination in plant cells.

J.R.: Construction

sequences. Davies,

of high copy yeast vectors

Methods

Enzymol.

J.W. and Stanley,

Genet.

using 2-pm circle

101 (1983) 307-325.

J.: Geminivirus

genes

and vectors.

Groncnborn,

B. and Matzeit,

mation:

V.: Plant gene vectors

plant viruses as vectors.

Cell Culture

and Somatic

and genetic transfor-

In: Schell, J. and Vasil, I.K. (Eds.),

Genetics

of Plants.

Academic

Press,

San

Diego, CA, 1989, pp. 69-100. Hanahan,

D.: Studies on transformation

cob with plasmids.

ofEscherichia

J. Mol. Biol. 166 (1983) 557-580. Laufs, J., Wirtz, U., Kammann, Czernilofsky, Acids

M., Matzeit,

V., Schaefer,

A.P., Baker, B. and Gronenborn,

vectors:

introduced

expression

into various

and excision

S., Schell, J.,

B.: Wheat dwarfvirus

of transposable

cereals by a viral replicon.

elements

Proc. Natl. Acad.

Sci. USA 87 (1990) 7752-7756. Lindsten,

K., Lindsten,

B., Abdelmoeti,

and some properties

Rothamsted, M.: Inhibition

by specific pBR322

E.F. and

Manual.

Abstracts,

Rotham-

of SV40 replication Nature

Sambrook,

Cold Spring

in simian cells

293 (1981) 79-81.

J.: Molecular

Harbor

Cloning.

A

Laboratory,

Cold Spring

P. and Berg, P.: Epstein-Barr

virus shuttle

NY, 1982.

Margolskee,

R.F., Kavathas,

vector for stable episomal in human Matzeit,

Conference

DNA sequences.

T., Fritsch,

Laboratory Harbor,

N.: Purification

1980, pp. 27-31.

Lusky, M. and Botchan, Maniatis,

M. and Juntti.

of wheat dwarf virus. Proc. 3rd Conf. Virus Dis.

in Europe,

Gramineae

sted (U.K.)

replication

of cDNA

expression

libraries

cells. Mol. Cell. Biol. 8 (1988) 2837-2847.

V., Schaefer,

Gronenborn,

S., Kammann,

B.: Wheat

dwarf

M., Schalk, virus vectors

foreign genes in ceils ofmonocotyledonous

H.-J., Schell, J. and replicate

and express

plants. Plant Cell 3 (1991)

247-258. Messing,

J.. Gronenborn,

Filamentous fragment Mullineaux,

B., Miiller-Hill,

coliphage

sense RNAs

transgenic

tobacco.

region in M 13 replicative

F. and Accotto,

form in vitro.

G.-P.: Processing

of Digifaitaria streak

Nucleic

of com-

virus in its host and in

Acids Res. 18 (1990) 7259-7265.

N., Byrne, J.C. and Howley,

P.M.: Transformation

tion in mouse cells of a bovine papillomavirus-pML2 that can be rescued

P.H.:

of a Hind11

Sci. USA 74 (1977) 3642-3646.

P.M., Guerineau,

plementary

B. and Hofschneider,

M 13 as a cloning vehicle: insertion

of the lac regulatory

Proc. Nat]. Acad.

Sarver, ACKNOWLEDGEMENTS

Trends

5 (1989) 77-81.

in bacteria.

and replicaplasmid

vector

Proc. Nat]. Acad. Sci. USA 79 (1982)

7147-7151.

We are indebted to Sabine Schaefer for skillful help and Udo Ringeisen for excellent photographical work. This work was supported by a grant from the Bundesministerium fur Forschung und Technologie to B.G.

Schalk,

H.-J., Matzeit,

V., Schiller,

B., Schell, J. and Gronenborn,

Wheat dwarf virus, a geminivirus ing for replication. Shepherd,

B.:

plants needs splic-

EMBO J. 8 (1989) 359-364.

R.J.: Biochemistry

The Biochemistry

of graminaceous

of DNA plant viruses. In: Marcus,

of Plants.

Academic

Press,

A. (Ed.),

San Diego, CA, 1989,

pp. 563-616. Stinchcomb, Eukaryotic NOTE

ADDED

AFTER

SUBMISSION

D.T., Thomas, DNA

M., Kelly, J., Selker,

segments

yeast. Proc. Natl. Acad.

capable

Messing,

J.: Replication

maize endosperm

R.W.:

replication

in

Sci. USA 77 (1980) 4559-4563.

Ugaki, M., Ueda, T., Timmermans,

After submission of this manuscript, the paper of Ugaki et al. (1991) was brought to our attention. They show that the combination of a recombinant WDV-neo genome with the P15A plasmid ori also leads to a functional shuttle vector.

E. and Davies,

of autonomous

M.C.P., Vieira, J., Elliston, K.O. and

of a geminivirus

cells. Nucleic

derived

shuttle

vector

in

Acids Res. 19 (1991) 37 l-377.

Vieira, J. and Messing, J.: The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.

Gene

19 (1982) 259-268.

Walden,

R. and

progress Woolston,

Schell,

and problems. C.J., Reynolds,

Replication coat protein

J.: Techniques

in plant

Eur. J. Biochcm. H.V.,

Stacey,

molecular

N.J. and

Mullincaux,

of wheat dwarf virus DNA in protoplasts mutants

17 (19X9) 6029-6041.

in protoplasts

biology

-

192 (1990) X13-573.

and plants.

P.M.:

and analysis

Nucleic

of

Acids Res.

Yanisch-Perron,

C., Vieira, J. and Messmg,

vectors

and host

pUCl9

vectors.

Zambryski, transfer

strains:

nucleotlde

J.: Improved

sequences

Ml3

of Ml3mplX

phage and

Gene 33 (1985) 109-I IY.

P.: Basic processes

underlying

to plant cells. Annu.

Agobacieriunz

Rev. Genct.

mediated

22 (198X) l-30.

DNA