E X T R A C H R O M O S O M A L F O R M S O F CLV D N A 1 IN T R A N S G E N I C P L A N T S A R E I N H E R I T E D B Y S Y M P T O M - F R E E P R O G E N Y
PETER MEYER', INGRID NIEDENHOF', IRIS HEIDMANN" and HEINZ SAEDLER b
• Maz-Delbrftck-Laboratorium im der MPG and bMax-Planck-Institutfiir Ziichtungsforschung, Carl-von-Linnd Weg 10 D-5000 K~ln 30 fF.R. GJ
(Received March 10th, 1989) (Revision received July llth, 1989) (Accepted July 11th, 1989)
Two full length copies of Cassava latent virus (CLV) DNA1 were cloned in head to taft arrangement on a plant expression vector to evaluate the potential of CLV for the development of an extraehromosomal vector system in plants. After direct transfer of the plasmid into protoplasts of N/cot/aria tabacum cv. Petit Havana SR1 extrachromosomal single-stranded (ss) and double-stranded (ds) forms of DNA1 appeared after the first cell division of protoplasts. The extrachromosomal copies could also be detected within transformants which has been regenerated from kanamycin-resistant calli. The CLV-harbouring trans- formants do not display any symptoms usually observed after CLV infection. Stable conservation of extrachromosomal DNA1 was observed in F1 plants derived from self-poUination and in plants regenerated from protoplasts of transformants. Our data show that dimer constructs of CLV DNA1 are attractive candidates for an extrachromosomal plant vector system.
Cassava l a t en t virus (CLV) is a m e m b e r of the geminiviruses , which are t r an smi t t ed by whitefl ies and which infect d icotyledonous plants [1]. The b ipar t i t e genome of CLV con- sists of two s ingle-s t randed DNA circles, DNA1 and DNA2, which are 2779 bp and 2724 bp in length and which contain a common region of approximate ly 200 bp, t ha t p robably contains the origin of repl icat ion [2]. Sys temic infection of N i c o t i a n a b e n t h a m i a n a with CLV requi res both DNA1 and DNA2 [3]. Similar data have been obta ined for ano ther b ipar t i t e gemini- virus, tomato golden mosaic virus (TGMV), where both components were also requ i red for infection of P e t u n i a h y b r i d a [4]. The repl icat ion mechanism of geminivi ruses is t hough t to be similar to tha t of single s t r anded bac te r iophage ~X174, as single s t r anded circles of the virus genome as well as double s t r anded supercoi led and open circular molecules are found [5,6]. Autonomous repl icat ion of TGMV DNA A,
which corresponds to CLV DNA1, has been r epo r t ed for t ransgenic plants containing dimer const ructs of the A component [4,7], however , no genet ic da ta on the s tabi l i ty of the free TGMV copies in la te r genera t ions has been repor ted . For CLV the appearance of f ree ds- and ss-forms of DNA1 could be de tec ted a f te r t r ans fe r of cloned DNA1 into pro toplas t s of N. b e n t h a m i a n a [8], bu t no stable t r ans fo rman t s have been r egene ra t ed . Autonomous ss- and ds- forms of CLV have been descr ibed for N. ben . t h a m i a n a af te r infection with a cloned f ragment of DNA2, t o g e th e r with a DNA1 con- s t ruc t in which a pa r t of the coding region for the coat prote in had been subs t i tu ted by the coding region of the CAT gene. The CAT gene was s tably re ta ined in the repl ica t ing DNA1 cons t ruc t and CAT act ivi ty could be measu red 10 days a f te r inoculation. The t r ans fec ted plants, however , show severe symptoms and the amount of CAT express ion cor responds with the seve r i ty of symptoms [9]. Our inten- tion was to use dimer cons t ruc ts of CLV DNA1
alone for direct transfer into plant protoplasts to avoid systemic infection caused by DNA2 functions. By this approach we could produce symptom-free transformants, which contain stable extrachromosomal copies of DNA1.
Mater ia l and M e t h o d s
Cons truction and preparation of plasmids A full size copy of CLV DNA1 was isolated
from pJS092 [2] by MluI restriction. The frag- ment was circularised by ligation, restricted with ClaI and cloned as a dimer in head to tail orientation into the unique ClaI site of pMP9 [10]. The resulting construct pCLVDim (Fig. 1) was isolated as described [11] from a dam* bac- terial strain.
Protoplast transformation and regeneration Ca2NOa/PEG-mediated DNA transfer was
performed with unsynchronized protoplasts as described previously [12]. Plasmid samples were analysed by gel electrophoresis prior to transformation to check for supercoiled confor- mation. Plants were regenerated on MS medium, supplemented with 75 mg/l kanamycinsulphate and 1 mg/l zeatin.
Eco RI
[
IIl
I
DNA isolation from leaves and protoplasts Leaf tissue (5--10 g) was ground in liquid
nitrogen and incubated in 10 ml 2o/o Cetyltrime- thylammoniumbromide (CTAB), 0.1 M T r i s - HC1, (pH 7.8), 20 mM EDTA, 1.4 M NaC1 at 65 °C for 15 min. After centrifugation at 5000 rev./ rain the supernatant was extracted twice with chloroform/isoamylalcohol (24:1). One volume of 1% CTAB, 10 mM EDTA, 50 mM Tris--HC1, pH 8 was added and incubated at room temper- ature for 30 rain. After centrifugation the pellet was dissolved in 1 ml 0.3 M NaAc and precipi- tated with two volumes of ethanol.
Freshly prepared protoplasts were lysed in 10 mM EDTA, 10 mM T r i s - H C l (pH 7), 1% SDS and incubated in 10 mg/l Proteinase K for 3 h at 50 °C. The solution was extracted twice in phenol/chloroform/isoamylalcohol (25:24:1), once with chloroform/isoamylalcohol (24:1) and precipitated with one volume of isopropanol.
DNA analysis DNA was analysed either undigested or
after restriction with 1 unit//~g restriction enzyme for 2--4 h. Samples were run in TBE buffer [13] and were transferred to hybond nylon membrane by the Southern blot tech- nique [14]. Filters were hybridized to oligomer primed ~ZP-probes [15], washing was performed in 0.1% SDS, 2 x SSC at 68°C.
Synchronisation of protoplasts Freshly isolated protoplasts were incubated
in 10 mg/1 aphidicolin for 3 days [10]. The S- phase arrest was released by two centrifuga- tion steps in 0.6 M mannitol and one floatation in 0.6 M sucrose.
R e s u l t s
$al I
Cl,
2779 bp
Fig. 1. P lasmid pCLVDim carr ies the full size DNA1 frag- m e n t of CLV cloned as a d imer in head to tail or ienta t ion.
Transformed protoplasts contain extrachromo- somal copies of DNA1
Protoplasts were transformed with pCLVDim, cultured for 3--6 days until the first cell divisions had occurred and DNA was iso- lated. To distinguish between introduced plas- mid DNA and newly replicated forms, the isolated DNA was analysed after DpnI or MboI
restriction. DpnI requires methylation of the adenosine-residue of its target sequence GATC [16], while MboI cleaves the same sequence only if the adenosine-residue is not methylated. Due to its propagation and adenosine methylation in E. coli, non-replicated plasmids can be digested by DpnI, but not by MboI (Fig. 2A). As shown in Fig. 2B new extrachromosomal copies of CLV DNA1 are detectable among the DNA reiso- lated from transformed protoplasts, which are susceptible to MboI digestion and which are resistant to DpnI. This suggests a loss of the original A-methylation due to replication of CLV monomers in the plant cell. Transforma- tion with plasmids carrying only a monomeric unit of CLV DNA1 never gave rise to extra- chromosomal copies after transformation (data not shown).
Transgenic plants contain eztrachzomosomal copies of DNA1
Kanamycin-resistant calli were selected from protoplasts transformed by pCLVDim and transgenic plants were raised. Although the transformants displayed a normal phenotype without any noticeable symptoms of CLV infec- tion, in two out of 10 transformants extrachro- mosomal copies of DNA1 could be detected. One plant, SRC, was characterized further. As shown in Fig. 3 the major forms of DNA1 are single stranded (ss) and supercoiled double stranded (sc). Upon $1 digestion the ss-form disappears and most of the sc-form is converted into the open circular (oc) and linear form (1). In some preparations we also detected extrachromosomal copies of higher molecular weight which are probably ss multimers.
To determine the number of integrated cop- ies in the genome, the extrachromosomal CLV- copies were separated from the genomic DNA of SRC by centrifugation in a CsCI gradient. Analysis of the purified genomic DNA showed that it contained only a very minor contamina- tion of residual extrachromosomal CLV copies (Fig. 4). The genomic DNA was digested with HindIII, which cuts only once in pCLVDim and which can therefore be used to assay the num- ber of integrated copies, as each HindIII-frag-
209
ment will extend from the plasmid-borne HindIII-site to a HindIII-site in the integration region and thus each independent integration of a copy of the transferred construct will create a different HindIII-fragment. If only parts of the construct are integrated which do not contain the plasmid-borne HindIII-site, HindIII-fragments will represent individual integration loci cut out of the genome. While hybridisation to the selectable marker gene revealed the integration of five copies of the NPTII gene, at least 15 copies of DNA1 are observed (Fig. 4). This argues for release of CLV monomers out of pCLVDim and their sub- sequent integration into the genome. If such a release takes place during the transformation procedure or later during the replication pro- cess of DNA1 remains to be analysed.
Conservation of extrachromosomal D NA l-copies
To evaluate the stability of extrachromoso- mal copies of CLV DNA1 nine plants were raised from individual protoplasts of SRC. Free copies of DNA1 were detectable in all proto- plast derived plants. Eight plants contained both ss- and ds-forms. In one plant only ds- forms were detectable (Fig. 5A). When F1- plants, derived from selfpoilination of SRC, were assayed, eight out of nine plants showed free forms of DNA1 at variable intensities (Fig. 5B).
Variation of free CL V copies within SRC To determine whether the considerable fluc-
tuations in the amount and composition of extrachromosomal copies of DNA1 reflect dif- ferences among different plants or differences within the development of each plant, protoplasts of SRC were synchronised into S- phase and DNA was analysed at different time- points after onset of replication. In four independent experiments extreme fluctuations were detectable both in the amount and composition of extrachromosomal CLV copies (Fig. 6). No consistant correlation in the appear- ance of free copies with the cell cycle was observed, but in all experiments free copies
210
t-"
I:}. a
A
O
B
o
D
_ _ v
23.0-
J
/t.3-
2.3- 2.0-
6.7-
Fig. 2. Release of extrachromosomal copies of CLV DNA1 after transfer into tobacco protoplasts. (AI pCLVDim was hybri- dized to CLV DNA1 undigested (ul or after digestion with MboI or DpnI. The plasmid is resistant to MboI cleavage and is com- pletely digested by DpnI. (B) DNA was isolated from protoplasts 5 days after pCLVDim transfer and the DNA was hybridized against DNA1 either uncut (u) or after MboI- or DpnI-treatment. Arrows indicate new extrachromosomal bands which are cleaved by MboI, but are resistant against DpnI. Size markers (given in kb) correspond to HindIILdigested ~-DNA.
211
A 13
u~
t - 0 Q . . ~
OC I
SC ' ~ - - SS
w
t - O
, - - CL a ~
tn o
Fig. 3. Analysis of extraehromosomal copies of CLV DNA1 in line SRC. Total DNA, which had been isolated from leaves of SRC, was separated without digestion (u) or after digestion with MboI, DpnI and Sl-nuclease. (A) The gel was blotted without denaturation to detect single stranded forms. After hybridisation to CLV DNA1 the ss form of DNA1 is detectable, which disappears after Sl-treatment. After DpnI- and MboI-treatment the intensity of the CLV specific singlestrand is reduced which is probably due to a weak endonuelease activity. (B) The gel was blotted after denaturation treatment to detect both ss- and ds-forms and was again hybridized to CLV DNA1. The double stranded supercoiled form (sc) which comigrates with the single stranded form is converted into the open circular form (oc) and the linear form (1) afte Sl-treatment and partly also after DpnIatigestion, due to a weak endonuclease activity in the preparation. The ds-forms are digested by MboI, while the ss-form is not effeeted. The high molecular weight chromosomal DNA is fully digested by MboI, but not after Sl-treatment or DpnI- digestion (the DpnI-lane contains less DNA than the other three lanes).
212
CLV U
CLV NPT]] Hind rll Hindm
23 -
9 . 6 - 6 . 6 -
4.3-
2.3- 2.0-
Fig. 4. Number of integrated copies of pCLVDim in the genome of SRC. Genomic DNA of SRC was separated from extrachromosomal copies of DNA1 by CsCl~entrifugation. Hybridisation of undigested DNA (u) to CLV DNA1 shows only a minor content of free DNA1 copies. The same prepa- ration was digested by HindIII and hybridized to DNA1 or NPTII. While five copies of the selectable marker gene are integrated, at least 15 copies of DNA1 are observed. The different intensities of hybridizing bands reflect the inte- gration of homologous segments which are deleted to differ- ent extents. Size markers correspond to HindIII~ligested LamdwDNA.
were clearly detectable directly after onset of replication and only minor amounts could be seen three hours after release of the S-phase block.
Discussion
Dimeric constructs of CLV DNA1 have been demonstrated to lead to the formation of extrachromosomal ss- and ds~opies of DNA1 after direct gene transfer into protoplasts. The free CLV-molecules appeared as soon as the first cell divisions had occurred and could later be found in transgenic plants where they were stably conserved in Fl-plants and in plants which were regenerated from individual proto- plasts of a transformant. Of considerable impor- tance is the fact that in none of these plants the usual symptoms were observed, such as chlorotic local lesions, systemic leaf curling and an irregular chlorotic line pattern along the midrib, as been described for CLV infection of Nicotiana tababum [17]. Our results are compa- rable to data reported for another member of the geminiviruses, TGMV, which also consists of a bipartite genome and for which it was dem- onstrated that DNA A encodes all viral func- tions that allow replication [18]. Substitution of the coat protein gene in DNA A with the neo gene from Tn5 resulted in replication of this enlarged construct, and in the formation of functional neomycin phosphotransferase [19]. When the coat protein coding sequence was substituted with sequences encoding CAT or GUS, the system could successfully be used as a transient expression system for both genes [20].
For CLV the appearance of monomeric DNA1 units has been demonstrated after uptake of cloned DNA1 constructs by proto- plasts of N. benthamiana [8], but no transgenic plants have been regenerated from these experiments. It was also shown that the coat protein I)NA sequence can be substituted by the CAT gene resulting in CAT activity ten days after inoculation of this construct together with DNA2, but severe symptoms appeared in the transfected plants [9]. Our data suggest that the propagation of extrachromoso- mal copies of CLV DNA1 is not linked to the development of symptoms, unless the symptom development is limited to so very few cells that it becomes undetectable.
B
,4
1 2
3 4
5 6
7 8
9
P 1
P 2
P 3
Sl
u u
u
-OC
-I
-$C
-SS
-OC
-I
-SC
-SS
Fig
. 5.
App
eara
nce
of f
ree
DN
A1
copi
es i
n pr
otop
last
-der
ived
pla
nts
and
Fl-
plan
ts (
A) D
NA
was
iso
late
d fr
om p
lant
s w
hich
had
bee
n ra
ised
fro
m i
ndiv
idua
l pr
otop
last
s of
SR
C. D
NA
was
hyb
ridi
zed
to D
NA
1 ei
ther
unt
reat
ed (
u) o
r af
ter
Sl-
trea
tmen
t. O
ne p
lant
, P1
, co
ntai
ns o
nly
ds-f
orm
s, w
hile
P2
and
P3
show
bot
h as
- an
d ds
-for
ms.
(b)
DN
A w
as e
xtra
cted
fro
m n
ine
diff
eren
t F
l-pl
ants
aft
er s
elf-
poll
inat
ion
of S
RC
. Var
iabl
e am
ount
s of
fre
e D
NA
1 co
pies
are
det
ecta
ble
in a
ll
but o
ne p
lant
. ~
o
A
B
C
3 2
1 0
2 1
0 3
2 1
0 3
D
2 0
OC
- O
C~
¸
OC
-
SC
- S
C-
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S
S-
Fig
. 6.
V
aria
tion
in
amou
nt a
nd c
ompo
siti
on o
f fr
ee D
NA
1 co
pies
. In
fou
r in
depe
nden
t ex
peri
men
ts (
A -
D)
prot
opla
sts
of S
RC
wer
e sy
nchr
oniz
ed i
nto
earl
y S
-pha
se a
nd D
NA
was
iso
late
d 0,
1, 2
and
3 h
aft
er r
elea
se o
f th
e ap
hidi
coli
n bl
ock.
Equ
al a
mou
nts
of D
NA
wer
e lo
aded
and
hyb
ridi
zed
to C
LV
DN
A1.
Str
ong
fluc
tuat
ions
can
be
obse
rved
in
the
cont
ent
of e
xtra
chro
mos
oma!
cop
ies
in t
he i
ndiv
idua
l ex
peri
men
ts,
how
ever
, in
all
fou
r ex
peri
men
ts f
ree
CL
V c
opie
s ar
e cl
earl
y de
tect
able
aft
er o
nset
of r
epli
cati
on (0
), bu
t no
t 3
h la
ter
(3).
The
ide
ntit
y of
the
diff
eren
t fo
rms
of f
ree
DN
A1
copi
es w
as d
educ
ed a
s di
scus
sed
in F
ig.
3.
It has been speculated that CLV replication is dependent upon host replication functions expressed during S-phase [8]. This assumption is supported by our observation that cell divi- sion was a prerequisite for the appearance of free DNA1 forms. The analysis of S-phase spe- cific production of free DNAl-copies showed no fully consistent correlation, but in all cases free CLV-forms were present shortly after the onset of the S-phase. The high fluctuations in the content of free DNAl-copies might be explained by a very fast turnover of these mole- cules. In most plants the ss-forms are predomi- nantly represented, which corresponds to the observation of other authors made for TGMV [4], but some exceptions are found where only ds-molecules are present. The fast turnover mentioned previously also accounts for the observed fluctuations in the ratio of ss- and ds- copies. The stable propagation of DNA1 con- structs in seed and protoplasts of symptom- free transformants might make the system suitable for extrachromosomal amplification and expression of genes which have been substituted for the coat protein. Interactions of different genes might be analysed after crosses or protoplast fusion of the individual transgenic lines. It has been argued that substitution of the coat protein by other sequences might be limited due to stringent size regulations of CLV [21]. Up to now, only DNA1 constructs 98 bp larger than the viral genome have been propa- gated successfully [22]. Such size limitations have not been observed for TGMV [19,20] and it remains to be seen what constraints are imposed on the use of DNA1 dimer constructs of CLV as a replacement vector system.
Acknowledgements
The authors wish to thank Drs. J.W. Davies and J. Stanley for providing plasmid pJS092 and J. Dangl for reading the manuscript. We also thank R. Heindrichs for support in the analysis of transgenic plants, T. Akmandor for technical assistance and F. Furkert, M. Kalda and D. Bock for illustrations and photos. This work was supported by the Bundesministerium fiir Forschung und Technologie (BCT0365 2-2/3).
215
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