Note: Descriptions are shown in the official language in which they were submitted.
~<r"ate:°~~
HOECHST AKTTENGESELLSCHAFT HOE 90/F 036 Dr. KH/rk
Description
Virus/herbicide-resistance genes, processes for the
preparation thereof and the use thereof
The synthesis of virus coat protein in plants leads to
an enhanced resistance of the plant to the corresponding
virus. European Patent Application 0 2~0 331, for eacam
ple, describes the preparation of plant cells which
contain such a coat protein.
Turner et al . [ EMBO J . 5 , 1181 ( 19 8 7 ) ] have carried out
the transformation of tobacco and tomato plants with a
chimeric gene which codes for the coat protein of alfalfa
mosaic virus. The progeny of these is transformed plants
which showed a significant reduction in the signs of
infection with the corresponding virus, and in some cases
even virus resistance.
It has now been found that such virus genes can be
combined with a herbicide-resistance gene, which facili-
rates the selection of the transger~ic plants. At the same
time, in practical field cultivation, the vitality of the
plants is increased by the virus tolerance, and an
improved plant protection is possible owing to the
herbicide-resistance gene. It has been generally observed
that herbicide application exerts a stimulating effect on
growth. The plant transformed according to the invention
shows an enhancement of this effect, which makes it
possible to achieve an improved plant yield.
Herbicide-resistance genes have already been disclosed.
German Offenlegungsschrift~ 37 16 309 describes the
selection of non-fungoid bacteria which are resistant to
phosphinothricin. The phosphinothricin-resistance gene
can be localized to a fragment 2 kb in size on the DNA of
these selectants.
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German Offenlegungsschrift 37 37 918 indicates a
way of synthesizing the phosphinothricin-resistance gene from
the genome of Streptomyces viridochromogenes. Incorporation
in gene structures with whose aid transformed plants become
resistant to the herbicide is likewise described therein.
The invention thus relates to a gene coding for a
virus-resistance combined with a herbicide-resistance.
According to one aspect of the present invention,
there is provided an isolated gene coding for a
phosphinothricin-resistance combined with an isolated gene
coding for a virus-resistance.
According to one other aspect of the present
invention, there is provided a plant cell transformed with a
DNA molecule consisting of a nucleotide sequence encoding a
protein conferring phosphinothricin-resistance and a
nucleotide sequence encoding a virus coat protein conferring
virus-resistance.
According to another aspect of the present
invention, there is provided a plant cell as described
herein, wherein the nucleotide sequence encoding the virus
coat protein can be obtained by cDNA cloning starting from
the RNA of cucumber mosaic virus, of alfalfa mosaic virus or
of brome mosaic virus.
According to still another aspect of the present
invention, there is provided a plant cell as described
herein, wherein the nucleotide sequence encoding a protein
conferring phosphinothricin-resistance is from Streptomyces.
According to yet another aspect of the present
invention, there is provided a plant cell as described herein
and expressing the respective nucleotide sequence.
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According to a further aspect of the present
invention, there is provided a process for the preparation of
transformed plants with improved plant yield, which are
transformed with a DNA molecule consisting of a nucleotide
sequence encoding a protein conferring phosphinothricin-
resistance and a nucleotide sequence encoding a virus coat
protein conferring virus-resistance, and which comprises
treating the virus- and herbicide-resistant regenerated
plants with phosphinothricin, and this treatment resulting in
improved plant yield.
According to yet a further aspect of the present
invention, there is provided a method for increasing the
yield from plants which are transformed with a DNA molecule
consisting of a nucleotide sequence encoding a protein
conferring phosphinothricin-resistance and a nucleotide
sequence encoding a virus coat protein conferring virus-
resistance, which comprises treating the plants with
phosphinothricin in the growth period.
According to still a further aspect of the present
invention, there is provided the use of a DNA molecule
consisting of a nucleotide sequence encoding a protein
conferring phosphinothricin-resistance and a nucleotide
sequence encoding a virus coat protein conferring virus-
resistance for increasing the yield from plants transformed
therewith.
The invention is described in detail hereinafter,
especially in its preferred embodiments. Furthermore, the
invention is defined by the contents of the claims.
The genes for virus-resistance, especially the
virus coat proteins, can be obtained starting from isolated
virus RNA by cDNA cloning in host organisms. The preferred
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starting material for this is the RNA of cucumber mosaic
virus, of alfalfa mosaic virus or of brome mosaic virus.
Herbicide-resistance genes can be isolated from
bacteria, for example of the genera Streptomyces or
Alcaligenes. Preferably used is the phosphinothricin-
resistance gene from Streptomyces viridochromogenes
(Wohlleben, W. et al, Gene 80, 25-57 (1988)), which can be
appropriately modified for expression in plants.
The genes are cloned and sequenced in each case
using the vectors pUCl9, pUCl8 or pBluescript (Stratagene,
Heidelberg, Product Information).
The gene is cloned in an intermediate vector with
plant promoter. Examples of such vectors are the plasmids
pPCV701 (Velten J. et al., EMBO J. 3, 2723-2730 (1984)), pNCN
(Fromm H. et al., PNAS 82, 5824-5826 (1985)), or pNOS (an, G.
et al., EMBO J. 4, 277-276 (1985)). Preferably used is the
vector pDH51 (Pietrzak, M. et al., NAR 14, 5857, (1986)) with
a 35S promoter, or the vector pNCN with a Nos promoter.
After subsequent transformation of E. coli, such
as, for example, E. coli MC 1061, DH1, DK1, GM48 or XL-1,
positive clones are identified by methods known per se
(Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982),
Molecular Cloning, a Laboratory Manual, 1st Edition, Cold
Spring Harbor, New York, U.S.A.), such as plasmid mini-
preparation and cleavage with an appropriate restriction
enzyme.
These positive clones are then subcloned together
into a binary plant vector. The plant vector which can be
employed is pGV3850 (Zambrysk, P. et al., EMBO J. 2, 2143-
2150 (1983)) or pOCAl8 (Olszewski, N., NAR 16, 10765-10782,
(1988)). pOCAl8 is preferably employed.
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The resulting binary plant vectors which contain
plant promoters with the attached DNA fragment for the
expression of virus coat protein and phosphinothricin-
resistance in the T-DNA are used to transform plants. This
can be carried out by techniques such as electroporation or
microinjection. Preferably employed is cocultivation of
protoplasts or transformation of leaf pieces with
Agrobacteria. For this, the plant vector construct is
transferred by transformation with purified DNA or, mediated
by a helper strain such as E. coli SM10 (Simon R. et al.,
Biotechnology 1, 784-791 (1983)), into Agrobacterium
tumefaciens such as A282 with a Ti plasmid via triparental
mating. Direct transformation and triparental mating were
carried out as described in "Plant Molecular Biology Manual"
(Kluwer Academic Publisher, Dardrech (1988)).
It is possible in principle to transform all plants
with the binary plant vectors carrying the DNA constructed
according to the invention. Dicotyledonous plants are
preferred, especially productive plants which produce or
store starch, carbohydrates, proteins or fats in utilizable
amounts in their organs, or which produce fruit and
vegetables or which provide spices, fibers and industrially
useful products or pharmaceuticals, dyes or waxes and,
moreover, fodder plants. As example mention
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may be made of tomato, strawberry, avocadoand plants
which bear tropical fruits, mango,
for example papaya,
but also pear, apple, nectarine, apricot or peach.
Further examples of plants to
be transfoaned are all
types of cereals, rape, bird rape... The transformed
cells are selected using a selection medium,cultured
to
a callus and regenerated to the plant on appropriate
an
medium (Shain M. et al., Theor. appl. Genet.~, 770-770
(1986)); Masson, J. et al., Plant Science ~,,'~,,167-176
( 1987 ) ) , Zhan g. et al. , Plant Mol. Hiol. ,~,,~,551-559
(1988); McGranaham G. et al., Bio/Technology 800-804
~,
(1988); Novak F. J. et al., Bio/Technology7, 154-159
(1989)).
The following examples serve to illustrate the invention
further.
8zamcples
1. Isolation of the virus coat protein gene
The virus was purified by modification of the method of
Lot, M. et al . , Anual Phytopath. ~, 25-32 ( 1972 ) . Alfalfa
was infected with alfalfa mosaic virus and, after
14 days, disrupted in the same volume of 0.5 M sodium
citrate (pH 6.5)/5 miK EDTA/0.5% thioglycolic acid. Then
1 volume of chlorofoan was added, and the mixture was
centrifuged at 12,000 x g for 10 min. The supernatant was
mixed with 10% PEG 6000 (~r/w) and stirred cautiously
overnight. Zt was then csntrifnged at 12,000 x g for
10 min and reauspendod in 50 ml of 5 mM sodium borate,
0.5 mM EDTA (pH 9). Triton g-100 (final concentrations
2%) was added and then the mixture was stirred for 30 min
and centrifuged at 19,000 x g for 15 min. The virus
pellet after centrifugation at 105,000 x g for Z h was
taken up in 5 mM borate buffer/0.5 mM EDTA (pH 9.0) and
subjected to a sucrose centrifuqation (5-25%).
Individual fractions from the gradient were analyzed on
an agarose gel in order to find the virus-containing
- 5 - cC~ ~., ~' , ~; ~.,. , ...
zone. The virus RNA was purified of coat protein by
phenol/SDS extraction (Peden, K.W. et al., Virology 53,
487-492 (1973). The RNA components were fractionated
using 2.8~ polyacrylamide with 40 mM tris acetate buffer
(pH 7.5) as described in Synous, R.H., Aust. J. Biol.
Sci. 31, 25-37 (1978). The RNA was removed from the gel
by electrophoresis in dialysis tubes and precipitated.
cDNA transcripts of RNA3 or RI~A4 were prepared as des-
cribed in Langenreis, K. et al., Plant Mol. Biol. 6,
281-288 (1986) using synthetic oligonucleotide primers
with 3'-complementary nucleotides to the template, each
of which had an Smal or PstI cleavage site at the 5' end.
The reactions for the cDNA synthesis were carried out in
accordance with the "Current Protocols in Mol. ~3iol. " ed.
Ausubel, F. et al., John Wiley and Sons.
The cDNA was cloned into the SmaI/PstT-cut p~JC 19 vector.
It was possible to delete the insertion again using
Smal/HindIII.
The method described above can equally be used to isolate
the CMV coat protein gene.
2. Isolation of the herbicide-resistance gene
A phosphinothricin-resistance gene with the following
sequence was synthesized in a synthesizer using the
phosphoamidite method.
y;s.~,.~y,.... ~,~;n
2. ~ t.g y
'~' GTC GAC ATG TCT CCG GAG AGG AG~a CCA GTT GAO ATT AGG CCA GCT
4 TAC AGA GGC CTC TCC TCT GG't CAA CTC TAA,TCC GGT CGA
~4 b~' 7~'81 fir,
ACA GCA GCT GAT ATG GCC GCG GTT TGT GIST ATC GTT AAC CAT TAC
TGT CGT CGA CTA TAC CGG CGC CAA ACA CTA TAG CAA TTG GTA ATO
99 1C~~ 117 1b
ATT GAG ACG TCT ACA GTG AAC TTT AGG ACA GAG CCA CAA ACA CCA
TAA CTC TGC AGA TGT CAC TTG AAA TCC TGT CfC GGT GTT TGT GGT
~. 44 1 ~._, 132 9.71
CRA GAG TGG ATT GAT GAT CTr'~ GAG AGG TTG CAA GAT ArA TAC CCT
GTT CTC ACC TAA CTA CTA GAT CTC TCC AAC GTT CTA TCT ATG GGA
189 199 d«7 ~i6
TGG TTO GTT GCT GAt3 GTT GA~3 GET GTT OTG GCT GGT ATT GCT TAC
ACC AAC CAA CGA CTC CAA CTC CCF~ CAA CAC CGA CC~1 TAA CGA ATG
"~4 '4C Wig: 261 27r,~
GCT GGG CCC TGG AAG GCT AGG ABC GCT TAC GAT TGG ACA GTT GAG
CGA CCC GGG ACC TTC CGA TCC TTG CGA ATG CTA ACC TGT CAA CTC
79 ~~18 .~~.97 Ct:W ~1~
AGT ACT OTT TAC GTG TCA CAT ArG CAT CAA AGG TTG ~3GC CTA GGA
TCA TGA CAA ATG CAC AGT GTA TCC GTA cTT TCC AAC CCG GAT CCT
4 ~ r~R ~ 4d agl ~ 6t t
TCC ACA TTG TAC ACA CAT TTO CTT Af~G TCT ATG GAG GCG CAA GGT
AGG TGT AAC ATG TGT DTA AAC GAA TTC AGA TAC CTC CGC CTT CCA
X69 ~71~ X87 ~9~ 4c;y
TTT AAG TCT GTG GTT GCT GTT ATA GGC CTT CCA AAr GAT CCA TCT
AAA TTC AOA CAC CAA CGA CAA TAT CCG GAA GGT T1'G CTA GGT AGA
414 4'~. 4.~ 441 4~~y
GTT AGG TTG CAT CAO GCT TTO OGA TAC ACA C~CC CGG GGT ACA TTG
CAA TCC AAC GTA CTC COA AAC CCT AT13 l~aT COG GCC CCA TGT AAC
4~9 469 47'? ~lg~ 4~~
CGC GCA GCT GAGA TAC AAG CAT G3T GG~4 T~3G CAT GA'T GTT GGT TTT
GCG CGT CGA CCT ATG TTC Gl'A CCA CC'T ACC OTA CTA CAA CCA A~1A
'5114 ~1~ '~,~~,;w '5~.,1 ~54r:~
TGG CAA AGG GAT TTT GAO TTY CCA GC'T CCT CCA AGO CCA GTT AGG
ACC GTT TCC CTA AAA CTC AAC GOT COA GGA GOT TCC GGT CAA TCC
:~49 ~~8
CCA OTT ACC CAO ATC TC,A O
GDT CAA TOO C3TC TAO ACY CAI CT~ ~'
This is a modification of the sequence for the acetyl-
transferase r~ene published by Wohlleben in Gene 7~D, 25-37
(1988).
It is likewise possible to examine a genomic DNA bank
from the Streptomyces viridochromogenes used by Wohlleben
in EMBL3 in E. coli for the acetylation of phosphino-
thricin. The acetylated product can be very easily
fractionated by thin-layer chromatography.
The gene was cloned in pUCl9 and sequenced. Expression in
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plants was carried out as SalI fragment.
3. Fusion of herbicide-resistance gene with Nos pro-
moter
The vector pNCN was digested with Bam/SalI, and the
resulting 2.5 by piece was isolated. The protruding ends
were digested off with mung bean nuclease. The acetyl
transferase gene was isolated as 0.5 by piece after SalI
digestion and filled in with Rlenow. After ligase, it was
possible to isolate positive clones by plasmid mini
preparations. The orientation was evident from a SalI/Bam
digestion.
4. Fusion of coat protein gene with 35S promoter
A fragment, 0.5 base-pairs long, from pAI RNA3 (the pUCl9
vector with coat protein gene insert) was isolated after
digestion with SmaI/HindIII. The protruding ends were
digested off by mung bean nuclease. The vector pDH 51 was
cut with XbaI, and ends were filled in with Klenow
polymerase. Fragment and vector were ligated and trans-
formed into MC 1061 (p35/AI). The same construction was
carried out with pCM RNA3 for the coat protein of CMV
(p35/CM).
5. Fusion of 35S/coat protein gene and nos/acetyl-
transferase gene
A 1.3 kb piece from the 35S/coat protein construct
(p35/AI, p35/CM) after EcoRI digestion was isolated from
a low melt agarose gel. The plant vector pOCA 18 was
digested with EcoRI and ligated to the 1.3 kbp DNA piece.
This pOCA/35 RNA3 vector was filled in with Klenow. A
2.5 kbp HindIII piece from nos/AC was, after Rlenow
treatment of the ends, inserted into the filled-in ClaI
site.
Constructions: pOCA/AcAI3
pOCA/AcCM3
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6. Transformation of Agrobacteria
The Agrobacterium strain pMP90RK was transformed with
pOCA/AcAI3 or pOCA/AcCM3 in triparental mating with SM10.
100 ~1 portions of bacteria from overnight cultures of
SM10, the MC 1061 carrying the construction, and the
Agrobacteria were spun down and suspended together in
30 ~1 of LB medium. These cells were placed on a small
circular filter on an LB plate without antibiotic. After
incubation at 37°C for 12 h, the filter was washed in
2.5 ml of 10 mM MgCl2, and aliquots thereof were selected
on LB plates containing rifampicin, tetracycline and
kanamycin. Positive colonies were identified by hybridi-
zation with 32P-labeled DNA of the genes.
7. Transformation of alfalfa
A modified version of the cocultivation method of
Marton S. et al., Nature 277, 129-130 (1979) was employed
for the transformation of alfalfa. Stalk sections about
1 cm long from sterile plants were placed in 40 ml of
sterile MS medium in Erlenmeyer flasks, and 11 ml of a
diluted overnight culture of the Agrobacteria (5 x 10'
cells/ml) were added. Incubation was continued at 25°C
for 3 days. The stalk segments were then washed three
times with sterile water and placed on MS medium contain-
ing 300 mg/1 carbamicillin and 100 mg/1 kanamycin. A
callus from which it was possible to regenerate whole
plants formed after 3 weeks.
8. Testing of the plants
The plants showed, after working up of RNA and hybridiza
tion with radiolabeled DNA of the genes, expression of AC
gene with alfalfa mosaic virus coat protein gene.
The plants grew on phosphinothricin-containing medium and
showed distinct tolerance after infection with alfalfa
mosaic virus.
