Note: Descriptions are shown in the official language in which they were submitted.
WOUND-STIMULATED DNA-SEQUENCE
FROM SOLANUM TUBEROSUM AND ITS USE
The invention relates to wound-stimulated DNA
from Solanum Tuberosum as well as parts thereof, the use
of such for the production of gene products in higher
plants using wound or pathogen attack, DNA-propagation
vectors containing the same, and plants or plant parts
containing the same.
It is known that the mechanical injury of
plant tissue can cause morphological and physiological
changes. Thus, the activity of various enzymes
increases after the wounding, for example of
phenylalanine-ammonialyase and peroxidases in potato
tubers, extensin in the storage tissue of carrots, fatty
acid synthetase in potato tubers and proteinase-
inhibitors in tomato and potato leaves. For some of
these enzymes, the increase in activity is associated
with increased mRNA amounts. One of the best understood
wound-induced genes is the proteinase-inhibitor in
tomatoes and potatoes; in wounded leaves, the mRNA for
this gene is significantly increased.
The wound-induced development of genes in
plants is of fundamental interest, because in this area
the promoters that become active are capable of
specifically activating the corresponding structure gene
after wounding. Promoters of this kind in connection
with other genes which, for example, code for
resistance, or code substances that are effective as
antibiotics, or promote the healing of wounding, are
naturally of great economic interest, if they can be
inserted into the genetic material of other plants.
It has now been found that relatively high
concentration of induced mRNA are specifically present
in wounded and/or microbially attacked potato plants.
2 ~aa2~a3~
This mRNA can be isolated and characterized. It does
not appear in healthy, uninjured plants.
Accordingly, in one aspect of the invention, DNA
sequences from potato plants which are stimulated
through injury and/or infection with pathogenic
microbes as well as parts of these sequences, such as
the promoter portion and the structural gene portion
are provided. In particular, the present invention
provides a DNA molecule having a nucleotide sequence as
set forth in Figure 6. Further, the use of these DNA
sequences for the creation of gene products in higher
plants using injury and/or pathogenic attack is
disclosed.
Furthermore, DNA propagation vectors into which
the DNA sequences defined above have been inserted are
provided. This invention also provides for plants or
plant materials which contain such a DNA sequence.
The expression "active region" of a gene, of its
promoter portion or in its structural gene portion is
to be understood to refer to those nucleotide sequences
which are unconditionally necessary in the promoter
portion of the gene for the activation of the
structural gene portion, and in the structural gene
portion for the development of an effective or active
gene product.
The expression "structural gene" is to be under-
stood to refer not only to the DNA-sequence from
Solanum Tuberosum in systems homologous to that herein
described but also structural genes from other sources,
thus from heterologous systems. An example would be
the CAT-, NPT- and GUS-structural genes, which can be
fused with the injury-stimulable promoter portion
described herein, and can be used for the development
Y.
of the respective structural gene in potato or foreign
plants.
~0~~~
The propagation vectors can be provided by any
suitable DNA-molecules which can be introduced into the
final bacterium. Typically, these would be plasmids as
are more fully described in the disclosure. The
insertion techniques are known to the person skilled in
the art.
The bacteria for the uptake and release of the
propagation vectors can be constituted by bacteria of
the genus Agrobacterium, in particular Agrobacterium
tumefaciens and Agrobacterium rhizogenes.
EP-A 122,79l describes a DNA-propagation
vector which contains T-DNA with a plant gene inserted
therein. This plant gene consists of a plant promoter
and a plant structural gene, wherein the plant promoter
is joined to the 5'-end of the plant structural gene,
and the plant structural gene is located behind the
plant promoter in the transcription direction. EP-A
122,791 provides a detailed description of processes and
steps for the insertion of genetic material in DNA-
propagation vectors, to which reference is now expressly
made.
Research has shown that Erwinia-bacteria are
latent in potato plants (Solanum tuberosum) and that
they will preferentially attack the stem and the tubers
of potatoes only after tissue has been wounded. This
was induced in potato tubers of the species Datura, by
slicing the tuber into discs and simultaneously
incubating with Erwinia carotovora of the species
atroseptica. As expected, these tubers exhibited a
severely macerated surface after eighteen hours of
incubation. The isolation of mRNA from the tissues
lying under the maceration was not possible with
~t~~Q~.
4
standard methods. The reasons for this were a high
contamination of the RNA with polysaccharides, poor
precipitation and redissolvement of the RNA, a high
level of RNA degradation, a small yield of RNA and
substantial labor and material expenditures. By
variations and new combinations of existing methods,
however, it was possible to develop a technique by which
large amounts of non-degraded RNA could be obtained with
relatively small material and time expenditures, even
from tissues containing polysaccharides. (Logemann et
al, Anal. Bioch. 163, 16, 1987).
On the basis of cleaned polyA+RNA from wounded
tubers of the potato species granola, incubated with
Erwinia, a cDNA-bank was built up. Using differential
colony hybridization techniques of 4000 cDNA-clones were
hybridized with radioactively labelled RNA from non-
wounded and Erwinia-wounded tubers. Two clones were
identified, here named wunl and wun2, of which the
complementary mRNA were induced by wounding of tubers of
various tetraploidal potato species as well as the
haploidal species AM 80/5793. Wun1-mRNA accumulated
within thirty minutes after mechanical wounding of a
tuber, and is thus involved in the primary processes
induced by wounding, whereas wun2 was induced 3.5 hours
after wounding. Twenty-four hours after wounding, both
clones showed even higher concentration of m-RNA. If in
addition to the mechanical wounding, the tuber is
incubated with Erwinia carotovora of the species
atroseptica, no alteration is found in the expresion
pattern. Altogether, the expression studies with tubers
reveal that wunl-mRNA and wun2-mRNA represent genes
which are not Erwinia-specifically induced, but are
stimulated by a11 processes which result in the
destruction of the tuber tissue.
i". ,
~~'
5
Wun1-mRNA accumulates in large quantities in
wounded potato tubers (quantity-wise comparable with
proteinase-inhibitor II-mRNA in non-wounded potato
tubers, compare Sanchez-Serrano et al, Mol. Gen. Genet.
2032, 15 (1986)); whereas in non-wounded tubers, it is
not detectable. It first appears thirty minutes after
wounding with a maximum between the fourth and twenty-
fourth hour after injury. In smaller amounts, it was
also detectable even after forty-eight hours.
The accumulation of wunl-mRNA and wun2-mRNA
after wounding is not limited to the tuber, but also
takes place in the leaves, stems and roots of various
tetraploidal potato plants with comparable kinetics and
intensity.
In contradistinction to wun2-mRNA, wunl-mRNA
is induced in leaves also in the absence of wounding,
when the leaves are sprayed with compatible phytophtora
infestans spores. This result shows that wunl can be
induced not only through mechanical wounding, but also
through the presence of (fungoid) pathogens.
There were differences in the development of
the wunl-gene, according to whether the tubers were
wounded under aerobic conditions (tuber slices were
sprayed with P-buffer and incubated for eighteen hours,
then exposed to the air) or were wounded under anaerobic
conditions (tuber slices were dipped in P-buffer and in
this oxygen-starved condition incubated for eighteen
hours). wunl-mRNA expression was significantly higher
in aerobically wounded tubers than in the corresponding
anaerobic situation.
For the use of promoters for the expression of
genes in accordance with the invention, it is important
whether the detectable wunl-mRNA or wunl-mRNA arises
through a new synthesis (transcriptional regulation), or
is constitutively expressed and stabilizes only in the
case of a wounding (post-transcriptional regulation).
"Run-off" transcription tests were able to
demonstrate transcriptional regulation for both wunl and
wun2, which means that the promoters of these genes are
responsible for the new synthesis in case of an
wounding.
Using known techniques, a series of wunl-
homologous cDNA-clones were obtained from the
tetraploidal potato "granola", from which some of them
were sequenced. By cloning wunl-25A2 in M13mp19-phages
in both orientations and partial exonucleaseIII
digestion, the nucleotide sequence was analysed in both
directions according to the dideoxy method.
Figure 1 shows the relative sizes of the
clones used for sequencing. The clone wunl-25A2 has
been cloned in two orientations in the PstI-site,
which, in respect to the position of the asymmetric XhoI
section position are called 24A2*MP19 and 25A2MP19.
Using successive exonucleaseIII-digestions, it was
possible to obtain deletion clones of different sizes,
which were available as clone 25A2*MPl9delta 1-3 and
25A2MP19delta 1-4 for sequencing.
Figure 2 shows the nucleotide sequence of the
cDNA-clone-wunl-25A2. Alternative open reading frames
are underlined.
The size of the cDNA-clone-wunl-25A2 is 711
bp. At the 3-end is a poly-A tail of 14 by (position
697-711). The largest open reading frames extends
r
~~~~ ~ ~_
7
over 105 aminoacids (position 121-438). Further, in 5'-
position are two short open reading frames which code
for three amino acids in position 23 and code for eight
amino acids in position 95 (underlined). The last two
mentioned open reading frames differ however from that
which codes the 105 amino acids. By referring back to
the genomic sequence data (Figure 6) the reading frame
coding for 105 amino acids is related to the wunl-
protein. The translational start point of the wunl-
protein TTTTTGATGCAA fits only to a lower extent the
consensus sequence for plant translation start
TAAACAATGGCT, as reported by Joshi, Nuc. Acids Res. 15,
6643, (1987).
Investigation of the genomic organization of
the gene coding for wunl-mRNA in the haploid potato line
AM80/5793 shows that this gene is available in only a
limited number of copies. The hybridization of
radioactively-marked wunl-cDNA against HindIII-, PstI-
and EcoRI-digested leaf-DNA leads, in the "Southern
Blot" analysis, to the definition of one strong and one
or two weak bands. The result of the "Southern Blot"
analysis of wunl- and wun2-cDNA is illustrated in Figure
3.
For the haploid potato line AM80/5793, the
"Southern Blot" analysis was able to identify only a few
genes responsible for the development of wunl- and wun2-
mRNA. The likelihood of obtaining an active gene and no
inactive pseudogene was particularly high here.
The presence of only one defined band in the
"Southern Blot" of the haploid potato line points to a
limited number of genes per genome. If this is
considered along with high and wound-specific expression
,... K
8
of wunl-mRNA in the haploid line and transcriptional
regulation, highly active genes become likely.
To build up a genomic bank, 10 micrograms of
EcoRI-digested DNA from AM80/5793 was ligated with
EcoRI-digested EMBL4-arms, and plated out on a C600-
bacteria-containing medium. Approximately 500,000
plaques were obtained which, considered statistically,
represent the genome of the potato. After the transfer
of these plaques to nitrocellulose, plaque hybridization
techniques with radioactively labelled wunl-cDNA
permitted the identification and purification of the
genomic clones wunl-22 and wunl-85.
The isolation of recombinant EMBL4-DNA from
wunl-22 and wunl-85, as well as the restriction mapping
and hybridization thereof with radioactive wunl-cDNA
gave the following organization.
The wunl-cDNA-equivalent fragment in wunl-85
has a size of 4 Kb. This exactly corresponds with the
fragment size (hybridizing with wunl-cDNA) of EcoRI-
digested DNA from the haploidal potato. On the basis of
the asymmetric XhoI-site in the 5'-region of the cDNA-
clone, the orientation of the gene in the 4 Kb fragment
could be established. According to this, a roughly 1 Kb
sized promoter region lies upstream of the wunl-gene,
whereas a rougly 2 Kb sized non-homologous portion is
located 3' from the gene. The 8 Kb-large EcoRI
fragment, also contained in wunl-85, could not be used
for further analysis of the wunl-gene. On the basis of
the total digestion of the potato DNA with EcoRI, it is
likely that during ligation, two fragments that did not
belong together were inserted into the EMBL4-vector,
these fragments being not functional related with each
other.
Figure 4 shows the arrangement of wunl in the
genomic clone wunl-85. Located on the 4 Kb-large EcoRI
fragment of the genomic clone wunl-85 are 1.0 Kb of
wunl-promoter, 0.8 Kb of the wunl gene and 2.0 Kb of the
3'-end.
Since in the restriction pattern no
differences emerged between wunl-85 and wunl-22, for
further analysis the 4 Kb fragment of wunl-85, was
ligated into EcoRI digested pUC8 (see Figure 4). For
the sequencing of the wunl promoter as well as the wunl
gene, there followed a further recloning of the 4 Kb
fragment in the EcoRI site of M13mp18. The
determination of its orientation was carried out with
control digestions using at the asymmetrically located
XhoI-site. The clones 85*mpl8 and 85mp18 represent both
orientations of the fragment. The digestion of these
plasmids with SphI and XbaI made it possible, with the
help of exonucleaseIII, to successively digest the 3'-
end of the wunl-gene of the clone 85mp18, and the 5'-end
of the wunl-gene of the clone 85*mpl8.
Figure 5 shows the result of the deletion
analysis of clone wunl-85 in schematic form.
The resulting deletion clone with different
fragment sizes was used for the sequencing. Generally,
it was possible to sequence the entire wunl-gene
bidirectionally, and, in addition, to analyze about 400
by of the 3'-end unidirectionally.
Figure 6 shows the nucleotide sequence of the
wunl promoter and gene from wunl-85. The CAAT-box,
TATA-box and Poly-A signal are indicated, and the
transcriptional start- and stop site are marked with
~:
to
arrows. In order to identify the precise
transcriptional start site of the wunl-gene, the method
of S1-nuclease-mapping was used. Figure 7 shows the
determination of the size of the S1-protected fragment
of the wunl gene. The hybridization with wunl-mRNA of
the region in pLS000 lying 5' from the XhoI site leads
to a 162-179 by long DNA-RNA-hybrid (TU), which is
protected from the single-strand specific S1-nuclease.
The actual transcriptional start site thus lies 162-197
by 5' from the XhoI section location (A,C,G,T = the
sequence arrangement for the sizing the DNA fragment).
If one begins with the longest obtained
fragment, then the transcription start begins 179 by
upstream from the XhoI-position, i.e. with the sequence
ACCATAC. This sequence agrees fully in the central
region (CAT) with the concensus sequence or
transcription start CTAATCA, discovered by Joshi et al
(1987). From the position of the transcription start,
further information is available (see Figure 6):
(1) Atposition 33, as seen from the transcription
start, a TATA-box CTATATATT is found which agrees well
with the concensus sequence TCACTATATATAG, as described
by Joshi et al (1987).
(2) The CAAT-box in the region between -60 and -80,
described by Benoist et al, Nuc. Acids Res. 8, l27-142
(1980), is found in position -58 (CAAACT) in the wunl-
promoter.
(3) The 5'-untranslated region of the wunl-gene has a
size of 217 by and, therefore, is relatively large.
(4) The wunl-mRNA coded gene is 794 by in size, which
11
very closely corresponds to the size of wunl-mRNA, on
the basis of the "Northern-Blot" analysis.
(5) 97 by of the 5'-untranslated region of the wunl
gene are missing in the cDNA-clone, wunl-25A2.
(6) Aside from the open reading frame already
determined in the cDNA-clone, no others were found in
the 5'-untranslated region of the genomic clone.
(7) The wunl-gene contains no introns.
Figure 8 shows the arrangement and position of
l0 important regions in the wunl-gene. The wunl-promoter
is marked in black, while the wunl-gene is hatched. The
important recognition sequences are boxed. The mRNA is
shown by a sinuous line, whereas the protein coding
region is marked with Xes. The sizes of the individual
regions are given in base pairs (bp).
Figure 9 gives the sequence comparison between
the cDNA-clone wunl-25A2 and the genomic clone wunl-85.
2368 by of the genomic clone wunl-5 were compared with
711 by of the cDNA clone wunl-25A2. Homologous base
pairs are marked by a vertical line. The missing
nucleotides are shown by a period. The two arrows
indicate the sequence of two 10-by direct in the cDNA-
clone.
Comparison analysis shows that the sequence of
the genomic clone wunl-85 of the haploid potato
AM80/5793 is up to 97~ homologous with the sequence of
the cDNA-clone wunl-25A2 of the tetraploidal potato
"Granola". Altogether, 11 base pair changes were
identified, of which five were located in the translated
region. A11 five base pair changes in the translated
,d
~ ~~~ ~ ,;~;
12
region resulted in no alteration of the amino acid
sequence. The additionally sequenced 400 nucleotides
downstream of the transcription stop exhibit no
remarkable regions.
The gene codes for a protein of 12000 Dalton,
which size can be approximately determined in hybrid-
released-translation-experiments. Computer evaluation
relative to the amino acid combination of the wunl
protein indicates that a very hydrophilic protein. The
amino acid sequence is seen in Figures 2 and 6.
The size of the wunl-protein, its hydrophilic
characteristics, the wound-inducibility in various
tissues and the inducibility by pathogens, can be
concluded on the basis of the fact that it belongs to
the PR-proteins. The name PR- or "Pathogenesis-
Related"-protein includes proteins of various plants
which are inducible, inter alia, by pathogen attack, and
in some cases show Chitinase-activity or Glucanase-
activity, i.e. the activity of enzymes which can destroy
the cell walls of the fungus.
Using the agrobacterium-transformation system
in transgenic tobacco plants, expression studies were
possible on mRNA-level, since tobacco-mRNA cross-
hybridizes only very weakly with wunl, and moreover does
not possess any wunl-homologous 5'-ends, as can be
gathered from S1-nuclease-mapping. Actually, a large
quantity of approximately 800 bp-large wunl-mRNA was
detected in transgenic tobacco plants in wounded leaves,
which on the one hand shows an active wunl-promoter, and
on the other indicates the use of the correct
transcription start point.
a::
,~,
13
A 4 Kb-fragment from pLS000, containing the
wunl-promoter, the complete wunl-gene, and 2 Kb of the
3'-region of the gene (wunl-wunl), was cloned (pLS001)
with its EcoRI-sites in the mobilization vector pMPK110,
which is necessary for the Agrobacterium tumefaciens
(At) transformation. After transfer to At3850km, the
tobacco line Wisconsin 38 (W38) was transformed with
this Agrobacterium on leaf discs. The small callus
appearing some three weeks after Kanamycin selection was
regenerated to the complete plant (LS1) through the use
of a shoot-induction medium.
Aside from the capability of growing on
Kanamycin, plants transformed with 3850Km have the
capability to synthesize nopaline, a property which
distinguishes them from untransformed plants (Zambryski
et al, EMBO J. 1 147-152 (1985)). Wunl-wunl transformed
plants, here called LS1-plants (4 = LS1-4; 6 = LS1-6),
were characterized by a high level of nopaline. By
contrast, no nopaline was detectable in untransformed
plants. Kanamycin-resistant and nopaline-positive LS1-
plants were transferred to the greenhouse and analyzed
on the DNA- and RNA-planes.
Figure 10 shows the construction of wunl-wunl-
fusions and wunl-CAT-fusions which were utilized for
expression studies on the basis of transient expression
and stable transformations.
(1) The creation of the plasmid pLS001:
Beginning with the plasmid pLS000 (see Figure 4), the 4
Kb-fragment which contains the wunl-promoter, the wunl-
gene and 2 Kb of the 3'-end was cloned (pLS001) within
the EcoRI-site of the vector pMPK110.
14
(2) Creation of the plasmid pLSOll:
The wunl-promoter, as well as 179 BP of the 5'-
untranslated wunl-gene region, were fused (pLS010) at
the XHOI site with the CAT-gene inclusive 3'-end.
Subcloning in the vector pMPK110 using PstI (pLS011) has
the advantage that this plasmid can be used for
transient studies in protoplasts and additionally can
behave as a mobilization plasmid for the transfer of
wunl-CAT-construction into the Agrobacterium 3850.
The "Southern Blot" analysis of EcoRI-digested
DNA from transgenic LS1-tobacco plants and untransformed
W38-tobacco plants resulted in a positive hybridization
through the use of 32P-radioactively labelled wunl-cDNA
probe, both in the DNAs of untransformed plants and the
DNAs of transformed plants. In both cases, a strong
band and weak band were recognized. However, in the
transgenic LS1-plants, there was additionally a 4 Kb-
large band, of which the size exactly corresponded to
the size of the EcoRI-insert from pLS000. By comparing
different DNA amounts, there appeared a copy number of
two to five of wunl-wunl in a11 tested LS1 plants. In
some LS1 plants, there were found additional bands in
high molecular regions, probably due to rearranged DNA
or undigested DNA.
A functional analysis of the wunl-wunl-DNA of
positive LS1 plants was possible on the RNA-level. Both
in wounded as well as in non-wounded, untransformed
tobacco plants, there was only a weak cross-
hybridization with a wunl-cDNA-probe. Additionally,
testing was carried out on isolated RNA from
untransformed tobacco plants against the 5'-region of
the wunl-gene containing the 5'-untranslated region and
the wunl-promoter (analogous to the fragment for
~~~"a~~,~~~'
determining transcription start for the wunl-gene) using
S1-analysis on homologous regions. This showed that no
wunl-homologous sequences were present, either in non-
wounded or in wounded untransformed tobacco leaves or
5 stems.
Out of 30 nopaline-positive LS1 plants studied
on the RNA-level, the "Northern Blot" experiment
detected 5 plants with high levels of 800 bp-large mRNA
hybridizing with wunl. An S1-analysis of the latter led
10 to evidence of two equally large DNA fragments,
corresponding in size with fragments from wounded potato
leaves. This was the proof to be wunl-homologous mRNA.
Several indicators supported the idea that wunl-
homologous mRNA is expressed to counteract large-scale
15 TMV attack.
One goal of the invention is the construction
and isolation of genes that have the ability to express
fused proteins having the desired qualities. For this,
it is necessary that the wunl-promoter be solely
responsible for the expression of the 3'-fused structure
gene, which is not always the case (e. g. proteinase-
inhibitor II).
Transcriptional fusions, consisting of the
wunl-promoter and 178 by of the 5'-untranslated gene,
were fused with various marker genes (wunl-CAT, wunl-
NPT, wunl-GUS), proof thereof being possible through
radioactive or fluorimetric methods. (GUS is the name
for !3-Glucuronidase (Jefferson et al, Proc. Nat. Acad.
Sci. 83, 8447-8451 (1987)).
The wunl-CAT construction was tested for
transient expression in potato protoplasts. This method
made it possible to analyze promoter activity within a
16
few days. For this, highly purified plasmid-DNA
containing the construction is transferred into the
protoplasten by known methods, remains there in stable
condition and can be transcribed and translated. The
requirements for expression is that the promoter is
functional, and the inducing factors are present in
protoplasts handled in this manner.
In these tests, the wunl-promoter, including
179 by of the 5'-untranslated region, was
transcriptionally fused with the
Chloramphenicolacetyltransferase-gene (CAT). The
construction of the wunl-CAT-fusion is shown in Figure
10.
The 35S-promoter of the pRT101-CAT plasmid was
removed using HincII/XhoI, and instead of this, the 1.0
Kb sized wunl-promoter together with 179 by of the 5'-
untranslated region was inserted using a filled-up
EcoRI-site and XhoI. This chimeric gene wunl-CAT was
able to be inserted into the PstI-site of the vector
pMPK110 (pLS011).
Large amounts of endogenous wunl-mRNA were
detected in the potato cell suspension used for
transient expression experiments, as well as in the
protoplasts derived therefrom. It was concluded that
the liquid-cultivation of cells, and in particular the
protoplasting step, produces a high stress in the cell,
which is indicated by a large yield of wunl-mRNA,
analogous to the process in the potato tuber.
For the transient analysis of the wunl-CAT
construction, 1 million protoplasts were isolated out of
a suspension culture of potato stems of the variety
~~
1~
Datura (D12), these being then transformed using the
CaCl2/PEG-method by adding 20 micrograms pLS011-DNA.
The CAT-analysis of such protoplasts
transformed with pLS011-DNA showed high CAT-activity. A
parallel test with a CAMV-35S-CAT-construction (35S-
promoter from the cauliflower-mosaic virus (CAMV))
(pRT101-CAT) showed approximately the same CAT activity,
whereas in a control test without the addition of DNA,
no CAT-activity was detected. This means that, in a
transient potato protoplast system, the activity of the
wunl-promoter is comparable to that of the 35S-promoter
and thus must be classified as very high. Up to the
present, the CAMV-promoter stands as the strongest known
promoter in plants.
The plasmid pLS011 (wunl-CAT), already used in
the transient system, was also used further for the
transformation of At 3850Km. 3850~::pLS011
Agrobacteria were used for the transformation of the
tobacco species Samsum NN (SNN) using the method of leaf
disc infection, and the resulting callus were
regenerated to plants (LS2).
By the use of this special tobacco species
that is resistant to tobacco mosaic virus (TMV), the
(suspected) inducibility of wunl through TMV should be
excluded. In LS2-plants, which were nopaline-positive
and kanamycin resistant, tests were made on the DNA-
level and for the presence of CAT-activity. PstI-
digested DNA from wunl-CAT transformed plants exhibited
a 2.2 Kb sized fragment hybridized with CAT-cDNA. This
exactly corresponded to the expected fragment size of
wunl-CAT. No hybridization could be detected in
untransformed plants. By comparing DNA-amounts 2 to 4
.s
18
copies per haploid genome were found in a11 positive
plants.
The functional analysis of LS2-plants
continued with the measurement of CAT-activity in
greenhouse plants and in plants of the F1-generation
grown in sterile culture. LS2-plants raised in the
greenhouse showed in their non-wounded leaves a slightly
increased CAT-activity, in comparison with the
background activity of untransformed tobacco leaves. In
wounded leaves an approximately 4-times higher CAT-
activity was noted by comparison with non-wounded
leaves. However, if an excess of boiled-down potato
tuber extract is added to the wounding condition, the
increase is 6-times higher than in the non-wounded
state. This effect was independent of the condition of
the tuber prior to extraction (non-wounded or wounded
tubers taken either fresh from the ground or from
storage). Each extract had the same inducing effect,
pointing to the permanent presence of an inductor in the
extract.
The cause of the relatively low inducibility
of wuni-CAT in transgenic tobacco plants is to be seen
in the activity of non-wounded leaves. In order to
eliminate possible causes determined by the greenhouse,
the seeds of LS2-plants were harvested, and then
germinated under sterile conditions. In young leaves of
kanamycin-resistant plants, a very weak CAT-activity was
measurable in the non-wounded condition, this being not
much different from the CAT-activity of untransformed
leaves. In wounded leaves, the CAT-activity rose at
least by a factor of 40. The addition of boiled-down
extracts from homogenized potato tubers to the wounding
condition led to a 60-times higher accumulation of CAT.
,,
19
Further, it is to be noted that with
increasing age of the plants, the activity of the wunl-
promoter in the leaves became higher and higher, such
that the strength equalled that of a transgenic CAMV-
CAT-plant. There is a possibility that the senescence
is also capable of inducing wunl.
With the help of the transient system, further
tests were made to determine in which plant species the
wunl-promoter is active, and correspondingly usable. It
turned out that this promoter is not only active in
potato protoplasts, tobacco protoplasts and parsley
protoplasts, but also showed high expression in rice
protoplasts similar to the activity in its homologous
system.
Figure 11 shows the creation of wunl-NPTII
constructs. Beginning from plasmid pLS001, the wunl-
promoter is cloned including 179 by of the 5'-
untranslated region at EcoRI-XhoI-blunt of an NPT-II
gene (Neomycinphosphotransferase-II). For improved
handling in transient experiments, the wunl-NPT-II
fusion was recloned in a high copy plasmid (pLS 020).
Using the wunl-NPTII construct, NPT-II
activity was found in tobacco and in parsley
protoplasts. Interestingly, wunl-dependent NPT-II
activity was also detected in rice protoplast (Oryza
sativa japonica c.v. Taipai), this activity being
comparable in its intensity with the wunl-NPTII activity
in potato protoplasts. Comparable transient expression
of wunl-NPTII and NOS-NPTII (pGV1103) in rice
protoplasts likewise showed similarly high values. In
this connection, transient expression in potato
protoplasts, similar to CAMV-35S-CAT were again
indicated. Generally speaking, these studies indicate
~~.~~~~-.
that the wunl-promoter is capable, at least to some
extent, of very high activity, both in homologous and in
heterologous systems. Neither in parsley-protoplasts
nor in tobacco-protoplasts nor in rice-protoplasts could
5 there be found mRNA hybridized with wunl. Thus one must
assume some factor stimulating the wunl-promoter, the
factor being equally available in a11 tested plant
species.
Generally speaking, a11 important DNA-regions
10 are available in the 5'-region of wunl, in order to be
able to express a subsequently fused gene in large
quantity after wound-induction.
Additionally, the functionality of wunl-CAT
and wunl-NPTII in diverse transient systems (potato,
15 parsley, tobacco, rice) as well as in stable transformed
tobacco, leads to the assumption of a similar molecular
background.
Within the framework of the above invention,
investigations further revealed that it can be
20 advantageous to fuse the wunl-promoter of this invention
with other promoters or enhancing elements.
An example is the fusing of the wunl-promoter
with the known TR-promoter of the Agrobacterium
tumefaciens TR-DNA.
The isolation of this TR-promoter from the
Agrobacterium tumefaciens TR-DNA is known from the work
of J. Velten and J. Schell "Selection-expression Vectors
for use in Genetic Transformation of Higher Plants",
published in Nucl. Acids Res., 13:6981-6997. From this
literature reference, it is also known that this
4;t,
21
promoter is responsible for the expression of Mannopine-
synthase, and shows the following characteristics:
(1) It is bidirectionally active, i.e. if structural
genes are fused to both sides, both can be read off in
the same regulation manner and intensity. In this
connection, reference should be made to the following:
Langridge, W. H. R., Fitzgerald, K.J., Koncz, C.,
Schell, J and Szalay, A.A. (1989). "Dual promoter of
Agrobacterium Tumefaciens Mannopine Synthase Genes is
regulated by Plant Growth Hormones". Proc. Natl. Acad.
Sc.i. USA, 86:3219-3223.
The orientation of the TR-promoter has been
determined as 1' and 2'.
(2) In transgenic plants, the TR-promoter can be
induced by wounding. The fusion of the TR2' with the
marker gene beta-galactosidase (beta-gal) leads to a
higher beta-gal-activity in wounded transgenic tobacco
plants than in non-wounded plants.
(3) The activity of the TR2'-promoter is primarily
found in the leaf veins, thus in the vascular system of
the leaf. For 2. and 3. reference is had to the
following: Teeri, T.H., Lehvaslaiho, H., Franck, M.,
Uotila, J., Heino, P., Palva, E.T., Van Montagu, M. and
Herrera-Estrella, L. (1989). "Gene fusions to lacZ
reveal new expression patterns of chimeric genes in
transgenic plants". EMBO, 8:343-350.
(4) The characteristics of the TR1'-promoter have not,
up to now, been very intensively researched. However,
it is known that the TR1'-promoter is approximately 4-7
times weaker than the TR2'-promoter, although aside from
this both of the promoters have similar characteristics.
22
Within the framework of researches aimed at a
qualitative and quantitative optimization of the wunl-
promoter, the TR-promoter was fused with the wunl-
promoter, specifically in the orientation TR1'-wunl
(Figure 12).
As evidence of the promoter activity, the
marker gene GUS (beta-Glucoronidase) was used (TR1'-
wunl-GUS). Using the Agrobacterium tumefaciens
transformation system, this construction was transformed
into tobacco and analyzed. The following
characteristics distinguish this tobacco plant from
wuni-GUS transgenic tobacco plants:
1. The tissue specificity had changed.
In wunl-GUS transgenic tobacco, the GUS-activity was
found primarily in the epidermis of leaves and stems.
In TR1'-GUS transgenic tobacco, the GUS activity was
primarily localized in the vascular system. There is
thus the possibility, depending on the scientific
requirements, to direct gene products to different
locations of the tissue. As against this, the wound-
inducibility of TR1'-wunl-GUS transgenic tobacco plants
is comparable with the wound-inducibility of wunl-GUS
transgenic tobacco plants.
2. Generally, the activity of the TR1'-wunl-promoter in
transgenic tobacco leaves and tobacco stems is
approximately 10 times higher than the activity of the
wunl-promoter. Hence, the TRl'-wunl-promoter appears to
be among the strongest promoters available for gene
expression in plants.
3. While promoter activity in transgenic dicotyl plants
currently presents little problem, there have been few
''
2 3 , ~.~ ~, ~~'
promoters up to now which are fully active in monocotyl
plants (to which the commercially important grain plants
belong). Transient development studies in rice
protoplasts (these serve as substitute for studies in
transgenic rice-plants, which are very difficult to
obtain) show that the TR1'-wunl-promoter has roughly 30
times higher activity than the wunl-promoter. This is
all the more remarkable because even the 35S-promoter
which is so strong in dicotyl plants is here 30 times
weaker than the TR1'-wunl-promoter.
Thus, the wunl-promoter, according to the invention,
can, for example, be altered or optimized in the
following characteristics, through fusion with the TR1'-
promoter:
(a) The specific activity of the wunl-promoter in the
leaf or stem epidermis of transgenic tobacco can be
targeted to the vascular system through fusion with the
TR1'-promoter;
(b) The capacity of the wunl-promoter for being induced
through wounding is not changed through fusion;
(c) The TR1'-wunl-promoter is approximately 10 times
stronger than the wunl-promoter alone, in transgenic
tobacco leaves and stems;
(d) In transient experiments with a substitute for
monocotyl plants (rice), the TR1'-wunl-promoter is
roughly 30 times stronger than the wunl-promoter.
In accordance with the invention, for example,
the expression of the wunl-promoter through fusion of
the same with an enhancing element of the CamV-35S-
promoter can be increased. Thus, for example, the
24
fusion of the enhancing element of CamV-35S-promoter at
the 5'-end of the wunl-promoter leads to an increased
activity of the wunl-promoter in transgenic tobacco and
potatoes.
Figure 12 shows the organization of the clones
wunl-GUS, TR1'-GUS and TR1'-wunlGUS.
Figure 13 shows the Northern-Blot-analysis of
wounded and non-wounded transgenic tobacco leaves. Non-
wounded (NW) and wounded (W) leaves of the transgenic
tobacco plants wunl-GUS, TR1'-wunl-GUS Nr.5 and Nr.ll
and 35S-GUS were used for the isolation of RNA. In each
case, 50 micrograms of the total RNA was separated gel-
electrophoretically using a 1.2% formaldehyde gel, then
transferred to a nylon membrane, then hybridized with a
radioactively labelled GUS-DNA probe, and visualized on
an autoradiogram. (A) shows a 2-day old exposure, (B)
shows a 6-hour old exposure.
The examples below serve to clarify the
invention:
Examples 1-8
Materials
Media
Culture media for bacteria:
The media used for the growth of bacteria were
established on the basis of data from Maniatis et al.,
"Molecular Cloning: A Laboratory Manual", Cold Spring
Harbour Laboratory, Cold Spring, Harbour, New York
(1982)
Plant Media:
The media utilized were derived from the media given by
Murashige and Skoog, Physiol. Plan. 15 473-497 (1962),
( MS ) .
4
3MS . MS + 3% Sucrose
3MSC . MS + 3% Sucrose, 500 micrograms/ml
Claforan
MSC10 . MS + 2% Sucrose. 500 micrograms/ml
5 Clarofan, 0.2 micrograms/ml NAA, 1
microgram/ml BAP
100 micrograms/ml Kanamycinsulphate
MSC15 . MS + 2% Sucrose, 500 micrograms/ml
Claforan, 100 micrograms/ml
10 Kanamycinsulphate
MS15 . MS + 2% Sucrose, 100 micrograms/ml
Kanamycin-sulphate
For solid media, 8g/1 of Bacto-Agar was added.
Strains and Vectors
15 E. coli-strain:
BMH 71-18: (lac-proAB), thi, supE:
F'(laciq. ZdeltaMl5,
proA+B+) (Messing et al.,
Proc. Nat. Acad. Sci.74,
20 3642-2646 (1977))
C 600 . - CR34 (Maniatis et
al. 1982)
GJ23 . AB1157 (R64drd11) (pGJ28)
(Van Haute et al., EMBO J.
25 2, 411-418, (1983))
Agrobacteria-strains . C58CIpGV3850~ (Zambryski
et al., EMBO J. 1, 147-
152, (l983))
Plasmids . pUC8 (Vieira and Messing,
Genes 19, 259-268 (1982))
pMPK110 (Eckes et al.,
Mol. Gen. Genet. 205, 14-
22 (1986))
26
pRT101-cat (Prols et al.,
Plant Cell Reports, in
print (1988))
pGV1103 (Rain et al., Mol.
Gen. Genet. 199, 166-168
(1985))
Phages . EMBL4 (Frischauf et al.,
J.Mol. Biol. 170, 827-842
(1983))
Ml3mpl8 (Yanisch-Perron et
al., Gene 33, 103-119,
(1985))
M13mp19 (Yanisch-Perron et
al., (1985))
Plants
Solanum tuberosum AM 80/5793 (haploid)
Berolina (tetraploid)
Datura (D12)
Granola
Nicotinia tabacum Wisconsin 38 (W38)
Samsum NN (SNN)
Oryza sativa japonica c.v. Taipai
A11 molecular biological standard methods,
such as restriction analysis, plasmid isolation, mini-
preparation of plasmid-DNA, transformation of bacteria
and so on, were carried out as described by Maniatis et
al., (1982) unless otherwise indicated.
Wounded Plant Tissue
Potato tuber material was cut into slices 3 mm
thick, and incubated in a phosphate solution (20mM
phosphate buffer, pH 7.0, with chloramphenicol (50
micrograms/ml)) for 18 hours at 28~C in the absence of
light. Leaf material, stem material and root material
was cut into small pieces and incubated under the same
~.m:~.".,
2~
conditions. After the completion of incubation, the
material was either directly processed or stored at
-70~C.
Non-Wounded Plant Tissue
Leaf material, stem material, root material
and tuber material was processed immediately after
removal from the living plant, or frozen in liquid
nitrogen and stored at -70~C.
Aerobic Wounding' of Potato Tubers
Treatment as described under "wounding", in
which during the incubation the tuber slices were only
lightly wetted with the phosphate buffer. After the
incubation, the potato surfaces were brown.
Anaerobic Wounding of Potato Tubers
Treatment as described under "wounding", in
which during the incubation the potato slices were fully
submerged in the phosphate buffer. After the
incubation, the tuber surfaces were bright yellow-white.
Development of wunl-mRNA in Pathogen-Attacked Potato
Leaves
Leaves of the species "Datum" were cut away
at the leaf stem and placed in water along with the
stem. Then the leaves were sprayed with a suspension
containing phytophtora-spores. At different times, the
complete RNA of a given leaf could be isolated and
investigated using the "Northern-Slotblot-Analysis".
Three different materials were used:
- Water, containing the spores of a Datura-compatible
phytophtora infestans species Pil:
- Water, with the spores of a Datura-incompatible
phytophtora infestans species Pi4;
- Water, without addition of spores (control)
Two hours after the treatment of the leaves
with water and spores of the incompatible phytophtora
species, densitometric evaluation of the strength of the
28
wunl-hybridization indicated a 4-times increase in the
amount of wunl-mRNA. From the fourth to the eighth
hour, the amount remained approximately the same,
whereupon it fell gradually up to the 30th hour back to
the 1.5 to 2-times value. The same development ratios
were found for wunl-mRNA when using water without spores
(control).
In the analogous trial using water and
compatible phytophtora spores, a 4-times increase in
wunl-mRNA was seen 2 hours after the attack, this factor
only slightly reducing up to the 10th hour. From the
16th hour at the latest, the factor increased to about
6-times the control value, and then constantly rose to
reach a factor of 9 after 30 hours.
Wounding with the addition of potato extract
Potato tubers were homogenized at 4~C without
the addition of buffers, and then centrifuged for 10
minutes at 10,000 rpm in an SS34-rotor.
The remaining material was cooked for 10
minutes and then again centrifuged. The clear
supernatant was added in the ratio of 1:10 to 20 mM
phosphate buffer, pH 7.0, and the tissues to be tested
were then incubated under the usual conditions.
Example 1
RNA-Isolation
The isolation of RNA from various organs of
the potato and the tobacco plant were carried out as
described by Logemann et al. in Anal. Biochem., 163:16-
20 (1987).
The selective concentration of PolyA+RNA was
done using mAP-papers (Werner et al., Analytical
Biochem. 141, 329=336 (1984)).
"Northern-Blot" Analysis
The RNA was separated electrophoretically on a
1.5~ formaldehyde-agarose-gel (Lehrach et al.,
29
Biochemistry 16, 4743 (1977)). As described by
Willmitzer et al., EMBO J. 1, 139-146 (1982), the RNA
was next transferred to nitrocellulose, fixed and
hybridized with 32P*radioactively marked cDNA, grown and
exposed.
DNA-Isolation
Nuclear DNA was obtained according to the method
of Bedbrook, PMB Newsletter II, 24 (l981) from potato
leaves and used for the cloning procedure in Lambda
Phages EMBL4. DNA from the transformed tissues was
isolated and purified with Triton-X-100~, SDS and
proteinase K (Wassenegger, Dissertation, Koln (1988)).
"Southern Blot" Analysis
DNA was electrophoretically separated on 0.8 to
1.2% Agarose gel, transferred to nitrocellulose and
fixed (Southern, J.Mol.Biol. 98, 503-517 (1975)), and
hybridized and grown as described by Willmitzer et al.
(1982) .
"Run-Off Experiments
A method which relatively accurately distinguishes
between these two regulation types (transcriptional and
translational regulatiion) is the "run-off" method. In
this method, cell nuclei from wounded and non-wounded
tubers are isolated, and newly synthesized mRNA there-
from is radioactively pulse-labeled and hybridized with
single strand DNA of the individual clones. In cell
nuclei from non-wounded clones, no wunl-mRNA was pro-
duced. By contrast, in cell nuclei from wounded
tubers, this material was produced in high quantity.
The isolation of cell nucleic from potato tubers,
as well as the subsequent in-vitro transcription, were
carried out in accordance with Willmitzer et al, Nucl.
Acids Res. 9, 19 (1981). The radioactively labeled
RNA-transcripts obtained by this
30 ~;~ ~~t
x.
method were hybridized with bidirectional M13 cDNA-
clones.
Protein Isolation and Splitting
The isolation of proteins from potato tubers
as well as the 1-dimensional and 2-dimensional gel-
electrophoretical separation thereof, has been described
by Mayer et al., Plant Cell Reports 6, 77-81, (1987).
Example 2
"Hybrid-released-translation"-Experiments
The 800 by fragment of the cDNA-clone wunl-
25A2 and the 700 by fragment of the cDNA-clone wun2-
29C12 were cloned in M13mp18 using the PstI sites. The
orientation of the insertions in the M13-clones were
determined by complementation analyses (Messing, in:
Genetic Engineering of Plants, (1983), Plenum Press, New
York, 1983). The identification of the M13-clone
containing the coding strand resulted from positive
hybridization with radioactively labelled polyA+RNA from
wounded potato tubers.
The isolation of single strand DNA from M13
phages (they contain the coding strand), the fixation of
this DNA on nitrocellulose and the hybridization with
potato tuber RNA, as well as the wash and release of
selected RNA, has been described by Maniatis et al
(1982). The selected RNA as well as the polyA+RNA
isolated from the potato tubers were translated in a
rabbit-reticulocyte-lysate (Pelham and Jackson, Eur. J,.
Biochem. 67, 247 (l976)) in the presence of 35S-
Methionin. The 1-dimensional and 2-dimensional
electrophoretic separation on a polyacrylamide-gel was
carried out according to Mayer et al (1987).
Example 3
Establishment and Screening of a genomic bank
Isolation of genomic DNA:
31
According to the methods of Bedbrook (1981),
isolated leaf DNA of the haploid line AM 80/5793 was
used as genomic potato DNA.
This DNA was fully digested with EcoRI.
Isolation of the phage-DNA:
EMBL4-DNA was cut into three fragments with
EcoRI, wherein after gel-electrophoretic separation and
subsequent fragment isolation, the two vector arms of
the middle fragment could be separated. In addition,
commercially available purified EMBL4-arms were also
used (Amersham).
Ligation and Loading:
After subsequent ligation of the EMBL4-arms
with the EcoRI-digested genomic DNA, the ligated, high-
molecular DNA was loaded in vitro into phage heads.(Hohn
and Murray, Proc. Nat. Acad. Sci. USA, 74, 3259-63,
(1977); Hohn, Meth. Enzym. 68, 299-309 (1979)). The
loading material derived from a "Lambda in vitro
packaging kit" of the company Amersham. The genomic
bank was plated out with a concentration of 25.000
plaques per plate (25X25cm).
Plaque hybridization:
The detection of cDNA-homologous Lambda-clones
resulted from a plaque hybridization according to Benton
and Davies, Science 196, 180 (1977) using a
radioactively labelled cDNA. Positively hybridising
plaques were isolated and again tested.
DNA-Preparation of Recombinant Phages:
20 ml of a C600-lysed bacterial culture was
mixed with chloroform in order to obtain, after
subsequent centrifugation, a bacteria-free supernatant.
The sedimentation of the phages from the supernatant was
32
carried out by a 4-hour centrifugation at 10,000 rpm.
The phage sediment was removed in 500 microlitres phage-
buffer (10 mM Tris-HC1, pH 8.0, 10 mM MgCl2) and treated
with DNase and RNase. After the extraction of the DNA
through repeated phenolysation, the phage DNA was EtOH-
precipitated, washed with 70% EtOH and removed in TE.
Example 4
Creation of deletion mutants after exonuclease-III-
treatment
The fragment to be investigated was cloned in
both orientations in M13mp19, in accordance with
Henikoff et al., Gene 28, 351-359 (1984). Since the
exonuclease III digests 5' overhanging ends, while the
3' overhanging ends remain, 20 microgram of the DNA to
be analyzed was digested by two restriction enzymes,
creating 5'-end and a 3'-end. Because the 5'-end was
located towards the fragment, an exonuclease-digestion
of the fragment could take place. Using repeated
stopping of this reaction, it was possible to isolate
200 by smaller fragments, of which the sticky ends were
changed into ligatable blunt ends through a subsequent
S1 treatment. Finally, there followed the
transformation of this DNA in BMH 7118-cells, and then
the single strand DNA isolation.
3 3 ~~
Seguencing~
The sequencing of single strand DNA was
carried out according to the chain interruption method
of Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463,
(1977). The separation of the reaction materials was
done on 6~ and 8~ sequence gel (Ansorge amd de Mayer, J.
Chromatogr. 202, 45-53 (l980); Ansorge and Barker, J.
Biochem. Biophys. Meth. 9, 33 (1984)).
Cloning and sequencing of wunl-25A2
The approximately 711 bp-sized insert of the
clone wunl-25A2 was cloned with its PstI-site in the
M13mp19-vector. The orientation of the insert was
analysed using the asymmetric XhoI-site. The clones
25A2-4-mpl9 and 25A-5-mpl9, differing in the orientation
of the insert, were digested with KpnI/XbaI, in order to
make possible a bidirectional exonuclease III digestion
of the fragment. Deletion clones of different sizes
could be obtained by successive stopping of the
exonuclease reaction. These deletion clones served as
purified single strand DNA for sequencing in accordance
with the method of Sanger et al (1977).
Example 5
S1-Analysis
The principle of S1-nuclease mapping for the
determination of transcription starting sites is based
on the concept that, through a hybridization of single
strand DNA from the 5'-region of the wunl-gene with
wunl-mRNA, only those regions are protected against
destruction by the single strand-specific nuclease S1,
which on the basis of its homology, can form a double
strand. The size of the protected DNA fragment can be
determined on a sequencing gel, and thus the
transcription start can be traced back.
~a
34
The determination of the transcription start
point was carried out according to Berk and Sharp, Proc.
Natl. Acad. Sci. USA 75, l274 (1987). For this, the 1.2
kb fragment was isolated from the plasmid pLS000 with
EcoRI and XhoI, and dephosphorylised with phosphatase.
Finally, there followed a radioactive labeling of the
5'OH-ends through the combination of polynucleotide-
kinase and y-32P-ATP. After denaturing the DNA-
fragments, they were hybridized with 50 micrograms of
whole RNA (hybridization buffer: 80~ formamide, 0.4 mM
NaCl, 40 mM PIPES pH 6.4 and 1 mM EDTA). This condition
favours RNA-DNA-hybrids in comparison to DNA-DNA hybrids
(Casey and Davidson, Nucl. Acids Res. 4, 1539-1552
(1977)). The hybridization temperature started near
80~C and was lowered overnight to 40~C. A subsequent
S1-nuclease digestion (120 U/ml) removed unpaired
strands. It was expected that the radioactively
labelled XhoI site located at the 5'-untranslated region
of the wunl-gene would be protected due to its homology
to mRNA. Finally, the S1-protected DNA strand was
electrophoretically separated on a sequencing gel, so
that the sequence of a known DNA-fragment would serve as
a size marker.
Example 6
Transient Expression in Potato Protoplasts
Quantities of 20 micrograms of highly purified
(CsCl-gradient) plasmid-DNA was transformed in potato
protoplasts treated with PEG/CaCl2 (Lipphardt,
Dissertation, Koln, 1988). The protoplasts were
isolated from a potato stem suspension culture of the
species Datura.
Transient Expression in Rice Protoplasts
20 micrograms of highly purified (CsCl-
gradient) plasmid DNA was transformed in accordance with
35
the modified method by Lorz et al., Mol. Gen., Genet.
199, l78-182 (l985). The concentration of polyethylene
glycol and osmoticum were altered.
Example 7
DNA-Transfer in Actrobacteria
Conjugation:
DNA cloned in E. coli was transferred using
the helper strains GJ23 into the Agrobacteria species
3850k according to the method described by Van Haute
et al. (1983).
DNA-analysis of Agrobacteria:
The monitoring of the DNA transfer into the
Agrobacterium was carried out through the isolation of
Agrobacteria DNA according to the method described by
Murray and Thompson, Nuc. Acid Res. 8, 4321-4325 (1980).
Restriction digestion of the DNA, the transfer to
nitrocellulose and the hybridization with the
corresponding radioactive probe, point out a successful
DNA-transfer into the Agrobacteria.
Example 8
Transformation of Tobacco
Growth of Agrobacteria:
The Agrobacteria 3850 ~ necessary for
infection, were grown in selective antibiotic media
(Zambryski et al., 1983), sedimented through
centrifugation and washed in a YEB-medium without
antibiotics. After another sedimentation and
resuspension in YEB-medium, the bacteria can be used for
infection.
Leaf Disc-Infection:
Sterile leaves of the tobacco lines SNN and
W38 were used for the leaf disc-infection. Leaf pieces
about 1 cm in size were sterilized by a 10-minuted
36
incubation in 0.1% HgCl2, 0.1% SDS, and then washed
three times in sterile water. There followed the
submersion in an Agrobacterium suspension as described
above, and the subsequent transfer to 3MS-medium. After
a two-day incubation with 16 hours under light and at
about 25~C to 27~C, the leaf pieces were washed several
times in liquid 3MS-medium, and transferred to 3MSC-
medium. After 4-6 weeks, shoots which emerged were
isolated and incubated on a 2MSC-medium. As further
evidence of a successful transformation, the nopaline
test was carried out.
Analysis of the Transformed Plants
Evidence of nopaline-synthase activity:
The detection of nopaline-synthase activity in
transformed plant leaves was carried out according to
Otten and Schilperoort, Biochem. Biophys. Acta 527, 497
(1978) .
Proof of NPT-II-Activity:
The NPT-II-activity in transformed plants was
established in accordance with Reiss et al., Gene 30,
217-223 (1984) and Schreier et al., EMBO J., 4, 25-32
(1985) .
Proof of CAT-Activity:
Chloramphenicol-acetyl-transferase (CAT)
activity was determined according to the method of
Velten and Schell, Nuc. Acids, Res. 13, 6981-6997 (1985)
and Herrera-Estrella et al., EMBO J. 2, 987 (1983).
Example 9
Optimization of the wunl-promoter by fusion with the TR-
promoter (TR1'wunl)
For the media used, reference may be had to
Examples 1-8.
2.1.4. Species and Vectors
Agrobacterial species: GV3101pmp90RK (Koncz,
C. and Schell, J. "The promoter of TL-DNA gene 5
controls the tissue specific expression of chimeric
genes carried by a novel type of Agrobacterium binary
vector" (1986), Mol. Gen. Gent. 204:383-396).
Plasmids:
pPCV720
35S-GUS = Prt99-GUS (Topfer, R., Schell, J.
and Steinhib, H.H. "Versatile
cloning vectors for transient gene
expression and direct gene transfer
in plant cells." (1988)
wunl-GUS
TR1'-wunl-GUS
TR1'-GUS
Plants:
Nicotinia tabacum Wisconsin 38 (W38)
Methods
A11 molecular biological standard methods, such
as, for example, Restriction analysis, Plasmid
Isolation, Mini-preparations of Plasmid DNA,
Transformation of Bacteria and so on, were carried out
as described by Maniatis et al., (1982), unless
otherwise indicated.
Injured and Uninjured Plant Tissue were obtained
as described in Examples 1-8.
Reference to Examples 1-8 can also be had in
connection with the RNA isolation, the "Northern Blot"
analysis, the DNA isolation, the transient expression
in rice protoplasts, the "Southern Blot" analysis and
the DNA-analysis of Agrobacteria
38
DNA-Transfer into Aarobacteria
The DNA cloned in E. coli was transferred into
the Agrobacteria species GV3101pmp90RK, in accordance
with the method described by Van Houte, E., Joos, H.,
Maes, M., Warren, G., Van Montagu, M., Schell, J.
"Intergenic transfer and exchange recombination of
restriction fragments cloned in pBR322: a novel
strategy for reversed genetics of Ti-plasmids of
Agrobacterium tumefaciens", EMBO J., 2:411-418.
Transformation of tobacco
Culture of Agrobacteria
The Agrobacteria GV3101pmp90RK, containing the
desired plasmid, were grown in a selective antibiotic
medium (Hygromycin), sedimented by centrifugation and
washed in YEB-medium (Maniatis et al, 1982) without
antibiotics. After further sedimentation and removal in
YEB-medium, the bacteria could be used for infection.
Leaf Disc-Infection
This was carried out as described in Example
8, however without the use of the nopaline test.
Analysis of the transformed plants
Proof of GUS-activity
A: The fluorometric analysis of beta-glucoronidase-
activity (GUS-activity) was carried out in accordance
with the methods of Jefferson, A.R., Kavanagh, T.A.,
Bevan, M.W. "Gus-Fusions: f3-Glucoronidase as a sensitive
and versatile gene fusion marker in higher plants", EMBO
J. 6:3901-3907 (1987).
B: Blue - staining of tissue in relation to GUS-
activity could be obtained using X-Gluc solution. X-
Gluc solution. 1-2mM X-Gluc (5-Bromo-4-chloro-3-
indolyl-glucuronide), and dimethyl formamide were
dissolved in 50 mM phosphate-buffer at pH 7Ø
- Wounded Tissues:
Thin tissue slices were incubated as described under
"Wounded Plant Tissues" and then stained overnight at
~,: a~~ ,~-,I ~ 'r~,' p
x.. 0.:J
39
37~C in X-Gluc solution. Finally, the chlorophyll was
removed by the addition of 100% methanol at 60~C.
- Non-wounded Tissues:
Thin tissue slices were incubated in X-Gluc solution
which additionally contained the translational inhibitor
Cycloheximide (1.8 mM). The incubation period and the
methanol treatment are the same as with the wounded
tissue.
- X-Gluc staining of Pollen:
Pollen was incubated overnight at 28~C in 30%
sucrose, leading to the development of pollen tubes.
Finally, a doubly concentrated 30% X-Gluc solution was
added and the pollen were incubated overnight at 28~C.
Alternatively, the pollen grains are incubated directly
in X-Gluc, without developing pollen tubes.
Results
Organization of TR1'-wunl-Gus, TR1'-GUS and wunl-GUS
(Figure 12)
wunl-GUS:
l022 by of the wunl-promoter and 178 by of the
wunl 5'-untranslated region were transcriptionally fused
with the beta-Glucuronidase-Gene (GUS). The poly-
adenylation signal (pA) of the nopaline-synthase-gene
was used (NOS) as a termination sequence.
TR1'-GUS:
The TRl'-promoter was fused transcriptionally
with the GUS-gene, and terminated by the pA-signal of
the NOS-gene.
TR1'-wunl-GUS:
Behind the 1'-promoter of the TR-promoter the
pA-signal of the octopine-synthase-gene (OCS) was cloned
in order to suppress a transcription through the wunl-
promoter. Behind the OCS pA-signal the wunl-promoter
(1022 bp) is located, including (in homology to the
wunl-GUS construction) 179 by of the untranslated 5'
region of the wunl-gene which are transcriptionally
3
~~;
fused with the GUS-gene. The pA-signal of the 35-S gene
serves as the termination sequence for the GUS-gene.
This construction (TR1'-wunl-GUS) is present in the
binary vector pPCV720, which can replicate in both E.
5 coli and in Agrobacteria (with the help of a helper
plasmid).
RNA-Analysis of TR1'-wunl-GUS transgenic tobacco
RNA from wounded and non-wounded leaves of
various TR1'-wunl-GUS transgenic tobacco plants, as well
10 as from wuni-GUS and 35S-GUS transgenic tobacco leaves,
was isolated and hybridized with radioactively labelled
GUS-DNA.
As is shown in the Northern-Blot in Figure 13,
1. The GUS-mRNA in transgenic TR1'-wunl-GUS-leaves is
15 exactly as large as in wunl-GUS transgenic leaves.
It appears that in both constructions, the same
transcription start was used.
2. The amount of RNA in wounded leaves, both in TR1'-
wunl-GUS-plants and in wunl-GUS-plants, is higher
20 than in non-wounded leaves. The wound-
inducibility already known for wunl-GUS was also
true for TR1'-wunl-GUS.
3. From an RNA quantity comparison, it was realized
that somewhat more RNA was to be found in wounded
25 TR1'-wunl-transgenic leaves than in 35S-GUS
transgenic leaves. Since the 35S-promoter is one
of the strongest promoters in plants, the TR1'-
wunl-promoter is estimated of similar strength.
Analvsis of the GUS-activity in transctenic tobacco
30 A quantitative analysis of GUS-activity of
transgenic TR1'-wunl-GUS plants shows that the TR1'-
wunl-promoter in transgenic leaves is inducible by
wounding at a factor of 2 times up to 13 times (on
average about six times). This observation is similar
35 to the wound inducibility of the wunl-promoter in
transgenic leaves (Logemann J., Lipphardt S., Lorz, H.,
h ~9 ~, ,G,
~a
41
Hauser, I., Willmitzer, L. and Schell J. "5'upstream
sequences from the wunl-gene are responsible for gene
activation by wounding in transgenic plants" The Plant
Cell 1:15l-158 (1989A)). Analogously to the trangenic
wunl-GUS tobacco plants, the wound inducibility in the
higher, younger leaves is the highest, while, in the
lower, older leaves it is the smallest. The reason for
this difference in inducibility is the increasing
activity, from top to bottom, of the TR1'1-wunl-promoter
in non-wounded tissue. In old leaves of old plants, the
activity of the TR1'-wunl-promoter in non-wounded
condition is already so high that wounding cannot induce
any further activity (analogous to wunl-GUS plants).
A relative comparison of promoter-activities
in transgenic tobacco leaves and stems shows that the
wunl-promoter is roughly 10 times stronger than the
TRl'-promoter. The TR1'-wunl-GUS promoter, on the
other hand, is 10 times stronger than the wunl-promoter.
Consequently, the TR1'-wunl-promoter is roughly 100
times stronger than the TR1'-promoter.
Localization of GUS-activity in transgenic tobacco
- Leaf, Stem:
The GUS-activity in the tissue slices from
wounded and non-wounded leaves and stems of wunl-GUS and
TR1'-wunl-GUS transgenic tobacco was localized by the
use of the substrate X-Gluc.
wunl-GUS:
As can be seen from the blue colored regions,
the activity of the wunl-promoter is limited primarily
to the epidermis (including trichome), and to a lesser
extent the vascular system of wounded leaves (W). In
non-wounded leaves (NW), on the other hand, only a small
wunl-promoter-activity can be seen in the epidermis, and
no activity in the vascular system. The same results
were obtained also with stem cross-sections.
y~ ~ ~; :'r~
42
Accordingly, the wunl-promoter activity is to be
regarded as epidermis-specific in leaves and stems.
In TR1'-wunl-GUS plants, an intensive blue
coloring in the vascular system of wounded plant slices
is recognized. In other regions (epidermis,
parenchyme), the coloring is weak. Non-wounded leaf
slices show a low level of blue coloring in the vascular
system, and no color in the epidermis. The same results
were found with stem cross sections.
This appears to suggest, in connection with
TR1'-wunl-GUS, a vascular-specific promoter in leaves
and stems.
Leaf cross-sections of TR1'-GUS plants were
used as control. Because of the weak promoter activity
of the TR1'-promoter (10 times weaker than the wunl-
promoter), blue staining was not found in either wounded
or non-wounded leaf cross sections.
- Anthers:
An X-Gluc staining of anther cross-sections as
well as of pollen shows the following characteristics,
which apply both for wunl-GUS and for TR1'-wunl-GUS:
A blue staining (and GUS-activity) was
detected in the stomium (the natural perforation
location of the anther in order to release pollen) and
in the pollen. In separated ungerminated pollen and
germinated pollen, there was found a blue staining in
the pollen grain and in the pollen tube.
Transient Expression with Rice-protoplasts
The constructions TR1'-wunl-GUS, wunl-GUS,
TR1'-GUS and 35S-GUS were used for studies of transient
development with rice protoplasts of the variety Oryza
sativa japonica c.v. Taipai. As shown in Table 1, the
activity of TR1'-GUS is not significantly different from
the control (GUS-activity of rice-protoplasts), which
were transformed without DNA). wunl-GUS and 35S-GUS
show a 2 times to 3 times higher activity than the
43
control. By contrast, TR1'-wunl-GUS DNA exhibits on
average a 57-times higher activity than the control, and
therefore is a highly active promoter in rice
protoplasts.
Table 1
Construction GUS-Activity Relative
nmol MU,/mgfmin GUS-Activity
TR-GUS 31 1.2
wun-GUS 59 2.3
TR-wun-GUS 1416 56.7
35S-GUS 66 2.6
Control 25 1.0
Legend for Table 1:
The given GUS-activity was based on 5 independent tests.
The results of Table 1 are seen through the
staining of the protoplasts with X-Gluc. Only
protoplasts transformed with TR1'-wunl-GUS show an
intensive blue staining.
