Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF INCREASING THE RESISTANCE OF CULTIVATED
PLANTS TO PHYTOPATHOGENIC FUNGI AND BACTERIA BY
METHODS OF MOLECULAR GENETICS
The present invention is a method of increasing the resistance of
crop plants to bacterial and fungal pathogens, wherein a plant is
generated by methods of molecular genetics in which the activity
of the enzyme flavanone 3-hydroxylase is reduced.
The method furthermore comprises inhibiting the activity of the
enzyme flavanone 3-hydroxylase fully or partially, permanently or
transiently, in the whole plant or in parts of the plant, by
methods of molecular biology (for example antisense construct,
cosuppression, the expression of specific antibodies, or the
expression of specific inhibitors).
The present invention furthermore relates to plants with an
increased resistance to bacterial and fungal pathogens, wherein
the activity of the enzyme flavanone 3-hydroxylase is reduced by
methods of molecular genetics.
The productivity of crop plants can be reduced in many ways by
stress factors. Stress factors which may be mentioned are, inter
olio, viral diseases, bacterial and fungal pathogens, harmful
insects, nematodes, slugs and snails, game damage, high,
moderately low and low temperatures, lack of water, unduly high
water content of the soil, soil salinification, unduly high
radiation intensity, unduly high ozone content, competition for
light, water and nutrients by the accompanying flora, inexpertly
applied herbicides, herbicides which have not been applied
optimally (in particular in fruit plantations), treatments with
herbicides, insecticides, fungicides, bioregulators or foliar
fertilizers of unduly low sensitivity, foliar applications of
crop protection products or fertilizers during intense
insolation.
Some of these problems caused by stressors can be minimized by
employing crop protection products, by using resistant plant
material or using suitable husbandry techniques. However, the
extent of possibilities is limited. Bacterioses, in particular,
can only be controlled with great difficulty, if at all. To do so
(for example when controlling fire blight in apples and pears),
antibiotics such as streptomycin or tetracyclins are employed,
which harbors the danger of a resistance developing, including
human pathogens. Moreover, for example fungal pathogens
frequently show adaptation to fungicides, so that the efficacy of
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the latter is diminished. A similar adaptation also exists in
"pathogen-resistant" plant breeding products generated by
conventional methods.
There is not only a demand for pathogen-resistant plants in
annual arable or horticultural crops, but also in valuable
perennial crops such as fruit and vines.
It is an object of the present invention to find a simple and
inexpensive method for permanently improving the resistance to
bacterial and fungal pathogens, in particular in crop plants.
We have found that this object is achieved, surprisingly, by
genetic engineering methods which are based on physiological
studies on growth regulators from the acylcyclohexanedione group
and with the aid of which crop plants can be generated which are
resistant to a series of phytopathogenic bacteria and fungi.
Acylcyclohexanediones such as prohexadione-Ca and
trinexapac-ethyl (previously known as cimectacarb) are employed
as bioregulators for inhibiting the longitudinal growth of
plants. The reason for their bioregulatory action is that they
block the biosynthesis of gibberellins, which promote
longitudinal growth. Owing to their structural relationship with
2-oxoglutaric acid, they inhibit certain dioxygenases which
require 2-oxoglutaric acid as co-substrate (Rademacher, W,
Biochemical effects of plant growth retardants, in: Plant
Biochemical Regulators, Gausman, HW (ed.), Marcel Dekker, Inc.,
New York, pp. 169-200 (1991)). It is known that such compounds
also engage in phenol metabolism and therefore can cause
inhibition of the anthocyanin production in several plant species
(Rademacher, W et al., The mode of action of
acylcyclohexanediones - a new type of growth retardant, in:
Progress in Plant Growth Regulation, Karssen, CM, van Loon, LC,
Vreugdenhil, D (eds.), Kluwer Academic Publishers, Dordrecht
(1992)). Such effects on the balance of phenolic constituents are
given as the cause for the side effect of prohexadione-Ca against
fire blight (Rademacher, W et al., Prohexadione-Ca - a new plant
growth regulator for apple with interesting biochemical features,
poster at the 25th Annual Meeting of the Plant Growth Regulation
Society of America, July 7-10, 1998, Chicago). A. Lux-Endrich
(PHD Thesis, Technical University of Munich at Weihenstephan,
1998) has found, during her studies on the mechanism of action of
prohexadione-Ca against fire blight, that, in apple cell
cultures, prohexadione-Ca causes an increase in phenolic
substance content-by several times, and that a series of phenols
which are otherwise not present is found. Within these studies,
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it was also found that the effect of prohexadione-Ca leads to
relatively large quantities of luteoliflavan and eriodictyol in
apple shoot tissue. Luteoliflavan does not normally occur in
apple tissue, and eriodictyol is present in minor amounts only as
an intermediate in the flavonoid metabolism. However, the
expected flavonoids catechin and cyanidin were not detectable in
the treated tissue, or only in markedly reduced quantities
(S. Rommelt et al., Paper presented at the 8th International
Workshop on Fire Blight, Kusadasi, Turkey, October 12-15, 1998).
It is an established observation that prohexadione-Ca,
trinexapac-ethyl and other acylcyclohexanediones inhibit
2-oxoglutaric acid-dependent hydroxylases which play an important
role in the metabolism of phenolic substances. These are
primarily chalcon synthetase (CHS) and flavanone 3-hydroxylase
(F3H) (W. Heller and G. Forkmann, Biosynthesis, in: The
Flavonoids, Harborne, JB (ed.), Chapman and Hall, New York,
1988). However, it cannot be excluded that acylcyclohexanediones
also inhibit other 2-oxoglutaric acid-dependent hydroxylases
which are hitherto unknown. Also, it seems to be obvious that a
lack of catechin, cyanidin and other flavonoid synthesis end
products is registered by the plant and that the activity of the
key enzyme phenylalanine ammonium-lyase (PAL) is increased via a
feedback mechanism. However, since CHS and F3H are still being
inihibited, these flavonoid end products cannot be produced, and
this leads to an increased production of luteoliflavan,
eriodictyol and other phenols (Figure 1).
Since the enzyme activity of the enzyme flavanone 3-hydroxylase
(F3H) is reduced, the flavonoids eriodictyol, proanthocyanidins
which are substituted on the C atom 3 by hydrogen, for example
luteoforol, luteoliflavan, apigeniflavan and tricetiflavan, and
homogeneous and heterogeneous oligomers and polymers of the
abovementioned and structurally related substances are produced
in greater quantities.
Increased concentrations of the phenols hydroxycinnamic acid
(p-coumaric acid, ferulic acid, sinapic acid), salicylic acid or
umbelliferone, including the homogeneous and heterogeneous
oligomers and polymers formed with them, are found in plants
after the enzyme activity of the enzyme flavanone 3-hydroxylase
(F3H) has been reduced. The concentration of the chalcones, for
example phloretin, and of the stilbenes, for example resveratrol,
are also elevated.
A reduction in the enzyme activity of the enzyme flavanone
3-hydroxylase also leads to an increased concentration of the
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glycosides of the flavonoids, of the phenolic compounds, of the
chalcones and of the stilbenes.
Based on these findings and conclusions, genetically modified
crop plants were generated in which F3H was reduced fully or
partially, permanently or transiently, in the whole plant or in
individual plant organs or tissues, by means of anti-sense
constructs, with the consequence that the phenolic compound
content in the entire plant is reduced. Subsequently, it was
possible to demonstrate experimentally that the resistance of
these plants to bacterial and/or fungal pathogens is increased.
As an alternative to the generation of plants whose flavonone
3-hydroxylase activity is reduced by means of antisense
technology, it is also possible to use other methods of molecular
genetics which are known from the literature in order to achieve
this effect, such as cosuppression, or the expression of specific
antibodies.
The method according to the invention for increasing the
resistance to attack by bacterial and fungal pathogens by
reducing the flavonone 3-hydroxylase enzyme activity can be
practiced successfully in the following crop plants: wheat,
barley, rye, oats, rice, maize, panic grasses, sugar cane,
bananas, tomatoes, tobacco, bell peppers, potatoes, oilseed rape,
sugar beet, Soya, cotton, tree fruit from the Rosaceae family,
such as apples and pears, plums, quetsch, peaches, nectarines and
cherries, and grapevines.
The method according to the invention is especially suitable for
increasing the resistance to Venturia inaequalis in apples and
pears and to Botrytis cinerea in grapevines.
Transgenic plants with reduced activity of the enzyme flavanone
3-hydroxylase, generated by the method described in the Examples,
surprisingly show an increased resistance to attack by
phytopathogenic bacteria. This was demonstrated with reference to
the attack by transgenic tomato plants whose flavanone
3-hydroxylase activity is reduced, by Clavibacter michiganensis
subsp. michiganensis (Cmm); see Example 3.
Plants whose flavanone 3-hydroxylase activity was reduced with
the aid of methods of molecular genetics also showed an increased
resistance to attack by Erwinia amylovora and other
phytopathogenic bacteria. The most important phytopathogenic
bacteria can be found in the publication "European Handbook of
Plant Diseases", Eds. Smith, I.M., Dunez, J., Lelliott, R.A.
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Phillips, D.H. and Archer, S.A. Blackwell Scientific
Publications, 1988.
In particular, the method according to the invention is suitable
5 for increasing the resistance to the following phytopathogenic
fungi
Erysiphe graminis (powdery mildew) on cereal
Erysiphe cichoracearum and Sphaerotheca fuliginea on cucurbites
Podosphaera leucotricha on apples,
Uncinula necator on grapevines,
Puccinia species on cereal,
Rhizoctonia species on cotton, rice and turf,
Ustilago species on cereals and sugar cane,
Venturia species (scab) on apples and pears,
Helminthosporium species on cereals,
Septoria species on wheat,
Botrytis cinerea (gray mold) on strawberries, vegetables,
ornamentals and grapevines,
Cercospora arachidicola on peanuts,
Pseudocercosporella herpotrichoides on wheat and barley,
Pyricularia oryzae on rice,
Phytophthora infestans on potatoes and tomatoes,
Plasmopara viticola on grapevines,
Pseudoperonospora species in hops and cucumbers,
Alternaria species on vegetables and fruit,
Mycosphaerella species in bananas and peanuts, and
Fusarium and Verticillium species in cereals, vegetables and
ornamentals.
Example 1
Cloning the gene of a flavanone 3-hydroxylase from Lycopersicon
esculentum Mill.cv. Moneymaker.
Ripe tomato fruits of Lycopersicon esculentum Mill.cv. Moneymaker
were washed, dried, and the pericarp was freed from seeds,
central columnella and woody parts by means of a sterile blade.
The pericarp (approx. 50 g) was frozen in liquid nitrogen. The
material was subsequently comminuted in a blender. In a
pre-cooled mortar, the comminuted material was treated with
100 ml of homogenization medium and mixed. The suspension was
then transferred into centrifuge tubes by squeezing it through
sterile gauze. Then, 1/10 volume 10~ SDS was added and the batch
was mixed thoroughly. After 10 minutes on ice, 1 volume of
phenol/chloroform-was added, and the centrifuge tube was sealed
and the contents mixed thoroughly. After centrifugation for 15
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minutes at 4000 rpm, the supernatant was transferred into a fresh
reaction container. Three more phenol/chloroform extractions and
one chloroform extraction followed. Then, 1 volume of 3 M NaAc
and 2.5 volumes of ethanol were added. The nucleic acids were
precipitated overnight at -20°C. Next morning, the nucleic acids
were pelleted in a refrigerated centrifuge (4°C) for 15 minutes at
10,000 rpm. The supernatant was discarded and the pellet was
resuspended in 5-10 ml of cold 3 M NaAc. This washing step was
repeated twice. The pellet was washed with 80$ ethanol. When
completely dry, the pellet was taken up in approx. 0.5 ml sterile
DEPC water, and the RNA concentration was determined
photometrically.
~.g of total RNA were first treated with 3.3 ~.1 of 3M sodium
15 acetate solution and 2 ~,1 of 1M magnesium sulfate solution, and
the mixture was made up with DEPC water to an end volume of
100 ~,1. One microliter of RNase-free DNase (Boehringer Mannheim)
was added, and the mixture was incubated for 45 minutes at 37~.
After the enzyme had been removed by extraction by shaking with
20 phenol/chloroform/isoamyl alcohol, the RNA was precipitated with
ethanol and the pellet was taken up in 100 ~1 DEPC water. 2.5 ~g
of RNA from this solution were transcribed into cDNA using a cDNA
kit (Gibco BRL).
Using amino acid sequences which were derived from cDNA clones
encoding for flavanone 3-hydroxylase, conserved regions in the
primary sequence were identified (Britsch et al., Eur. ,7.
Biochem. 217, 745 -754 (1993), and these form the basis for the
design of degenerated PCR oligonucleotides. The 5'-
oligonucleotide was determined using the peptide sequence SRWPDK
(aminoacid 147-152 in the Petunia hybrida sequence FL3H PETHY)
and had the following sequence:
5'-TCI (A/C) G (A/G) TGG CC(A/C/G) GA (C/T) AA (A/G) CC-3.
The sequence of the oligonucleotide derived using the peptide
sequence DHQAW (amino acid 276281 in the Petunia hybrida
sequence FL3H PETHY) was as follows: 5'-CTT CAC ACA (C/G/T) GC
(C/T) TG (A/G)TG (A/G)TC-3.
The PCR reaction was carried out using the Perkin-Elmer tTth
polymerase following the manufacturer's instructions. 1/8 of the
cDNA was employed as template (corresponding to 0.3 ~.g of RNA).
The PCR program was as follows:
30 cycles
94 degrees 4 sec
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40 degrees 30 sec
72 degrees 2 min
72 degrees 10 min
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The fragment was cloned into the vector pGEM-T by Promega
following the manufacturer's instructions.
The correctness of the fragment was checked by sequencing. Using
the restriction cleavage sites Ncol and Pstl, which exist in the
polylinker of vector pGEM-T, the PCR fragment was isolated, and
the overhangs were made blunt-ended using T4 polymerase. This
fragment was cloned into an SmaI (blunt-)ended vector pBinAR
(Hofgen and Willmitzer, Plant Sci. 66: 221 -230 (1990)) (see
Figure 2). This vector contains the CaMV (cauliflower mosaic
virus) 35S promotor (Franck et al., Cell 21: 285 - 294 (1980))
and the octopine synthase gene termination signal (Gielen et al.,
EMBO J. 3: 835 - 846( 1984)). In plants, this vector mediates
resistance to the antibiotic kanamycin. The resulting DNA
constructs contained the PCR fragment in sense and antisense
orientation. The antisense construct was employed for generating
transgenic plants.
Figure 2: Fragment A (529 bp) contains the CaMV 35S promotor
(nucleotide 6909 to 7437 of cauliflower mosaic virus). Fragment B
contains the fragment of the F3H gene in antisense orientation.
Fragment C (192 bp) contains the termination signal of the
octopine synthase gene .
Cloning of a larger cDNA fragment of the Lycopersicon esculentum
Mill.cv. Moneymaker flavanone 3-hydroxylase using the 5'RACE
system.
A second antisense construct using a larger F3H fragment should
be generated so as to exclude failure to generate plants with a
reduced mRNA flow equilibrium quantity of F3H due to the small
size of the F3H PCR fragment used in zhe antisense construct.
To clone a larger F3H fragment, the 5'RACE method (system for
rapid amplification of cDNA ends) was used.
Extending the F3H PCR fragment by the 5'RACE method using the
5'RACE system for rapid amplification of cDNA ends, Version 2-0
by Life TechnologiesTM.
Total RNA was isolated from ripe tomato fruits of Lycopersicun
esculentum Mill.cv. Moneymaker (see above).
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cDNA first strand synthesis was carried out following the
manufacturer's instructions using the GSP-1 (gene-specific
primer) 5'-TTCACCACTGCCTGGTGGTCC-3'. Following RNase digestion,
the cDNA was purified following the manufacturer's instructions
using the GlassMAx spin system from Life TechnologiesTM.
Following the manufacturer's instructions, a cytosin homopolymer
was added to the 3' end of the purified single-stranded F3H cDNA
using terminal deoxynucleotidyl transferase.
5'-extended F3H cDNA was amplified using a second gene-specific
primer (GSP-2) which binds in the 3' region upstream of the GSP-1
recognition sequence, thus allowing a "nested" PCR. The 5' primer
used was the "5'RACE abridged anchor primer" which was provided
by the manufacturer and which is complementary to the
homopolymeric dC tail of the cDNA.
The cDNA fragment amplified in this way and termed F3Hextendea was
cloned into the pGEM-T vector by Promega following the
manufacturer's instructions.
The identity of the cDNA was confirmed by sequencing.
The F3Hextenaea cDNA fragment was isolated using the restriction
cleavage sites Ncol and PstI, which are present in the polylinker
of the vector pGEM-T, and the overhangs were made blunt-ended
using T4-polymerase. This fragment was cloned into an SmaI
(blunt) Vector pBinAR (Hofgen and Willmitzer, 1990) (see
Figure 3). This vector contains the CaMV (cauliflower mosaic
virus) 35S promotor (Franck et al., 1980) and the octopine
synthase gene termination signal (Gielen et al., 1984). In
plants, this vector mediates resistance to the antibiotic
kanamycin. The DNA constructs obtained contained the PCR fragment
in sense and antisense orientation. The antisense construct was
employed for generating transgenic plants.
Figure 3: Fragment A (529 bp) contains the CaMV 35S promotor
(nucleotides 6909 to 7437 of the cauliflower mosaic virus).
Fragment B contains the fragment of the F3H gene in antisense
orientation. Fragment C (192 bp) contains the octopine synthase
gene termination signal.
005050060 CA 02340329 2001-02-15
Example 2
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Generation of transgenic Lycopersicon esculentum Mill.cv.
Moneymaker expressing a flavanone 3-hydroxylase subfragment in
antisense orientation.
The method according to Ling et al., Plant Cell Report 17, 843 -
847 ( 1998 ) was used. Cultivation was carried out at approx. 22°C
in a 16-h - light / 8-h - dark regime.
Tomato seeds (Lycopersicon esculentum Mill. cv. Moneymaker) were
sterilized by incubation for 10 minutes in 4g sodium hypochlorite
solution, subsequently washed 3 - 4 times with sterile distilled
water and placed on MS medium supplemented with 3% sucrose, pH
6.1, for germination. After a germination time of 7 - 10 days,
the cotyledons were ready for use in transformation.
Day 1: Petri dishes containing "MSBN" medium were overlaid with
1.5 ml of an approximately 10-day-old tobacco suspension culture.
The plates were covered with film and incubated at room
temperature until the next day.
Day 2: Sterile filter paper was placed in such a way on the
plates overlaid with the tobacco suspension culture that air
bubbles were absent. The cotyledons, which had been dissected
crosswise, were placed on this filter paper upside down. The
Petri dishes were incubated for 3 days in the culture room.
Day 5: The agrobacterial culture (LBA4404) was sedimented by
centrifuging for 10 minutes at approx. 3000 g and resuspended in
MS medium so that the OD was 0.3. The cotyledon sections were
placed into this suspension and incubated with gentle shaking for
30 minutes at room temperature. Thereafter, the cotyledon
sections were dried a little on sterile filter paper and returned
to their starting plates to continue cocultivation for 3 days in
the culture room.
Day 8: The cocultivated cotyledon sections were placed on
MSZ2K50+13 and incubated for the next 4 weeks in the culture room.
Then, they were subcultured.
Shoots which formed were transferred to root induction medium.
After successful rooting, the plants were ready to be testing and
transferred to the greenhouse.
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Example 3
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Transgenic tomato plants with reduced flavanone 3-hydroxylase
activity infected with Clavibacter michiganensis subsp.
5 michiganensis (Cmm).
Cmm was grown for 2 days at 28°C on yeast-dextrose-Ca agar (YDC).
The bacteria were rinsed with sterile water, and their cell
density was determined. For inoculation, the cell density was
10 brought to 106 cells / ml using sterile water. The injections were
carried out using 20-gage hypodermic needles filled with the
bacterial suspension. They were given into the leaf axil of the
uppermost fully developed leaf of young plants having a total of
3-4 leaves. The infection was evaluated by assessing the
phenotype which developed.
While over 75% of the leaves were wilted in wild-type plants, a
significantly lower degree of wilting was found in the transgenic
tomato plants.
Example 4
Test for the increase in the resistance to attack by Phytophthora
infestans in tomatoes with flavanone 3-hydroxylase in antisense
orientation.
The leaves of tomato plants cd. "Moneymaker" which were not
genetically modified or genetically modified according to the
invention were infected with an aqueous zoospore suspension of
Phytophthora infestans one week after they had reached the 4-leaf
stage. Then, the plants were placed into a chamber with 100%
atmospheric humidity at temperatures between 16 and 18~C. After 6
days, the brown rot on the control plants which had not been
genetically modified had developed to a high degree. Tomato
plants which expressed an antisense construct of flavanone
3-hydroxylase showed a considerably lower level of Phytophthora
infestans infection than the control.
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