JP2003505016A - Methods for increasing the resistance of cultivated plants to phytopathogenic fungi and bacteria using molecular genetics techniques - Google Patents

Abstract

  (57) [Summary] Claims are directed to a method for increasing the resistance of cultivated plants to bacterial and fungal pathogens, which comprises producing a plant with reduced activity of the enzyme flavanone 3-hydroxylase by molecular genetics techniques.

Description

Detailed Description of the Invention

[0001] The present invention increases the resistance of cultivated plants to bacterial and fungal pathogens, which is characterized by producing a plant in which the activity of the enzyme flavanone 3-hydroxylase is reduced by a method of molecular genetics. Regarding how to make.

Further, the present method provides a method for determining the activity of the enzyme flavanone 3-hydroxylase in whole or a part of a plant by means of molecular biology (eg, antisense construct, co-suppression, expression of specific antibody, Expression of a specific inhibitor), either completely or partially, permanently or temporarily.

The invention further relates to plants with increased resistance to bacterial and fungal pathogens, characterized in that the enzymatic activity of the enzyme flavanone 3-hydroxylase is reduced by means of molecular genetics techniques.

The productivity of cultivated plants can be reduced in various ways by stressors. Stress factors may include, among others: viral diseases, bacterial and fungal pathogens, harmful insects, nematodes, slugs, and snails, as well as feeding by wildlife, and high and low temperatures, and Cold temperature, lack of water, excessive moisture content of soil, excessive soil salinity, excessive radiation intensity, excessive ozone content, competition for light, water, and nutrients by coexisting flora, immature practices Treatment with herbicides applied in, and not applied optimally (particularly fruit plantations), and herbicides, insecticides, fungicides, bioregulators, or foliar fertilizers with much lower selectivity. , Application to crop protection agents or fertilizer leaves during strong sunshine.

Some of the problems caused by stressors can be minimized by using crop protection products, using resistant plant material, and using suitable agricultural techniques. . However, the possible range is limited. Bacterial diseases in particular are very difficult to control, even if they can be suppressed. Antibiotics such as streptomycin and tetracycline are used to provide such control (eg, to control burns in apples and pears), which may lead to resistance, including human pathogens. Be sexual. In addition, fungal pathogens, for example, often show adaptation to fungicides, reducing their effectiveness. Similar indications are "pathogen resistant" created by conventional methods
It also occurs in breeding plants.

There is a demand for pathogen-resistant plants not only in annual crops or horticultural crops but also in valuable perennial plants such as fruits 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, especially in cultivated plants.

The present inventors have surprisingly achieved this object by a genetic engineering method based on physiological research on growth regulators derived from the acylcyclohexanedione group, and utilize the method. Have found that it is possible to produce plants that are resistant to a range of phytopathogenic bacteria and fungi.

Acylcyclohexanediones, such as prohexadione-calcium and trinexapac-ethyl (formerly known as Simectacarb), are used as bioregulators to suppress the vertical growth of plants. They exhibit bioregulatory effects because they interfere with the biosynthesis of gibberellin, which promotes longitudinal growth. Due to their structural association with 2-oxoglutarate, they inhibit certain dioxygenases that require 2-oxoglutarate as a co-substrate [Rademacher
, W, Biochemical effects of plant growth retardants, Plant Biochemical R
Among egurators, Gausman, HW (ed.), Marcel Dekker, Inc., New York, pp. 169-200
(1991)]. It is known that such compounds are also involved in phenol metabolism and therefore can inhibit anthocyanin production in various plant species [Ra
demacher, W. et al., The mode of action of cylcyclohexanediones-a new type
of growth retardant, Progress in Plant Growth Regulation, Karssen, CM,
van Loon, LC, Vreugdenhil, D (ed.), Kluwer Academic Publishers, Dordrecht
(1992)]. Such effects on the balance of the phenolic component are thought to be responsible for the side effects of prohexadione-calcium on burns [Radema
cher, W. et al., prohexadione-calcium-a new plant growth regulator for appl
e with interesting biochemical features, 25 ^th Annual Meeting of the Plan
t Growth Regulation Society of America, Poster Exhibited at Chicago, July 7-10, 1998]. A. Lux-Endrich (Technical University of Munich, Weih
Ph.D. dissertation at enstephan, 1998) showed a several-fold increase in the content of phenolic substances in apple cell cultures during the study of the mechanism of action of prohexadione-calcium on burn injury caused by prohexadione-calcium. However, in the process, they found that a series of phenols that are not normally present are found.
It was also found in these studies that prohexadione-calcium produced relatively high amounts of luteoliflavan and eriodictyol in shoot tissues of apple. Luteoli flavan is not normally present in apple tissue, and eriodictyol is only present in small amounts only as an intermediate in flavonoid metabolism. However, in the treated tissue, the expected flavonoids and is catechin and or cyanidin was undetectable, or was only present at a significantly lower amount [S. Rommelt et ^{al, 8 th International Workshop on Fire Blight} , Kusadas
i, Turkey, document submitted 12-15 October 1998].

[0010] Prohexadione-calcium, trinexapac-ethyl and other acylcyclohexanediones are now found to inhibit 2-oxoglutarate-dependent hydroxylases, which play important roles in the metabolism of phenolic substances. ing. These are primarily chalcone synthetase (CHS) and flavanone 3-hydroxylase (F3H) [W. Heller and G. Forkmann, Biosynthesis, The
In Flavonoids, Harborne, JB (eds.), Chapman and Hall, New York, 1998].
However, it cannot be ignored that acylcyclohexanedione also inhibits other as yet unknown 2-oxoglutarate-dependent hydroxylases. Furthermore, it appears that deficiencies in catechin, cyanidin, and other end products of flavonoid synthesis appear in plants, and that the activity of the key enzyme phenylalanine ammonium lyase (PAL) is increased by a feedback mechanism. . However, due to the continued inhibition of CHS and F3H, these flavonoid end products could not be produced, resulting in increased production of luteoliflavan, eriodictythiol and other phenols (FIG. 1).

Due to the reduced enzymatic activity of the enzyme flavanone 3-hydroxylase (F3H), flavonoids eriodictyol, proanthocyanidins (where carbon atoms 3 have been replaced by hydrogen), such as luteoferol, luteoliflavan, Higher amounts of apigeniflavan and tricetiflavan, as well as homogeneous and heterogeneous oligomers and polymers of the above structurally related substances are produced.

[0012] In plants, after the enzymatic activity of the enzyme flavanone 3-hydroxylase (F3H) is decreased, the phenol hydroxycinnamic acid (p-coumaric acid, ferulic acid, sinapinic acid), salicylic acid, or umbelliferone ( Increasing concentrations of homogenous or heterogeneous oligomers and polymers produced from them) are seen. Similarly, the concentrations of chalcones (eg phloretin) and stilbenes (eg resveratrol) are increased.

Since the enzymatic activity of the enzyme flavanone 3-hydroxylase is reduced, the concentrations of flavonoid glycosides, phenolic compounds, chalcones and stilbenes are also increased.

Based on these findings and inferences, F3H activity in whole plants or individual plant organs or tissues can be fully or partially demonstrated by antisense constructs.
Genetically modified cultivated plants were produced that were permanently or temporarily reduced, resulting in a reduced content of phenolic compounds in the whole plant. Subsequently, it could be shown experimentally that the plants have an increased resistance to bacterial and / or fungal pathogens.

As an alternative to the production of plants with reduced flavanone 3-hydroxylase activity using antisense techniques, molecular genetics known from the literature such as co-suppression or expression of specific antibodies have been used. It is also possible to achieve this effect using other techniques.

The method of the invention for increasing resistance to attack of bacterial and fungal pathogens by reducing flavanone 3-hydroxylase enzyme activity can be successfully carried out in the following cultivated plants: wheat. , Barley, rye, oats, rice, corn, panicgrass, sugar cane, bananas, tomatoes, tobacco, chili peppers, potatoes, rape, sugar beet, soybeans, cotton, apples and pears, plums, plums, peaches, nectarines, and Rose-like tree-like fruits such as cherries, and vines.

The method of the invention is particularly intended to increase resistance to Venturia inaequalis in apples and pears, and to Botrytis cinerea in grapevine. It is suitable.

Transgenic plants with reduced enzyme flavanone 3-hydroxylase activity, produced by the method described in the Examples, surprisingly have increased resistance to attack by phytopathogenic bacteria. Is shown. This was demonstrated with respect to resistance to attack by Clavibacter michiganensis subsp. Misiganensis (Cmm) by transgenic tomatoes with reduced flavanone 3-hydroxylase activity (see Example 3). thing).

Plants whose flavanone 3-hydroxylase activity has been reduced using molecular genetics techniques also have increased resistance to attack by Erwinia amylovora and other phytopathogenic bacteria. I showed that. The most important phytopathogenic bacteria are the publication "European Handbook of Plant Diseases" (edited by Smit
h, IM, Dunez, J., Lelliott, RA Phillips, DH and Archer, SA Bla
ckwell Scientific Publications, 1988).

In particular, the method of the invention is suitable for increasing the resistance to the following phytopathogenic fungi: Erysiphe graminis in cereals;
Erysiphe cichoracearum and Spherotheca fuliginea in Cucurbitaceae; Podosphaera leucotricha in apples; Uncinula necator in vines; Species; Rhizoctonia species in cotton, rice, and turf; Ustilago species in cereals and sugar cane; Venturia species in apple and pear (black spot fungus); Helmintosporium in cereals (Helminthosporium) species; Septoria species in wheat; Botrytis cinerea (Bo) in strawberries, vegetables, ornamentals, and vines
trytis cinerea; Gray mold); Cercospora arachidicola in peanut;
Pseudocercosporera herpotrichoeides in wheat and barley
(Pseudocercosporella herpotrichoides); Pyricularia oryzae in rice; Phytophthor infestans (Phytophthor) in potato and tomato
a infestans; Plasmopara viticola in vines; Pseudoperonospora species in hops and cucumbers; Alternaria species in vegetables and fruits; Mycosphaerella in bananas and peanuts Species, and Fusarium and Verticillium species in cereals, vegetables, and ornamental plants.

Example 1 Cloning of the gene for flavanone 3 -hydroxylase from tomato (Lycopersicon esculentum Mill; variety Moneymaker) Ripe tomato fruits of tomato (Lycopersicon esculentum Mill; variety Moneymaker) were washed, drained and sterilized with a sterile knife. The pericarp was used to separate the seed, the central columella and the xylem. Pericarp (about 50 g) was frozen in liquid nitrogen. The material was then chopped with a blender. 100 m of the chopped material in a pre-cooled mortar
l of homogenizing solvent and mixed. The suspension was then transferred to a centrifuge flask by squeezing through sterile gauze. Then 1/10 volume of 10% SDS was added and the material was mixed well. After 10 minutes on ice, 1 volume of phenol / chloroform was added, the centrifuge flask was sealed and the contents were mixed well. 1 at 4000 rpm
After centrifugation for 5 minutes, the supernatant was transferred to a new reaction vessel. This was followed by another 3 phenol / chloroform extractions and 1 chloroform extraction.
Then 1 volume of 3M NaAc and 2.5 volumes of ethanol were added. Nucleic acid -20 ℃
It was allowed to settle overnight. The 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 resuspended in 5-10 ml cold 3M NaAc. This washing step was repeated twice. The pellet was washed with 80% ethanol. Once completely dry, the pellet was taken up in about 0.5 ml of sterile DEPC water and RNA concentration was measured spectrophotometrically.

20 μg of total RNA was first treated with 3.3 μl of 3M sodium acetate solution, 2 μl of 1M magnesium sulfate and the mixture was made up to a final volume of 100 μl with DEPC water. 1
μl RNase-free DNase (Boehringer Mannheim) was added to this and the mixture was incubated at 37 ° C.
Incubated for 45 minutes. The enzyme was removed by adding phenol / chloroform / isoamyl alcohol and shaking to extract, then RNA was precipitated with ethanol and the pellet was taken up in 100 μl of DEPC water. 2.5 μg RNA from this solution
It was transcribed using a cDNA kit (Gibco BRL) to obtain cDNA.

Amino acid sequences derived from a cDNA clone encoding flavanone 3-hydroxylase were used to identify conserved regions within the primary sequence [Britsch et al., Eur. J. Bioch.
em. 217, 745-754 (1993)], which served as the basis for designing degenerate PCR oligonucleotides. Peptide sequence SRWPDK (Petunia hybrid (Pet
unia hybrida) The 5'oligonucleotide was determined using amino acids 147 to 152) in the sequence FL3H PETHY and had the following sequence.

[0024]   5'-TCI (A / C) G (A / G) TGG CC (A / C / G) GA (C / T) AA (A / G) CC-3

The sequence of the oligonucleotides derived by using the peptide sequence DHQAVV (amino acids 276-281 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.

PCR reactions were performed using Perkin Elmer's tTth polymerase according to the manufacturer's instructions. The template used was 1/8 of cDNA (corresponding to 0.3 μg of RNA). The PCR program was as follows.

[0027]   30 cycles   94 ° C, 4 seconds   40 ° C, 30 seconds   72 ° C, 2 minutes   72 ° C, 10 minutes

This fragment was cloned into the vector pGEM-T from Promega according to the manufacturer's instructions.

The fragment was confirmed to be correct by sequencing. Restriction cleavage sites NcoI and PstI
(Present in the polylinker of vector pGEM-T) is used to isolate the PCR fragment,
The overhang was made blunt with T4-polymerase. This fragment was digested with SmaI (blunt) vector pBinAR [Hofgen and Willmitzer, Plant Sci. 66: 221-230 (1990)].
(See Figure 2). This vector contains a CaMV (cauliflower mosaic virus) 35S promoter [Franck et al., Cell 21: 285-294 (1980)] and a termination signal derived from the octopine synthase gene [Gielen et al., EMBO J. 3: 835-.
846 (1984)]. This vector mediates resistance to the antibiotic kanamycin in plants. The resulting DNA construct contained the PCR fragment in sense and antisense orientations. The antisense construct was used for the production of transgenic plants.

FIG. 2: Fragment A (529 bp) contains the CaMV 35S promoter (nucleotides 6909-7437 of the cauliflower mosaic virus). Fragment B contains a fragment of the F3H gene in the antisense orientation. Fragment C (192 bp) contains the termination signal for the octopine synthase gene.

The tomato using 5'RACE system; Preparation of plant mRNA flow equilibrium amount of cloning F3H larger cDNA fragment (Lycopersicon esculentum Mill cultivar Moneymaker) or al flavanone 3-hydroxylase is reduced, To avoid going wrong due to the small size of the F3H PCR fragment used in the antisense construct, a second antisense construct with a larger F3H fragment should be made.

The 5'RACE method (a system for rapid amplification of cDNA ends) was used for cloning larger F3H fragments.

5'RACE system for rapid amplification of cDNA ends (Version 2-0 of Life Technologies ^™ )
The extended total RNA of F3H PCR fragment by 5'RACE method was isolated from ripe tomato fruit of tomato (Lycopersicon esculentum Mill; variety Moneymaker) (see above).

The synthesis of the first strand of the cDNA is performed by GSP-1 (gene-specific primer) 5'-TTCACCACTGCCTGG
Performed according to the manufacturer's instructions using TGGTCC-3 '. After digestion with RNase, the cDNA
Was purified using the GlassMAX spin system from Life Technologies ^™ according to the manufacturer's instructions.

Cytosine homopolymer was added to the 3'end of the purified single-stranded F3H cDNA using terminal deoxynucleotidyl transferase according to the manufacturer's instructions.

The 5′-extended F3H cDNA was labeled with the second gene-specific primer (GSP-2) (this is GSP-1).
It was attached to the region 3'upstream of the recognition sequence, thereby allowing a "nested" PCR to be performed). The 5'primer used was the "5'RACE Cleavage Anchor Primer", which was provided by the manufacturer and the cDNA
Is complementary to the homopolymeric dC tail of.

The cDNA thus amplified was named F3H _extended and cloned into the vector pGEM-T of Promega according to the manufacturer's instructions.

[0038]   The identity of the cDNAs was confirmed by sequencing.

The F3H _extended cDNA fragment was isolated using the restriction cleavage sites NcoI and PstI (present within the polylinker of vector pGEM-T) and its overhangs made blunt-ended using T4-polymerase. This fragment was cloned into the SmaI (blunt) cut vector pBinAR (Hofgen and Willmitzer, 1990) (see Figure 3). This vector is Ca
It contains the MV (cauliflower mosaic virus) 35S promoter (Franck et al., 1980) and the termination signal from the octopine synthase gene (Gielen et al., 1984). This vector mediates resistance to the antibiotic kanamycin in plants. The resulting DNA construct contained the PCR fragment in sense and antisense orientations. The antisense construct was used for the production of transgenic plants.

FIG. 3: Fragment A (529 bp) contains the CaMV 35S promoter (nucleotides 6909-7437 of the cauliflower mosaic virus). Fragment B contains a fragment of the F3H gene in the antisense orientation. Fragment C (192 bp) contains the termination signal for the octopine synthase gene.

The transgenic tomatoes expressing partial fragments of Example 2 flavanone 3-hydroxylase in antisense orientation; method used for manufacturing a (Lycopersicon esculentum Mill cultivar Moneymaker) is, Ling et al., Plant Cell Report 17, 843- 847 (1998). Cultivation was carried out at about 22 ° C. under the control system of 16 hours-light / 8 hours-dark.

Tomato seeds (Lycopersicon esculentum Mill; variety Moneymaker) are sterilized by incubation in a 4% strength sodium hypochlorite solution for 10 minutes,
Subsequently, it was washed 3 to 4 times with sterile distilled water, and 3% sucrose was added for germination.
It was placed on MS medium (pH 6.1). After a germination period of 7-10 days, cotyledons were prepared for use in transformation.

Day 1: The petri dish containing "MSBN" medium was overlaid with 1.5 ml of approximately 10 day old tobacco suspension culture. The plate was covered with film and incubated at room temperature until the next day.

Day 2: Sterile filter paper was placed on the plate overlaid with the tobacco suspension culture to prevent air bubbles from forming. The cotyledons cut laterally were placed upside down on the filter paper. The dish was incubated in the culture chamber for 3 days.

Day 5: Agrobacterium culture (LBA4404) was precipitated by centrifugation at approximately 3000 g for 10 minutes and resuspended in MS medium to an OD of 0.3.
Cotyledon sections were placed on this suspension and incubated for 30 minutes at room temperature with gentle shaking. Then, the cotyledon pieces were dried to some extent on sterile filter paper, returned to the original plate, and co-culture was continued in the culture chamber for 3 days.

Day 8: Co-cultured cotyledonary sections were mounted on MSZ2K50 + β and incubated in the culture chamber for an additional 4 weeks. Then, they were subcultured.

[0047]   The formed shoots were transferred to root induction medium.

[0048]   Once rooted successfully, the plants were tested and transferred to the greenhouse.

Example 3 Flavoviruses infected with Claviobacter misiganensis subspecies misiganensis (Cmm)
Transgenic tomato Cmm having reduced rabanone 3-hydroxylase activity was cultured on yeast-dextrose-Ca agar (YDC) at 28 ° C for 2 days.
The bacteria were rinsed with sterile water and the cell density was measured. For inoculation, the cell density was adjusted to 10 ⁶ cells / ml with sterile water. Injections were made using a 20 gauge hypodermic needle, which was filled with the bacterial suspension. The liquor was applied to the top axils of fully grown leaves of young plants with a total of 3-4 leaves. Infection was assessed by judging the phenotype that appeared.

It was found that over 75% of the leaves were wilted in wild type plants, but the wilting rate was significantly lower in transgenic tomatoes.

[0051] in tomato with Example 4 flavanone 3-hydroxylase in the antisense orientation was genetically modified in accordance with the test the present invention relates to increased resistance to attack by full Itofutora infestans, or modified non Tomato Variety "Mone
The leaves of "ymaker" were infected with an aqueous zoospore suspension of Phytophthora infectans one week after the plant reached the 4-leaf stage. The plant is then 16
Placed in a chamber at 100% ambient humidity at a temperature of ~ 18 ° C. After 6 days, a high proportion of red rot developed in control plants that were not genetically modified. Tomatoes expressing the flavanone 3-hydroxylase antisense construct were shown to be significantly less susceptible to Phytophthora infestans compared to controls.

[Brief description of drawings]

FIG. 1 shows that inhibition of flavanone 3-hydroxylase does not produce flavonoid end products such as catechin and cyanidin, but increases phenol production such as luteoliflavan.

FIG. 2: Fragment A (529 bp) contains the CaMV 35S promoter (nucleotides 6909-7437 of the cauliflower mosaic virus). Fragment B contains a fragment of the F3H gene in the antisense orientation. Fragment C (192 bp) contains the termination signal for the octopine synthase gene.

FIG. 3: Fragment A (529 bp) contains the CaMV 35S promoter (cauliflower mosaic virus nucleotides 6909-7437). Fragment B contains a fragment of the F3H gene in the antisense orientation. Fragment C (192 bp) contains the termination signal for the octopine synthase gene.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, C H, CN, CR, CU, CZ, DE, DK, DM, DZ , EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, K G, KP, KR, KZ, LC, LK, LR, LS, LT , LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, S D, SE, SG, SI, SK, SL, TJ, TM, TR , TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Ammelmann, Eberhard             Federal Republic of Germany D-64646             Penheim, von-gagern-stra             2 (72) Inventor Jabs, Torsten             Federal Republic of Germany D-67105             Verstatt, Greiwitzel             Strasse 3 (72) Inventor Harbors, Karin             Germany D-06484 Que             Drinberg, Am Heinge 6 F term (reference) 2B030 AB04 AD04 CA15 CA17 CA19                 4B024 AA08 BA07 CA04 DA01 EA04                       GA11 GA17                 4B065 AA89X AA89Y AB01 BA02                       CA53

Claims (6)

[Claims] 1. A method for increasing the resistance of a cultivated plant to bacterial and fungal pathogens, which comprises producing a plant in which the activity of the enzyme flavanone 3-hydroxylase is reduced by a method of molecular genetics. . 2. The activity of the enzyme flavanone 3-hydroxylase in the whole or part of the plant body is measured by a method of molecular biology (for example, antisense construct, co-suppression, expression of specific antibody, or specific inhibitor). 3. The method according to claim 1, characterized in that it is completely or partially, permanently or transiently inhibited by the expression of. 3. The cultivated plant is wheat, barley, rye, oats, rice, corn, panic glass, sugar cane, banana, tomato, tobacco, pepper, potato, rape, sugar beet, soybean, cotton, and apple and pear. The method according to claim 1 or 2, which is a tree-like fruit of the family Rosaceae such as peach, plum, peach, nectarine, and cherry, and a vine. 4. The method according to any one of claims 1 to 3, which increases resistance to Venturia inaequalis in apples and pears. 5. In the vines, Botrytis cinere (Botrytis cinere
4. The method according to any one of claims 1 to 3, which increases the resistance to a). 6. A plant with increased resistance to bacterial and fungal pathogens, characterized in that the activity of the enzyme flavanone 3-hydroxylase has been reduced by means of molecular genetics.