WO2000078981A1

WO2000078981A1 - Method of increasing the resistance of cultivated plants to phytopathogenic fungi and bacteria by methods of molecular genetics

Info

Publication number: WO2000078981A1
Application number: PCT/EP2000/005259
Authority: WO
Inventors: Wilhelm Rademacher; John-Bryan Speakman; Eberhard Ammermann; Thorsten Jabs; Karin Herbers
Original assignee: Basf Aktiengesellschaft
Priority date: 1999-06-17
Filing date: 2000-06-07
Publication date: 2000-12-28
Also published as: ZA200101327B; IL141249A0; PL346058A1; HUP0103259A2; AU5970500A; AR024382A1; KR20010113630A; BR0006873A; DE19927575A1; HUP0103259A3; TR200100561T1; CA2340329A1; JP2003505016A; EP1102856A1

Abstract

The invention relates to a method of increasing the resistance of cultivated plants to bacterial and fungal pathogens by producing a plant by means of molecular genetics in which the activity of the enzyme flavonone-3-hydroxylase is reduced.

Description

Process for increasing the resistance of crop plants to phytopathogenic fungi and bacteria using molecular genetic methods

description

The present invention relates to a method for increasing the resistance of crop plants to bacterial and fungal pathogens, characterized in that a plant is produced using molecular genetic methods in which the activity of the enzyme flavanone-3-hydroxylase is reduced.

The method is further characterized in that the enzyme flavanone-3-hydroxylase by molecular biological methods (eg anti-sense construct, co-suppression, the expression of specific antibodies or the expression of specific inhibitors) in whole or in part, continuously or temporarily, in the entire plant or in parts of the plant is inhibited in its activity.

The present invention furthermore relates to plants with increased resistance to bacterial and fungal pathogens, characterized in that the activity of the enzyme flavanone-3-hydroxylase is reduced by molecular genetic methods.

The productivity of crops can be reduced in many ways by stress factors. These include: viral diseases, bacterial and fungal pathogens, damaging insects, nematodes, snails, bite, heat, cool, cold, lack of water, too high water content in the soil, salinity, too high radiation intensity, too high ozone content, competition for light , Water and nutrients due to accompanying flora, improper or inapplicable herbicide applications (especially in fruit crops), treatments with herbicides, insecticides, fungicides, bioregulators or foliar fertilizers with insufficient selectivity, foliar applications of plant protection products or fertilizers during intensive sun exposure.

A number of these problems caused by stressors can be minimized through the use of plant protection products, through the use of resistant plant material or through suitable cultivation techniques. However, the scope of these options is limited. Bacteriosis in particular are very difficult to control, if at all. Antibiotics such as streptomycin or tetracyclines are used (for example to combat fire blight in apples and pears) used, which also harbors the risk of developing resistance in the case of human pathogens. Furthermore, fungal pathogens, for example, frequently adapt to fungicides so that their effectiveness wears off. A similar adaptation also exists for "pathogen-resistant" breeds that are produced by conventional methods.

There is a need for plants that are resistant to pathogens not only in annual crops or horticultural crops, but also in valuable permanent crops such as fruit and wine.

The object of the invention was to find a simple and inexpensive method for permanently improving the resistance 15 to bacterial and fungal pathogens, in particular in crop plants.

Based on physiological studies with growth regulators from the group of acylcyclohexadiones, 20 surprisingly genetic engineering methods have now become available which can be used to produce crop plants that are resistant to a number of phytopathogenic bacteria and fungi.

Acylcyclohexadiones such as prohexadione-Ca and trinexapac-ethyl (äl-

25 tere designation: Cimectacarb) are used as bioregulators to inhibit plant growth. Their bioregulatory effect arises from the fact that they block the biosynthesis of gibberellins that promote length growth. They inhibit due to their structural relationship

30 2-oxoglutaric acid certain dioxygenases that 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 in the

35 Interfering with phenol metabolism and thus can inhibit anthocyanin formation 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

40 Academic Publishers, Dordrecht (1992)). Such effects on the household of phenolic ingredients are said to be the cause of 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 on the

45 25 ^th Annual Meeting of the Plant Growth Regulation Society of America, 7th-10th July 1998, Chicago). A. Lux-Endrich (dissertation Technical University of Munich in Weihenstephan, 1998) takes place in the In the course of their investigations into the mechanism of action of prohexadione-Ca against fire blight, that in cell cultures of apple by prohexadione-Ca there is a multiple increase in the content of phenolic substances and that a number of otherwise non-existing phenols occur. In the course of these investigations, it was also found that under the influence of prohexadione-Ca, relatively high amounts of luteoliflavan and eriodyctiol occur in apple shoot tissue. Luteoliflavan does not normally occur in apple tissue and eriodyctiol occurs as an intermediate of the flavonoid substance echse only in small amounts. The expected flavonoids catechin and cyanidin were not detectable in the treated tissue or occurred only in significantly reduced amounts (p Ro melt et al, lecture 8 ^th International Workshop on Fire Blight, Kusadasi, Turkey, 12th-15th October over 1998).

It can be considered certain that prohexadione-Ca, trinexapac-ethyl and other acylcyclohexadiones inhibit 2-oxoglutaric acid-dependent hydroxylases, which are important in the metabolism of phenolic substances. These are primarily chalcone 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 acylcyclohexadiones also inhibit other, previously unknown, 2-oxoglutaric acid-dependent hydroxylases. It should also be obvious that a lack of catechin, cyanidine or other end products of flavonoid synthesis is registered by the plant and that the activity of the key enzyme phenylalanine ammonium lyase (PAL) is increased via a feedback mechanism. However, by continuing to inhibit CHS and F3H, these flavonoid end products cannot be formed and there is an increased formation of luteoliflavan, eriodyctiol and other phenols (Figure 1).

By reducing the enzyme activity of the enzyme flavanon-3-hydroxylase (F3H) the flavonoids eriodictyol, proanthocyanidins, which are substituted on the C atom 3 with hydrogen, e.g. Luteforol, luteoliflavan, apigeniflavan and tricetiflavan, as well as homogeneous and heterogeneous oligomers and polymers are increasingly formed from the above-mentioned and structurally related substances.

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 from them, are reduced after reducing the enzyme activity of the enzyme flavanone-3-hydroxylase (F3H ) in plants detected. The concentration of the chalcones, such as phloretin, and the stilbene, such as resveratrol, also increases.

The concentration of the glycosides of the flavonoids, the phenolic compounds, the chalcones and the stilbene is also increased by reducing the enzyme activity of the enzyme flavanon-3-hydroxy-lase.

On the basis of these findings and conclusions, genetically modified crop plants were produced in which F3H was reduced in its activities in whole or in part, permanently or temporarily, by means of anti-sense constructs, in the entire plant or in individual plant organs or tissues, with the result that the content of phenolic compounds in the whole plant is reduced. It could then be shown experimentally that these plants are increased in their resistance to bacterial and / or fungal pathogens.

As an alternative to the production of plants which are reduced in their flavonone-3-hydroxylase activity with the aid of anti-sense technology, other molecular genetic methods known from the literature, such as co-suppression or the expression of specific antibodies, can also be used to achieve this effect.

The process 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 successfully carried out on the following crop plants: wheat, barley, rye, oats, rice, corn, millet, sugar cane, Banana, tomato, tobacco, bell pepper, potato, rapeseed, sugar beet, soya, cotton, fruit trees from the rosacea family, such as apple and pear, plum, plum, peach, nectarine and cherry, and grapevines.

The method according to the invention is particularly suitable for increasing the resistance to Venturia inaequalis in apple and pear and to Botrytis cinerea in grapevines.

Transgenic plants with reduced activity of the enzyme flavonon-3-hydroxylase produced by the method described in the examples surprisingly show an increased resistance to attack by phytopathogenic bacteria. This could be done using the example of the infestation of transgenic tomato plants whose flavanone 3-hydroxylase activity is reduced. with Clavibacter michiganensis subsp. michiganensis (Cmm) are shown, see example 3

Plants whose flavanone 3-hydroxylase was reduced with the help of molecular genetic methods 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. Phillips, D.H. and Archer, S.A. Blackwell Scientific Publications, 1988.

The method according to the invention is particularly suitable for increasing the resistance to the following phytopathogenic fungi:

Erysiphe graminis (powdery mildew) on cereals Erysiphe cichoracearum and Sphaerotheca fuliginea on pumpkin plants Podosphaera leucotricha on apples, Uncinula necator on vines, Puccinia species on cereals,

Rhizoctonia species on cotton, rice and lawn, 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, ornamental plants and vines, Cercospora arachidicola on peanuts, Pseudocercosporella herpotrichoides on wheat and barley, Pyricularia oryzae on rice,

Phytophthora infestans on potatoes and tomatoes, Plasmopara viticola on vines, Pseudoperonospora species in hops and cucumbers, Alternaria species on vegetables and fruits,

Mycosphaerella species in bananas and peanuts and Fusarium and Verticillium species in cereals, vegetables and ornamental plants.

example 1

Cloning of a Flavanone-3-hydroxylase gene from Lycopersicon esculentum Mill.cv. Moneymaker.

Ripe tomato fruits from Lycopersicon esculentum Mill.cv. Moneymakers were washed, dried and, using a sterile blade, the pericarp of seeds, middle columnella and wooden parts freed. The pericarp (approx. 50 g) was frozen in liquid nitrogen. The material was then crushed in a mixer. The comminuted material was mixed with 100 ml homogenizing medium in a pre-cooled mortar. The suspension was then transferred to centrifuge cups by pressing through sterile gauze cloths. Then 1/10 vol 10% SDS was added and mixed well. After 10 minutes on ice, 1 volume of phenol / chloroform was added, the centrifuge cup closed and mixed well. After centrifugation at 4000 rpm for 15 minutes, the supernatant was transferred to a new reaction vessel. This was followed by three further phenol / chloroform extractions and one chloroform extraction. In the following, 1 vol 3 M NaAC and 2.5 vol ethanol were added. The nucleic acids were precipitated overnight at -20 ° C. The next morning, the nucleic acids were pelleted for 15 minutes at 10,000 rpm in a refrigerated centrifuge (4 ° C). The supernatant was discarded and the pellet resuspended in 5 ml of cold 3 M NaAc. This washing step was repeated twice. The pellet was washed with 80% ethanol. The completely dried pellet was taken up in about 0.5 ml of sterile DEPC water and the RNA concentration was determined photometrically.

20 μg of total RNA was first mixed with 3.3 μl of 3M sodium acetate solution, 2 μl of IM magnesium sulfate solution and made up to 100 μl of final volume with DEPC water. In addition, a microliter

Rnase free Dnase (Boehringer Mannheim) given and incubated for 45 min at 37 ° degrees. After removing the enzyme by shaking with phenol / chloroform / isoamyl alcohol, the RNA was precipitated with ethanol and the pellet was taken up in 100 μl DEPC water. 2.5 μg RNA from this solution were transcribed into cDNA using a cDNA kit (Gibco BRL).

Using amino acid sequences derived from cDNA clones coding for flavanone-3-hydroxylase, conserved regions in the primary sequence could be identified (Britsch et al., Eur. J. Biochem. 217, 745-754 (1993), the served as the basis for the design of degenerate PCR oligonucleotides The 5 'oligonucleotide was determined using the peptide sequence SRWPDK (amino acid 147-152 in the sequence FL3H PETHY from Petunia hybrida) 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 sequence FL3H PETHY from Petunia hybridaj was as follows: 5'-CTT CAC ACA (C / G / T) GC (C / T) TG (A / G) G (A / G) TC-3.5. 5

The PCR reaction was carried out using the Perkin-Elmer tTth polymerase according to the manufacturer's instructions. 1/8 of the cDNA was used as template (corresponds to 0.3 μg RNA). The PCR program was: 10

30 cycles

94 degrees 4 sec

40 degrees 30 sec

72 degrees 2 min

15 72 degrees 10 min

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

20 The correctness of the fragment was checked by sequencing. The PCR fragment was isolated using the restriction sites Ncol and Pstl present in the polylinker of the vector pGEM-T and the protruding ends were blunt-ended using the T4 polymerase. This fragment

25 was cloned into a Smal (blunt) cut vector pBinAR (Höfgen and Willmitzer, Plant Sei. 66: 221-230 (1990)) (see Figure 2). This contains the 35S promoter of the CaMV (cauliflower mosaic virus) (Franck et al., Cell 21: 285-294 (1980)) and the termination signal of the octopine synthase gene (Gielen et al.,

30 EMBO J. 3: 835-846 (1984)). This vector mediates resistance to the antibiotic kanamycin in plants. The DNA constructs obtained contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants.

35

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

40 phase gens.

Cloning of a larger cDNA fragment of Flavanone-3-hydroxylase from Lycopersicon esculentum Mill.cv. Moneymaker using the 5 'RACE system. 5 In order to rule out that the generation of plants with a reduced mRNA flow equilibrium amount of the F3H is not successful due to the small size of the F3H PCR fragment used in the antisense construct, a second antisense construct should be generated using a larger F3H fragment.

The 5'RACE method (System for Rapid amplification of cDNA ends) was used to clone a larger fragment of the F3H.

Extension of the F3H PCR fragment by the 5 'RACE method using the 5' RACE System for rapid amplification of cDNA ends, Version 2-0 from Life Tecgnologies ™.

From ripe tomato fruits from Lycopersicon esculentum Mill.cv. Moneymaker total RNA was isolated (see above).

The cDNA first strand synthesis was carried out according to the manufacturer's instructions using the GSP-1 (gene-specific primer) 5'-TTCAC-CACTGCCTGGTGGTCC-3 '. Following RNase digestion, the cDNA was purified using the GlassMAX spin system from Life Tecgnologies ™ according to the manufacturer's instructions.

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

The 5 'extended F3H cDNA was amplified using a second gene-specific primer (GSP-2) which binds in the region 3' before the GSP-1 recognition sequence and thus enables a "nested" PCR Manufacturer supplied "5'RACE abrided anchor primer" used, which is complementary to the homopolymeric dC tail of the cDNA.

The cDNA fragment amplified in this way and designated as FSH _extended was cloned into the vector pGEM-T from Promega according to the manufacturer's instructions.

The identity of the cDNA was confirmed by sequencing.

The F3Heχ _en de _c - cDNA fragment was isolated using the restriction sites Ncol and Pstl present in the polylinker of the vector pGEM-T and the protruding ends were transferred using the T4 polymerase and blunt ends. This fragment was cloned into a Smal (blunt) cut vector pBinAR (Höfgen and Willmitzer, 1990) (see Figure 3). This contains the 35S promoter of the CaMV (cauliflower mosaic virus) (Franck et al., 1980) and the termination signal of the octopine synthase gene (Gielen et al., 1984). This vector mediates resistance to the antibiotic kanamycin in plants. The DNA constructs obtained contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants.

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

Example 2

Production of transgenic Lycopersicon esculentum Mill.cv. Moneymakers who express a partial fragment of flavanone 3-hydroxylase in an antisense orientation.

The method according to Ling et al., Plant Cell Report 17, 843-847 (1998) was used. The cultivation takes place at approx. 22 ° C under a 16 h light / 8 h dark regime.

Tomato seeds (Lycopersicon esculentum Mill. Cv. Moneymaker) were incubated for 10 minutes in 4% sodium hypochlorite solution, then washed 3-4 times with sterile distilled water and on MS medium with 3% sucrose, pH 6.1 Germination designed. After a germination period of 7-10 d, the cotyledons could be used for the transformation.

Day 1: Petri dishes with the medium "MSBN" were overlaid with 1.5 ml of an approximately 10 day old tobacco suspension culture. The plates were covered with foil and incubated at room temperature until the next day.

Day 2: Sterile filter paper was placed on the plates coated with the tobacco suspension culture without air bubbles. The cross-cut cotyledons were placed on top with the top down. The petri dishes were incubated for 3 days in the culture room.

Day 5: The agrobacterial culture (LBA4404) was sedimented by centrifugation at approx. 3000 g for 10 min and resuspended in MS medium so that the OD was 0.3. The cotyledon fragments were added to this suspension, which were incubated with gentle shaking for 30 minutes at room temperature. The cotyledon fragments were then placed on sterile filter paper a little dried and put back on their original plates to continue cocultivation for 3 days in the culture room.

Day 8: The cocultivated pieces of cotyledon were placed on MSZ2K50 + ß and incubated for the next 4 weeks in the culture room. Subcultivation then took place.

Rungs that formed were placed on root induction medium.

After successful rooting, the plants could be tested and transferred to the greenhouse.

Example 3

Transgenic tomato plants with reduced flavanone 3-hydroxylase activity were treated with Clavibacter michiganensis subsp. michiganensis (Cmm) infected.

Cmm was cultured on yeast dextrose Ca agar (YDC) at 28 ° C for 2 days. The bacteria were washed off with sterile water and their cell density was determined. For inoculation, the cell density was adjusted to 10 ⁶ cells / ml with sterile water. The injections were carried out with injection needles (No. 20), which were filled with the bacterial suspension. They were done in the

Leaf axil of the top fully developed leaf of young plants, which had a total of 3-4 leaves. The infection was evaluated by assessing the developing phenotype.

In contrast, while more than 75% of the leaves had withered in wild-type plants, the degree of withering was significantly lower in the transgenic tomato plants.

Example 4

Test for increased resistance to attack by Phytophthora infestans in tomatoes with flavanon-3-hydroxylase in an antisense orientation.

The leaves of non-genetically modified or genetically modified tomato plants of the "Monymaker" variety were infected one week after the 4-leaf stage with an aqueous suspension of zoospores from Phytophthora infestans. The plants were then placed in a water vapor - saturated chamber at temperatures between 16 and 18 ° C. After 6 days, the late blight on the genetic strongly unmodified control plants. Tomato plants which expressed an antisense construct of flavanon-3-hydroxylase showed a significantly lower infection with Phytophthora infestans than the control.

Claims

claims

1. A process for increasing the resistance of crop plants to bacterial and fungal pathogens, characterized in that a plant is produced using molecular genetic methods in which the activity of the enzyme flavanon-3-hydroxylase is reduced.

2. The method according to claim 1, characterized in that the enzyme flavanone-3-hydroxylase by molecular biological methods (eg anti-sense construct, co-suppression, the expression of specific antibodies or the expression of specific inhibitors) in whole or in part, continuously or before - is temporarily inhibited in its activity in the entire plant or in parts of the plant.

3. Process according to claims 1 and 2, characterized in that the crops are wheat, barley, rye, oats, rice, corn, millet, sugar cane, banana, tomato, tobacco, bell pepper, potato, rapeseed, sugar beet, Soy, cotton, fruit trees from the rosaceae family, such as apple and pear, plum, plum, peach, nectarine and cherry as well as grapevines.

4. The method according to claims 1-3, characterized in that the resistance to Venturia inaequalis in apple and pear is increased.

5. The method according to claims 1-3, characterized in that the resistance to botrytis cinerea is increased in grapevine.

6. Plant with increased resistance to bacterial and fungal pathogens, characterized in that the activity of the enzyme flavanone-3-hydroxylase is reduced by molecular genetic methods.