AU5970500A - Method of increasing the resistance of cultivated plants to phytopathogenic fungi and bacteria by methods of molecular genetics - Google Patents

Description

Method of increasing the resistance of crop plants to phytopathogenic fungi and bacteria with the aid of methods of molecular genetics 5 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. 10 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, 15 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 20 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 25 alia, 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 30 light, water and nutrients by the accompanying flora, herbicides to be [sic] applied inexpertly, herbicides to be [sic] applied optimally (in particular in fruit plantations), treatments with herbicides, insecticides, fungicides, bioregulators or foliar fertilizers of unduly low sensitivity, foliar applications of 35 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 40 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, 45 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 2 the latter is diminished. A similar adaptation also exists in "pathogen-resistant" plant breeding products generated by conventional methods. 5 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 10 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 15 studies on growth regulators from the acylcyclohexadione [sic] group and with the aid of which crop plants can be generated which are resistant to a series of phytopathogenic bacteria and fungi. 20 Acylcyclohexadiones [sic] 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 25 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., 30 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: 35 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 40 growth regulator for apple with interesting biochemical features, poster at the 2 5 th 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 45 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 3 which are otherwise not present is found. Within these studies, 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 5 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. R6mmelt et al., Paper presented at the 8th International 10 Workshop on Fire Blight, Kusadasi, Turkey, October 12-15, 1998). It is an established observation that prohexadione-Ca, trinexapac-ethyl and other acylcyclohexadiones [sic] inhibit 2-oxoglutaric acid-dependent hydroxylases which play an important 15 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 acylcyclohexadiones 20 [sic] 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 25 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). 30 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 35 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 40 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, 45 are also elevated.

4 A reduction in the enzyme activity of the enzyme flavanone 3-hydroxylase also leads to an increased concentration of the glycosides of the flavonoids, of the phenolic compounds, of the chalcones and of the stilbenes. 5 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 10 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. 15 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 20 antibodies. The method according to the invention for increasing the resistance to attack by bacterial and fungal pathogens by reducing the flavonone (sic] 3-hydroxylase enzyme activity can be 25 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 30 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. 35 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 40 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 [lacuna] was reduced with 45 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 5 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. 5 In particular, the method according to the invention is suitable for increasing the resistance to the following phytopathogenic fungi: 10 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, 15 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, 20 Botrytis cinerea (gray mold) on strawberries, vegetables, ornamentals and grapevines, Cercospora arachidicola on peanuts, Pseudocercosporella herpotrichoides on wheat and barley, Pyricularia oryzae on rice, 25 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 30 Fusarium and Verticillium species in cereals, vegetables and ornamentals. Example 1 35 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, 40 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 45 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 6 phenol/chloroform was added, and the centrifuge tube was sealed and the contents mixed thoroughly. After centrifugation for 15 minutes at 4000 rpm, the supernatant was transferred into a fresh reaction container. Three more phenol/chloroform extractions and 5 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 0 C. Next morning, the nucleic acids were pelleted in a refrigerated centrifuge (4 0 C) for 15 minutes at 10,000 rpm. The supernatant was discarded and the pellet was 10 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. 15 20 g of total RNA were first treated with 3.3 1 of 3M sodium acetate solution and 2 l 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) 20 was added, and the mixture was incubated for 45 minutes at 370 degerees [sic]. After the enzyme had been removed by extraction by shaking with 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 25 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. J. 30 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: 35 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 DHQAVV (amino acid 276281 in the Petunia hybrida 40 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 45 cDNA was employed as template (corresponding to 0.3 Rg of RNA). The PCR program was as follows: 7 30 cycles 94 degrees 4 sec 40 degrees 30 sec 72 degrees 2 min 5 72 degrees 10 min The fragment was cloned into the vector pGEM-T by Promega following the manufacturer's instructions. 10 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 Smal [sic] (blunt-)ended vector 15 pBinAR (Hbfgen and Willmitzer, Plant Sci. 66: 221 -230 (1990)) (see Figure 2). This vector contains the CaMV (cauliflower mosaic virus) 355 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 20 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. 25 Figure 2: Fragment A (529 bp) contains the CaMV 35S promotor (nucleotide 6909 to 7437 of cauliflower mosaic virus). Fragment B [lacuna] the fragment of the F3H gene in antisense orientation. Fragment C (192 bp) contains the termination signal of the octopine synthase gene 30 Cloning of a larger cDNA fragment of the Lycopersicon esculentum Mill.cv. Moneymaker flavanone 3-hydroxylase using the 5'RACE system. 35 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 the antisense construct. 40 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 45 by Life TechnologiesTM.

8 Total RNA was isolated from ripe tomato fruits of Lycopersicon esculentum Mill.cv. Moneymaker (see above). cDNA first strand synthesis was carried out following the 5 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. 10 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 15 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 abrided [sic] anchor primer" which was provided by the manufacturer and which is complementary to the homopolymeric dC tail of the cDNA. 20 The cDNA fragment amplified in this way and termed F 3 Hextended was cloned into the pGEM-T vector by Promega following the manufacturer's instructions. 25 The identity of the cDNA was confirmed by sequencing. The F 3 Hextended cDNA fragment was isolated using the restriction cleavage sites Ncol [sic] and Pstl [sic], which are present in the polylinker of the vector pGEM-T, and the overhangs were made 30 blunt-ended using T4-polymerase. This fragment was cloned into an Smal [sic] (blunt) Vector pBinAR (H6fgen 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 35 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. 40 Figure 3: Fragment A (529 bp) contains the CaMV 35S promotor (nucleotides 6909 to 7437 of the cauliflower mosaic virus). Fragment B [lacuna] the fragment of the F3H gene in antisense orientation. Fragment C (192 bp) contains the octopine synthase gene termination signal. 45 9 Example 2 Generation of transgenic Lycopersicon esculentum Mill.cv. Moneymaker expressing a flavanone 3-hydroxylase subfragment in 5 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 0 C in a 16-h - light / 8-h - dark regime. 10 Tomato seeds (Lycopersicon esculentum Mill. cv. Moneymaker) were incubated [sic] by incubation for 10 minutes in 4% sodium hypochlorite solution, subsequently washed 3 - 4 times with sterile distilled water and placed on MS medium supplemented with 15 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. 20 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 25 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 30 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 35 to their starting plates to continue cocultivation for 3 days in the culture room. Day 8: The cocultivated cotyledon sections were placed on MSZ2K50+B and incubated for the next 4 weeks in the culture room. 40 Then, they were subcultured. Shoots which formed were transferred to root induction medium. After successful rooting, the plants were ready to be testing and 45 transferred to the greenhouse.

10 Example 3 Transgenic tomato plants with reduced flavanone 3-hydroxylase activity infected with Clavibacter michiganensis subsp. 5 michiganensis (Cmm). Cmm was grown for 2 days at 280C 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 15 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. 20 Example 4 Test for the increase in the resistance to attack by Phytophthora infestans in tomatoes with flavanone 3-hydroxylase in antisense 25 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 30 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 180C. After 6 days, the brown rot on the control plants which had not been genetically modified had developed to a high degree. Tomato 35 plants which expressed an antisense construct of flavanone 3-hydroxylase showed a considerably lower level of Phytophthora infestans infection than the control. 40 45

Claims (6)

1. A method of increasing the resistance of crop plants to 5 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.

2. A method as claimed in claim 1, wherein the activity of the 10 enzyme flavanone 3-hydroxylase is fully or partially, permanently or transiently, in the whole plant or in parts of the plant, inhibited by methods of molecular biology (for example antisense construct, cosuppression, the expression of specific antibodies or the expression of specific 15 inhibitors).

3. A method as claimed in claim 1 or 2, wherein the crop plants are wheat, barley, rye, oats, rice, maize, panic grasses, sugar cane, bananas, tomatoes, tobacco, bell peppers, 20 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.

4. A method as claimed in claims 1 - 3, wherein the resistance 25 to Venturia inaequalis in apples and pears is increased.

5. A method as claimed in claims 1 - 3, wherein the resistance to Botrytis cinerea in grapevines is increased. 30

6. A plant 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. 35 40 45