MXPA01001608A - Method of increasing the resistance of cultivated plants to phytopathogenic fungi and bacteria by methods of molecular genetics - Google Patents

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

METHOD TO INCREASE THE RESISTANCE OF FORAGE PLANTS TO PHYTOPATHOGENIC FUNGI AND BACTERIA WITH THE AID OF METHODS OF MOLECULAR GENETICS The present invention is a method for increasing the resistance of forage plants to bacterial and fungal pathogens, wherein a plant is generated by molecular genetic methods in which the activity of enzymatic flavanone 3-hydroxylase is reduced. The method further comprises inhibiting the activity of the enzymatic flavanone 3-hydroxylase completely or partially, permanently or temporarily, throughout the plant or in parts of the plant, by molecular biology methods (e.g. antisense constructions, cosuppression, the expression of specific antibodies, or the expression of specific inhibitors). The present invention also relates to plants with an increased resistance to bacterial and fungal pathogens, where the activity of enzymatic flavanone 3-hydroxylase is reduced by molecular genetic methods. The productivity of forage plants can be reduced in many ways by stress factors. The stress factors that can be mentioned are, among other things, viral diseases, bacterial pathogens and ¿¿¿? ¿¿¿¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¿ fungi, harmful insects, nematodes, slugs and snails, hunting damage, high temperatures, moderately low and low, lack of water, moderately high water content of the soil, soil salinification, moderately high radiation intensity, moderately ozone content elevated, competition for light, water and nutrients for the associated flora, herbicides applied in an inexperienced way, herbicides that have not been applied properly (in particular in fruit plantations), treatments with herbicides, insecticides, fungicides, bioregulators, or foliar fertilizers of moderately low sensitivity, foliar applications of products or crop protection fertilizers during intense isolation. Some of these problems caused by the stress factors can be reduced by applying crop protection products, by using resistant plant material or by using suitable conservation techniques. However, the scope of possibilities is limited. Bacteriosis, in particular, can only be controlled with great difficulty, if there is one. To do so (for example when controlling the fire blight in apple and pear trees), antibiotics such as streptomycin or tetracyclines are used, which harbors the danger of developing resistance, including human pathogens. On the other hand, for example, fungal pathogens often show *. * ?; a »gi» "^ a * ^. * ^ i ^ &^ P« ii? í-Sas ^ - & a¡s ^ adaptation to fungicides, so that the effectiveness of the above is diminished. Similar adaptation also exists in the reproductive products of "resistant-pathogenic" plants generated by conventional methods 5. There is not only a demand for resistant-pathogenic plants in annual arable or horticultural crops, but also in valuable perennial crops such as fruits and vegetables. It is an object of the present invention to find a simple and inexpensive method to permanently improve resistance to bacterial and fungal pathogens, particularly in forage plants. this object, by means of genetic engineering methods which are based on physiological studies on growth regulators from the acylcyclohexanedione group and with the help of forage plants that can be generated, which are resistant to a series of bacteria and fungi phytopathogenic Acylcyclohexandiones such as prohexadione-Ca and trinexapac-ethyl (previously known as cimectacarb) are used as bioregulators to inhibit the longitudinal growth of the plants. The reason for his bioregulatory action is that they block the biosynthesis of ^ - ~ ^ ll-É-g3É_i gibberellins, which promote longitudinal growth. Due to their structural relationship with 2-oxobutaric acid, they inhibit certain dioxygenases that require 2-oxobutaric acid as a co-substrate (Rademacher,, Biochemical effects of plant growth retardants, in: Plant Biochemical Regulators, Gausman, H (ed.), Marcel Dekker, Inc., New York, pp. 169-200 (1991)). It is known that such compounds also bind in phenol metabolism and therefore can cause the inhibition of anthocyanin in various 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 other phenolic constituents are given as a cause for the side effect of prohexadione-Ca against fire blight (Rademacher, et al., Pr © hexadione-Ca - a new plant growth regulator for apple with interisting 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 his studies on the mechanism of action of prohexadione-Ca against fire blight, that, in cell cultures of apples, prohexadione -Ca causes an increase in the content of phenolic substance on many occasions, and that a series of phenols that are otherwise not present were found. Within these studies, it was also found that the effect of prohexadione-Ca leads to relatively large amounts of luteoliflavana and eriodictyol in the shoot tissue of the apple tree. Luteoliflavana does not normally occur in the apple tissue, and eriodicyol is present in minor amounts only as an intermediary in flavonoid metabolism. However, the expected flavonoid catechin and cyanidin 10 were not detected in the treated tissue, or only in markedly reduced amounts (S. Rommelt et al., Paper presented at the 8 th International orshop on Fire Blight, Kusadasi, Turkey, October 12 -15, 1998). 15 Is it an established observation that prohexadione- Ca, trinexapac-ethyl and other acylcyclohexandiones inhibit hydroxylases? They depend on 2-oxoglutaric acid that play an important role in the metabolism of phenolic substances. These are mainly chalcone synthetase (CHS) and flavanone 3-hydroxylase (F3H) (Heller and G. Forkmann, Biosynthesis, in: The Flavonoids, Harborne, JB (ed.), Chapman and Hall, New York, 1988 ). However, it can not be excluded that acylcyclohexandiones also inhibit other hydroxylases that depend on 2-oxoglutaric acid which are unknown ^ di ^^^^ é (st ^^ f ^ &.Mé ^ iíí ^ s ^^^ B ^ tí ^? 1fr ^ ff - ^ ^ S ^^^^^ 1 in the present. It is obvious that a lack of catechin, cyanidin and other final products of flavonoid synthesis is registered by the plant and that the activity of key enzyme phenylalanine ammonium lyase (PAL) is increased by a feedback mechanism. that CHS and F3H are still being inhibited, these final flavonoid products can not be produced, and this leads to an increased production of luteoliflavan, and eriodicyol and other phenols (Figure 1). - enzymatic flavanone hydroxylase (F3H), flavonoid eriodictyol, proanthocyanidins that are replaced on the 3 C atoms by hydrogen, for example, luteoforol, luteoliflavana, apigeniflavana and tricetiflavana, and oligomers and homogeneous and heterogeneous polymers of the aforementioned and structurally related substances are produced in larger amounts. w Increased hydroxycinnamic acid concentrations of phenols (p-coumaric acid, ferulic acid, sinapic acid), salicylic acid or unbelliferone, including the homogeneous and heterogeneous oligomers and polymers formed therewith, are found in plants after the activity of the enzymatic flavanone 3-hydroxylase (F3H) has been reduced. It also rises the concentration of chalcones, for example floretin, and -jjgM-t- «lg-a? B - * t- M-at-a« ^^ .t,. ". ^ ...» - ». ^ .i ^ aasff4 ^ Ü" ^ - l - ^ a- ^ ¿> of stilbenes, for example resveratrol. A reduction in the enzymatic flavanone 3-hydroxylase activity also leads to an increased concentration of the glycosides of flavonoids, phenolic compounds, chalcones and stilbenes. Based on these findings and conclusions, genetically modified forage plants were generated in which F3H was completely or partially reduced, permanently or temporarily, in the whole plant or in individual plant organisms or tissues, by means of antisense constructions. , with the sequence that the content of the phenolic compound in the whole 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 for the generation of plants whose flavanone 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 a cosuppression, or the expression of specific antibodies. The method according to the invention for increasing the resistance to attack of bacterial and fungal pathogens by reducing the activity of enzymatic flavanone 3-hydroxylase can be practiced successfully in the following forage plants: wheat, barley, rye, oats, rice, corn, panic grass, sugar cane, bananas, tomatoes, tobacco, bell peppers, potatoes, oilseed rape, sugar beet, soybeans, cotton, fruit of the tree of the family of the Rosaceae, such as apples and pears, plums, quetsches (plum brandy), peaches, nectarines and cherries, and grapevines. The method according to the invention is especially suitable for increasing resistance to Venturia inaequalis in apples and pears and to Botrytis cinerea in vines. The transgenic plants with reduced activity of the enzymatic flavanone 3-hydroxylase, generated by the method described in the Examples, surprisingly show an increased resistance to attack by the phytopathogenic bacteria. This was demonstrated with reference to 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 molecular genetic methods that also showed 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, IM, Dunez, J., Lelliot, RA Phillips, DH and Archer, SA Blackwell Scientific Publications, 1988. In particular, the method according to the invention is suitable for increasing resistance to the following phytopathogenic fungi: Erysiphe graminis (powdery mildew) in cereal Erysiphe cichoracearum and Sphaerotheca fuligina in cucurbits Podosphaera leucotricha in apples, Uncinula necator in vines, Puccinia species in cereal, Rhizoctonia species in cotton, rice and grass, Ustilago species in cereals and sugar cane, Venturia species (pulp) in apples and pears, Helminthosporium species in cereals, Septoria species in wheat Botrytis cinerea (botrytis) in strawberries, vegetables, ornamentals and vines, Cercospora arachidicola in peanuts, Pseudocercosporella herpotrichoides in wheat and barley, Pyricularia oryzae in rice, Phytophthora infestans in potatoes and tomatoes, Plasmopara viticola in vines, 8 Pseudoperonospora species in hops and gherkins, Alternating species in vegetables and fruits, Mycosphaerella species in bananas and cachuates, and Fusarium and Verticillium species in cereals, vegetables and Ornamental Example 1 The manipulation of the gene for a flavanone 3-hydroxylase from Lycopersicon esculentum Mill.cv. Moneymaker The ripe tomato fruits of Lycopersicon esculentum Mill.cv. Moneymaker was washed, dried, and the pericarp was freed from the seeds, central axis and wooded parts by means of a sterile knife. The pericarp (approximately 50 g) was frozen in liquid nitrogen. The material was subsequently ground in a mixer. In a pre-cooled mortar, the ground material was treated with 100 ml of the homogenization medium and mixed. The suspension was then transferred into centrifuge tubes by squeezing it through a sterile gauze. Then, 1/10 of 10% of the volume of SDS was added and the bath was mixed thoroughly. After 10 minutes on ice, a volume of 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 new reaction vessel. Three extractions of phenol / cfbroformo and a chloroform extraction followed. Then, a volume of 3 M NaAc and 2.5 volumes of ethanol were added. The nucleic acids were precipitated overnight at -20 ° C. 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 was resuspended in 5-10 ml of 3 M cold NaAc. This washing step was repeated twice. The pellet was washed with 80% ethanol. When completely dried, the pellet was extracted in approximately 0.5 ml of sterile DEPC water, and the concentration of RNA was determined photometrically. 20 μg of total RNA was first treated with 3.3 μl of sodium acetate solution and 2 μl of the 1M magnesium sulfate solution, and the mixture was placed with DEPC water to a final volume of 100 μl. One microliter of RNase-free DNase (Boehringer Mannheim) was added, and the mixture was incubated for 45 minutes at 37 °. After the enzyme has been removed by stirring with phenol / chloroform / isoamyl alcohol, the RNA was precipitated with ethanol and the pellet was extracted in 100 μl of DEPC water. 2.5 μg of RNA from this solution was transcribed into cDNA using a cDNA kit (Gibco BRL). Using the amino acid sequences that were derived from the cDNA clones encoding flavanone 3-hydroxylase, regions conserved in the main sequence were identified (Britsch et al., Eur. J. Biochem. 217, 745-754 (1993), and these form the basis for the design of the degenerate PCR oligonucleotides.The 5'-oligonucleotide was determined using the peptide sequence SR PDK (amino acids 147-152 in the hybrid sequence Petunia FL3H PETHY) and had the following sequence: -TCI (A / C) G (A / G) TGG CC (A / C / G) GA (C / T) AA (A / G) CC-3 The sequence of the derived oligonucleotide using the peptide sequence THQAVV ( amino acid 276281 in the hybrid sequence Petunia FL3H PETHY) was as follows: 5'-CTT CAC ACA (C / G / T) GC (C / T) TG (A / G) TG (A / G) TC-3. PCR reaction was carried out using Perkin-Elmer tTth polymerase following the manufacturer's instructions, 1/8 of the cDNA was used as a standard (corresponding to 0.3 μg of RNA). PCR was as follows: 30 cyclest 94 degrees 4 seconds 40 degrees 30 seconds 72 degrees 2 minutes 72 degrees 10 minutes The fragment was cloned into the vector pGEM -T by Promega following the manufacturer's instructions. The accuracy of the fragment was reviewed by sequencing Using the Ncol and Pctl restriction cleavage sites, which exist in the pGEM-T vector polylinker, the PCR fragment was isolated, the projections were blunted at the ends using T4 polymerase. This fragment was cloned into an end vector (blunted) Smal pBinAR (Hofgen and Willmitzer, Plant Sci 66: 221-230 (1990)) (see Figure 2). This vector contains the 35 S CaMV promoter (cauliflower mosaic virus) (Franck et al., Cell 21: 285-294 (1980)) and the octopine synthase 10 gene termination signal (Gielen et al., EMBO J. 3: 835-846 (1984)). In plants, this vector mediates resistance to antibiotic kanamycin. The resulting DNA constructs contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants. Figure 2: Fragment A (529 bp) contains the 35S CaMV promoter (nucleotide 6909 to 7437 of cauliflower mosaic virus). "Fragment B contains the fragment of the F3H gene in antisense orientation. Fragment C (192 bp) 20 contains the signal terminating the octopine synthase gene. The manipulation of a longer cDNA fragment of the flavanone 3-hydroxylase from Lycopersicon esculentum Mill.cv. Moneymaker using the 5 'RACE system. A second antisense construct using a longer F3H fragment must be generated to exclude the failure ~ ^^^^ a ^ a ^^ M ^ a- ^ ga ^^ '^^^ feA ^^ g ^^ Sfe ^ it ^^^ -. • Jj ^^? ^ s ^ ssa ir ^^ to generate plants with an amount of reduced mRNA flux balance of F3H due to the small size of the F3H PCR fragment used in the antisense construct. 5 To clone a longer F3H fragment, the 5 'RACE method (system for rapid extension of cDNA ends) was used. Extending the F3H PCR fragment by the 5 'RACE method using the 5' RACE system for rapid amplification 10 of cDNA ends, Version 2 ~ 0 by Life Technologies ™. Total RNA was isolated from the mature tomato fruits of Lycopersicon esculentum Mill.cv. Moneymaker (see above). The first synthesis of cDNA strain was carried out following the manufacturer's instructions using the GSP-m 1 (gene-specific primer) 5 '-TTCACCACTGCCTGGTGGTCC-3'. After RNAse digestion, the cDNA was purified following the manufacturer's instructions using the Life Technologies ™ GlassMAX rotational system. Following the manufacturer's instructions, a cytosine homopolymer was added to the 3 'end of the purified single strain F3H cDNA using the terminal deoxynucleotide transferase. The extended F3H 51 DNA was amplified using a second specific gene primer (GSP-2) which is tA - ^ 'w-tw links to the 5' end < in the 3 'region of the GSP-1 recognition sequence, thus allowing a "nested" PCR. The 5 'primer used was the' 5 'RACE shortened anchor primer which was provided by the manufacturer and which is complementary to the homopolymer dC termination of the cDNA. The cDNA fragment amplified in this way and called extended F3H was cloned into the pGEM-T vector by Promega following the manufacturer's instructions. The identity of the cDNA was confirmed by sequencing. The extended F3H cDNA fragment was isolated using the Ncol and PstI restriction cleavage sites, which are presented in the polylinker of vector pGEM-T, and the projections were blunted at the ends using T4 polymerase. This fragment was cloned into a pBinAR Smal (blunt) vector (Hofinger and Willmitzer, (1990) (see Figure 3) .This vector contains the 35 S CaMV promoter (cauliflower mosaic virus) (Franck et al., ( 1980) and the octopine synthase gene termination signal (Gielen et al., 1984) In plants, this vector mediates the resistance to the antibiotic kanamycin The obtained DNA constructs 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 CaMV promoter (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 signal terminating the octopine synthase gene. Example 2 The generation of Lycopersicon esculentum Mill.cv. Transgenic moneymaker that expresses a subfragment of flavanone 3-hydroxylase in the antisense orientation. The method according to Ling et al., Plant Cell Report 17, 843-847 (1998) was used. The cultivation was carried out at approximately 22 ° C in a 16-h-light / 8-h-dark regime. Tomato seeds (Lycopersicon esculentum Milli. cv. Moneymaker) were sterilized 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 3% sucrose, pH 6.1, for germination. After a germination time of 7-10 days, the cotyledons were ready for use in the transformation. Day 1: The Petri dishes that contain the medium "MSBN" were overlaid with 1.5 ml of an approximately 10-day tobacco suspension culture. Dishes ** £ i¡ && e? > t > They were covered with film and incubated at room temperature until the next day. Day 2: The sterile filter paper was placed in such a manner on the overlays with the tobacco suspension culture that the air bubbles were absent. The cotyledons, which had been sectioned diagonally, were placed on this filter paper face down. The Petri dishes were incubated for 3 days in the culture room. Day 5: The agrobacterial culture (LBA4404) was pelleted by centrifugation for 10 minutes at approximately 3000 g and resuspended in MS medium so that the OD was 0.3. The cotyledon sections were placed inside this suspension and incubated with moderate agitation for 30 minutes at room temperature. Then, the cotyledon sections were dried a little on sterile filter paper and returned to their initial dishes to continue cocultivation for 3 days in the culture room. Day 8: Cocultivated cotyledon sections were placed on MSZ2K50 + β and incubated for the next 4 weeks in the culture room. Then they were subcultured. The suckers formed were transferred to the root induction medium. After successfully rooting, the plants were ready to be tested and transferred to the greenhouse. Example 3 Transgenic tomato plants with reduced flavanone 3-hydroxylase activity were infected with Clavibacter michiganensis subsp. Michiganensis (Cmm). The Cmm grew for 2 days at 28 ° C on yeast-dextrose-Ca agar (YDC). The bacteria were raised with sterile water and their cell density was determined. For inoculation, the cell density was brought to 106 cells / ml using sterile water. The injections were carried out using 20 gauge hypodermic needles filled with bacterial suspension. They were given in the armpit of the fully developed uppermost leaf of young plants that has a total of 3-4 leaves. The infection was assessed by assessing the phenotype that developed. While more than 75% of the leaves withered in wild type plants, a significantly lower degree of wilting was found in the transgenic tomato plants. Example 4 The test for the increase in resistance to attack by Phytophthora infestans in tomatoes with flavanone 3-hydroxylase in antisense orientation. The leaves of tomato plants cd. "Moneymaker" that were not genetically modified or that were genetically modified according to the invention were infected with a suspension of aqueous zoospora of the Phytophthora infestans one week after they reached the 4-leaf stage. Afterwards, the plants were placed inside a chamber with 100% atmospheric humidity at temperatures between 16 and 18 ° C. After 6 days, the brown root on control plants that had not been genetically modified had developed to a high degree. Tomato plants that expressed an antisense construction of flavanone 3-hydroxylase showed a significantly lower level of Phytophthora infestans infection than the control.

Claims (6)

1. CLAIMS 1. A method to increase the resistance of forage plants to bacterial and fungal pathogens, where a plant is generated by genetic methods 5 molecular in which the activity of the enzymatic flavanone-3-hydroxylase is reduced.
2. 2. The method as claimed in claim 1, wherein the activity of the enzymatic flavanone 3-hydroxylase is completely or partially, in the form 10 permanently or temporarily, in the whole plant or in parts of the plant, inhibited by molecular biology methods (for example antisense construction, cosuppression, the expression of specific antibodies or the expression of specific inhibitors).
3. 3. The method as claimed in claim 1 or 2, wherein the forage plants are wheat, barley, rye, oats, rice, corn, panic grass, sugar cane, bananas, tomatoes, tobacco, bell peppers, potatoes, oily rapeseed, sugar beet, soybeans, cotton, 20 fruit of the tree of the Rosaceae family, such as apples and pears, plums, quetsches (plum brandy), peaches, nectarines and cherries, and vines.
4. 4. The method as claimed in claims 1-3, wherein the resistance to 25 the Venturia inaequalis in apples and pears. ^ * *? hMm * J ^ ~ «- ^^^ - ^ - ^ i ^ y..if ^^^ .. ^. ^ -. ^. ^^
5. 5. The method as claimed in claims 1- 3, where resistance to Botrytis cinerea is increased in vines.
6. 6. A plant with increased resistance to bacterial and fungal pathogens, where the activity of enzymatic flavanone 3-hydroxylase is reduced by molecular genetic methods. ? I? i t-fflMitiii? i? fíii T J m iililif I Miii i "flirt