DE19927575A1 - Increasing resistance of crop plants to fungal or bacterial pathogens, by engineering a reduction in activity of flavanone-3-hydroxylase - Google Patents

Abstract

Increasing (M1) the resistance of crop plants to bacterial or fungal pathogens by genetically engineering plants to have reduced activity of flavanone-3-hydroxylase (I), is new. An Independent claim is also included for plants produced by (M1).

Description

The present invention is a method for increasing the resilience of crops to bacterial and fungal pathogens, characterized in that with moleku largenetic methods a plant is produced in the 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 constructs, co-suppression, expression specifi shear antibody or the expression of specific inhibitors) in whole or in part, permanently or temporarily, in the ge velvet plant or in parts of the plant in its activity ge is inhibited.

Another object of the present invention are plants with increased resistance to bacterial and fungal Pa thogens, characterized in that by molecular genetic Methods the activity of the enzyme flavanone-3-hydroxylase redu is decorated.

The productivity of crops can be varied in many ways be reduced by stress factors. To call here are below other: viral diseases, bacterial and fungal pathogens, damaging insects, nematodes, snails, game bites, heat, Cool, cold, lack of water, too high water content of the soil, Bo salinization, too high radiation intensity, too high ozone content, Competition for light, water and nutrients through accompanying flora, improper or not optimally applied herbicide application (especially in fruit crops), treatments with herbicides, Insecticides, fungicides, bioregulators or foliar fertilizers from to low selectivity, foliar applications of crop protection or fertilizer during intense sunlight.

A number of these problems caused by stressors can through the use of pesticides, through the use resistant plant material or suitable growing techniques mi be optimized. However, the scope of these possibilities is be borders. In particular, bacteriosens are, if anything, very much hard to fight. In this case (eg against fire blight at Ap fel and pear) antibiotics such as streptomycin or tetracyclines  used, which is the risk of resistance even in human pathogenic. Furthermore, z. B. fungal patho often adapt to fungicides, so that their effectiveness subsides. A similar adaptation exists even with "pathogenresi stents ", which are produced by conventional methods be presented.

A need for plants that are resistant to pathogens are not just annual or arable neric cultures, but also in valuable permanent crops such as Fruit and wine.

The object of the invention was a simple and inexpensive Process for permanently improving the resistance against bacterial and fungal pathogens, especially in Kul turplants.

Based on physiological studies with growth Regulators from the group of acylcyclohexandione were now Surprisingly, genetic engineering methods available with the Help produce crops that are against a series of phytopathogenic bacteria and fungi are resistant.

Acylcyclohexanediones such as prohexadione-Ca and trinexapacethyl (former term: Cimectacarb) are used as bioregulators for inhibition of plant growth. Their bioreguatory effect is due to the fact that they block the biosynthesis of length-growth-promoting gibberellins. Due to their structural similarity to 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 Regulator, Gaus man, HW (ed.), Marcel Dekker, Inc., New York, pp. 169-200 (1991)). It is known that such compounds also intervene in the metabolism of phenols and thus in several Pflan zenarten can cause an inhibition of Anthocyanbildung (Rade macher, W et al., The mode of action of acylcyclohexanediones - a new type of growth retardant, in Progress in Plant Growth Regu lation, Karssen, CM, by Loon, LC, Vreugdenhil, D (eds.), Kluwer Academic Publishers, Dordrecht (1992)). Such effects on the phenolic content are reported as the cause of the side-effect of prohexadione-Ca against fire blight (Rade macher, W et al., Prohexadione-Ca - a new plant growth regulator for apple with interesting biochemical features, poster on the 25 ^th Annual Meeting of the Plant Growth Regulation Society of America, 7-10 July 1998, Chicago). A. Lux-Endrich (Dissertation Technical University of Munich in Weihenstephan, 1998) found in the course of their studies on the mechanism of action of prohexadione-Ca against fire blight that there is a multiple increase in the content of phenolic substances in cell cultures of apple by Pro hexadione-Ca and that a number of otherwise non-existent phenols occurs. In the context of these investigations it was further found that relatively high levels of luteoliflavan and eryodyctiol occur in shoot tissue of apple under the influence of prohexadione-Ca. Luteoliflavan is not normally found in apple tissue, and erythodyctiol occurs only in small amounts as an intermediate in flavonoid metabolism. However, the expected flavonoids catechin and cyanidin were undetectable in the treated tissue or occurred only in significantly reduced amounts (S. Römmelt et al., Lecture 8 ^th International Workshop on Fire Blight, Kusadasi, Turkey, 12.-15. October 1998).

It can be taken for granted that prohexadione-Ca, Trinexapac ethyl and other acylcyclohexanediones inhibit 2-oxoglutaric acid-dependent hydroxylases, which are important in the metabolism of phenolic sub stances. These are primarily chal consynthetase (CHS) and flavanone 3-hydroxylase (F3H) (W. Heller and G. Forkmann, Biosynthesis, in: The Flavonoids, Har borne, JB (ed.), Chapman and Hall, New York, 1988). However, it can not ever be ruled out that acylcyclohexanediones also inhibit further, as yet unknown, 2-oxoglutaric acid-dependent hydroxylases. It should also be apparent that a lack of catechol, cyanidin or other end products of the flavonoid synthesis is registered by the plant and that via a feedback mechanism, the activity of the key enzyme Phenylala ninammoniumlyase (PAL) is increased. However, with the continued inhibition of CHS and F3H, these flavoniodic end products can not be formed and luteoliflavan, eryodyctiol, and other phenols are increasingly formed ( Figure 1).

By reducing the enzyme activity of the enzyme flavanone-3-hy droxylase (F3H) are the flavonoids eriodictyol, proanthocyani dines which are substituted on the C-atom 3 by hydrogen, z. B. Lu teoforol, luteoliflavan, apigeniflavan and tricetiflavan, as well as homogeneous and heterogeneous oligomers and polymers from the mentioned and structurally related substances increasingly formed.

Increased concentrations of phenols hydroxycinnamic acids (p-coumar acid, ferulic acid, sinapinic acid), salicylic acid or umbellife ron, including the homogeneous and het formed from them Rogenic oligomers and polymers are used after reduction of the enzyme activity of the enzyme flavanone-3-hydroxylase (F3H) in plants  detected. Likewise, the concentration of chalcone increases, such as B. phloretin, and the stilbenes, such as. B. Resveratrol.

By reducing the enzyme activity of the enzyme flavanone-3-hydroxy The concentration of glycosides in flavonoids, phenolic compounds, chalcone and stilbene increased.

Based on these findings and conclusions gen produced technically altered crops in which F3H by Anti-sense constructs in whole or in part, permanently or before passing, throughout the plants or in individual plants organs or tissues was reduced in their activities with the consequence that the content of phenolic compounds in the whole plant is reduced. Experimental could then be shown be that these plants in their resistance to bacterial and / or fungal pathogens are increased.

Alternatively to the production of plants, with the help of anti sense technology in its flavonone-3-hydorxylase activity redu are graced, other literature known molecular genetic methods such as co-suppression or expression of use specific antibodies to achieve this effect chen.

The inventive method for increasing the resistance force against infestation with bacterial and fungal pathogens Reduction of flavonone-3-hydroxylase enzyme activity can be successful rich in the following crops: Wheat, Ger ste, rye, oats, rice, corn, millet, sugar cane, banana, to mate, tobacco, paprika, potato, oilseed rape, sugar beet, soybean, tree Wool, fruit trees of the family Rosacea, such as apple and Pear, plum, plum, peach, nectarine and cherry as well Grapevines.

Particularly suitable is the inventive method for increasing resistance to Venturia inaequalis in apple and Pear and Botrytis cinerea in grapevines.

Transgenic plants with reduced activity of the enzyme flava non-3-hydroxylase prepared according to the in the examples be Surprisingly, the method described shows an increased Wi Resistance to infestation with phytopathogenic bacteria rien. This could be exemplified by the infestation of transgenic tomatoes plants whose flavanone 3-hydroxylase activity is reduced,  with Clavibacter michiganensis subsp. michiganensis (C mm) see example 3.

Plants whose flavanone 3-hydroxylase with the help of molecular genetic methods was reduced, also showed an increased Resistance to infestation with Erwinia amylovora and others phytopathogenic bacteria. The most important phytopathogenic bacteria can be found in the publication "European Handbook of Plant Di Smiths, I.M., Dunez, J., Lelliott, R.A. Phillips, D.H. and Archer, S.A. Blackwell Scientific Publications, 1988, be removed.

In particular, the method according to the invention is suitable for increasing the resistance to the following phytopathogenic fungi:
Erysiphe graminis (powdery mildew) on cereals
Erysiphe cichoracearum and Sphaerotheca fuliginea on cucurbits
Podosphaera leucotricha on apples,
Uncinula necator on vines,
Puccinia species on cereals,
Rhizoctonia species on cotton, rice and turf,
Ustilago species on cereals and sugarcane,
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; Fusarium and Verticillium species in cereals, vegetables and ornamental plants.

example 1

Cloning of the gene of a flavanone-3-hydroxylase Lycopersicon esculentum Mill.cv. Moneymaker.

Ripe tomato fruits from Lycopersicon esculentum Mill.cv. Mo Neymakers were washed, dried and sterilized by means of a sterile Blade the pericarp of seeds, middle columnar and wooden parts  freed. The pericarp (about 50 g) was in liquid nitrogen frozen. The material was then crushed in a blender kleinert. The crushed material was in a pre-cooled Mortar mixed with 100 ml homogenization medium and mixed. The suspension was then transferred to centrifuge beakers by it was pressed through sterile cheesecloths. Subsequently was 1/10 vol 10% SDS added and mixed well. After 10 minutes on ice, 1 volume of phenol / chloroform was added, the centrifuge closed and well mixed. After 15 minutes Zen tritugation at 4000 rpm, the supernatant was in a new Reakti transferred onsgefäß. It was followed by three more phenol / chloro form extractions and a chloroform extraction. Hereinafter 1 volume of 3M NaAC and 2.5 volumes of ethanol were added. The precipitation the nucleic acids were overnight at -20 ° C. The next Mor The nucleic acids were incubated at 10,000 rpm for 15 minutes in the Refrigerated centrifuge (4 ° C) pelleted. The supernatant was discarded and The pellet is resuspended in 5-10 ml of cold 3 M NaAc. This Washing step was repeated twice. The pellet was washed with Washed 80% ethanol. The completely dried pellet was taken up in about 0.5 ml of sterile DEPC water and the RNA Concentration determined photometrically.

20 μg of total RNA were first mixed with 3.3 μl 3 M sodium acetate Lö solution, 2 .mu.l 1 M magnesium sulfate solution and 100 .mu.l end Volume filled up with DEPC water. This was a microliter Rnase-free DNase (Boehringer Mannheim) and 45 min at 37 ° Degree incubated. After removing the enzyme by shaking out with Phenol / chloroform / isoamyl alcohol was the RNA ge with ethanol drops and the pellet is taken up in 100 μl of DEPC water. 2.5 μg RNA from this solution were analyzed by means of a cDNA kit (Gibco BRL) in rewritten cDNA.

Using amino acid sequences derived from cDNA clones coding for Flava none-3 hydroxylase, conserved regions in the primary sequence could be identified (Britsch et al., Eur. J. Biochem., 217, 745-754 (1993) The basis for the design of degenerate PCR oligo nucleotides 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 using the peptide sequence DHQAW (Amino acid 276281 in the sequence FL3H PETHY from Petunia hybrida) oligonucleotide read 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 according to the manufacturer's instructions. 1/8 of the cDNA was used as template (corresponding to 0.3 μg RNA). The PCR program was:

AL = L <30 cycles 94 degrees 4 sec 40 degrees 30 sec 72 degrees 2 min 72 degrees 10 min

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

The correctness of the fragment was checked by sequencing. The PCR fragment was isolated using the restriction sites Ncol and PstI present in the polylinker of vector pGEM-T and the protruding ends were transformed using T4 polymerase and blunt-ended. This fragment was cloned into a Smal (blunt) cut vector pBinAR (Hofgen and Willmitzer, Plant Sci. 66: 221-230 (1990)) (see Figure 2). This contains the 35S promoter of the CaMV (flower kohl mosaic virus) (Franck et al., Cell 21: 285-294 (1980)) and the termination signal of the octopine synthase gene (Gielen et al., EMBO J. 3: 835-846 (1984)). This vector mediates in plant resistance to the antibiotic kanamycin. The resulting DNA constructs contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants.

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

Cloning of a larger cDNA fragment of flavanone-3-Hydro xylase from Lycopersicon esculentum Mill.cv. Moneymaker under Use of the 5'RACESystem.  

To rule out that the production of plants with reduce mRNA steady-state amount of F3H due to the low Size of the F3H PCR fragment used in the antisense construct not successful, should be a second antisense construct be generated using a larger F3H fragment.

For the purpose of cloning a larger fragment of F3H was the 5'RACE method (System for Rapid amplification of cDNA ends) applied.

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 of Life Tecgnologies ™.

From ripe tomato fruits of Lycopersicon esculentum Mill.cv.Mo neymaker total RNA was isolated (see above).

The cDNA first strand synthesis was according to the manufacturer Use of the GSP-1 (Gene Specific Primer) 5'-TTCAC CACTGCCTGGTGGTCC-3 '. Following a Rnase Ver the cDNA was synthesized using the GlassMAX spin system of Life Tecgnologies ™ according to the manufacturer's instructions.

At the 3 'end of the purified single-stranded F3H cDNA using the terminal deoxynucleotide transferase ge according to the manufacturer's instructions added a cytosine homopolymer.

Amplification of the 5 'extended F3H cDNA was performed under Use of a second gene specific primer (GSP-2) in the Area 3 'before the GSP-1 recognition sequence binds and thus a "nested" PCR enabled. As 5'Primer was the manufacturer supplied "5'RACE abrided anchor primer" used the komple to the homopolymeric dC tail of the cDNA.

The thus amplified and designated as F3H _extended cDNA fragment was cloned according to the manufacturer's instructions in the vector pGEM-T Promega.

The identity of the cDNA was confirmed by sequencing.

The F3H _extended cDNA fragment was isolated using the restriction sites Ncol and PstI present in the poly left of the vector pGEM-T and the protruding ends were transformed using the T4 polymerase and blunt ends. This fragment was cloned into a Smal (blunt) cut vector pBinAR (Hofgen and Willmitzer, 1990) (see Figure). The sera contains the 35S promoter of 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 resulting DNA constructs contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants.

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

Example 2

Production of Transgenic Lycopersicon esculentum Mill.cv. Moneyma ker which is a partial fragment of flavanone-3-hydroxylase in antisense Express orientation.

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

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

Day 1: Petri dishes containing the medium "MSBN" were mixed with 1.5 ml of a Overcoated about 10 d old tobacco suspension culture. The plates were covered with foil and until the next day at Raumtem incubated.

Day 2: On the tobacco suspension culture coated plat In this case, sterile filter paper was applied without air bubbles. Thereon the cross-cut cotyledons receded with the top side placed on the bottom. The Petri dishes were cultured for 3 days room incubated.

Day 5: The culture of agrobacteria (LBA4404) was determined by Zentri Fungation sedimented at about 3000 g for 10 min and in MS medium resuspended so that the OD is 0.3. In this suspension the cotyledons were added, which under light shake were incubated for 30 minutes at room temperature. to closing the cotyledons were on sterile Filterpa  pierced a bit and returned to its original location for the continuation of cocultivation for 3 days in culture Renraum laid.

Day 8: The cocultivated cotyledons were assayed for MSZ2K50 + β and incubated in the culture room for the next 4 weeks. Then the subcultivation took place.

Forming shoots were placed on root induction medium.

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

Example 3

Transgenic tomato plants with reduced flavanone 3-hydroxylase Activity was detected with Clavibacter michiganensis subsp. michiganen sis (Cmm) infected.

Cmm was cultured on yeast dextrose Ca agar (YDC) at 28 ° C for 2 days fourth. The bacteria were washed off with sterile water and determines their cell density. Inoculation was the cell density adjusted to 106 cells / ml with sterile water. The injection NEN were using hypodermic needles (# 20) with the bacteria suspension filled goods were carried out. They took place in the Blattachsel of the uppermost fully developed leaf young Pflan zen, which had a total of 3-4 leaves. The evaluation of In Fektion was made by assessing the developing pheno type.

While in wild-type plants more than 75% of the leaves wither In contrast, in the transgenic tomato plants, a si Significantly lower Verwelkungsgrad recorded.

Example 4

Test for increased resistance to infestation with Phytophthora infestans in tomatoes with flavanone 3-hydroxylase in Antisense orientation.

The leaves of non-genetically modified or fiction according to genetically modified tomato plants of the variety "Mo neymaker "was given a week after the 4-leaf stage with one aqueous zoospore suspension of Phytophthora infestans infi ed. Subsequently, the plants were in a steam saturated chamber at temperatures between 16 and 180 ° up provides. After 6 days, the blight on the gentech  unmodified control plants are so strongly developed. Tomato plants containing an antisense construct of flavanone-3-hydro xylase expressed a significantly lower infestation Phytophthora infestans as the control.

Claims (6)

1. A method for increasing the resistance of Kulturpflan zen against bacterial and fungal pathogens, characterized in that by molecular genetic methods, a plant is produced in which the activity of the enzyme flavanone-3-hydroxylase is reduced. 2. The method according to claim 1, characterized in that the Enzyme Flavanon-3-hydroxylase by molecular biological Ver drive (eg anti-sense constructs, co-suppression, ex expression of specific antibody or expression specifi shear inhibitors) in whole or in part, on-going or before passing, throughout the plant or in parts of the Plant is inhibited in its activity. 3. Process according to claims 1 and 2, characterized net, that the crops are wheat, barley, Rye, oats, rice, corn, millet, sugarcane, banana, tomato, Tobacco, peppers, potatoes, rapeseed, sugarbeet, soya, cotton, Fruit trees from the family of the Rosacea, such as apple and Pear, plum, plum, peach, nectarine and cherry as well as grapevines. 4. Process according to claims 1-3, characterized in that that the resistance to Venturia inaequalis in apple and pear is increased. 5. Process according to claims 1-3, characterized that the resistance to Botrytis cinerea in grapevines is increased. 6. Plant with increased resistance to bacterial and fungal pathogens, characterized in that by mole Kulargenetische methods the activity of the enzyme Flava non-3-hydroxylase is reduced.