Polypeptides against plant pathogenic fungi
10266842 ยท 2019-04-23
Assignee
Inventors
Cpc classification
International classification
C12N15/82
CHEMISTRY; METALLURGY
C07K14/00
CHEMISTRY; METALLURGY
Abstract
The present invention discloses polypeptides comprising an amino acid sequence being identical with at least 12 contiguous amino acid residues of SEQ ID No 2. The polypeptides according to the invention are effective against fungi, especially against fungi causing plant diseases, and against fungi colonizing agricultural products. The invention further discloses processes for preparing such polypeptides, and nucleic acids coding for such polypeptides. In addition, the invention relates to processes and preparations for treating plants using the polypeptides according to the invention, and to the use of the nucleic acids according to the invention for producing crops that are protected against damage from fungi.
Claims
1. A polypeptide comprising an amino acid sequence identical with SEQ ID NO: 1 or SEQ ID NO: 2, wherein (i) the N-terminal end is derivatized by partial alkylation, complete alkylation, or acylation, (ii) the C-terminal end is derivatized by amidation or esterification, (iii) the peptide chain is derivatized by PEGylation or HESylation, or (iv) a combination thereof.
2. The polypeptide according to claim 1, which leads to a half-maximum inhibition of the spore germination of Fusarium graminearum at a concentration of 1 to 1000 M.
3. The polypeptide according to claim 1, which is effective against the growth of organisms of the genera Fusarium and/or Phytophthora.
4. The polypeptide according to claim 1, having the following sequence TABLE-US-00004 (SEQIDNO:2) Z.sub.1-QHGYGAGGHGQQGYGSQHSSHAPQGGHVVREQGFSGHVHE QQAGHHHEAGHHEQAGHHEQSGQQVHGQGHGYK-Z.sub.2, where Z.sub.1 is the N-terminal end of the polypeptide, the derivatized N-terminal amino group of the polypeptide, or a chain of up to ten arbitrary amino acids; and Z.sub.2 is the C-terminal end of the polypeptide, the derivatized C-terminal carboxy group of the polypeptide, or a chain of up to ten arbitrary amino acids; wherein at least one of Z.sub.1 or Z.sub.2 is derivatized or a chain of up to ten arbitrary amino acids.
5. The polypeptide according to claim 4, wherein Z.sub.2 has an amino acid sequence of SHGY-Z.sub.3, wherein Z.sub.3 the C-terminal end of the polypeptide, the derivatized C-terminal carboxy group of the polypeptide, or a chain of up to six arbitrary amino acids, and wherein Z.sub.1 is derivatized or has a chain of up to ten arbitrary amino acids or Z.sub.3 is derivatized or has a chain of up to six amino acids.
6. The polypeptide according to claim 1, wherein the polypeptide has D-amino acids in part, D-amino acids completely, or a D-retro-inverso peptide structure.
7. A polynucleotide coding for the polypeptide according to claim 1.
8. A method of using the polypeptide according to claim 1 the method comprising applying the polypeptide to a plant for controlling fungi or oomycetes.
9. A cell, except for wild type cells of Lucilia sericata, wherein said cell contains a nucleic acid for the expression of a polypeptide according to claim 1.
10. A transgenic crop containing a cell according to claim 9.
11. The polypeptide according to claim 1, which leads to a half-maximum inhibition of the spore germination of Fusarium graminearum at a concentration of 2 M.
12. The method according to claim 8, wherein the fungi cause plant diseases.
13. The method according to claim 8, wherein the fungi are selected from the group consisting of the genus Fusarium and the genera Phytophthora, and the oomycetes are from the class Peronosporomycetes.
Description
(1) The invention also relates to a transgenic crop containing a cell according to the invention.
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(12) According to the invention, the term fungicidal activity is understood to mean the killing of fungi, the inhibition of fungal growth, and the prevention of the germination of fungal spores. These effects can be completely or partially pronounced.
(13) According to the invention, the term control of fungi is understood to mean the killing of fungi, or the inhibition of the growth of existing fungi as well as the prevention of fungal colonization, and the prevention of the germination of fungal spores.
(14) According to the invention, fungi is also understood to mean those organisms that are colloquially referred to as fungi (or molds), even though this does not correctly reflect the scientific evidence relating to phylogenesis. In particular, fungi according to the invention is intended to include representatives of the class Peronosporomycetes (former designations Oomycota or Oomycetes), including the genus Phytophthora.
(15) The polypeptides according to the invention are suitable for controlling fungi that cause health-related or economical damage.
(16) In a preferred embodiment of the invention, the polypeptides according to the invention are employed for controlling fungi that cause plant diseases or damage in the storage of agricultural products.
(17) In another preferred embodiment of the invention, the polypeptides according to the invention are employed for controlling fungi of the genera Phytophthora and Fusarium.
(18) The polypeptides according to the invention can be used in a pure form or in the form of different formulations for controlling fungi. For external application, the polypeptides can be diluted to form a liquid solution or suspension containing from 0.01 to 30 mg/ml of the respective polypeptide, or mixed with an extender solid for application as a dust or powder. Methods for adapting common methods for application to particular crops and pathogens are known in the literature (Methods for Evaluating Pesticides for Control of Plant Pathogens, K. D. Hickey, Ed., The American Phytopathological Society, 1986). Methods for application include the singular or periodically performed aqueous and non-aqueous spraying of plants or plant parts, seed coating, and the incorporation in spraying systems. Auxiliary additives that may be added to the formulation include stabilizers, agents for improving the dissolving performance, and wetting agents, and also agents that allow microencapsulation.
(19) For a particularly effective control of fungi, the polypeptides according to the invention can be employed in combination with other fungicidally active substances. Also, combination with bactericidal, antiviral, nematocidal, insecticidal and other active substances common in plant protection is possible. Combination with fertilizers, plant hormones and growth regulators is also possible. One possibility of controlling fungi by means of the polypeptides according to the invention is to genetically engineer plants by introducing polynucleotide sequences coding for such polypeptides into their genetic material by a process known as transformation. Methods for preparing such genetically engineered plants according to the invention are known to the skilled person (Methods for Plant Molecular Biology, A. Weissbach, H. Weissbach, Eds., Saunders College Publishing/Harcourt Brace, June 1988; Methods in plant molecular biology and biotechnology, B. R. Glick, J. E. Thompson, CRC Press, Boca Raton, Fla., 1993). In an advantageous variant of the invention, artificial gene constructs of suitably modified vectors are incorporated into plants, plant parts or plant cells by bombardment with DNA-coated microparticles, by the so-called floral dip method, or by Agrobacterium-mediated transformation. Other possible methods include, for example, microinjection, chemical permeabilization, electroporation, and protoplast fusion with DNA-containing units, such as cells, minicells, protoplasts, or liposomes. In another advantageous variant of the invention, a gene coding for a polypeptide according to the invention is prepared synthetically, in which the nucleotide triplets, which code for one amino acid each, are adapted to the preferred codon usage of the genetically engineered plant. Further, it is possible to place the foreign gene to be transferred under the control of a promoter that is activatable by injury and auxine activity according to a known method, whereby an enhanced expression after fungal colonization is achieved (Rahnamaeian. Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens. J Exp Bot. 2009, 60: 4105-14). In an advantageous embodiment of the invention, corn and/or potato plants are genetically engineered to be able to produce the polypeptides according to the invention.
(20) A polypeptide corresponding to SEQ ID No. 1 or No. 2 can be used for controlling fungi. In addition, it is also possible to employ polypeptides or oligopeptides resulting from truncation of the sequence or the addition of further amino acid residues.
(21) Further, it is possible to employ variants of the polypeptide that have additional sequence elements having been added, for example, in order to achieve a higher yield in the preparation in a recombinant form, or a facilitated purification.
(22) In some cases, it may be advantageous to employ oligopeptides consisting of at least twelve contiguous amino acid residues of SEQ ID No. 1. In addition, it may be advantageous to remove individual amino acid residues, or replace them by the residues of different amino acids. The insertion of additional amino acid residues is also possible. Such changes are also possible when proceeding from truncated or extended variants of the polypeptide. In particular, it may be advantageous to replace individual amino acid residues by those having similar physico-chemical properties. Thus, mainly residues of the amino acids are mutually interchangeable within the following groups: Arginine and lysine; glutamic acid and aspartic acid; glutamine, asparagine and threonine; glycine, alanine and proline; leucine, isoleucine and valine; tyrosine, phenylalanine and tryptophan; serine and threonine.
(23) The identification of polypeptides that are derived from SEQ ID No. 1 can be effected, for example, by the screening of genomic and/or cDNA gene libraries using known methods, which are described, for example, in Sambrook and Russell 2001 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NJ. The gene libraries can be prepared, for example, using bacteria or bacteriophages as receptor organisms. Also, the gene libraries could be in the form of sequence data electronically stored on suitable media, especially if such data were produced by means of the techniques known as Next Generation Sequencing without molecular cloning of nucleic acid molecules. As probes for the screening, there can be employed, in particular, nucleotide sequences corresponding to the complementary strand of SEQ ID No. 3 or SEQ ID No. 4, or of fragments of these sequences. The sequences of these probes may vary within the scope of the degeneracy of the genetic code, and may also contain nucleosides such as inosine, which do not naturally occur in protein-encoding nucleic acids, in order to enhance the capability of hydrogen bonding.
(24) The screening may also be effected using antibodies raised against polypeptides corresponding to SEQ ID No. 1, SEQ ID No. 2, or fragments of these sequences.
(25) Because of their comparatively small size, the polypeptides according to the invention can be prepared by methods of chemical peptide synthesis (Example 1). This may involve the use of known solid-phase methods, for example, according to B. Merrifield. The synthesis may be effected manually or with the aid of an automated peptide synthesizer using Fmoc or Boc protective group strategy. It is possible to synthesize the polypeptides in smaller fragments, which are subsequently linked together.
(26) The preparation of the polypeptides according to the invention may also be effected by the heterologous expression of suitable nucleic acid constructs in different receptor organisms and receptor cells. The required methods of genetic engineering are described in Sambrook and Russell 2001 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NJ. Prokaryotic systems (for example, Escherichia coli or Pseudomonas fluorescens) or eukaryotic systems (for example, insect cells, plant cells or mammal cells) may be employed.
(27) In a preferred embodiment of the invention, the preparation is effected in E. coli as a fusion polypeptide with thioredoxin and a hexahistidine sequence, wherein a specific protease recognition sequence is provided for the cleavage of the polypeptide according to the invention (Examples 2, 3 and 4).
(28) The polypeptides according to the invention obtained by heterologous expression can be purified from cell lysates or supernatants of the genetically engineered organisms or cells by a number of known methods. Particularly suitable methods include immobilized metal ion affinity chromatography, anion-exchange chromatography, and reverse-phase chromatography (Example 3).
EXAMPLE 1
Synthetic Preparation of the Polypeptide LserFCP1-77
(29) The polypeptide having the amino acid sequence according to SEQ ID No. 1 was prepared completely by solid-phase synthesis on a polymeric support resin. An analysis of the product by reverse-phase chromatography (column: Alltech Alltima C18 4.6250 mm, Fischer Scientific) with an ascending methanol gradient in water yielded an essentially homogeneous peak, so that a purity of at least 80% could be assumed (
EXAMPLE 2
Construction of Plasmids for the Recombinant Preparation of LserFCP1-77 and LserFCP1-73 in Escherichia coli
(30) For the heterologous expression in E. coli, a synthetic gene was prepared that codes for the sequence of the polypeptide LserFCP1-77 (SEQ ID No. 1). The codon usage was adapted to that of the receptor organism E. coli K12. The gene synthesis was performed by the company Eurofins MWG Operon (Anzingerstr. 7a, 85560 Ebersberg, Germany) on a contract basis. Further sequences enabling the insertion into the vector pASK-IBA33plus were added to the coding sequence at the 5 and 3 terminals. Thus, the complete synthetically prepared polynucleotide had the sequence as shown in SEQ ID No. 4. The synthetic gene was inserted into the vector pASK-IBA33plus in an oriented way by using two BsaI restriction endonuclease cleaving sites. The thus obtained construct was used for the transformation of E. coli cells of the strains TOP10 and BL21.
(31) In further experiments proceeding from the construct based on pASK-IBA33plus, the synthetic gene was recloned into the vector pET-32a(+). The recloning was performed to obtain a plasmid coding for a fusion polypeptide consisting of thioredoxin, a hexahistidine sequence, a protease recognition sequence, and the polypeptide LserFCP1-77. Two variants of this plasmid were prepared; in one, the encoded recognition sequence was specific for enterokinase, and in the other, for coagulation factor Xa. Accordingly, the plasmids were designated as pET-32-FCP-EK and pET-32-FCP-Xa.
(32) For the construction of these plasmids, the synthetic gene was amplified by PCR, wherein the additional sequences required for the insertion into the new vector were added with the primers. pET-32-FCP-EK was constructed using the forward primer 1 consisting of a KpnI restriction site, a linking sequence, a sequence coding for the enterokinase recognition sequence, and a specific sequence coding for the first amino acid residues of the polypeptide LserFCP1-77. pET-32-FCP-Xa was constructed using the forward primer 2 consisting of a KpnI restriction site, a linking sequence, a sequence coding for the factor Xa recognition sequence, and a specific sequence coding for the first amino acid residues of the polypeptide LserFCP1-77. For both constructs, the reverse primer 3 consisting of an EcoRI restriction site, a stop codon and a specific sequence coding for the last amino acid residues of the polypeptide LserFCP1-77 was used.
(33) TABLE-US-00002 Primer1(forward): (SEQIDNO:5) 5-tgaggtaccgacgacgacgacaagcagcatggctatggagcgg-3 Primer2(forward): (SEQIDNO:6) 5-tgaggtaccggtggtggctccggtattgagggtgccagatggcta tggagcgg-3 Primer3(reverse): (SEQIDNO:7) 5-tcagaattttaatacccgtggatttgtag-3
(34) The PCR products were inserted through the KpnI and EcoRI restriction sites into the vector pET-32a(+), and the constructs obtained were used for the transformation of E. coli cells of the strain BL21(DE3) (Novagen/Merck). The identification of transformed cells was effected by selection on ampicillin-containing nutrient medium. In its genomic DNA, the E. coli strain employed contains a lysogenic lambda phage on which a gene coding for T7 RNA polymerase is under the control of a lacUV5 promoter. The expression of the T7 RNA polymerase gene and thus the expression of the foreign gene being under the control of a T7 promoter on the expression plasmid is induced by adding isopropyl-beta-D-thiogalactopyranoside (IPTG). The formation of the corresponding fusion polypeptide at different times after the induction was detected by SDS PAGE followed by Coomassie blue staining (
EXAMPLE 3
Production of the Recombinant Polypeptide LserFCP1-77 in Escherichia coli
(35) E. coli cells of the strain BL21(DE3) (Novagen/Merck) were transformed with the plasmid pET-32-FCP-Xa constructed as described in Example 2, and cultured in six one-liter Erlenmeyer flasks with baffles, each of which contained 400 ml of LB medium supplemented with 300 mg/l ampicillin, at 37 C. with shaking at 250 rpm. After an absorption value of 0.4 at 600 nm as observed by turbidity measurement had been achieved, the induction was effected by adding 1 mM IPTG. After the culture had been continued for 3 hours, the bacterial cells were harvested by centrifugation (10,000g, 10 min, 4 C.). The pellet was resuspended in 200 ml of buffer A (100 mM NaCl, 30 mM Tris, pH 7.5), and the cells were lysed by shear forces in a high-pressure homogenizer (Microfluidizer M110PS, Microfluidics, 30 Ossipee Road, Newton, Mass. 02464 U.S.A.). After centrifugation (70,000g, 30 min, 4 C.), the supernatant was filtered through a 0.22 mm membrane. The cell lysate was charged at a flow rate of 4 ml/min onto an immobilized metal ion affinity chromatographic column loaded with Co.sup.2+ ions (16100 mm, TALON Superflow Resin, Clontech Laboratories, 1290 Terra Bella Avenue, Mountain View, Calif. 94043, U.S.A.). The column had previously been equilibrated with buffer A. After the sample had been charged, the column was washed with buffer A until the detection at 280 nm yielded a constant value. The elution was effected with a step gradient from 30 mM to 100 mM imidazole in buffer A. The main quantity of the fusion polypeptide was eluted with the 100 mM imidazole step. An analysis by SDS PAGE followed by Coomassie blue staining yielded a purity of more than 90% (
(36) The fusion polypeptide was rebuffered by gel permeation chromatography (HiPrep Desalting Column 26/10, GE Healthcare, Buckinghamshire, UK) in 10 mM NaCl, 10 mM Tris, pH 7.5. Ten micrograms of the fusion polypeptide was incubated with 500 units of factor Xa (Merck) in 10 ml of cleavage buffer (100 mM Tris-HCl, 20 mM NaCl, pH 7.5) for 16 h at room temperature. The reaction product was charged at a flow rate of 1 ml/min onto a strong ion-exchange column (MonoQ 5/50 GL, GE Healthcare) equilibrated with 10 mM Tris-HCl, pH 8, and the column was washed with the same buffer. The elution was effected with a gradient from 0 to 300 mM NaCl over 20 minutes. An analysis by SDS PAGE showed that the polypeptide LserFCP1-77, which was obtained as a cleaving product and eluted at 11 min, was completely separated from the remaining polypeptide components (
EXAMPLE 4
Production of the Recombinant Polypeptide LserFCP1-73 in Escherichia coli
(37) E. coli cells of the strain BL21(DE3) (Novagen/Merck) were transformed with the plasmid pET-32-FCP-EK constructed as described in Example 2. The culturing of the cells and the processing of the cell lysate were performed by analogy with the operations described in Example 3, except that 0.1 units of enterokinase (Novagen) were employed for cleaving the fusion polypeptide. In this case too, it was possible to separate the target molecule by anion-exchange chromatography (
EXAMPLE 5
Determination of the Activity of LserFCP1-77 Against Fusarium graminearum
(38) Fusarium graminearum (Strain IFA 65)
(39) Source of supply: Interuniversitary Department for Agricultural Biotechnology of Tulln, Austria
(40) Reference: Steiner B, Kurz H, Lemmens M, Buerstmayr H. Differential gene expression of related wheat lines with contrasting levels of head blight resistance after Fusarium graminearum inoculation. Theor Appl Genet. 2009 February; 118(4): 753-64.
(41) Fusarium graminearum was cultured on SNA-agar plates at room temperature to sporulation. The spores were swept off the plate with water, adjusted to a density of 20,000 spores/ml, and stored at 4 C. until further use. For performing the tests, 0.05 ml each of the spore suspension was combined with 0.05 ml each of differently concentrated LserFCP1-77 solutions in the wells of a 96-well microtitration plate. After incubation for 24 h at room temperature, the spores were examined microscopically for germination using objective lenses with 4 fold and 10 fold magnifications. The result was documented photographically (
EXAMPLE 6
Determination of the Activity of LserFCP1-73 Against Fusarium graminearum
(42) The effectiveness of LserFCP1-73 at a concentration of 100 M was determined as compared to a water control by means of the test system described in Example 5. A complete inhibition of spore germination by LserFCP1-73 was observed (
EXAMPLE 7
Determination of the Activity of LserFCP1-77 Against Phytophthora parasitica
(43) Phytophthora parasitica (Isolate 329)
(44) Source of supply: INRA (institut national de la recherche agronomique), France Reference: Keller H, Pamboukdjian N, Ponchet M, Poupet A, Delon R, Verrier J L, Roby D, Ricci P. Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance. Plant Cell. 1999 February; 11(2): 223-35.
(45) Phytophthora parasitica was cultured on Rye B agar plates for 8 days at 25 C. The sporangia were washed off with water, and the sporangia suspension obtained was incubated for 4 h at 4 C. to induce the formation of zoospores. After 1:50 dilution in RPMI 1640 medium, the spore density was determined and adjusted to 20,000 spores/ml. The further performance of the test was effected by using the methods described in Example 5. The result was also documented photographically (
SEQUENCE LISTINGS
(46) The amino acids were abbreviated according to the IUPAC nomenclature as follows: alanine A, arginine R, asparagine N, aspartic acid E, cysteine C, glutamic acid D, glutamine Q, glycine G, histidine H, isoleucine I, leucine L, lysine K, methionine M, phenylalanine F, proline P, serine S, threonine T, tryptophan W, tyrosine Y, valine V
(47) TABLE-US-00003 SEQIDNO:1-LserFCP1-77 QHGYGAGGHGQQGYGSQHSSHAPQGGHVVREQGFSGHVHEQQAGHHHEAG HHEQAGHHEQSGQQVHGQGHGYKSHGY(SEQIDNO:1) SEQIDNO:2-LserFCP1-73 QHGYGAGGHGQQGYGSQHSSHAPQGGHVVREQGFSGHVHEQQAGHHHEAG HHEQAGHHEQSGQQVHGQGHGYK(SEQIDNO:2) SEQIDNO:3-L.sericatacDNAcodingfor LserFCP1-77 5-CAACACGGCTATGGTGCCGGTGGCCATGGCCAACAAGGCTATGGTAG CCAACATAGCAGTCATGCTCCCCAAGGTGGACATGTTGTCCGTGAACAAG GTTTTAGTGGTCATGTTCATGAACAACAGGCTGGGCATCATCATGAAGCT GGCCATCATGAGCAAGCTGGTCATCATGAACAATCTGGTCAACAAGTTCA TGGTCAAGGTCATGGCTATAAAAGTCATGGTTAT-3 (SEQIDNO:3) SEQIDNO:4-syntheticgenewithE.coiladapted codonusagecodingforLserFCP1-77 Thenon-codingsequencesegmentsaddedfor cloningareprintedinobliquecharacters. 5-ATGGTAGGTCTCAAATGCAGCATGGCTATGGAGCGGGT GGACATGGCCAGCAGGGTTACGGCTCTCAGCACAGC AGTCATGCTCCGCAAGGTGGCCATGTCGTTCGCGAA CAGGGCTTTTCCGGTCACGTACACGAGCAGCAAGCA GGCCATCACCATGAAGCCGGCCATCACGAACAAGCG GGTCACCATGAGGAGTCAGGGCAGCAAGTGCATGGG CAAGGTCATGGCTACAAATCGCACGGGTAT TAAAGCGCTGAGACCTACCAT-3 (SEQIDNO:4)