Plant protection and/or plant growth promotion system

Abstract

The invention relates to a plant protection and/or plant growth promotion system, comprising at least one anchor peptide for increased binding to a plant part, a support function, which is indirectly or directly bound to the anchor peptide, and an active substance for protecting the plant and/or promoting plant growth and yield.

Claims

1. A plant protection and/or plant growth promotion system, comprising at least one anchor peptide for increased binding to a plant organ or a plant part, the anchor peptide being from 10 to 100 amino acids long and comprising an amino acid sequence selected from SEQ ID NOs: 1-48 or an amino acid sequence at least 80% identical thereto, a support function, which is indirectly or directly bound to the anchor peptide, and an active substance for protecting the plant and/or promoting plant growth and/or yield, wherein the active substance is physically or chemically bound to the support function and wherein the support function serves to connect the active substance with the anchor peptide.

2. The plant protection and/or plant growth promotion system according to claim 1, wherein the active substance is selected from the group consisting of: a pesticide; an herbicide; a bactericide; a fungicide; a safener; a beneficial organism that has a positive effect on the resistance, the disease resistance, the growth and/or the yield of plants; and a plant growth agent.

3. The plant protection and/or plant growth promotion system according to claim 1, wherein the support function is selected from the group consisting of a nanogel, a microgel, a polysome, a polymerosome, a synthosome, a poly-amino acid-spacer, a colloidosome and a cubosome.

4. The plant protection and/or plant growth promotion system according to claim 1, wherein the anchor peptide binds to a leaf, trunk, stem, root, fruit, seed, bud, blossom and/or tuber of a plant.

5. The plant protection and/or plant growth promotion system according to claim 1, wherein the support function is a nano- or microgel and configured such that the active substance either remains on the support function or is released, wherein the release is direct, time delayed, and/or stimulation dependent.

6. The plant protection and/or plant growth promotion system according to claim 5, wherein the support function is stimulation dependently released and the stimulus is selected from the group consisting of pH, temperature, humidity, light (including UV light), duration of irradiation, electric pulses, magnetic pulses, elicitors, enzymatic reactions, microbe associated molecular patterns, pathogen associated molecular patterns, damage associated molecular patterns, and herbivore associated molecular patterns.

7. The plant protection and/or plant growth promotion system according to claim 1, wherein the system is a fusion protein.

8. The plant protection and/or plant growth promotion system according to claim 7, wherein the support function is a spacer of 2-100 amino acids.

9. A method for protecting plants and/or promoting plant growth, comprising the following steps: a) providing a plant protection and/or plant growth promotion system according to claim 1, and b) applying the plant protection and/or plant growth promotion system onto a plant.

10. The plant protection and/or plant growth promotion system according to claim 1, wherein the support function is selected from the group consisting of a nanogel, a microgel, a polysome, a polymerosome, a synthosome, a poly-amino acid-spacer, a colloidosome and a cubosome.

11. The plant protection and/or plant growth promotion system according to claim 2, wherein the support function is selected from the group consisting of a nanogel, a microgel, a polysome, a polymerosome, a synthosome, a poly-amino acid-spacer, a colloidosome and a cubosome.

12. The plant protection and/or plant growth promotion system according to claim 1, wherein the anchor peptide binds to a leaf, trunk, stem, root, fruit, seed, bud, blossom and/or tuber of a plant.

13. The plant protection and/or plant growth promotion system according to claim 2, wherein the anchor peptide binds to a leaf, trunk, stem, root, fruit, seed, bud, blossom and/or tuber of a plant.

14. The plant protection and/or plant growth promotion system according to claim 3, wherein the anchor peptide binds to a leaf, trunk, stem, root, fruit, seed, bud, blossom and/or tuber of a plant.

15. The plant protection and/or plant growth promotion system according to claim 1, wherein the system is a fusion protein.

16. The plant protection and/or plant growth promotion system according to claim 2, wherein the system is a fusion protein.

17. The plant protection and/or plant growth promotion system according to claim 3, wherein the system is a fusion protein.

18. The plant protection and/or plant growth promotion system according to claim 4, wherein the system is a fusion protein.

19. The plant protection and/or plant growth promotion system according to claim 18, wherein the support function is a spacer of 2-100 amino acids.

20. The plant protection and/or plant growth promotion system according to claim 1, wherein the anchor peptide comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-48.

Description

EXAMPLES

Example 1—Wash-Off Test

(1) The wash-off test is used to test whether the anchor peptide provides for an improved binding of the plant protection and/or plant growth promoting system according to the present invention: 1. The plant protection and/or plant growth promotion system to be tested (sample) and molecules of the same composition but without the section of the anchor peptide being present (comparative sample), are provided. 2. Procedure a) An incubation is carried out for five minutes at room temperature on the plant part to be tested for the sample and the comparative sample. b) Subsequently, it is washed with Tris/HCl buffer 50 mM Tris/HCl buffer, pH 8.0 for three times for five minutes each. c) Subsequently, the concentration of the sample and the comparative sample on the respective plant part is determined using a suitable detection method. An improved binding within the meaning of the present invention is at least present, when at least 50% more of the sample remains on the plant than of the comparative sample. If necessary, the test plant is, in case of doubt, a soybean plant (Glycine max) or a corn plant (Phocea) or wheat (Triticum L). Preferably, the above-mentioned soybean plant and the corn plant are the test plants (as examples for single and dicotyledonous plants). In case of doubt, all plant parts of the comparative plant are tested to determine whether there is an increased binding to one of the plant parts. Here, in particular, the following plant parts are to be mentioned: leaf, leaf stem, trunk, stem, root, fruit, bud and blossom. Preferably, an increased binding in the sense of the present invention is present when the sample, compared to comparative sample, is washed off in the wash-off test from at least one of said plant parts by at least 50% less.

Example 2—Preferred Anchor Peptides

(2) TABLE-US-00001 TABLE 1 Possible anchor peptide sequences: Peptide Amino acid sequence Antifungal AGCIKNGGRCNASAGPPYCCSSYCFQIAGQSYGVCKNR SEQ 1 protein 1 Cg-Def GFGCPGNQLKCNNHCKSISCRAGYCDAATLWLRCTCTD SEQ 2 CNGKK Dermaseptin GMWSKIKNAGKAAKAAAKAAGKAALGAVSEAM SEQ 3 DA4 Defensin VTCDLLSFEAKGFAANHSLCAAHCLAIGRRGGSCERGVC SEQ 4 ICRR hDermcidin SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVH SEQ 5 DVKDVLDSV Pelovaterin DDTPSSRCGSGGWGPCLPIVDLLCIVHVTVGCSGGFGC SEQ 6 CRIG Thanatin GSKKPVPIIYCNRRTGKCQRM SEQ 7 Androctonin RSVCRQIKICRRRGGCYYKCTNRPY SEQ 8 TAP_20_N NPVSCVRNKGICVPIRCPGNMKQIGTCVGRAVKCCRKK SEQ 9 PgD5 MEKRMGSLSVLFLLLVLVTSFEMQVEGRMCESQSHKFK SEQ 10 GYCASSSNCKVVCQTEKFLTGSCRDTHFGNRRCFCEKP C Alpha- KSCCRSTLGRNCYNLCRARGAQKLCAGVCRCKISSGLS SEQ 11 purothionin CPKGFPK Carnobacter- VNYGNGVSCSKTKCSVNWGQAFQERYTAGINSFVSGVA SEQ 12 iocin B2 SGAGSIGRRP MMGP1 MLWSASMRIFASAFSTRGLGTRMLMYCSLPSRCWRK SEQ 13 Macaque DSHEERHHGRHGHHKYGRKFHEKHHSHRGYRSNYLYD SEQ 14 histatin N MiAMP1 SAFTVWSGPGCNNRAERYSKCGCSAIHQKGGYDFSYTG SEQ 15 QTAALYNQAGCSGVAHTRFGSSARACNPFGWKSIFIQC MBP-1 RSGRGECRRQCLRRHEGQPWETQECMRRCRRRG SEQ 16 RNase 7 KPKGMTSSQWFKIQHMQPSPQACNSAMKNINKHTKRCK SEQ 17 DLNTFLHEPFSSVAATCQTPKIACKNGDKNCHQSHGAVS LTMCKLTSGKYPNCRYKEKRQNKSYVVACKPPQKKDSQ QFHLVPVHLDRVL Shepherin I GYGGHGGHGGHGGHGGHGGHGHGGGGHG SEQ 18 Snakin-1 GSNFCDSKCKLRCSKAGLADRCLKYCGICCEECKCVPS SEQ 19 GTYGNKHECPCYRDKKNSKGKSKCP Stomoxyn RGFRKHFNKLVKKVKHTISETAHVAKDTAVIAGSGAAVVA SEQ 20 AT VarvF GVPICGETCTLGTCYTAGCSCSWPVCTRN SEQ 21 VrD1 RTCMIKKEGWGKCLIDTTCAHSCKNRGYIGGNCKGMTR SEQ 22 TCYCLVNC European FVPYNPPRPYQSKPFPSFPGHGPFNPKIQWPYPLPNPG SEQ 23 bumblebee H abaecin Papiliocin RWKIFKKIEKVGRNVRDGIIKAGPAVAVVGQAATVVK SEQ 24 BM Moricin AKIPIKAIKTVGKAVGKGLRAINIASTANDVFNFLKPKKRKA SEQ 25 Carnocyclin A LVAYGIAQGTAEKVVSLINAGLTVGSIISILGGVTVGLSGVF SEQ 26 TAVKAAIAKQGIKKAIQL Curvacin A ARSYGNGVYCNNKKCWVNRGEATQSIIGGMISGWASGL SEQ 27 AGM Lactoferricin B FKCRRWQWRMKKLGAPSITCVRRAF SEQ 28 Magainin 2 GIGKFLHSAKKFGKAFVGEIMNS SEQ 29 Pleurocidin GWGSFFKKAAHVGKHVGKAALTHYL SEQ 30 Psoriasin MSNTQAERSIIGMIDMFHKYTRRDDKIDKPSLLTMMKENF SEQ 31 PNFLSACDKKGTNYLADVFEKKDKNEDKKIDFSEFLSLLG DIATDYHKQSHGAAPCSGGSQ Spinigerin HVDKKVADKVLLLKQLRIMRLLTRL SEQ 32 Acanthoscurrin 1 DVYKGGGGGRYGGGRYGGGGGYGGGLGGGGLGGGG SEQ 33 LGGGKGLGGGGLGGGGLGGGGLGGGGLGGGKGLGGG GLGGGGLGGGGLGGGGLGGGKGLGGGGLGGGGLGGG RGGGYGGGGGYGGGYGGGYGGGKYKG ALF-L DGIWTQLIFTLVKNLATLWQSGDFQFLDHECHYRIKPTFR SEQ 34 RLKWKYKGKFWCPSWTSITGRATKSSRSGAVEHSVRNF VGQAKSSGLITQRQAEQFISQYN BDEF_TACTR NPLIPAIYIGATVGPSVWAYLVALVGAAAVTAANIRRASSD SEQ 35 NHSCAGNRGWCRSKCFRHEYVDTYYSAVCGRYFCCRS R PP102 GSCSCSGTISPYGLRTCRATKTKPSHPTTKETHPQTLPT SEQ 36 Tachystatin A2 YSRCQLQGFNCVVRSYGLPTIPCCRGLTCRSYFPGSTYG SEQ 37 RCQRY CecropinA- KWKLFKKIKFLHSAKKF SEQ 38 Magainin2 Plantaricin A KSSAYSLQMGATAIKQVKKLFKKWGW SEQ 39 Ocellatin-F1 GVVDILKGAAKDIAGHLASKVMNKL SEQ 40 PgD5 MEKRMGSLSVLFLLLVLVTSFEMQVEGRMCESQSHKFK SEQ 41 GYCASSSNCKVVCQTEKFLTGSCRDTHFGNRRCFCEKP C TAP 20N NPVSCVRNKGICVPIRCPGNMKQIGTCVGRAVKCCRKK SEQ 42 Androctonin RSVCRQIKICRRRGGCYYKCTNRPY SEQ 43 Adenoregulin GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAVQ SEQ 44 Cathelicidin-BF KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF SEQ 45 Cecropin A KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK SEQ 46 Lcl AIKLVQSPNGNFAASFVLDGTKWIFKSKYYDSSKGYWVG SEQ 47 IYEVWDRK Reutericin-6 IYWIADQFGIHLATGTARKLLDAMASGASLGTAFAAILGVT SEQ 48 LPAWALAAAGALGATAA

(3) The anchor peptides mentioned are particularly suitable for the plant protection and/or growth promotion systems according to the invention. In this case, the binding of the anchor peptide to the residual molecule (in particular directly or indirectly to the support) is carried out by a skilled person according to the prior art.

Example 3

(4) General Protocol for the Expression of a Plant Protection and/or Plant Growth Promotion System According to the Invention in the Form of a Fusion Protein:

(5) Principle: Expression of Fusion Proteins in Small Amounts

(6) The gene encoding the plant protection and/or plant growth promotion system according to the invention, including the anchor peptide, is introduced into a pET28 expression vector. The expression strain is E. coli Bl21-Gold.

(7) Procedure:

(8) 1. Plating the E. coli on an agar plate. Growth overnight at 37° C. 2. Extracting a single colony from the plate to inoculate a pre-culture of 10 mL of LBKan medium, incubating overnight at 37° C. with 200 rpm. 3. Preparing 10 mL of TB-medium (80 mL solution A+20 mL solution B) per strain, adding Kanamycin. 4. Checking the OD.sub.600 of the pre-culture; inoculating the main culture with x ml pre-culture, x=2.5/OD.sub.pre-culture 5. Checking the OD.sub.600 of the main culture (aim: at least 0.025). 6. Incubating the main culture at 37° C., 200 rpm, for 4 hours. 7. Adding 100 μL 0.1 M IPTG. 8. Incubating the main culture at 16° C., 200 rpm, for 48 hours. 9. Harvesting the cells by means of centrifugation, 30 min at 4° C., 4000 rpm (Eppendorf 5810 R) 10. The cells can be stored at −20° C. until their use.

(9) The chemicals are obtained from Sigma Aldrich.

(10) The used solutions are, in detail:

(11) LB-medium: Trypton (10 g/L), yeast extract (5 g/L) NaCl (10 g/L)

(12) The LB-medium is autoclaved for twenty minutes at 121° C.

(13) For 100 mL LB-medium of the pre-culture (LBKan), 100 μL sterile filtrated Kanamycinsulfate (100 mg/mL in ddH2O) were added.

(14) TB-Medium:

(15) Solution A:

(16) Peptone 12 g, yeast extract 24 g, glycerol 4 g, filled up to 800 mL with dH.sub.2O and autoclaved at 121° C. for 20 minutes.

(17) Solution B:

(18) K.sub.2HPO.sub.4 (12.5 g) and KH.sub.2PO.sub.4 (2.31 g) were filled up to 200 mL with dH.sub.2O and were autoclaved at 121° C. for 20 minutes.

(19) For the preparation of TB-medium with Kanamycin (TBkan), 100 μL sterile filtrated Kanamycinsulfate (100 mg/mL in ddH2O) were added to 200 mL TB-medium.

Example 4—Purification of Fusion Proteins

(20) By means of affinity chromatography, proteins are separated on the basis of a reversible interaction between a protein and a specific ligand bound to a chromatography matrix. This technique provides high selectivity, high resolution and usually a large capacity for the protein of interest. The technique can be used to separate active biomolecules from denatured or functionally altered forms or to isolate pure substances that are present in low concentration in large volumes of raw samples or to remove specific impurities.

(21) Procedure:

(22) 1. A cell pellet (for example one of Example 3) is taken up with buffer A (see below), which has been admixed with 0.1 mM PMSF and 0.1 mM DTT just before use. 6 mug cells are used. 2. The cell disintegration is performed in a high-pressure homogenizer, 3 times at 1500 bar. 3. Subsequently, it is centrifuged for 15 min at 4° C. and 4000 rpm. 4. It follows a centrifugation for 30 min at 4° C. and 10000 rpm (Sorvall centrifuge). If the supernatant is not clear, it is again centrifuged for 15 min at the same conditions. 5. Subsequently, the supernatant is filtered with 0.45 μm cellulose acetate-membrane filters (not sterile) (VWR). 6. It follows a purification via His-tag using ÄKTA prime and a NiNTA column (5 mL). 7. Equilibration of the column: The column is washed for 5 times with ddH2O Tube B is filled with buffer B (see below) Tube A is filled with buffer A and then washed into the column with 2 bed volume buffer A (0% buffer B) 8. Application of the clear supernatant: A flow rate of 2 mL/min at a maximum pressure of 0.6 MPa is set The column is equilibrated with buffer A The starting gradient is 100 mL 0-50% buffer B The eluates are collected in 2-5 mL fractions The fractions are checked via SDS-Page It is dialyzed against buffer C at 4° C. for 24 hours The purity is determined via SDS-Page and the concentration is determined via BCA-assay 0.1 mM PMSF, 0.1 mM DTT und EDTA—solution are added Optionally, the solution is frozen at −20° C. and, optionally, subsequently lyophilized The lyophilized fusion protein can be stored at −20° C.
Note: The chemicals are supplied by Sigma Aldrich, ÄKTA prime was used.
Solutions:

(23) Buffer A (50 mM Tris-buffer, 5 mM imidazole, pH 8.0): 4.44 g/L Tris HCl, 2.65 g/L Tris-base und 0.34 g/L imidazole are diluted with dH2O.

(24) Buffer B (50 mM Tris-buffer, 500 mM imidazole, pH 8.0): 4.44 g/L Tris HCl and 2.65 g/L Tris-Base und 34.04 g/L imidazole are mixed in dH2O.

(25) Buffer C (50 mM Tris-buffer, pH 8.0): 4.44 g/L Tris HCl and 2.65 g/L Tris-base are diluted with dH2O.

Example 5

(26) Mode II for a Plant Protection and/or Plant Growth Promotion System According to the Invention:

(27) For improved detectability, the active substance (e.g., an antimicrobial peptide) is replaced by eGFP in this model.

(28) FIG. 1 schematically shows the polypeptide chain of a fusion protein according to the invention.

(29) The plant protection and/or plant growth promotion system according to the invention consists of a His-tag for the purification (optional), a part of green fluorescent protein (eGFP) as a model for the active substance and for a simple detection of the support function in the form of a 10× Alanine spacer with an optional protease cleavage site (TEV) (10×Ala-TEV) as well as the anchor peptide, either Cecropion A (CecA) or LCl.

(30) The corresponding amino acid sequences are as follows:

(31) TABLE-US-00002 SEQ 50: His-eGFP-10xAla-TEV-CecA MGSSHHHHHHSSGLVPRGSHMVSKGEELFTGVVPILVELDGDVNGHKFSV SGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMK QHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGID FKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGI TLGMDELYKAAAAAAAAAAENLYFQGKWKLFKKIEKVGQNIRDGIIKAGP AVAVVGQATQIAK SEQ 51: His-eGFP-10xAla-TEV-LCI MGSSHHHHHHSSGLVPRGSHMVSKGEELFTGVVPILVELDGDVNGHKFSV SGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMK QHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGID FKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGI TLGMDELYKAAAAAAAAAAENLYFQGAIKLVQSPNGNFAASFVLDGTKWI FKSKYYDSSKGYWVGIYEVWDRK The SEQ 52 MGSSHHHHHHSSGLVPRGSH represents the His-tag, The SEQ 53 MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT YGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAM PEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILG HKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPI GDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK represents the eGFP The SEQ 54 AAAAAAAAAA represents the alanine spacer and the SEQ 55 ENLYFQG represents the TEV-cleavage site. The remainder of the sequence is the CecA - or the LCI sequence (cf., Example 2).

Example 6

(32) Application Example of the Model Fusion Protein:

(33) The DNA sequence for the model fusion protein was inserted into a pET28 expression vector and expressed according to Example 3. After obtaining and purifying the fusion proteins (cf., Example 4), they were applied to soya leaves.

(34) The fusion protein according to SEQ 51 (LCl-eGFP) was used as sample in a concentration of 1 mg/mL and the same sequence without the anchor peptide sequence, also expressed according to Example 3, was used as comparative sample. The comparative sample was also applied in a concentration of 1 mg/mL.

(35) The incubation was carried out according to the wash-off test, cf., Example 1. After carrying out the wash-off test according to Example 1, a large number of fluorescence signals were observed in the fluorescence microscope only on the soybean leaf treated with the sample (model fusion protein), whereas fluorescence was no longer detectable on the leaf treated with the comparative sample.

Example 7

(36) Example for Binding Anchor Peptide Sequences with a Sequence Variance of the LCl Sequence:

(37) TABLE-US-00003 (LCI WT) SEQ 56 AIKLVQSPNGNFAASFVLDGTKWIFKSKYYDSSKGYWVGIYEVWDRK (LCI KR1) SEQ 57 AIKLVQSLNGNFAARFVLDGTKWIFKCKYYDSSKGYWVGIYEVWGRK (LCI KR2) SEQ 58 AIRLVQSQKCNFAASFVLGGTKWTFKGKYYDSSKDYWVGIYEVRVRK (LCI KR3) SEQ 59 AIKQVQSPNGNFAASFVLDGTKWIFKSKYYDSSRGYWVGIYVVWDRK (LCI KR4) SEQ 60 AIKLVHSPNGNFAASFVLDGTKWIFKSKFYDSSKGYWVGTYEVWARK