Hypersensitivity reaction elicitor peptides and uses thereof

Abstract

Hypersensitive response eliciting peptides exhibiting improved solubility, stability, resistance to chemical degradation, or a combination of these properties are disclosed. Also disclosed are uses of these peptides or fusion polypeptides or DNA constructs encoding the same for modulating plant biochemical signaling, conferring disease resistance to a plant, enhancing plant growth, conferring tolerance to a plant to a biotic stress, conferring tolerance and resistance to a plant to an abiotic stress, conferring desiccation resistance to a cutting taken from an ornamental plant, conferring postharvest disease resistance or postharvest desiccation resistance to a fruit or vegetable, or enhancing the postharvest life of a fruit or vegetable.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201580064625.7, filed on October 1, 2015, entitled "Allergic Response Evotron Peptide and Its Use Therein," the original application being the national phase application of International Application No. PCT / US2015 / 053387. This international application claims priority to U.S. Provisional Patent Application Serial No. 62 / 058,535, filed on October 1, 2014, and U.S. Provisional Patent Application Serial No. 62 / 140,789, filed on March 31, 2015, each of which is incorporated herein by reference in its entirety. Invention Field This invention relates to novel allergic response elicitor peptides and their use in inducing active plant responses, particularly including enhanced growth, disease resistance, pest or insect resistance, and stress resistance. Background of the Invention The identification and isolation of harpin proteins stemmed from fundamental research at Cornell University aimed at understanding how plant pathogens interact with plants. The first line of defense is the hypersensitive response (HR), the localized death of plant cells at the site of infection. Cell death creates a physical barrier to pathogen movement, and in some plants, dead cells can release compounds toxic to invading pathogens. Studies have shown that pathogens may possess a single factor responsible for triggering HR. The fundamental goal of the Cornell research was to identify the specific bacterial proteins responsible for eliciting HR. The target proteins are known to be encoded by one of a class of bacterial genes called the hypersensitive response and pathogenicity (hrp) gene cluster. The analysis of *Hyperpyrethrum pearense*, the bacterium that causes fire blight in pear and apple trees, revealed this process. Erwinia amylovora, The hrp cluster in Ea) was identified, and a single protein that elicits HR in some plants was identified. This protein was named harpin (and later referred to as harpin). Ea And name the corresponding genes. hrpN This is the first instance of such a protein and gene identified from all bacterial species.

[0002] Already from Erwinia spp. ( Erwinia ), Pseudomonas spp. Pseudomonas ), Rolstonia ( Ralstonia Flavobacterium ( Xanthomonas ) and Pantotheca ( Pantoea Many different harpin proteins have been identified through various methods. At the primary amino acid sequence level, these different harpin proteins share common biochemical and biophysical characteristics and biological functions. Based on their unique properties, harpin proteins are often considered a separate class of proteins in the literature.

[0003] Following its identification and isolation, harpin was later found to induce disease resistance and increase plant growth in plants. An important early finding was that application of purified harpin protein confers resistance to subsequent pathogen attacks in plants, and that the application site is located far from the injection site. This suggests that harpin protein can trigger systemically acquired resistance (SAR), a plant defense mechanism that provides resistance to a wide range of viral, bacterial, and fungal pathogens.

[0004] In crop protection, a combination of methods to improve plant health is always needed. Healthier plants are desired because they produce better yields and / or better plant or crop quality. Healthier plants are also better resistant to biotic and abiotic stresses. High resistance to biotic stresses, in turn, allows growers to reduce the amount of pesticides applied, thereby slowing the development of resistance to various pesticides.

[0005] harpin It is a fusion protein derived from several different harpins. Harpins have been confirmed. It inhibits nematode egg production, enhances plant growth, quality, and yield, and increases plant vigor. Its amino acid and nucleotide sequences are described in detail in U.S. Patent Application Publication No. 2010 / 0043095.

[0006] To date, harpin and harpin Harpin protein has traditionally been produced and used in agricultural and horticultural applications as a powdered solid coated on starch. This has limited the use and versatility of harpin protein, as liquid suspensions of powdered harpin protein in water have an effective lifespan of only 48–72 hours before significant degradation and loss of activity occur. Another problem with harpin solutions is protein solubility and stability.

[0007] The aim is to identify synthetic and derived harpin peptides that are readily soluble in aqueous solutions, stable, resistant to chemical degradation, and effectively induce allergic reactions in plants.

[0008] The present invention aims to overcome these and other limitations in the art. Invention Overview The first aspect of the invention relates to an isolated peptide having the amino acid sequence (L / I / V / F)-XX-(L / I / V / F)-(L / I)-XX-(L / I / V / F)-(L / I / V / A)-XX-(L / I)-(L / I / V / F) (SEQ ID NO: 93). The peptide is free of cysteine ​​and methionine; each X at positions 2 and 6 is optional and, when present, is any amino acid; and each X at positions 3, 7, 10, and 11 is any amino acid. In one embodiment, only one X at positions 2 and 6 is optional. In some embodiments, SEQ ID NO: 93 may also include additional amino acid residues between hydrophobic duplexes (as shown, two of L / I / V / F / A). In some embodiments, the isolated peptide further includes a hydrophilic amino acid sequence located at the N-terminus or C-terminus of SEQ ID NO: 93.

[0009] A second aspect of the invention relates to an isolated peptide having the amino acid sequence (L / I / V / F)-XX-(L / I / V / F)-(L / I)-XX-(L / I / V / F)-(L / I / V / A)-XX-(L / I)-(L / I / V / F) (SEQ ID NO: 93). The peptide is free of cysteine ​​and methionine; each X at positions 2, 6, and 10 is optional and, when present, is any amino acid; and each X at positions 3, 7, and 11 is any amino acid. In one embodiment, only one X at positions 2, 6, and 10 is optional. In some embodiments, SEQ ID NO: 93 may also include additional amino acid residues between hydrophobic duplexes (as shown, two of L / I / V / F / A). In some embodiments, the isolated peptide further includes a hydrophilic amino acid sequence located at the N-terminus or C-terminus of SEQ ID NO: 93.

[0010] A third aspect of the invention relates to an isolated peptide having the amino acid sequence XXGISEKXXXXXXXXXXXXXXXX (SEQ ID NO: 1, common to P1 / P4). in X at position 1 is optional and can be S, N, D, isoD, G, A or S; X at position 2 is optional and can be Q, E, g-glutamic acid, G, A or S; X at position 8 is Q, E, γ-glutamic acid, G, A, or S; X at position 9 is L, I, F, or V; X at position 10 is optional and can be D or isoD; X at position 11 is Q, E, γ-glutamic acid, G, A, or S; X at position 12 is M, L, I, or F; X at position 13 is M, L, or I; X at position 14 is optional and can be any hydrophilic amino acid, preferably C, S, T, A, D, isoD, K or Q; X at position 15 is Q, E, γ-glutamic acid, G, A, S, K, or I; X at position 16 is M, L, I, V, or F; X at position 17 is M, L, I, A, or V; X at position 18 is Q, E, γ-glutamic acid, G, A, S, M, T, or K; X at position 19 is A, D, isoD, S, V, T, K, R, E, H, or G; X at position 20 is M, L, or I; X at position 21 is M, L, I, V, S, or F; X at position 22 represents Q, E, γ-glutamic acid, G, A, and S. X at position 23 is P, Q, E, γ-glutamic acid, G, A, or S; and The isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence. In some embodiments, the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence.

[0011] An exemplary family of peptides according to a third aspect of the present invention has the amino acid sequence SXGISEKXXDXXXXXXXXAXXXP (SEQ ID NO: 2, P4 common), wherein X at position 2 is Q, E, γ-glutamic acid, G, A, or S; X at position 8 is Q, E, γ-glutamic acid, G, A, or S; X at position 9 is L, A, D, isoD, I, V, or F; X at position 11 is Q, E, γ-glutamic acid, G, A, or S; X at position 12 is L, D, isoD, I, or F; X at position 13 is L, I, V, or F; X at position 14 can be any hydrophilic amino acid, preferably C, S or T, S or T or only S; X at position 15 is Q, E, γ-glutamic acid, G, A, S, K, or I; X at position 16 is L, A, I, V, M, or F; X at position 17 is I, S, or F; X at position 18 is Q, E, γ-glutamic acid, G, A, or S; X at position 20 is L, I, V, or F; X at position 21 is either L or F; and X at position 22 is Q, E, g-glutamic acid, G, A, or S.

[0012] In some embodiments, these peptides according to the third aspect of the invention also satisfy the structural features defining peptides according to the first or second aspect of the invention.

[0013] Another exemplary family of peptides according to a third aspect of the invention has the amino acid sequence XXGISEKXLDXLLTXLIXALLXX (SEQ ID NO: 3, common to P1), wherein X at position 1 is N, D, isoD, G, A, or S; X at position 2 is Q, E, γ-glutamic acid, G, A, or S; X at position 8 is Q, E, γ-glutamic acid, G, A, or S; X at position 11 is Q, E, γ-glutamic acid, G, A, or S; X at position 15 is Q, E, γ-glutamic acid, G, A, or S; X at position 18 is M, T, K, E, γ-glutamic acid, G, A, or S; X at position 22 is Q, E, γ-glutamic acid, G, A, or S; and X at position 23 is Q, E, g-glutamic acid, G, A, or S.

[0014] In some embodiments, these peptides according to the third aspect of the invention also satisfy the structural features defining peptides according to the first or second aspect of the invention.

[0015] A fourth aspect of the invention relates to an isolated peptide having the following amino acid sequence: (i) KPXDSXSXIAKLISXLIXSLLX (SEQ ID NO: 47, shared by P15b / P20), where X at position 3 is N, D, or isoD; X at position 6 is Q, E, γ-glutamic acid, G, A, or S; X at position 8 is N, D, or isoD; X at position 15 is optional and can be any amino acid; X at position 18 is M, E, γ-glutamic acid, G, A, S, T, or K; and X at position 22 is optional and can be Q, E, γ-glutamic acid, G, A, or S; or (ii) IAKLISXLIXSLLX (SEQ ID NO: 12, P15 / 20 min co-occurring), of which The X at position 7 is optional and can be any amino acid; X at position 10 is M, E, γ-glutamic acid, G, A, S, T, or K; and X at position 14 is optional and can be Q, E, g-glutamic acid, G, A or S.

[0016] In some embodiments, the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence. In some embodiments, the peptide according to the fourth aspect of the invention also satisfies the structural features defined for a peptide according to the first or second aspect of the invention.

[0017] A fifth aspect of the invention relates to an isolated peptide having the following amino acid sequence: (i) PSPXTXXLXXIVGXILXAXN (SEQ ID NO: 66, common to P6 / 6a), where X at position 4 is either F or Y; X at position 6 is Q, E, γ-glutamic acid, G, A, or S; X at position 7 is optional and can be L, M, E, γ-glutamic acid, G, A, S, T or K; X at position 9 is M, E, γ-glutamic acid, G, A, S, T, or K; X at position 10 is either H or N; X at position 14 is E, γ-glutamic acid, D, or isoD; X at position 17 is Q, E, γ-glutamic acid, G, A, or S; and X at position 19 is Q, E, γ-glutamic acid, G, A, or S; or (ii) XTXXLXXIVGXIL (SEQ ID NO: 135, P6 / 6a min co-occurring), of which X at position 1 is either F or Y; X at position 3 is Q, E, γ-glutamic acid, G, A, or S; X at position 4 is optional and, according to one embodiment, may be M, E, γ-glutamic acid, G, A, S, T, or K; or, according to another embodiment, may be L; X at position 6 is M, E, γ-glutamic acid, G, A, S, T, or K; X at position 7 is either H or N; and X at position 11 is E, γ-glutamic acid, D, or isoD; The isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence. In some embodiments, the peptide according to the fifth aspect of the invention also satisfies the structural features defined for a peptide according to the first or second aspect of the invention.

[0018] A sixth aspect of the invention relates to an isolated peptide having the following amino acid sequence: (i) XXXXXXLXXLLXXLVXLLK (SEQ ID NO: 13, P14d common), where X at position 1 can be: Q, N, D, E, g-glutamic acid, isoD, or S; X at position 2 could be: D, E, γ-glutamic acid, or isoD; X at position 3 can be: P, D, E, isoD, or γ-glutamic acid; X at position 4 can be M, A, S, D, E, isoD, or γ-glutamic acid. X at position 5 can be Q, E, or g-glutamic acid; X at position 6 can be A, E, or g-glutamic acid; X at position 8 can be M, L, E, Q, D, N, G, A, S, isoD, or γ-glutamic acid; X at position 9 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 12 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 13 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; and X at position 16 can be K, Q, N, E, D, R, G, A, or S; or (ii) LXXLLXXLVXLLK (SEQ ID NO: 14, P14d min common), where X at position 2 can be M, L, E, Q, D, N, G, A, S, isoD, or γ-glutamic acid; X at position 3 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 6 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 7 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; and X at position 10 can be K, Q, N, E, D, R, G, A, or S.

[0019] In some embodiments, the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence. In some embodiments, the peptide according to the sixth aspect of the invention also satisfies the structural features defined for a peptide according to the first or second aspect of the invention.

[0020] A seventh aspect of the invention relates to an isolated peptide having the following amino acid sequence: (i) LXXL(L / M)XILXXLV (SEQ ID NO: 16, P25 common), where X at position 2 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 3 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 6 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 9 can be E, γ-glutamic acid, D, isoD, Q, N, T, S, A, or G; and X at position 10 can be A, G, S, T, E, γ-glutamic acid, D, isoD, Q, or N; or (ii) LXXVLXXL(L / M)XILXXLV (SEQ ID NO: 17, P25 common), of which X at position 2 can be T, S, A, G, D, isoD, E, γ-glutamic acid, Q, or N; X at position 3 can be G, T, S, A, D, isoD, E, γ-glutamic acid, Q, or N; X at position 6 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 7 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 10 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 13 can be E, g-glutamic acid, D, isoD, Q, N, T, S, A, or G; X at position 14 can be A, G, S, T, E, γ-glutamic acid, D, isoD, Q, or N; and The V at position 16 is optional.

[0021] In some embodiments, the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence. In some embodiments, the peptide according to the seventh aspect of the invention also satisfies the structural features defined for a peptide according to the first or second aspect of the invention.

[0022] The eighth aspect of the present invention relates to an isolated peptide having the following amino acid sequence: (i) XXXXXXXXXXX(L / M)XXLLXXLLXXLLXXX (SEQ ID NO: 21, P17 / 18), where X at position 1 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 2 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 3 can be any amino acid, but is preferably P, Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 4 can be any amino acid, but is preferably I, Q, S, E, γ-glutamic acid, A, T, G, D, N, isoD, K, or R; X at position 5 can be any amino acid, but D, isoD, S, E, γ-glutamic acid, A, T, G, N, Q, K or R are preferred. X at position 6 can be any amino acid, but is preferably R, Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, or K; X at position 7 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 8 can be any amino acid, but is preferably T, Q, S, E, γ-glutamic acid, A, G, D, isoD, N, K, or R; X at position 9 can be any amino acid, but is preferably I, Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 10 can be any amino acid, but is preferably E, γ-glutamic acid, Q, S, A, T, G, D, isoD, N, K or R; X at position 11 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 13 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 14 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 17 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 18 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 21 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N or R; X at position 22 can be any amino acid, but is preferably S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 25 can be any amino acid, but is preferably S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 26 can be any amino acid, but is preferably P, S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K, or R; and X at position 27 can be any amino acid, but is preferably Q, S, A, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; or (ii) (L / M)XXLLXXLLXXLL (SEQ ID NO: 25, P17 / 18 min total), of which X at position 2 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 3 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 6 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 7 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 10 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or R; and X at position 11 can be any amino acid, but is preferably S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; The isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence. In some embodiments, the peptide according to the eighth aspect of the invention also satisfies the structural features defined for a peptide according to the first or second aspect of the invention.

[0023] A ninth aspect of the present invention relates to an isolated peptide having the following amino acid sequence: XLXX(L / M)LXLIXX(L / I / V / F / M)(L / I / V / F / M) (SEQ ID NO: 26, P19 common), where X at position 1 is optional and can be L, I, V, F or M; X at position 3 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or R; X at position 4 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 7 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or R; X at position 10 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K, or R; and X at position 11 can be any amino acid, but is preferably R, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or K.

[0024] In some embodiments, the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence. In some embodiments, the peptide according to the ninth aspect of the invention also satisfies the structural features defined for a peptide according to the first or second aspect of the invention.

[0025] A tenth aspect of the present invention relates to an isolated peptide comprising the following amino acid sequence: (L / M)XXLLX(L / M)FXXI(L / M)XX (SEQ ID NO: 15, P3min total) where X at position 2 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 3 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 6 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 9 can be E, γ-glutamic acid, D, isoD, Q, N, T, S, A, or G; X at position 10 can be A, G, S, T, E, γ-glutamic acid, D, isoD, Q, or N; X at position 13 can be Q, N, E, γ-glutamic acid, D, isoD, T, S, A, or G; and X at position 14 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G.

[0026] In some embodiments, the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence. In some embodiments, the peptide according to the tenth aspect of the invention also satisfies the structural features defined for a peptide according to the first or second aspect of the invention.

[0027] The eleventh aspect of the present invention relates to a fusion protein comprising one of the peptides of the first to eleventh aspects of the present invention, together with a purification tag, a solubility tag, or one or more of a second peptide according to one of the first to tenth aspects of the present invention.

[0028] The twelfth aspect of the present invention relates to a composition comprising one or more peptides according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth aspects of the present invention, or a fusion protein according to the eleventh aspect of the present invention, and a carrier.

[0029] The thirteenth aspect of the present invention relates to a method for conferring disease resistance to plants. This method comprises: applying to a plant or plant seed or the site where the plant is growing or intended to grow an effective amount of an isolated peptide according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect of the present invention, a fusion protein according to the eleventh aspect of the present invention, or a composition according to the twelfth aspect of the present invention, wherein said application effectively confers disease resistance.

[0030] The fourteenth aspect of the present invention relates to a method for enhancing plant growth. This method comprises: applying to a plant or plant seed or the site where the plant is growing or intended to grow an effective amount of an isolated peptide according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect of the present invention, a fusion protein according to the eleventh aspect of the present invention, or a composition according to the twelfth aspect of the present invention, wherein said application effectively enhances plant growth.

[0031] The fifteenth aspect of the present invention relates to a method for increasing the tolerance and resistance of plants to biotic stress factors. This method comprises: applying to a plant, plant seeds, or the site where the plant grows or is intended to grow an effective amount of an isolated peptide according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect of the present invention, a fusion protein according to the eleventh aspect of the present invention, or a composition according to the twelfth aspect of the present invention, wherein said application effectively increases the tolerance and resistance of the plant to biotic stress factors selected from insects, spiders, nematodes, weeds, and combinations thereof.

[0032] The sixteenth aspect of the present invention relates to a method for increasing the tolerance of plants to abiotic stresses. This method comprises: applying to a plant, plant seeds, or the site where the plant grows or is intended to grow an effective amount of an isolated peptide according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect of the present invention, a fusion protein according to the eleventh aspect of the present invention, or a composition according to the twelfth aspect of the present invention, wherein said application effectively increases the plant's tolerance to abiotic stressors selected from salt stress, water stress (including drought and flood), ozone stress, heavy metal stress, low temperature stress, high temperature stress, nutrient stress (phosphate, potassium, nitrogen deficiency), bleaching, and light-induced stress, and combinations thereof.

[0033] The seventeenth aspect of the present invention relates to a method for imparting anti-drying properties to cuttings taken from ornamental plants. This method comprises applying to the plant or the site where the plant grows an isolated peptide according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect of the present invention, a fusion protein according to the eleventh aspect of the present invention, or a composition according to the twelfth aspect of the present invention, wherein said application effectively imparts anti-drying properties to the cuttings taken from the ornamental plant.

[0034] The eighteenth aspect of the present invention relates to a method for conferring postharvest disease resistance or postharvest desiccation resistance on fruits or vegetables. This method comprises: applying to a plant containing the fruit or vegetable or the site where the plant grows an effective amount of an isolated peptide according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect of the present invention, a fusion protein according to the eleventh aspect of the present invention, or a composition according to the twelfth aspect of the present invention; or applying to harvested fruits or vegetables an effective amount of an isolated peptide or composition, wherein the application effectively confers postharvest disease resistance or desiccation resistance on the fruits or vegetables.

[0035] The nineteenth aspect of the present invention relates to a method for enhancing the ripening life of fruits or vegetables. This method comprises: applying to a plant containing the fruit or vegetable or the site where the plant grows an effective amount of an isolated peptide according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect of the present invention, a fusion protein according to the eleventh aspect of the present invention, or a composition according to the twelfth aspect of the present invention; or applying to harvested fruits or vegetables an effective amount of an isolated peptide or composition, wherein the application effectively enhances the ripening life of the fruits or vegetables.

[0036] A twentieth aspect of the invention relates to a method for regulating one or more biological signal transduction processes in a plant. This method comprises: applying to the plant or the site of plant growth an effective amount of an isolated peptide according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect of the invention, a fusion protein according to the eleventh aspect of the invention, or a composition according to the twelfth aspect of the invention, wherein said application effectively regulates one or more biochemical signal transduction processes.

[0037] The twenty-first aspect of the present invention relates to a DNA construct comprising a first nucleic acid molecule encoding a polypeptide according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspects of the present invention or a fusion protein according to the eleventh aspect of the present invention; and a promoter-effective nucleic acid molecule operatively coupled to the first nucleic acid molecule. This aspect of the present invention also covers recombinant expression vectors containing the DNA construct, recombinant host cells containing the DNA construct, and transgenic plants or plant seeds comprising recombinant plant cells of the present invention (containing the DNA construct).

[0038] A twenty-second aspect of the invention relates to a method for conferring disease resistance, enhancing plant growth, conferring tolerance and resistance to biotic stresses, conferring tolerance to abiotic stresses, or modulating plant biochemical signal transduction. This method includes providing a transgenic plant transformed with a DNA construct according to a twenty-first aspect of the invention; and growing the plant under conditions that effectively allow the DNA construct to express the peptide or fusion polypeptide to confer disease resistance, enhance plant growth, confer tolerance to biotic stresses, confer tolerance to abiotic stresses, or modulate biochemical signal transduction in the transgenic plant.

[0039] The twenty-third aspect of the present invention relates to a method for conferring desiccation resistance on cuttings taken from ornamental plants, postharvest disease resistance or postharvest desiccation resistance on fruits or vegetables, or enhancing the maturity lifespan of fruits or vegetables. The method includes providing a transgenic plant transformed with a DNA construct comprising a first nucleic acid molecule encoding a polypeptide or a fusion protein according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect of the present invention; and growing the plant under conditions that effectively allow the DNA construct to express the peptide or fusion polypeptide to confer desiccation resistance on cuttings taken from transgenic ornamental plants, postharvest disease resistance or desiccation resistance on fruits or vegetables taken from transgenic plants, or enhancing the maturity lifespan of fruits or vegetables taken from transgenic plants.

[0040] A twenty-fourth aspect of the present invention relates to a method for conferring disease resistance, enhancing plant growth, conferring tolerance and resistance to biotic stresses, conferring tolerance to abiotic stresses, or modulating biochemical signal transduction in plants. This method includes providing transgenic plant seeds transformed with a DNA construct according to a twenty-first aspect of the present invention; planting the transgenic plant seeds in soil; and propagating the transgenic plant from the transgenic plant seeds to allow the DNA construct to express the peptide or the fusion polypeptide to confer disease resistance, enhance plant growth, confer tolerance to biotic stresses, or confer tolerance to abiotic stresses.

[0041] A twenty-fifth aspect of the present invention relates to a method for conferring desiccation resistance on cuttings taken from ornamental plants, postharvest disease resistance or postharvest desiccation resistance on fruits or vegetables, or enhancing the maturity lifespan of fruits or vegetables. The method includes providing transgenic plant seeds transformed with a DNA construct according to a twenty-first aspect of the present invention; planting the transgenic plant seeds in soil; and propagating the transgenic plant from the transgenic plant seeds to allow the DNA construct to express the peptide or the fusion polypeptide to confer desiccation resistance on cuttings taken from transgenic ornamental plants, postharvest disease resistance or desiccation resistance on fruits or vegetables taken from transgenic plants, or enhancing the maturity lifespan of fruits or vegetables taken from transgenic plants.

[0042] By providing HR-stimulating peptides that exhibit enhanced solubility, stability, resistance to chemical degradation, or a combination of these properties, growers will have greater flexibility in preparing, treating, and delivering effective amounts of compositions containing these HR-stimulating peptides to their plants in the field or greenhouse. Simplifying the application process for growers will result in higher compliance and, consequently, improved outcomes regarding one or more of the following: disease resistance, enhanced growth, tolerance and resistance to biotic stresses, tolerance to abiotic stresses, desiccation resistance of cuttings from ornamental plants, postharvest disease resistance or desiccation resistance of fruits or vegetables harvested from the plant, and / or increased fruit or vegetable maturity lifespan of fruits or vegetables harvested from the plant. These and other benefits are described herein. Brief description of the attached diagram Figure 1 Solubility and stability tests of peptide P1 and its mutants in deionized water are shown. The following peptides are shown: P1 (SEQ ID NO: 4); P1-18A (SEQ ID NO: 44); P1-18K (SEQ ID NO: 45); and P1-18T (SEQ ID NO: 42). Curves 1* are normalized to 100% of P1 at day 1; 1** are raw P1 data.

[0043] Figure 2 Solubility and stability tests of peptide P1 and its mutants in 50 mM citrate (pH 5.6) are shown. The following peptides are shown: P1, P1-18A, P1-18K, and P1-18T. Curves 1* are normalized to 100% for P1 at day 1; 1** are raw P1 data.

[0044] Figure 3 Solubility and stability tests of peptide P1 and its mutants in 50 mM MES (pH 6) are shown. The following peptides are shown: P1, P1-18A, P1-18K, and P1-18T. Curves 1* are normalized to 100% for P1 at day 1; 1** are raw P1 data.

[0045] Figure 4 Solubility and stability tests of peptide P1 and its mutants in 50 mM MOPS (pH 6.5) are shown. The following peptides are shown: P1, P1-18A, P1-18K, and P1-18T.

[0046] Figure 5 Solubility and stability tests of peptide P1 and its mutants in 50 mM citrate (pH 7.2) are shown. The following peptides are shown: P1, P1-18A, P1-18K, and P1-18T.

[0047] Figure 6 Solubility and stability tests of peptide P1 and its mutants in 50 mM EDDS (pH 7.3) are shown. The following peptides are shown: P1, P1-18A, P1-18K, and P1-18T.

[0048] Figure 7 Solubility and stability tests of peptide P1 and its mutant are shown in 50 mM imidazole (pH 7.5). The following peptides are shown: P1, P1-18A, P1-18K, and P1-18T.

[0049] Figure 8 Solubility and stability tests of peptide P1 and its mutant in 50 mM EDTA (pH 8) are shown. The following peptides are shown: P1, P1-18A, P1-18K, and P1-18T.

[0050] Figure 9 Solubility and stability tests of peptide P1 and its mutant in phosphate (pH 8.0) are shown. The following peptides are shown: P1, P1-18A, P1-18K, and P1-18T.

[0051] Figure 10 Solubility and stability tests of peptide P1 and its mutants in 50 mM TES (pH 8.0) are shown. The following peptides are shown: P1, P1-18A, P1-18K, and P1-18T.

[0052] Figure 11 Solubility and stability tests of peptide P4 and its mutant in deionized water are shown. The following peptides are shown: P1, P1-18A, P1-18K, and P1-18T.

[0053] Figure 12 Solubility and stability tests of peptide P4 and its mutants in 50 mM citrate (pH 5.6) are shown. The following peptides are shown: P4 (SEQ ID NO: 5); P4-14A (SEQ ID NO: 136); P4-14D (SEQ ID NO: 137); P4-14K (SEQ ID NO: 138); P4-14Q (SEQ ID NO: 139); and P4-14S (SEQ ID NO: 6).

[0054] Figure 13 Solubility and stability tests of peptide P4 and its mutants in 50 mM MES (pH 6) are shown. The following peptides are shown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

[0055] Figure 14 Solubility and stability tests of peptide P4 and its mutants in 50 mM MOPS (pH 6.5) are shown. The following peptides are shown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

[0056] Figure 15 Solubility and stability tests of peptide P4 and its mutants are shown in 50 mM citrate (pH 7.2). The following peptides are shown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

[0057] Figure 16 Solubility and stability tests of peptide P4 and its mutants in 50 mM EDDS (pH 7.3) are shown. The following peptides are shown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

[0058] Figure 17 Solubility and stability tests of peptide P4 and its mutants in 50 mM imidazole (pH 7.5) are shown. The following peptides are shown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

[0059] Figure 18 Solubility and stability tests of peptide P4 and its mutants in 50 mM EDTA (pH 8) are shown. The following peptides are shown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

[0060] Figure 19 Solubility and stability tests of peptide P4 and its mutants in phosphate (pH 8) are shown. The following peptides are shown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

[0061] Figure 20 Solubility and stability tests of peptide P4 and its mutants in 50 mM TES (pH 8) are shown. The following peptides are shown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

[0062] Figure 21This study compares the stability of peptide P4 and its mutant in solutions of 30% isopropanol, 5 mM DTPA, 0.5% sodium thiosulfate, and 50 mM TES (pH 8). The following peptides are shown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S. Figure 22 This diagram shows a comparison of the stability of various peptides dissolved in 50 mM TES (pH 8). The following peptides are shown: P1 (SEQ ID NO: 4); P4-14S (SEQ ID NO: 6); P1-1S (SEQ ID NO: 109); P1-14S (SEQ ID NO: 110); P1-18Q (SEQ ID NO: 115); and P1-23P (SEQ ID NO: 118).

[0063] Figure 23 The solubility and stability of peptide P18 (SEQ ID NO: 83) in MES (pH 6), MOPS (pH 6.5), EDDS (pH 7.3), imidazole (pH 7.5) or EDTA (pH 8) are shown in the test.

[0064] Figure 24 Solubility and stability tests of peptide P18 and its mutant in 50 mM EDTA (pH 8) are shown. The following peptides are shown: P18 (SEQ ID NO: 83), P18-1 (SEQ ID NO: 163), and P18-4 (SEQ ID NO: 164).

[0065] Figure 25 Stability tests of peptides P19 (SEQ ID NO: 89) and P19-20L (SEQ ID NO: 90) mutants dissolved in 50 mM citrate (pH 7.2) are shown.

[0066] Figure 26 Stability tests of peptides P19 (SEQ ID NO: 89) and P19-20L (SEQ ID NO: 90) mutants dissolved in 50 mM TES (pH 8.0) are shown. Invention Details One aspect of the present invention relates to novel peptides having the ability to induce an allergic response in plants and promote active plant responses that provide one or more of the following properties: disease resistance, enhanced growth, tolerance and resistance to biotic stresses, tolerance to abiotic stresses, desiccation resistance of cuttings taken from ornamental plants, postharvest disease resistance or desiccation resistance of fruits or vegetables harvested from plants, and / or increased fruit or vegetable maturity lifespan of fruits or vegetables harvested from plants.

[0067] As used herein, naturally occurring amino acids are identified throughout using the common three-letter and / or single-letter abbreviations corresponding to the amino acid's common name, according to the following list: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). These abbreviations are accepted in the peptide field and recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Naturally occurring variants of amino acids include, but are not limited to, γ-glutamic acid (g-Glu) and isoaspartic acid (iso-Asp or isoD).

[0068] The term "amino acid" also includes analogues, derivatives, and homologs of any specific amino acid mentioned herein, as well as C-terminal or N-terminal protected amino acid derivatives (e.g., modified with N-terminal, C-terminal, or side-chain protecting groups, including but not limited to acetylation, formylation, methylation, amidation, esterification, PEGylation, and lipid augmentation). Non-naturally occurring amino acids are well known and can be introduced into the peptides of the present invention using solid-phase synthesis as described below. Furthermore, the term "amino acid" includes D- and L-amino acids. Therefore, amino acids identified herein by their names, three-letter or single-letter symbols, but not specifically identified as having a D or L configuration, should be understood as presumably having either a D or L configuration. In one embodiment, the peptide comprises all L-amino acids.

[0069] In some embodiments, the peptide is identified as consisting "of" the listed sequences, in which case the peptide comprises only the listed amino acid sequences, without any additional amino acids at its N-terminus or C-terminus. Where the listed sequences are in the form of common sequences, and one or more indicated X or Xaa residues can be any of one or more amino acids, then a peptide consisting of such listed sequences comprises multiple peptide sequences.

[0070] In some other embodiments, the identified peptide is "substantially composed" of the listed sequences, in which case the peptide comprises one or more of the listed amino acid sequences, optionally with one or more additional amino acids at its N-terminus and / or C-terminus, said additional amino acids not substantially altering one or more of the following properties: (i) the ability of the peptide to induce an allergic response in plants, (ii) the solubility of the peptide in water or aqueous solutions, (iii) the stability of the peptide dissolved in water or aqueous solutions at 50°C over a period of time (e.g., 3 weeks), and (iv) the stability of the peptide at 50°C in the presence of biocides (e.g., Proxel). ® Resistance of peptides to chemical degradation over a period of time (e.g., 3 weeks) in the presence of an aqueous buffer solution of GXL.

[0071] In short, peptide stability and resistance to chemical degradation can be assessed using peptide samples with initial purities of at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98%. For water stability, the peptide is dissolved directly in deionized water. For chemical degradation testing, the peptide is dissolved in an aqueous solution containing 50 mM pH buffer and 0.25% Proxel GXL. Exemplary pH buffers include, but are not limited to, (i) citrate pH 5.6; (ii) MES pH 6.2; (iii) MOPS pH 6.5; (iv) imidazole pH 7.0; (v) citrate pH 7.2; (vi) EDDS, pH 7.3; (vii) EDTA pH 8.0; (viii) sodium phosphate pH 8.0; or (ix) TES pH 8.0. First, the peptide was dissolved in an aqueous solution with a concentration of 0.5 mg / ml. The sample was incubated at 50°C to allow for accelerated degradation. The initial peptide sample was then removed, diluted 10x with water, and analyzed by reversed-phase HPLC. In short, 20... Samples were injected into the solvent stream of the HPLC instrument and analyzed on a C18 HPLC column (YMC ProPack C18, YMC, Japan or C18 Stablebond, Agilent Technologies, USA) using a triethylamine phosphate gradient in water / acetonitrile or a 0.1% TFA gradient in water / 0.1% TFA in acetonitrile to separate different peptide species. Eluted peptides were monitored by UV absorbance at 218 nm and quantified based on peak area. The peak area of ​​the initial peptide sample was processed into a standard for relative quantification in subsequent runs. Each peptide sample was measured and analyzed by HPLC as described above at regular intervals (e.g., 1, 3, 7, 10, 14, 17, and 21 days). If necessary to observe degradation (i.e., when the peptide exhibits high chemical stability), the protocol can be extended for several weeks to observe degradation. Quantifications of subsequent peptide runs are expressed as a percentage of the original (day 0) HPLC results.

[0072] Peptides that are at least partially soluble in water or aqueous solutions exhibit solubility greater than 0.1 mg / ml, preferably at least about 1.0 mg / ml, at least about 2.0 mg / ml, at least about 3.0 mg / ml, or at least about 4.0 mg / ml. In some embodiments, the peptides exhibit high solubility in water or aqueous solutions, with solubility of at least about 5.0 mg / ml, at least about 10.0 mg / ml, at least about 15.0 mg / ml, or at least about 20 mg / ml.

[0073] A peptide stable in water or an aqueous solution, when incubated at 50°C for a specified period of time, exhibits at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, or at least about 90% of the original peptide concentration. In some embodiments, the specified period of time is 3 days, 7 days, 14 days, 21 days, 28 days, one month, two months, or three months.

[0074] The chemically resistant peptides, when incubated at 50°C for a specified period of time, exhibit at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, or at least about 90% of the original peptide concentration. In some embodiments, the specified period of time is 3 days, 7 days, 14 days, 21 days, 28 days, one month, two months, or three months.

[0075] The property of a peptide to induce or not induce an allergic reaction after infiltration or application to plant tissue can be measured by applying the peptide, in dry powder or solution form, to plants, particularly but not exclusively to plant leaves. Application rates include 1-500 μg / ml (for liquid solutions) and 0.0001-0.5% (w / w, for powder applications). Exemplary applications of peptides in solution form are described in the accompanying examples. Plants exhibiting extensive, macroscopically visible cell death, accompanied by wilting and browning of affected tissues within 48 hours, are considered HR-positive (“HR+”). Plants not exhibiting significant wilting or macroscopically observable tissue death are considered HR-negative (“HR-”).

[0076] In some embodiments, a substantial change in one or more properties is intended to mean a change of less than 20%, less than 15%, less than 10%, or less than 5% in the listed properties when comparing a peptide having the one or more additional amino acids to another identical peptide without the one or more additional amino acids. In some embodiments, the number of additional amino acids at the N-terminus or C-terminus is up to 20 amino acids at one or both ends, up to 15 amino acids at one or both ends, up to 10 amino acids at one or both ends, up to 7 amino acids at one or both ends, up to 5 amino acids at one or both ends, or up to 3 amino acids at one or both ends. Further, if the listed sequences are in the form of a common sequence, where one or more indicated X or Xaa residues can be any of one or more amino acids, then a peptide substantially composed of such listed sequences comprises multiple peptide sequences, regardless of any additional variations in such sequences due to the presence of additional amino acids at its N-terminus and / or C-terminus.

[0077] In various embodiments of the invention, the disclosed peptide may include a hydrophilic amino acid sequence, for example, at the N-terminus or C-terminus of a designated peptide sequence. The hydrophilic amino acid sequence is at least 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length and includes hydrophilic amino acid residues that contribute to the hydrophilic sequence adjacent to the amino acid sequence of the designated peptide (i.e., the peptide that induces an active plant response). Different methods have been used in the art to calculate the relative hydrophobicity / hydrophilicity of amino acid residues and proteins (Kyte et al., “A Simple Method for Displaying the Hydropathic Character of a Protein”). J. Mol. Biol.157: 105-32(1982); Eisenberg D, “Three-dimensional Structure of Membrane and SurfaceProteins,” Ann. Rev. Biochem. 53: 595-623 (1984); Rose et al., "Hydrogen Bonding, Hydrophobicity, Packing, and Protein Folding," Annu. Rev. Biomol. Struct. 22:381-415 (1993); Kauzmann, “Some Factors in the Interpretation of ProteinDenaturation,” Advanced Protein Chem. 14: 1-63 (1959), which is incorporated herein by reference in its entirety. Any of these hydrophobicity scales may be used for the purposes of this invention; however, the Kyte-Doolittle hydrophobicity scale is probably the most commonly referenced. These hydrophilicity scales provide a ranking list of the relative hydrophobicity of amino acid residues. For example, amino acids that contribute to hydrophilicity include Arg(R), Lys(K), Asp(D), Glu(E), Gln(Q), Asn(N), and His(H), and (albeit to a lesser extent) Ser(S), Thr(T), Gly(G), Pro(P), Tyr(Y), and Trp(W). For example, polyglutamic acid sequences can be used to enhance the solubility of proteins and other drug molecules (Lilie et al., Biological Chemistry 394(8):995-1004(2013); Li et al., Cancer Research 58: 2404-2409 (1998), each of which is incorporated herein by reference in its entirety.

[0078] The "hydrophilicity index" of a protein or amino acid sequence is a numerical value representing its average hydrophilicity or hydrophobicity. A negative hydrophilicity index defines the hydrophilicity of the target amino acid sequence. The hydrophilicity index is directly proportional to the hydrophilicity of the target amino acid sequence; therefore, the more negative the index, the higher the hydrophilicity. In some embodiments, the hydrophilicity index of the added hydrophilic amino acid sequence is less than 0, -0.4, -0.9, -1.3, -1.6, -3.5, -3.9, or -4.5. In some embodiments, the hydrophilicity index of the resulting whole peptide will be less than 0.3, 0.2, 0.1, or 0.0, preferably less than -0.1, -0.2, -0.3, or -0.4, more preferably less than -0.5, -0.6, -0.7, -0.8, -0.9, or -1.0.

[0079] In the peptides of this invention, for the peptide as a whole or for added hydrophilic amino acid sequences, amino acids that contribute to the hydrophilicity index include Arg(R), Lys(K), Asp(D), Glu(E), Gln(Q), Asn(N), His(H), Ser(S), Thr(T), Gly(G), Pro(P), Tyr(Y), and Trp(W). Preferably, Asp(D), Glu(E), Gln(Q), Asn(N), or variants thereof. Exemplary variants include γ-glutamic acid (for Glu) and isoaspartic acid (or isoD) (for Asp).

[0080] As used herein, in this and other aspects of the invention, the term "hydrophobic amino acid" is intended to refer to an amino acid whose hydrophilicity index contributes to the hydrophobicity of a specified amino acid sequence. For a peptide as a whole or a particular amino acid sequence thereof, amino acids that contribute to the hydrophobicity index include Ile(I), Val(V), Leu(L), Phe(F), Cys(C), Met(M), and Ala(A). In some embodiments, the term "hydrophobic amino acid" may refer to any one of Ile(I), Val(V), Leu(L), Phe(F), Cys(C), Met(M), and Ala(A); or alternatively, any one of Ile(I), Val(V), Leu(L), Phe(F), and Ala(A). In some other embodiments, the term "hydrophobic amino acid" may refer to any one of Ile(I), Val(V), Leu(L), and Phe(F).

[0081] As used herein, “non-hydrophobic amino acid” is intended to refer to an amino acid that is hydrophilic (or non-hydrophobic) on a hydrophobicity scale identified above. The term generally refers to those amino acids that contribute to the hydrophilicity index for a peptide as a whole or for an added hydrophilic amino acid sequence.

[0082] In one aspect of the invention, the peptide comprises the following amino acid sequence: (L / I / V / F)-XX-(L / I / V / F)-(L / I)-XX-(L / I / V / F)-(L / I / V / A)-XX-(L / I)-(L / I / V / F) (SEQ ID NO: 93) The peptides described herein do not contain cysteine ​​and methionine; each X at positions 2 and 6 is optional and, when present, is any amino acid, including any naturally occurring amino acid; and each X at positions 3, 7, 10, and 11 is any amino acid, including any naturally occurring amino acid.

[0083] In a relevant aspect of the invention, the peptide comprises the amino acid sequence of SEQ ID NO: 93 (as shown above), wherein the peptide is free of cysteine ​​and methionine; each X at positions 2, 6 and 10 is optional and, when present, is any amino acid, including any naturally occurring amino acid; and each X at positions 3, 7 and 11 is any amino acid, including any naturally occurring amino acid.

[0084] According to one embodiment, one or more X's at positions 2, 6, and 10 are absent (i.e., the vacancy between the first hydrophobic amino acid and the first hydrophobic amino acid duplex is reduced from two to one amino acid residue and / or the vacancy between the first and second hydrophobic amino acid duplexes is reduced from two to one amino acid residue and / or the vacancy between the second and third hydrophobic amino acid duplexes is reduced from two to one amino acid residue). In this embodiment, it is anticipated that these peptides do not include amino acids at only one of positions 2, 6, and 10.

[0085] In an alternative implementation, X is present at both positions 2 and 6 (i.e., the vacancy between the hydrophobic amino acids is maintained at two amino acid residues at both positions).

[0086] In another embodiment, X is present at each of positions 2, 6 and 10 (i.e., the vacancy between the hydrophobic amino acids is maintained at two amino acid residues at each position).

[0087] In some embodiments, SEQ ID NO: 93 may also include an additional amino acid residue between the hydrophobic duplexes (as shown, two of L / I / V / F / A), and the additional amino acid may be any amino acid. In these embodiments, the vacancy preceding the first hydrophobic duplex is three amino acids, the vacancy between the first and second hydrophobic duplexes is three amino acids, the vacancy between the second and third hydrophobic duplexes is three amino acids, or a combination thereof.

[0088] In this embodiment, the peptide length is less than 100 amino acids, or optionally less than 90, 80, 70, 60, or about 50 amino acids. In some embodiments, the peptide length is between 13 and about 50 amino acids.

[0089] In the above embodiments, X (if present) at positions 2, 3, 6, 7, 10, and 11 of SEQ ID NO: 93 can be any amino acid, and in some embodiments these residues are actually hydrophilic. As mentioned above, these hydrophilic amino acids include Arg(R), Lys(K), Asp(D), Glu(E), Gln(Q), Asn(N), His(H), Ser(S), Thr(T), Gly(G), Pro(P), Tyr(Y), and Trp(W). Preferably, Asp(D), Glu(E), Gln(Q), Asn(N), or variants thereof. Exemplary variants include γ-glutamic acid (for Glu) and isoaspartic acid (or isoD) (for Asp).

[0090] In this embodiment, the isolated peptide is stable when dissolved in water; as described above, it is resistant to chemical degradation under aqueous conditions in the presence of a pH buffer and a biocide; and / or its solubility in aqueous solution is at least about 1.0 mg / ml.

[0091] Another aspect of the present invention relates to an isolated peptide having the following amino acid sequence: XXGISEKXXXXXXXXXXXXXXXX (SEQ ID NO: 1, shared by P1 / P4), where X at position 1 is optional and can be S, N, D, isoD, G, A or S; X at position 2 is optional and can be Q, E, g-glutamic acid, G, A or S; X at position 8 is Q, E, γ-glutamic acid, G, A, or S; X at position 9 is L, I, F, or V; X at position 10 is optional and can be D or isoD; X at position 11 is Q, E, γ-glutamic acid, G, A, or S; X at position 12 is M, L, I, or F; X at position 13 is M, L, or I; X at position 14 is optional and can be any hydrophilic amino acid, preferably C, S, T, A, D, isoD, K or Q; X at position 15 is Q, E, γ-glutamic acid, G, A, S, K, or I; X at position 16 is M, L, I, V, or F; X at position 17 is M, L, I, A, or V; X at position 18 is Q, E, γ-glutamic acid, G, A, S, M, T, or K; X at position 19 is A, D, isoD, S, V, T, K, R, E, γ-glutamic acid, H, or G; X at position 20 is M, L, or I; X at position 21 is M, L, I, V, S, or F; X at position 22 represents Q, E, γ-glutamic acid, G, A, and S. X at position 23 is P, Q, E, γ-glutamic acid, G, A, or S; and The isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence. In some embodiments, the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence.

[0092] In some embodiments, these peptides according to the second aspect of the invention also satisfy the structural features of the peptide defining SEQ ID NO: 93, in which case methionine and cysteine ​​residues are absent.

[0093] In this embodiment, for comparing the properties of the peptides of the present invention, the corresponding wild-type amino acid sequence is a polypeptide comprising or composed of the following amino acid sequence: NQGISEKQLDQLLTQLIMALLQQ (P1, SEQ ID NO: 4) or SQGISEKQLDQLLCQLIQALL (amino acids 1-21 of SEQ ID NO: 5, P4). P1 (SEQ ID NO: 4) is derived from the full-length protein of Xanthomonas harpin HpaG (Kim et al., “Mutational Analysis of Xanthomonas Harpin HpaG Identifies a Key Functional Region That Elicits the Hypersensitive Response in Nonhost Plants”). J. Bacteriol. 186(18):6239-6247(2004), which is incorporated herein by reference in its entirety. P4 (SEQ ID NO: 5) is derived from the rice bacterial blight pathogen ( Xanthomonas oryzae pv. oryzae ) full-length harpin (Ji et al., "Two Coiled-Coil Regions ofXanthomonas oryzae pv. Oryzae Harpin Differ in Oligomerization andHypersensitive Response Induction," Amino Acids 40:381-392 (2011), which is incorporated in its entirety by reference.

[0094] In this embodiment, the isolated peptide is stable when dissolved in water; as described above, it is resistant to chemical degradation under aqueous conditions in the presence of a pH buffer and a biocide; and / or its solubility in aqueous solution is at least about 1.0 mg / ml.

[0095] According to this second aspect, the peptide length is preferably less than about 100 amino acids, or optionally less than 90, 80, 70, 60, or about 50 amino acids. In some embodiments, the peptide length is between 23 and about 50 amino acids.

[0096] An exemplary family of peptides according to a second aspect of the present invention has the following amino acid sequence: SXGISEKXXDXXXXXXXXAXXXP (SEQ ID NO: 2, P4 shared), of which X at position 2 is Q, E, γ-glutamic acid, G, A, or S; X at position 8 is Q, E, γ-glutamic acid, G, A, or S; X at position 9 is L, A, D, isoD, I, V, or F; X at position 11 is Q, E, γ-glutamic acid, G, A, or S; X at position 12 is L, D, isoD, I, or F; X at position 13 is L, I, V, or F; X at position 14 can be any hydrophilic amino acid, preferably C, S or T, S or T or only S; X at position 15 is Q, E, γ-glutamic acid, G, A, S, K, or I. X at position 16 is L, A, I, V, M, or F. X at position 17 is I, S, or F. X at position 18 is Q, E, γ-glutamic acid, G, A, or S; X at position 20 is L, I, V, or F; X at position 21 is either L or F; and X at position 22 is Q, E, g-glutamic acid, G, A, or S.

[0097] In some embodiments, these peptides according to SEQ ID NO: 2 also satisfy the structural features defining the peptide of SEQ ID NO: 93, in which case methionine and cysteine ​​residues are absent. Therefore, in these embodiments, X at position 14 is S or T, preferably S.

[0098] Table 1 below identifies exemplary peptides that share a common structure with SEQ ID NO: 2 or originate from SEQ ID NO: 2 and satisfy SEQ ID NO: 93: Table 1: Peptide variants of peptide P4 (SEQ ID NO: 5) Table 1 (continued) The peptides selected in Table 1 include solubility tags indicated by italic printing, including SEEEEE, SEE, EEEEE, EE, RRRRRGG, KKKKKGG, GGKKKKKK, and GGRRRRR. This paper also anticipates including peptides with the sequences shown in Table 1 but lacking these specific solubility tags (or having different solubility tags).

[0099] As mentioned above, peptide P4 (SEQ ID NO: 5) is derived from the harpin of *Xanthomonas oryzae* pv. oryzae, the pathogen of rice bacterial blight. (Ji et al., "Two Coiled-Coil Regions of *Xanthomonas oryzae* pv. oryzae Harpin Differ in Oligomerization and Hypersensitive Response Induction,") Amino Acids 40:381-392 (2011), which is incorporated herein by reference in its entirety. Ji et al. disclosed a fragment of this harpin having the amino acid sequence SQGISEKQLDQLLCQLIQALL (i.e., amino acids 1-21 of SEQ ID NO: 5). In some embodiments, the isolated peptide comprising the amino acid sequence of SEQ ID NO: 5 is a peptide with a total length of less than about 100 amino acids (i.e., from 23 amino acids to about 100 amino acids). In some other embodiments, the isolated peptide consists essentially of SEQ ID NO: 5, wherein in another embodiment the isolated peptide consists entirely of SEQ ID NO: 5.

[0100] Another exemplary family of peptides according to the second aspect of the present invention has the following amino acid sequence: XXGISEKXLDXLLTXLIXALLXX (SEQ ID NO: 3, P1 common), where X at position 1 is N, D, isoD, G, A, or S; X at position 2 is Q, E, γ-glutamic acid, G, A, or S; X at position 8 is Q, E, γ-glutamic acid, G, A, or S; X at position 11 is Q, E, γ-glutamic acid, G, A, or S; X at position 15 is Q, E, γ-glutamic acid, G, A, or S; X at position 18 is M, T, K, E, γ-glutamic acid, G, A, or S; X at position 22 is Q, E, γ-glutamic acid, G, A, or S; and X at position 23 is Q, E, g-glutamic acid, G, A, or S.

[0101] In some embodiments, these peptides according to SEQ ID NO: 3 also satisfy the structural features defining the peptide of SEQ ID NO: 93, in which case methionine and cysteine ​​residues are absent. Therefore, in those embodiments, X at position 18 is T, K, E, γ-glutamic acid, G, A, or S.

[0102] In some embodiments, the peptide sharing the structure of SEQ ID NO: 3 has at least one residue different from Gln(Q) at positions 2, 8, 11, 15, 22, and 23 of SEQ ID NO: 3, namely E, γ-glutamic acid, G, A, or S. In some embodiments, two or more residues at positions 2, 8, 11, 15, 22, and 23 of SEQ ID NO: 3 are different from Gln(Q), including three, four, five, or all six of these residues being different from Gln(Q).

[0103] Table 2 below identifies exemplary peptides that share a common structure with SEQ ID NO: 3 or originate from SEQ ID NO: 3 and satisfy SEQ ID NO: 93: Table 2: Peptide variants of peptide P1 (SEQ ID NO: 4) The peptides selected for Table 2 include those with solubility tags indicated by italic printing, including SEEEEE. This document also anticipates peptides containing the sequences shown in Table 2, but lacking this specific solubility tag (or having a different solubility tag).

[0104] As mentioned above, the peptide in SEQ ID NO: 4 is derived from the harpin of *Xanthomonas oryzae* pv. oryzae, the causal agent of rice bacterial blight. (Ji et al., "Two Coiled-Coil Regions of *Xanthomonas oryzae* pv. oryzae Harpin Differ in Oligomerization and Hypersensitive Response Induction,") Amino Acids 40:381-392 (2011), which is incorporated in its entirety by reference.

[0105] Another aspect of the invention relates to an isolated peptide having the following amino acid sequence: (i) KPXDSXSXIAKLISXLIXSLLX (SEQ ID NO: 47, shared by P15b / P20), where X at position 3 is N, D, or isoD; X at position 6 is Q, E, γ-glutamic acid, G, A, or S; X at position 8 is N, D, or isoD; X at position 15 is optional and can be any amino acid; X at position 18 is M, E, γ-glutamic acid, G, A, S, T, or K; and X at position 22 is optional and can be Q, E, γ-glutamic acid, G, A, or S; or (ii) IAKLISXLIXSLLX (SEQ ID NO: 12, P15 / 20 min co-occurring), of which The X at position 7 is optional and can be any amino acid; X at position 10 is M, E, γ-glutamic acid, G, A, S, T, or K; and X at position 14 is optional and can be Q, E, g-glutamic acid, G, A or S.

[0106] In some embodiments, these peptides according to SEQ ID NO: 47 or 12 also satisfy the structural features defining the peptide of SEQ ID NO: 93, in which case methionine and cysteine ​​residues are absent. Therefore, in those embodiments, the X at position 15 of SEQ ID NO: 47 is different from M, and the X at position 18 of SEQ ID NO: 47 is E, γ-glutamic acid, G, A, S, T, or K. Similarly, the X at position 7 of SEQ ID NO: 12 is different from M, and the X at position 10 of SEQ ID NO: 47 is E, γ-glutamic acid, G, A, S, T, or K.

[0107] According to this third aspect, the length of the peptide is preferably less than about 100 amino acids, or optionally less than 90, 80, 70, 60, or about 50 amino acids. In some embodiments, the length of the peptide is between 20 and 44 amino acids.

[0108] In some embodiments, the peptide sharing the structure of SEQ ID NO: 47 has at least one residue at positions 6 and 22 of SEQ ID NO: 47 that is different from Gln(Q), namely E, γ-glutamic acid, G, A, or S. In some embodiments, both residues at positions 6 and 22 of SEQ ID NO: 47 are different from Gln(Q), or the residue at position 6 is different from Gln(Q), while the residue at position 22 is absent.

[0109] Table 3 below identifies exemplary peptides that share a common structure with SEQ ID NO: 47 or 12, or that originate from SEQ ID NO: 47 and 12 and satisfy a common structure of SEQ ID NO: 93: Table 3: Peptide variants common to peptide P15 / P20 (SEQ ID NO: 47 or 12) The peptides selected for Table 3 include those with solubility tags indicated by italic printing, including SEEEEE. This document also anticipates including peptides with the sequences shown in Table 3 but lacking this specific solubility tag (or having a different solubility tag).

[0110] In this implementation scheme, the corresponding wild-type amino acid sequence corresponds to the *Pseudomonas syringae* identified in Fan et al.'s PCT application WO 01 / 98501, which is incorporated herein by reference. Pseudomonas syringae Amino acids 52 to 96 of the HrpW sequence. For comparison of the properties of the peptides of the present invention, polypeptides comprising the amino acid sequence of SEQ ID NO: 48 or peptides consisting of the amino acid sequence of SEQ ID NO: 48 are intended to be used as references.

[0111] In some embodiments, the peptide comprises one or more mutations relative to the corresponding wild-type amino acid sequence of SEQ ID NO: 48. These one or more mutations include deletions or substitutions relative to SEQ ID NO: 48. In some embodiments, the one or more mutations improve the solubility, stability, and / or resistance to chemical degradation of the isolated peptide in aqueous solution relative to the corresponding wild-type amino acid sequence comprising SEQ ID NO: 48 or a polypeptide thereof. In this embodiment, the isolated peptide is stable when dissolved in water; resistant to chemical degradation under aqueous conditions in the presence of a pH buffer and a biocide, as described above; and / or has a solubility in aqueous solution of at least about 1.0 mg / ml.

[0112] In some embodiments, the isolated peptide containing the amino acid sequence of SEQ ID NO: 47 is a peptide with a total length between 20 and 36 amino acids and is substantially composed of SEQ ID NO: 49, SEQ ID NO: 63 or SEQ ID NO: 64, while in another embodiment, the isolated peptide is composed of SEQ ID NO: 49, SEQ ID NO: 63 or SEQ ID NO: 64.

[0113] In some embodiments, the isolated peptide comprising the amino acid sequence of SEQ ID NO: 47 is a peptide with a total length of less than 100 amino acids and the amino acid sequence includes SEQ ID NO: 65. In some embodiments, the amino acid sequence of the peptide consists essentially of SEQ ID NO: 65, while in another embodiment, the isolated peptide consists entirely of SEQ ID NO: 65.

[0114] Another aspect of the invention relates to an isolated peptide having the following amino acid sequence: (i) PSPXTXXLXXIVGXILXAXN (SEQ ID NO: 66, common to P6 / 6a), where X at position 4 is either F or Y; X at position 6 is Q, E, γ-glutamic acid, G, A, or S; X at position 7 is optional and, according to one embodiment, may be M, E, γ-glutamic acid, G, A, S, T, or K; or, according to another embodiment, may be L; X at position 9 is M, E, γ-glutamic acid, G, A, S, T, or K; X at position 10 is either H or N; X at position 14 is E, γ-glutamic acid, D, or isoD; X at position 17 is Q, E, γ-glutamic acid, G, A, or S; and X at position 19 is Q, E, γ-glutamic acid, G, A, or S; or (ii) XTXXLXXIVGXIL (SEQ ID NO: 135, P6 / 6a min co-occurring), of which X at position 1 is either F or Y; X at position 3 is Q, E, γ-glutamic acid, G, A, or S; X at position 4 is optional and, according to one embodiment, may be M, E, γ-glutamic acid, G, A, S, T, or K; or, according to another embodiment, may be L; X at position 6 is M, E, γ-glutamic acid, G, A, S, T, or K; X at position 7 is either H or N; and X at position 11 is E, γ-glutamic acid, D, or isoD; The isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence. In some embodiments, the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence.

[0115] The wild-type sequence corresponds to amino acid 85-105 of the full-length harpin of *Xanthomonas oryzae* pv. *Oryzae* (Ji et al., "Two Coiled-Coil Regions of *Xanthomonas oryzae* pv. *Oryzae* Harpin Differ in Oligomerization and Hypersensitive Response Induction") Amino Acids 40:381-392 (2011), which is incorporated herein by reference in its entirety. This contrasting wild-type sequence is a peptide consisting of the amino acid sequence PSPFTQMLMHIVGEILQAQNG (SEQ ID NO: 153).

[0116] In some embodiments, the peptide according to this aspect of the invention is not composed of the amino acid sequence PSPFTQMLMHIVGEILQAQN (P6a, SEQ ID NO: 67) corresponding to amino acid 85-104 of the full-length harpin of *Xanthomonas oryzae* pv. *Oryzae* (Ji et al., “Two Coiled-Coil Regions of *Xanthomonas oryzae* pv. *Oryzae* Harpin Differ in Oligomerization and Hypersensitive Response Induction”). Amino Acids 40:381-392 (2011), which is incorporated in its entirety by reference.

[0117] In some embodiments, the peptide in this respect does not contain the peptide sequence of motif 2 as described in U.S. Patent No. 8,440,881, which is defined as (P / A / V)S(P / Q / A)(F / L / Y)TQ(M / A)LM(H / N / Q)IV(G / M)(E / D / Q), SEQ ID NO: 154. For example, when all other aligned residues match the sequence of motif 2, the peptide according to SEQ ID NO: 66 does not include a peptide having M / A / T at position 7; or when all other aligned residues match the sequence of motif 2, the peptide according to SEQ ID NO: 66 does not include a peptide having H / N at position 10; or when all other aligned residues match the sequence of motif 2, the peptide according to SEQ ID NO: 66 does not include a peptide having E / D at position 14. Similarly, when all other aligned residues match the sequence of motif 2, the peptide according to SEQ ID NO: 135 does not include the peptide with M / A / T at position 4; or when all other aligned residues match the sequence of motif 2, the peptide according to SEQ ID NO: 135 does not include the peptide with H / N at position 7; or when all other aligned residues match the sequence of motif 2, the peptide according to SEQ ID NO: 135 does not include the peptide with E / D at position 11.

[0118] In some embodiments, the peptide comprises one or more mutations relative to the corresponding wild-type amino acid sequence of SEQ ID NO: 153. These one or more mutations include deletions or substitutions relative to SEQ ID NO: 153. In some embodiments, the one or more mutations improve the solubility, stability, and / or resistance to chemical degradation of the isolated peptide in aqueous solution relative to the corresponding wild-type amino acid sequence comprising SEQ ID NO: 153 or a polypeptide thereof.

[0119] The peptide length is preferably less than about 100 amino acids, or optionally less than 90, 80, 70, 60, or about 50 amino acids. In some embodiments, the peptide length is between 19 and 50 amino acids.

[0120] In some embodiments, the peptides according to SEQ ID NO: 66 and 135 also satisfy the structural features defining the peptide of SEQ ID NO: 93, in which case methionine and cysteine ​​residues are absent. For example, in the methionine-free peptide according to SEQ ID NO: 66, X at position 7, if present, is E, γ-glutamic acid, G, A, S, T, K, or L; and X at position 9 is E, γ-glutamic acid, G, A, S, T, or K. Similarly, for the methionine-free peptide according to SEQ ID NO: 135, X at position 4, if present, is E, γ-glutamic acid, G, A, S, T, K, or L; and X at position 6 is E, γ-glutamic acid, G, A, S, T, or K.

[0121] In some embodiments, peptides sharing the structure of SEQ ID NO: 66 have at least one residue at positions 6, 17, and 19 of SEQ ID NO: 66 that is different from Gln(Q), namely E, γ-glutamic acid, G, A, or S. In some embodiments, two or three residues at positions 6, 17, and 19 of SEQ ID NO: 66 are different from Gln(Q). Similarly, for peptides sharing the structure of SEQ ID NO: 135, according to one embodiment, these peptides have a residue at position 6 that is different from Gln(Q), namely E, γ-glutamic acid, G, A, or S.

[0122] Table 4 below identifies exemplary peptides that share a common structure with SEQ ID NO: 66 or 135, or that originate from one of SEQ ID NO: 66 and 135 and satisfy SEQ ID NO: 93: Table 4: Peptide variants common to peptide P6 / P6b (SEQ ID NO: 66 or 135) The peptides selected in Table 4 include solubility tags indicated by italic printing, including SEE, SEEE, and SEEEEE. This document also anticipates including peptides with the sequences shown in Table 4 but lacking these specific solubility tags (or having different solubility tags).

[0123] Another aspect of the present invention relates to a peptide having the following amino acid sequence: (i) XXXXXXXXXXX(L / M)XXLLXXLLXXLLXXX (SEQ ID NO: 18, P17 / 18), where X at position 1 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 2 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 3 can be any amino acid, but is preferably P, Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 4 can be any amino acid, but is preferably I, Q, S, E, γ-glutamic acid, A, T, G, D, N, isoD, K, or R; X at position 5 can be any amino acid, but D, isoD, S, E, γ-glutamic acid, A, T, G, N, Q, K or R are preferred. X at position 6 can be any amino acid, but is preferably R, Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, or K; X at position 7 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 8 can be any amino acid, but is preferably T, Q, S, E, γ-glutamic acid, A, G, D, isoD, N, K, or R; X at position 9 can be any amino acid, but is preferably I, Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 10 can be any amino acid, but is preferably E, γ-glutamic acid, Q, S, A, T, G, D, isoD, N, K or R; X at position 11 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 13 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 14 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 17 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 18 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 21 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N or R; X at position 22 can be any amino acid, but is preferably S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 25 can be any amino acid, but is preferably S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 26 can be any amino acid, but is preferably P, S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K, or R; and X at position 27 can be any amino acid, but is preferably Q, S, A, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; or (ii) (L / M)XXLLXXLLXXLL (SEQ ID NO: 25, P17 / 18 min total), of which X at position 2 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 3 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 6 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 7 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 10 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or R; and X at position 11 can be any amino acid, but is preferably S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R.

[0124] In some embodiments, the peptide comprises one or more mutations relative to the corresponding wild-type amino acid sequence of *Pyracantha fortuneana* HrpW. These one or more mutations include deletions or substitutions relative to the wild-type HrpW sequence. In some embodiments, the one or more mutations improve the solubility, stability, and / or resistance to chemical degradation of the isolated peptide in aqueous solution relative to the corresponding wild-type amino acid sequence of *Pyracantha fortuneana* HrpW or a polypeptide composed thereof.

[0125] Based on this, and by referencing Fan et al.'s PCT application WO 01 / 98501, which was incorporated in its entirety, HrpW was identified.Ea The two allergic response-excited domains. The first domain extends from amino acid 5 to amino acid 64, specifically from HrpW. Ea The amino acid 31 extends to amino acid 57. The second domain extends from amino acid 103 to amino acid 146, particularly from HrpW. Ea Amino acid 116 extends to amino acid 140. Although Fan et al. described it this way, this reference only identified HrpW. Ea A single peptide fragment, which is a peptide composed of 10 to 59 amino acids.

[0126] The wild-type sequence corresponds to amino acids 10 to 59 of the full-length *Pyrus pyrifolia* HrpW sequence identified in PCT application WO 01 / 98501 by Fan et al., which is hereby incorporated by reference in its entirety. For the purpose of comparing the properties of the peptides of the present invention, a peptide consisting of amino acids 10 to 59 of *Pyrus pyrifolia* HrpW is intended to be used as a reference.

[0127] In some embodiments, the peptide in this respect is not composed of the amino acid sequence TSSSPGLFQSGGDNGLGGHNANSALGQQPIDRQTIEQMAQLLAELLKSLL (SEQ ID NO: 162) corresponding to amino acids 10 to 59 of the full-length pear fire blight pathogen HrpW (Fan et al. PCT application WO 01 / 98501, which is incorporated herein by reference in its entirety).

[0128] In some embodiments, the peptide comprises one or more mutations relative to the corresponding wild-type amino acid sequence of SEQ ID NO: 162. These one or more mutations include deletions or substitutions relative to SEQ ID NO: 162. In some embodiments, the one or more mutations improve the solubility, stability, and / or resistance to chemical degradation of the isolated peptide in aqueous solution relative to the corresponding wild-type amino acid sequence comprising SEQ ID NO: 162 or a polypeptide thereof.

[0129] According to this fourth aspect, the length of the peptide is preferably less than about 100 amino acids, or optionally less than 90, 80, 70, 60, or about 50 amino acids. In some embodiments, the length of the peptide is between 13 and 50 amino acids, or even between 13 and 40 amino acids.

[0130] In some embodiments, the peptides according to SEQ ID NO: 18 and 25 also satisfy the structural features defining the peptide of SEQ ID NO: 93, in which case methionine and cysteine ​​residues are absent. For example, when the peptide containing SEQ ID NO: 18 does not contain a methionine amino acid residue, the amino acid at position 12 is L. Similarly, when the peptide containing SEQ ID NO: 25 does not contain a methionine amino acid residue, the amino acid at position 1 is L.

[0131] In some other embodiments, one or more of amino acids 1 to 11 and / or 25 to 27 are absent in the isolated peptide of SEQ ID NO:18. For example, peptides lacking amino acids 25 to 27 exhibit increased stability relative to the wild-type sequence.

[0132] Table 5 below identifies exemplary peptides that share a common structure with or are derived from one of SEQ ID NO: 18 or 25, or that satisfy a common structure of SEQ ID NO: 93: Table 5: Peptide variants shared by peptide P17 / P18 (SEQ ID NO: 18 or 25) The peptides selected for Table 5 include solubility tags indicated by italic printing, including SEEEEE. This document also anticipates including peptides with the sequences shown in Table 5 but lacking these specific solubility tags (or having different solubility tags).

[0133] In this embodiment, the wild-type amino acid sequence corresponds to amino acids 10 to 59 of the *Pyrus pyrifolia* HrpW sequence identified thereunder in PCT application WO 01 / 98501 by Fan et al., which is hereby incorporated in its entirety by reference. For comparison of the properties of the peptides of the present invention, a peptide consisting of amino acids 10 to 59 of *Pyrus pyrifolia* HrpW is intended to be used as a reference.

[0134] Another aspect of the present invention relates to peptides having the following amino acid sequence: XLXX(L / M)LXLIXX(L / I / V / F / M)(L / I / V / F / M) (SEQ ID NO: 26, P19 common), where X at position 1 is optional and can be L, I, V, F or M; X at position 3 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or R; X at position 4 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 7 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or R; X at position 10 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K, or R; and X at position 11 can be any amino acid, but is preferably R, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or K.

[0135] As mentioned above, HrpW was identified by referencing Fan et al.'s PCT application WO 01 / 98501, which was incorporated in its entirety. Ea The two allergic response excitation domains, one of which extends from amino acid 103 to amino acid 146, particularly from HrpW Ea Amino acid 116 extends to amino acid 140. Although Fan et al. described it this way, this reference did not identify HrpW containing this domain. Ea peptide fragments.

[0136] The wild-type sequence corresponds to amino acids 116 to 140 of the full-length *Pyrus pyrifolia* HrpW sequence identified in PCT application WO 01 / 98501 by Fan et al., which is hereby incorporated by reference in its entirety. For comparison of the properties of the peptides of the present invention, a peptide consisting of amino acids 116 to 140 of *Pyrus pyrifolia* HrpW is intended to be used as a reference.

[0137] In some embodiments, the peptide in this respect is not composed of the amino acid sequence ITPDGQGGGQIGDNPLLKAMLKLIA (SEQ ID NO: 89) corresponding to amino acid 116 to 140 of the full-length pear fire blight pathogen HrpW (PCT application WO 01 / 98501 by Fan et al., which is incorporated herein by reference in its entirety).

[0138] In some embodiments, the peptide comprises one or more mutations relative to the corresponding wild-type amino acid sequence of SEQ ID NO: 89. These one or more mutations include deletions or substitutions relative to SEQ ID NO: 89. In some embodiments, the one or more mutations improve the solubility, stability, and / or resistance to chemical degradation of the isolated peptide in aqueous solution relative to the corresponding wild-type amino acid sequence comprising SEQ ID NO: 89 or a polypeptide thereof.

[0139] The peptide length is preferably less than about 100 amino acids, or optionally less than 90, 80, 70, 60, or about 50 amino acids. In some embodiments, the peptide length is between 18 and 50 amino acids.

[0140] Table 6 below identifies exemplary peptides that share a common structure with or originate from SEQ ID NO: 26 and satisfy the common structure of SEQ ID NO: 93: Table 6: Common peptide variants of peptide P19 (SEQ ID NO: 26) The peptides selected in Table 6 include those with solubility tags indicated by italic printing, including SEEEEE. This document also anticipates including peptides with sequences shown in Table 6 but lacking that specific solubility tag (or having a different solubility tag).

[0141] Some peptides in Table 6 also satisfy the structural features of the peptide defined by SEQ ID NO: 93, in which case methionine and cysteine ​​residues are absent. When these peptides also satisfy the restrictions of SEQ ID NO: 93, amino acid residue 1 of SEQ ID NO: 26, if present, is L, I, V, or F; amino acid 5 of SEQ ID NO: 26 is L; and amino acids 12 and 13 of SEQ ID NO: 26 are independently L, I, V, or F.

[0142] Another aspect of the present invention relates to a peptide having the following amino acid sequence: (i) XXXXXXLXXLLXXLVXLLK (SEQ ID NO: 13, P14d common), where X at position 1 can be: Q, N, D, E, g-glutamic acid, isoD, or S; X at position 2 could be: D, E, γ-glutamic acid, or isoD; X at position 3 can be: P, D, E, isoD, or γ-glutamic acid; X at position 4 can be M, A, S, D, E, isoD, or γ-glutamic acid. X at position 5 can be Q, E, or g-glutamic acid; X at position 6 can be A, E, or g-glutamic acid; X at position 8 can be M, L, E, Q, D, N, G, A, S, isoD, or γ-glutamic acid; X at position 9 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 12 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 13 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; and X at position 16 can be K, Q, N, E, D, R, G, A, or S; or (ii) LXXLLXXLVXLLK (SEQ ID NO: 14, P14d min common), where X at position 2 can be M, L, E, Q, D, N, G, A, S, isoD, or γ-glutamic acid; X at position 3 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 6 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 7 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; and X at position 10 can be K, Q, N, E, D, R, G, A, or S.

[0143] In some embodiments, the peptide is relative to Ralstonia solanacearum (Ralstonia solanacearum). Ralstonia solanacearum (formerly known as Ralstonia solanacearum) Pseudomonas solanacearum The corresponding wild-type amino acid sequence of PopA includes one or more mutations. These one or more mutations include deletions or substitutions relative to the wild-type PopA sequence. In some embodiments, the one or more mutations improve the solubility, stability, and / or resistance to chemical degradation of the isolated peptide in aqueous solution relative to the corresponding wild-type amino acid sequence of Ralstonia solanacearum PopA or a polypeptide composed therefrom.

[0144] The wild-type sequence corresponds to amino acids 92 to 125 of the PopA sequence of Ralstonia solanacearum (formerly known as Ralstonia solanacearum) identified in PCT application WO 01 / 98501 by Fan et al., which is hereby incorporated in its entirety by reference. For the purpose of comparing the properties of the peptides of the present invention, the wild-type peptide of Fan et al., consisting of amino acids 92 to 125 of PopA from Ralstonia solanacearum, is intended to be used as a reference.

[0145] In some embodiments, the peptide in this aspect is not composed of the amino acid sequence QAPQSANKTGNVDDANNQDPMQALMQLLEDLVKL (SEQ ID NO: 174) corresponding to amino acids 92 to 125 of Ralstonia solanacearum PopA (see PCT application WO 01 / 98501 by Fan et al., which is incorporated herein by reference in its entirety).

[0146] The peptide length is preferably less than about 100 amino acids, or optionally less than 90, 80, 70, 60, or about 50 amino acids. In some embodiments, the peptide length is between 12 and 50 amino acids.

[0147] Table 7 below identifies exemplary peptides that share a common structure with SEQ ID NO: 13 or 14, or that originate from SEQ ID NO: 13 and satisfy a common structure with SEQ ID NO: 93: Table 7: Peptide variants of peptide P14d (SEQ ID NO: 13) The peptides selected for Table 7 include solubility tags indicated by italic printing, including SEEEEE. This document also anticipates including peptides with the sequences shown in Table 7 but lacking these specific solubility tags (or having different solubility tags).

[0148] It is noteworthy that the C-terminal lysine residue appears to be essential for HR induced by the p14d variant. This is a slight deviation from the standard sequence of SEQ ID NO: 93. Without being bound by belief, it is believed that the C-terminal lysine may be essential due to a single hydrophilic amino acid between two hydrophobic duplex sequences within the p14d variant (LVKLL).

[0149] Some peptides in this respect also satisfy the structural features defining the peptide of SEQ ID NO: 93, in which case methionine and cysteine ​​residues are absent. For example, for the peptide containing SEQ ID NO: 13, amino acid residue 4 of SEQ ID NO: 13 is A, S, D, isoD, E, or γ-glutamic acid, and amino acid residue 8 of SEQ ID NO: 13 is L, E, γ-glutamic acid, Q, D, isoD, N, G, A, or S. Similarly, for the peptide containing SEQ ID NO: 14, the amino acid residue at position 2 is L, E, γ-glutamic acid, Q, D, isoD, N, G, A, or S.

[0150] Another aspect of the present invention relates to a peptide having the following amino acid sequence: (i) LXXL(L / M)XILXXLV (SEQ ID NO: 16, P25 total) X at position 2 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 3 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 6 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 9 can be E, γ-glutamic acid, D, isoD, Q, N, T, S, A, or G; and X at position 10 can be A, G, S, T, E, γ-glutamic acid, D, isoD, Q, or N; or (ii) LXXVLXXL(L / M)XILXXLV (SEQ ID NO: 17, P25 total) of which X at position 2 can be T, S, A, G, D, isoD, E, γ-glutamic acid, Q, or N; X at position 3 can be G, T, S, A, D, isoD, E, γ-glutamic acid, Q, or N; X at position 6 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 7 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 10 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 13 can be E, g-glutamic acid, D, isoD, Q, N, T, S, A, or G; X at position 14 can be A, G, S, T, E, γ-glutamic acid, D, isoD, Q, or N; and The V at position 16 is optional.

[0151] In some embodiments, the peptide includes one or more mutations relative to the corresponding wild-type amino acid sequence of Ralstonia solanacearum (formerly known as Ralstonia solanacearum) PopA. These one or more mutations include deletions or substitutions relative to the wild-type PopA sequence. In some embodiments, the one or more mutations improve the solubility, stability, and / or resistance to chemical degradation of the isolated peptide in aqueous solution relative to the corresponding wild-type amino acid sequence comprising Ralstonia solanacearum PopA or a polypeptide thereof.

[0152] The wild-type sequence corresponds to amino acids 206 to 260 of the PopA sequence of Ralstonia solanacearum (formerly known as Ralstonia solanacearum) identified as an allergic reaction domain in PCT application WO 01 / 98501 by Fan et al., which is hereby incorporated in its entirety by reference. For comparison of the properties of the peptides of the present invention, the wild-type peptide of Fan et al., consisting of amino acids 206 to 260 of PopA from Ralstonia solanacearum, is intended to be used as a reference.

[0153] In some embodiments, the peptide in this aspect is not composed of the amino acid sequence NGADGGNGVNGNQANGPQNAGDVNGANGADDGSEDQGGLTGVLQKLMKILNALVQ (SEQ ID NO:179) corresponding to amino acids 206 to 260 of PopA in Ralstonia solanacearum (see PCT application WO 01 / 98501 by Fan et al., which is incorporated herein by reference in its entirety).

[0154] The peptide length is preferably less than about 100 amino acids, or optionally less than 90, 80, 70, 60, or about 50 amino acids. In some embodiments, the peptide length is between 12 and 50 amino acids.

[0155] Table 8 below identifies exemplary peptides that share a common structure with one of SEQ ID NO: 16 or 17, or that originate from SEQ ID NO: 16 or 17 and satisfy a common structure of SEQ ID NO: 93: Table 8: Peptide variants of peptides P2 (SEQ ID NO: 180) and P25 (SEQ ID NO: 182) [*]=N terminal sequence NGADGGNGVNGNQANGPQNAGDVNG The peptides selected for Table 8 include solubility tags indicated by italic printing, including SEEEEE. This document also anticipates including peptides with the sequences shown in Table 8 but lacking these specific solubility tags (or having different solubility tags).

[0156] Notably, many of these derived peptides in Table 8 include repetitive LT sequences not observed in the wild-type sequence. However, it should be noted that these sequences require a larger hydrophobic sequence compared to SEQ ID NO: 93 to elicit an allergic reaction. Without being bound by belief, this is believed to be due to the presence of the amino acid valine instead of leucine in the sequence and the presence of only a single hydrophilic amino acid (LLKIL) between the hydrophobic duplexes. While these changes are detrimental to HR, their effects can be reversed by adding additional hydrophobic residues (…KIL versus…KILEALV or…KILNALV) to the C-terminus of the peptide.

[0157] Some peptides in this respect also satisfy the structural features defining the peptide of SEQ ID NO: 93, in which case methionine and cysteine ​​residues are absent. For example, for the peptide containing SEQ ID NO: 16, amino acid residue 5 is L; and for the peptide containing SEQ ID NO: 17, amino acid residue 9 is L.

[0158] Another aspect of the present invention relates to a peptide having the following amino acid sequence: (i) (L / M)XXLLX(L / M)FXXI(L / M)XX (SEQ ID NO: 15, P3min total) where X at position 2 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 3 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 6 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 9 can be E, γ-glutamic acid, D, isoD, Q, N, T, S, A, or G; X at position 10 can be A, G, S, T, E, γ-glutamic acid, D, isoD, Q, or N; X at position 13 can be Q, N, E, γ-glutamic acid, D, isoD, T, S, A, or G; and X at position 14 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G.

[0159] In some embodiments, the peptide comprises one or more mutations relative to the corresponding wild-type amino acid sequence of *HrpN* pear fire blight pathogen. These one or more mutations include deletions or substitutions relative to the wild-type HrpN sequence. In some embodiments, the one or more mutations improve the solubility, stability, and / or resistance to chemical degradation of the isolated peptide in aqueous solution relative to the corresponding wild-type amino acid sequence of *HrpN* pear fire blight pathogen or a polypeptide composed thereof.

[0160] The wild-type sequence corresponds to amino acids 137 to 180 or 150 to 180 of the *HrpN* sequence of *Pyrus pyrifolia* identified in U.S. Patent No. 7,132,525 to Wei et al., which is incorporated herein by reference in its entirety. HrpN peptides containing amino acids 137 to 180 were identified as anaphylactic triggering fragments, while HrpN peptides containing amino acids 150 to 180 could not be expressed or tested. For comparison of the properties of the peptides of the present invention, the wild-type peptides of Wei et al., consisting of amino acids 137 to 180 or 150 to 180 of *HrpN*, are intended to be used as a reference.

[0161] In some embodiments, the peptide in this aspect is not composed of the S-terminus of amino acids 137 to 180 corresponding to HrpN of pear fire blight pathogen. 137 TSQNDDSTSGTDS 150TSDSSDPMQQLLKMFSEIMQSLFGDGQDGT 180 The amino acid sequence of (SEQ ID NO: 230) (see U.S. Patent No. 7,132,525 to Wei et al., which is incorporated herein by reference in its entirety) or the peptide composition of 31 amino acids corresponding to amino acids 150 to 180.

[0162] The peptide length is preferably less than about 100 amino acids, or optionally less than 90, 80, 70, 60, 50, 40, or 30 amino acids. In some embodiments, the peptide length is between 12 and 30 amino acids.

[0163] Table 9 below identifies exemplary peptides that share a common structure with SEQ ID NO: 15 or originate from SEQ ID NO: 15 and satisfy SEQ ID NO: 93: Table 9: Peptide variants common to peptide P3 (SEQ ID NO: 15) The peptides selected in Table 9 include solubility tags indicated by italic printing, including SEEE, SEEEEE, and EEEE. This document also anticipates including peptides with sequences shown in Table 9 but lacking these specific solubility tags (or possessing different solubility tags).

[0164] It is noteworthy that the smallest P3 sequence requires a longer sequence than the smallest HR box sequence of SEQ ID NO: 93. Without being bound by belief, this is believed to be due to the presence of two phenylalanine residues in the hydrophobic sequence.

[0165] Some peptides in this respect also satisfy the structural features defining the peptide of SEQ ID NO: 93, in which case methionine and cysteine ​​residues are absent. For example, for the peptide containing SEQ ID NO: 15, amino acid residues 1, 7, and 12 are L.

[0166] Based on the publicly disclosed common sequence (SEQ ID NO: 93), it is possible to generate novel peptide sequences with predicted HR activity that deviate significantly from bacterial protein sequences. These peptides may contain hydrophilic residues optimized for maximum solubility and chemical stability. In a preferred embodiment, these hydrophilic residues are glutamic acid. Lysine and arginine are also possible choices; however, large amounts of these residues will induce toxic reactions in plants.

[0167] In addition to the aforementioned peptides modeled (and modified) based on sequences naturally present in large HR-excitation proteins, the present invention also contemplates fully synthetic peptides satisfying the common sequence of SEQ ID NO: 93. Ideally, these synthetic peptides comprise a number of strongly hydrophilic amino acids spanning the hydrophobic residues specified by SEQ ID NO: 93. Exemplary synthetic peptides are listed in Table 10 below. These peptides contain the essential hydrophobic peptides associated with HR excitation. The inserted hydrophilic residues are selected for maximum solubility, preferably having charged amino acids. Uncharged amino acids may be used, but a large proportion of uncharged amino acids can cause the resulting peptides to aggregate in solution and form precipitates or gels. Glutamic acid is preferred due to its superior chemical stability compared to aspartic acid. Although lysine and arginine have even better solubility characteristics, polycations produce toxic reactions in the test plants. Therefore, sequences rich in arginine, such as P30-1 (SEQ ID NO: 211), should be avoided.

[0168] Table 10: Other HR box peptides The peptides selected for Table 10 include solubility tags indicated by italic printing, including SEE, EE, DD, or EEE. This document also anticipates including peptides with sequences shown in Table 10 but lacking these specific solubility tags (or having different solubility tags).

[0169] The isolated peptides of this invention can also be presented in the form of fusion peptides, which further include a second amino acid sequence coupled to the peptide of this invention via a peptide bond. The second amino acid sequence can be a purification tag, such as polyhistidine (His6-), glutathione S-transferase (GST-), or maltose-binding protein (MBP-), which facilitates purification but can subsequently be removed, i.e., cleaved from the peptide after recovery. A protease-specific cleavage site or a chemically specific cleavage site (i.e., in a cleavable linker sequence) can be introduced between the purification tag and the desired peptide. Protease-specific cleavage sites are well-known in the literature and include, but are not limited to, the enterokinase-specific cleavage site (Asp)4-Lys cleaved after lysine; the factor Xa-specific cleavage site Ile-(Glu or Asp)-Gly-Arg cleaved after arginine; the trypsin-specific cleavage site cleaved after Lys and Arg; and the Genenase™ I-specific cleavage site Pro-Gly-Ala-Ala-His-Tyr. Chemicals and their specific cleavage sites include, but are not limited to, cyanogen bromide (CNBr), which cleaves at methionine (Met) residues; BNPS-skatole, which cleaves at tryptophan (Trp) residues; formic acid, which cleaves at the aspartic-proline (Asp-Pro) peptide bond; hydroxylamine, which cleaves at the asparagine-glycine (Asn-Gly) peptide bond; and 2-nitro-5-cyanothiobenzoic acid (NTCB), which cleaves at the cysteine ​​(Cys) residue (see Crimmins et al., “Chemical Cleavage of Proteins in Solution,”). Curr. Protocol. Protein Sci. (Chapter 11: Unit 11.4 (2005), which is incorporated herein by reference in its entirety). To use one of these cleavage methods, it may be necessary to remove unwanted cleavage sites from the desired peptide sequence through mutation. For example, to achieve compatibility with trypsin, p4-7E-cR (SEQ ID NO: 40) has been mutated: the lysine residue at position 7 is mutated to glutamic acid and a C-terminal arginine is added to present a product theoretically cleaved by trypsin. Similarly, p19-5 (SEQ ID NO: 168) contains the sequence 'NP', which can be cleaved under acidic conditions. Mutating these residues to 'DE' in p19-5a (SEQ ID NO: 169) prevents this particular cleavage mechanism. The desired peptide product can be further purified to remove the purification tag of the cleavage.

[0170] The isolated peptides of the present invention may also be presented in the form of fusion peptides comprising multiple peptide sequences of the present invention linked together by linker sequences, which may or may not be in the form of cleavable amino acid sequences of the type described above. Such multimeric fusion proteins may or may not include a purification tag. In one embodiment, each monomeric sequence may include a purification tag linked to the peptide of the present invention via a first cleavable peptide sequence; and several monomeric sequences may be linked to adjacent monomeric sequences via a second cleavable peptide sequence. Therefore, after expression of the multimeric fusion protein, i.e., in host cells, the recovered fusion protein can be treated with a protease or a chemical that effectively cleaves the second cleavable peptide sequence, thereby releasing the individual monomeric peptide sequence containing the purification tag. After affinity purification, the recovered monomeric peptide sequence can be treated with a protease or a chemical that effectively cleaves the first cleavable peptide sequence, thereby releasing the purification tag from the target peptide. The latter can be further purified using gel filtration and / or HPLC as described below.

[0171] According to one method, the peptides of the present invention can be synthesized using standard peptide synthesis procedures. These include FMOC (9-fluorenylmethoxy-carbonyl) and tBoc (tert-butoxy-carbonyl) synthesis schemes, which can be performed on automated solid-phase peptide synthesis instruments, including but not limited to Applied Biosystems 431 A, 433 A synthesizers and Peptide Technologies Symphony or large-scale Sonata or CEM Liberty automated solid-phase peptide synthesizers. The use of alternative peptide synthesis instruments is also anticipated. Peptides prepared using solid-phase synthesis are recovered in substantially pure form.

[0172] The peptides of the present invention can also be prepared by using a recombinant expression system, followed by isolation and purification of the recombinant peptide. Typically, this involves inserting the encoding nucleic acid molecule into an expression system in which the molecule is heterologous (i.e., generally not present). One or more desired nucleic acid molecules encoding the peptides of the present invention can be inserted into a vector. The heterologous nucleic acid molecule is inserted into the expression system or vector with an appropriate meaningful (5'–3') orientation and correct reading frame relative to the promoter and any other 5' and 3' regulatory molecules.

[0173] Table 11 below includes representative nucleotide sequences used for expression in bacterial and plant hosts: Table 11 Based on the knowledge of the encoded amino acid sequences listed in this article and the desired transgenic organisms, it is possible to generate additional codon-optimized DNA and RNA sequences using only conventional techniques.

[0174] The expression (including transcription and translation) of the peptides or fusion polypeptides of the present invention by the DNA construct can be regulated in terms of expression level, tissue type of expression, and / or developmental stage of expression. Many heterologous regulatory sequences (e.g., promoters and enhancers) can be used to control the expression of the DNA construct. These include constitutive, inducible, and tunable promoters, as well as promoters and enhancers that control expression in a tissue- or spatiotemporally specific manner. Exemplary constitutive promoters include the raspberry E4 promoter (U.S. Patents 5,783,393 and 5,783,394, each of which is incorporated herein by reference in its entirety), the caustic solanine synthase (NOS) promoter (Ebert et al., Proc. Natl. Acad. Sci. (USA) 84:5745-5749 (1987), which is incorporated hereby by reference), octopus alkaloid synthase (OCS) promoter (which is carried on the oncogenic plasmid of Agrobacterium tumefaciens), caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324 (1987), which is incorporated here by reference (and CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985), which is incorporated herein by reference in its entirety; the 35S promoter of Scrophularia mosaic virus (US Patent No. 5,378,619, which is incorporated herein by reference in its entirety); the photoinducible promoter derived from the small subunit of ribulose-1,5-bisphosphonate carboxylase (ssRUBISCO); the Adh promoter (Walker et al., Proc. Natl. Acad. Sci. (USA) 84:6624-6628 (1987), which is incorporated here by reference), sucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci. (USA) 87:4144-4148 (1990), which is based on this by incorporating the whole reference), R gene complex promoter (Chandler et al., Plant Cell 1:1175-1183 (1989), which was incorporated in its entirety through citation), chlorophyll a / b binding protein gene promoter, CsVMV promoter (Verdaguer et al., Plant Mol Biol., 37:1055-1067 (1998), which is incorporated herein by reference in its entirety) and the melon actin promoter (PCT Publication No. WO00 / 56863, which is incorporated herein by reference in its entirety). Exemplary tissue-specific promoters include the tomato E4 and E8 promoters (US Patent No. 5,859,330, which is incorporated herein by reference in its entirety) and the tomato 2AII gene promoter (Van Haaren et al., Plant Mol Bio., 21:625-640 (1993), which is incorporated hereby by reference in its entirety.

[0175] In a preferred embodiment, the expression of the DNA construct is controlled by regulatory sequences from genes whose expression is associated with early seed and / or embryonic development. In fact, in a preferred embodiment, the promoter used is a seed-enhancing promoter. Examples of such promoters include 5' regulatory regions from genes such as napin (Kridl et al.). Seed Sci. Res . 1:209:219 (1991), which is incorporated here by reference), globulin (Belanger and Kriz, Genet 129: 863-872 (1991), GenBank accession number L22295, each of which is incorporated herein by reference in its entirety), γ-corn protein Z 27 (Lopes et al., Mol Gen Genet . 247:603-613 (1995), which is incorporated herein by reference in its entirety, L3 oleosin promoter (US Patent No. 6,433,252, which is incorporated herein by reference in its entirety), phaseolin (Bustos et al., Plant Cell 1(9):839-853 (1989), which is incorporated herein by reference in its entirety, arcelin5 (US Patent Application Publication No. 2003 / 0046727, which is incorporated herein by reference in its entirety), soybean 7S promoter, 7Sa promoter (US Patent Application Publication No. 2003 / 0093828, which is incorporated herein by reference in its entirety), soybean 7S With the conglycinin promoter and 7S Promoters (Beachy, etc.) EMBO J. 4:3047 (1985); Schuler et al. Nucleic Acid Res. 10(24):8225-8244 (1982), each of which is incorporated in whole by reference), soybean trypsin inhibitors (Riggs et al., Plant Cell 1(6):609-621 (1989), which is incorporated here by reference (ACP (Baerson et al., Plant Mol. Biol ., 22(2):255-267 (1993), which is incorporated here by reference), stearoyl-ACP desaturase (Slocombe et al, Plant Physiol. 104(4):167-176 (1994), which is incorporated here by reference (soybeans) Accompanied by the a' subunit of soybean globulin (Chen et al., Proc. Natl. Acad. Sci. 83:8560-8564 (1986), which is incorporated herein by reference in its entirety, fava bean (vicia faba) USP (US Patent Application Publication No. 2003 / 229918, which is incorporated herein by reference in its entirety) and maize L3 oleogen protein promoter (Hong et al., Plant Mol. Biol ., 34(3):549-555 (1997), which is incorporated hereby by reference in its entirety.

[0176] Nucleic acid molecules encoding the peptides of the present invention can be prepared via solid-phase synthesis using, for example, the phosphoramidite method and phosphoramidite building blocks derived from protected 2′-deoxynucleosides. To obtain the desired oligonucleotides, the building blocks are sequentially coupled to the growing oligonucleotide chains in the desired order of the product sequence. After chain assembly is complete, the product is released from the solid phase into solution for deprotection, collection, and typically purification using HPLC. Solid-phase synthesis is suitable for preparing oligonucleotides up to about 200 nt in length, encoding peptides of about 65 amino acids or fewer. The ends of the synthesized oligonucleotides can be designed to include specific restriction enzyme cleavage sites to facilitate the ligation of the synthesized oligonucleotides into expression vectors.

[0177] For longer peptides, oligonucleotides can be prepared via solid-phase synthesis, and then various techniques can be used to link the synthesized oligonucleotide sequences together. This review summarizes recombinant techniques for constructing complete synthetic genes, for example, in Hughes et al., “Chapter Twelve – Gene Synthesis: Methods and Applications,” Methods in Enzymology In 498:277-309 (2011), it was incorporated in its entirety by reference.

[0178] Once a suitable expression vector has been selected, the desired nucleic acid sequence is cloned into the vector using standard cloning procedures in the art, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, NY (1989), or U.S. Patent No. 4,237,224 to Cohen and Boyer, which is incorporated herein by reference in its entirety. The vector is then introduced into a suitable host.

[0179] The peptides of the present invention can be recombinantly expressed using various host-vector systems. Primarily, the vector system must be compatible with the host used. Host-vector systems include, but are not limited to, the following: bacteria transformed with phage DNA, plasmid DNA, or copious DNA; microorganisms such as yeast containing a yeast vector; mammalian cell systems infected with viruses (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with viruses (e.g., baculovirus); and Agrobacterium (… Agrobacterium Infected plant cells. The expression elements of these vectors differ in their strength and specificity. Depending on the host-vector system used, any of a number of suitable transcriptional and translational elements can be used to achieve this and other aspects of the invention.

[0180] Purified peptides can be obtained by several methods. Preferably, peptides in purified form (preferably with a purity of at least about 80% or 85%, more preferably at least about 90% or 95%) are produced using conventional techniques. Depending on whether the recombinant host cells secrete the peptide into the growth medium (see U.S. Patent No. 6,596,509 to Bauer et al., which is incorporated herein by reference in its entirety), the peptide can be separated and purified by centrifugation (to separate the cellular components from the supernatant containing the secreted peptide), followed by continuous ammonium sulfate precipitation of the supernatant. The peptide-containing component is subjected to gel filtration in a suitably sized dextran or polyacrylamide column to separate the peptide from other proteins. If necessary, the peptide component can be further purified by HPLC.

[0181] Alternatively, if the target peptide is not secreted, it can be isolated from the recombinant cells using standard isolation and purification protocols. This involves disrupting the cells (e.g., by sonication, freezing, Freund's press, etc.) and then recovering the peptide from the cell debris. Purification can be achieved using the centrifugation, precipitation, and purification procedures described above. Using the purification label described above can simplify this process.

[0182] In some embodiments, purification is not required. When purification is not performed, cell-free lysates can be recovered after centrifugation to remove cell debris. The resulting cell-free lysates may be heat-treated for a sufficient time to inactivate any native proteases in the recovered components, for example, at 100°C for 10 minutes. If desired, one or more of biocides, protease inhibitors, and nonionic surfactants may be introduced into such cell-free formulations (see Wei’s U.S. Application Publication No. 20100043095, which is incorporated herein by reference in its entirety).

[0183] Once the peptides of the present invention are recovered, they can be used to prepare compositions comprising a carrier and one or more additives selected from the group consisting of: fungicides or biocides, protease inhibitors, nonionic surfactants, fertilizers, herbicides, insecticides, fungicides, nematicides, bio-inoculation agents, plant regulators, and mixtures thereof.

[0184] In some embodiments, the composition comprises peptides greater than about 1 nM, greater than about 10 nM, greater than about 20 nM, greater than about 30 nM, greater than about 40 nM, greater than about 50 nM, greater than about 60 nM, greater than about 70 nM, greater than about 80 nM, greater than about 90 nM, greater than about 100 nM, greater than about 150 nM, greater than about 200 nM, or greater than about 250 nM. In some embodiments, the composition comprises peptides less than about 1 nM. For example, certain peptides may be present at concentrations below about 2 ng / ml, below about 1.75 ng / ml, below about 1.5 ng / ml, below about 1.25 ng / ml, below about 1.0 ng / ml, below about 0.75 ng / ml, below about 0.5 ng / ml, below about 0.25 ng / ml, or even below about 0.1 ng / ml.

[0185] Suitable carriers include water, optional aqueous solutions containing one or more co-solvents, slurries, and solid carrier particles. Exemplary solid carriers include mineral clays such as silicates, silica gel, talc, kaolin, limestone, lime, chalk, red basalt, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, abrasive synthetic materials, and plant-derived products such as cereal flour, bark flour, wood flour and nut shell flour, cellulose flour, starch and starch derivatives, as well as other monosaccharides, disaccharides, and polysaccharides.

[0186] Suitable fertilizers include, but are not limited to, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, and combinations thereof.

[0187] Suitable insecticides include, but are not limited to, neonicotinoids such as imidacloprid, clothianidin, and thiamethoxam; organophosphates such as chlorpyrifos and malathion; pyrethroids such as permethrin; other natural insecticides such as nicotine, nornicotine, and pyrethrin; and carbamates such as aldicarb, carbofuran, and carbaryl. Arbaryl; macrolide members, such as various abamectin, avermectin, and ivermectin products; diamide members, such as chlorantraniliprole, cyantraniliprole, and flubendiamide; chitin synthesis inhibitors, especially those of the benzoylurea class, such as lufenuron and diflubenzuron; and any combination thereof, including combinations of two or more, three or more, or four or more insecticides. Additional insecticides are listed in the Compendium of Pesticide Common Names, a database operated by Alan Wood and available electronically at alanwood.net.

[0188] Suitable fungicides include, but are not limited to, members of the methoxyacrylate class, such as azoxystrobin, piraclostrobin, trifloxystrobin, picoxystrobin, and fluoxastrobin; and members of the triazole class, such as ipconazole, metconazole, tebuconazole, triticonazole, tetraconazole, difenoconazole, flutriafol, propiconazole, and prothioconazole. Succinate dehydrogenases, such as carboxin, fluxapyroxad, boscalid, and sedaxane; phenylamides, such as metalaxyl, mefenoxam, benalaxyl, and oxadiyxl; phenylpyrroles, such as fludioxonil; phthalimides, such as captan; dithiocarbamates, such as mancozeb and thiram; benzimidazoles, such as thiabendazole; and any combination thereof, including combinations of two or more, three or more, or four or more fungicides. Additional fungicides are listed in the Generic Names of Pesticides database operated by Alan Wood and available electronically at alanwood.net.

[0189] Suitable nematicides include, but are not limited to, carbamate chemicals such as aldoxycarb, oxamyl, furadan, and cleothocarb; and organophosphate chemicals such as thionazin, ethoprophos, fenamiphos, fensulfothion, terbufos, isazofos, and ebufos. Additional nematicides are listed in the Generic Names of Pesticides Database, which is a database operated by Alan Wood and available electronically at alanwood.net.

[0190] Suitable fungicides include, but are not limited to, those based on dichlorophenol and benzyl alcohol hemiacetal (Proxel® from ICI, Acticide® RS from Thor Chemie, and Kathon® MK from Rohm & Haas) and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones (Acticide® MBS from Thor Chemie; Proxel® GXL from ICI). Additional fungicides are listed in the Generic Names of Pesticides Directory, which is part of a database operated by Alan Wood and is available electronically at alanwood.net.

[0191] Suitable inoculants include, but are not limited to, species of the genus *Syntrophus* (including *Syntrophus*). Bradyrhizobium spp. ), especially slow-growing soybean rhizobia ( Bradyrhizobium japonicum (BASF Vault® products), Bacillus subtilis ( Bacillus subtilis ), Bacillus stolonifer ( Bacillus firmus ), Bacillus pumilus ( Bacillus pumilis Streptomyces lidii ( Streptomyces lydicus Trichoderma species ( Trichoderma spp. ), species of Pasteurella ( Pasteuria spp. (and other cultures of root nodule cells (BASF Nodulator® and Rhizo-Flo®) and any combination thereof, including combinations of two or more, three or more, or four or more inoculants.

[0192] Plant regulators are natural or synthetic chemical substances that stimulate or inhibit biochemical signal transduction in plants. These are typically, but not always, recognized by receptors on the cell surface, triggering a cascade of reactions within the cell. Suitable plant regulators include, but are not limited to, ethephon; ethylene; salicylic acid; acetylsalicylic acid; jasmonic acid; methyl jasmonate; dihydrojasmonate; chitin; chitosan; abscisic acid; any auxin compound or inhibitor, including but not limited to (4-chlorophenoxy)acetic acid, (2,4-dichlorophenoxy)acetic acid, and 2,3,5-triiodobenzoic acid; any cytokinin, including but not limited to kinetin and zeatin; gibberellin; brassinolide; and any combination thereof, including combinations of two or more, three or more, or four or more regulators.

[0193] Other suitable additives include buffers, wetting agents, coating agents, and abrasives. These substances can be used to facilitate the application of the compositions according to the invention. Additionally, the compositions can be applied to plant seeds together with other conventional seed formulations and treatments, including clays and polysaccharides.

[0194] Compositions or systems for plant seed treatment include: one or more peptides of the present invention, preferably but not exclusively one of P1, P4-14S, P6a, P14d, P15a, P18, P19, or P25, in combination with one or more insecticides, nematicides, fungicides, other inoculants, or other plant regulators, including combinations of multiple insecticides or multiple nematicides, multiple fungicides, multiple other inoculants, or multiple plant regulators. Suitable insecticides, nematicides, fungicides, inoculants, and plant regulators for these combined treatments include those identified above. These compositions are presented as a single composition during seed treatment. Conversely, systems for seed treatment may involve multiple treatments, for example, using a composition containing the peptides in one treatment and compositions containing one or more of the insecticides, nematicides, fungicides, plant regulators, and / or fungicides in different treatments. In the latter embodiment, the two treatments are performed at approximately the same time, i.e., before or approximately at planting.

[0195] One such example includes one or more peptides of the present invention, including (but not limited to) one of P1, P4-14S, P6a, P14d, P15a, P18, P19 or P25, in combination with Poncho™ (thiamethoxam), available from Bayer Crop Science; Poncho™ VOTiVO (thiamethoxam and Bacillus thuringiensis bionematicide), available from Bayer Crop Science; and Gaucho™ (imidacloprid), available from Bayer Crop Science.

[0196] Another example includes one or more peptides of the present invention, including (but not limited to) one of P1, P4-14S, P6a, P14d, P15a, P18, P19, or P25, in combination with Cruiser™ (thiamethoxam), CruiserMaxx™ (thiamethoxam, metalaxyl, and fludioxonil), Cruiser Extreme™ (thiamethoxam, metalaxyl, fludioxonil, and azoxystrobin), Avicta™ (thiamethoxam and avermectin), and Avicta™ Complete (thiamethoxam, avermectin, and containing Pasteurella multocida). Pasteuria nishizawaeClariva Complete™ bio-inoculation agent (Pn1) and Avicta Complete™ Corn (thiamethoxam, metalaxyl, fludioxonil, azoxystrobin, thiabendazole and abamectin) available from Syngenta.

[0197] Another example includes one or more peptides of the present invention, including (but not limited to) one of P1, P4-14S, P6a, P14d, P15a, P18, P19 or P25, in combination with Vault Liquid plus Integral (MBI 600 inoculant for slow-growing rhizobia species and Bacillus subtilis strains) available from BASF, Vault NP (slow-growing soybean rhizobia inoculant) available from BASF and Subtilex NG (Bacillus subtilis bioinoculant) available from BASF.

[0198] This invention also relates to methods for conferring disease resistance to plants, enhancing plant growth, achieving pest control, conferring tolerance to biotic or abiotic stresses to plants, and / or regulating plant biochemical signaling. These methods involve applying an effective amount of the isolated peptides or compositions of this invention to a plant or plant seed or the site of growth or intended growth of said plant. Upon such application, the peptides contact the cells of the plant or plant seed and induce disease resistance, enhanced growth, tolerance to biotic stresses, tolerance to abiotic stresses, or altered biochemical signaling in the plant or plant grown from plant seeds. Optionally, the peptides or compositions of this invention can be applied to plants such that seeds recovered from such plants themselves can confer disease resistance in the plant, enhance plant growth, influence insect control, confer tolerance to biotic or abiotic stresses, and / or regulate biochemical signaling, regulating maturity.

[0199] In these embodiments, it may also be possible to selectively apply the isolated peptides or compositions of the present invention to the plants, seeds, or sites thereon. For example, for fields known to contain high nematode content, plants or seeds intended for growth in such fields, or the field (site), may be selectively treated by applying the isolated peptides or compositions of the present invention as described herein; while for plants or seeds grown in fields containing low nematode content, such treatment may not be necessary. Similarly, for fields with reduced irrigation, plants or seeds intended for growth in such fields, or the field (site), may be selectively treated by applying the isolated peptides or compositions of the present invention as described herein; while for plants or seeds grown in well-irrigated fields, such treatment may not be necessary. Likewise, for flood-prone fields, plants or seeds intended for growth in such fields, or the field (site), may be selectively treated by applying the isolated peptides or compositions of the present invention as described herein; while for plants or seeds grown in fields less prone to flooding, such treatment may not be necessary. As another example of such selection, for fields vulnerable to insect attack at certain times of the growing season, the plants or seeds to be grown in such fields, or the field (site), may be selectively treated by applying isolated peptides or compositions of the present invention as described herein; while the same fields may be left untreated during ineffective times of the growing season, or other fields less susceptible to such attacks may be left untreated. Such selection steps can be performed when practicing each of the methods of application described herein, namely, conferring plant resistance, enhancing plant growth, achieving pest control (including insects and nematodes), conferring plant tolerance to biotic or abiotic stresses, and / or modulating plant biochemical signal transduction.

[0200] As an alternative to applying isolated peptides or compositions containing isolated peptides to plants or plant seeds to confer plant disease resistance, promote plant growth, control insects, confer stress resistance and / or regulate biochemical signaling, transgenic plants or plant seeds may be utilized. When using transgenic plants, this involves providing transgenic plants transformed with DNA molecules encoding the peptides of the present invention and allowing the plants to grow under conditions that effectively allow the DNA molecules to confer plant disease resistance, enhance plant growth, control insects, confer tolerance to biotic or abiotic stresses and / or regulate biochemical signaling. Optionally, seeds of transgenic plants transformed with DNA molecules encoding the peptides of the present invention may be provided and planted in soil. The planted plants are then propagated from the planted seeds under conditions that effectively allow the DNA molecules to express the peptides and thereby confer transgenic plant disease resistance, enhance plant growth, control insects, confer tolerance to biotic or abiotic stresses and / or regulate biochemical signaling.

[0201] This invention also relates to improving the desiccation resistance of cuttings taken from ornamental plants, the postharvest disease resistance or desiccation resistance of fruits or vegetables harvested from plants, and / or methods for increasing the maturity life of fruits or vegetables harvested from plants. These methods involve applying an effective amount of the isolated peptides of the invention or the compositions according to the invention to the plant or the site of plant growth. Upon such application, the peptides contact the cells of the plant or plant seeds and induce desiccation resistance in cuttings taken from ornamental plants, postharvest disease resistance or desiccation resistance in fruits or vegetables harvested from plants, and / or induce an increased maturity life of fruits or vegetables harvested from plants. Optionally, an effective amount of the isolated peptides of the invention or the compositions according to the invention may be applied to the harvested fruits or vegetables. Upon such application, the peptides contact the cells of the harvested fruits or vegetables and induce postharvest disease resistance or desiccation resistance in the treated fruits or vegetables, and / or induce an increased maturity life of the treated fruits or vegetables.

[0202] As an alternative to applying isolated peptides or compositions containing isolated peptides to plants or plant seeds to induce desiccation resistance in cuttings taken from ornamental plants, postharvest disease resistance or desiccation resistance in fruits or vegetables harvested from the plants, and / or to induce increased fruit or vegetable maturity, transgenic plants or plant seeds may be used. When using transgenic plants, this involves providing transgenic plants transformed with DNA molecules encoding the peptides of the present invention and allowing the plants to grow under conditions that effectively permit the DNA molecules to induce desiccation resistance in cuttings taken from ornamental plants, postharvest disease resistance or desiccation resistance in fruits or vegetables harvested from the transgenic plants, and / or to induce increased fruit or vegetable maturity. Optionally, seeds of transgenic plants transformed with DNA molecules encoding the peptides of the present invention may be provided and planted in soil. Then, the plant is propagated from seeds under conditions that effectively allow the DNA molecule to express the peptide and thereby induce drought resistance in cuttings taken from ornamental plants, induce postharvest disease resistance or drought resistance in fruits or vegetables harvested from transgenic plants, and / or induce increased fruit or vegetable maturity lifespan in fruits or vegetables harvested from transgenic plants.

[0203] In these embodiments, genetically modified plants or seeds may also be selected for the implementation of the invention. For example, for fields known to contain high nematode levels, genetically modified plants or seeds may be selectively grown in such fields; while non-genetically modified plants or seeds may be grown in fields containing low nematode levels. Similarly, for fields with reduced irrigation, genetically modified plants or seeds may be selectively grown in such fields; while non-genetically modified plants or seeds may be grown in fields with adequate irrigation. Likewise, for fields prone to flooding, genetically modified plants or seeds may be grown in such fields; while non-genetically modified plants or seeds may be grown in fields less prone to flooding. As another example of such selection, for fields susceptible to insect attack at certain times of the growing season, genetically modified plants or seeds may be selectively grown in such fields; while non-genetically modified plants or seeds may be grown in fields less susceptible to such insect attack. Such selection steps can be performed when practicing each of the uses described herein, namely, conferring plant disease resistance, enhancing plant growth, achieving pest control (including insects and nematodes), conferring plant tolerance to biotic or abiotic stresses, and / or modulating plant biochemical signal transduction.

[0204] This invention also relates to improving the desiccation resistance of cuttings taken from ornamental plants, the postharvest disease resistance or desiccation resistance of fruits or vegetables harvested from plants, and / or methods for increasing the maturity life of fruits or vegetables harvested from plants. These methods involve applying an effective amount of the isolated peptides of the invention or the compositions according to the invention to the plant or the site of plant growth. Upon such application, the peptides contact the cells of the plant or plant seeds and induce desiccation resistance in cuttings taken from ornamental plants, postharvest disease resistance or desiccation resistance in fruits or vegetables harvested from plants, and / or induce an increased maturity life of fruits or vegetables harvested from plants. Optionally, an effective amount of the isolated peptides of the invention or the compositions according to the invention may be applied to the harvested fruits or vegetables. Upon such application, the peptides contact the cells of the harvested fruits or vegetables and induce postharvest disease resistance or desiccation resistance in the treated fruits or vegetables, and / or induce an increased maturity life of the treated fruits or vegetables.

[0205] In these embodiments, the plants, cuttings, fruits, vegetables, or sites to which the isolated peptides or compositions of the present invention are applied may also be selectively treated. For example, cuttings, fruits, or vegetables harvested for long-distance transport or long-term storage may be selectively treated with the isolated peptides or compositions of the present invention as described herein; while cuttings, fruits, or vegetables harvested locally and intended for consumption with substantially no storage period may be excluded from such treatment.

[0206] As an alternative to applying isolated peptides or compositions containing isolated peptides to plants or plant seeds to induce desiccation resistance in cuttings taken from ornamental plants, postharvest disease resistance or desiccation resistance in fruits or vegetables harvested from the plants, and / or to induce increased fruit or vegetable maturity, transgenic plants or plant seeds may be used. When using transgenic plants, this involves providing transgenic plants transformed with DNA molecules encoding the peptides of the present invention and allowing the plants to grow under conditions that effectively permit the DNA molecules to induce desiccation resistance in cuttings taken from ornamental plants, postharvest disease resistance or desiccation resistance in fruits or vegetables harvested from the transgenic plants, and / or to induce increased fruit or vegetable maturity. Optionally, seeds of transgenic plants transformed with DNA molecules encoding the peptides of the present invention may be provided and planted in soil. Then, the plant is propagated from seeds under conditions that effectively allow the DNA molecule to express the peptide and thereby induce drought resistance in cuttings taken from ornamental plants, induce postharvest disease resistance or drought resistance in fruits or vegetables harvested from transgenic plants, and / or induce increased fruit or vegetable maturity lifespan in fruits or vegetables harvested from transgenic plants.

[0207] In these embodiments, genetically modified plants or plant seeds may also be selected for the purpose of realizing the invention. For example, when it is known that the harvested cuttings, fruits, or vegetables are intended to be transported long distances or stored for a long period after harvesting, genetically modified plants or plant seeds may be selected for growth; while when it is known that the harvested cuttings, fruits, or vegetables are intended to be transported locally and / or consumed, with substantially no storage period, non-genetically modified plants or plant seeds may be selected for growth.

[0208] Suitable plants include both dicotyledons and monocotyledons, including agricultural, afforestation, ornamental, and horticultural plants, whether in natural or genetically modified forms. Exemplary plants include, but are not limited to, alfalfa, apple trees, apricot trees, asparagus, avocado trees, banana trees, barley, legumes, beech (Fagus spec.), begonia, birch, blackberry, blueberry, cabbage, camphor, mustard, carrot, castor bean, cherry tree, cinnamon tree, citrus tree, cocoa bean, coffee, corn, cotton, cucumber, gourd, eucalyptus, fir, flax, fodder beet, fuchsia, garlic, geranium, grape, peanut, hemp, hop, juneberry, mustard (Brassica juncea), jute, lentils, lettuce, flaxseed, melon, mustard, nectarine, oak, oats, oil palm, oilseed rape, olive, onion, pepper, pea, peach, pear, geranium, pepper, petunia, pine (Pinus spec.) (sp.)), plum trees, poplar trees (Populus spec.)), pome fruits, potatoes, rapeseed, raspberries, rice, rubber trees, rye, sorghum, soybeans, spinach, spruce, pumpkins, strawberries, sugar beets, sugarcane, sunflowers, tea trees, teak, tobacco, tomatoes, black wheat, turf, watermelons, wheat, and willow trees (Salix spec.)), Arabidopsis thaliana, African violets (Saintpaulia), poinsettias, chrysanthemums, carnations, and zinnias.

[0209] Altered biochemical signal transduction includes the enhancement of certain plant biochemical pathways and the weakening of others. Biochemical signal transduction pathways that can be altered according to the present invention include gene expression and protein production, metabolite generation, and signal molecule / secondary metabolite generation. Exemplary biochemical signal transduction pathways and their alterations include, but are not limited to, induction of nitric oxide production, peroxide production, and other secondary metabolites; activation of the ethylene signal transduction pathway and induction of ethylene-responsive gene expression (see Dong et al.). Plant Phys. 136:3628-3638(2004); Li et al., Planta 239:831-46 (2014); Chang et al. PLoS One 10,e0125498 (2015), each of which is incorporated herein by reference in its entirety; activation and induction of salicylic acid response gene expression in the salicylic acid signaling pathway (see Dong et al., Plant J.20:207-215 (1999), which is incorporated herein by reference in its entirety; activation of the abscisic acid pathway and induction of abscisic acid response gene expression (see Dong et al., Planta 221: 313-327 (2005), which is incorporated herein by reference in its entirety; activating and inducing gibberellin response gene expression in the gibberellin signaling pathway (see Li et al., Planta 239:831-46 (2014), which is incorporated herein by reference in its entirety; antagonism of jasmonic acid signaling and inhibition of jasmonic acid response gene expression (see Dong et al., Plant Phys. 136:3628-3638 (2004), which is incorporated herein by reference in its entirety; inducing protease inhibitor expression (see Laluk and Mengiste, Plant J. 68:480-494 (2011); Xia et al., Chin. Sci. Bull 56: 2351-2358 (2011), each of which is incorporated herein by reference in its entirety; induces the generation of reactive oxygen species in plant tissues; induces the generation of immune-related and antimicrobial peptides, such as, but not limited to, peroxidases, superoxide dismutases, chitinases, and β-glucanases (Wang et al., J. Agric. Food Chem. 59:12527-12533 ​​(2011), which is incorporated herein by reference in its entirety; and the induction of extended protein gene expression and generation (see Li et al., Planta 239:831-46 (2014), which is incorporated herein by reference in its entirety.

[0210] While resistance may not confer absolute immunity to infection, it reduces the severity of disease and delays symptom development. The number and size of lesions, as well as the extent of fungal pathogen spore formation, are all reduced. This method of conferring resistance has the potential to treat previously untreatable diseases, systemic diseases that cannot be treated alone due to cost, and to avoid the use of infectious agents or environmental hazards.

[0211] The method for conferring plant resistance to pathogens according to the present invention is used to confer resistance to various pathogens, including viruses, bacteria, and fungi. In particular, resistance to the following viruses can be achieved by the method of the present invention: tobacco mosaic virus and tomato mosaic virus. The present invention can also confer resistance to plants, especially to the following bacteria: pathogenic Pseudomonas species (…). Pseudomonas spp.), pathogenic Erwinia species ( Erwinia spp. ), pathogenic Flavobacterium species ( Xanthomonas spp.) and pathogenic Ralstonia species ( RalstoniaPlants can be made particularly resistant to the following fungi by using the methods of the present invention: Fusarium species (spp.). Fusarium spp.) and species of the genus Phytophthora ( Phytophthora spp.).

[0212] Regarding the use of the peptides or compositions of the present invention for enhancing plant growth, various forms of plant growth enhancement or promotion can be achieved. This can occur as early as when plant growth begins from seed or later during the plant's lifespan. For example, the plant growth benefits according to the invention encompass higher yields, enhanced plant vigor, enhanced seedling (i.e., post-germination) vigor, increased plant weight, increased biomass, increased number of flowers per plant, higher grain and / or fruit yields, increased seed yields, increased percentage of germinating seeds, increased germination rate, increased plant size, reduced plant height (for wheat), higher biomass, more and larger fruits, earlier fruit coloring, earlier germination, earlier fruit and plant maturity, more tillers or lateral branches, larger leaves, delayed leaf senescence, increased branch growth, increased root growth, altered root / branch allocation, increased protein content, increased oil content, increased carbohydrate content, increased pigment content, increased chlorophyll content, increased total photosynthesis, increased photosynthetic efficiency, reduced respiration (lower O2 usage), compensation for yield-reducing treatments, enhanced stem durability (and resistance to lodging), enhanced root durability (and resistance to root lodging), better plant growth under low light conditions, and combinations thereof. Therefore, the invention provides growers with significant economic benefits. For example, early germination and early maturity allow crops to grow in areas where a short growing season would otherwise hinder their growth. Increased seed germination percentage leads to improved crop stands and more efficient seed use. Higher yields, increased size, and enhanced biomass production allow for greater returns from a given plot of land.

[0213] Regarding the use of the peptides or compositions of the present invention for the control of pests (including but not limited to insects and nematodes that are biological stressors), such pest control encompasses preventing pests from contacting plants to which the peptides or compositions of the present invention have been applied, preventing direct damage to the plants through feeding injury, keeping pests away from such plants, killing pests that approach such plants, interfering with the larvae of insects feeding on such plants, preventing insects from colonizing host plants, preventing colonizing insects from releasing plant toxins, interfering with oviposition on host plants, and so on. The present invention also prevents subsequent diseases of plants caused by pest infestation.

[0214] This invention is effective against various insects (biological stressors). The European corn borer is a major pest of corn (dent corn and sweet corn), but it also feeds on more than 200 plant species, including green beans, cowpeas, and kidney beans, as well as edible soybeans, peppers, potatoes, and tomatoes, plus many weed species. Other insect larvae that damage various vegetable crops include the following feeding pests: beet armyworm, white spot moth, cotton bollworm, armyworm, diamondback moth, cabbage root maggot, onion maggot, seed fly, pickle worm (melon worm), pepper maggot, and tomato codling moth. In general, this type of pest represents one of the most economically important pests for vegetable production worldwide. This invention is also effective against nematodes (another economically important biological stressor). Soybean heterodermal nematode (soybean cyst nematode) Heterodera glycines) ) is a major pest of soybeans. Kidney-shaped nematodes (Kidney-shaped nematodes (Kidney-shaped nematodes) Rotylenchulus reniformis) The root-knot nematode is a major pest of cotton and can also parasitize other crop species, especially soybeans and corn. Other nematode pests include those of the genus *Root-knot Nematodes*. Meloidogyne Root-knot nematodes (especially in corn, wheat, and barley), Heterodera nematodes ( Heterodera ) of the gramineous cyst nematode (especially in soybeans, wheat, and barley), and the genus *Brietonia* (…). Pratylenchus Root-rot nematodes, granular nematodes ( Anguina Seed gall nematodes (especially in wheat, barley, and rye) and stem nematodes ( Ditylenchus Stem nematodes. Other biological stressors include spiders, weeds, and combinations thereof.

[0215] Regarding the use of the peptides or compositions of the present invention for conferring resistance to abiotic stresses on plants, such abiotic stresses encompass any environmental factors that adversely affect plant physiology and development. Examples of such environmental stresses include climate-related stresses (e.g., drought, floods, frost, low temperatures, high temperatures, excessive light, and insufficient light) and air pollution stresses (e.g., carbon dioxide, carbon monoxide, sulfur dioxide, NO). x This invention relates to the healing response to damage caused by various environmental stresses, including those caused by hydrocarbons, ozone, ultraviolet radiation, acid rain, chemicals (e.g., pesticides, fungicides, herbicides, heavy metals), nutrient stresses (e.g., excess or deficiency of fertilizers, micronutrients, macronutrients, particularly potassium, nitrogen derivatives, and phosphorus derivatives), and the improvement of healing responses to such damage. The peptides of this invention confer resistance in plants to these forms of environmental stress.

[0216] Another aspect of the invention relates to the use of the peptide of the invention as a safener in combination with one or more active agents (i.e., in a composition or in separate compositions) for the control of aquatic plants in water bodies, as described in Mann’s U.S. Publication No. 20150218099, which is incorporated herein by reference in its entirety.

[0217] Another aspect of the invention relates to the use of the peptide of the invention as a plant fortifier in a composition for application to plants grown under conditions of reduced irrigation, said composition further comprising at least one antioxidant and at least one radiation management agent, and optionally at least one plant growth regulator, as described in U.S. Publication No. 20130116119 by Rees et al., which is incorporated herein by reference in its entirety.

[0218] When treating all or part of a plant, including leaves, stems, roots, propagules (e.g., cuttings), fruits, etc., the methods of application of the peptides or compositions of the present invention can be carried out through a variety of procedures. This may (but does not necessarily) involve the infiltration of the peptides into the plant. Suitable application methods include high-pressure or low-pressure spraying, injection, and leaf abrasion in the immediate vicinity of peptide application. When treating plant seeds, according to the application embodiments of the present invention, allergic response elicitor proteins or polypeptides can be applied by low-pressure or high-pressure spraying, coating, immersion (e.g., soaking), or injection. Other suitable application procedures will be contemplated by those skilled in the art, as long as they enable contact between the allergic response elicitor polypeptides or proteins and the plant or plant seed cells. Once treated with the peptides or compositions of the present invention, the seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants. After plants are propagated from seeds treated according to the present invention, the plants can be treated once or multiple times with the peptides or compositions of the present invention to confer disease resistance, enhance plant growth, control insects on the plants, confer tolerance to biotic or abiotic stresses, improve the drought resistance of cuttings, confer postharvest disease resistance or drought resistance to harvested fruits or vegetables, and / or improve the maturity life of harvested fruits or vegetables.

[0219] The peptides or compositions of the present invention can be applied to plants or plant seeds, either alone or in combination with other substances, according to the present invention. Optionally, the peptides or compositions can be applied to plants alone, while other substances are applied at different times.

[0220] In alternative embodiments of the invention involving the use of transgenic plants and transgenic seeds, it is not necessary to apply the peptides of the invention topically to the plants or seeds. Instead, transgenic plants are produced according to procedures known in the art, using DNA molecules encoding the peptides of the invention. Vectors suitable for expression in plants (i.e., containing translational and transcriptional control sequences operable in plants) can be mechanically transferred by direct microinjection into plant cells using micropipettes. Crossway, Mol. Gen. Genetics , 202:179-85 (1985), which is incorporated herein by reference in its entirety. Polyethylene glycol can also be used to transfer genetic material into plant cells. Krens et al., Nature, 296:72-74 (1982), which is incorporated in its entirety by reference.

[0221] Another method of transforming plant cells with a gene encoding the peptide of the present invention is particle bombardment of the host cell (also known as gene gun transformation). This can be achieved in one of several ways. The first involves propelling inert or bioactive particles at the cell site. This technique is described in U.S. Patents 4,945,050, 5,036,006, and 5,100,792 to Sanford et al., which are incorporated herein by reference. Typically, this procedure involves propelling inert or bioactive particles at the cell site under conditions that allow them to effectively penetrate the cell's outer surface and be incorporated into its interior. When using inert particles, the vector can be introduced into the cell by coating the particles with a carrier containing heterologous DNA. Optionally, the target cell can be surrounded by the carrier, such that the carrier is carried into the cell by the particle tail. Bioactive particles (e.g., dried bacterial cells containing a carrier and heterologous DNA) can also be propelled into plant cells.

[0222] Another approach introduced is the fusion of protoplasts with other entities, such as microcells, cells, lysosomes, or other fusible lipid surfaces. Fraley et al. Proc. Natl. Acad. Sci. USA , 79:1859-63 (1982), which is incorporated in its entirety by reference.

[0223] DNA molecules can also be introduced into plant cells via electroporation. Fromm et al., Proc. Natl. Acad. Sci. USA , 82:5824 (1985), which is incorporated herein by reference in its entirety. In this technique, plant protoplasts are electroporated in the presence of a plasmid containing an expression cassette. High-field electrical pulses reversibly permeate the biomembrane, allowing the introduction of the plasmid. Electroporated plant protoplasts reform their cell walls, divide, and regenerate.

[0224] Another method to introduce DNA molecules into plant cells is to infect the plant cells with *Agrobacterium tumefaciens* or *Agrobacterium rhizogenes* that have previously been transformed with that gene. A. rhizogenesUnder suitable conditions known in the art, transformed plant cells are allowed to grow to form shoots and roots, and further develop into plants. Typically, this procedure involves inoculating plant tissue with a bacterial suspension and incubating the tissue at 25–28°C on an antibiotic-free regeneration medium for 48–72 hours. *Agrobacterium* is a representative genus of the Gram-negative rhizobium family. Its species are responsible for crown gall (*Agrobacterium tumefaciens*) and root rot (*Agrobacterium rhizogenes*). Plant cells within crown galls and root rot are induced to produce an amino acid derivative called opine, which is metabolized solely by the bacteria. The bacterial gene responsible for opine expression is a convenient source of control elements for chimeric expression cassettes. Furthermore, the determination of the presence of opine can be used to identify transformed tissues. Heterologous gene sequences can be introduced into suitable plant cells using the *Ti* plasmid of *Agrobacterium tumefaciens* or the *Ri* plasmid of *Agrobacterium rhizogenes*. The *Ti* or *Ri* plasmid spreads into plant cells upon infection with *Agrobacterium* and is stably integrated into the plant genome. J. Schell, Science , 237:1176-83(1987), which is incorporated in its entirety by reference.

[0225] After transformation, the transformed plant cells must regenerate. Plant regeneration from cultured protoplasts was demonstrated by Evans et al. Handbook of Plant Cell Cultures Volume 1: (MacMillan Publishing Co., New York, 1983); and Nasil IR (eds.), Cell Culture and Somatic Cell Genetics of Plants As described in Acad. Press, Orlando, Vol. 1, 1984 and Vol. 11 (1986), which are incorporated herein by reference in their entirety.

[0226] Almost all plants are known to regenerate from cultured cells or tissues. The regeneration process varies depending on the plant species, but typically begins with a suspension of transformed protoplasts or Petri plates containing transformed explants. Callus forms, and shoots can be induced from the callus, subsequently leading to root formation. Optionally, embryo formation can be induced in the callus. These embryos germinate to form plants, just like natural embryos. Culture media usually contain various amino acids and hormones, such as auxins and cytokinins. Adding glutamate and proline to the culture medium is also beneficial, especially for species such as maize and alfalfa. Effective regeneration will depend on the culture medium, genotype, and culture history. If these three variables are controlled, regeneration is generally reproducible and repeatable.

[0227] Once the expression cassette is stably incorporated into a transgenic plant, it can be transferred to other plants through sexual hybridization. Depending on the species to be hybridized, any of many standard breeding techniques can be used.

[0228] Once this type of transgenic plant is produced, the plant itself can be cultivated according to conventional procedures. The presence of genes encoding allergic response elicitors results in disease resistance, enhanced plant growth, control of insects on the plant, tolerance to abiotic or biotic stresses, increased drought resistance in cuttings, postharvest disease resistance or drought resistance in harvested fruits or vegetables, and / or increased fruit or vegetable maturity lifespan for harvested fruits or vegetables.

[0229] Optionally, genetically modified seeds can be recovered from genetically modified plants. These seeds can then be planted in soil and cultured using conventional procedures to produce genetically modified plants. Genetically modified plants are propagated from planted genetically modified seeds under conditions that effectively confer plant disease resistance, enhance plant growth, control insects, confer tolerance to abiotic or biotic stresses, improve the drought resistance of cuttings, confer postharvest disease resistance or drought resistance in harvested fruits or vegetables, and / or confer an increased fruit or vegetable maturity lifespan.

[0230] When using transgenic plants and plant seeds according to the present invention, they may also be treated with the same substances used to treat plants and seeds treated with the peptides or compositions of the present invention. These other substances, including the peptides or compositions of the present invention, may be applied to transgenic plants and plant seeds via the procedures mentioned above, including high-pressure or low-pressure spraying, injection, coating, and immersion. Similarly, after plants have been propagated from transgenic plant seeds, they may be treated with one or more applications of the peptides or compositions of the present invention to confer disease resistance, enhance growth, control insects, confer tolerance to abiotic or biotic stresses, confer desiccation resistance to cuttings, confer postharvest disease resistance or desiccation resistance in harvested fruits or vegetables, and / or confer an increased fruit or vegetable maturity lifespan in harvested fruits or vegetables.

[0231] Such genetically modified plants can also be treated with conventional plant treatment agents, such as fungicides or biocides, protease inhibitors, nonionic surfactants, fertilizers, herbicides, insecticides, fungicides, nematicides, biological inoculants, plant regulators and mixtures thereof, as described above.

[0232] This application also includes at least the following implementation schemes: Implementation Scheme 1. An isolated peptide comprising the amino acid sequence (L / I / V / F)-XX-(L / I / V / F)-(L / I)-XX-(L / I / V / F)-(L / I / V / A)-XX-(L / I)-(L / I / V / F) (SEQ ID NO: 93) in The peptide does not contain cysteine ​​or methionine; Each X at positions 2, 6, and 10 is optional and, when present, is any amino acid; and Each X at positions 3, 7, and 11 represents any amino acid.

[0233] Implementation Scheme 2. The isolated peptide according to Implementation Scheme 1, wherein the length of the peptide is less than 100 amino acids.

[0234] Implementation Scheme 3. The isolated peptide according to Implementation Scheme 2, wherein the length of the peptide is between 13 and 50 amino acids.

[0235] Implementation Scheme 4. The isolated peptide according to Implementation Scheme 1, wherein the isolated peptide is stable when dissolved in water or an aqueous solution.

[0236] Implementation Scheme 5. The isolated peptide according to Implementation Scheme 1, wherein the isolated peptide is resistant to chemical degradation when dissolved in an aqueous buffer solution containing a biocide.

[0237] Implementation Scheme 6. The isolated peptide according to Implementation Scheme 1, wherein the isolated peptide has a solubility of more than about 0.1% in water or aqueous solution.

[0238] Implementation Scheme 7. The peptide isolated according to Implementation Scheme 1, wherein one or both X's at positions 2 and 6 are absent.

[0239] Implementation Scheme 8. The peptide isolated according to Implementation Scheme 1, wherein both X's at positions 2 and 6 are present.

[0240] Implementation Scheme 9. The isolated peptide according to Implementation Scheme 1 or 7, wherein X at position 10 is absent.

[0241] Implementation Scheme 10. The isolated peptide according to Implementation Scheme 1 or 8, wherein X is present at position 10.

[0242] Implementation Scheme 11. The peptide isolated according to Implementation Scheme 1, wherein Each X at positions 2 and 6, when present, is a polar or charged amino acid; and Each X at positions 3, 7, 10, and 11 is a polar or charged amino acid.

[0243] Implementation Scheme 12. The peptide isolated according to Implementation Scheme 1, wherein Each X at positions 2 and 6, when present, is selected from R, K, D, isoD, E, N, Q, H, S, T, Y, W, G, A, and g-glutamic acid; and Each X at positions 3, 7, 10, and 11 is selected from R, K, D, isoD, E, N, Q, H, S, T, Y, W, G, A, and g-glutamic acid.

[0244] Implementation Scheme 13. The peptide isolated according to Implementation Scheme 1, wherein Each X at positions 2 and 6, when present, is selected from D, isoD, E, and γ-glutamic acid; and Each X at positions 3, 7, 10, and 11 is selected from D, isoD, E, and g-glutamic acid.

[0245] Implementation Scheme 14. The isolated peptide according to Implementation Scheme 13, wherein the peptide comprises the following amino acid sequence: SEELEELLEELIEELL,SEQ ID NO: 189, LEELLEELIEELLEE, SEQ ID NO: 190, LEELLEELIEELL, SEQ ID NO: 210, LEELLEELLEELLEE,SEQID NO: 213, LEQLLEDLVELLEEE, SEQ ID NO: 215, LEELLEDLVELLEEE, SEQ ID NO: 216, LEELLEELVELLEEE, SEQ ID NO: 217, LEELLELFEEILEELFE, SEQ ID NO: 218, LEELLKLFEEILEELFEE, SEQ ID NO: 219, IEELIELIEELLEE, SEQ ID NO: 220, IEELIEELIEELLEE, SEQ ID NO: 221, LEELLELIERLLEE, SEQ ID NO: 223, or LEELLELIEELLEE, SEQ ID NO: 225.

[0246] Implementation Scheme 15. The peptide isolated according to Implementation Scheme 1 further comprises a hydrophilic amino acid sequence at the N-terminus or C-terminus of SEQ ID NO: 93.

[0247] Implementation Scheme 16. The isolated peptide according to Implementation Scheme 15, wherein the peptide comprises an amino acid sequence of one of SEQ ID NO: 31, 33, 126, 134, 141, 149, 150, 161, 166, 167, 168-173, 178, 187-189, 200-202, 206-209, 231 or 232.

[0248] Implementation Scheme 17. The isolated peptide according to Implementation Scheme 1, wherein the peptide comprises the following amino acid sequence: SEEEEE LDQLLSQLIQALL (SEQ ID NO: 33) or residues 7-19 thereof; KQLDQLLSQLIQALLQP (SEQ ID NO: 94); EKQLDQLLSQLIQALLQP (SEQ ID NO: 95); SEKQLDQLLSQLIQALLQP (SEQ ID NO: 96); GISEKQLDQLLSQLIQALLQP (SEQ ID NO: 97); SQGISEKQLQLLSQLIQALLQP (SEQ ID NO: 132); SQGISEKQLDQLLQLIQALLQP (SEQ ID NO: 133); SQGISEKQALDQLLSQLIQALLQP (SEQ ID NO: 140); SEEEEE LDQLLTQLIEALL (SEQ ID NO: 141) or residues 7-19 thereof; SEEEEE IAKLISALIESLLE (SEQ ID NO: 150) or residues 7-20 thereof; SEEEEE LAQLLAQLLKSLL (SEQ ID NO: 167) or residues 7-19 thereof; SEEEEE LKALLKLIARLL (SEQ ID NO: 173) or residues 7-18 thereof; SEEEEE ALEQLLEDLVKLLK (SEQ ID NO: 178) or residues 7-20 thereof; SEEEEELTGVLQKLLKILEAL (SEQ ID NO: 188) or residues 7 - 21 thereof; SEELEELLEELIEELL (SEQ ID NO: 189); LEELLEELIEELLEE (SEQ ID NO: 190); SEEEEELTLTGVLQKLLKILEA (SEQ ID NO: 200) or residues 7 - 22 thereof; SEEEEEVLQKLLKILEALV (SEQ ID NO: 201) or residues 7 - 19 thereof; SEEEEELQKLLKILEALVQ (SEQ ID NO: 202) or residues 7 - 19 thereof; LEELLEELIEELL (SEQ ID NO: 210); LEELLEELLEELLEE (SEQ ID NO: 213); LEQLLEDLVELLEEE (SEQ ID NO: 215); LEELLEDLVELLEEE (SEQ ID NO: 216); LEELLEELVELLEEE (SEQ ID NO: 217); LEELLELFEEILEELFEE (SEQ ID NO: 218); LEELLKLFEEILEELFEE (SEQ ID NO: 219); IEELIELIEELLEE (SEQ ID NO: 220); IEELIEELIEELLEE (SEQ ID NO: 221); LEELLELIERLLEE (SEQ ID NO: 223); LEELLELIEELLEE (SEQ ID NO: 225); LEQLLEDLVKLLKEE (SEQ ID NO: 214); LEELLKLIERLLEE (SEQ ID NO: 222); or LEELLKLIEELLEE (SEQ ID NO: 224).

[0249] Implementation Scheme 18. An isolated peptide comprising the amino acid sequence XXGISEKXXXXXXXXXXXXXXXX (SEQ ID NO: 1, common to P1 / P4), wherein X at position 1 is optional and can be S, N, D, isoD, G, A or S; X at position 2 is optional and can be Q, E, g-glutamic acid, G, A or S; X at position 8 is Q, E, γ-glutamic acid, G, A, or S; X at position 9 is L, I, F, or V; X at position 10 is optional and can be D or isoD; X at position 11 is Q, E, γ-glutamic acid, G, A, or S; X at position 12 is M, L, I, or F; X at position 13 is M, L, or I; X at position 14 is optional and can be any hydrophilic amino acid; X at position 15 is Q, E, γ-glutamic acid, G, A, S, K, or I; X at position 16 is M, L, I, V, or F; X at position 17 is M, L, I, A, or V; X at position 18 is Q, E, γ-glutamic acid, G, A, S, M, T, or K; X at position 19 is A, D, isoD, S, V, T, K, R, E, H, or G; X at position 20 is M, L, or I; X at position 21 is M, L, I, V, S, or F; X at position 22 represents Q, E, γ-glutamic acid, G, A, and S. X at position 23 is P, Q, E, γ-glutamic acid, G, A, or S; and The isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the stability or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence.

[0250] Implementation Scheme 19. The isolated peptide according to Implementation Scheme 18, wherein the peptide is not composed of NQGISEKQLDQLLTQLIMALLQQ (P1, SEQ ID NO: 4).

[0251] Implementation Scheme 20. The isolated peptide according to Implementation Scheme 18, wherein the peptide is not composed of SQGISEKQLDQLLCQLIQALLQP (P4, SEQ ID NO: 5).

[0252] Implementation Scheme 21. The isolated peptide according to Implementation Scheme 18, wherein the polypeptide comprising the corresponding wild-type amino acid sequence is composed of SEQ ID NO: 4 or 5, and the isolated peptide is more stable in water or aqueous solution than the polypeptide of SEQ ID NO: 4 or 5.

[0253] Implementation Scheme 22. The isolated peptide according to Implementation Scheme 18, wherein the polypeptide containing the corresponding wild-type amino acid sequence is SEQ ID NO: 4 or 5, and the isolated peptide is more resistant to chemical degradation than the polypeptide of SEQ ID NO: 4 or 5 when dissolved in an aqueous buffer solution containing a biocide.

[0254] Implementation Scheme 23. The isolated peptide according to Implementation Scheme 18, wherein the peptide comprises the following amino acid sequence: SXGISEKXXDXXXXXXXXAXXXP (SEQ ID NO: 2, P4 shared), of which X at position 2 is Q, E, γ-glutamic acid, G, A, or S; X at position 8 is Q, E, γ-glutamic acid, G, A, or S; X at position 9 is L, A, D, isoD, I, V, or F; X at position 11 is Q, E, γ-glutamic acid, G, A, or S; X at position 12 is L, D, isoD, I, or F; X at position 13 is L, I, V, or F; X at position 14 can be any hydrophilic amino acid; X at position 15 is Q, E, γ-glutamic acid, G, A, S, K, or I; X at position 16 is L, A, I, V, M, or F; X at position 17 is I, S, or F; X at position 18 is Q, E, γ-glutamic acid, G, A, or S; X at position 20 is L, I, V, or F; X at position 21 is either L or F; and X at position 22 is Q, E, g-glutamic acid, G, A, or S.

[0255] Implementation Scheme 24. The peptide isolated according to Implementation Scheme 23, wherein X at position 14 is S.

[0256] Implementation Scheme 25. The isolated peptide according to Implementation Scheme 23, wherein the peptide comprises an amino acid sequence of one of SEQ ID NO: 6-11, 19, 20, 22-24, 27-30, 32, 34, 37, 40, 98-108, 111-114, 116, 117, 119-125, 127-131, 136-139, 191, and 196.

[0257] Implementation Scheme 26. The isolated peptide according to Implementation Scheme 18, wherein the peptide comprises the following amino acid sequence: XXGISEKXLDXLLTXLIXALLXX (SEQ ID NO: 3, P1 common), where X at position 1 is N, D, isoD, G, A, or S; X at position 2 is Q, E, γ-glutamic acid, G, A, or S; X at position 8 is Q, E, γ-glutamic acid, G, A, or S; X at position 11 is Q, E, γ-glutamic acid, G, A, or S; X at position 15 is Q, E, γ-glutamic acid, G, A, or S; X at position 18 is M, T, K, E, γ-glutamic acid, G, A, or S; X at position 22 is Q, E, γ-glutamic acid, G, A, or S; and X at position 23 is Q, E, g-glutamic acid, G, A, or S.

[0258] Implementation Scheme 27. The isolated peptide according to Implementation Scheme 26, wherein for at least one of positions 2, 8, 11, 15, 22 and 23 of SEQ ID NO: 3, X is E, g-glutamic acid, G, A or S.

[0259] Implementation Scheme 28. The isolated peptide according to Implementation Scheme 26, wherein the peptide comprises an amino acid sequence of one of SEQ ID NO: 41-46, 109, 110, 115 and 118.

[0260] Implementation Scheme 29. The isolated peptide according to Implementation Scheme 18, wherein the length of the peptide is less than 100 amino acids.

[0261] Implementation Scheme 30. The isolated peptide according to Implementation Scheme 29, wherein the length of the peptide is between 23 and 50 peptides.

[0262] Implementation Scheme 31. The isolated peptide according to Implementation Scheme 18, wherein the peptide consists substantially of the listed amino acid sequence.

[0263] Implementation Scheme 32. The isolated peptide according to Implementation Scheme 18, wherein the peptide consists of the listed amino acid sequences.

[0264] Implementation Scheme 33. An isolated peptide of less than 100 amino acids, said isolated peptide comprising the amino acid sequence of SEQ ID NO: 5.

[0265] Implementation Scheme 34. The isolated peptide according to Implementation Scheme 33, wherein the isolated peptide is substantially composed of SEQ ID NO: 5.

[0266] Implementation Scheme 35. The isolated peptide according to Implementation Scheme 33, wherein the isolated peptide comprises SEQ ID NO:5.

[0267] Implementation Scheme 36. An isolated peptide comprising the following amino acid sequence (i) KPXDSXSXIAKLISXLIXSLLX (SEQ ID NO: 47, shared by P15b / P20), where X at position 3 is N, D, or isoD; X at position 6 is Q, E, γ-glutamic acid, G, A, or S; X at position 8 is N, D, or isoD; X at position 15 is optional and can be any amino acid; X at position 18 is M, E, γ-glutamic acid, G, A, S, T, or K; and X at position 22 is optional and can be Q, E, γ-glutamic acid, G, A, or S; or (ii) IAKLISXLIXSLLX (SEQ ID NO: 12, P15 / 20 min co-occurring), of which The X at position 7 is optional and can be any amino acid; X at position 10 is M, E, γ-glutamic acid, G, A, S, T, or K; and X at position 14 is optional and can be Q, E, g-glutamic acid, G, A or S.

[0268] The isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the stability or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence.

[0269] Implementation Scheme 37. The isolated peptide according to Implementation Scheme 36, wherein the isolated peptide is less than 100 amino acids in length.

[0270] Implementation Scheme 38. The isolated peptide according to Implementation Scheme 36, wherein the polypeptide comprising the corresponding wild-type amino acid sequence is SEQ ID NO: 48, and the isolated peptide is more stable in water or aqueous solution than the polypeptide of SEQ ID NO: 48.

[0271] Implementation Scheme 39. The isolated peptide according to Implementation Scheme 36, wherein the polypeptide comprising the corresponding wild-type amino acid sequence is SEQ ID NO: 48, and the isolated peptide is more resistant to chemical degradation than the polypeptide of SEQ ID NO: 48 when dissolved in an aqueous buffer solution containing a biocide.

[0272] Implementation Scheme 40. The isolated peptide according to Implementation Scheme 36, wherein the peptide comprises an amino acid sequence of one of SEQ ID NO: 49-65, 142-144, 151 or 152.

[0273] Implementation Scheme 41. The isolated peptide according to Implementation Scheme 36, wherein the peptide consists substantially of the listed amino acid sequence.

[0274] Implementation Scheme 42. The isolated peptide according to Implementation Scheme 36, wherein the peptide consists of the listed amino acid sequences.

[0275] Implementation Scheme 43. An isolated peptide comprising 22 to 36 amino acids, wherein the peptide is substantially composed of the amino acids of SEQ ID NO: 49, SEQ ID NO: 63 or SEQ ID NO: 64.

[0276] Implementation Scheme 44. An isolated peptide comprising the amino acid sequence of SEQ ID NO: 65.

[0277] Implementation Scheme 45. An isolated peptide comprising the following amino acid sequence (i) PSPXTXXLXXIVGXILXAXN (SEQ ID NO: 66, peptide 6 / 6a co-occurring), in which X at position 4 is either F or Y; X at position 6 is Q, E, γ-glutamic acid, G, A, or S; X at position 7 is optional and can be M, E, γ-glutamic acid, G, A, S, T or K; X at position 9 is M, E, γ-glutamic acid, G, A, S, T, or K; X at position 10 is either H or N; X at position 14 is E, γ-glutamic acid, D, or isoD; X at position 17 is Q, E, γ-glutamic acid, G, A, or S; and X at position 19 is Q, E, γ-glutamic acid, G, A, or S; or (ii) XTXXLXXIVGXIL (SEQ ID NO: 135, P6 / 6a min co-occurring), of which X at position 1 is either F or Y; X at position 3 is Q, E, γ-glutamic acid, G, A, or S; X at position 4 is optional and, according to one embodiment, may be M, E, γ-glutamic acid, G, A, S, T, or K; or, according to another embodiment, may be L; X at position 6 is M, E, γ-glutamic acid, G, A, S, T, or K; X at position 7 is either H or N; and X at position 11 is E, γ-glutamic acid, D, or isoD; The isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the stability or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence.

[0278] Implementation Scheme 46. The isolated peptide according to Implementation Scheme 45, wherein the peptide is not composed of PSPFTQMLMHIVGEILQAQN (P6a, SEQ ID NO: 67).

[0279] Implementation Scheme 47. The isolated peptide according to Implementation Scheme 45, wherein the polypeptide comprising the corresponding wild-type amino acid sequence is SEQ ID NO: 67, and the isolated peptide is more stable in water or aqueous solution than the polypeptide of SEQ ID NO: 67.

[0280] Implementation Scheme 48. The isolated peptide according to Implementation Scheme 45, wherein the polypeptide comprising the corresponding wild-type amino acid sequence is SEQ ID NO: 67, and the isolated peptide is more resistant to chemical degradation than the polypeptide of SEQ ID NO: 67 when dissolved in an aqueous buffer solution containing a biocide.

[0281] Implementation Scheme 49. The isolated peptide according to Implementation Scheme 45, wherein the peptide comprises an amino acid sequence of one of SEQ ID NO: 68-80, 155-161, 197 and 198.

[0282] Implementation Scheme 50. The isolated peptide according to Implementation Scheme 45, wherein the length of the peptide is less than 100 amino acids.

[0283] Implementation Scheme 51. The isolated peptide according to Implementation Scheme 45, wherein the length of the peptide is between 20 and 50 peptides.

[0284] Implementation Scheme 52. The isolated peptide according to Implementation Scheme 45, wherein the peptide consists substantially of the listed amino acid sequence.

[0285] Implementation Scheme 53. The isolated peptide according to Implementation Scheme 45, wherein the peptide consists of the listed amino acid sequence.

[0286] Implementation Scheme 54. The isolated peptide according to Implementation Scheme 45, wherein the peptide does not contain SEQ ID NO:154.

[0287] Implementation Scheme 55. The isolated peptide according to Implementation Scheme 45, wherein the peptide comprises the amino acid sequence PSPXTXXLXXIVGXILXAXN (SEQ ID NO: 66, common to peptide 6 / 6a).

[0288] Implementation Scheme 56. The isolated peptide according to Implementation Scheme 45, wherein the peptide comprises the amino acid sequence XTXXLXXIVGXIL (SEQ ID NO: 135, P6 / 6a min common).

[0289] Implementation Scheme 57. An isolated peptide comprising the following amino acid sequence (i) LXXL(L / M)XILXXLV (SEQ ID NO: 16, P25 total) X at position 2 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 3 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 6 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 9 can be E, γ-glutamic acid, D, isoD, Q, N, T, S, A, or G; and X at position 10 can be A, G, S, T, E, γ-glutamic acid, D, isoD, Q, or N; or (ii) LXXVLXXL(L / M)XILXXLV (SEQ ID NO: 17, P25 total) of which X at position 2 can be T, S, A, G, D, isoD, E, γ-glutamic acid, Q, or N; X at position 3 can be G, T, S, A, D, isoD, E, γ-glutamic acid, Q, or N; X at position 6 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 7 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 10 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 13 can be E, g-glutamic acid, D, isoD, Q, N, T, S, A, or G; X at position 14 can be A, G, S, T, E, γ-glutamic acid, D, isoD, Q, or N; and The V at position 16 is optional.

[0290] Implementation Scheme 58. The isolated peptide according to Implementation Scheme 57, wherein the peptide is not composed of SEQ ID NO: 179.

[0291] Implementation Scheme 59. The isolated peptide according to Implementation Scheme 57, wherein the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, the polypeptide containing the corresponding wild-type amino acid sequence is SEQ ID NO: 179, and the isolated peptide is more stable in water or aqueous solution than the polypeptide of SEQ ID NO: 179.

[0292] Implementation Scheme 60. The isolated peptide according to Implementation Scheme 57, wherein the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, the polypeptide containing the corresponding wild-type amino acid sequence is SEQ ID NO: 179, and the isolated peptide is more resistant to chemical degradation than the polypeptide of SEQ ID NO: 179 when dissolved in an aqueous buffer solution containing a biocide.

[0293] Implementation Scheme 61. The isolated peptide according to Implementation Scheme 57, wherein the peptide comprises the amino acid sequence LXXL(L / M)XILXXLV (SEQ ID NO: 16, P25 common).

[0294] Implementation Scheme 62. The isolated peptide according to Implementation Scheme 57, wherein the peptide comprises the amino acid sequence LXXVLXXL(L / M)XILXXLV (SEQ ID NO: 17, P25 common).

[0295] Implementation Scheme 63. The isolated peptide according to Implementation Scheme 57, wherein the peptide comprises the amino acid sequence LXXVLXXL(L / M)XILXXL (SEQ ID NO: 17, P25 common).

[0296] Implementation Scheme 64. The isolated peptide according to Implementation Scheme 57, wherein the peptide comprises an amino acid sequence of one of SEQ ID NO: 180-188.

[0297] Implementation Scheme 65. The isolated peptide according to Implementation Scheme 57, wherein the length of the peptide is less than 100 amino acids.

[0298] Implementation Scheme 66. The isolated peptide according to Implementation Scheme 57, wherein the length of the peptide is between 20 and 50 peptides.

[0299] Implementation Scheme 67. An isolated peptide comprising the following amino acid sequence (i) XXXXXXXXXXX(L / M)XXLLXXLLXXLLXXX (SEQ ID NO: 18, P17 / 18), where X at position 1 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 2 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 3 can be any amino acid, but is preferably P, Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 4 can be any amino acid, but is preferably I, Q, S, E, γ-glutamic acid, A, T, G, D, N, isoD, K, or R; X at position 5 can be any amino acid, but D, isoD, S, E, γ-glutamic acid, A, T, G, N, Q, K or R are preferred. X at position 6 can be any amino acid, but is preferably R, Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, or K; X at position 7 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 8 can be any amino acid, but is preferably T, Q, S, E, γ-glutamic acid, A, G, D, isoD, N, K, or R; X at position 9 can be any amino acid, but is preferably I, Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 10 can be any amino acid, but is preferably E, γ-glutamic acid, Q, S, A, T, G, D, isoD, N, K or R; X at position 11 can be any amino acid, but is preferably Q, S, E, γ-glutamic acid, A, T, G, D, isoD, N, K, or R; X at position 13 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 14 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 17 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 18 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 21 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N or R; X at position 22 can be any amino acid, but is preferably S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 25 can be any amino acid, but is preferably S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 26 can be any amino acid, but is preferably P, S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K, or R; and X at position 27 can be any amino acid, but is preferably Q, S, A, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; or (ii) (L / M)XXLLXXLLXXLL (SEQ ID NO: 25, P17 / 18 min total), of which X at position 2 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 3 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 6 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 7 can be any amino acid, but is preferably Q, A, S, T, G, D, isoD, E, γ-glutamic acid, N, K, or R; X at position 10 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or R; and X at position 11 can be any amino acid, but is preferably S, A, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; The isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, and the one or more mutations improve the water solubility, stability, or resistance to chemical degradation of the isolated peptide relative to a polypeptide containing the corresponding wild-type amino acid sequence.

[0300] Implementation Scheme 68. The isolated peptide according to Implementation Scheme 67, wherein the peptide is not composed of SEQ ID NO: 162.

[0301] Implementation Scheme 69. The isolated peptide according to Implementation Scheme 67, wherein the polypeptide comprising the corresponding wild-type amino acid sequence is SEQ ID NO: 162, and the isolated peptide is more stable in water or aqueous solution than the polypeptide of SEQ ID NO: 162.

[0302] Implementation Scheme 70. The isolated peptide according to Implementation Scheme 67, wherein the polypeptide comprising the corresponding wild-type amino acid sequence is SEQ ID NO: 162, and the isolated peptide is more resistant to chemical degradation than the polypeptide of SEQ ID NO: 162 when dissolved in an aqueous buffer solution containing a biocide.

[0303] Implementation Scheme 71. The isolated peptide according to Implementation Scheme 67, wherein the peptide comprises an amino acid sequence of one of SEQ ID NO: 84-88, 163-167, 228, 229 and 231.

[0304] Implementation Scheme 72. The isolated peptide according to Implementation Scheme 67, wherein the peptide comprises the amino acid sequence XXXXXXXXXXX(L / M)XXLLXXLLXXLLXXX (SEQ ID NO: 18, P17 / 18 common).

[0305] Implementation Scheme 73. The peptide isolated according to Implementation Scheme 67, wherein the peptide comprises the amino acid sequence (L / M)XXLLXXLLXXLL (SEQ ID NO: 25, P17 / 18 min common).

[0306] Implementation Scheme 74. The isolated peptide according to Implementation Scheme 67, wherein the length of the peptide is less than 100 amino acids.

[0307] Implementation Scheme 75. The isolated peptide according to Implementation Scheme 67, wherein the length of the peptide is between 20 and 50 peptides.

[0308] Implementation Scheme 76. An isolated peptide comprising the amino acid sequence XLXX(L / M)LXLIXX(L / I / V / F / M)(L / I / V / F / M) (SEQ ID NO: 26, P19 common), wherein X at position 1 is optional and can be L, I, V, F or M; X at position 3 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or R; X at position 4 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K or R; X at position 7 can be any amino acid, but is preferably K, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or R; X at position 10 can be any amino acid, but is preferably A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, K, or R; and X at position 11 can be any amino acid, but is preferably R, A, S, T, G, D, isoD, E, γ-glutamic acid, Q, N, or K.

[0309] Implementation Scheme 77. The isolated peptide according to Implementation Scheme 76, wherein the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, the polypeptide containing the corresponding wild-type amino acid sequence is SEQ ID NO: 89, and the isolated peptide is more stable in water or aqueous solution than the polypeptide of SEQ ID NO: 89.

[0310] Implementation Scheme 78. The isolated peptide according to Implementation Scheme 76, wherein the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, the polypeptide containing the corresponding wild-type amino acid sequence is SEQ ID NO: 89, and the isolated peptide is more resistant to chemical degradation than the polypeptide of SEQ ID NO: 89 when dissolved in an aqueous buffer solution containing a biocide.

[0311] Implementation Scheme 79. The isolated peptide according to Implementation Scheme 76, wherein the peptide comprises an amino acid sequence of one of SEQ ID NO: 90-92, 168-173 and 226.

[0312] Implementation Scheme 80. The isolated peptide according to Implementation Scheme 76, wherein the length of the peptide is less than 100 amino acids.

[0313] Implementation Scheme 81. The isolated peptide according to Implementation Scheme 76, wherein the length of the peptide is between 20 and 50 peptides.

[0314] Implementation Scheme 82. The isolated peptide according to Implementation Scheme 76, wherein the amino acid residue at position 1 is present.

[0315] Implementation Scheme 83. An isolated peptide comprising the following amino acid sequence (i) XXXXXXLXXLLXXLVXLLK (SEQ ID NO: 13, P14d common), where X at position 1 can be: Q, N, D, E, g-glutamic acid, isoD, or S; X at position 2 could be: D, E, γ-glutamic acid, or isoD; X at position 3 can be: P, D, E, isoD, or γ-glutamic acid; X at position 4 can be M, A, S, D, E, isoD, or γ-glutamic acid. X at position 5 can be Q, E, or g-glutamic acid; X at position 6 can be A, E, or g-glutamic acid; X at position 8 can be M, L, E, Q, D, N, G, A, S, isoD, or γ-glutamic acid; X at position 9 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 12 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 13 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; and X at position 16 can be K, Q, N, E, D, R, G, A, or S; or (ii) LXXLLXXLVXLLK (SEQ ID NO: 14, P14d min common), where X at position 2 can be M, L, E, Q, D, N, G, A, S, isoD, or γ-glutamic acid; X at position 3 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 6 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; X at position 7 can be Q, N, E, D, G, A, S, isoD, or γ-glutamic acid; and X at position 10 can be K, Q, N, E, D, R, G, A, or S.

[0316] Implementation Scheme 84. The isolated peptide according to Implementation Scheme 83, wherein the peptide is not composed of SEQ ID NO: 174.

[0317] Implementation Scheme 85. The isolated peptide according to Implementation Scheme 83, wherein the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, the polypeptide containing the corresponding wild-type amino acid sequence is SEQ ID NO: 174, and the isolated peptide is more stable in water or aqueous solution than the polypeptide of SEQ ID NO: 174.

[0318] Implementation Scheme 86. The isolated peptide according to Implementation Scheme 83, wherein the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, the polypeptide containing the corresponding wild-type amino acid sequence is SEQ ID NO: 174, and the isolated peptide is more resistant to chemical degradation than the polypeptide of SEQ ID NO: 174 when dissolved in an aqueous buffer solution containing a biocide.

[0319] Implementation Scheme 87. The isolated peptide according to Implementation Scheme 83, wherein the peptide comprises an amino acid sequence of one of SEQ ID NO: 175-178 and 199.

[0320] Implementation Scheme 88. The isolated peptide according to Implementation Scheme 83, wherein the peptide comprises the amino acid sequence XXXXXXLXXLLXXLVXLLK (SEQ ID NO: 13, P14d common).

[0321] Implementation Scheme 89. The isolated peptide according to Implementation Scheme 83, wherein the peptide comprises the amino acid sequence LXXLLXXLVXLLK (SEQ ID NO: 14, common to P14d min).

[0322] Implementation Scheme 90. The isolated peptide according to Implementation Scheme 83, wherein the length of the peptide is less than 100 amino acids.

[0323] Implementation Scheme 91. The isolated peptide according to Implementation Scheme 83, wherein the length of the peptide is between 12 and 50 peptides.

[0324] Implementation Scheme 92. An isolated peptide comprising the following amino acid sequence (i) (L / M)XXLLX(L / M)FXXI(L / M)XX (SEQ ID NO: 15, P3min total) where X at position 2 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 3 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 6 can be K, Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G; X at position 9 can be E, γ-glutamic acid, D, isoD, Q, N, T, S, A, or G; X at position 10 can be A, G, S, T, E, γ-glutamic acid, D, isoD, Q, or N; X at position 13 can be Q, N, E, γ-glutamic acid, D, isoD, T, S, A, or G; and X at position 14 can be Q, N, E, g-glutamic acid, D, isoD, T, S, A, or G.

[0325] Implementation Scheme 93. The isolated peptide according to Implementation Scheme 92, wherein the peptide is not composed of SEQ ID NO: 230 or the C-terminal 31 amino acid portion thereof.

[0326] Implementation Scheme 94. The isolated peptide according to Implementation Scheme 92, wherein the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, the polypeptide containing the corresponding wild-type amino acid sequence is SEQ ID NO: 230, and the isolated peptide is more stable in water or aqueous solution than the polypeptide of SEQ ID NO: 230.

[0327] Implementation Scheme 95. The isolated peptide according to Implementation Scheme 92, wherein the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, the polypeptide containing the corresponding wild-type amino acid sequence is SEQ ID NO: 230, and the isolated peptide is more resistant to chemical degradation than the polypeptide of SEQ ID NO: 230 when dissolved in an aqueous buffer solution containing a biocide.

[0328] Implementation Scheme 96. The isolated peptide according to Implementation Scheme 92, wherein the isolated peptide contains one or more mutations relative to the corresponding wild-type amino acid sequence, the polypeptide containing the corresponding wild-type amino acid sequence is SEQ ID NO: 230, and the isolated peptide is more soluble in aqueous solution than the polypeptide of SEQ ID NO: 230.

[0329] Implementation Scheme 97. The isolated peptide according to Implementation Scheme 92, wherein the peptide is composed of SEQ ID NO: 204.

[0330] Implementation Scheme 98. The isolated peptide according to Implementation Scheme 92, wherein the peptide comprises an amino acid sequence of one of SEQ ID NO: 205-209.

[0331] Implementation Scheme 99. The isolated peptide according to Implementation Scheme 92, wherein the length of the peptide is less than 100 amino acids.

[0332] Implementation Scheme 100. The isolated peptide according to Implementation Scheme 92, wherein the length of the peptide is between 12 and 30 peptides.

[0333] Implementation Scheme 101. The isolated peptide according to any one of Implementation Schemes 1 to 100, wherein the peptide has a purity of at least 90%.

[0334] Implementation Scheme 102. The isolated peptide according to any one of Implementation Schemes 1 to 30, 33, 36 to 40, 44 to 51, 54 to 96 and 98 to 100, wherein the peptide is a fusion polypeptide comprising a second amino acid sequence coupled to the amino acid sequence via a peptide bond.

[0335] Implementation Scheme 103. The isolated peptide according to Implementation Scheme 102, wherein the second amino acid sequence includes a purification tag.

[0336] Implementation Scheme 104. The isolated peptide according to Implementation Scheme 103, wherein the second amino acid sequence further comprises a cleavable linker sequence between the purification tag and the amino acid sequence.

[0337] Implementation Scheme 105. The isolated peptide according to Implementation Scheme 102, wherein the peptide is a fusion polypeptide comprising a first amino acid sequence of the peptide linked to a second amino acid sequence of the peptide.

[0338] Implementation Scheme 106. The isolated peptide according to Implementation Scheme 102, wherein the second amino acid sequence comprises an N-terminal or C-terminal hydrophilic amino acid sequence.

[0339] Implementation Scheme 107. The isolated peptide according to Implementation Scheme 106, wherein the hydrophilic amino acid sequence comprises a plurality of Glu (E) amino acid residues.

[0340] Implementation Scheme 108. A fusion polypeptide comprising a plurality of amino acid sequences linked together in tandem, each of the plurality of amino acid sequences constituting a peptide according to one of Implementation Schemes 1 to 30, 33, 36 to 40, 44 to 51, 54 to 96 and 98 to 100.

[0341] Implementation Scheme 109. The fusion polypeptide according to Implementation Scheme 108, wherein the plurality of amino acid sequences are linked together by a cleavable linker sequence.

[0342] Implementation Scheme 110. The fusion polypeptide according to Implementation Scheme 108, wherein each of the plurality of amino acid sequences comprises a purified tag, an N-terminal or C-terminal hydrophilic amino acid sequence, or both.

[0343] Implementation Scheme 111. A composition comprising one or more peptides according to any one of Implementation Schemes 1 to 107 or a fusion polypeptide according to any one of Implementation Schemes 108 to 110 and a carrier.

[0344] Implementation Scheme 112. The composition according to Implementation Scheme 111, wherein the composition is a clarified cell extract.

[0345] Implementation Scheme 113. The composition according to Implementation Scheme 111 further comprises an additive selected from fertilizers, herbicides, insecticides, fungicides, nematicides, bactericides, biological inoculants, plant regulators, and mixtures thereof.

[0346] Implementation Scheme 114. The composition according to Implementation Scheme 113, wherein the insecticide is a neonicotinoid insecticide, an organophosphate insecticide, a pyrethroid insecticide, a macrolide insecticide, a carbamate insecticide, a diamide insecticide, an abamectin insecticide, a chitin synthesis inhibitor, or any combination thereof.

[0347] Implementation Scheme 115. The composition according to Implementation Scheme 113, wherein the fungicide is a methoxyacrylate fungicide, a triazole fungicide, a succinate dehydrogenase fungicide, a phenylamide fungicide, a phenylpyrrole fungicide, a phthalimide fungicide, a dithiocarbamate fungicide, a benzimidazole fungicide, or any combination thereof.

[0348] Implementation Scheme 116. The composition according to Implementation Scheme 113, wherein the nematicide is a carbamate nematicide.

[0349] Implementation Scheme 117. The composition according to Implementation Scheme 113, wherein the bactericide is a dichlorophenol and benzyl alcohol hemiacetal bactericide, an isothiazolinone bactericide, or a combination thereof.

[0350] Implementation Scheme 118. The composition according to Implementation Scheme 113, wherein the biological inoculant is a species of Rhizobium, Bacillus, Streptomyces, Trichoderma, Pasteurella or any combination thereof.

[0351] Implementation Scheme 119. The composition according to Implementation Scheme 113, wherein the composition comprises: One or more of peptides P1, P4-14S, P6a, P14d, P15a, P18, P19, or P25; and Thiamethoxam, a combination of thiamethoxam and Bacillus thuringiensis, imidacloprid or a combination of imidacloprid and Bacillus thuringiensis.

[0352] Implementation Scheme 120. The composition according to Implementation Scheme 113, wherein the composition comprises: One or more of peptides P1, P4-14S, P6a, P14d, P15a, P18, P19, or P25; and Thiamethoxam; combinations of thiamethoxam, metalaxyl, and fludioxonil; combinations of thiamethoxam, metalaxyl, fludioxonil, and azoxystrobin; combinations of thiamethoxam and abamectin; combinations of thiamethoxam, abamectin, and Pasteurella nematicides; or combinations of thiamethoxam, metalaxyl, fludioxonil, azoxystrobin, thiabendazole, and abamectin.

[0353] Implementation Scheme 121. The composition according to Implementation Scheme 113, wherein the composition comprises: One or more of peptides P1, P4-14S, P6a, P14d, P15a, P18, P19, or P25; and Biological inoculants containing species of the genera *Staphylococcus*, *Bacillus*, and combinations thereof.

[0354] Implementation Scheme 122. The composition according to Implementation Scheme 111, wherein the carrier is an aqueous carrier.

[0355] Implementation Scheme 123. The composition according to Implementation Scheme 122, wherein the aqueous carrier further comprises one or more of a biocide, a protease inhibitor, a nonionic surfactant, or a combination thereof.

[0356] Implementation Scheme 124. The composition according to Implementation Scheme 111, wherein the carrier is a solid carrier in the form of particles.

[0357] Implementation Scheme 125. The composition according to Implementation Scheme 124, wherein the solid carrier is a dry powder.

[0358] Implementation Scheme 126. A method for conferring disease resistance to plants, comprising: Applying an effective amount of an isolated peptide according to one of embodiments 1 to 107, a fusion polypeptide according to one of embodiments 108 to 110, or a composition according to one of embodiments 111 to 125 to a plant or plant seed or the site where the plant is growing or is intended to grow, wherein the application effectively confers disease resistance.

[0359] Implementation Scheme 127. The method according to Implementation Scheme 126, wherein the disease is a viral disease, a bacterial disease, or a fungal disease.

[0360] Implementation Scheme 128. The method according to Implementation Scheme 126, wherein the application is carried out using plants.

[0361] Implementation Scheme 129. The method according to Implementation Scheme 128, wherein the plant is tolerant to at least one herbicide.

[0362] Implementation Scheme 130. The method according to Implementation Scheme 126, wherein the application is carried out using plant seeds, the method further comprising planting the seeds treated with the peptide or composition in natural or artificial soil, and propagating plants from the seeds planted in the soil.

[0363] Implementation Scheme 131. The method according to Implementation Scheme 126, wherein the application is performed at the site where the plant is growing or is expected to grow.

[0364] Implementation Scheme 132. The method according to Implementation Scheme 126, wherein the plant is selected from agricultural, afforestation, ornamental and horticultural plants, each in its natural or genetically modified form.

[0365] Implementation Scheme 133. The method according to Implementation Scheme 126, wherein the plant is a genetically modified plant.

[0366] Implementation Scheme 134. The method according to Implementation Scheme 126, wherein the plants to be treated are selected from alfalfa, apple trees, apricot trees, asparagus, avocado trees, banana trees, barley, beans, beech (beech species), begonia, birch, blackberry, blueberry, cabbage, camphor, mustard, carrot, castor bean, cherry tree, cinnamon tree, citrus tree, cocoa bean, coffee, corn, cotton, cucumber, gourd, eucalyptus, fir, flax, fodder beet, fuchsia, garlic, geranium, grape, peanut, hemp, hops, iris, mustard (mustard greens) Vegetables include rapeseed, jute, lentils, lettuce, flaxseed, melon, mustard, oak, oats, oil palm, oilseed rape, olive, onion, pepper, pea, peach, pear, geranium, pepper, petunia, pine (Pinus species), poplar (Populus species), pome fruit, potato, rapeseed, raspberry, rice, rubber tree, rye, sorghum, soybean, spinach, spruce, pumpkin, strawberry, sugar beet, sugarcane, sunflower, tea tree, teak, tobacco, tomato, black wheat, turf, watermelon, wheat, and willow (Salix species).

[0367] Implementation Scheme 135. A method for enhancing plant growth, comprising: Applying an effective amount of an isolated peptide according to one of embodiments 1 to 107, a fusion polypeptide according to one of embodiments 108 to 110, or a composition according to one of embodiments 111 to 125 to a plant or plant seed or the site where the plant is growing or is expected to grow, wherein the application effectively enhances plant growth.

[0368] Implementation Scheme 136. The method according to Implementation Scheme 135, wherein the enhanced growth includes increased plant vigor, increased plant weight, increased biomass, increased number of flowers per plant, higher grain and / or fruit yield, more tillers or lateral branches, larger leaves, increased branch growth, increased protein content, increased oil content, increased starch content, increased pigment content, increased chlorophyll content, and combinations thereof.

[0369] Implementation Scheme 137. The method according to Implementation Scheme 135, wherein the application is carried out using plants.

[0370] Implementation Scheme 138. The method according to Implementation Scheme 137, wherein the plant is tolerant to at least one herbicide.

[0371] Implementation Scheme 139. The method according to Implementation Scheme 135, wherein the application is carried out using plant seeds, the method further comprising planting the seeds treated with the peptide or composition in natural or artificial soil, and propagating plants from the seeds planted in the soil.

[0372] Implementation Scheme 140. The method according to Implementation Scheme 135, wherein the application is performed at the site where the plant is growing or is intended to grow.

[0373] Implementation Scheme 141. The method according to Implementation Scheme 135, wherein the plant is selected from agricultural, afforestation, ornamental and horticultural plants, each in its natural or genetically modified form.

[0374] Implementation Scheme 142. The method according to Implementation Scheme 135, wherein the plant is a genetically modified plant.

[0375] Implementation Scheme 143. The method according to Implementation Scheme 135, wherein the plants to be treated are selected from alfalfa, apple trees, apricot trees, asparagus, avocado trees, banana trees, barley, beans, beech (beech species), begonia, birch, blackberry, blueberry, cabbage, camphor, mustard, carrot, castor bean, cherry tree, cinnamon tree, citrus tree, cocoa bean, coffee, corn, cotton, cucumber, gourd, eucalyptus, fir, flax, fodder beet, fuchsia, garlic, geranium, grape, peanut, hemp, hops, iris, mustard (mustard greens) Vegetables include rapeseed, jute, lentils, lettuce, flaxseed, melon, mustard, oak, oats, oil palm, oilseed rape, olive, onion, pepper, pea, peach, pear, geranium, pepper, petunia, pine (Pinus species), poplar (Populus species), pome fruit, potato, rapeseed, raspberry, rice, rubber tree, rye, sorghum, soybean, spinach, spruce, pumpkin, strawberry, sugar beet, sugarcane, sunflower, tea tree, teak, tobacco, tomato, black wheat, turf, watermelon, wheat, and willow (Salix species).

[0376] Implementation Scheme 144. A method for increasing the tolerance of plants to biotic stress, comprising: Applying an effective amount of an isolated peptide according to one of embodiments 1 to 107, a fusion polypeptide according to one of embodiments 108 to 110, or a composition according to one of embodiments 111 to 125 to a plant or plant seed or the site where the plant is growing or is intended to grow, wherein the application effectively increases the plant’s tolerance to biological stressors selected from insects, spiders, nematodes, weeds, and combinations thereof.

[0377] Implementation Scheme 145. The method according to Implementation Scheme 144, wherein the application is carried out using plants.

[0378] Implementation Scheme 146. The method according to Implementation Scheme 145, wherein the plant is tolerant to at least one herbicide.

[0379] Implementation Scheme 147. The method according to Implementation Scheme 144, wherein the application is carried out using plant seeds, the method further comprising planting the seeds treated with the peptide or composition in natural or artificial soil, and propagating plants from the seeds planted in the soil.

[0380] Implementation Scheme 148. The method according to Implementation Scheme 144, wherein the application is performed at the site where the plant grows or is expected to grow.

[0381] Implementation Scheme 149. The method according to Implementation Scheme 144, wherein the plant is selected from agricultural, afforestation, ornamental and horticultural plants, each in its natural or genetically modified form.

[0382] Implementation Scheme 150. The method according to Implementation Scheme 144, wherein the plant is a genetically modified plant.

[0383] Implementation Scheme 151. The method according to Implementation Scheme 144, wherein the plants to be treated are selected from alfalfa, apple trees, apricot trees, asparagus, avocado trees, banana trees, barley, beans, beech (beech species), begonia, birch, blackberry, blueberry, cabbage, camphor, mustard, carrot, castor bean, cherry tree, cinnamon tree, citrus tree, cocoa bean, coffee, corn, cotton, cucumber, gourd, eucalyptus, fir, flax, fodder beet, fuchsia, garlic, geranium, grape, peanut, hemp, hops, iris, mustard (mustard greens) Vegetables include rapeseed, jute, lentils, lettuce, flaxseed, melon, mustard, oak, oats, oil palm, oilseed rape, olive, onion, pepper, pea, peach, pear, geranium, pepper, petunia, pine (Pinus species), poplar (Populus species), pome fruit, potato, rapeseed, raspberry, rice, rubber tree, rye, sorghum, soybean, spinach, spruce, pumpkin, strawberry, sugar beet, sugarcane, sunflower, tea tree, teak, tobacco, tomato, black wheat, turf, watermelon, wheat, and willow (Salix species).

[0384] Implementation Scheme 152. A method for increasing plant tolerance to abiotic stress, comprising: Applying an effective amount of an isolated peptide according to one of embodiments 1 to 107, a fusion polypeptide according to one of embodiments 108 to 110, or a composition according to one of embodiments 111 to 125 to a plant or plant seed or the site where the plant is growing or is expected to grow, wherein the application effectively increases the plant’s tolerance to abiotic stressors selected from salt stress, water stress, ozone stress, heavy metal stress, low temperature stress, high temperature stress, nutrient stress, and combinations thereof.

[0385] Implementation Scheme 153. The method according to Implementation Scheme 152, wherein the application is carried out using plants.

[0386] Implementation Scheme 154. The method according to Implementation Scheme 153, wherein the plant is tolerant to at least one herbicide.

[0387] Implementation Scheme 155. The method according to Implementation Scheme 152, wherein the application is carried out using plant seeds, the method further comprising planting the seeds treated with the peptide or composition in natural or artificial soil, and propagating plants from the seeds planted in the soil.

[0388] Implementation Scheme 156. The method according to Implementation Scheme 152, wherein the application is performed at the site where the plant grows or is expected to grow.

[0389] Implementation Scheme 157. The method according to Implementation Scheme 152, wherein the plant is selected from agricultural, afforestation, ornamental and horticultural plants, each in its natural or genetically modified form.

[0390] Implementation Scheme 158. The method according to Implementation Scheme 152, wherein the plant is a genetically modified plant.

[0391] Implementation Scheme 159. The method according to Implementation Scheme 152, wherein the plants to be treated are selected from alfalfa, apple trees, apricot trees, asparagus, avocado trees, banana trees, barley, beans, beech (beech species), begonia, birch, blackberry, blueberry, cabbage, camphor, mustard, carrot, castor bean, cherry tree, cinnamon tree, citrus tree, cocoa bean, coffee, corn, cotton, cucumber, gourd, eucalyptus, fir, flax, fodder beet, fuchsia, garlic, geranium, grape, peanut, hemp, hops, iris, mustard (mustard greens) Vegetables include rapeseed, jute, lentils, lettuce, flaxseed, melon, mustard, oak, oats, oil palm, oilseed rape, olive, onion, pepper, pea, peach, pear, geranium, pepper, petunia, pine (Pinus species), poplar (Populus species), pome fruit, potato, rapeseed, raspberry, rice, rubber tree, rye, sorghum, soybean, spinach, spruce, pumpkin, strawberry, sugar beet, sugarcane, sunflower, tea tree, teak, tobacco, tomato, black wheat, turf, watermelon, wheat, and willow (Salix species).

[0392] Implementation Scheme 160. A method for imparting desiccation resistance to cuttings taken from ornamental plants, comprising: Applying an effective amount of the isolated peptide according to one of embodiments 1 to 107, the fusion polypeptide according to one of embodiments 108 to 110, or the composition according to one of embodiments 111 to 125 to the plant or the site where the plant grows, wherein the application effectively imparts anti-drying properties to the cuttings taken from the ornamental plant.

[0393] Implementation Scheme 161. The method according to Implementation Scheme 160, wherein the application is carried out using ornamental plants.

[0394] Implementation Scheme 162. The method according to Implementation Scheme 160, wherein the application is performed at the location where the ornamental plant grows.

[0395] Implementation Scheme 163. The method according to Implementation Scheme 160, wherein the ornamental plant is a genetically modified ornamental plant.

[0396] Implementation Scheme 164. The method according to Implementation Scheme 160, wherein the plants to be treated are selected from beech (beech species), begonia, birch, ornamental cabbage, fir, fuchsia, garlic, geranium, oak, ornamental onion, geranium, petunia, pine (pine species), poplar (poplar species), sunflower, teak, tobacco, turf, and willow (willow species).

[0397] Implementation Scheme 165. A method for conferring postharvest disease resistance or postharvest drought resistance to fruits or vegetables, comprising: Applying an effective amount of the isolated peptide according to any one of embodiments 1 to 107, the fusion polypeptide according to any one of embodiments 108 to 110, or the composition according to any one of embodiments 111 to 125 to a plant containing fruit or vegetable or the site where said plant grows, or Apply an effective amount of the isolated peptide or the composition to harvested fruits or vegetables. The application of the above-mentioned substance effectively imparts postharvest disease resistance or postharvest drought resistance to the fruit or vegetable.

[0398] Implementation Scheme 166. The method according to Implementation Scheme 165, wherein the application is carried out using plants.

[0399] Implementation Scheme 167. The method according to Implementation Scheme 166, wherein the plant is tolerant to at least one herbicide.

[0400] Implementation Scheme 168. The method according to Implementation Scheme 165, wherein the application is performed at the site where the plant grows.

[0401] Implementation Scheme 169. The method according to Implementation Scheme 165, wherein the application is carried out using harvested fruits or vegetables.

[0402] Implementation Scheme 170. The method according to Implementation Scheme 165, wherein the plant is a genetically modified plant.

[0403] Implementation Scheme 171. The method according to Implementation Scheme 165, wherein the plants are selected from apple trees, apricot trees, asparagus, avocado trees, banana trees, blackberries, blueberries, cabbage, carrots, cherry trees, citrus trees, corn, cucumbers, gourds, fodder beets, garlic, grapes, dandelion, mustard (mustard-type rapeseed), lettuce, melons, mustard, olives, onions, peas, peach trees, pear trees, peppers, pome fruits, potatoes, rapeseed, raspberries, spinach, pumpkins, strawberries, sugar beets, sugarcane, tea trees, tomatoes, black wheat, and watermelons.

[0404] Implementation Scheme 172. A method for enhancing the ripening life of fruits or vegetables, comprising: Applying an effective amount of the isolated peptide according to any one of embodiments 1 to 107, the fusion polypeptide according to any one of embodiments 108 to 110, or the composition according to any one of embodiments 111 to 125 to a plant containing fruit or vegetable or the site where said plant grows, or Apply an effective amount of the isolated peptide or the composition to harvested fruits or vegetables. The application of these substances effectively enhances the ripening life of fruits or vegetables.

[0405] Implementation Scheme 173. The method according to Implementation Scheme 172, wherein the application is carried out using plants.

[0406] Implementation Scheme 174. The method according to Implementation Scheme 172, wherein the plant is tolerant to at least one herbicide.

[0407] Implementation Scheme 175. The method according to Implementation Scheme 172, wherein the application is performed at the site where the plant grows.

[0408] Implementation Scheme 176. The method according to Implementation Scheme 172, wherein the application is carried out using harvested fruits or vegetables.

[0409] Implementation Scheme 177. The method according to Implementation Scheme 172, wherein the plant is a genetically modified plant.

[0410] Implementation Scheme 178. The method according to Implementation Scheme 172, wherein the plants are selected from apple trees, apricot trees, asparagus, avocado trees, banana trees, blackberries, blueberries, cabbage, carrots, cherry trees, citrus trees, corn, cucumbers, gourds, fodder beets, garlic, grapes, dandelion, mustard (mustard-type rapeseed), lettuce, melons, mustard, olives, onions, peas, peach trees, pear trees, peppers, pome fruits, potatoes, rapeseed, raspberries, spinach, pumpkins, strawberries, sugar beets, sugarcane, tea trees, tomatoes, black wheat, and watermelons.

[0411] Implementation Scheme 179. A method for regulating plant biochemical signal transduction, comprising: Applying an effective amount of an isolated peptide according to one of embodiments 1 to 107, a fusion polypeptide according to one of embodiments 108 to 110, or a composition according to one of embodiments 111 to 125 to a plant or plant seed or the site where the plant is growing or is intended to grow, wherein the application effectively modulates plant biochemical signal transduction.

[0412] Implementation Scheme 180. The method according to Implementation Scheme 179, wherein the plant biochemical signal transduction is selected from the induction of nitric oxide production, peroxide production, or secondary metabolites; agonistic regulation of the ethylene signal transduction pathway and induction of ethylene response gene expression; agonistic regulation of the salicylic acid signal transduction pathway and induction of salicylic acid response gene expression; agonistic regulation of the abscisic acid pathway and induction of abscisic acid response gene expression; agonistic regulation of the gibberellin signal transduction pathway and induction of gibberellin response gene expression; antagonistic regulation of jasmonic acid signal transduction and inhibition of jasmonic acid response gene expression; induction of protease inhibitor expression; induction of reactive oxygen species production in plant tissues; induction of immune-related and antimicrobial peptide production; and induction of extended protein gene expression and production.

[0413] Implementation Scheme 181. A DNA construct comprising a first nucleic acid molecule encoding a peptide isolated from a polypeptide according to any one of Implementation Schemes 1 to 107 or a fusion polypeptide according to any one of Implementation Schemes 108 to 110, and a promoter-effective nucleic acid molecule operatively coupled to the first nucleic acid molecule.

[0414] Implementation Scheme 182. A recombinant expression vector comprising the DNA construct according to Implementation Scheme 181.

[0415] Implementation Scheme 183. A recombinant host cell comprising the DNA construct according to Implementation Scheme 181.

[0416] Implementation Scheme 184. The recombinant host cell according to Implementation Scheme 183, wherein the recombinant host cell is a plant protoplast.

[0417] Implementation Scheme 185. The recombinant host cell according to Implementation Scheme 183, wherein the recombinant host cell is a bacterium.

[0418] Implementation Scheme 186. A transgenic plant comprising a recombinant host cell as described in Implementation Scheme 183.

[0419] Implementation Scheme 187. A transgenic plant seed comprising a recombinant host cell as described in Implementation Scheme 183.

[0420] Implementation Scheme 188. A transgenic plant comprising a DNA construct according to Implementation Scheme 181.

[0421] Implementation Scheme 189. A transgenic plant seed comprising a DNA construct according to Implementation Scheme 181.

[0422] Implementation Scheme 190. A method for conferring disease resistance to plants, comprising: Provide transgenic plants transformed with the DNA construct described in embodiment 181; and The plant is grown under conditions that effectively allow the DNA construct to express the peptide or the fusion polypeptide to confer disease resistance.

[0423] Implementation Scheme 191. A method for enhancing plant growth, comprising: Provide transgenic plants transformed with the DNA construct described in embodiment 181; and To enhance plant growth, the plant is grown under conditions that effectively allow the DNA construct to express the peptide or the fusion polypeptide.

[0424] Implementation Scheme 192. A method for conferring disease resistance to plants, comprising: Provide transgenic plants transformed with the DNA construct described in embodiment 181; and The plant is grown under conditions that effectively allow the DNA construct to express the peptide or the fusion polypeptide to confer disease resistance.

[0425] Implementation Scheme 193. A method for increasing the tolerance of plants to biotic stress, comprising: Provide transgenic plants transformed with the DNA construct described in embodiment 181; and The plant is grown under conditions that effectively allow the DNA construct to express the peptide or the fusion polypeptide in order to increase the plant's tolerance to biological stressors selected from insects, spiders, nematodes, weeds, and combinations thereof.

[0426] Implementation Scheme 194. A method for increasing plant tolerance to abiotic stress, comprising: Provide transgenic plants transformed with the DNA construct described in embodiment 181; and The plant is grown under conditions that effectively allow the DNA construct to express the peptide or the fusion polypeptide in order to increase the plant's tolerance to abiotic stress factors selected from salt stress, drought stress, ozone stress, heavy metal stress, and low temperature stress, and combinations thereof.

[0427] Implementation Scheme 195. A method for imparting desiccation resistance to cuttings taken from ornamental plants, comprising: Provide transgenic ornamental plants transformed with the DNA construct described in embodiment 181; and The plant is grown under conditions that effectively allow the DNA construct to express the peptide or the fusion polypeptide to confer drought resistance to cuttings taken from the transgenic ornamental plant.

[0428] Implementation Scheme 196. A method for conferring postharvest disease resistance or postharvest desiccation resistance to fruits or vegetables, comprising: Provide transgenic plants transformed with the DNA construct described in embodiment 181; and The plant is grown under conditions that effectively allow the DNA construct to express the peptide or the fusion polypeptide to confer postharvest disease resistance or desiccation resistance to fruits or vegetables derived from the transgenic plant.

[0429] Implementation Scheme 197. A method for enhancing the ripening life of fruits or vegetables, comprising: Provide transgenic plants transformed with the DNA construct described in embodiment 181; and The plant is grown under conditions that effectively allow the DNA construct to express the peptide or the fusion polypeptide in order to enhance the ripening life of fruits or vegetables derived from the transgenic plant.

[0430] Implementation Scheme 198. A method for conferring disease resistance to plants, comprising: Provide transgenic plant seeds transformed with the DNA construct described in embodiment 181; Plant the genetically modified plant seeds in the soil; and Transgenic plants are propagated from the seeds of the transgenic plant to allow the DNA construct to express the peptide or the fusion polypeptide to confer disease resistance.

[0431] Implementation Scheme 199. A method for enhancing plant growth, comprising: Provide transgenic plant seeds transformed with the DNA construct described in embodiment 181; Plant the genetically modified plant seeds in the soil; and Transgenic plants are propagated from the seeds of the transgenic plant to allow the DNA construct to express the peptide or the fusion polypeptide to enhance plant growth.

[0432] Implementation Scheme 200. A method for conferring disease resistance to plants, comprising: Provide transgenic plant seeds transformed with the DNA construct described in embodiment 181; Plant the genetically modified plant seeds in the soil; and Transgenic plants are propagated from the seeds of the transgenic plant to allow the DNA construct to express the peptide or the fusion polypeptide to confer disease resistance.

[0433] Implementation Scheme 201. A method for increasing the tolerance of plants to biotic stress, comprising: Provide transgenic plant seeds transformed with the DNA construct described in embodiment 181; Plant the genetically modified plant seeds in the soil; and Transgenic plants are propagated from the seeds of the transgenic plants to allow the DNA construct to express the peptide or the fusion polypeptide to increase the plant’s tolerance to biological stressors selected from insects, spiders, nematodes, weeds and combinations thereof.

[0434] Implementation Scheme 202. A method for increasing plant tolerance to abiotic stress, comprising: Provide transgenic plant seeds transformed with the DNA construct described in embodiment 181; Plant the genetically modified plant seeds in the soil; and Transgenic plants are propagated from the seeds of the transgenic plants to allow the DNA construct to express the peptide or the fusion polypeptide to increase the plant’s tolerance to abiotic stress factors selected from salt stress, drought stress, ozone stress, heavy metal stress, and low temperature stress and combinations thereof.

[0435] Implementation Scheme 203. A method for imparting desiccation resistance to cuttings taken from ornamental plants, comprising: Provide transgenic ornamental plant seeds transformed with the DNA construct described in embodiment 181; Plant the genetically modified ornamental plant seeds in the soil; and Transgenic ornamental plants are propagated from the seeds of the transgenic ornamental plant to allow the DNA construct to express the peptide or the fusion polypeptide to impart desiccation resistance to cuttings taken from the transgenic ornamental plant.

[0436] Implementation Scheme 204. A method for conferring postharvest disease resistance or postharvest desiccation resistance to fruits or vegetables, comprising: Provide transgenic plant seeds transformed with the DNA construct described in embodiment 181; Plant the genetically modified plant seeds in the soil; and Transgenic plants are propagated from the seeds of the transgenic plant to allow the DNA construct to express the peptide or the fusion polypeptide to confer postharvest disease resistance or desiccation resistance to fruits or vegetables derived from the transgenic plant.

[0437] Implementation Scheme 205. A method for enhancing the ripening life of fruits or vegetables, comprising: Provide transgenic plant seeds transformed with the DNA construct described in embodiment 181; Plant the genetically modified plant seeds in the soil; and Transgenic plants are propagated from the seeds of the transgenic plant to allow the DNA construct to express the peptide or the fusion polypeptide to enhance the ripening life of fruits or vegetables derived from the transgenic plant.

[0438] Implementation Scheme 206. A method for regulating plant biochemical signal transduction, comprising: Provide transgenic plant seeds transformed with the DNA construct described in embodiment 181; Plant the genetically modified plant seeds in the soil; and Transgenic plants are propagated from the seeds of the transgenic plant to allow the DNA construct to express the peptide or the fusion polypeptide to regulate plant biochemical signal transduction.

[0439] Implementation Scheme 207. The method according to Implementation Scheme 206, wherein the plant biochemical signal transduction is selected from the induction of nitric oxide production, peroxide production, or secondary metabolites; agonistic regulation of the ethylene signal transduction pathway and induction of ethylene response gene expression; agonistic regulation of the salicylic acid signal transduction pathway and induction of salicylic acid response gene expression; agonistic regulation of the abscisic acid pathway and induction of abscisic acid response gene expression; agonistic regulation of the gibberellin signal transduction pathway and induction of gibberellin response gene expression; antagonistic regulation of jasmonic acid signal transduction and inhibition of jasmonic acid response gene expression; induction of protease inhibitor expression; induction of reactive oxygen species production in plant tissues; induction of immune-related and antimicrobial peptide production; and induction of extended protein gene expression and production.

[0440] Example The following examples are provided to illustrate embodiments of the invention, but are by no means intended to limit its scope.

[0441] Example 1 - Development of the "HR box" peptide of SEQ ID NO: 93 The HR box was initially developed based on the examination of many sequences that induce anaphylactic responses (P1, SEQ ID NO: 4; P4, SEQ ID NO: 5; and P15, SEQ ID NO: 64, etc.). Repeated sequences of leucine and isoleucine residues were identified. P4 was selected as a representative sequence, serving as the basis for mutation studies that will reveal the sequence determinants of HR activation. HRs in tobacco, such as Wei, were tested. ScienceAs stated in 257:85-88 (1992), it is incorporated herein by reference in its entirety. In short, according to 500... The peptide was dissolved in an aqueous solution at a concentration of g / ml. Four consecutive dilutions with equal volumes of water yielded concentrations of 500, 250, 125, 62.5, and 31.25 g / ml. Peptide samples were prepared using g / ml peptide solutions. Red-flowered tobacco plants aged 5-7 weeks (before flowering) were used. Nicotiana tabacum Cultivated Xanthi plants. Leaves were gently punctured in the middle of the leaf panel using a toothpick. Peptide solutions were then infused into the wounds via a needle-less syringe to fill the leaf surface. Each peptide sample was infused into the leaves of two different plants. Leaves were observed over the next 48 hours and lesions typical of wilting, browning, and programmed cell death were scored. These mutation studies had three main objectives: (1) to increase the solution stability of the peptides; (2) to generate disruptive mutations to examine the residues most important for HR excitation; and (3) to generate conserved mutations to identify the degree of specificity for specific amino acids.

[0442] The peptides are evaluated for one or more of the following: solubility, stability against chemical degradation, effect of filler on solution stability, oxidative protection, and solution stability studies.

[0443] Solubility was assessed by generating a pure, chemically synthesized peptide in 0.2% AI (active ingredient) solution in deionized water and observing evidence of precipitation at room temperature over 48 hours. P1 (SEQ ID NO: 4) was largely insoluble in water. However, several mutants with glutamine residues replacing glutamate residues (P1-2E, 8E, 11E, 15E, 18E, SEQ ID NO: 46) were soluble. P4 (SEQ ID NO: 5) and P4-14S (SEQ ID NO: 6) were also completely soluble.

[0444] Further experiments were conducted to better quantify peptide solubility. 20-50 mg of pure peptide was mixed with 0.25 ml of water, and increasing amounts of water were added until the peptide dissolved. These experiments estimated the solubility of P1 (SEQ ID NO: 4) to be <1 mg / ml, P4 (SEQ ID NO: 5) to be 100 mg / ml, and P1-18K (SEQ ID NO: 45) to be 20 mg / ml.

[0445] Pure, chemically synthesized peptides were produced by reacting a 0.2% AI solution in deionized water with Proxel at a weight / volume ratio of 0.25%. ®GXL (a biocidal agent) and eight buffers at 50 mM (individually): MES pH 5.6, MOPS pH 6.5, citrate pH 7.2, EDDS pH 7.3, EDTA pH 8, phosphate pH 8, imidazole pH 8, and TES pH 8 were used to assess the stability against chemical degradation in various pH buffers. Evidence of degradation of the solutions over several weeks at high temperature (50°C) was observed on HPLC (loss of peptide signal over time relative to the sample at 0). P1-2E, -8E, -11E, -15E, and -18E (SEQ ID NO: 46) were more stable than P1 (SEQ ID NO: 4) (over 80% more for 40 days vs. 20 days), and P4-14S (SEQ ID NO: 6) was significantly more stable than P4 (SEQ ID NO: 5) (over 80% more for 35 days vs. 3 days). Following this order, the best buffers for P1 and P4-14S were TES pH 8 and citrate pH 7.2. P1 precipitation was observed after several days. The other peptides (P1-2E, -8E, -11E, -15E, -18E; P4 and P4-14s) remained in solution.

[0446] The effect of fillers on the chemical degradation of P1 and P1-2E, -8E, -11E, -15E, and -18E was evaluated by generating pure, chemically synthesized peptides in 0.2% AI solutions of trehalose, maltodextrin, sucrose, or talc (formulated separately) in 50 mM TES solution at pH 8.0 in water and in 20% (w / v) filler solutions of trehalose, maltodextrin, sucrose, or talc (formulated separately). Evidence of degradation over time at high temperature (50 °C) in these solutions was observed on HPLC (the percentage loss of peptide signal over time relative to the sample at 0 °C). After 6 days of incubation, the concentration of P1 in all mixtures decreased to less than 60% of the original peptide concentration. Conversely, the concentrations of p1-2E, -8E, -11E, -15E, and -18E in all samples remained above 80% of the original concentration for at least 14 days. For P1-2E, -8E, -11E, -15E, and -18E, the best filler is talc (over 80% after 44 days).

[0447] The stability of pure, chemically synthesized peptides was studied by preparing them in 0.2% AI solution in deionized water, 50 mM TES buffer, 0.25% Proxel GXL, and 30% isopropanol. The loss of peptide signal over time relative to the sample at 0 was analyzed by HPLC. The maximum lifetime of P1-2E, -8E, -11E, -15E, and -18E was over 80% at 45 days. The maximum lifetime of P4-14S was over 80% at 140 days.

[0448] Solution stability mutations: Solution stability was increased by selecting peptide sequences that did not contain methionine residues (P4, SEQ ID NO: 5). However, this peptide contained cysteine ​​residues, resulting in extremely poor stability. This cysteine ​​mutation was converted to a conserved substitution of serine (chemically, sulfur to oxygen) to produce P4-14s (SEQ ID NO: 6), which retains its ability to excite HR. It was later confirmed (as mentioned above) that P4-14s is a highly stable peptide. Other studies have substituted one or more glutamine residues with glutamic acid residues to reduce the likelihood of deamidation in solution. Specifically, variants of P1, designated P1-2E,8E,11E,15E,18E (SEQ ID NO: 46), contain these mutations at positions 2, 8, 11, 15, and 18. These peptides exhibit improved solubility and stability compared to P1.

[0449] Based on the stable P4-14s backbone (SEQ ID NO: 6), disruptive single mutations were introduced at specific residues in this sequence. In the case of leucine residues, these mutations were alanine (smaller and less hydrophobic side chain, moderately disruptive mutation) and / or aspartic (negatively charged side chain, highly disruptive mutation). Depending on the identity of the amino acid in question, the inserted sequence was mutated to have a negative charge (aspartic or glutamic acid), a hydrophobic side chain (valine), a minimal side chain (alanine), or a small polar side chain (serine). The activation of allergic reactions by these mutant peptides was tested. Additional mutations were selected based on the initial HR results. Additionally, the spacing between leucine / isoleucine residues was estimated by deleting a single residue between leucine repeat sequences (denoted by 'del') or inserting an alanine residue between leucine repeat sequences (denoted by iA).

[0450] For those amino acids important for HR activation, more conserved mutations are selected to determine the specificity of the interaction. Leucine residues are mutated to isoleucine, valine, phenylalanine, or tyrosine, the latter two being less conserved. HR activation of these mutants is then tested as described above.

[0451] The results of these mutation studies are summarized in Table 12 below: Table 12: Summary of Mutation and HR Activation Results Table 12 shows the sequence of P4-14s along with all mutations tested at each position. Mutations that do not interfere with the allergic reaction are listed in the "HR-positive mutations" row. Mutations that reduce the severity of the allergic reaction are shown in the row labeled "weak HR mutations". Mutations that eliminate the allergic reaction are shown in the red row labeled "HR-negative mutations". The symbols dN2 and dN4 represent the deletion of 2 or 4 residues from the start of the peptide, respectively; dC2 represents the deletion of 2 residues from the end of the peptide; del indicates the deletion of residues at that position; and iA represents the insertion of an alanine residue before that position.

[0452] Example 2 - Solubility and stability of P1 and mutant peptides As described above, the solution stability and chemical compatibility of P1 and its P1-derived sequences with a mutation at position 18 (methionine replaced by alanine, threonine, or lysine) were evaluated after 14 days. Notably, P1 exhibited solubility issues at lower pH levels (in deionized water, in 50 mM citrate at pH 5.6, and in 50 mM MES at pH 6.0). Under these conditions, the peptide concentration increased after incubation at 50°C for 24 hours. Notably, mutant peptides generally did not exhibit this problem. Figure 1-3 As shown, the data were normalized to 100% peptides at day 1 (shown as peptide 1* in the legend, and the original peptide 1 data are marked with double asterisks **). In the stability test for water solubility ( Figure 1 Peptide 1 is moderately stable but exhibits solubility issues. The 18K and 18A mutants show slightly higher stability (10-25% after 14 days). It dissolves in slightly acidic citrate buffer. Figure 2 P1 exhibited both solubility and stability issues. After 14 days, P1 was undetectable in solution by HPLC. Conversely, the 18T and 18K mutants maintained 80% of their original concentrations, and the 18A mutant maintained approximately 60% of its original concentration. Figure 3 As shown, in 50 mM MES at pH 6.0, P1 exhibited stronger solubility issues, with a 50% increase in concentration after 24 hours of incubation at 50°C. However, P1 showed better stability than the mutant (10-30% after 14 days). In citrate at pH 7.2 ( Figure 6 P1 did not exhibit solubility issues but showed weak stability (20% of the original concentration after 7 days at 50°C). In contrast, the 18K and 18T mutants showed >60% stability after 14 days. (In 50 mM EDDS at pH 7.3) Figure 7 Peptide 1 exhibited weak stability, retaining only 10% of the original material after 7 days. In contrast, the mutant retained at least 50% of the starting material after 14 days. (In 50 mM imidazole at pH 8.0) Figure 8 Peptide 1 exhibited particularly weak stability, decreasing to less than 10% of its original concentration after only 3 days. In contrast, all mutants showed greater stability, with the 18K and 18T mutants retaining 60-75% of their original composition after 14 days. Peptide 1 showed even better stability in a solution of 50 mM EDTA at pH 8.0. Figure 9 The performance of the 18K and 18T mutants matched that of the 18A mutant. However, the 18K and 18T mutants showed better stability (15-20% improvement) after incubation at 50°C for 14 days. When dissolved in phosphate at pH 8.0... Figure 10 While peptide 1 did exhibit solubility issues (some turbidity in solution), it appeared to be more stable than the mutant. In a solution of 50 mM TES at pH 8.0 (…), Figure 11 Peptide 1 was over 90% degraded after incubation at 80°C for one week. In contrast, the 18T, 18K, and 18A mutants showed better performance (71%, 58%, and 47% retention, respectively, after incubation at 50°C for 14 days).

[0453] Generally, peptide 1 exhibits solubility problems or weak stability in various buffer solutions. This is resolved by mutating methionine to other residues. For stability, larger residues (threonine and lysine) generally appear to be preferred over alanine.

[0454] Example 3 - Solubility and stability of P4 and mutant peptides As described above, the solution stability and chemical compatibility of P4 and the P4-derived sequence with mutations at position 14 (cysteine ​​replaced by alanine / A, aspartic acid / D, lysine / K, glutamine / Q, and serine / S) were evaluated after 14 days. Generally, peptide 4 exhibits extremely weak stability due to the presence of cysteine. Figure 12-21 Less than one day later, the original P4 HPLC peak was not detected in the sample. In contrast, all mutants exhibited better stability. Most of these retained at least 50% of the original substance for 14 days at 50°C. Generally, the peptide 4 mutants showed better stability at higher pH values ​​(>7.0). Notably, p4-14s could consistently exhibit 90% stability after 14 days, depending on the conditions. All mutant peptides exhibited hypersensitive reactions upon infiltration into tobacco leaves (as in Example 1).

[0455] Example 4 - Comparison of the stability of peptide 1 and peptide 4 Although peptides 1 and 4 exhibit high sequence similarity, the stabilizing mutant of peptide 4 is more stable than the p1 mutant. A series of mutations were generated in p1 to confer stability similar to p4-14s. These are: p1-1S (SEQ ID NO: 109, Table 1), p1-14S (SEQ ID NO: 110, Table 1), p1-18Q (SEQ ID NO: 115, Table 1), and p1-23P (SEQ ID NO: 118, Table 1). These peptides, along with p1 and p4-14s, were dissolved in 30% isopropanol, 5 mM DTPA, and 50 mM TES at pH 8.0 and their stability was tested at 50 °C. Similar stability was observed for p4-14S and p1-1S, indicating that the N-terminal amino acid has a strong influence on peptide stability.

[0456] Example 5 - Solubility of P15b and mutant Initial results indicated that p15b had solubility issues. It exhibited relatively high hydrophobicity (0.19). At 0.2% w / v, it was partially soluble in water and insoluble in 50 mM citrate at approximately pH 5.2, citrate at pH 7.0, phosphate at pH 7.0 (verified), TES at pH 8.0, EDTA at pH 8.0, and EDDS at pH 7.0. It was at least partially soluble in 50 mM MES at pH 6.0 and MOPS at pH 6.5. However, p15a was more readily soluble in aqueous solution. Its solubility in 50 mM TES at pH 8.0 was >10 mg / ml. Additional p15 variants, including polyglutamic acid solubility tags, were synthesized (p15-59G and p15-59, SEQ ID NO: 149 and 150, respectively). When p15-59 is dissolved in 50 mM TES at pH 8.0, it exhibits a solubility of >10 mg / ml (1% w / v).

[0457] Stability of Examples 6-P17 / P18 and Variants As described above, the stability and chemical compatibility of P18 (SEQ ID NO: 83) were tested using different pH buffers. P18 exhibited relatively weak stability in an aqueous buffer at 50°C. Most samples degraded to 20% of their original concentration within 3 days. One exception was a 50 mM EDTA solution, which degraded to 35% after 7 days. Figure 24 A mutation of methionine at position 12 to leucine (in P18-4, SEQ ID NO: 164) resulted in moderate stabilization: stability was 60% after 14 days. Notably, truncation of the last 3 amino acids from the C-terminus (P18-1, SEQ ID NO: 163) also resulted in a significant increase in stability (stability >90% after 14 days).

[0458] Stability of Examples 7-P19 and Variants Generally, P19 (SEQ ID NO: 89) exhibits relatively high stability under various conditions, with stability >80% after 14 days at 50°C. Exceptions include the peptide dissolved in water alone (52%) or 50 mM TES at pH 8.0 (62%). A mutation of a methionine residue at position 12 to leucine (P19-20L, SEQ ID NO: 90) results in a moderate increase in stability when dissolved in 50 mM citrate at pH 7.2 or 50 mM TES at pH 8.0. Figure 25 and 26 When using buffers with lower pH (5.5-7.0), P19 and P19-20L showed similar performance; over 80% of the peptides were retained for 14 days.

[0459] Example 8 - Stability of P14d, P14e, and P14f The P14d sequence (SEQ ID NO: 175) is derived from the popA sequence of *Ralstonia solanacearum*. It is consistent with the HR-box motif and induces HR in tobacco leaves. Mutations of methionine residues yield stable peptides P14e (SEQ ID NO: 176) and P14f (SEQ ID NO: 177). The mutant peptides exhibit >85% stability at 50°C (in 50 mM TES, pH 8.0, and 30% isopropanol) for >50 days. During the same time period, P14d exhibits approximately 50% chemical stability.

[0460] Example 9 - Growth Test For the growth trials, corn and soybean seeds were planted in flat plots, with two seeds per cell in each flat plot within the greenhouse facility. Seeds were allowed to germinate, and smaller plants were removed, leaving one plant per cell. Plant height was initially measured once the first true leaf was fully expanded and the second leaf began to unfold. This was done by stretching the tallest leaf upwards and measuring the distance to the soil. Peptides were dissolved in water at a specified concentration (see below). Plants were then treated with foliar spray using widely available spray bottles until the liquid dripped from the leaves. Four plots of flat land were treated for each case (peptide or control), with 14 plants in each. Corn and soybean were treated as shown in Table 13 and compared with matched water-treated control plants. Plants were allowed to grow for 14 days. Plant height was measured again and compared to the initial height to quantify growth. In some cases, plants were allowed to grow without watering for 2–4 days until wilting and drought stress occurred. At this point, the aboveground parts of the plants were harvested and weighed to determine fresh weight. Finally, the aboveground material was dried at 70°C for 48 hours and weighed to determine dry biomass. The results of these growth experiments are shown in Table 13. Growth, dry biomass, and fresh weight were calculated as a percentage increase compared to the water-treated control.

[0461] Table 13: Results of the growth experiment ND = Not measured Several peptides tested showed increased growth and / or biomass in maize and soybean. Notably, while P15b did not induce a significant growth or dry biomass phenotype, it did induce an increase in fresh biomass, indicating enhanced water uptake or retention. This is an indicator of drought tolerance in those treated plants. Another peptide, P30-3, was observed to induce increases in growth, fresh weight, and dry biomass.

[0462] Example 10 - Minimum sequence required for HR reaction Having identified the most critical residues for triggering an allergic response, we designed additional mutants to test the minimal peptide sequence responsible for this behavior. Recognizing that solubility would be an issue for the minimal peptide due to the hydrophobic nature of the core HR sequence (containing 7 leucine or isoleucine residues, totaling 13 residues), we added hydrophilic sequences to many peptides, giving them a hydrophobicity of approximately -0.2 on the Kyte-Doolittle scale.

[0463] Initially, polylysine or polyarginine sequences were used, i.e., P4 with an N-linked poly-R or poly-K sequence, or a C-linked poly-R or poly-K sequence (SEQ ID NO: 35, 36, 38, 39). However, upon penetration into tobacco leaves, these peptides caused atypical necrotizing lesions in HR. This led to the hypothesis that polycationic sequences induce a toxic reaction upon penetration into tobacco leaves. Similar necrotizing lesions were observed when polylysine and polyarginine alone penetrated into the leaves. Therefore, testing on peptides containing sequences that enhance cationic solubility was discontinued. Notably, HR+ peptides may contain one or two cationic amino acids, but a larger number of positive charges appears to be detrimental. As an alternative to cationic peptides, polyanionic peptides, particularly polyglutamic acid, were considered. Polyglutamic acid was chosen because aspartic acid is more likely to isomerize to isoaspartic acid, and serine was added to the N-terminus to eliminate the formation of pyroglutamic acid at the N-terminus of the peptide. Adding glutamic acid residues to the C-terminus of the peptide is also reasonable.

[0464] As described in Example #1, an allergy test was performed. For P4, the smallest variant peptide that elicits HR is P4-poly-E-min3 (SEQ ID NO: 33). For P1, the smallest variant peptide that elicits HR is P1-poly-E-min3 (SEQ ID NO: 141). For P18, the smallest variant peptide that elicits HR is P18-7 (SEQ ID NO: 167). For P19, the smallest variant peptide that elicits HR is P19-8 (SEQ ID NO: 173). For P15, the smallest variant peptide that elicits HR is P15-59 (SEQ ID NO: 150). For P14d, the smallest variant peptide that elicits HR is P14-30 (SEQ ID NO: 178). For P25, the smallest variant peptide that elicits HR is P25-11 (SEQ ID NO: 188). Additionally, a minimum peptide sequence was generated, incorporating leucine repeats and glutamic acid residues specific to the HR box at variable positions to increase solubility. These sequences are: P30-2 (SEELEELLEELIEELL, SEQ ID NO: 189), P30-3 (LEELLEELIEELLEE, SEQ ID NO: 190), and P30-4 (LEELLEELIEELL, SEQ ID NO: 210). These minimal HR cassette sequences are soluble in 50 mM TES >5 mg / ml and produce an HR response when infiltrated into tobacco leaves.

[0465] Similarly, based on the hydrophobic backbone sequences of P3, P25, P14, P15, and P19, additional peptides were developed to enhance solubility. These are listed in Table 10 above.

[0466] Based on the previously described properties of harpin and HR+ peptides, these new peptides are expected to have a wide range of biological activities, including inducing resistance to TMV, resistance to nematodes, enhanced stress and drought resistance, enhanced growth and increased yield, as described in PCT application WO 01 / 98501 by Fan et al., which is incorporated herein by reference in its entirety.

[0467] Example 11 - Peptide P1 derivatives that elicit HR responses in tobacco Variants of P1 were subjected to HR testing (as described in Example 1) to determine the minimum sequence required for HR and to identify important residues. The peptides in Table 14 below were determined to be positive for HR: Table 14 Example 12 - Peptide P3 derivatives that induce HR response in tobacco Variants of P3 were subjected to HR testing (as described in Example 1) to determine the minimum sequence required for HR and to identify important residues. The peptides in Table 15 below were determined to be positive for HR: Table 15 It is noteworthy that for effective HR excitation, P3 appears to require a sequence longer than the minimum HR box repeat sequence. This is likely due to the suboptimal phenylalanine residue and the presence of only a single K residue separating the hydrophobic residues present in this sequence (LLKLF in P3 and its variants). However, it is important to note that P3-6 and P3-7 are expected to elicit HR, and additional hydrophobic residues are not absolutely necessary.

[0468] Example 13 - Peptide P25 derivatives that induce HR response in tobacco Variants of P25 were subjected to HR testing (as described in Example 1) to determine the minimum sequence required for HR and to identify important residues. The peptides in Table 16 below were determined to be positive for HR.

[0469] Table 16 Importantly, it should be noted that the P25 variant appears to require a sequence larger than the smallest HR concordant sequence (SEQ ID NO:93) to elicit HR. This could be due to the presence of a valine residue at the preferred leucine position or due to a single hydrophilic residue (LLKIL) between the hydrophobic repeat sequences. Although P25-15, P25-16, and P25-17 are included for HR+, they exhibit extremely weak hypersensitive responses at the highest application rates, observed only in some tobacco plants. Notably, as the bioresponse to P25-15 suggests, the additional sequence contents do not appear to require leucine / isoleucine / valine residues.

[0470] Example 14 - Peptide P14d derivatives that elicit HR responses in tobacco Variants of P14 were subjected to HR testing (as described in Example 1) to determine the minimum sequence required for HR and to identify important residues. The peptides in Table 17 below were determined to be positive for HR.

[0471] Table 17 Importantly, it should be noted that the P14d variant appears to require a sequence larger than the minimum HR concordance sequence (SEQ ID NO:93) to activate HR. Specifically, the addition of a C-terminal lysine residue appears to be required for activity. This is likely due to the presence of a single hydrophilic residue (LVKLL) in the hydrophobic repeat sequence.

[0472] Example 15 - Peptide P15 / P20 derivatives that induce HR response in tobacco The variants of P15 / P20 were subjected to HR testing (as described in Example 1) to determine the minimum sequence required for HR and to identify important residues. The peptides in Table 18 below were determined to be positive for HR.

[0473] Table 18 Example 16 - Peptide P17 / P18 derivatives that elicit HR responses in tobacco Variants of P17 and P18 were subjected to HR testing (as described in Example 1) to determine the minimum sequence required for HR and to identify important residues. The peptides in Table 19 below were determined to be positive for HR.

[0474] Table 19 [*]=TSSSPGLFQSGGDNGLGGHNANSALG's N-terminal sequence Example 17 - Peptide P19 derivatives that induce HR response in tobacco Variants of P19 were subjected to HR testing (as described in Example 1) to determine the minimum sequence required for HR and to identify important residues. The peptides in Table 20 below were determined to be positive for HR.

[0475] Table 20 It is important to note that although P19 and P19-1 exhibit HR, they are not perfectly identical to the shared HR box sequence (SEQ ID NO: 93). However, adding neighboring sequences to P19-2 and P19-3 produces sequences identical to the shared sequence. It is likely that the additional isoleucine residues in P19 and P19-1 (N-terminal isoleucine and IGDN sequence) enhance the propensity for HR activation.

[0476] Example 18 - Induced resistance in tobacco to tobacco mosaic virus infection The test peptide was used to induce resistance to Tobacco Mosaic Virus (TMV) in tobacco. Briefly, three 6-8 week old tobacco plants were selected for each group (sample and control). The bottom leaves of the plants were covered and the plants were sprayed with a solution of water (negative control), the peptide, or Proact (positive control). The spray was applied until the leaves were completely wetted, as indicated by dripping liquid from the leaves. The plants were then allowed to dry and the leaf covering was removed.

[0477] Three days after treatment, diatomaceous earth was gently sprinkled on the previously covered leaves and the opposite leaves, followed by application of 20 μL of a 1.7 μg / ml purified tobacco mosaic virus solution. The TMV solution was then spread across the leaf surface by gently rubbing the solution and diatomaceous earth through the leaf surface. Two minutes after inoculation, the diatomaceous earth was rinsed off the leaves with water. Three days after TMV inoculation, the leaves were scored based on the observed number of TMV lesions. The leaves were also scored for signs of hypersensitive reaction, including yellowing and wilting of affected leaves.

[0478] The efficacy described in Table 21 refers to the percentage reduction in TMV lesions relative to UTC in treated plants. A reduction in TMV on covered leaves indicates a systemic immune response in the plant, while a reduction on uncovered leaves indicates a local response. An asterisk indicates a p-value < 0.05 from a t-test.

[0479] Table 21: Summary of TMV Resistance Generally, peptides that elicit an allergic response in tobacco also confer strong resistance to TMV. These peptides provide resistance in treated leaves. However, they also induce “systemically acquired resistance,” where an immune response in one part of the plant triggers signaling that enhances immunity in other parts. This is evidenced by reduced TMV infection in shaded leaves that were not directly treated with the peptides. Peptides that elicit particularly strong immune responses include some of the smallest HR box peptide sequences: P14d, P25-11, and P30-3.

[0480] Example 19 - Effects of peptide seed treatment on root and shoot growth The biological effects of the peptides on the allocation of growth resources to branches (aboveground) and roots (belowground) were tested. The peptides were dissolved in 100 ml of deionized water at 0.2, 2, or 5 µg / ml concentrations. Corn or soybean seeds were then soaked in the peptide solution for one hour. Untreated control (UTC) plants were soaked in deionized water. 300 ml clear plastic beverage cups (Solo®, DartContainer Corporation) were prepared for planting, with a cross drawn on the bottom dividing the bottom into four equal sections. The cups were then filled with sieved ¼” Sunshine Mix #1 soil (SunGro Horticulture). 100 ml of water was added to the soil. The treated seeds were then planted by gently pressing them into the top of the soil. The seeds were then covered with 50 ml of loose soil. The seeds were allowed to germinate and grow for 12–14 days.

[0481] The length of the branch was measured from the soil to the slightly open tip of the highest leaf of each plant. Plants that failed to germinate or showed signs of stunted growth were removed from the experiment. Stunting was defined as the lack of fully unfolded true leaves at the time of data collection or, by visual inspection, true leaves being less than half the average leaf area of ​​the treatment group. Typically, 30 seeds were planted in each treatment group, and 15–25 plants were used for data collection.

[0482] Root growth is estimated by counting the number of times primary roots cross the quarter mark at the bottom of the cup. These are often observed along the circumference of the bottom of the cup, although some are visible along the sides of the container and counted as if they extend vertically across the quarter mark. This number divided by 4 gives the root growth index. This index is found to be about 90% correlated with the total measured primary root length (the sum of the lengths of all primary roots measured directly after rinsing the soil from the rootstock).

[0483] Table 22: Summary of Root and Shoot Growth Having described the basic concept of the invention in this way, it will be quite apparent to those skilled in the art that the detailed disclosure above is intended to be presented by way of example only and not as limiting. Various changes, modifications, and alterations will occur and are intended to be made by those skilled in the art, although not expressly stated herein. These changes, modifications, and alterations are intended to be presented herein and are within the spirit and scope of the invention. Furthermore, the order of the listed processing elements or sequences, or the use of numbers, letters, or other names, is therefore not intended to limit the claimed method to any order other than that specified in the claims. Therefore, the invention is limited only to the following claims and their equivalents.

Claims

1. An isolated peptide comprising the amino acid sequence (L / I / V / F)-XX-(L / I / V / F)-(L / I)-XX-(L / I / V / F)-(L / I / V / A)-XX-(L / I)-(L / I / V / F) (SEQ ID NO: 93) in The peptide does not contain cysteine ​​or methionine; Each X at positions 2, 6, and 10 is optional and, when present, is any amino acid; and Each X at positions 3, 7, and 11 represents any amino acid.

2. The isolated peptide according to claim 1, wherein the length of the peptide is less than 100 amino acids.

3. The isolated peptide according to claim 2, wherein the length of the peptide is between 13 and 50 amino acids.

4. The isolated peptide according to claim 1, wherein the isolated peptide is stable when dissolved in water or an aqueous solution.

5. The isolated peptide according to claim 1, wherein the isolated peptide is resistant to chemical degradation when dissolved in an aqueous buffer solution containing a biocide.

6. The isolated peptide according to claim 1, wherein the isolated peptide has a solubility of more than about 0.1% in water or an aqueous solution.

7. The isolated peptide according to claim 1, wherein one or both X's at positions 2 and 6 are absent.

8. The isolated peptide according to claim 1, wherein both X's at positions 2 and 6 are present.

9. The isolated peptide according to claim 1 or 7, wherein X at position 10 is absent.

10. The isolated peptide according to claim 1 or 8, wherein X is present at position 10.

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