Pvd1 variant polypeptides for pest control

Abstract

New fungicidal peptides, polypeptides, proteins, and nucleotides; their expression in culture and plants; methods of producing the peptides, polypeptides, proteins, and nucleotides; new processes; new production techniques; new formulations; and new organisms, are disclosed. The present disclosure is also related to a novel type of peptide named PvD1 Variant Polypeptides (PVPs) that are a non-naturally occurring, modified-form of the peptide, PvD1. The present disclosure provides, PVPs, genes encoding PVPs; various formulations and methods for using the same that are useful for the control of pathogenic microbes. Further, the present invention relates to novel, engineered, non-naturally occurring PVPs to prevent and treat colonization and attack of plants and plant parts from pathogenic microbes by treating said plants and plant parts, e.g. seeds with effective concentrations of the PVPs disclosed herein.

Description

Docket No. 277702-548911 PvD1 VARIANT POLYPEPTIDES FOR PEST CONTROL CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the priority benefit under 35 U.S.C. §l19(e) of U.S. Provisional Application No.63 / 508,787 filed on June 16, 2023, the contents of the aforementioned application is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] This application incorporates by reference in its entirety the Sequence Listing entitled “277702-548911.xml” (41,619 bytes bytes), which was created on June 12, 2024, and filed electronically herewith. TECHNICAL FIELD

[0003] The present disclosure provides fungicidal proteins, nucleotides, peptides, their expression in plants, methods of producing the peptides, new formulations, and methods for the control of pathogenic microbes are described. BACKGROUND

[0004] The lives and livelihoods of millions of people depend on several agriculturally important crops. Deleterious microbes that threaten these agriculturally important crops represent a dire threat to global food security and the economy.

[0005] Microbial infections, e.g., fungal infections, are a particular problem in damp climates, and are of major concern during crop storage; indeed, the degree and severity of fungal infections can be exacerbated by modern growing methods—as harvesting and storage systems frequently provide a favorable environment for these plant pathogens. Adding to the problems caused by microbial infections are the rich diversity of pathogenic microbes (e.g., fungi, bacteria, oomycetes, etc.) that can contribute to plant disease. World-wide international travel has aided in spreading these deleterious microbes to parts of the planet where native plants have evolved no defenses. Moreover, the emphasis on intensive monoculture practices of commercially relevant crops in concert with traditional disease-mitigation strategies has allowed pathogenic microbes to become resistant and thrive.

[0006] The incidence of plant diseases has traditionally been controlled by agronomic practices that include crop rotation, the use of agrochemicals, and conventional breeding techniques. The use of chemicals to control plant pathogens, however, increases costs to farmers and causes harmful effects on the ecosystem. Accordingly, consumers and government regulators alike are becoming increasingly concerned with the environmental hazards associated with the production and use of synthetic agrochemicals for protecting plants from pathogens. Thus, there is a significant need for novel alternatives for the control of plant pathogens that56330291.3- 1 -Docket No. 277702-548911 possess a lower risk of pollution and environmental hazards than is characteristic of traditional agrochemical-based methods.

[0007] Accordingly, there is a need for alternative, environmentally friendly antimicrobial agents and compositions to protect economically important commodity and staple calorie crops from the threats posed by pathogenic microbes. SUMMARY

[0008] The present disclosure describes a PvD1 variant polypeptide (PVP) having fungicidal activity against one or more pathogenic microbes. Here, the PVP comprises an amino acid sequence comprising or consisting of: (i) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2- G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; or (ii) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to an amino acid sequence of any one of SEQ ID NOs: 4-23, or an agriculturally acceptable salt thereof.

[0009] In various embodiments, the PVP comprises or consists of an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequenceaccording to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C- D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof.

[0010] In various embodiments, the PVP comprises or consists of an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to any one PVPs as set forth in SEQ ID NOs: 4-23.

[0011] In various embodiments, the PVP comprises or consists of an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to any one PVPs as set forth in SEQ ID NOs: 5-23.

[0012] In various embodiments, the PVP consists of an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to any one PVPs as set forth in SEQ ID NOs: 5-23.56330291.3- 2 -Docket No. 277702-548911

[0013] In addition, the present disclosure describes a composition comprising a PVP, or a PVP-fungicidal protein, or combinations thereof, and an excipient.

[0014] The present disclosure describes a polynucleotide , or a complementary nucleotide sequence thereof, operable to encode a PVP, where the PVP comprises an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C- D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M. In some embodiments, X2 is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, X1 is G or A, X2 is A or K, and X3is F or L. In another preferred embodiments, X1is G and X2is A. In another preferred embodiment, X1 is G or A, X2 is K or A, and X3 is F or L. In a more preferred embodiment, the present disclosure provides a polynucleotide , or a complementary nucleotide sequence thereof, the polynucleotide being operable to encode a PVP, where the PVP consists of an amino acid sequence according to any one of SEQ ID NOs: 5-23.

[0015] The present disclosure describes a plant, plant tissue, plant cell, plant seed, or part thereof, comprising one or more PVPs, or a polynucleotide encoding the same, said PVP comprising an amino acid sequence that is at least 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2- G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof. In some embodiments, X2is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, X1is G or A, X2is A or K, and X3is F or L. In another preferred embodiments, X1 is G and X2 is A.

[0016] In addition, the present disclosure describes a method of producing a PVP, the method comprising: preparing a vector comprising a first expression cassette comprising a polynucleotide operable to encode a PVP, and / or a complementary nucleotide sequence thereof, said PVP comprising an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L- A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C- T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof. In some embodiments, X2 is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2is K or A. In further preferred embodiments, X1is G or A, X2is A or K, and X3is F or56330291.3- 3 -Docket No. 277702-548911 L. In another preferred embodiments, X1 is G and X2 is A, and introducing the vector into a yeast strain; and growing the yeast strain in a growth medium under conditions operable to enable expression of the PVP and secretion into the growth medium.

[0017] In addition, the present disclosure describes a method for protecting a plant from pathogenic microbes, the method comprising: providing a plant that expresses a PVP, or a polynucleotide encoding the same.

[0018] Furthermore, the present disclosure describes a method for controlling pathogenic microbes comprising, providing to said pathogenic microbe a transgenic plant that comprises in its genome a stably incorporated expression cassette, wherein said stably incorporated expression cassette comprises a polynucleotide operable to encode a PVP.

[0019] The present disclosure describes a method of combating, controlling, or inhibiting a pest comprising, applying a pesticidally effective amount of the composition consisting of a PVP, a PVP-fungicidal protein or peptide, or combinations thereof, and an excipient, to the locus of the pest, or to a plant or animal susceptible to an attack by the pest.

[0020] In addition, the present disclosure describes a vector comprising a polynucleotide operable to encode a PVP having an amino acid sequence with at least 95%, or 96%, or 97%, or 98%, or 99% or at least 100% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 1 and 4-23.

[0021] In related embodiments, the present disclosure provides a polynucleotide operable to encode a PVP, wherein the PVP comprises or consists of, an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to any one of SEQ ID NO: 1, wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or SEQ ID NOs: 4-23; or an agriculturally acceptable salt thereof, or a complementary nucleotide sequence thereof. Preferably, the polynucleotide encodes a PVP wherein X2 is A or K and X1 is G or A and X3 is F or L. In still further preferred embodiments, the polynucleotide encodes a PVP consisting of an amino sequence as set forth in any one of SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof.

[0022] The present disclosure provides a plant, plant tissue, plant cell, plant seed, or part thereof, comprising one or more PVPs, or a polynucleotide encoding the same, wherein the PVP expressed in the plant, plant tissue, plant cell, plant seed, or part thereof, comprises or consists of an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to an amino acid sequence, according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3- T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3is F, L, G, A,56330291.3- 4 -Docket No. 277702-548911 V, I or M; or an amino acid sequence of any one of SEQ ID NOs: 4-23, or SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof, and optionally, wherein the PVP comprises from one to three amino acid conservative substitutions relative to the sequence of a PVP as set forth in SEQ ID NO:1, with the proviso that the conservative amino acid substitution does not occur at X1, X2and X3.

[0023] The present disclosure also describes a yeast strain comprising a first expression cassette comprising a polynucleotide operable to encode a PVP, said PVP comprising: (i) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T- T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; or (ii) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to any one of SEQ ID NOs: 4-23. In some embodiments, with regards to Formula (I), in some embodiments, X2is A or V or G. In a preferred embodiment, X1is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, X1 is G or A, X2 is A or K, and X3is F or L. In another preferred embodiment, X1is G and X2is A.

[0024] In some aspects, the present disclosure provides for a PVP having an amino acid sequence in accordance with Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C- D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof. In some embodiments, X2is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, X1is G or A, X2is A or K, and X3is F or L. In another preferred embodiments, X1 is G and X2 is A. In related embodiments, a PVP of Formula (I) further comprises a PVP having the amino acid sequence of Formula (I) as described herein, said PVP having one to three conservative amino acid substitutions along the entire length of the PVP amino acid sequence of Formula (I), with the exception of the first amino acid residue X1, the 12thamino acid residue X2 and the 16thamino acid X3 of Formula (I). In still further preferred embodiments, the present disclosure provides a PVP consisting of an amino sequence as set forth in any one of SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG.1 depicts a graph showing the results of an alanine scanning mutagenesis of PvD1a (SEQ ID NO: 4).56330291.3- 5 -Docket No. 277702-548911

[0026] FIG.2 depicts a graph showing the results of an alanine scanning mutagenesis of PvD1a (SEQ ID NO: 4). Residues revealed to improve PvD1a activity 2.5 fold are: G1, K2, T3, E5, D9 and T18 (indicated with stars).

[0027] FIG.3 depicts a graph showing the results of an alanine scanning mutagenesis of PvD1a (SEQ ID NO: 4). Here, mutant strains having a mutation at position N6, K12, T17, K46 of PvD1a results in an improvement of yield relative to the PvD1a strain.

[0028] FIGs.4A-4E depict chromatograms showing mutations in the amino acid sequence of PvD1a at positions K12A, P14A, K26A and K46A.4A shows degradation of PvD1a. 4B shows the degradation of the PvD1M10 mutant having a K12A mutation. 4C shows the degradation of the PvD1M12 mutant having a P14A mutation.4D shows the degradation of the PvD1M38 mutant having a K46A mutation.4E shows the degradation of the PvD1M21 mutant having a K26A mutation.

[0029] FIGs.5A-5D depict chromatograms showing mutations in the amino acid sequence of PvD1a at positions K12A with further mutations at T3.5A shows degradation of the M58 mutant having a having a K12A and T3A mutation. 5B shows degradation of the M60 mutant having a having a K12A and T3D mutation.5C shows degradation of the M63 mutant having a having a K12A and T3H mutation.5D shows degradation of PvD1a.

[0030] FIG.6 shows the next generation sequencing results of 720 PvD1a variants. Here, the total number of variants was 720 individual single mutants pooled together. As shown here, the distribution indicates non-bias of each mutant. The PvD1a variant pool DNA was transformed into K. lactis for PvD1a single mutant expression.

[0031] FIG.7A shows a 3D model of a PvD1a variant peptide having a F16L amino acid substitution.

[0032] FIG.7B depicts a graph showing gene productivity for PvD1a. Gene productivity compares peptide expression yield per integrated gene copy, and was determined based on the slope obtained when measuring plots of peptide yield vs. integrated gene copy number. Here, gene productivity was determined from the slope of yields-vs-GeneCopy from qPCR with force fitting passing origin.

[0033] FIG.7C depicts a graph showing gene productivity for PvD1M88, having an F16L mutation relative to PvD1a. Gene productivity compares peptide expression yield per integrated gene copy, and was determined based on the slope obtained when measuring plots of peptide yield vs. integrated gene copy number. Here, gene productivity was determined from the slope of yields-vs-GeneCopy from qPCR with force fitting passing origin. Overall, F16L gene productivity (slope = 159.65) was improved comparing to PvD1a (slope = 140.39).56330291.3- 6 -Docket No. 277702-548911

[0034] FIG.8 depicts a graph showing gene productivity for PvD1a and WT-PvD1. Gene productivity compares peptide expression yield per integrated gene copy, and was determined based on the slope obtained when measuring plots of peptide yield vs. integrated gene copy number.

[0035] FIGs.9A-9B show LC-MS chromatograms for WT-PvD1 and PvD1a, respectively. As shown in 9A, WT-PvD1 has two peaks, wherein the peak on the left (10 – PvD1-Pk1 – 2.928) is WT-PvD1, and the peak on the right (11 – PvD1-Pk2 – 3.050) represents a peptide species with a molecular weight that is 755 Daltons more that WT-PvD1; this resulted from incompletely processed α-mating factor (α-MF) and PvD1 fusion protein during expression and secretion process in K. lactis. As shown in 9B, , the PvD1 variant peptide (PVP), PvD1a (SEQ ID NO: 4), shows a single purified peak—indicating that addition of Glycine at N- terminus of WT-PvD1 improves the expression efficiency.

[0036] FIG.10 depicts a graph showing the peptide expression yield of PVPs M68-M84 and PvD1a.

[0037] FIG.11 depicts a graph showing gene productivity for PVPs M68-M84 and PvD1a. Gene productivity compares peptide expression yield per integrated gene copy, and was determined based on the slope obtained when measuring plots of peptide yield vs. integrated gene copy number.

[0038] FIG.12 shows a dose response curve for (1) control (synthetic) PvD1a; (2) control (synthetic) WT-PvD1; (3) expressed WT-PvD1 peak 1; and (4) expressed WT-PvD1 peak 2. As shown here, the IC50 against Monilinia fructicola for control synthetic WT-PvD1 was 0.8 ppm, for PvD1a was 0.85, for the expressed WT PvD1 Peak 1 was 1.1258 ppm; and expressed WT PvD1 Peak 2 was 1.077 ppm.

[0039] FIG.13A depicts a dose-response curve showing the growth of Botrytis cinerea when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. Here, the amount of EDTA required to achieve IC50in Botrytis cinerea cells was 4.4611 µM. The box on the left provides an estimate of the EDTA dose required for targeted inhibition based on this experimental IC50as determined in the dose response curve; this estimation in turn helps to determine the EDTA dose required for combination bioassay, i.e., doses less than IC50 but having some level of effective inhibition on microbial growth. The box on the right shows the actual EDTA doses used in this experiment, and show the estimated inhibition (%).

[0040] FIG.13B depicts a dose-response curve showing the growth of Botrytis cinerea when treated with native PVD1, and as measured via relative OD600 (%) after 96 hours. Here, the amount of PVD1 required to achieve IC50 in Botrytis cinerea cells was 58.555 ppm.56330291.3- 7 -Docket No. 277702-548911

[0041] FIG.13C depicts an isobologram showing the effect of native PVD1 and EDTA on B. cinerea cells.

[0042] FIG.14A depicts a dose-response curve showing the growth of Monilinia fructicola when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. Here, the amount of EDTA required to achieve IC50 in Monilinia fructicola cells was 5.8344 µM. The box on the left provides an estimate of the EDTA dose required for targeted inhibition based on this experimental IC50 as determined in the dose response curve; this estimation in turn helps to determine the EDTA dose required for combination bioassay, i.e., doses less than IC50 but having some level of effective inhibition on microbial growth. The box on the right shows the actual EDTA doses used in this experiment, and show the estimated inhibition (%).

[0043] FIG.14B depicts a dose-response curve showing the growth of Monilinia fructicola when treated with native PVD1, and as measured via relative OD600 (%) after 96 hours. Here, the amount of PVD1 required to achieve IC50in Monilinia fructicola cells was 0.93984 ppm.

[0044] FIG.14C depicts an isobologram showing the effect of native PVD1 and EDTA on Monilinia fructicola cells.

[0045] FIG.15A depicts a dose-response curve showing the growth of Phytophthora infestans when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. Here, the amount of EDTA required to achieve IC50in Phytophthora infestans cells was 14.584 ppm. The box on the left provides an estimate of the EDTA dose required for targeted inhibition based on this experimental IC50 as determined in the dose response curve; this estimation in turn helps to determine the EDTA dose required for combination bioassay, i.e., doses less than IC50but having some level of effective inhibition on microbial growth. The box on the right shows the actual EDTA doses used in this experiment, and show the estimated inhibition (%).

[0046] FIG.15B depicts a dose-response curve showing the growth of Phytophthora infestans when treated with native PVD1, and as measured via relative OD600 (%) after 96 hours. Here, the amount of PVD1 required to achieve IC50 in Phytophthora infestans cells was 152.5 ppm.

[0047] FIG.15C depicts an isobologram showing the effect of native PVD1 and EDTA on Phytophthora infestans cells.

[0048] FIG.16A depicts a dose-response curve showing the growth of Cercospora sojina when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. Here, the amount of EDTA required to achieve IC50 in Cercospora sojina cells was 93.491 ppm. The box on the left provides an estimate of the EDTA dose required for targeted inhibition based on this experimental IC50as determined in the dose response curve; this estimation in turn helps to56330291.3- 8 -Docket No. 277702-548911 determine the EDTA dose required for combination bioassay, i.e., doses less than IC50 but having some level of effective inhibition on microbial growth. The box on the right shows the actual EDTA doses used in this experiment, and show the estimated inhibition (%).

[0049] FIG.16B depicts a dose-response curve showing the growth of Cercospora sojina when treated with native PVD1, and as measured via relative OD600 (%) after 96 hours. Here, the amount of PVD1 required to achieve IC50in Cercospora sojina cells was 302.82 ppm.

[0050] FIG.16C depicts an isobologram showing the effect of native PVD1 and EDTA on Cercospora sojina cells.

[0051] FIG.17A depicts a dose-response curve showing the growth of Fusarium graminearum when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. Here, the amount of EDTA required to achieve IC50in Fusarium graminearum cells was 54.782 ppm. The box on the left provides an estimate of the EDTA dose required for targeted inhibition based on this experimental IC50as determined in the dose response curve; this estimation in turn helps to determine the EDTA dose required for combination bioassay, i.e., doses less than IC50 but having some level of effective inhibition on microbial growth.

[0052] FIG.17B depicts a dose-response curve showing the growth of Fusarium graminearum when treated with native PVD1, and as measured via relative OD600 (%) after 96 hours. Here, the amount of PVD1 required to achieve IC50 in Fusarium graminearum cells was 17.35 ppm.

[0053] FIG.17C depicts an isobologram showing the effect of native PVD1 and EDTA on Fusarium graminearum cells.

[0054] FIG.18A depicts a dose-response curve showing the growth of Aspergillus niger when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. Here, the amount of EDTA required to achieve IC50in Aspergillus niger cells was 21.184 ppm. The box on the left provides an estimate of the EDTA dose required for targeted inhibition based on this experimental IC50as determined in the dose response curve; this estimation in turn helps to determine the EDTA dose required for combination bioassay, i.e., doses less than IC50 but having some level of effective inhibition on microbial growth. The box on the right shows the actual EDTA doses used in this experiment, and show the estimated inhibition (%).

[0055] FIG.18B depicts a dose-response curve showing the growth of Aspergillus niger when treated with native PVD1, and as measured via relative OD600 (%) after 96 hours.

[0056] FIG.18C depicts an isobologram showing the effect of native PVD1 and EDTA on Aspergillus niger cells.56330291.3- 9 -Docket No. 277702-548911

[0057] FIG.19A depicts a dose-response curve showing the growth of Botrytis cinerea when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. The box shows the doses of EDTA evaluated, and the corresponding percent inhibition (%).

[0058] FIG.19B depicts a dose-response curve showing the growth of Botrytis cinerea when treated with PVD1a, and as measured via relative OD600 (%) after 96 hours.

[0059] FIG.19C depicts an isobologram showing the effect of PVD1a and EDTA on B. cinerea cells.

[0060] FIG.20A depicts a dose-response curve showing the growth of Monilinia fructicola when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. The box shows the doses of EDTA evaluated, and the corresponding percent inhibition (%).

[0061] FIG.20B depicts a dose-response curve showing the growth of Monilinia fructicola when treated with PVD1a, and as measured via relative OD600 (%) after 96 hours.

[0062] FIG.20C depicts an isobologram showing the effect of PVD1a and EDTA on Monilinia fructicola cells.

[0063] FIG.21A depicts a dose-response curve showing the growth of Phytophthora infestans when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. The box on the left provides an estimate of the EDTA dose required for targeted inhibition based on this experimental IC50 as determined in the dose response curve; this estimation in turn helps to determine the EDTA dose required for combination bioassay, i.e., doses less than IC50but having some level of effective inhibition on microbial growth. The box on the right shows the actual EDTA doses used in this experiment, and show the estimated inhibition (%).

[0064] FIG.21B depicts a dose-response curve showing the growth of Phytophthora infestans when treated with PVD1a, and as measured via relative OD600 (%) after 96 hours.

[0065] FIG.21C depicts an isobologram showing the effect of PVD1a and EDTA on Phytophthora infestans cells.

[0066] FIG.22A depicts a dose-response curve showing the growth of Cercospora sojina when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. The box on the left provides an estimate of the EDTA dose required for targeted inhibition based on this experimental IC50 as determined in the dose response curve; this estimation in turn helps to determine the EDTA dose required for combination bioassay, i.e., doses less than IC50 but having some level of effective inhibition on microbial growth. The box on the right shows the actual EDTA doses used in this experiment, and show the estimated inhibition (%).

[0067] FIG.22B depicts a dose-response curve showing the growth of Cercospora sojina when treated with PVD1a, and as measured via relative OD600 (%) after 96 hours.56330291.3- 10 -Docket No. 277702-548911

[0068] FIG.22C depicts an isobologram showing the effect of PVD1a and EDTA on Cercospora sojina cells.

[0069] FIG.23A depicts a dose-response curve showing the growth of Fusarium graminearum when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. The box shows the doses of EDTA evaluated, and the corresponding percent inhibition (%).

[0070] FIG.23B depicts a dose-response curve showing the growth of Fusarium graminearum when treated with PVD1a, and as measured via relative OD600 (%) after 96 hours.

[0071] FIG.23C depicts an isobologram showing the effect of PVD1a and EDTA on Fusarium graminearum cells.

[0072] FIG.24A depicts a dose-response curve showing the growth of Aspergillus niger when treated with EDTA, and as measured via relative OD600 (%) after 96 hours. The box shows the doses of EDTA evaluated, and the corresponding percent inhibition (%).

[0073] FIG.24B depicts a dose-response curve showing the growth of Aspergillus niger when treated with PVD1a, and as measured via relative OD600 (%) after 96 hours.

[0074] FIG.24C depicts an isobologram showing the effect of PVD1a and EDTA on Aspergillus niger cells.

[0075] FIG.25 depicts a line graph showing the expression results of two control PvD1 strains versus two PVPs (PvD1E1 and PvD1E2).

[0076] FIG.26 depicts a chromatogram of the four strains using an equivalent volume of culture medium.

[0077] FIG.27 depicts a line graph of gene productivity of two control PvD1 strains versus two PVPs (PvD1E1 and PvD1E2).

[0078] FIG.28 depicts IC50 actifungal activity charts depicting the antifungal activity of two control PvD1 strains versus two PVPs (PvD1E1 (SEQ ID NO: 22) and PvD1E2 (SEQ ID NO: 23)). DETAILED DESCRIPTION

[0079] DEFINITIONS

[0080] The term “5’-end” and “3’-end” refers to the directionality, i.e., the end-to-end orientation of a nucleotide polymer (e.g., DNA). The 5’-end of a polynucleotide is the end of the polynucleotide that has the fifth carbon.

[0081] “5’- and 3’-homology arms” or “5’ and 3’ arms” or “left and right arms” refers to the polynucleotide sequences in a vector and / or targeting vector that homologously recombine with the target genome sequence and / or endogenous gene of interest in the host organism in order to achieve successful genetic modification of the host organism’s chromosomal locus.56330291.3- 11 -Docket No. 277702-548911

[0082] The term "about" as used herein means within ± 10%, preferably ± 5% of a given value, for example the term "about" as applied to the value: 2, contemplates a range from 1.8 to 2.2, or a range from 2.1 to 1.9 and all values and integers therebetween.

[0083] “ADN1 promoter” refers to the DNA segment comprised of the promoter sequence derived from the Schizosaccharomyces pombe adhesion defective protein 1 gene.

[0084] “Affect” refers to how a something influences another thing, e.g., how a peptide, polypeptide, protein, drug, or chemical influences an pathogenic microbe, e.g., a fungal pest.

[0085] “Alignment” refers to a method of comparing two or more sequences (e.g., nucleotide, polynucleotide, amino acid, peptide, polypeptide, or protein sequences) for the purpose of determining their relationship to each other. Alignments are typically performed by computer programs that apply various algorithms, however, it is also possible to perform an alignment by hand. Alignment programs typically iterate through potential alignments of sequences and score the alignments using substitution tables, employing a variety of strategies to reach a potential optimal alignment score. Commonly-used alignment algorithms include, but are not limited to, CLUSTALW (see Thompson J. D., Higgins D. G., Gibson T. J., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research 22: 4673-4680, 1994); CLUSTALV (see Larkin M. A., et al., CLUSTALW2, ClustalW and ClustalX version 2, Bioinformatics 23(21): 2947-2948, 2007); Mafft; Kalign; ProbCons; and T-Coffee (see Notredame et al., T-Coffee: A novel method for multiple sequence alignments, Journal of Molecular Biology 302: 205-217, 2000). Exemplary programs that implement one or more of the foregoing algorithms include, but are not limited to, MegAlign from DNAStar (DNAStar, Inc. 3801 Regent St. Madison, Wis.53705), MUSCLE, T-Coffee, CLUSTALX, CLUSTALV, JalView, Phylip, and Discovery Studio from Accelrys (Accelrys, Inc., 10188 Telesis Ct, Suite 100, San Diego, Calif.92121). In some embodiments, an alignment will introduce “phase shifts” and / or “gaps” into one or both of the sequences being compared in order to maximize the similarity between the two sequences, and scoring refers to the process of quantitatively expressing the relatedness of the aligned sequences.

[0086] “Alpha-MF signal” or “αMF secretion signal” refers to a protein that directs nascent recombinant polypeptides to the secretory pathway.

[0087] “Agent” refers to one or more chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryote organisms, or eukaryote organisms, and agents produced therefrom.56330291.3- 12 -Docket No. 277702-548911

[0088] “Agriculturally-acceptable carrier” covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation.

[0089] “Agroinfection” means a plant transformation method where DNA is introduced into a plant cell by using Agrobacteria A. tumefaciens or A. rhizogenes.

[0090] “BAAS” means barley alpha-amylase signal peptide, and is an example of an ERSP. One example of a BAAS is a BAAS having the amino acid sequence of SEQ ID NO: 29 (NCBI Accession No. AAA32925.1).

[0091] “Biomass” refers to any measured plant product.

[0092] “Binary vector” or “binary expression vector” means an expression vector which can replicate itself in both E. coli strains and Agrobacterium strains. Also, the vector contains a region of DNA (often referred to as t-DNA) bracketed by left and right border sequences that is recognized by virulence genes to be copied and delivered into a plant cell by Agrobacterium.

[0093] “bp” or “base pair” refers to a molecule comprising two chemical bases bonded to one another forming a. For example, a DNA molecule consists of two winding strands, wherein each strand has a backbone made of an alternating deoxyribose and phosphate groups. Attached to each deoxyribose is one of four bases, i.e., adenine (A), cytosine (C), guanine (G), or thymine (T), wherein adenine forms a base pair with thymine, and cytosine forms a base pair with guanine.

[0094] “C-terminal” refers to the free carboxyl group (i.e., -COOH) that is positioned on the terminal end of a polypeptide.

[0095] “cDNA” or “copy DNA” or “complementary DNA” refers to a molecule that is complementary to a molecule of RNA. In some embodiments, cDNA may be either single- stranded or double-stranded. In some embodiments, cDNA can be a double-stranded DNA synthesized from a single stranded RNA template in a reaction catalyzed by a reverse transcriptase. In yet other embodiments, “cDNA” refers to all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3’ and 5’ non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns removed by nuclear RNA splicing, to create a continuous open reading frame encoding the protein. In some embodiments, “cDNA” refers to a DNA that is complementary to and derived from an mRNA template.

[0096] “Cleavable Linker” see Linker.

[0097] “Cloning” refers to the process and / or methods concerning the insertion of a DNA segment (e.g., usually a gene of interest, for example PVP) from one source and recombining it with a DNA segment from another source (e.g., usually a vector, for example, a plasmid) and56330291.3- 13 -Docket No. 277702-548911 directing the recombined DNA, or “recombinant DNA” to replicate, usually by transforming the recombined DNA into a bacteria or yeast host.

[0098] “Codon optimization” refers to the production of a gene in which one or more endogenous, native, and / or wild-type codons are replaced with codons that ultimately still code for the same amino acid, but that are of preference in the corresponding host.

[0099] “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides as understood by those of skill in the art. Thus, two sequences are “complementary” to one another if they are capable of hybridizing to one another to form a stable anti-parallel, double-stranded nucleic acid structure. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions. Thus, the polynucleotide whose sequence 5’-TATAC- 3’ is complementary to a polynucleotide whose sequence is 5’-GTATA-3’.

[0100] “Conditioned medium” means the cell culture medium which has been used by cells and is enriched with cell derived materials but does not contain cells.

[0101] “Copy number” refers to the number of identical copies of a vector, an expression cassette, an amplification unit, a gene or indeed any defined nucleotide sequence, that are present in a host cell at any time. For example, in some embodiments, a gene or another defined chromosomal nucleotide sequence may be present in one, two, or more copies on the chromosome. An autonomously replicating vector may be present in one, or several hundred copies per host cell.

[0102] “Culture” or “cell culture” refers to the maintenance of cells in an artificial, in vitro environment.

[0103] “Culturing” refers to the propagation of organisms on or in various kinds of media. For example, the term “culturing” can mean growing a population of cells under suitable conditions in a liquid or solid medium. In some embodiments, culturing refers to fermentative recombinant production of a heterologous polypeptide of interest and / or other desired end products (typically in a vessel or reactor).

[0104] “Cystine” refers to an oxidized cysteine-dimer. Cystines are sulfur-containing amino acids obtained via the oxidation of two cysteine molecules, and are linked with a disulfide bond.

[0105] “Defined medium” means a medium that is composed of known chemical components but does not contain crude proteinaceous extracts or by-products such as yeast extract or peptone.56330291.3- 14 -Docket No. 277702-548911

[0106] “Degeneracy” or “codon degeneracy” refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a protein or polypeptide can vary due to degeneracies. As a result of the degeneracy of the genetic code, many nucleic acid sequences can encode a given polypeptide with a particular activity; such functionally equivalent variants are contemplated herein.

[0107] “Disulfide bond” or “disulfide bridges” refers to a covalent bond between two cysteine amino acids derived by the coupling of two thiol groups on their side chains. In some embodiments, a disulfide bond occurs via the oxidative folding of two different thiol groups (- SH) present in a polypeptide, e.g., a CRIP. In some embodiments, a polypeptide can comprise at least six different thiol groups (i.e., six cysteine residues each containing a thiol group); thus, in some embodiments, a polypeptide can form zero, one, two, three, or more intramolecular disulfide bonds.

[0108] “Disulfide bond connectivity” or “disulfide bond linkage pattern” refers to the linking pattern of disulfide bonds and cysteine residues. In some embodiments, a CRIP with the preferred ICK architecture comprises six conserved cysteine residues (numbered I-VI) that form three disulfide bonds with the following disulfide bond connectivities: CIand CIV; CIIand CV; and CIIIand CVI. In some embodiments, the disulfide bonding connectivity is topologically constant, meaning the disulfide bonds can only be changed by unlinking one or more disulfides such as using redox conditions.

[0109] “Double expression cassette” refers to two PVP expression cassette s contained on the same vector.

[0110] “Double transgene peptide expression vector” or “double transgene expression vector” means a yeast expression vector that contains two copies of the PVP expression cassette.

[0111] “DNA” refers to deoxyribonucleic acid, comprising a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]), which can be arranged in single-stranded or double-stranded form. For example, one or more nucleotides creates a polynucleotide.

[0112] “dNTPs” refers to the nucleoside triphosphates that compose DNA and RNA.

[0113] “ELISA” or “iELISA” means an assay protocol in which the samples are fixed to the surface of a plate and then detected as follows: a primary antibody is applied followed by a secondary antibody conjugated to an enzyme which converts a colorless substrate to colored substrate which can be detected and quantified across samples. During the protocol, antibodies are washed away such that only those that bind to their epitopes remain for detection. The samples, in our hands, are predominantly proteins, and ELISA allows for the quantification of the amount of protein recovered.56330291.3- 15 -Docket No. 277702-548911

[0114] “Endogenous” refers to a polynucleotide, peptide, polypeptide, protein, or process that naturally occurs and / or exists in an organism, e.g., a molecule or activity that is already present in the host cell before a particular genetic manipulation.

[0115] “Enhancer element” refers to a DNA sequence operably linked to a promoter, which can exert increased transcription activity on the promoter relative to the transcription activity that results from the promoter in the absence of the enhancer element.

[0116] “Engineered” or “engineered protein” refers to refers to a non-naturally-occurring peptide, polypeptide, or protein (e.g., engineered PVP). As used herein, the term “engineered” encompasses, for example, a polypeptide that comprises one or more changes, including additions, deletions, and / or substitutions, relative to a naturally occurring polypeptide, wherein such changes were introduced, e.g., by recombinant DNA techniques. The term “engineered” also encompasses a peptide, polypeptide, or protein that comprises, consists essentially of, or consists of: an amino acid sequence generated by humans; an artificial peptide, polypeptide, or protein; a fusion protein; and / or and a chimeric polypeptide; a nucleotide sequence generated by humans; an artificial nucleotide, polynucleotide, DNA, RNA, or gene; a polynucleotide encoding a fusion protein; and / or and a polynucleotide encoding a chimeric polypeptide. Once expressed, engineered peptides, polypeptides, and / or proteins can be purified according to standard procedures known to one of ordinary skill in the art, e.g., including but not limited to: ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. In some embodiments, engineered proteins may be produced by any means, including, for example, peptide, polypeptide, or protein synthesis. In some embodiments, an engineered protein can be a non-naturally occurring cysteine-rich fungicidal protein (CRIP).

[0117] “Expression cassette” refers to a segment of DNA that contains one or more (1) promoter and / or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; and / or (3) the DNA sequence of interest, for example, a polynucleotide encoding a PVP. Additional elements that can included in an expression cassette are cis-acting elements such as an internal ribosome entry site (IRES); introns; and posttranscriptional regulatory elements.

[0118] “ER” or “Endoplasmic reticulum” is a subcellular organelle common to all eukaryotes where some post translation modification processes occur.

[0119] “ERSP” or “Endoplasmic reticulum signal peptide” is an N-terminus sequence of amino acids that—during protein translation of the mRNA molecule encoding a PVP—is recognized and bound by a host cell signal-recognition particle, which moves the protein translation ribosome / mRNA complex to the ER in the cytoplasm. The result is the protein translation is paused until it docks with the ER where it continues and the resulting protein is injected into the ER.56330291.3- 16 -Docket No. 277702-548911

[0120] “ersp” refers to a polynucleotide encoding the peptide, ERSP.

[0121] “ER trafficking” means transportation of a cell expressed protein into ER for post-translational modification, sorting and transportation.

[0122] “FECT” means a transient plant expression system using Foxtail mosaic virus with elimination of coating protein gene and triple gene block.

[0123] As used herein, the term “fungicidal” is generally used to refer to the ability of a polypeptide or protein used herein, to increase mortality or inhibit growth rate of pathogenic microbes, for example, fungal pests.

[0124] “Fungicidal activity” means that upon or after exposing the pathogenic microbe to compounds, agents, or peptides, the pathogenic microbe either dies stops or slows its movement; stops or slows its feeding; stops or slows its growth; fails to divide and / or reproduce.

[0125] “GFP” means a green fluorescent protein from the jellyfish, Aequorea victoria.

[0126] “Growth medium” refers to a nutrient medium used for growing cells in vitro.

[0127] “Gut” as used herein can refer to any organ, structure, tissue, cell, extracellular matrix, and / or space comprising the gut, for example: the foregut, e.g., mouth, pharynx, esophagus, crop, proventriculus, or crop; the midgut, e.g., midgut caecum, ventriculus; the hindgut, e.g., pylorum, ileum, rectum or anus; the peritrophic membrane; microvilli; the basement membrane; the muscle layer; Malpighian tubules; or rectal ampulla.

[0128] “Homologous” refers to Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ×100. Homologous refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology.

[0129] The term “homology,” when used in relation to nucleic acids, refers to a degree of complementarity. There may be partial homology, or complete homology and thus identical.56330291.3- 17 -Docket No. 277702-548911 “Sequence identity” refers to a measure of relatedness between two or more nucleic acids, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues that are identical and in the same relative positions in their respective larger sequences.

[0130] “Identity” refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing said sequences. The term “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by any one of the myriad methods known to those having ordinary skill in the art, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994:, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the disclosures of which are incorporated herein by reference in their entireties. Furthermore, methods to determine identity and similarity are codified in publicly available computer programs. For example in some embodiments, methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol.215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.20894; Altschul, S., et al., J. Mol. Biol.215: 403-410 (1990), the disclosures of which are incorporated herein by reference in their entireties.

[0131] “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0132] “Inactive” refers to a condition wherein something is not in a state of use, e.g., lying dormant and / or not working. For example, when used in the context of a gene or when referring to a gene, the term inactive means said gene is no longer actively synthesizing a gene product, having said gene product translated into a protein, or otherwise having the gene perform its normal function. For example, in some embodiments, the term inactive can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications); interference with non-coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference56330291.3- 18 -Docket No. 277702-548911 with translation; protein folding; translocation; protein transport; and / or inhibition and / or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.

[0133] “Inoperable” refers to the condition of a thing not functioning, malfunctioning, or no longer able to function. For example, when used in the context of a gene or when referring to a gene, the term inoperable means said gene is no longer able to operate as it normally would, either permanently or transiently. For example, “inoperable,” in some embodiments, means that a gene is no longer able to synthesize a gene product, having said gene product translated into a protein, or is otherwise unable to gene perform its normal function. For example, in some embodiments, the term inoperable can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications); interference with non-coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and / or inhibition and / or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.

[0134] “Integrative expression vector” or “integrative vector” means a yeast expression vector which can insert itself into a specific locus of the yeast cell genome and stably becomes a part of the yeast genome.

[0135] “Intervening linker” refers to a short peptide sequence in the protein separating different parts of the protein, or a short DNA sequence that is placed in the reading frame in the ORF to separate the upstream and downstream DNA sequences. For example, in some embodiments, an intervening linker may be used allowing proteins to achieve their independent secondary and tertiary structure formation during translation. In some embodiments, the intervening linker can be either resistant or susceptible to cleavage in plant cellular environments, or in the pathogenic microbe.

[0136] “Isolated” refers to separating a thing and / or a component from its natural environment, e.g., a toxin isolated from a given genus or species means that toxin is separated from its natural environment.

[0137] “kb” refers to kilobase, i.e., 1000 bases. As used herein, the term “kb” means a length of nucleic acid molecules. For example, 1 kb refers to a nucleic acid molecule that is 1000 nucleotides long. A length of double-stranded DNA that is 1 kb long, contains two thousand nucleotides (i.e., one thousand on each strand). Alternatively, a length of single-stranded RNA that is 1 kb long, contains one thousand nucleotides.56330291.3- 19 -Docket No. 277702-548911

[0138] “kDa” refers to kilodalton, a unit equaling 1,000 daltons; a “Dalton” or “dalton” is a unit of molecular weight (MW).

[0139] “Knock in” or “knock-in” or “knocks-in” or “knocking-in” refers to the replacement of an endogenous gene with an exogenous or heterologous gene, or part thereof,. For example, in some embodiments, the term “knock-in” refers to the introduction of a nucleic acid sequence encoding a desired protein to a target gene locus by homologous recombination, thereby causing the expression of the desired protein. In some embodiments, a “knock-in” mutation can modify a gene sequence to create a loss-of-function or gain-of-function mutation. The term “knock-in” can refer to the procedure by which a exogenous or heterologous polynucleotide sequence or fragment thereof is introduced into the genome, (e.g., “they performed a knock-in” or “they knocked-in the heterologous gene”), or the resulting cell and / or organism (e.g., “ the cell is a “knock-in” or “the animal is a “knock-in”).

[0140] “Knock out” or “knockout” or “knock-out” or “knocks-out” or “knocking-out” refers to a partial or complete suppression of the expression gene product (e.g., mRNA) of a protein encoded by an endogenous DNA sequence in a cell. In some embodiments, the “knock- out” can be effectuated by targeted deletion of a whole gene, or part of a gene encoding a peptide, polypeptide, or protein. As a result, the deletion may render a gene inactive, partially inactive, inoperable, partly inoperable, or otherwise reduce the expression of the gene or its products in any cell in the whole organism and / or cell in which it is normally expressed. The term “knock-out” can refer to the procedure by which an endogenous gene is made completely or partially inactive or inoperable (e.g., “they performed a knock-out” or “they knocked-out the endogenous gene”), or the resulting cell and / or organism (e.g., “ the cell is a “knock-out” or “the animal is a “knock-out”).

[0141] “Knockdown dose 50” or “KD50” refers to the median dose required to cause paralysis or cessation of movement in 50% of a population, for example a population of Musca domestica (common housefly) and / or Aedes aegypti (mosquito).

[0142] “l” or “linker” refers to a nucleotide encoding intervening linker peptide.

[0143] in the proper context refers to an intervening linker peptide, which links a translational stabilizing protein (STA) with an additional polypeptide, e.g., a PVP, and / or multiple PVPs. When referring to amino acids, “L” can also mean leucine.

[0144] “LAC4 promoter” or “Lac4 promoter” or “pLac4” refers to a DNA segment comprised of the promoter sequence derived from the K. lactis β-galactosidase gene. The LAC4 promoters is strong and inducible reporter that is used to drive expression of exogenous genes transformed into yeast.56330291.3- 20 -Docket No. 277702-548911

[0145] “LAC4 terminator” or “Lac4 terminator” refers to a DNA segment comprised of the transcriptional terminator sequence derived from the K. lactis β-galactosidase gene.

[0146] “LD20” refers to a dose required to kill 20% of a population.

[0147] “LD50” refers to lethal dose 50 which means the dose required to kill 50% of a population.

[0148] “Linker” or “LINKER” or “peptide linker” or “L” or “intervening linker” refers to a short peptide sequence operable to link two peptides together. Linker can also refer to a short DNA sequence that is placed in the reading frame of an ORF to separate an upstream and downstream DNA sequences. In some embodiments, a linker can be cleavable by an pathogenic microbe protease. In some embodiments, a linker may allow proteins to achieve their independent secondary and tertiary structure formation during translation. In some embodiments, the linker can be either resistant or susceptible to cleavage in plant cellular environments, in the pathogenic microbe. In some embodiments, a linker can be cleaved by a protease, e.g., in some embodiments, a linker can be cleaved by a plant protease (e.g., papain, bromelain, ficin, actinidin, zingibain, and / or cardosins), a fungal protease, a vertebrate protease, an invertebrate protease, a bacteria protease, a mammal protease, a reptile protease, or an avian protease. In some embodiments, a linker can be cleavable or non-cleavable. In some embodiments, a linker comprises a binary or tertiary region, wherein each region is cleavable by at least two types of proteases: one of which is a pathogenic microbe protease and the other one of which is a human protease. In some embodiments, a linker can have one of (at least) three roles: to cleave in the pathogenic microbe environment, to cleave in the plant cell, or to be designed not to intentionally cleave.

[0149] “Medium” (plural “media”) refers to a nutritive solution for culturing cells in cell culture.

[0150] “MOA” refers to mechanism of action.

[0151] “Molecular weight (MW)” refers to the mass or weight of a molecule, and is typically measured in “daltons (Da)” or kilodaltons (kDa). In some embodiments, MW can be calculated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical ultracentrifugation, or light scattering. In some embodiments, the SDS-PAGE method is as follows: the sample of interest is separated on a gel with a set of molecular weight standards. The sample is run, and the gel is then processed with a desired stain, followed by destaining for about 2 to 14 hours. The next step is to determine the relative migration distance (Rf) of the standards and protein of interest. The migration distance can be determined using the following equation: Rf = (migration distance of the protein) / (Migration distance of the dye front). Next, the logarithm of the MW can be determined based on the values obtained for the bands in56330291.3- 21 -Docket No. 277702-548911 the standard; e.g., in some embodiments, the logarithm of the molecular weight of an SDS- denatured polypeptide and its relative migration distance (Rf) is plotted into a graph. After plotting the graph, interpolating the value derived will provide the molecular weight of the unknown protein band.

[0152] “Motif” refers to a polynucleotide or polypeptide sequence that is implicated in having some biological significance and / or exerts some effect or is involved in some biological process.

[0153] “Multiple cloning site” or “MCS” refers to a segment of DNA found on a vector that contains numerous restriction sites in which a DNA sequence of interest can be inserted.

[0154] “Mutant” refers to an organism, DNA sequence, peptide sequence, or polypeptide sequence, that has an alteration (for example, in the DNA sequence), which causes said organism and / or sequence to be different from the naturally occurring or wild-type organism and / or sequence. For example, a wild-type PvD1 polypeptide can be altered resulting in a non-naturally occurring PVP.

[0155] “N-terminal” refers to the free amine group (i.e., -NH2) that is positioned on beginning or start of a polypeptide.

[0156] “NCBI” refers to the National Center for Biotechnology Information.

[0157] “nm” refers to nanometers.

[0158] “Non-Polar amino acid” is an amino acid that is weakly hydrophobic and includes glycine, alanine, proline, valine, leucine, isoleucine, phenylalanine and methionine. Glycine or gly is the most preferred non-polar amino acid for the dipeptides of this invention.

[0159] “Normalized peptide yield” means the peptide yield in the conditioned medium divided by the corresponding cell density at the point the peptide yield is measured. The peptide yield can be represented by the mass of the produced peptide in a unit of volume, for example, mg per liter or mg / L, or by the UV absorbance peak area of the produced peptide in the HPLC chromatograph, for example, mAu.sec. The cell density can be represented by visible light absorbance of the culture at wavelength of 600 nm (OD600).

[0160] “OD” refers to optical density. Typically, OD is measured using a spectrophotometer.

[0161] “OD660nm” or “OD660nm” refers to optical densities at 660 nanometers (nm).

[0162] “One letter code” means the peptide sequence which is listed in its one letter code to distinguish the various amino acids in the primary structure of a protein: alanine=A, arginine=R, asparagine=N, aspartic acid=D, asparagine or aspartic acid=B, cysteine=C, glutamic acid=E, glutamine=Q, glutamine or glutamic acid=Z, glycine=G, histidine=H, isoleucine=I,56330291.3- 22 -Docket No. 277702-548911 leucine=L, lysine=K, methionine=M, phenylalanine=F, proline=P, serine=S, threonine=T, tryptophan=W, tyrosine=Y, and valine=V.

[0163] “Operable” refers to the ability to be used, the ability to do something, and / or the ability to accomplish some function or result. For example, in some embodiments, “operable” refers to the ability of a polynucleotide, DNA sequence, RNA sequence, or other nucleotide sequence or gene to encode a peptide, polypeptide, and / or protein. For example, in some embodiments, a polynucleotide may be operable to encode a protein, which means that the polynucleotide contains information that imbues it with the ability to create a protein (e.g., by transcribing mRNA, which is in turn translated to protein).

[0164] “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, in some embodiments, operably linked can refer to two or more DNA, peptide, or polypeptide sequences. In other embodiments, operably linked can mean that the two adjacent DNA sequences are placed together such that the transcriptional activation of one DNA sequence can act on the other DNA sequence. In yet other embodiments, the term “operably linked” can refer to two or more peptides and / or polypeptides, wherein said two or more peptides and / or polypeptides are connected in such a way as to yield a single polypeptide chain; alternatively, the term operably linked can refer to two or more peptides that are connected in such a way that one peptide exerts some effect on the other. In yet other embodiments, operably linked can refer to two adjacent DNA sequences are placed together such that the transcriptional activation of one can act on the other.

[0165] “ORF” or “open reading frame” or “coding sequence” refers to a polynucleotide or nucleic acid sequence that can be transcribed and translated (e.g., in the case of DNA) or translated (e.g., in the case of mRNA) into a polypeptide, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A transcription termination sequence will usually be located 3’ to the coding sequence. A coding sequence may be flanked on the 5’ and / or 3’ ends by untranslated regions. In some embodiments, an ORF is a continuous stretch of codons that begins with a start codon (usually AUG) and ends at a stop codon (usually UAA, UAG or UGA). An ATG codon (AUG in terms of RNA). In other embodiments, an ORF can be length of RNA or DNA sequence, between a translation start signal (e.g., AUG or ATG) and any one or more of the known termination codons, wherein said length of RNA or DNA sequence encodes one or more polypeptide sequences. In some other embodiments, an ORF can be a DNA sequence encoding a protein56330291.3- 23 -Docket No. 277702-548911 which begins with an ATG start codon and ends with a TGA, TAA or TAG stop codon. ORF can also mean the translated protein that the DNA encodes.

[0166] “Out-recombined” or “out-recombination” refers to the removal of a gene and / or polynucleotide sequence (e.g., an endogenous gene) that is flanked by two site-specific recombination sites (e.g., the 5’- and 3’- nucleotide sequence of a target gene that is homologous to the homology arms of a target vector) during in vivo homologous recombination. See “knockout.”

[0167] “Peptide expression cassette” or “expression cassette” means a DNA sequence which is composed of all the DNA elements necessary to complete transcription of an fungicidal protein in a biological expression system. In the described methods herein, it includes a transcription promoter, a DNA sequence to encode an α-mating factor signal sequence, a cleavage site, an fungicidal protein transgene, a stop codon and a transcription terminator.

[0168] “Peptide expression vector” means a host organism expression vector which contains a heterologous peptide transgene.

[0169] “Peptide expression yeast strain”, “peptide expression strain” or “peptide production strain” means a yeast strain which can produce a heterologous peptide.

[0170] “Peptide Linker” see Linker.

[0171] “Peptide subunit” refers to an amino acid sequence upstream, downstream, and / or between one or more cysteine residues in a peptide, polypeptide, or protein. In some embodiments, a peptide subunit is upstream, downstream, and / or between cysteine residues in an engineered, non-naturally occurring CRIP with an ICK architecture according to Formula (II). In some embodiments, a peptide subunit can have a length of 1 to 13 amino acid residues. In yet other embodiments, a peptide subunit can have a length of 13 or more amino acid residues. In some embodiments, peptide subunits in a CRIP comprising the preferred ICK architecture of Formula (II) are designated as NE, L1, L2, L3, L4, L5, and CE.

[0172] “Peptide transgene” or “fungicidal peptide transgene” or “fungicidal protein transgene” or “PvD1 variant transgene” refers to a DNA sequence that encodes a PVP and can be translated in a biological expression system.

[0173] “Peptide yield” means the fungicidal peptide concentration in the conditioned medium which is produced from the cells of a peptide expression yeast strain. It can be represented by the mass of the produced peptide in a unit of volume, for example, mg per liter or mg / L, or by the UV absorbance peak area of the produced peptide in the HPLC chromatograph, for example, mAu.sec.

[0174] “Pest” includes, but is not limited to: pathogenic microbes, which may include: fungi, yeast, bacteria, protozoans, and the like.56330291.3- 24 -Docket No. 277702-548911

[0175] “Pesticidally-effective amount” refers to an amount of the PVP that is able to bring about death to at least one pest, or to noticeably reduce pest growth, feeding, or normal physiological development. This amount will vary depending on such factors as, for example, the specific target pests to be controlled, the specific environment, location, plant, crop, or agricultural site to be treated, the environmental conditions, and the method, rate, concentration, stability, and quantity of application of the pesticidally-effective polypeptide composition. The formulations may also vary with respect to climatic conditions, environmental considerations, and / or frequency of application and / or severity of pest infestation.

[0176] “Pharmaceutically acceptable salt” refers to a compound that is modified by making acid or base salts thereof.

[0177] “Plant” shall mean whole plants, plant tissues, plant cells, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

[0178] “Plant transgenic protein” means a protein from a heterologous species that is expressed in a plant after the DNA or RNA encoding it was delivered into one or more of the plant cells.

[0179] “Plant-incorporated protectant” or “PIP” means an fungicidal protein produced by transgenic plants, and the genetic material necessary for the plant to produce the protein.

[0180] “Plant cleavable linker” means a cleavable linker peptide, or a nucleotide encoding a cleavable linker peptide, which contains a plant protease recognition site and can be cleaved during the protein expression process in the plant cell.

[0181] “Plant regeneration media” means any media that contains the necessary elements and vitamins for plant growth and plant hormones necessary to promote regeneration of a cell into an embryo which can germinate and generate a plantlet derived from tissue culture. Often the media contains a selectable agent to which the transgenic cells express a selection gene that confers resistance to the agent.

[0182] “Plasmid” refers to a DNA segment that acts as a carrier for a gene of interest (e.g., PVP) and, when transformed or transfected into an organism, can replicate and express the DNA sequence contained within the plasmid independently of the host organism. Plasmids are a type of vector, and can be “cloning vectors” (i.e., simple plasmids used to clone a DNA fragment and / or select a host population carrying the plasmid via some selection indicator) or “expression plasmids” (i.e., plasmids used to produce large amounts of polynucleotides and / or polypeptides).

[0183] “Polar amino acid” is an amino acid that is polar and includes serine, threonine, cysteine, asparagine, glutamine, histidine, tryptophan and tyrosine; preferred polar amino acids56330291.3- 25 -Docket No. 277702-548911 are serine, threonine, cysteine, asparagine and glutamine; with serine being most highly preferred.

[0184] “Polynucleotide” refers to a polymeric-form of nucleotides (e.g., ribonucleotides, deoxyribonucleotides, or analogs thereof) of any length; e.g., a sequence of two or more ribonucleotides or deoxyribonucleotides. As used herein, the term “polynucleotide” includes double- and single-stranded DNA, as well as double- and single-stranded RNA; it also includes modified and unmodified forms of a polynucleotide (modifications to and of a polynucleotide, for example, can include methylation, phosphorylation, and / or capping). In some embodiments, a polynucleotide can be one of the following: a gene or gene fragment (for example, a probe, primer, EST, or SAGE tag); genomic DNA; genomic DNA fragment; exon; intron; messenger RNA (mRNA); transfer RNA; ribosomal RNA; ribozyme; cDNA; recombinant polynucleotide; branched polynucleotide; plasmid; vector; isolated DNA of any sequence; isolated RNA of any sequence; nucleic acid probe; primer or amplified copy of any of the foregoing.

[0185] In yet other embodiments, a polynucleotide can refer to a polymeric-form of nucleotides operable to encode the open reading frame of a gene.

[0186] In some embodiments, a polynucleotide can refer to cDNA.

[0187] In some embodiments, polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The structure of a polynucleotide can also be referenced to by its 5’- or 3’- end or terminus, which indicates the directionality of the polynucleotide. Adjacent nucleotides in a single-strand of polynucleotides are typically joined by a phosphodiester bond between their 3’ and 5’ carbons. However, different internucleotide linkages could also be used, such as linkages that include a methylene, phosphoramidate linkages, etc. This means that the respective 5’ and 3’ carbons can be exposed at either end of the polynucleotide, which may be called the 5’ and 3’ ends or termini. The 5’ and 3’ ends can also be called the phosphoryl (PO4) and hydroxyl (OH) ends, respectively, because of the chemical groups attached to those ends. The term polynucleotide also refers to both double- and single- stranded molecules. Unless otherwise specified or required, any embodiment that makes or uses a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0188] In some embodiments, a polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs (including nucleotides with non-natural bases, nucleotides with modified natural bases such as aza- or deaza-purines, etc.). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.56330291.3- 26 -Docket No. 277702-548911

[0189] In some embodiments, a polynucleotide can also be further modified after polymerization, such as by conjugation with a labeling component. Additionally, the sequence of nucleotides in a polynucleotide can be interrupted by non-nucleotide components. One or more ends of the polynucleotide can be protected or otherwise modified to prevent that end from interacting in a particular way (e.g. forming a covalent bond) with other polynucleotides.

[0190] In some embodiments, a polynucleotide can be composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T). Uracil (U) can also be present, for example, as a natural replacement for thymine when the polynucleotide is RNA. Uracil can also be used in DNA. Thus, the term “sequence” refers to the alphabetical representation of a polynucleotide or any nucleic acid molecule, including natural and non- natural bases.

[0191] The term “RNA molecule” or ribonucleic acid molecule refers to a polynucleotide having a ribose sugar rather than deoxyribose sugar and typically uracil rather than thymine as one of the pyrimidine bases. An RNA molecule of the invention is generally single-stranded, but can also be double-stranded. In the context of an RNA molecule from an RNA sample, the RNA molecule can include the single-stranded molecules transcribed from DNA in the cell nucleus, mitochondrion or chloroplast, which have a linear sequence of nucleotide bases that is complementary to the DNA strand from which it is transcribed.

[0192] In some embodiments, a polynucleotide can further comprise one or more heterologous regulatory elements. For example, in some embodiments, the regulatory element is one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; or combinations thereof.

[0193] “Post-transcriptional regulatory elements” are DNA segments and / or mechanisms that affect mRNA after it has been transcribed. Mechanisms of post-transcriptional mechanisms include splicing events; capping, splicing, and addition of a Poly (A) tail, and other mechanisms known to those having ordinary skill in the art.

[0194] “Promoter” refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene.

[0195] “Protein” has the same meaning as “peptide” and / or “polypeptide” in this document.

[0196] “PvD1” or “WT-PvD1” or “native PvD1” refers to the peptide produced by the microorganism Phaseolus vulgaris and has the Gen Bank Accession number ADR30066.1, also known as defensin D1 (Phaseolus vulgaris) having the amino acid sequence of SEQ ID NO: 256330291.3- 27 -Docket No. 277702-548911 and more specifically the PvD1 mature peptide having the amino acid sequence: KTCENLADTYKGPCFTTGSCDDHCKNKEHLRSGRCRDDFRCWCTKNC (SEQ ID NO: 3).

[0197] “PVP” or “PvD1 variant polynucleotide” or “variant PvD1 polynucleotide” refers to a polynucleotide sequence operable to encodes a PVP. The term “PvD1 variant polynucleotide” when used to describe the PvD1 variant polynucleotide sequence contained in a PVP ORF, its inclusion in a vector, and / or when describing the polynucleotides encoding an fungicidal protein, is described as “pvp” and / or “PVP”.

[0198] “PVP” or “PvD1 Variant Polypeptides” refer to peptide, polypeptide, or protein mutants or variants having 45 to 55 contiguous amino acids that differ from the full length amino acid sequence of wild-type mature PvD1 (KT CENLADTYKG PCFTTGSCDD HCKNKEHLRS GRCRDDFRCW CTKNC isolated from Phaseolus vulgaris -SEQ ID NO: 3)for example, in some embodiments, this variance can be one to five amino acid substitutions, amino acid deletions / insertions, and / or a mutations or variance to a polynucleotide operable to encode the wild-type mature PvD1 of SEQ ID NO: 3. A full length PVP will not have the amino acid sequence of SEQ ID NO: 3 in its entirety. The result of this variation is a non-naturally occurring polypeptide and / or polynucleotide sequence encoding the same that enginereed peptide that possesses fungicidal activity against one or more pathogenic microbe species. When referring to a PVP, or a polynucleotide encoding a PVP, in each case, variants may have an amino acid sequence having at least 95%, or 96%, or 97%, or 98%, or 99% (i.e. less than 100%) identity to any PVP noted herein, the contemplated genera of PVPs excludes and explicity disclaims the wild-type PvD1 peptide having an entire or full length amino acid sequence of SEQ ID NOs: 2 or 3. All PVPs disclosed herein are synthetic and do not occur in nature.

[0199] “PVP expression cassette” refers to one or more regulatory elements such as promoters; enhancer elements; mRNA stabilizing polyadenylation signal; an internal ribosome entry site (IRES); introns; post-transcriptional regulatory elements; and a polynucleotide operable to encode a PVP, e.g., a PVP ORF. For example, one example of a PVP expression cassette is one or more segments of DNA that contains a polynucleotide segment operable to express a PVP, a ADH1 promoter, a LAC4 terminator, and an alpha-MF secretory signal. A PVP expression cassette contains all of the nucleic acids necessary to encode a PVP or a PVP- fungicidal protein or peptide.

[0200] “PVP ORF” refers to a polynucleotide operable to encode a PVP, or a PVP- fungicidal protein or peptide.

[0201] “PVP ORF diagram” refers to the composition of one or more PVP ORFs, as written out in diagram or equation form. For example, a “PVP ORF diagram” can be written out as using acronyms or short-hand references to the DNA segments contained within the56330291.3- 28 -Docket No. 277702-548911 expression ORF. Accordingly, in one example, a “PVP ORF diagram” may describe the polynucleotide segments encoding the ERSP, LINKER, STA, and PVP, by diagramming in equation form the DNA segments as “ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide); “linker” or “L” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide); “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), and“PVP” (i.e., the sequence encoding a PVP), respectively. An example of a PVP ORF diagram is “ersp-sta-(linkeri-PVPj)N,” or “ersp-(PVPj-linkeri)N-sta” and / or any combination of the DNA segments thereof.

[0202] “PVP-fungicidal protein or peptide” or “PVP-fungicidal polypeptide” or “fungicidal protein” or “fungicidal polypeptide” refers to any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement, comprising: (1) at least one PVP, or two or more PVPs; and (2) additional peptides, polypeptides, or proteins. For example, in some embodiments, these additional peptides, polypeptides, or proteins have the ability to increase the mortality and / or inhibit the growth of pathogenic microbes when the pathogenic microbes are exposed to a PVP-fungicidal protein or peptide, relative to a PVP alone; increase the expression of said PVP-fungicidal protein or peptide, e.g., in a host cell or an expression system; and / or affect the post-translational processing of the PVP-fungicidal protein or peptide. In some embodiments, a PVP-fungicidal protein can be a polymer comprising two or more PVPs. In some embodiments, a PVP-fungicidal protein can be a polymer comprising two or more PVPs, wherein the PVPs are operably linked via a linker peptide, e.g., a cleavable and / or non-cleavable linker. In some embodiments, a PVP-fungicidal protein can refer to a one or more PVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an fungal cleavable or fungal non-cleavable linker (L); and / or any other combination thereof. In some embodiments, a PVP- fungicidal protein can be a non- naturally occurring protein comprising (1) a PVP; and (2) additional peptides, polypeptides, or proteins, e.g., an ERSP; a linker; a STA; a UBI; or a histidine tag or similar marker.

[0203] A “PVP construct” refers to the three-dimensional arrangement / orientation of peptides, polypeptides, and / or motifs of operably linked polypeptide segments (e.g., a PVP- fungicidal protein or peptide). For example, a PVP ORF can include one or more of the following components or motifs: a PVP; an endoplasmic reticulum signal peptide (ERSP); a linker peptide (L); a translational stabilizing protein (STA); or any combination thereof. And, as used herein, the term “PVP construct” is used to describe the designation and / or orientation of the structural motif. In other words, the PVP construct describes the arrangement and orientation of the components or motifs contained within a given PVP ORF. For example, in some embodiments, a PVP construct describes, without limitation, the orientation of one of the56330291.3- 29 -Docket No. 277702-548911 following PVP-fungicidal protein or peptides: ERSP-PVP; ERSP-(PVP)N; ERSP-PVP-L; ERSP- (PVP)N-L; ERSP-(PVP-L)N; ERSP-L-PVP; ERSP-L-(PVP)N; ERSP-(L-PVP)N; ERSP-STA- PVP; ERSP-STA-(PVP)N; ERSP-PVP-STA; ERSP-(PVP)N-STA; ERSP-(STA-PVP)N; ERSP- (PVP-STA)N; ERSP-L-PVP-STA; ERSP-L-STA-PVP; ERSP-L-(PVP-STA)N; ERSP-L-(STA- PVP)N; ERSP-L-(PVP)N-STA; ERSP-(L-PVP)N-STA; ERSP-(L-STA-PVP)N; ERSP-(L-PVP- STA)N; ERSP-(L-STA)N-PVP; ERSP-(L-PVP)N-STA; ERSP-STA-L-PVP; ERSP-STA-PVP-L; ERSP-STA-L-(PVP)N; ERSP-(STA-L)N-PVP; ERSP-STA-(L-PVP)N; ERSP-(STA-L-PVP)N; ERSP-STA-(PVP)N-L; ERSP-STA-(PVP-L)N; ERSP-(STA-PVP)N-L; ERSP-(STA-PVP-L)N; ERSP-PVP-L-STA; ERSP-PVP-STA-L; ERSP-(PVP)N-STA-L ERSP-(PVP-L)N-STA; ERSP- (PVP-STA)N-L; ERSP-(PVP-L-STA)N; or ERSP-(PVP-STA-L)N; wherein N is an integer ranging from 1 to 200. See also “Structural motif.”

[0204] “Ratio” refers to the quantitative relation between two amounts showing the number of times one value contains or is contained within the other.

[0205] “Reading frame” refers to one of the six possible reading frames, three in each direction, of the double stranded DNA molecule. The reading frame that is used determines which codons are used to encode amino acids within the coding sequence of a DNA molecule. In some embodiments, a reading frame is a way of dividing the sequence of nucleotides in a polynucleotide and / or nucleic acid (e.g., DNA or RNA) into a set of consecutive, non- overlapping triplets.

[0206] “Recombinant DNA” or “rDNA” refers to DNA that is comprised of two or more different DNA segments.

[0207] “Recombinant vector” means a DNA plasmid vector into which foreign DNA has been inserted.

[0208] “Regulatory elements” refers to a genetic element that controls some aspect of the expression and / or processing of nucleic acid sequences. For example, in some embodiments, a regulatory element can be found at the transcriptional and post-transcriptional level. Regulatory elements can be cis-regulatory elements (CREs), or trans-regulatory elements (TREs). In some embodiments, a regulatory element can be one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; and / or other elements that influence gene expression, for example, in a tissue-specific manner; temporal-dependent manner; to increase or decrease expression; and / or to cause constitutive expression.

[0209] “Restriction enzyme” or “restriction endonuclease” refers to an enzyme that cleaves DNA at a specified restriction site. For example, a restriction enzyme can cleave a56330291.3- 30 -Docket No. 277702-548911 plasmid at an EcoRI, SacII or BstXI restriction site allowing the plasmid to be linearized, and the DNA of interest to be ligated.

[0210] “Restriction site” refers to a location on DNA comprising a sequence of 4 to 8 nucleotides, and whose sequence is recognized by a particular restriction enzyme.

[0211] “Selection gene” means a gene which confers an advantage for a genetically modified organism to grow under the selective pressure.

[0212] “Serovar” or “serotype” refers to a group of closely related microorganisms distinguished by a characteristic set of antigens. In some embodiments, a serovar is an antigenically and serologically distinct variety of microorganism

[0213] “sp.” refers to species.

[0214] “ssp.” or “subsp.” refers to subspecies.

[0215] “Subcloning” or “subcloned” refers to the process of transferring DNA from one vector to another, usually advantageous vector. For example, polynucleotide encoding a mutant PVP can be subcloned into a pLB102 plasmid subsequent to selection of yeast colonies transformed with pKLAC1 plasmids.

[0216] “SSI” is an acronym that is context dependent. In some contexts, it can refer to “site-specific integration,” which is used to refer to a sequence that will permit in vivo homologous recombination to occur at a specific site within a host organism’s genome. Thus, in some embodiments, the term “site-specific integration” refers to the process directing a transgene to a target site in a host-organism’s genome, allowing the integration of genes of interest into pre-selected genome locations of a host-organism. However, in other contexts, SSI can refer to “surface spraying indoors,” which is a technique of applying a variable volume sprayable volume of an fungicide onto surfaces where vectors rest, such as on walls, windows, floors and ceilings.

[0217] “STA” or “Translational stabilizing protein” or “stabilizing domain” or “stabilizing protein” (used interchangeably herein) means a peptide or protein with sufficient tertiary structure that it can accumulate in a cell without being targeted by the cellular process of protein degradation. The protein can be between 5 and 50 amino acids long. The translational stabilizing protein is coded by a DNA sequence for a protein that is operably linked with a sequence encoding an fungicidal protein or a PVP in the ORF. The operably-linked STA can either be upstream or downstream of the PVP and can have any intervening sequence between the two sequences (STA and PVP) as long as the intervening sequence does not result in a frame shift of either DNA sequence. The translational stabilizing protein can also have an activity which increases delivery of the PVP.

[0218] “sta” means a nucleotide encoding a translational stabilizing protein.56330291.3- 31 -Docket No. 277702-548911

[0219] “Structural motif” refers to the three-dimensional arrangement of peptides and / or polypeptides, and / or the arrangement of operably linked polypeptide segments. For example, the polypeptide comprising ERSP-STA-L-PVP has an ERSP motif, an STA motif, a LINKER motif, and a PVP polypeptide motif.

[0220] “Toxin” refers to a venom and / or a poison, especially a protein or conjugated protein produced by certain animals, higher plants, and pathogenic bacteria. Generally, the term “toxin” is reserved natural products, e.g., molecules and peptides found in scorpions, spiders, snakes, poisonous mushrooms, etc., whereas the term “toxicant” is reserved for man-made products and / or artificial products e.g., man-made chemical pesticides. However, as used herein, the terms “toxin” and “toxicant” are used synonymously

[0221] “Transfection” and “transformation” both refer to the process of introducing exogenous and / or heterologous DNA or RNA (e.g., a vector containing a polynucleotide that encodes a PVP) into a host organism (e.g., a prokaryote or a eukaryote). Generally, those having ordinary skill in the art sometimes reserve the term “transformation” to describe processes where exogenous and / or heterologous DNA or RNA are introduced into a bacterial cell; and reserve the term “transfection” for processes that describe the introduction of exogenous and / or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the term “transformation” and “transfection” are used synonymously, regardless of whether a process describes the introduction exogenous and / or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).

[0222] “Transgene” means a heterologous and / or exogenous DNA sequence encoding a protein which is transformed into a plant.

[0223] “Transgenic host cell” or “host cell” means a cell which is transformed with a gene and has been selected for its transgenic status via an additional selection gene.

[0224] “Transgenic plant” means a plant that has been derived from a single cell that was transformed with foreign DNA such that every cell in the plant contains that transgene.

[0225] “Transient expression system” means an Agrobacterium tumefaciens-based system which delivers DNA encoding a disarmed plant virus into a plant cell where it is expressed. The plant virus has been engineered to express a protein of interest at high concentrations, up to 40% of the TSP.

[0226] “Triple expression cassette refers to three PVP expression cassette s contained on the same vector.

[0227] “TRBO” means a transient plant expression system using Tobacco mosaic virus with removal of the viral coating protein gene.56330291.3- 32 -Docket No. 277702-548911

[0228] “Trypsin cleavage” means an in vitro assay that uses the protease enzyme trypsin (which recognizes exposed lysine and arginine amino acid residues) to separate a cleavable linker at that cleavage site. It also means the act of the trypsin enzyme cleaving that site.

[0229] “TSP” or “total soluble protein” means the total amount of protein that can be extracted from a plant tissue sample and solubilized into the extraction buffer.

[0230] “var.” refers to varietas or variety. The term “var.” is used to indicate a taxonomic category that ranks below the species level and / or subspecies (where present). In some embodiments, the term “var.” represents members differing from others of the same subspecies or species in minor but permanent or heritable characteristics.

[0231] “Variant” or “variant sequence” or “variant peptide” or “variant thereof” refers to an amino acid sequence that possesses one or more amino acid substitutions or modifications (e.g., deletion or addition). In some embodiments, the one or more amino acid substitutions or modifications can be conservative; here, such a conservative amino acid substitution and / or modification in a “variant” does not substantially diminish the activity of the variant in relation to its non-varied form. For example, in some embodiments, a “variant” possesses one or more conservative amino acid substitutions when compared to a peptide with a disclosed and / or claimed sequence, as indicated by a SEQ ID NO. For example, in some embodiments, the phrase: “a PVP or a variant thereof,” refers to a PVP or a PVP-variant, with one or more amino acid additions, deletions, and / or substitution that does not substantially diminish the activity of the PVP-variant in relation to its non-varied, PVP form

[0232] “Vector” refers to the DNA segment that accepts a foreign gene of interest (e.g., PVP). The gene of interest is known as an “insert” or “transgene.”

[0233] “Wild type” or “WT” refer to the phenotype and / or genotype (i.e., the appearance or sequence) of an organism, polynucleotide sequence, and / or polypeptide sequence, as it is found and / or observed in its naturally occurring state or condition.

[0234] “Yeast expression vector” or “expression vector” or “vector” means a plasmid which can introduce a heterologous gene and / or expression cassette into yeast cells to be transcribed and translated.

[0235] “Yield” refers to the production of a peptide, and increased yields can mean increased amounts of production, increased rates of production, and an increased average or median yield and increased frequency at higher yields. The term “yield” when used in reference to plant crop growth and / or production, as in “yield of the plant” refers to the quality and / or quantity of biomass produced by the plant.

[0236] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of56330291.3- 33 -Docket No. 277702-548911 compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0237] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, solid phase and liquid nucleic acid synthesis, peptide synthesis in solution, solid phase peptide synthesis, immunology, cell culture, and formulation. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp1-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series; J. F. Ramalho Ortigao, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun.73336-342; Merrifield, R. B. (1963). J. Am. Chem. Soc.85, 2149-2154; Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, 3. eds.), vol.2, pp.1-284, Academic Press, New York.12. Wiinsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Muler, E., ed.), vol.15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res.25, 449-474; Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000); each of these references are incorporated herein by reference in their entireties.

[0238] Throughout this specification, unless the context requires otherwise, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

[0239] WILD-TYPE PvD1 AND PVPS56330291.3- 34 -Docket No. 277702-548911

[0240] Plant defensins are antimicrobial peptides recognized as part of the armamentarium of plant innate immune system. They are small and basic peptides of 45–54 amino acid residues comprised in a three-dimensional structure formed by three anti-parallel β- strands and one α-helix which is stabilized by four disulfide bonds. The four disulfide bonds form a cysteine-stabilized α-helix β-strand motif, common to these peptides. Plant defensins, like insect and mammal defensins, possess antimicrobial activity. In plant defensins, this activity is directed mainly against fungi, including agronomic important plant fungal pathogens belonging to the genera Fusarium, Alternaria and Verticillium, besides some bacteria are also inhibited.

[0241] Others have demonstrated that these peptides play an important role in the protection of radish seeds during germination. Furthermore, transgenic expression of defensin demonstrated that they enhance resistance against fungal plant pathogens, not only in greenhouse experiments, but also under field conditions. Two defensins peptides had their receptors identified on the plasma membrane of fungi. They bind to sphingolipids (glucosylceramide and mannosyldiinositolphosphoryl-ceramide) of fungal membranes, causing them an insurmountable injury. During the inhibition process, an interaction between the peptide and the sphingolipid occurs at a first moment, and then the peptide became inserted into the plasma membrane, causing its permeabilization. However, until recently, it is not known whether the arresting of spawned growth is due just to membrane permeabilization or whether the defensins interact with an intracellular target. Recently, the surmised defensin intracellular target was reported as being the cyclin F for the Pisum sativum defensin 1 (Psd1). Psd1 interacts with the cyclin F and is localized in the nuclei of fungal hyphae, in vivo. This interaction and its localization were related to cause interference in the cell cycle, as confirmed by the model system of interkinetic nuclear migration in the mouse retinal neuroblast.

[0242] The wild-type PvD1 polypeptide exemplified in SEQ ID NO:2 includes a signal peptide region and a propeptide region. Following polypeptide processing, the mature wild-type PvD1 polypeptide possesses an amino acid sequence of “KTCENLADTYKGPCFTTGSCDDHCKNKEHLRSGRCRDDFRCWCTKNC” (SEQ ID NO:3).

[0243] PvD1 Variant Polypeptides (PVPs), or agriculturally acceptable salts thereof, are mutants or variants that differ from the wild-type mature PvD1 (SEQ ID NO:3), e.g., in some embodiments, this variance can be one or more amino acid substitutions, amino acid deletions / insertions, or a change to the polynucleotide sequence encoding the wild-type PvD1. The result of this variation is a non-naturally occurring polypeptide and / or polynucleotide sequence encoding the same, that possesses fungicidal activity against one or more pathogenic microbe species, or have mutations in the amino acid sequence that enables the PVP to be expressed in culture recombinantly (for example recombinant yeast culture conditions), in56330291.3- 35 -Docket No. 277702-548911 greater quantities relative to wild-type PvD1 of SEQ ID NO: 3 cultured under the same conditions.

[0244] The present disclosure describes a PvD1 variant polypeptide (PVP) having fungicidal activity against one or more pathogenic microbe species. Here, the PVP comprises or consists of: (i) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T- Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; or (ii) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to an amino acid sequence of any one of SEQ ID NOs: 4-23, or an agriculturally acceptable salt thereof.

[0245] In some embodiments, the present disclosure describes a PvD1 variant polypeptide (PVP) having fungicidal activity against one or more pathogenic microbe species, wherein the PVP comprises an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to any one of SEQ ID NOs: 5-23; or an agriculturally acceptable salt thereof.

[0246] The present disclosure describes a PvD1 variant polypeptide (PVP) having fungicidal activity against one or more pathogenic microbe species. Here, the PVP comprises an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T- T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof.

[0247] In some embodiments, a PVP in accordance with Formula (I) comprises X2 is A or V or G. In a preferred embodiment, a preferred PVP comprises the amino acid sequence of SEQ ID NO: 1, wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, a preferred PVP comprises the amino acid sequence of SEQ ID NO: 1, wherein X1 is G or A, X2 is A or K, and X3 is F or L. In another preferred embodiment, a preferred PVP comprises the amino acid sequence of SEQ ID NO: 1, wherein, X1is G and X2is A, and X3 is F or L.

[0248] In addition, the present disclosure describes a PVP comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 4-23, or SEQ ID NOs: 5-23.

[0249] In addition, the present disclosure describes a PVP having an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino56330291.3- 36 -Docket No. 277702-548911 acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C- D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; for example, wherein, X2is A or V or G; or wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A; or wherein X1 is G or A, X2 is A or K, and X3is F or L; or wherein, X1is G and X2is A, X3is F or L.

[0250] In some aspects, the present disclosure provides for a PVP having an amino acid sequence in accordance with Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C- D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; or wherein X2is A or V or G; or wherein, X1is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A; or wherein X1 is G or A, X2 is A or K, and X3 is F or L; or wherein X1is G and X2is A, and X3is F or L.

[0251] In related embodiments, a PVP having an amino acid sequence of any one of SEQ ID NOs: 4-23, wherein said PVP has one to three conservative amino acid substitutions along the entire length of the PVP amino acid sequence. In some embodiments, a PVP of Formula (I) may have one to three conservative amino acid substitutions along the entire length of the PVP amino acid sequence, with the exception of the first amino acid residue X1, the 12th amino acid residue X2and the 16th amino acid X3of Formula (I).

[0252] In addition, the present disclosure describes a composition consisting of a PVP described herein, a PVP-fungicidal protein (PVC-antimicrobial protein used interchangeably herein) or PVP-polypeptide, or combinations thereof, and at least one excipient.

[0253] The present disclosure describes a polynucleotide operable to encode a PVP, or a complementary nucleotide sequence thereof, said PVP comprising or consisting of, an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to an amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T- G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; or a polynucleotide operable to encode a PVP comprising or consisting of an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% according SEQ ID NOs: 4-23; or an agriculturally acceptable salt thereof, or a complementary nucleotide sequence thereof.

[0254] In some embodiments, the polynucleotide encodes a PVP wherein X2 is A or K and X1 is G or A and X3 is F or L. In some embodiments, X2 is A or V or G. In a preferred embodiment, X1is G, A, V, L, I, F, T, Y, N, Q or D and X2is K or A. In further preferred56330291.3- 37 -Docket No. 277702-548911 embodiments, X1 is G or A, X2 is A or K, and X3 is F or L. In another preferred embodiment, X1 is G and X2is A, X3is F or L, or a complementary nucleotide sequence thereof.

[0255] In some embodiments, the present disclosure provides a polynucleotide operable to encode a PVP, the PVP comprising or consisting of an amino sequence as set forth in any one of SEQ ID NOs: 4-23, or an agriculturally acceptable salt thereof.

[0256] In some embodiments, the present disclosure provides a polynucleotide operable to encode a PVP, the PVP comprising or consisting of an amino sequence as set forth in any one of SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof.

[0257] In addition, the present disclosure describes a method of producing a PVP, the method comprising: preparing a vector comprising a first expression cassette comprising a polynucleotide operable to encode a PVP, and / or a complementary nucleotide sequence thereof, said polynucleotide encodes: (i) a PVP, or complementary nucleotide sequence thereof, said PVP comprising an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T- Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N- C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or (ii) a PVP comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 4-23. In some embodiments, X2 is A or V or G. In a preferred embodiment, X1is G, A, V, L, I, F, T, Y, N, Q or D and X2is K or A. In further preferred embodiments, X1is G or A, X2is A or K, and X3is F or L. In another preferred embodiment, X1is G and X2 is A, X3 is F or L, and introducing the vector into a yeast strain; and growing the yeast strain in a growth medium under conditions operable to enable expression of the PVP and secretion into the growth medium.

[0258] In addition, the present disclosure describes a method for protecting a plant from pathogenic microbes, the method comprising: providing a plant that expresses a PVP, or a polynucleotide encoding the same.

[0259] Furthermore, the present disclosure describes a method for controlling pathogenic microbes comprising, providing to said pathogenic microbe, a transgenic plant or plant part, for example, a seed, that comprises in its genome a stably incorporated expression cassette, wherein said stably incorporated expression cassette comprises a polynucleotide operable to encode a PVP.

[0260] The present disclosure describes a method of combating, controlling, or inhibiting a pest comprising, applying a pesticidally effective amount of the composition consisting of a PVP, a PVP-fungicidal protein or peptide, or combinations thereof, and an excipient, to the locus of the pest, or to a plant or animal susceptible to an attack by the pest.56330291.3- 38 -Docket No. 277702-548911

[0261] In addition, the present disclosure describes a vector comprising a polynucleotide operable to encode a PVP having an amino acid sequence with at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 1 and 4-23.

[0262] In some embodiments, the present disclosure provides a vector comprising a polynucleotide operable to encode a PVP having an amino acid sequence with at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 5-23.

[0263] The present disclosure also describes a yeast strain comprising a first expression cassette comprising a polynucleotide operable to encode a PVP, said PVP comprising: (i) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T- T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; or (ii) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to any one of SEQ ID NOs: 4-23. In some embodiments, with regards to PVPs encoded by a polynucleotide in accordance with Formula (I), X2 is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2is K or A. In further preferred embodiments, X1is G or A, X2is A or K, and X3is F or L. In another preferred embodiment, X1is G and X2is A, X3is F or L.

[0264] In some aspects, the present disclosure provides for a PVP having an amino acid sequence in accordance with Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C- D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof. In some embodiments, X2is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, X1is G or A, X2is A or K, and X3is F or L. In another preferred embodiment, a polynucleotide is opeable to encode a PVP in accordance with Formula (I) wherein, X1 is G and X2 is A, and X3 is F or L. In related embodiments, a polynucleotide is contemplated for expression of a PVP of Formula (I), as described herein, said PVP having one to three conservative amino acid substitutions along the entire length of the PVP amino acid sequence of Formula (I), with the exception of the first amino acid residue X1, the 12thamino acid residue X2 and the 16thamino acid X3 of Formula (I).56330291.3- 39 -Docket No. 277702-548911

[0265] In some embodiments, a polynucleotide encodes a PVP, comprising an amino acid sequence that is at least at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T- G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof. In some embodiments, X2 is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, X1 is G or A, X2 is A or K, and X3 is F or L. In another preferred embodiments, X1is G and X2is A. In related embodiments, a PVP of Formula (I) further comprises a PVP having the amino acid sequence of Formula (I) as described herein, said PVP having one to three conservative amino acid substitutions along the entire length of the PVP amino acid sequence of Formula (I), with the exception of the first amino acid residue X1, the 12thamino acid residue X2and the 16thamino acid X3of Formula (I).

[0266] In some embodiments, a polynucleotide encodes a PVP, comprising or consisting of an amino acid sequence that is at least at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino sequence as set forth in any one of SEQ ID NOs: 4-23, or SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof.

[0267] In some embodiments, a PVP can be a homopolymer or heteropolymer of two or more PVPs, wherein the amino acid sequence of each PVP is the same or different.

[0268] In some embodiments, a PVP can be a fused protein comprising two or more PVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each PVP may be the same or different. And, in some embodiments, the linker is cleavable inside the pathogenic microbe.

[0269] In some embodiments, the PVP can be combined with one or more additional peptides and / or produces. For example, a PVP can be part of a composition comprising a PVP as described herein, and an excipient.

[0270] In some embodiments, a PVP can be encoded by a polynucleotide. For example, a polynucleotide operable to encode a PVP, said PVP comprising an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence56330291.3- 40 -Docket No. 277702-548911 according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N- K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof. In some embodiments, X2is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, X1is G or A, X2is A or K, and X3is F or L. In another preferred embodiments, X1is G and X2 is A.

[0271] In related embodiments, a PVP of Formula (I) or a PVP in accordance with one of SEQ ID NO: 4-23, further comprises a PVP having one to three conservative amino acid substitutions along the entire length of the PVP amino acid sequence, with the exception of the first amino acid residue G and the 12th amino acid residue X1of Formula (I).

[0272] In yet other embodiments, the polynucleotide encodes a PVP having an amino sequence that has at least 95% or at least 96%, or 97%, or 98%, or 99%, or 100% sequence identity as set forth in any one of SEQ ID NOs: 1, 4-23, or an agriculturally acceptable salt thereof, with the proviso that the entire amino acid sequence of the PVP is not wild-type PvD1 (SEQ ID NO: 3).

[0273] In some embodiments, a plant, plant tissue, plant cell, plant seed, or part thereof can comprise one or more PVPs as described herein, or a polynucleotide encoding a PVP as described herein.

[0274] In some embodiments, a PVP can be produced by a method comprising: (a) preparing a vector comprising a first expression cassette comprising a polynucleotide operable to express a PVP or complementary nucleotide sequence thereof, said PVP comprising or consisting of an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence according to: (i) a PVP according to Formula (I): X1- K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D- F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or (ii) a PVP comprising or consisting of an amino acid sequence as provided in any one of SEQ ID NOs: 4-23, or an agriculturally acceptable salt thereof; (b) introducing the vector into a yeast strain; and (c) growing the yeast strain in a growth medium under conditions operable to enable expression of the PVP and secretion into the growth medium.

[0275] In some embodiments, the vector is a plasmid comprising an alpha-MF signal. In other embodiments, the vector is transformed into a yeast strain. For example, in some56330291.3- 41 -Docket No. 277702-548911 embodiments, the yeast strain is selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces. In some embodiments, the yeast strain is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris. For example, in some embodiments, the yeast strain is Kluyveromyces lactis.

[0276] In some embodiments, expression of the PVP provides a yield of: at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, at least 190 mg / L 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L of PVP per liter of medium. For example, in some embodiments, expression of the PVP provides a yield of at least 100 mg / L of PVP per liter of medium.

[0277] In some embodiments, expression of the PVP in the medium results in the expression of a single PVP in the medium.

[0278] In some embodiments, expression of the PVP in the medium results in the expression of a PVP polymer comprising two or more PVP polypeptides in the medium.

[0279] In some embodiments, the vector comprises two or three expression cassettes, each expression cassette operable to encode the PVP of the first expression cassette. In some embodiments, the vector comprises two or three expression cassettes, each expression cassette operable to encode the PVP of the first expression cassette, or a PVP of a different expression cassette. In some embodiments, the expression cassette is operable to encode a PVP as set forth in any one of SEQ ID NOs: 1, and 4-23, or an agriculturally acceptable salt thereof.

[0280] In some embodiments, a PVP comprises a polypeptide having an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to any one of the amino acid sequences listed in Table 1, or an agriculturally acceptable salt thereof, or wherein56330291.3- 42 -Docket No. 277702-548911 the PVP of Formula (I), including the PVPs of SEQ ID NOs: 1, and 4-23, or preferably SEQ ID NOs: 5-23, all of the foregoing PVPs may be substituted with one to three conservative substituted amino acids along the entire length of the PVP amino acid sequence, with the exception of the first amino acid residue X1, is G, A, V, L, I, F, T, Y, N, Q or D and the 12th amino acid residue X2 of Formula (I) is A, K, V, G, L or I, and X3 is is F, L, G, A, V, I or M; and wherein the PVP retains fungicidal activity as measured using the fungicidal assays as exemplified in the present disclosure and examples set forth herein.

[0281] Table 1. Exemplary PvD1 Variant Polypeptides (PVPs) including shorthand name, SEQ ID NO, and full amino acid sequence listing. SEQ PvD1 ID Variant Amino Acid Sequence C

[0282] In various embodiments, polynucleotides encoding PVPs can be used to transform plant cells, yeast cells, or bacteria cells. In some embodiments, the fungicidal PVP transgenic proteins may be formulated into compositions that can be sprayed or otherwise applied in any manner known to those skilled in the art to the surface of plants or parts thereof. Accordingly, DNA constructs are provided herein, operable to encode one or more PVPs under the appropriate conditions in a host cell, for example, a plant cell. Methods for controlling a pest infection by a parasitic pathogenic microbe of a plant cell comprises administering or introducing a56330291.3- 43 -Docket No. 277702-548911 polynucleotide encoding an PVP as described herein to a plant, plant tissue, or a plant cell by recombinant techniques and growing said recombinantly altered plant, plant tissue or plant cell in a field exposed to the pest. Alternatively, PVPs can be formulated into a sprayable composition consisting of a PVP and an excipient, and applied directly to susceptible plants by direct application, such that upon ingestion of the PVP by the infectious pathogenic microbe results in a deleterious effect.

[0283] In some embodiments, the PVP may comprise an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 1, and 4-23, or an agriculturally acceptable salt thereof.

[0284] In some embodiments, the PVP may comprise an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 4-23, or an agriculturally acceptable salt thereof.

[0285] In some preferred embodiments, the PVP may comprise an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof.

[0286] In some embodiments, the present disclosure provides a polynucleotide operable to encode a PVP having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 1, and 4-23, or a PVP of any one of SEQ ID NOs: 5-23, each of the foregoing PVPs substituted with one to three conservative substituted amino acids along the entire length of the PVP amino acid sequence, with the proviso that the first amino acid residue X1of Formula (I), is G, A, V, L, I, F, T, Y, N, Q or D and the 12th amino acid residue X2 of Formula (I) is A, K, V, G, L or I, and the amino acid residue X3 of Formula (I) is F, L, G, A, V, I or M; and wherein the PVP retains fungicidal activity as measured using the fungicidal assays as exemplified in the present disclosure and examples set forth herein, or a complementary nucleic acid sequence thereof.

[0287] In some embodiments, a vector can comprise a polynucleotide operable to encode a PVP, wherein the PVP comprises or consists of an amino acid sequence having at least .56330291.3- 44 -Docket No. 277702-548911

[0288] In some embodiments, a vector can comprise a polynucleotide operable to encode a PVP having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 4-23, or a PVP of any one of SEQ ID NOs: 5-23, each of the foregoing PVPs optionally substituted with one to three conservative substituted amino acids along the entire length of the PVP amino acid sequence.

[0289] PVP-fungicidal peptides and proteins

[0290] In some embodiments, a PVP can be any peptide, polypeptide, amino acid sequence, configuration, construct or arrangement that is capable or operable to kill or impair the growth or metabolism of at least one species of fungus described herein. In some embodiments, a PVP protein comprises: (1) at least one PVP, or two or more PVPs; and (2) at least one additional peptide, polypeptide, or protein. For example, in some embodiments, these additional peptides, polypeptides, or proteins may have the ability to increase the mortality and / or inhibit the growth of fungal microorganisms exposed to the PVP-fungicidal protein or peptide, relative to the PVP alone; increase the expression of the PVP-fungicidal protein or peptide, e.g., in a host cell; and / or affect the post-translational processing of the PVP-fungicidal protein or peptide.

[0291] In some embodiments, a PVP-fungicidal protein can be a polymer comprising two or more PVPs. In yet other embodiments, a PVP-fungicidal protein can be a polymer comprising two or more PVPs, wherein the PVPs are operably linked via a linker peptide, e.g., a cleavable and / or non-cleavable linker.

[0292] In some embodiments, a PVP-fungicidal protein can refer to a one or more PVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an fungal cleavable or fungal non-cleavable linker (L); and / or any other combination thereof.

[0293] In some embodiments, a PVP-fungicidal protein can be a polymer of amino acids that when properly folded or in its most natural thermodynamic state exerts an fungicidal activity against one or more pathogenic microbes. For example, in some embodiments, a PVP-fungicidal protein can be a polymer comprising two or more PVPs that are different. In other embodiments, an fungicidal protein can be a polymer of two or more PVPs that are the same.

[0294] In yet other embodiments, a PVP-fungicidal protein can comprise one or more PVPs, and one or more peptides, polypeptides, or proteins, that may assist in the PVP-fungicidal protein or peptide’s folding.

[0295] In some embodiments, a PVP-fungicidal protein can comprise one or more PVPs, and one or more peptides, polypeptides, or proteins, wherein the one or more peptides,56330291.3- 45 -Docket No. 277702-548911 polypeptides, or proteins are protein tags that help stability or solubility. In other embodiments, the peptides, polypeptides, or proteins can be protein tags that aid in affinity purification.

[0296] In some embodiments, a PVP-fungicidal protein can refer to a one or more PVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an fungal cleavable or fungal non-cleavable linker; one or more heterologous peptides; one or more additional polypeptides; and / or any other combination thereof. In some embodiments, an fungicidal protein can comprise a one or more PVPs as disclosed herein.

[0297] In some embodiments, a PVP-fungicidal protein can comprise a PVP homopolymer, e.g., two or more PVP monomers that are the same PVP. In some embodiments, the fungicidal PVP protein can comprise a PVP heteropolymer, e.g., two or more PVP monomers, wherein the PVP monomers are different.

[0298] In some embodiments, a PVP-fungicidal protein can comprise one or more PVPs having an amino acid sequence set forth in SEQ ID NOs: 1, or 4-23, or an agriculturally acceptable salt thereof. In some embodiments, the PVP-fungicidal protein may comprise a PVP as described herein, for example a PVP having an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to of any one of SEQ ID NOs: 1, or 4-23, including a PVP of SEQ ID NOs: 1, and 4-23, substituted with one to three conservative substituted amino acids along the entire length of the PVP amino acid sequence, with the proviso that the first amino acid residue X1, is G, A, V, L, I, F, T, Y, N, Q or D and the 12th amino acid residue X2of Formula (I) is A, K, V, G, L or I, and X3is is F, L, G, A, V, I or M; and retains fungicidal activity as measured using the fungicidal assays as exemplified in the present disclosure and examples set forth herein or an agriculturally acceptable salt thereof.

[0299] In some embodiments, a PVP-fungicidal protein can comprise one or more PVPs having an amino acid sequence set forth in SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof. In some embodiments, the PVP-fungicidal protein may comprise a PVP as described herein, for example a PVP having an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to of any one of SEQ ID NOs: 5- 23, substituted with one to three conservative substituted amino acids along the entire length of the PVP amino acid sequence, and retains fungicidal activity as measured using the fungicidal assays as exemplified in the present disclosure and examples set forth herein or an agriculturally acceptable salt thereof.56330291.3- 46 -Docket No. 277702-548911

[0300] Exemplary methods for the generation of cleavable and non-cleavable linkers can be found in U.S. Patent Application No.15 / 727,277; and PCT Application No. PCT / US2013 / 030042, the disclosures of which are incorporated herein by reference in their entireties.

[0301] METHODS FOR PRODUCING A PVP

[0302] Isolating and mutating wild-type PvD1

[0303] In various illustrative embodiments, an PVP can be obtained by creating a mutation in the wild-type PvD1 polynucleotide sequence; inserting that PvD1 variant polynucleotide (PVP) sequence into the appropriate vector; transforming a host organism in such a way that the polynucleotide encoding a PVP is expressed; culturing the host organism to generate the desired amount of PVP; and then purifying the PVP from in and / or around host organism.

[0304] Wild-type PvD1 toxins can be isolated from Phaseolus vulgaris (cv. Pérola) seeds.

[0305] A wild-type PvD1 polynucleotide sequence can be obtained by screening a genomic library using primer probes directed to the PvD1 polynucleotide sequence. Alternatively, wild-type PvD1 polynucleotide sequence and / or PVP polynucleotide sequences can be chemically synthesized. For example, a wild-type PvD1 polynucleotide sequence and / or PVP polynucleotide sequence can be generated using the oligonucleotide synthesis methods such as the phosphoramidite; triester, phosphite, or H-Phosphonate methods (see Engels, J. W. and Uhlmann, E. (1989), Gene Synthesis [New Synthetic Methods (77)]. Angew. Chem. Int. Ed. Engl., 28: 716–734, the disclosure of which is incorporated herein by reference in its entirety).

[0306] Chemically synthesizing PVP polynucleotides

[0307] In some embodiments, the polynucleotide sequence encoding the PVP can be chemically synthesized using commercially available polynucleotide synthesis services such as those offered by Genewiz® (e.g., TurboGENETM; PriorityGENE; and FragmentGENE), or Sigma-Aldrich® (e.g., Custom DNA and RNA Oligos Design and Order Custom DNA Oligos). Exemplary method for generating DNA and or custom chemically synthesized polynucleotides are well known in the art, and are illustratively provided in U.S. Patent No.5,736,135, Serial No. 08 / 389,615, filed on Feb.13, 1995, the disclosure of which is incorporated herein by reference in its entirety. See also Agarwal, et al., Chemical synthesis of polynucleotides. Angew Chem Int Ed Engl.1972 Jun; 11(6):451-9; Ohtsuka et al., Recent developments in the chemical synthesis of polynucleotides. Nucleic Acids Res.1982 Nov 11; 10(21): 6553–6570; Sondek & Shortle. A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites. Proc Natl Acad Sci U S A.1992 Apr 15; 89(8): 3581–3585; Beaucage S. L., et al., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite56330291.3- 47 -Docket No. 277702-548911 Approach. Tetrahedron, Elsevier Science Publishers, Amsterdam, NL, vol.48, No.12, 1992, pp. 2223-2311; Agrawal (1993) Protocols for Oligonucleotides and Analogs: Synthesis and Properties; Methods in Molecular Biology Vol.20, the disclosure of which is incorporated herein by reference in its entirety.

[0308] Producing a mutation in wild-type PvD1 polynucleotide sequence can be achieved by various means that are well known to those having ordinary skill in the art. Methods of mutagenesis include Kunkel’s method; cassette mutagenesis; PCR site-directed mutagenesis; the “perfect murder” technique (delitto perfetto); direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker; direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker using long homologous regions; transplacement “pop-in pop-out” method; and CRISPR-Cas 9. Exemplary methods of site- directed mutagenesis can be found in Ruvkun & Ausubel, A general method for site-directed mutagenesis in prokaryotes. Nature.1981 Jan 1; 289(5793):85-8; Wallace et al., Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA. Nucleic Acids Res.1981 Aug 11; 9(15):3647-56; Dalbadie- McFarland et al., Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc Natl Acad Sci U S A.1982 Nov; 79(21):6409-13; Bachman. Site-directed mutagenesis. Methods Enzymol.2013; 529:241-8; Carey et al., PCR-mediated site- directed mutagenesis. Cold Spring Harb Protoc.2013 Aug 1; 2013(8):738-42; and Cong et al., Multiplex genome engineering using CRISPR / Cas systems. Science.2013 Feb 15; 339(6121):819-23, the disclosures of all of the aforementioned references are incorporated herein by reference in their entireties.

[0309] Chemically synthesizing polynucleotides allows for a DNA sequence to be generated that is tailored to produce a desired polypeptide based on the arrangement of nucleotides within said sequence (i.e., the arrangement of cytosine [C], guanine [G], adenine [A] or thymine [T] molecules); the mRNA sequence that is transcribed from the chemically synthesized DNA polynucleotide can be translated to a sequence of amino acids, each amino acid corresponding to a codon in the mRNA sequence. Accordingly, the amino acid composition of a polypeptide chain that is translated from an mRNA sequence can be altered by changing the underlying codon that determines which of the 20 amino acids will be added to the growing polypeptide; thus, mutations in the DNA such as insertions, substitutions, deletions, and frameshifts may cause amino acid insertions, substitutions, or deletions, depending on the underlying codon.

[0310] In some embodiments, a polynucleotide can be chemically synthesized, wherein said polynucleotide harbors one or more mutations. In some embodiments, an mRNA can be56330291.3- 48 -Docket No. 277702-548911 created from the template DNA sequence. In yet other embodiments, the mRNA can be cloned and transformed into a competent cell.

[0311] Recombinant expression, vectors and transformation

[0312] Obtaining a PVP from a chemically synthesized DNA polynucleotide sequence and / or a wild-type DNA polynucleotide sequence that has been altered via mutagenesis can be achieved by cloning the DNA sequence into an appropriate vector. There are a variety of expression vectors available, host organisms, and cloning strategies known to those having ordinary skill in the art. For example, the vector can be a plasmid, which can introduce a heterologous gene and / or expression cassette into yeast cells to be transcribed and translated. The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A vector may contain “vector elements” such as an origin of replication (ORI); a gene that confers antibiotic resistance to allow for selection; multiple cloning sites; a promoter region; a selection marker for non-bacterial transfection; and a primer binding site. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al., 1989 and Ausubel et al., 1996, both incorporated herein by reference in their entireties. In addition to encoding an PVP polynucleotide, a vector may encode a targeting molecule. A targeting molecule is one that directs the desired nucleic acid to a particular tissue, cell, or other location.

[0313] In some embodiments, a polynucleotide operable to encode a PVP or a PVP- fungicidal protein can be transformed into a host cell.

[0314] In some embodiments, a polynucleotide operable to encode a PVP or a PVP- fungicidal protein can be cloned into a vector, and transformed into a host cell.

[0315] In some embodiments, a PVP ORF can be transformed into a host cell.

[0316] In addition to a polynucleotide sequence operable to encode a PVP (e.g., a PVP ORF) or a PVP-fungicidal protein or peptide, additional DNA segments known as regulatory elements can be cloned into a vector that allow for enhanced expression of the foreign DNA or transgene; examples of such additional DNA segments include (1) promoters, terminators, and / or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; (3) an internal ribosome entry site (IRES); (4) introns; and (5) post-transcriptional regulatory elements. The56330291.3- 49 -Docket No. 277702-548911 combination of a DNA segment of interest (e.g., PVP) with any one of the foregoing cis-acting elements is called an “expression cassette.”

[0317] In some embodiments, an expression cassette or PVP expression cassette can contain one or more PVPs, and / or one or more PVP-fungicidal protein or peptides.

[0318] In some embodiments, an expression cassette or PVP expression cassette can contain one or more PVPs, and / or one or more PVP-fungicidal protein or peptides, and one or more additional regulatory elements such as: (1) promoters, terminators, and / or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; (3) an internal ribosome entry site (IRES); (4) introns; and (5) post-transcriptional regulatory elements.

[0319] In some embodiments, a single expression cassette can contain one or more of the aforementioned regulatory elements, and a polynucleotide operable to express a PVP. For example, in some embodiments, a PVP expression cassette can comprise polynucleotide operable to express an PVP, and an α-MF signal; Kex2 site; LAC4 terminator; ADN1 promoter; and an acetamidase (amdS) selection marker—flanked by LAC4 promoters on the 5’-end and 3’- end.

[0320] In some embodiments, there can be numerous expression cassettes cloned into a vector. For example, in some embodiments, there can be a first expression cassette comprising a polynucleotide operable to express a PVP. In alternative embodiments, there are two expression cassettes operable to encode a PVP (i.e., a double expression cassette). In other embodiments, there are three expression cassettes operable to encode a PVP (i.e., a triple expression cassette).

[0321] In some embodiments, a double expression cassette can be generated by subcloning a second PVP expression cassette into a vector containing a first PVP expression cassette.

[0322] In some embodiments, a triple expression cassette can be generated by subcloning a third PVP expression cassette into a vector containing a first and a second PVP expression cassette.

[0323] In some embodiments, a PVP polynucleotide can be cloned into a vector using a variety of cloning strategies, and commercial cloning kits and materials readily available to those having ordinary skill in the art. For example, the PVP polynucleotide can be cloned into a vector using such strategies as the SnapFast; Gateway; TOPO; Gibson; LIC; InFusionHD; or Electra strategies. There are numerous commercially available vectors that can be used to produce PVP. For example, a PVP polynucleotide can be generated using polymerase chain reaction (PCR), and combined with a pCRTMII-TOPO vector, or a PCRTM2.1-TOPO® vector (commercially available as the TOPO® TA Cloning ® Kit from Invitrogen) for 5 minutes at room temperature; the TOPO® reaction can then be transformed into competent cells, which can subsequently be56330291.3- 50 -Docket No. 277702-548911 selected based on color change (see Janke et al., A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast. 2004 Aug; 21(11):947-62; see also, Adams et al. Methods in Yeast Genetics. Cold Spring Harbor, NY, 1997, the disclosure of which is incorporated herein by reference in its entirety).

[0324] In some embodiments, a polynucleotide encoding a PVP can be cloned into a vector such as a plasmid, cosmid, virus (bacteriophage, animal viruses, and plant viruses), and / or artificial chromosome (e.g., YACs).

[0325] In some embodiments, a polynucleotide encoding a PVP can be inserted into a vector, for example, a plasmid vector using E. coli as a host, by performing the following: digesting about 2 to 5 μg of vector DNA using the restriction enzymes necessary to allow the DNA segment of interest to be inserted, followed by overnight incubation to accomplish complete digestion (alkaline phosphatase may be used to dephosphorylate the 5’-end in order to avoid self-ligation / recircularization); gel purify the digested vector. Next, amplify the DNA segment of interest, for example, a polynucleotide encoding an PVP, via PCR, and remove any excess enzymes, primers, unincorporated dNTPs, short-failed PCR products, and / or salts from the PCR reaction using techniques known to those having ordinary skill in the art (e.g., by using a PCR clean-up kit). Ligate the DNA segment of interest to the vector by creating a mixture comprising: about 20 ng of vector; about 100 to 1,000 ng or DNA segment of interest; 2 μL 10x buffer (i.e., 30 mM Tris-HCl 4 mM MgCl2, 26 μM NAD, 1 mM DTT, 50 μg / ml BSA, pH 8, stored at 25°C); 1 μL T4 DNA ligase; all brought to a total volume of 20 μL by adding H2O. The ligation reaction mixture can then be incubated at room temperature for 2 hours, or at 16°C for an overnight incubation. The ligation reaction (i.e., about 1 μL) can then be transformed to competent cell, for example, by using electroporation or chemical methods, and a colony PCR can then be performed to identify vectors containing the DNA segment of interest.

[0326] In some embodiments a polynucleotide encoding a PVP (e.g., a PVP ORF), along with other DNA segments together composing a PVP expression cassette can be designed for secretion from host yeast cells. An illustrative method of designing a PVP expression cassette is as follows: the cassette can begin with a signal peptide sequence, followed by a DNA sequence encoding a Kex2 cleavage site (Lysine-Arginine), and subsequently followed by the PVP polynucleotide transgene (PVP ORF), with the addition of glycine-serine codons at the 5’-end, and finally a stop codon at the 3’-end. All these elements will then be expressed to a fusion peptide in yeast cells as a single open reading frame (ORF). An α-mating factor (αMF) signal sequence is most frequently used to facilitate metabolic processing of the recombinant fungicidal peptides through the endogenous secretion pathway of the recombinant yeast, i.e. the expressed fusion peptide will typically enter the Endoplasmic Reticulum, wherein the α -mating factor56330291.3- 51 -Docket No. 277702-548911 signal sequence is removed by signal peptidase activity, and then the resulting pro-fungicidal peptide will be trafficked to the Golgi Apparatus, in which the Lysine-Arginine dipeptide mentioned above is completely removed by Kex2 endoprotease, after which the mature, polypeptide (i.e., PVP), is secreted out of the cells.

[0327] In some embodiments, polypeptide expression levels in recombinant yeast cells can be enhanced by optimizing the codons based on the specific host yeast species. Naturally occurring frequencies of codons observed in endogenous open reading frames of a given host organism need not necessarily be optimized for high efficiency expression. Furthermore, different yeast species (for example, Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, etc.) have different optimal codons for high efficiency expression. Hence, codon optimization should be considered for the PVP expression cassette, including the sequence elements encoding the signal sequence, the Kex2 cleavage site and the PVP, because they are initially translated as one fusion peptide in the recombinant yeast cells.

[0328] In some embodiments, a codon-optimized PVP expression cassette can be ligated into a yeast-specific expression vectors for yeast expression. There are many expression vectors available for yeast expression, including episomal vectors and integrative vectors, and they are usually designed for specific yeast strains. One should carefully choose the appropriate expression vector in view of the specific yeast expression system which will be used for the peptide production. In some embodiments, integrative vectors can be used, which integrate into chromosomes of the transformed yeast cells and remain stable through cycles of cell division and proliferation. The integrative DNA sequences are homologous to targeted genomic DNA loci in the transformed yeast species, and such integrative sequences include pLAC4, 25S rDNA, pAOX1, and TRP2, etc. The locations of fungicidal peptide transgenes can be adjacent to the integrative DNA sequence (Insertion vectors) or within the integrative DNA sequence (replacement vectors).

[0329] In some embodiments, the expression vectors or cloning vectors can contain E. coli elements for DNA preparation in E. coli, for example, E. coli replication origin, antibiotic selection marker, etc. In some embodiments, vectors can contain an array of the sequence elements needed for expression of the transgene of interest, for example, transcriptional promoters, terminators, yeast selection markers, integrative DNA sequences homologous to host yeast DNA, etc. There are many suitable yeast promoters available, including natural and engineered promoters, for example, yeast promoters such as pLAC4, pAOX1, pUPP, pADH1, pTEF, pGal1, etc., and others, can be used in some embodiments.

[0330] In some embodiments, selection methods such as acetamide prototrophy selection; zeocin-resistance selection; geneticin-resistance selection; nourseothricin-resistance56330291.3- 52 -Docket No. 277702-548911 selection; uracil deficiency selection; and / or other selection methods may be used. For example, in some embodiments, the Aspergillus nidulans amdS gene can be used as selectable marker. Exemplary methods for the use of selectable markers can be found in U.S. Patent Nos.6,548,285 (filed Apr.3, 1997); 6,165,715 (filed June 22, 1998); and 6,110,707 (filed Jan.17, 1997), the disclosures of which are incorporated herein by reference in its entirety.

[0331] In some embodiments, a polynucleotide encoding a PVP can be inserted into a pKLAC1 vector. The pKLAC1 is commercially available from New England Biolabs® Inc., (item no. (NEB #E1000). The pKLAC1 is designed to accomplish high-level expression of recombinant protein (e.g., PVP) in the yeast Kluyveromyces lactis. The pKLAC1 plasmid can be ordered alone, or as part of a K. lactis Protein Expression Kit. The pKLAC1 plasmid can be linearized using the SacII or BstXI restriction enzymes, and possesses a MCS downstream of an αMF secretion signal. The αMF secretion signal directs recombinant proteins to the secretory pathway, which is then subsequently cleaved via Kex2 resulting in peptide of interest, for example, a PVP. Kex2 is a calcium-dependent serine protease, which is involved in activating proproteins of the secretory pathway, and is commercially available (PeproTech®; item no.450- 45).

[0332] In some embodiments, a polynucleotide encoding a PVP can be inserted into a pLB102 plasmid, or subcloned into a pLB102 plasmid subsequent to selection of yeast colonies transformed with pKLAC1 plasmids ligated with polynucleotide encoding a PVP. Yeast, for example K. lactis, transformed with a pKLAC1 plasmids ligated with polynucleotide encoding a PVP can be selected based on acetamidase (amdS), which allows transformed yeast cells to grow in YCB medium containing acetamide as its only nitrogen source. Once positive yeast colonies transformed with a pKLAC1 plasmids ligated with polynucleotide encoding a PVP are identified.

[0333] In some embodiments, a polynucleotide encoding a PVP can be inserted into other commercially available plasmids and / or vectors that are readily available to those having skill in the art, e.g., plasmids are available from Addgene (a non-profit plasmid repository); GenScript®; Takara®; Qiagen®; and PromegaTM.

[0334] In some embodiments, a yeast cell transformed with one or more PVP expression cassettes can produce PVP in a yeast culture with a yield of: at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, at least 190 mg / L 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,00056330291.3- 53 -Docket No. 277702-548911 mg / L, at least 5,500 mg / L, at least at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L of PVP per liter of medium.

[0335] In some embodiments, a culture of K. lactis transformed with one or more PVP expressions cassettes, can produce PVP in a yeast culture with a yield of: at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, at least 190 mg / L 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L of PVP per liter of growth medium containing: (1) MSM media recipe: 2 g / L sodium citrate dihydrate; 1 g / L calcium sulfate dihydrate (0.79 g / L anhydrous calcium sulfate); 42.9g / L potassium phosphate monobasic; 5.17g / L ammonium sulfate; 14.33 g / L potassium sulfate; 11.7 g / L magnesium sulfate heptahydrate; 2 mL / L PTM1trace salt solution; 0.4 ppm biotin (from 500X, 200 ppm stock); 1-2% pure glycerol or other carbon source. (2) PTM1 trace salts solution: Cupric sulfate-5H2O 6.0 g; Sodium iodide 0.08 g; Manganese sulfate- H2O 3.0 g; Sodium molybdate-2H2O 0.2 g; Boric Acid 0.02 g; Cobalt chloride 0.5 g; Zinc chloride 20.0 g; Ferrous sulfate-7H2O 65.0 g; Biotin 0.2 g; Sulfuric Acid 5.0 ml; add Water to a final volume of 1 liter. An illustrative composition for K. lactis defined medium (DMSor) is as follows: 11.83 g / L KH2PO4, 2.299 g / L K2HPO4, 20 g / L of a fermentable sugar, e.g., galactose, maltose, latotriose, sucrose, fructose or glucose and / or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, 1 g / L MgSO4.7H2O, 10 g / L (NH4)SO4, 0.33 g / L CaCl2.2H2O, 1 g / L NaCl, 1 g / L KCl, 5 mg / L56330291.3- 54 -Docket No. 277702-548911 CuSO4.5H2O, 30 mg / L MnSO4.H2O, 10 mg / L, ZnCl2, 1 mg / L KI, 2 mg / L CoCl2.6H2O, 8mg / L Na2MoO4.2H2O, 0.4 mg / L H3BO3,15 mg / L FeCl3.6H2O, 0.8 mg / L biotin, 20 mg / L Ca- pantothenate, 15 mg / L thiamine, 16 mg / L myo-inositol, 10 mg / L nicotinic acid, and 4 mg / L pyridoxine; a selection marker, and culturing under conditions that enable optimum expression.

[0336] In some embodiments, one or more expression cassettes comprising a polynucleotide operable to express a PVP can be inserted into a vector, resulting in a yield of about 100 mg / L of PVP (supernatant of yeast fermentation broth). For example, in some embodiments, two expression cassettes comprising a polynucleotide operable to express a PVP can be inserted into a vector, for example a pKS022 plasmid, resulting in a yield of about 2 g / L of PVP (supernatant of yeast fermentation broth). Alternatively, in some embodiments, three expression cassettes comprising a polynucleotide operable to express a PVP can be inserted into a vector, for example a pLB103bT plasmid.

[0337] In some embodiments, multiple PVP expression cassettes can be transfected into yeast in order to enable integration of one or more copies of the optimized PVP transgene into the K. lactis genome. An exemplary method of introducing multiple PVP expression cassettes into a K. lactis genome is as follows: a PVP expression cassette DNA sequence is synthesized, comprising an intact LAC4 promoter element, a codon-optimized PVP ORF element and a pLAC4 terminator element; the intact expression cassette is ligated into the pLB103b vector between Sal I and Kpn I restriction sites, downstream of the pLAC4 terminator of pLB10V5, resulting in the double transgene PVP expression vector, pKS022; the double transgene vectors, pKS022, are then linearized using Sac II restriction endonuclease and transformed into YCT306 strain of K. lactis by electroporation. The resulting yeast colonies are then grown on YCB agar plate supplemented with 5 mM acetamide, which only the acetamidase-expressing cells could use efficiently as a metabolic source of nitrogen. To evaluate the yeast colonies, about 100 to 400 colonies can be picked from the pKS022 yeast plates. Inoculates from the colonies are each cultured in 2.2 mL of the defined K. lactis media with 2% sugar alcohol added as a carbon source. Cultures are incubated at 23.5°C, with shaking at 280 rpm, for six days, at which point cell densities in the cultures will reach their maximum levels as indicated by light absorbance at 600 nm (OD600). Cells are then removed from the cultures by centrifugation at 4,000 rpm for 10 minutes, and the resulting supernatants (conditioned media) are filtered through 0.2 μM membranes for HPLC yield analysis.

[0338] Chemically synthesizing PVPs

[0339] Peptide synthesis or the chemical synthesis or peptides and / or polypeptides can be used to generate PVPs: these methods can be performed by those having ordinary skill in the art, and / or through the use of commercial vendors (e.g., GenScript®; Piscataway, New Jersey). For56330291.3- 55 -Docket No. 277702-548911 example, in some embodiments, chemical peptide synthesis can be achieved using Liquid phase peptide synthesis (LPPS), or solid phase peptide synthesis (SPPS).

[0340] In some embodiments, peptide synthesis can generally be achieved by using a strategy wherein the coupling the carboxyl group of a subsequent amino acid to the N-terminus of a preceding amino acid generates the nascent polypeptide chain—a process that is opposite to the type of polypeptide synthesis that occurs in nature.

[0341] Peptide deprotection is an important first step in the chemical synthesis of polypeptides. Peptide deprotection is the process in which the reactive groups of amino acids are blocked through the use of chemicals in order to prevent said amino acid’s functional group from taking part in an unwanted or non-specific reaction or side reaction; in other words, the amino acids are “protected” from taking part in these undesirable reactions.

[0342] Prior to synthesizing the peptide chain, the amino acids must be “deprotected” to allow the chain to form (i.e., amino acids to bind). Chemicals used to protect the N-termini include 9-fluorenylmethoxycarbonyl (Fmoc), and tert-butoxycarbonyl (Boc), each of which can be removed via the use of a mild base (e.g., piperidine) and a moderately strong acid (e.g., trifluoracetic acid (TFA)), respectively.

[0343] The C-terminus protectant required is dependent on the type of chemical peptide synthesis strategy used: e.g., LPPS requires protection of the C-terminal amino acid, whereas SPPS does not owing to the solid support which acts as the protecting group. Side chain amino acids require the use of several different protecting groups that vary based on the individual peptide sequence and N-terminal protection strategy; typically, however, the protecting group used for side chain amino acids are based on the tert-butyl (tBu) or benzyl (Bzl) protecting groups.

[0344] Amino acid coupling is the next step in a peptide synthesis procedure. To effectuate amino acid coupling, the incoming amino acid’s C-terminal carboxylic acid must be activated: this can be accomplished using carbodiimides such as diisopropylcarbodiimide (DIC), or dicyclohexylcarbodiimide (DCC), which react with the incoming amino acid’s carboxyl group to form an O-acylisourea intermediate. The O-acylisourea intermediate is subsequently displaced via nucleophilic attack via the primary amino group on the N-terminus of the growing peptide chain. The reactive intermediate generated by carbodiimides can result in the racemization of amino acids. To avoid racemization of the amino acids, reagents such as 1-hydroxybenzotriazole (HOBt) are added in order to react with the O-acylisourea intermediate. Other couple agents that may be used include 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate56330291.3- 56 -Docket No. 277702-548911 (BOP), with the additional activating bases. Finally, following amino acid deprotection and coupling,

[0345] At the end of the synthesis process, removal of the protecting groups from the polypeptide must occur—a process that usually occurs through acidolysis. Determining which reagent is required for peptide cleavage is a function of the protection scheme used and overall synthesis method. For example, in some embodiments, hydrogen bromide (HBr); hydrogen fluoride (HF); or trifluoromethane sulfonic acid (TFMSA) can be used to cleave Bzl and Boc groups. Alternatively, in other embodiments, a less strong acid such as TFA can effectuate acidolysis of tBut and Fmoc groups. Finally, peptides can be purified based on the peptide’s physiochemical characteristics (e.g., charge, size, hydrophobicity, etc.). Techniques that can be used to purify peptides include Purification techniques include Reverse-phase chromatography (RPC); Size-exclusion chromatography; Partition chromatography; High-performance liquid chromatography (HPLC); and Ion exchange chromatography (IEC).

[0346] Exemplary methods of peptide synthesis can be found in Anderson G. W. and McGregor A. C. (1957) T-butyloxycarbonylamino acids and their use in peptide synthesis. Journal of the American Chemical Society.79, 6180-3; Carpino L. A. (1957) Oxidative reactions of hydrazines. Iv. Elimination of nitrogen from 1, 1-disubstituted-2-arenesulfonhydrazides1-4. Journal of the American Chemical Society.79, 4427-31; McKay F. C. and Albertson N. F. (1957) New amine-masking groups for peptide synthesis. Journal of the American Chemical Society.79, 4686-90; Merrifield R. B. (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society.85, 2149-54; Carpino L. A. and Han G. Y. (1972) 9-fluorenylmethoxycarbonyl amino-protecting group. The Journal of Organic Chemistry. 37, 3404-21; and A Lloyd-Williams P. et al. (1997) Chemical approaches to the synthesis of peptides and proteins. Boca Raton: CRC Press. 278; U.S. Patent Nos: 3,714,140 (filed Mar.16, 1971); 4,411,994 (filed June 8, 1978); 7,785,832 (filed Jan.20, 2006); 8,314,208 (filed Feb.10, 2006); and 10,442,834 (filed Oct., 2, 2015); and United States Patent Application 2005 / 0165215 (filed Dec.23, 2004), the disclosures of which are incorporated herein by reference in their entirety.

[0347] CELL CULTURE AND TRANSFORMATION TECHNIQUES

[0348] The terms “transformation” and “transfection” both describe the process of introducing exogenous and / or heterologous DNA or RNA to a host organism. Generally, those having ordinary skill in the art sometimes reserve the term “transformation” to describe processes where exogenous and / or heterologous DNA or RNA are introduced into a bacterial cell; and reserve the term “transfection” for processes that describe the introduction of exogenous and / or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the56330291.3- 57 -Docket No. 277702-548911 term “transformation” and “transfection” are used synonymously, regardless of whether a process describes the introduction exogenous and / or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).

[0349] In some embodiments, a host cell can be transformed with a polynucleotide operable to encode a PVP.

[0350] In some embodiments, a vector containing a PVP expression cassette can be cloned into an expression plasmid and transformed into a host cell. In some embodiments, the yeast cell can any one of those yeast cells described herein.

[0351] In some embodiments, a host cell can be transformed using the following methods: electroporation; cell squeezing; microinjection; impalefection; the use of hydrostatic pressure; sonoporation; optical transfection; continuous infusion; lipofection; through the use of viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus; the chemical phosphate method; endocytosis via DEAE-dextran or polyethylenimine (PEI); protoplast fusion; hydrodynamic deliver; magnetofection; nucleoinfection; and / or others. Exemplary methods regarding transfection and / or transformation techniques can be found in Makrides (2003), Gene Transfer and Expression in Mammalian Cells, Elvesier; Wong, TK & Neumann, E. Electric field mediated gene transfer. Biochem. Biophys. Res. Commun.107, 584– 587 (1982); Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol.2003 May; CHAPTER: Unit–9.3; Kim & Eberwine, Mammalian cell transfection: the present and the future. Anal Bioanal Chem.2010 Aug; 397(8): 3173–3178, each of these references are incorporated herein by reference in their entireties.

[0352] In some embodiments, electroporation can be used transform a cell with one or more PVP expression cassettes, which can produce PVP in a yeast culture with a yield of: at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, at least 190 mg / L 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,00056330291.3- 58 -Docket No. 277702-548911 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L of PVP per liter of medium.

[0353] Electroporation is a technique in which electricity is applied to cells causing the cell membrane to become permeable; this in turn allows exogenous DNA to be introduced into the cells. Electroporation is readily known to those having ordinary skill in the art, and the tools and devices required to achieve electroporation are commercially available (e.g., Gene Pulser Xcell™ Electroporation Systems, Bio-Rad®; Neon® Transfection System for Electroporation, Thermo-Fisher Scientific; and other tools and / or devices). Exemplary methods of electroporation are illustrated in Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol.2003 May; CHAPTER: Unit–9.3; Saito (2015) Electroporation Methods in Neuroscience. Springer press; Pakhomov et al., (2017) Advanced Electroporation Techniques in Biology and Medicine. Taylor & Francis; the disclosure of which is incorporated herein by reference in its entirety.

[0354] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding a PVP into yeast, for example, in some embodiments, a PVP expression cassette cloned into a plasmid, and transformed into yeast cells via electroporation.

[0355] In some embodiments, a PVP expression cassette cloned into a plasmid, and transformed a yeast cell via electroporation can be accomplished by inoculating about 10-200 mL of yeast extract peptone dextrose (YEPD) with a suitable yeast species, for example, Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris, etc., and incubate on a shaker at 30°C until the early exponential phase of yeast culture (e.g. about 0.6 to 2 x 108cells / mL); harvesting the yeast in sterile centrifuge tube and centrifuging at 3000 rpm for 5 minutes at 4°C (note: keep cells chilled during the procedure) washing cells with 40 mL of ice cold, sterile deionized water, and pelleting the cells a 23,000 rpm for 5 minutes; repeating the wash step, and the resuspending the cells in 20 mL of 1M fermentable sugar, e.g. galactose, maltose, latotriose, sucrose, fructose or glucose and / or sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, followed by spinning down at 3,000 rpm for 5 minutes; resuspending the cells with proper volume of ice cold 1M fermentable sugar, e.g. galactose, maltose, latotriose, sucrose, fructose or glucose and / or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol to final cell density of 3x109cell / mL; (1.5x109cell / mL to 6x109cell / mL are acceptable cell densities); mixing 40 µl of the yeast suspension with about 1-4 µl (at a concentration of 100-300ng / µl) of the vector containing a linear polynucleotide encoding a PVP (~1 µg) in a prechilled 0.2 cm electroporation cuvette (note: ensure the sample is in contact with both sides of the aluminum cuvette); providing a single56330291.3- 59 -Docket No. 277702-548911 pulse at 2000 V, for optimal time constant of 5 ms of the RC circuit, the cells was then let recovered in 0.5 ml YED and 0.5mL 1M fermentable sugar, e.g. galactose, maltose, latotriose, sucrose, fructose or glucose and / or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol mixture, and then spreading onto selective plates.

[0356] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding a PVP into yeast, for example, a PVP cloned into a plasmid, and transformed into K. lactis cells via electroporation, can be accomplished by inoculating about 10-200 mL of yeast extract peptone dextrose (YEPD) incubating on a shaker at 30°C until the early exponential phase of yeast culture (e.g. about 0.6 to 2 x 108cells / mL); harvesting the yeast in sterile centrifuge tube and centrifuging at 3000 rpm for 5 minutes at 4°C (note: keep cells chilled during the procedure) washing cells with 40 mL of ice cold, sterile deionized water, and pelleting the cells a 23,000 rpm for 5 minutes; repeating the wash step, and the resuspending the cells in 20 mL of 1M fermentable sugar, e.g. galactose, maltose, latotriose, sucrose, fructose or glucose and / or sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, followed by spinning down at 3,000 rpm for 5 minutes; resuspending the cells with proper volume of ice cold 1M fermentable sugar, e.g. galactose, maltose, latotriose, sucrose, fructose or glucose and / or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol to final cell density of 3x109cell / mL; mixing 40 µl of the yeast suspension with about 1-4 µl of the vector containing a linear polynucleotide encoding a PVP (~1 µg) in a prechilled 0.2 cm electroporation cuvette (note: ensure the sample is in contact with both sides of the aluminum cuvette); providing a single pulse at 2000 V, for optimal time constant of 5 ms of the RC circuit, the cells was then let recovered in 0.5 ml YED and 0.5mL 1M fermentable sugar, e.g. galactose, maltose, latotriose, sucrose, fructose or glucose and / or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol mixture, and then spreading onto selective plates.

[0357] In some embodiments, using the illustrated methods described herein, i.e., vectors of the present invention utilizing yeast, and methods transformation and fermentation, may result in production of PVP in amounts of: at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, at least 190 mg / L 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least56330291.3- 60 -Docket No. 277702-548911 at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L of PVP per liter of medium.

[0358] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding a PVP into plant protoplasts by incubating sterile plant material in a protoplast solution (e.g., around 8 mL of 10 mM 2-[N-morpholino]ethanesulfonic acid (MES), pH 5.5; 0.01% (w / v) pectylase; 1% (w / v) macerozyme; 40 mM CaCl2; and 0.4 M mannitol) and adding the mixture to a rotary shaker for about 3 to 6 hours at 30°C to produce protoplasts; removing debris via 80-μm-mesh nylon screen filtration; rinsing the screen with about 4 ml plant electroporation buffer (e.g., 5 mM CaCl2; 0.4 M mannitol; and PBS); combining the protoplasts in a sterile 15 mL conical centrifuge tube, and then centrifuging at about 300 × g for about 5 minutes; subsequent to centrifugation, discarding the supernatant and washing with 5 mL of plant electroporation buffer; resuspending the protoplasts in plant electroporation buffer at about 1.5 x 106to 2 x 106protoplasts per mL of liquid; transferring about 0.5-mL of the protoplast suspension into one or more electroporation cuvettes, set on ice, and adding the vector (note: for stable transformation, the vector should be linearized using anyone of the restriction methods described above, and about 1 to 10 μg of vector may be used; for transient expression, the vector may be retained in its supercoiled state, and about 10 to 40 μg of vector may be used); mixing the vector and protoplast suspension; placing the cuvette into the electroporation apparatus, and shocking for one or more times at about 1 to 2 kV (a 3- to 25-μF capacitance may be used initially while optimizing the reaction); returning the cuvette to ice; diluting the transformed cells 20-fold in complete medium; and harvesting the protoplasts after about 48 hours.

[0359] Host cells

[0360] The methods, compositions, PVPs, and PVP-fungicidal protein or polypeptides of the present invention may be implemented in any cell type, e.g., a eukaryotic or prokaryotic cell.

[0361] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein is a prokaryote. For example, in some embodiments, the host cell may be an Archaebacteria or Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria,56330291.3- 61 -Docket No. 277702-548911 Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus.

[0362] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be a unicellular cell. For example, in some embodiments, the host cell may be bacterial cells such as gram positive bacteria.

[0363] In some embodiments, the host cell may be a bacteria selected from the following genera consisting of: Candidatus Chloracidobacterium, Arthrobacter, Corynebacterium, Frankia, Micrococcus, Mycobacterium, Propionibacterium, Streptomyces, Aquifex Bacteroides, Porphyromonas, Bacteroides, Porphyromonas, Flavobacterium, Chlamydia, Prosthecobacter, Verrucomicrobium, Chloroflexus, Chroococcus, Merismopedia, Synechococcus, Anabaena, Nostoc, Spirulina, Trichodesmium, Pleurocapsa, Prochlorococcus, Prochloron, Bacillus, Listeria, Staphylococcus, Clostridium, Dehalobacter, Epulopiscium, Ruminococcus, Enterococcus, Lactobacillus, Streptococcus, Erysipelothrix, Mycoplasma, Leptospirillum, Nitrospira, Thermodesulfobacterium, Gemmata, Pirellula, Planctomyces, Caulobacter, Agrobacterium, Bradyrhizobium, Brucella, Methylobacterium, Prosthecomicrobium, Rhizobium, Rhodopseudomonas, Sinorhizobium, Rhodobacter, Roseobacter, Acetobacter, Rhodospirillum, Rickettsia, Rickettsia conorii, Mitochondria, Wolbachia, Erythrobacter, Erythromicrobium, Sphingomonas, Alcaligenes, Burkholderia, Leptothrix, Sphaerotilus, Thiobacillus, Neisseria, Nitrosomonas, Gallionella, Spirillum, Azoarcus, Aeromonas, Succinomonas, Succinivibrio, Ruminobacter, Nitrosococcus, Thiocapsa, Enterobacter, Escherichia, Klebsiella, Salmonella, Shigella, Wigglesworthia, Yersinia, Coxiella, Legionella, Halomonas, Pasteurella, Acinetobacter, Azotobacter, Pseudomonas, Psychrobacter, Beggiatoa, Thiomargarita, Vibrio, Xanthomonas, Bdellovibrio, Campylobacter, Helicobacter, Myxococcus, Desulfosarcina, Geobacter, Desulfuromonas, Borrelia, Leptospira, Treponema, Petrotoga, Thermotoga, Deinococcus, or Thermus.

[0364] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be selected from one of the following bacteria species: Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptomyces lividans, Streptomyces murinus, Streptomyces coelicolor, Streptomyces albicans, Streptomyces griseus, Streptomyces plicatosporus, Escherichia albertii, Escherichia blattae, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia senegalensis, Escherichia vulneris, Pseudomonas abietaniphila, Pseudomonas agarici, Pseudomonas agarolyticus, Pseudomonas alcaliphila, Pseudomonas alginovora, Pseudomonas andersonii, Pseudomonas antarctica,56330291.3- 62 -Docket No. 277702-548911 Pseudomonas asplenii, Pseudomonas azelaica, Pseudomonas batumici, Pseudomonas borealis, Pseudomonas brassicacearum, Pseudomonas chloritidismutans, Pseudomonas cremoricolorata, Pseudomonas diterpeniphila, Pseudomonas filiscindens, Pseudomonas frederiksbergensis, Pseudomonas gingeri, Pseudomonas graminis, Pseudomonas grimontii, Pseudomonas halodenitrificans, Pseudomonas halophila, Pseudomonas hibiscicola, Pseudomonas hydrogenovora, Pseudomonas indica, Pseudomonas japonica, Pseudomonas jessenii, Pseudomonas kilonensis, Pseudomonas koreensis, Pseudomonas lini, Pseudomonas lurida, Pseudomonas lutea, Pseudomonas marginata, Pseudomonas meridiana, Pseudomonas mesoacidophila, Pseudomonas pachastrellae, Pseudomonas palleroniana, Pseudomonas parafulva, Pseudomonas pavonanceae, Pseudomonas proteolyica, Pseudomonas psychrophila, Pseudomonas psychrotolerans, Pseudomonas pudica, Pseudomonas rathonis, Pseudomonas reactans, Pseudomonas rhizosphaerae, Pseudomonas salmononii, Pseudomonas thermaerum, Pseudomonas thermocarboxydovorans, Pseudomonas thermotolerans, Pseudomonas thivervalensis, Pseudomonas umsongensis, Pseudomonas vancouverensis, Pseudomonas wisconsinensis, Pseudomonas xanthomarina Pseudomonas xiamenensis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas anguilliseptica, Pseudomonas citronellolis, Pseudomonas flavescens, Pseudomonas jinjuensis, Pseudomonas mendocina, Pseudomonas nitroreducens, Pseudomonas oleovorans, Pseudomonas pseudoalcaligenes, Pseudomonas resinovorans, Pseudomonas straminae, Pseudomonas aurantiaca, Pseudomonas chlororaphis, Pseudomonas fragi, Pseudomonas lundensis, Pseudomonas taetrolens Pseudomonas azotoformans, Pseudomonas brenneri, Pseudomonas cedrina, Pseudomonas congelans, Pseudomonas corrugata, Pseudomonas costantinii, Pseudomonas extremorientalis, Pseudomonas fluorescens, Pseudomonas fulgida, Pseudomonas gessardii, Pseudomonas libanensis, Pseudomonas mandelii, Pseudomonas marginalis, Pseudomonas mediterranea, Pseudomonas migulae, Pseudomonas mucidolens, Pseudomonas orientalis, Pseudomonas poae, Pseudomonas rhodesiae, Pseudomonas synxantha, Pseudomonas tolaasii, Pseudomonas trivialis, Pseudomonas veronii Pseudomonas denitrificans, Pseudomonas pertucinogena, Pseudomonas fulva, Pseudomonas monteilii, Pseudomonas mosselii, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas balearica, Pseudomonas luteola, or Pseudomonas stutzeri. Pseudomonas avellanae, Pseudomonas cannabina, Pseudomonas caricapapyae, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas fuscovaginae, Pseudomonas tremae, or Pseudomonas viridiflava

[0365] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein can be eukaryote.56330291.3- 63 -Docket No. 277702-548911

[0366] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be a cell belonging to the clades: Opisthokonta; Viridiplantae (e.g., algae and plant); Amebozoa; Cercozoa; Alveolata; Marine flagellates; Heterokonta; Discicristata; or Excavata.

[0367] In some embodiments, the procedures and methods described here can be accomplished using a host cell that is, e.g., a Metazoan, a Choanoflagellata, or a fungi.

[0368] In some embodiments, the procedures and methods described here can be accomplished using a host cell that is a fungi. For example, in some embodiments, the host cell may be a cell belonging to the eukaryote phyla: Ascomycota, Basidiomycota, Chytridiomycota, Microsporidia, or Zygomycota

[0369] In some embodiments, the procedures and methods described here can be accomplished using a host cell that is a fungi belonging to one of the following genera: Aspergillus, Cladosporium, Magnaporthe, Morchella, Neurospora, Penicillium, Saccharomyces, Cryptococcus, or Ustilago.

[0370] In some embodiments, the procedures and methods described here can be accomplished using a host cell that is a fungi belonging to one of the following species: Saccharomyces cerevisiae, Saccharomyces boulardi, Saccharomyces uvarum; Aspergillus flavus, A. terreus, A. awamori; Cladosporium elatum, Cladosporium Herbarum, Cladosporium Sphaerospermum, and Cladosporium Cladosporioides; Magnaporthe grise, Magnaporthe oryzae, Magnaporthe rhizophila; Morchella deliciosa, Morchella esculenta, Morchella conica; Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma; Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

[0371] In some embodiments, the procedures and methods described here can be accomplished using a host cell that is a Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, or Pichia pastoris.

[0372] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be a fungi belonging to one of the following genera: Aspergillus, Cladosporium, Magnaporthe, Morchella, Neurospora, Penicillium, Saccharomyces, Cryptococcus, or Ustilago.

[0373] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be a member of the Saccharomycetaceae family. For example, in some embodiments, the host cell may be one of the following genera within the Saccharomycetaceae family: Brettanomyces, Candida, Citeromyces, Cyniclomyces, Debaryomyces, Issatchenkia, Kazachstania, Kluyveromyces, Komagataella, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Vanderwaltozyma, Torulaspora, Williopsis, Zygosaccharomyces, or Zygotorulaspora.56330291.3- 64 -Docket No. 277702-548911

[0374] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be one of the following: Aspergillus flavus, Aspergillus terreus, Aspergillus awamori, Cladosporium elatum, Cladosporium Herbarum, Cladosporium Sphaerospermum, Cladosporium cladosporioides, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthe rhizophila, Morchella deliciosa, Morchella esculenta, Morchella conica, Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma, Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

[0375] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be a species within the Candida genus. For example, the host cell may be one of the following: Candida albicans, Candida ascalaphidarum, Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida auris, Candida blankii, Candida blattae, Candida bracarensis, Candida bromeliacearum, Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydalis, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida humilis, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, or Candida kefyr.

[0376] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be a species within the Kluyveromyces genus. For example, the host cell may be one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces nonfermentans, or Kluyveromyces wickerhamii.

[0377] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be a species within the Pichia genus. For example, the host cell may be one of the following: Pichia farinose, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia methanolica, or Pichia subpelliculosa.

[0378] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be a species within the Saccharomyces genus. For example, the host cell may be one of the following: Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var boulardii, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguous, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces56330291.3- 65 -Docket No. 277702-548911 spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, or Saccharomyces zonatus.

[0379] In some embodiments, the host cell used to produce a PVP or PVP-fungicidal protein may be one of the following: Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, Schizosaccharomyces pombe, or Hansenula anomala.

[0380] The use of yeast cells as a host organism to generate recombinant PVP is an exceptional method, well known to those having ordinary skill in the art. In some embodiments, the methods and compositions described herein can be performed with any species of yeast, including but not limited to any species of the genus Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces and the species Saccharomyces includes any species of Saccharomyces, for example Saccharomyces cerevisiae species selected from following strains: INVSc1, YNN27, S150-2B, W303-1B, CG25, W3124, JRY188, BJ5464, AH22, GRF18, W303-1A and BJ3505. In some embodiments, members of the Pichia species including any species of Pichia, for example the Pichia species, Pichia pastoris, for example, the Pichia pastoris is selected from following strains: Bg08, Y-11430, X-33, GS115, GS190, JC220, JC254, GS200, JC227, JC300, JC301, JC302, JC303, JC304, JC305, JC306, JC307, JC308, YJN165, KM71, MC100-3, SMD1163, SMD1165, SMD1168, GS241, MS105, any pep4 knock- out strain and any prb1 knock-out strain, as well as Pichia pastoris selected from following strains: Bg08, X-33, SMD1168 and KM71. In some embodiments, any Kluyveromyces species can be used to accomplish the methods described here, including any species of Kluyveromyces, for example, Kluyveromyces lactis, and we teach that the stain of Kluyveromyces lactis can be but is not required to be selected from following strains: GG799, YCT306, YCT284, YCT389, YCT390, YCT569, YCT598, NRRL Y-1140, MW98-8C, MS1, CBS293.91, Y721, MD2 / 1, PM6-7A, WM37, K6, K7, 22AR1, 22A295-1, SD11, MG1 / 2, MSK110, JA6, CMK5, HP101, HP108 and PM6-3C, in addition to Kluyveromyces lactis species is selected from GG799, YCT306 and NRRL Y-1140.

[0381] In some embodiments, the host cell used to produce a PVP or a PVP-fungicidal protein can be an Aspergillus oryzae.

[0382] In some embodiments, the host cell used to produce a PVP or a PVP-fungicidal protein can be an Aspergillus japonicas.

[0383] In some embodiments, the host cell used to produce a PVP or a PVP-fungicidal protein can be an Aspergillus niger.

[0384] In some embodiments, the host cell used to produce a PVP or a PVP-fungicidal protein can be a Bacillus licheniformis.56330291.3- 66 -Docket No. 277702-548911

[0385] In some embodiments, the host cell used to produce a PVP or a PVP-fungicidal protein can be a Bacillus subtilis.

[0386] In some embodiments, the host cell used to produce a PVP or a PVP-fungicidal protein can be a Trichoderma reesei.

[0387] In some embodiments, the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Hansenula species including any species of Hansenula and preferably Hansenula polymorpha. In some embodiments, the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Yarrowia species for example, Yarrowia lipolytica. In some embodiments, the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Schizosaccharomyces species including any species of Schizosaccharomyces and preferably Schizosaccharomyces pombe.

[0388] In some embodiments, yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, and others, can be used as a host organism. Yeast cell culture techniques are well known to those having ordinary skill in the art. Exemplary methods of yeast cell culture can be found in Evans, Yeast Protocols. Springer (1996); Bill, Recombinant Protein Production in Yeast. Springer (2012); Hagan et al., Fission Yeast: A Laboratory Manual, CSH Press (2016); Konishi et al., Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in pre-culture. Biosci Biotechnol Biochem.2014; 78(6):1090-3; Dymond, Saccharomyces cerevisiae growth media. Methods Enzymol.2013; 533:191-204; Looke et al., Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques.2011 May; 50(5):325-8; and Romanos et al., Culture of yeast for the production of heterologous proteins. Curr Protoc Cell Biol.2014 Sep 2; 64:20.9.1- 16, the disclosure of which is incorporated herein by reference in its entirety.

[0389] Recipes for yeast cell fermentation media and stocks are described as follows: (1) MSM media recipe: 2 g / L sodium citrate dihydrate; 1 g / L calcium sulfate dihydrate (0.79 g / L anhydrous calcium sulfate); 42.9g / L potassium phosphate monobasic; 5.17g / L ammonium sulfate; 14.33 g / L potassium sulfate; 11.7 g / L magnesium sulfate heptahydrate; 2 mL / L PTM1trace salt solution; 0.4 ppm biotin (from 500X, 200 ppm stock); 1-2% pure glycerol or other carbon source. (2) PTM1 trace salts solution: Cupric sulfate-5H2O 6.0 g; Sodium iodide 0.08 g; Manganese sulfate-H2O 3.0 g; Sodium molybdate-2H2O 0.2 g; Boric Acid 0.02 g; Cobalt chloride 0.5 g; Zinc chloride 20.0 g; Ferrous sulfate-7H2O 65.0 g; Biotin 0.2 g; Sulfuric Acid 5.0 ml; add Water to a final volume of 1 liter. An illustrative composition for K. lactis defined medium (DMSor) is as follows: 11.83 g / L KH2PO4, 2.299 g / L K2HPO4, 20 g / L of a fermentable56330291.3- 67 -Docket No. 277702-548911 sugar, e.g., galactose, maltose, latotriose, sucrose, fructose or glucose and / or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, 1 g / L MgSO4.7H2O, 10 g / L (NH4)SO4, 0.33 g / L CaCl2.2H2O, 1 g / L NaCl, 1 g / L KCl, 5 mg / L CuSO4.5H2O, 30 mg / L MnSO4.H2O, 10 mg / L, ZnCl2, 1 mg / L KI, 2 mg / L CoCl2.6H2O, 8mg / L Na2MoO4.2H2O, 0.4 mg / L H3BO3,15 mg / L FeCl3.6H2O, 0.8 mg / L biotin, 20 mg / L Ca- pantothenate, 15 mg / L thiamine, 16 mg / L myo-inositol, 10 mg / L nicotinic acid, and 4 mg / L pyridoxine.

[0390] Yeast cells can be cultured in 48-well Deep-well plates, sealed after inoculation with sterile, air-permeable cover. Colonies of yeast, for example, K. lactis cultured on plates can be picked and inoculated the deep-well plates with 2.2 mL media per well, composed of DMSor. Inoculated deep-well plates can be grown for 6 days at 23.5˚C with 280 rpm shaking in a refrigerated incubator-shaker. On day 6 post-inoculation, conditioned media should be harvested by centrifugation at 4000 rpm for 10 minutes, followed by filtration using filter plate with 0.22 µM membrane, with filtered media are subject to HPLC analyses.

[0391] In some embodiments, a yeast strain can be produced by (a) preparing a vector comprising a first expression cassette comprising a polynucleotide operable to express a PVP or complementary nucleotide sequence thereof, said PVP comprising an amino acid sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2- G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof. In some embodiments, X2is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, X1is G or A, X2is A or K, and X3is F or L. In another preferred embodiments, X1 is G and X2 is A and X3 is F or L; or an agriculturally acceptable salt thereof; (b) introducing the vector into a yeast strain; and (c) growing the yeast strain in a growth medium under conditions operable to enable expression of the PVP and secretion into the growth medium.

[0392] In some embodiments, a yeast strain can be produced by (a) preparing a vector comprising a first expression cassette comprising a polynucleotide operable to express a PVP or complementary nucleotide sequence thereof, said PVP comprising an amino acid sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% identical to: (i) an amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y- X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X356330291.3- 68 -Docket No. 277702-548911 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; or (ii) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to any one of SEQ ID NOs: 4-23. In some embodiments, with regards to Formula (I) the PVP of the present disclosure comprises an amino acid sequence wherein X1is G or A, X2 is A or K, and X3 is F or L. In some embodiments, a yeast strain can be produced by (a) preparing a vector comprising a first expression cassette comprising a polynucleotide operable to express a PVP or complementary nucleotide sequence thereof, said PVP comprising an amino acid sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof.

[0001] In some embodiments, the yeast strain comprises a polynucleotide operable to encode a PVP, wherein the PVP comprises an amino sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% identical to: (i) an amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C- D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof, or wherein X2is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, X1is G or A, X2is A or K, and X3 is F or L. In another preferred embodiments, X1 is G and X2 is A and X3 is F or L; or an agriculturally acceptable salt thereof; (b) introducing the vector into a yeast strain; and (c) growing the yeast strain in a growth medium under conditions operable to enable expression of the PVP and secretion into the growth medium. In some embodiments, the yeast strain comprises a polynucleotide operable to encode a PVP, wherein the PVP comprises an amino sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% identical to any one amino acid sequence of SEQ ID Nos: 4-23, or an agriculturally acceptable salt thereof. In some embodiments, the yeast strain comprises a polynucleotide operable to encode a PVP, wherein the PVP comprises or consists of an amino sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% identical to any one amino acid sequence of SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof.

[0002] In some embodiments, a yeast strain can be produced by (a) preparing a vector comprising a first expression cassette comprising a polynucleotide operable to express a PVP or complementary nucleotide sequence thereof, said PVP comprising an amino acid sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-56330291.3- 69 -Docket No. 277702-548911 G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof. In some embodiments, X2 is A or V or G. In a preferred embodiment, X1is G, A, V, L, I, F, T, Y, N, Q or D and X2is K or A. In further preferred embodiments, X1 is G or A, X2 is A or K, and X3 is F or L. In another preferred embodiments, X1is G and X2is A and X3is F or L; or an agriculturally acceptable salt thereof; (b) introducing the vector into a yeast strain; and (c) growing the yeast strain in a growth medium under conditions operable to enable expression of the PVP and secretion into the growth medium.

[0003] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or peptide, wherein the PVP comprises, or consists of an amino sequence as set forth in any one of SEQ ID NOs: 1, or 4-23, or an agriculturally acceptable salt thereof.

[0004] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or peptide, wherein the PVP comprises, or consists of an amino sequence as set forth in any one of SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof.

[0005] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or polypeptide, wherein the PVP is a homopolymer or heteropolymer of two or more PVPs, wherein the amino acid sequence of each PVP is the same or different.

[0006] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or peptide, wherein the PVP is a fused protein comprising two or more PVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each PVP may be the same or different.

[0007] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or polypeptide, wherein the linker is cleavable inside the pathogenic microbe

[0008] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or peptide, wherein the vector is a plasmid comprising an alpha-MF signal.

[0009] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or peptide, wherein the vector is transformed into a yeast strain.

[0010] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or peptide, wherein the yeast strain is selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces.

[0011] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or peptide, wherein the yeast strain is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.56330291.3- 70 -Docket No. 277702-548911

[0012] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or peptide, wherein the yeast strain is Kluyveromyces lactis.

[0013] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or peptide, wherein expression of the PVP provides a yield of at least: 70 mg / L, 80 mg / L, 90 mg / L, 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L, 160 mg / L, 170 mg / L, 180 mg / L, 190 mg / L 200 mg / L, 500 mg / L, 750 mg / L, 1,000 mg / L, 1,250 mg / L, 1,500 mg / L, 1,750 mg / L or at least 20,000 mg / L of PVP per liter of medium.

[0014] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or polypeptide, wherein expression of the PVP provides a yield of at least 100 mg / L of PVP per liter of medium.

[0015] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or polypeptide, wherein expression of the PVP in the medium results in the expression of a single PVP in the medium.

[0016] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or polypeptide, wherein expression of the PVP in the medium results in the expression of a PVP polymer comprising two or more PVP polypeptides in the medium.

[0017] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or polypeptide, wherein the vector comprises two or three expression cassettes, each expression cassette operable to encode the PVP of the first expression cassette.

[0018] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or polypeptide, wherein the vector comprises two or three expression cassettes, each expression cassette operable to encode the PVP of the first expression cassette, or a PVP of a different expression cassette.

[0019] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or polypeptide, wherein the expression cassette is operable to encode a PVP as set forth in any one of SEQ ID NOs: 1, or 4-23, or an agriculturally acceptable salt thereof.

[0020] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or polypeptide, wherein the expression cassette is operable to encode a PVP as set forth in any one of SEQ ID NOs: 4-23, or an agriculturally acceptable salt thereof.

[0021] In some embodiments, a yeast strain can be operable to express a PVP or PVP- fungicidal protein or polypeptide, wherein the expression cassette is operable to encode a PVP as set forth in any one of SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof.

[0022] Any of the aforementioned methods, and / or any of the methods described herein, can be used to produce one or more of the PVPs or PVP-fungicidal protein or peptides as described herein. For example, any of the methods described herein can be used to produce one56330291.3- 71 -Docket No. 277702-548911 or more of the PVPs described in the present disclosure, e.g., PVPs having the amino acid sequence of SEQ ID NOs: 1, or 4-23, which are likewise described herein.

[0023] Yeast transformation, PVP purification, and analysis

[0024] An exemplary method of yeast transformation is as follows: the expression vectors carrying a PVP ORF are transformed into yeast cells. First, the expression vectors are usually linearized by specific restriction enzyme cleavage to facilitate chromosomal integration via homologous recombination. The linear expression vector is then transformed into yeast cells by a chemical or electroporation method of transformation and integrated into the targeted locus of the yeast genome by homologous recombination. The integration can happen at the same chromosomal locus multiple times; therefore, the genome of a transformed yeast cell can contain multiple copies of PVP expression cassettes. The successfully transformed yeast cells can be identified using growth conditions that favor a selective marker engineered into the expression vector and co-integrated into yeast chromosomes with the PVP ORF; examples of such markers include, but are not limited to, acetamide prototrophy, zeocin resistance, geneticin resistance, nourseothricin resistance, and uracil prototrophy.

[0025] Due to the influence of unpredictable and variable factors—such as epigenetic modification of genes and networks of genes, and variation in the number of integration events that occur in individual cells in a population undergoing a transformation procedure—individual yeast colonies of a given transformation process will differ in their capacities to produce a PVP ORF. Therefore, transgenic yeast colonies carrying the PVP transgenes should be screened for high yield strains. Two effective methods for such screening—each dependent on growth of small-scale cultures of the transgenic yeast to provide conditioned media samples for subsequent analysis—use reverse-phase HPLC or housefly injection procedures to analyze conditioned media samples from the positive transgenic yeast colonies.

[0026] The transgenic yeast cultures can be performed using 14 mL round bottom polypropylene culture tubes with 5 to 10 mL defined medium added to each tube, or in 48-well deep well culture plates with 2.2 mL defined medium added to each well. The defined medium, not containing crude proteinaceous extracts or by-products such as yeast extract or peptone, is used for the cultures to reduce the protein background in the conditioned media harvested for the later screening steps. The cultures are performed at the optimal temperature, for example, 23.5°C for K. lactis, for about 5-6 days, until the maximum cell density is reached. PVPs will now be produced by the transformed yeast cells and secreted out of cells to the growth medium. To prepare samples for the screening, cells are removed from the cultures by centrifugation and the supernatants are collected as the conditioned media, which are then cleaned by filtration through 0.22 µm filter membrane and then made ready for strain screening.56330291.3- 72 -Docket No. 277702-548911

[0027] In some embodiments, positive yeast colonies transformed with PVP can be screened via reverse-phase HPLC (rpHPLC) screening of putative yeast colonies. In this screening method, an HPLC analytic column with bonded phase of C18 can be used. Acetonitrile and water are used as mobile phase solvents, and a UV absorbance detector set at 220 nm is used for the peptide detection. Appropriate amounts of the conditioned medium samples are loaded into the rpHPLC system and eluted with a linear gradient of mobile phase solvents. The corresponding peak area of the fungicidal peptide in the HPLC chromatograph is used to quantify the PVP concentrations in the conditioned media. Known amounts of pure PVP are run through the same rpHPLC column with the same HPLC protocol to confirm the retention time of the peptide and to produce a standard peptide HPLC curve for the quantification.

[0028] An exemplary reverse-phase HPLC screening process of positive K. lactis cells is as follows: a PVP ORF can be inserted into the expression vector, pKLAC1, and transformed into the K. lactis strain, YCT306, from New England Biolabs, Ipswich, MA, USA. pKLAC1 vector is an integrative expression vector. Once the PVP transgenes were cloned into pKLAC1 and transformed into YCT306, their expression was controlled by the LAC4 promoter. The resulting transformed colonies produced pre-propeptides comprising an α-mating factor signal peptide, a Kex2 cleavage site and mature PVPs. The α-Mating factor signal peptide guides the pre-propeptides to enter the endogenous secretion pathway, and mature PVPs are released into the growth media.

[0029] In some embodiments, codon optimization for PVP expression can be performed in two rounds, for example, in the first round, based on some common features of high expression DNA sequences, multiple variants of the PVP ORF, expressing an α-Mating factor signal peptide, a Kex2 cleavage site and the PVP, are designed and their expression levels are evaluated in the YCT306 strain of K. lactis, resulting in an initial K. lactis expression algorithm; in a second round of optimization, additional variant PVP ORFs can be designed based on the initial K. lactis expression algorithm to further fine-tuned the K. lactis expression algorithm, and identify the best ORF for PVP expression in K. lactis. In some embodiments, the resulting DNA sequence from the foregoing optimization can have an open reading frame encoding an α-MF signal peptide, a Kex2 cleavage site and a PVP, which can be cloned into the pKLAC1 vector using Hind III and Not I restriction sites, resulting in PVP expression vectors.

[0030] In some embodiments, the yeast, Pichia pastoris, can be transformed with a PVP expression cassette. An exemplary method for transforming P. pastoris is as follows: yeast vectors can be used to transform a PVP expression cassette into P. pastoris. The vectors can be obtained from commercial vendors known to those having ordinary skill in the art. In some embodiments, the vectors can be integrative vectors, and may use the uracil56330291.3- 73 -Docket No. 277702-548911 phosphoribosyltransferase promoter (pUPP) to enhance the heterologous transgene expression. In some embodiments, the vectors may offer different selection strategies; e.g., in some embodiments, the only difference between the vectors can be that one vector may provide G418 resistance to the host yeast, while the other vector may provide Zeocin resistance. In some embodiments, pairs of complementary oligonucleotides, encoding the PVP may be designed and synthesized for subcloning into the two yeast expression vectors. Hybridization reactions can be performed by mixing the corresponding complementary oligonucleotides to a final concentration of 20 µM in 30 mM NaCl, 10 mM Tris-Cl (all final concentrations), pH 8, and then incubating at 95°C for 20 min, followed by a 9-hour incubation starting at 92°C and ending at 17°C, with 3 °C drops in temperature every 20 min. The hybridization reactions will result in DNA fragments encoding PVP. The two P. pastoris vectors can be digested with BsaI-HF restriction enzymes, and the double stranded DNA products of the reactions are then subcloned into the linearized P. pastoris vectors using standard procedures. Following verification of the sequences of the subclones, plasmid aliquots can be transfected by electroporation into a P. pastoris strain (e.g., Bg08). The resulting transformed yeast, can be selected based on resistance (e.g., in this example, to Zeocin or G418) conferred by elements engineered into the vectors.

[0031] Culture and fermentation conditions

[0032] Cell culture techniques are well-known in the art. In some embodiments, the culture method and / or materials will necessarily require adaption based on the host cell selected; and, such adaptions (e.g., modifying pH, temperature, medium contents, and the like) are well known to those having ordinary skill in the art. In some embodiments, any known culture technique may be employed to produce an PVP or PVP-antimicrobial protein of the present disclosure.

[0033] Exemplary culture methods are provided in U.S. Patent Nos. 3,933,590; 3,946,780; 4,988,623; 5,153,131; 5,153,133; 5,155,034; 5,316,905; 5,330,908; 6,159,724; 7,419,801; 9,320,816; 9,714,408; and 10,563,169; the disclosures of which are incorporated herein by reference in their entireties.

[0034] Yeast culture

[0035] Yeast cell culture techniques are well known to those having ordinary skill in the art. Exemplary methods of yeast cell culture can be found in Evans, Yeast Protocols. Springer (1996); Bill, Recombinant Protein Production in Yeast. Springer (2012); Hagan et al., Fission Yeast: A Laboratory Manual, CSH Press (2016); Konishi et al., Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in pre-culture. Biosci Biotechnol Biochem.2014; 78(6):1090-3; Dymond, Saccharomyces cerevisiae growth media. Methods Enzymol.2013; 533:191-204; Looke et al., Extraction of genomic DNA from56330291.3- 74 -Docket No. 277702-548911 yeasts for PCR-based applications. Biotechniques.2011 May; 50(5):325-8; and Romanos et al., Culture of yeast for the production of heterologous proteins. Curr Protoc Cell Biol.2014 Sep 2; 64:20.9.1-16, the disclosure of which is incorporated herein by reference in its entirety.

[0036] Yeast can be cultured in a variety of media, e.g., in some embodiments, yeast can be cultured in minimal medium; YPD medium; yeast synthetic drop-out medium; Yeast Nitrogen Base (YNB with or without amino acids); YEPD medium; ADE D medium; ADE DS" medium; LEU D medium; HIS D medium; or Mineral salts medium.

[0037] In some embodiments, yeast can be cultured in minimal medium. In some embodiments, minimal medium ingredients can comprise: 2% Sugar; Phosphate Buffer, pH 6.0; Magnesium Sulfate; Calcium Chloride; Ammonium Sulfate; Sodium Chloride; Potassium Chloride; Copper Sulfate; Manganese Sulfate; Zinc Chloride; Potassium Iodide; Cobalt Chloride; Sodium Molybdate; Boric Acid; Iron Chloride; Biotin; Calcium pantothenate; Thiamine; Myo-inositol; Nicotinic Acid; and Pyridoxine.

[0038] In some embodiments, yeast can be cultured in YPD medium. YPD medium comprises a bacteriological peptone, yeast extract, and glucose.

[0039] In some embodiments, yeast can be cultured in yeast synthetic drop-out medium, which can be used to differentiate auxotrophic mutant strains that cannot grow without a specific medium component transformed with a plasmid that allows said transformant to grow on a medium lacking the required component.

[0040] In some embodiments, yeast can be cultured using Yeast Nitrogen Base (YNB with or without amino acids), which comprises nitrogen, vitamins, trace elements, and salts.

[0041] In some embodiments, the medium can be YEPD medium, e.g., a medium comprising 2% D-glucose, 2% BACTO Peptone (Difco Laboratories, Detroit, MI), 1% BACTO yeast extract (Difco), 0.004% adenine, and 0.006% L-leucine; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol

[0042] In some embodiments, the medium can be ADE D medium, e.g., a medium comprising 0.056%-Ade-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, and 0.5% 200× tryptophan, threonine solution; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol

[0043] In some embodiments, the medium can be ADE DS" medium, e.g., a medium comprising 0.056%-Ade-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, 0.5% 200× tryptophan, threonine solution, and 18.22% D-sorbitol; or, a variation thereof, wherein the carbon source is entirely a sugar alcohol, e.g., glycerol or sorbitol

[0044] In some embodiments, the medium can be LEU D medium e.g., a medium comprising 0.052%-Leu-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2%56330291.3- 75 -Docket No. 277702-548911 D-glucose, and 0.5% 200× tryptophan, threonine solution; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol.

[0045] In some embodiments, the medium can be HIS D medium, e.g., a medium comprising 0.052%-His-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D- glucose, and 0.5% 200× tryptophan, threonine solution; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol.

[0046] In some embodiments, a mineral salts medium can be used. Mineral salts media consists of mineral salts and a carbon source such as, e.g., glucose, sucrose, or glycerol. Examples of mineral salts media include, e.g., M9 medium, Pseudomonas medium (ATCC 179), and Davis and Mingioli medium. See, Davis & Mingioli (1950) J. Bact.60:17-28. The mineral salts used to make mineral salts media include those selected from among, e.g., potassium phosphates, ammonium sulfate or chloride, magnesium sulfate or chloride, and trace minerals such as calcium chloride, borate, and sulfates of iron, copper, manganese, and zinc. Typically, no organic nitrogen source, such as peptone, tryptone, amino acids, or a yeast extract, is included in a mineral salts medium. Instead, an inorganic nitrogen source is used and this may be selected from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia. A mineral salts medium will typically contain glucose or glycerol as the carbon source.

[0047] In comparison to mineral salts media, minimal media can also contain mineral salts and a carbon source, but can be supplemented with, e.g., low levels of amino acids, vitamins, peptones, or other ingredients, though these are added at very minimal levels. Media can be prepared using the methods described in the art, e.g., in U.S. Pat. App. Pub. No. 2006 / 0040352, the disclosure of which is incorporated herein by reference in its entirety. Details of cultivation procedures and mineral salts media useful in the methods of the present disclosure are described by Riesenberg, D et al., 1991, “High cell density cultivation of Escherichia coli at controlled specific growth rate,” J. Biotechnol.20 (1):17-27.

[0048] In some embodiments, Kluyveromyces lactis are grown in minimal media supplemented with 2% glucose, galactose, sorbitol, or glycerol as the sole carbon source. Cultures are incubated at 30ºC until mid-log phase (24-48 hours) for β-galactosidase measurements, or for 6 days at 23.5ºC for heterologous protein expression.

[0049] In some embodiments, yeast cells can be cultured in 48-well Deep-well plates, sealed after inoculation with sterile, air-permeable cover. Colonies of yeast, for example, K. lactis cultured on plates can be picked and inoculated the deep-well plates with 2.2 mL media per well, composed of DMSor. Inoculated deep-well plates can be grown for 6 days at 23.5˚C with 280 rpm shaking in a refrigerated incubator-shaker. On day 6 post-inoculation, conditioned56330291.3- 76 -Docket No. 277702-548911 media should be harvested by centrifugation at 4000 rpm for 10 minutes, followed by filtration using filter plate with 0.22 µM membrane, with filtered media are subject to HPLC analyses.

[0050] In some embodiments, yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, and others, can be used as a host organism, and / or the yeast to be modified using the methods described herein.

[0051] Temperature and pH conditions will vary depending on the stage of culture and the host cell species selected. Variables such as temperature and pH in cell culture are readily known to those having ordinary skill in the art.

[0052] The pH level is important in the culturing of yeast. One of skill in the art will appreciate that the culturing process includes not only the start of the yeast culture but the maintenance of the culture as well. The yeast culture may be started at any pH level, however, since the media of a yeast culture tends to become more acidic (i.e., lowering the pH) over time, care must be taken to monitor the pH level during the culturing process.

[0053] In some embodiments of the invention, the yeast is grown in a medium at a pH level that is dictated based on the species of yeast used, the stage of culture, and / or the temperature. Thus, in some embodiments, the pH level can fall within a range from about 2 to about 10. Those having ordinary skill in the art will recognize that the optimum pH for most microorganisms is near the neutral point (pH 7.0). However, in some embodiments, some fungal species prefer an acidic environment: accordingly, in some embodiments, the pH can range from 2 to 6.5. In some embodiments, the pH can range from about 4 to about 4.5. Some fungal species (e.g., molds) can grow can grow in a pH of from about 2 to about 8.5, but favor an acid pH. See Mountney & Gould, Practical food microbiology and technology.1988. Ed.3; and Pena et al., Effects of high medium pH on growth, metabolism and transport in Saccharomyces cerevisiae. FEMS Yeast Res.2015 Mar;15(2):fou005.

[0054] In other embodiments, the pH is about 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2, 6.1 to 6.3, 6.2 to 6.5, 6.4 to 6.7, 6.5 to 6.8, 6.6 to 6.9, 6.7 to 7.0, 6.8 to 7.1, 6.9 to 7.2, 7.0 to 7.3, 7.1 to 7.4, 7.2 to 7.5, 7.3 to 7.6, 7.4 to 7.7, 7.5 to 7.8, 7.6 to 7.9, 7.7 to 8.0, 7.8 to 8.1, 7.9 to 8.2, 8.0 to 8.3, 8.1 to 8.4, 8.2 to 8.5, 8.3 to 8.6, 8.4 to 8.7, or 8.5 to 8.8.

[0055] In some embodiments, the pH of the medium can be at least 5.5. In other aspects, the medium can have a pH level of about 5.5. In other aspects, the medium can have a pH level of between 4 and 8. In some cases, the culture is maintained at a pH level of between 5.5 and 8. In other aspects, the medium has a pH level of between 6 and 8. In some cases, medium has a pH level that is maintained at a pH level of between 6 and 8. In some embodiments, the yeast is grown and / or maintained at a pH level of between 6.1 and 8.1. In some embodiments, the yeast is grown and / or maintained at a pH level of between 6.2 and 8.2. In some56330291.3- 77 -Docket No. 277702-548911 embodiments, the yeast is grown and / or maintained at a pH level of between 6.3 and 8.3. In some embodiments, the yeast is grown and / or maintained at a pH level of between 6.4 and 8.4. In some embodiments, the yeast is grown and / or maintained at a pH level of between 5.5 and 8.5. In some embodiments, the yeast is grown and / or maintained at a pH level of between 6.5 and 8.5. In some embodiments, the yeast is grown at a pH level of about 5.6, 5.7, 5.8 or 5.9. In some embodiments, the yeast is grown at a pH level of about 6. In some embodiments, the yeast is grown at a pH level of about 6.5. In some embodiments, the yeast is grown at a pH level of about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. In some embodiments, the yeast is grown at a pH level of about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the yeast is grown at a level of above 8.

[0056] In some embodiments, the pH of the medium can range from a pH of 2 to 8.5. In certain embodiments, the pH is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.

[0057] Exemplary methods of yeast culture can be found in U.S. Patent No.5,436,136, entitled “Repressible yeast promoters” (filed 12 / 20 / 1991; assignee Ciba-Geigy Corporation); U.S. Patent No.6,645,739, entitled “Yeast expression systems, methods of producing polypeptides in yeast, and compositions relating to same” (filed 07 / 26 / 2001; assignee Phoenix Pharmacologies, Inc., Lexington, KY); and U.S. Patent No.10,023,836, entitled “Medium for yeasts” (filed 08 / 23 / 2013; assignee Yamaguchi University); the disclosures of which are incorporated herein by reference in their entireties.

[0058] Fermentation

[0059] The present disclosure contemplates the culture of host organisms in any fermentation format. For example, batch, fed-batch, semi-continuous, and continuous fermentation modes may be employed herein.

[0060] Fermentation may be performed at any scale. The methods and techniques contemplated according to the present disclosure are useful for recombinant protein expression at any scale. Thus, in some embodiments, e.g., microliter-scale, milliliter scale, centiliter scale, and deciliter scale fermentation volumes may be used, and 1 Liter scale and larger fermentation volumes can be used.

[0061] In some embodiments, the fermentation volume is at or above about 1 Liter. For example, in some embodiments, the fermentation volume is about 1 liter to about 100 liters. In some embodiments, the fermentation volume is about 1 liter, about 2 liters, about 3 liters, about 4 liters, about 5 liters, about 6 liters, about 7 liters, about 8 liters, about 9 liters, or about 10 liters. In some embodiments, the fermentation volume is about 1 liter to about 5 liters, about 1 liter to56330291.3- 78 -Docket No. 277702-548911 about 10 liters, about 1 liter to about 25 liters, about 1 liter to about 50 liters, about 1 liter to about 75 liters, about 10 liters to about 25 liters, about 25 liters to about 50 liters, or about 50 liters to about 100 liters In other embodiments, the fermentation volume is at or above 5 Liters, 10 Liters, 15 Liters, 20 Liters, 25 Liters, 50 Liters, 75 Liters, 100 Liters, 200 Liters, 500 Liters, 1,000 Liters, 2,000 Liters, 5,000 Liters, 10,000 Liters, or 50,000 Liters.

[0062] In some embodiments, the fermentation medium can be a nutrient solution used for growing and or maintaining cells. Without limitation, this solution ordinarily provides at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbon source, e.g., glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids; (3) vitamins and / or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.

[0063] In some embodiments, the fermentation medium can be the same as the cell culture medium or any other media described herein. In some embodiments, the fermentation medium can be different from the cell culture medium. In some embodiments, the fermentation medium can be modified in order to accommodate the large-scale production of proteins.

[0064] In some embodiments, the fermentation medium can be supplemented electively with one or more components from any of the following categories: (1) hormones and other growth factors such as, serum, insulin, transferrin, and the like; (2) salts, for example, magnesium, calcium, and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases such as, adenosine, thymidine, etc.; (5) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (6) antibiotics, such as gentamycin; and (7) cell protective agents, for example pluronic polyol.

[0065] In some embodiments, the pH of the fermentation medium can be maintained using pH buffers and methods known to those of skill in the art. Control of pH during fermentation can also can be achieved using aqueous ammonia. In some embodiments, the pH of the fermentation medium will be selected based on the preferred pH of the organism used. Thus, in some embodiments, and depending on the host cell and temperature, the pH can range from about to 1 to about 10.

[0066] In some embodiments, the pH of the fermentation medium can range from a pH of 2 to 8.5. In certain embodiments, the pH is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.56330291.3- 79 -Docket No. 277702-548911

[0067] In other embodiments, the pH is about 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2, 6.1 to 6.3, 6.2 to 6.5, 6.4 to 6.7, 6.5 to 6.8, 6.6 to 6.9, 6.7 to 7.0, 6.8 to 7.1, 6.9 to 7.2, 7.0 to 7.3, 7.1 to 7.4, 7.2 to 7.5, 7.3 to 7.6, 7.4 to 7.7, 7.5 to 7.8, 7.6 to 7.9, 7.7 to 8.0, 7.8 to 8.1, 7.9 to 8.2, 8.0 to 8.3, 8.1 to 8.4, 8.2 to 8.5, 8.3 to 8.6, 8.4 to 8.7, or 8.5 to 8.8

[0068] In some embodiments, e.g., where Escherichia coli (E. coli) is used, the optimal pH range is between 6.5 and 7.5, depending on the temperature.

[0069] In other embodiments, e.g., where a yeast strain is used, the pH can range from about 4.0 to 8.0.

[0070] In some embodiments, neutral pH, i.e., a pH of about 7.0 can be used.

[0071] Those having ordinary skill in the art will recognize that during fermentation, the pH levels may drift as result of conversion and production of substrates and metabolic compounds.

[0072] In some embodiments, the fermentation medium can be supplemented with a buffer or other chemical in order to avoid changes to the pH. For example, in some embodiments, the addition of Ca(OH)2, CaCO3, NaOH, or NH4OH can be added to the fermentation medium to neutralize the production of acidic compounds that occur, e.g., in some yeast species during industrial processes.

[0073] Temperature is another important consideration in the fermentation process; and, like pH considerations, temperature will depend on the type of host cell selected.

[0074] In some embodiments, the fermentation temperature is maintained at about 4°C. to about 42°C. In certain embodiments, the fermentation temperature is about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, or about 42°C.

[0075] In other embodiments, the fermentation temperature is maintained at about 25°C to about 27°C, about 25°C to about 28°C, about 25°C to about 29°C, about 25°C to about 30°C, about 25°C to about 31°C, about 25°C to about 32°C, about 25°C to about 33°C, about 26°C to about 28°C, about 26°C to about 29°C, about 26°C to about 30°C, about 26°C to about 31°C, about 26°C to about 32°C, about 27°C to about 29°C, about 27°C to about 30°C, about 27°C to about 31°C, about 27°C to about 32°C, about 26°C to about 33°C, about 28°C to about 30°C, about 28°C to about 31°C, about 28°C to about 32°C, about 29°C to about 31°C, about 29°C to about 32°C, about 29°C to about 33°C, about 30°C to about 32°C, about 30°C to about 33°C,56330291.3- 80 -Docket No. 277702-548911 about 31°C to about 33°C, about 31°C to about 32°C, about 30°C to about 33°C, or about 32°C to about 33°C

[0076] In other embodiments, the temperature is changed during fermentation, e.g., depending on the stage of fermentation.

[0077] Fermentation can be achieved with a variety of microorganisms known to those having ordinary skill in the art. Suitable microorganisms for up-scaled production of an PVP or PVP-antimicrobial protein include any microorganism listed herein. In some embodiments, non- limiting examples of microorganisms include strains of the genus Saccharomyces spp. (including, but not limited to, S. cerevisiae (baker's yeast), S. distaticus, S. uvarum), or the genus Kluyveromyces, (including, but not limited to, K. marxianus, K. fragilis). See, e.g., Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212.

[0078] Fermentation medium may be selected depending on the host cell and / or needs of the end-user. Any necessary supplements besides, e.g., carbon, may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.

[0079] Yeast Fermentation

[0080] Fermentation methods using yeast are well known to those having ordinary skill in the art. In some embodiments, batch fermentation can be used according to the methods provided herein; in other embodiments, continuous fermentation procedures can be used.

[0081] In some embodiments, the batch method of fermentation can be used to produce PVPs of the present disclosure. Briefly, the batch method of fermentation refers to a type of fermentation that is performed with a closed system, wherein the composition of the medium is determined at the beginning of the fermentation and is not subject to artificial alterations during the fermentation (i.e., the medium is inoculated with one or more yeast cells at the start of fermentation, and fermentation is allowed to proceed, uninterrupted by the user). Typically, in batch fermentation systems, the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures, yeast cells pass through a static lag phase to a high growth log phase, and, finally, to a stationary phase, in which the growth rate is diminished or stopped. If untreated, yeast cells in the stationary phase will eventually die. In a batch method, yeast cells in log phase generally are responsible for the bulk of synthesis of end product.

[0082] In some embodiments, fed-batch fermentation can be used to produce PVPs of the present disclosure. Briefly, fed-batch fermentation is similar to typical batch method (described above), however, the substrate in the fed-batch method is added in increments as the56330291.3- 81 -Docket No. 277702-548911 fermentation progresses. Fed-batch fermentation is useful when catabolite repression may inhibit yeast cell metabolism, and when it is desirable to have limited amounts of substrate in the medium. Generally, the measurement of the substrate concentration in a fed-batch system is estimated on the basis of the changes of measurable factors reflecting metabolism, such as pH, dissolved oxygen, the partial pressure of waste gases (e.g., CO2), and the like.

[0083] In some embodiments, the fed-batch fermentation procedure can be used to produce PVPs as follows: culturing a production organism (e.g., a modified yeast cell) in a 10 L bioreactor sparged with an N2 / CO2 mixture, using 5 L broth containing 5 g / L potassium phosphate, 2.5 g / L ammonium chloride, 0.5 g / L magnesium sulfate, and 30 g / L corn steep liquor, and an initial first and second carbon source concentration of 20 g / L. As the modified yeast cells grow and utilize the carbon sources, additional 70% carbon source mixture is then fed into the bioreactor at a rate approximately balancing carbon source consumption. The temperature of the bioreactor is generally maintained at 30° C. Growth continues for approximately 24 hours or more, and the heterologous peptides reach a desired concentration, e.g., with the cell density being between about 5 and 10 g / L. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit such as a centrifuge to remove cells and cell debris, and the fermentation broth can be transferred to a product separations unit. Isolation of the heterologous peptides can take place by standard separations procedures well known in the art.

[0084] In some embodiments, continuous fermentation can be used to produce PVPs of the present disclosure. Briefly, continuous fermentation refers to fermentation with an open system, wherein a fermentation medium is added continuously to a bioreactor, and an approximately equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a high density, in which yeast cells are primarily in log phase growth. Typically, continuous fermentation methods are performed to maintain steady state growth conditions, and yeast cell loss, due to medium withdrawal, should be balanced against the cell growth rate in the fermentation.

[0085] In some embodiments, the continuous fermentation method can be used to produce PVPs as follows: a modified yeast strain can be cultured using a bioreactor apparatus and a medium composition, albeit where the initial first and second carbon source is about, e.g., 30-50 g / L. When the carbon source is exhausted, feed medium of the same composition is supplied continuously at a rate of between about 0.5 L / hr and 1 L / hr, and liquid is withdrawn at the same rate. The heterologous peptide concentration in the bioreactor generally remains constant along with the cell density. Temperature is generally maintained at 30° C., and the pH is generally maintained at about 4.5 using concentrated NaOH and HCl, as required.56330291.3- 82 -Docket No. 277702-548911

[0086] In some embodiments, when producing PVPs, the bioreactor can be operated continuously, for example, for about one month, with samples taken every day or as needed to assure consistency of the target chemical compound concentration. In continuous mode, fermenter contents are constantly removed as new feed medium is supplied. The exit stream, containing cells, medium, and heterologous peptides, can then be subjected to a continuous product separations procedure, with or without removing cells and cell debris, and can be performed by continuous separations methods well known in the art to separate organic products from peptides of interest.

[0087] In some embodiments, a yeast cell operable to express an PVP or PVP- antimicrobial protein can be grown, e.g., using a fed batch process in aerobic bioreactor. Briefly, reactors are filled to about 20% to about 70% capacity with medium comprising a carbon source and other reagents. Temperature and pH is maintained using one or more chemicals as described herein. Oxygen level is maintained by sparging air intermittently in concert with agitation.

[0088] For example, in some embodiments, the present disclosure provides a method of using a fed batch process in aerobic bioreactor, wherein the reactor is filled to about 20%; 21%; 22%; 23%; 24%; 25%; 26%; 27%; 28%; 29%; 30%; 31%; 32%; 33%; 34%; 35%; 36%; 37%; 38%; 39%; 40%; 41%; 42%; 43%; 44%; 45%; 46%; 47%; 48%; 49%; 50%; 51%; 52%; 53%; 54%; 55%; 56%; 57%; 58%; 59%; 60%; 61%; 62%; 63%; 64%; 65%; 66%; 67%; 68%; 69%; or 70% capacity.

[0089] In some embodiments, the present disclosure provides a fed batch fermentation method using an aerobic bioreactor to produce PVPs, wherein the medium is a rich culture medium. For example, in some embodiments, the carbon source can be glucose, sorbitol, or lactose.

[0090] In some embodiments, the amount of glucose can be about 2 g / L; 3 g / L; 4 g / L; 5 g / L; 6 g / L; 7 g / L; 8 g / L; 9 g / L; 10 g / L; 11 g / L; 12 g / L; 13 g / L; 14 g / L; 15 g / L; 16 g / L; 17 g / L; 18 g / L; 19 g / L; 20 g / L; 21 g / L; 22 g / L; 23 g / L; 24 g / L; 25 g / L; 26 g / L; 27 g / L; 28 g / L; 29 g / L; or 30 g / L of the medium.

[0091] In some embodiments, the amount of sorbitol can be about 2 g / L; 3 g / L; 4 g / L; 5 g / L; 6 g / L; 7 g / L; 8 g / L; 9 g / L; 10 g / L; 11 g / L; 12 g / L; 13 g / L; 14 g / L; 15 g / L; 16 g / L; 17 g / L; 18 g / L; 19 g / L; 20 g / L; 21 g / L; 22 g / L; 23 g / L; 24 g / L; 25 g / L; 26 g / L; 27 g / L; 28 g / L; 29 g / L; or 30 g / L of the medium.

[0092] In some embodiments, the amount of lactose can be about 2 g / L; 3 g / L; 4 g / L; 5 g / L; 6 g / L; 7 g / L; 8 g / L; 9 g / L; 10 g / L; 11 g / L; 12 g / L; 13 g / L; 14 g / L; 15 g / L; 16 g / L; 17 g / L; 18 g / L; 19 g / L; 20 g / L; 21 g / L; 22 g / L; 23 g / L; 24 g / L; 25 g / L; 26 g / L; 27 g / L; 28 g / L; 29 g / L; or 30 g / L of the medium.56330291.3- 83 -Docket No. 277702-548911

[0093] In some embodiments, the present disclosure provides a fed batch fermentation method using an aerobic bioreactor, wherein the medium is supplemented with one or more of phosphoric acid, calcium sulfate, potassium sulfate, magnesium sulfate heptahydrate, potassium hydroxide, and / or corn steep liquor.

[0094] In some embodiments, the medium can be supplemented with phosphoric acid in an amount of about 2 g / L; 3 g / L; 4 g / L; 5 g / L; 6 g / L; 7 g / L; 8 g / L; 9 g / L; 10 g / L; 11 g / L; 12 g / L; 13 g / L; 14 g / L; 15 g / L; 16 g / L; 17 g / L; 18 g / L; 19 g / L; 20 g / L; 21 g / L; 22 g / L; 23 g / L; 24 g / L; 25 g / L; 26 g / L; 27 g / L; 28 g / L; 29 g / L; or 30 g / L to the medium.

[0095] In some embodiments, the medium can be supplemented with calcium sulfate in an amount of about 0.05 g / L; 0.15 g / L; 0.25 g / L; 0.35 g / L; 0.45 g / L; 0.55 g / L; 0.65 g / L; 0.75 g / L; 0.85 g / L; 0.95 g / L; 1.05 g / L; 1.15 g / L; 1.25 g / L; 1.35 g / L; 1.45 g / L; 1.55 g / L; 1.65 g / L; 1.75 g / L; 1.85 g / L; 1.95 g / L; 2.05 g / L; 2.15 g / L; 2.25 g / L; 2.35 g / L; 2.45 g / L; 2.55 g / L; 2.65 g / L; 2.75 g / L; 2.85 g / L; or 2.95 g / L to the medium.

[0096] In some embodiments, the medium can be supplemented with potassium sulfate in an amount of about 2 g / L; 2.5 g / L; 3 g / L; 3.5 g / L; 4 g / L; 4.5 g / L; 5 g / L; 5.5 g / L; 6 g / L; 6.5 g / L; 7 g / L; 7.5 g / L; 8 g / L; 8.5 g / L; 9 g / L; 9.5 g / L; 10 g / L; 10.5 g / L; 11 g / L; 11.5 g / L; 12 g / L; 12.5 g / L; 13 g / L; 13.5 g / L; 14 g / L; 14.5 g / L; 15 g / L; 15.5 g / L; 16 g / L; 16.5 g / L; 17 g / L; 17.5 g / L; 18 g / L; 18.5 g / L; 19 g / L; 19.5 g / L; or 20 g / L to the medium.

[0097] In some embodiments, the medium can be supplemented with magnesium sulfate heptahydrate in an amount of about 0.25 g / L; 0.5 g / L; 0.75 g / L; 1 g / L; 1.25 g / L; 1.5 g / L; 1.75 g / L; 2 g / L; 2.25 g / L; 2.5 g / L; 2.75 g / L; 3 g / L; 3.25 g / L; 3.5 g / L; 3.75 g / L; 4 g / L; 4.25 g / L; 4.5 g / L; 4.75 g / L; 5 g / L; 5.25 g / L; 5.5 g / L; 5.75 g / L; 6 g / L; 6.25 g / L; 6.5 g / L; 6.75 g / L; 7 g / L; 7.25 g / L; 7.5 g / L; 7.75 g / L; 8 g / L; 8.25 g / L; 8.5 g / L; 8.75 g / L; 9 g / L; 9.25 g / L; 9.5 g / L; 9.75 g / L; 10 g / L; 10.25 g / L; 10.5 g / L; 10.75 g / L; 11 g / L; 11.25 g / L; 11.5 g / L; 11.75 g / L; 12 g / L; 12.25 g / L; 12.5 g / L; 12.75 g / L; 13 g / L; 13.25 g / L; 13.5 g / L; 13.75 g / L; 14 g / L; 14.25 g / L; 14.5 g / L; 14.75 g / L; or 15 g / L to the medium.

[0098] In some embodiments, the medium can be supplemented with potassium hydroxide in an amount of about 0.25 g / L; 0.5 g / L; 0.75 g / L; 1 g / L; 1.25 g / L; 1.5 g / L; 1.75 g / L; 2 g / L; 2.25 g / L; 2.5 g / L; 2.75 g / L; 3 g / L; 3.25 g / L; 3.5 g / L; 3.75 g / L; 4 g / L; 4.25 g / L; 4.5 g / L; 4.75 g / L; 5 g / L; 5.25 g / L; 5.5 g / L; 5.75 g / L; 6 g / L; 6.25 g / L; 6.5 g / L; 6.75 g / L; or 7 g / L to the medium.

[0099] In some embodiments, the medium can be supplemented with corn steep liquor in an amount of about 5 g / L; 6 g / L; 7 g / L; 8 g / L; 9 g / L; 10 g / L; 11 g / L; 12 g / L; 13 g / L; 14 g / L; 15 g / L; 16 g / L; 17 g / L; 18 g / L; 19 g / L; 20 g / L; 21 g / L; 22 g / L; 23 g / L; 24 g / L; 25 g / L; 26 g / L; 27 g / L; 28 g / L; 29 g / L; 30 g / L; 31 g / L; 32 g / L; 33 g / L; 34 g / L; 35 g / L; 36 g / L; 37 g / L; 38 g / L; 3956330291.3- 84 -Docket No. 277702-548911 g / L; 40 g / L; 41 g / L; 42 g / L; 43 g / L; 44 g / L; 45 g / L; 46 g / L; 47 g / L; 48 g / L; 49 g / L; 50 g / L; 51 g / L; 52 g / L; 53 g / L; 54 g / L; 55 g / L; 56 g / L; 57 g / L; 58 g / L; 59 g / L; 60 g / L; 61 g / L; 62 g / L; 63 g / L; 64 g / L; 65 g / L; 66 g / L; 67 g / L; 68 g / L; 69 g / L; or 70 g / L to the medium.

[0100] In some embodiments, the temperature of the reactor can be maintained between about 15°C and about 45°C. In some embodiments, the reactor can have a temperature of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0101] In some embodiments, the pH can have a level of about 3 to about 6. In some embodiments, the pH can be 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

[0102] In some embodiments, the pH can be maintained at a constant level via the addition of one or more chemicals. For example, in some embodiments, ammonium hydroxide can be added to maintain pH. In some embodiments, ammonium hydroxide can be added to a level of ammonium hydroxide in the medium that is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, of ammonium hydroxide

[0103] In some embodiments, oxygen levels can be maintained by sparging. For example, in some embodiments, dissolved oxygen can be maintained at a constant level by sparging air between 0.5-1.5 volume / volume / min and by increasing agitation to maintain a set point of 10-30%.

[0104] In some embodiments, inoculation of the reactor can be accomplished based on an overnight seed culture comprising from about 2.5 g / L to about 50 g / L of a carbon source, e.g., glucose, sorbitol, or lactose. In some embodiments, the overnight seed culture can comprise corn steep liquor, e.g., from about 2.5 g / L to about 50 g / L of corn steep liquor.

[0105] In some embodiments, the inoculation percentage can range from about 5-20% of initial fill volume. Following inoculation, the reactor can be fed with from about a 50% to about an 80% solution of the selected carbon source up until the reactor is filled and / or the desired supernatant peptide concentration is achieved. In some embodiments, the time required to fill the reactor can range from about 86 hours to about 160 hours. In some embodiments, the quantity required to reach the desired peptide concentration can range from about 0.8 g / L to about 1.2 g / L. Upon completion of the fermentation, the contents can be passed through a cell separation unit and optionally concentrated, depending on intended use of the material.

[0106] Additional recipes for yeast fermentation media are provided herein.

[0107] Recipes for yeast cell fermentation media and stocks are described as follows: (1) MSM media recipe: 2 g / L sodium citrate dihydrate; 1 g / L calcium sulfate dihydrate (0.79 g / L anhydrous calcium sulfate); 42.9g / L potassium phosphate monobasic; 5.17g / L ammonium56330291.3- 85 -Docket No. 277702-548911 sulfate; 14.33 g / L potassium sulfate; 11.7 g / L magnesium sulfate heptahydrate; 2 mL / L PTM1trace salt solution; 0.4 ppm biotin (from 500X, 200 ppm stock); 1-2% pure glycerol or other carbon source. (2) PTM1 trace salts solution: Cupric sulfate-5H2O 6.0 g; Sodium iodide 0.08 g; Manganese sulfate-H2O 3.0 g; Sodium molybdate-2H2O 0.2 g; Boric Acid 0.02 g; Cobalt chloride 0.5 g; Zinc chloride 20.0 g; Ferrous sulfate-7H2O 65.0 g; Biotin 0.2 g; Sulfuric Acid 5.0 ml; add Water to a final volume of 1 liter. An illustrative composition for K. lactis defined medium (DMSor) is as follows: 11.83 g / L KH2PO4, 2.299 g / L K2HPO4, 20 g / L of a fermentable sugar, e.g., galactose, maltose, latotriose, sucrose, fructose or glucose and / or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, 1 g / L MgSO4.7H2O, 10 g / L (NH4)SO4, 0.33 g / L CaCl2.2H2O, 1 g / L NaCl, 1 g / L KCl, 5 mg / L CuSO4.5H2O, 30 mg / L MnSO4.H2O, 10 mg / L, ZnCl2, 1 mg / L KI, 2 mg / L CoCl2.6H2O, 8mg / L Na2MoO4.2H2O, 0.4 mg / L H3BO3,15 mg / L FeCl3.6H2O, 0.8 mg / L biotin, 20 mg / L Ca- pantothenate, 15 mg / L thiamine, 16 mg / L myo-inositol, 10 mg / L nicotinic acid, and 4 mg / L pyridoxine.

[0108] Yeast peptide yield screening and evaluation

[0109] In some embodiments, PVP or PVP-fungicidal proteinyield can be evaluated using an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm, C18 reverse-phase analytical HPLC column and an auto-injector. An illustrative use of the Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm, C18 reverse-phase analytical HPLC column and an auto-injector is as follows: filtered conditioned media samples from transformed K. lactis cells are analyzed using Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm, C18 reverse-phase analytical HPLC column and an auto-injector by analyzing HPLC grade water and acetonitrile containing 0.1% trifluoroacetic acid, constituting the two mobile phase solvents used for the HPLC analyses; the peak areas of both the PVP or PVP-fungicidal proteinare analyzed using HPLC chromatographs, and then used to calculate the peptide concentration in the conditioned media, which can be further normalized to the corresponding final cell densities (as determined by OD600 measurements) as normalized peptide yield.

[0110] In some embodiments, positive yeast colonies transformed with PVP or PVP- fungicidal protein can be screened using a housefly injection assay. PVP or PVP-fungicidal protein can paralyze / kill houseflies when injected in measured doses through the body wall of the dorsal thorax. The efficacy of the PVP or PVP-fungicidal protein can be defined by the median paralysis / lethal dose of the peptide (PD50 / LD50), which causes 50% knock-down ratio or mortality of the injected houseflies respectively. The pure PVP or PVP-fungicidal proteinis normally used in the housefly injection assay to generate a standard dose-response curve, from56330291.3- 86 -Docket No. 277702-548911 which a PD50 / LD50 value can be determined. Using a PD50 / LD50 value from the analysis of a standard dose-response curve of the pure PVP or PVP-fungicidal protein or peptide, quantification of the PVP or PVP-fungicidal proteinproduced by the transformed yeast can be achieved using a housefly injection assay performed with serial dilutions of the corresponding conditioned media.

[0111] An exemplary housefly injection bioassay is as follows: conditioned media is serially diluted to generate full dose-response curves from the housefly injection bioassay. Before injection, adult houseflies (Musca domestica) are immobilized with CO2, and 12-18 mg houseflies are selected for injection. A microapplicator, loaded with a 1 cc syringe and 30-gauge needle, is used to inject 0.5 µL per fly, doses of serially diluted conditioned media samples into houseflies through the body wall of the dorsal thorax. The injected houseflies are placed into closed containers with moist filter paper and breathing holes on the lids, and they are examined by knock-down ratio or by mortality scoring at 24 hours post-injection. Normalized yields are calculated. Peptide yield means the peptide concentration in the conditioned media in units of mg / L. However, peptide yields are not always sufficient to accurately compare the strain production rate. Individual strains may have different growth rates, hence when a culture is harvested, different cultures may vary in cell density. A culture with a high cell density may produce a higher concentration of the peptide in the media, even though the peptide production rate of the strain is lower than another strain which has a higher production rate. Accordingly, the term “normalized yield” is created by dividing the peptide yield with the cell density in the corresponding culture and this allows a better comparison of the peptide production rate between strains. The cell density is represented by the light absorbance at 600 nm with a unit of “A” (Absorbance unit).

[0112] Screening yeast colonies that have undergone a transformation with PVP or PVP- fungicidal protein can identify the high yield yeast strains from hundreds of potential colonies. These strains can be fermented in bioreactor to achieve at least up to 4 g / L or at least up to 3 g / L or at least up to 2 g / L yield of the PVP or PVP-fungicidal proteinwhen using optimized fermentation media and fermentation conditions described herein. The higher rates of production (expressed in mg / L) can be anywhere from about 100 mg / L to about 100,000 mg / L; or from about 100 mg / L to about 90, 000 mg / L; or from about 100 mg / L to about 80,000 mg / L; or from about 100 mg / L to about 70,000 mg / L; or from about 100 mg / L to about 60,000 mg / L; or from about 100 mg / L to about 50,000 mg / L; or from about 100 mg / L to about 40,000 mg / L; or from about 100 mg / L to about 30,000 mg / L; or from about 100 mg / L to about 20,000 mg / L; or from about 100 mg / L to about 17,500 mg / L; or from about 100 mg / L to about 15,000 mg / L; or from about 100 mg / L to about 12,500 mg / L; or from about 100 mg / L to about 10,000 mg / L; or from56330291.3- 87 -Docket No. 277702-548911 about 100 mg / L to about 9,000 mg / L; or from about 100 mg / L to about 8,000 mg / L; or from about 100 mg / L to about 7,000 mg / L; or from about 100 mg / L to about 6,000 mg / L; or from about 100 mg / L to about 5,000 mg / L; or from about 100 mg / L to about 3,000 mg / L; or from about 100 mg / L to 2,000 mg / L; or from about 100 mg / L to 1,500 mg / L; or from about 100 mg / L to 1,000 mg / L; or from about 100 mg / L to 750 mg / L; or from about 100 mg / L to 500 mg / L; or from about 150 mg / L to 100,000 mg / L; or from about 200 mg / L to 100,000 mg / L; or from about 300 mg / L to 100,000 mg / L; or from about 400 mg / L to 100,000 mg / L; or from about 500 mg / L to 100,000 mg / L; or from about 750 mg / L to 100,000 mg / L; or from about 1,000 mg / L to 100,000 mg / L; or from about 1,250 mg / L to 100,000 mg / L; or from about 1,500 mg / L to 100,000 mg / L; or from about 2,000 mg / L to 100,000 mg / L; or from about 2,500 mg / L to 100,000 mg / L; or from about 3,000 mg / L to 100,000 mg / L; or from about 3,500 mg / L to 100,000 mg / L; or from about 4,000 mg / L to 100,000 mg / L; or from about 4,500 mg / L to 100,000 mg / L; or from about 5,000 mg / L to 100,000 mg / L; or from about 6,000 mg / L to 100,000 mg / L; or from about 7,000 mg / L to 100,000 mg / L; or from about 8,000 mg / L to 100,000 mg / L; or from about 9,000 mg / L to 100,000 mg / L; or from about 10,000 mg / L to 100,000 mg / L; or from about 12,500 mg / L to 100,000 mg / L; or from about 15,000 mg / L to 100,000 mg / L; or from about 17,500 mg / L to 100,000 mg / L; or from about 20,000 mg / L to 100,000 mg / L; or from about 30,000 mg / L to 100,000 mg / L; or from about 40,000 mg / L to 100,000 mg / L; or from about 50,000 mg / L to 100,000 mg / L; or from about 60,000 mg / L to 100,000 mg / L; or from about 70,000 mg / L to 100,000 mg / L; or from about 80,000 mg / L to 100,000 mg / L; or from about 90,000 mg / L to 100,000 mg / L; or any range of any value provided or even greater yields than can be achieved with a peptide before conversion, using the same or similar production methods that were used to produce the peptide before conversion.

[0113] PVP INCORPORATION INTO PLANTS OR PARTS THEREOF

[0114] The PVPs described herein, and / or an fungicidal protein comprising at least one PVP as described herein, can be incorporated into plants, plant tissues, plant cells, plant seeds, and / or plant parts thereof, for either the stable, or transient expression of a PVP or a PVP- fungicidal protein or peptide, and / or a polynucleotide sequence encoding the same.

[0115] In some embodiments, the PVP or PVP-fungicidal protein can be incorporated into a plant using recombinant techniques known in the art. In some embodiments, the PVP or PVP-fungicidal protein may be in the form of an fungicidal protein which may comprise one or more PVP monomers.

[0116] As used herein, with respect to transgenic plants, plant tissues, plant cells, and plant seeds, the term “PVP” also encompasses a PVP-fungicidal protein or peptide, and a “PVP56330291.3- 88 -Docket No. 277702-548911 polynucleotide” is similarly also used to encompass a polynucleotide or group of polynucleotides operable to express and / or encode an fungicidal protein comprising one or more PVPs.

[0117] The goal of incorporating a PVP into plants is to deliver PVPs and / or PVP- fungicidal protein or peptides to the pest via the pathogenic microbe’s consumption of the transgenic PVP expressed in a plant tissue consumed by the pathogenic microbe. Upon the consumption of the PVP by the pathogenic microbe from its food (e.g., via a pathogenic microbe feeding upon a transgenic plant transformed with a PVP), the consumed PVP may have the ability to inhibit the growth, impair the movement, or even kill the pathogenic microbe. Accordingly, transgenic plants expressing a PVP polynucleotide and / or a PVP polypeptide may express said PVP polynucleotide / polypeptide in a variety of plant tissues, including but not limited to: the epidermis (e.g., mesophyll); periderm; phloem; xylem; parenchyma; collenchyma; sclerenchyma; and primary and secondary meristematic tissues. For example, in some embodiments, a polynucleotide sequence encoding a PVP can be operably linked to a regulatory region containing a phosphoenolpyruvate carboxylase promoter, resulting in the expression of a PVP in a plant’s mesophyll tissue.

[0118] Transgenic plants expressing a PVP and / or a polynucleotide operable to express PVP can be generated by any one of the various methods and protocols well known to those having ordinary skill in the art; such methods of the invention do not require that a particular method for introducing a nucleotide construct to a plant be used, only that the nucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. “Transgenic plants” or “transformed plants” or “stably transformed” plants or cells or tissues refers to plants that have incorporated or integrated exogenous nucleic acid sequences or DNA fragments into the plant cell. These nucleic acid sequences include those that are exogenous, or not present in the untransformed plant cell, as well as those that may be endogenous, or present in the untransformed plant cell. “Heterologous” generally refers to the nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like.

[0119] Transformation of plant cells can be accomplished by one of several techniques known in the art. Typically, a construct that expresses an exogenous or heterologous peptide or polypeptide of interest (e.g., a PVP), would contain a promoter to drive transcription of the gene, as well as a 3’ untranslated region to allow transcription termination and polyadenylation. The design and organization of such constructs is well known in the art. In some embodiments, a56330291.3- 89 -Docket No. 277702-548911 gene can be engineered such that the resulting peptide is secreted, or otherwise targeted within the plant cell to a specific region and / or organelle. For example, the gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum. It may also be preferable to engineer the plant expression cassette to contain an intron, such that mRNA processing of the intron is required for expression.

[0120] Typically, a plant expression cassette can be inserted into a plant transformation vector. This plant transformation vector may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as “binary vectors.” Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a “gene of interest” (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the PVP are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). Several types of Agrobacterium strains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

[0121] In general, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass. Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of56330291.3- 90 -Docket No. 277702-548911 selecting agent. The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods for generating transgenic plants are found in Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120. Because the transformed material contains many cells, both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non- transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants.

[0122] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Generation of transgenic plants may be performed by one of several methods, including, but not limited to, microinjection, electroporation, direct gene transfer, introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium- mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, ballistic particle acceleration, aerosol beam transformation, Lec1 transformation, and various other non-particle direct-mediated methods to transfer DNA. Exemplary transformation protocols are disclosed in U.S. Published Application No.20010026941; U.S. Pat. No. 4,945,050; International Publication No. WO 91 / 00915; and U.S. Published Application No. 2002015066, the disclosures of which are incorporated herein by reference in their entireties.

[0123] Chloroplasts can also be readily transformed, and methods concerning the transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J.12:601-606, the disclosure of which is incorporated herein by reference in its entirety. The method of chloroplast transformation relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0124] Following integration of heterologous foreign DNA into plant cells, one having ordinary skill may then apply a maximum threshold level of appropriate selection56330291.3- 91 -Docket No. 277702-548911 chemical / reagent (e.g., an antibiotic) in the medium to kill the untransformed cells, and separate and grow the putatively transformed cells that survive from this selection treatment by transferring said surviving cells regularly to a fresh medium. By continuous passage and challenge with appropriate selection, an artisan identifies and proliferates the cells that are transformed with the plasmid vector. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.

[0125] The cells that have been transformed may be grown into plants in accordance with conventional methods known to those having ordinary skill in the art. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84, the disclosure of which is incorporated herein by reference in its entirety. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present disclosure provides transformed seed (also referred to as “transgenic seed”) having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

[0126] In various embodiments, the present disclosure provides a PVP-fungicidal protein or peptide, that act as substrates for pathogenic microbe proteinases, proteases and peptidases (collectively referred to herein as “proteases”) as described above.

[0127] In some embodiments, transgenic plants or parts thereof, that may be receptive to the expression of PVPs can include: alfalfa, banana, barley, bean, broccoli, cabbage, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and a wheat plant.

[0128] In some embodiments the transgenic plant may be grown from cells that were initially transformed with the DNA constructs described herein. In other embodiments, the transgenic plant may express the encoded PVP in a specific tissue, or plant part, for example, a leaf, a stem a flower, a sepal, a fruit, a root, a seed, or combinations thereof.56330291.3- 92 -Docket No. 277702-548911

[0129] In some embodiments, the plant, plant tissue, plant cell, plant seed, or part thereof, can be transformed with a PVP or a polynucleotide encoding the same, wherein the has the amino acid sequence that is at least 95% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R- S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof. In some embodiments, X2 is A or V or G. In a preferred embodiment, X1 is G, A, V, L, I, F, T, Y, N, Q or D and X2 is K or A. In further preferred embodiments, X1 is G or A, X2 is A or K, and X3 is F or L. In another preferred embodiments, X1 is G and X2 is A; or an agriculturally acceptable salt thereof.

[0130] In some embodiments, the plant, plant tissue, plant cell, or plant seed can be transformed with a PVP wherein the PVP has an amino acid sequence of any of the aforementioned PVPs (e.g., one or more the PVPs enumerated in Table 1), or a polynucleotide encoding the same.

[0131] In some embodiments, the plant, plant tissue, plant cell, or plant seed can be transformed with a PVP having an amino acid sequence selected from the group consisting of SEQ NOs: 5-53, or a polynucleotide encoding the same.

[0132] In some embodiments, the plant, plant tissue, plant cell, or plant seed can be transformed with a PVP wherein the PVP is a homopolymer or heteropolymer of two or more PVP polypeptides, wherein the amino acid sequence of each PVP is the same or different, or a polynucleotide encoding the same.

[0133] Any of the aforementioned methods, and / or any of the methods described herein, can be used to incorporate one or more of the PVPs or PVP-fungicidal protein or peptides as described herein, into plants or plant parts thereof. For example, any of the methods described herein can be used to incorporate into plants one or more of the PVPs described in the present disclosure, e.g., PVPs having the amino acid sequence of SEQ ID NOs: 1, or 4-23, which are likewise described herein.

[0134] Polynucleotide incorporation into plants, the proteins expressed therefrom

[0135] A challenge regarding the expression of heterogeneous polypeptides in transgenic plants is maintaining the desired effect (e.g., fungicidal activity) of the introduced polypeptide upon expression in the host organism; one way to maintain such an effect is to increase the chance of proper protein folding through the use of an operably linked Endoplasmic Reticulum Signal Peptide (ERSP). Another method to maintain the effect of a transgenic protein is to incorporate a Translational Stabilizing Protein (STA).56330291.3- 93 -Docket No. 277702-548911

[0136] Plants can be transiently or stably transfected with the DNA sequence that encodes a PVP or a PVP-fungicidal proteincomprising one or more PVPs, using any of the transfection methods described above. Alternatively, plants can be transfected with a polynucleotide that encodes a PVP, wherein said PVP is operably linked to a polynucleotide operable to encode an Endoplasmic Reticulum Signal Peptide (ERSP); linker, Translational Stabilizing Protein (STA); or combination thereof. For example, in some embodiments, a transgenic plant or plant genome can be transformed with a polynucleotide sequence that encodes the Endoplasmic Reticulum Signal Peptide (ERSP); PVP; and / or intervening linker peptide (LINKER or L), thus causing mRNA transcribed from the heterogeneous DNA to be expressed in the transformed plant, and subsequently, said mRNA to be translated into a peptide.

[0137] Endoplasmic Reticulum Signal Peptide (ERSP)

[0138] The subcellular targeting of a recombinant protein to the ER can be achieved through the use of an ERSP operably linked to said recombinant protein; this allows for the correct assembly and / or folding of such proteins, and the high level accumulation of these recombinant proteins in plants. Exemplary methods concerning the compartmentalization of host proteins into intracellular storage are disclosed in McCormick et al., Proc. Natl. Acad. Sci. USA 96(2):703-708, 1999; Staub et al., Nature Biotechnology 18:333-338, 2000; Conrad et al., Plant Mol. Biol.38:101-109, 1998; and Stoger et al., Plant Mol. Biol.42:583-590, 2000, the disclosures of which are incorporated herein by reference in their entireties. Accordingly, one way to achieve the correct assembly and / or folding of recombinant proteins, is to operably link an endoplasmic reticulum signal peptide (ERSP) to the recombinant protein of interest.

[0139] In some embodiments, a peptide comprising an Endoplasmic Reticulum Signal Peptide (ERSP) can be operably linked to a PVP (designated as ERSP-PVP), wherein said ERSP is the N-terminal of said peptide. In some embodiments, the ERSP peptide is between 3 to 60 amino acids in length, between 5 to 50 amino acids in length, between 20 to 30 amino acids in length.

[0140] In some embodiments, PVP ORF starts with an ersp at its 5’-end. For the PVP to be properly folded and functional when it is expressed from a transgenic plant, it must have an ersp nucleotide fused in frame with the polynucleotide encoding a PVP. During the cellular translation process, translated ERSP can direct the PVP being translated to insert into the Endoplasmic Reticulum (ER) of the plant cell by binding with a cellular component called a signal-recognition particle. Within the ER the ERSP peptide is cleaved by signal peptidase and the PVP is released into the ER, where the PVP is properly folded during the post-translation modification process, for example, the formation of disulfide bonds. Without any additional retention protein signals, the protein is transported through the ER to the Golgi apparatus, where56330291.3- 94 -Docket No. 277702-548911 it is finally secreted outside the plasma membrane and into the apoplastic space. PVP can accumulate at apoplastic space efficiently to reach the fungicidal dose in plants.

[0141] The ERSP peptide is at the N-terminal region of the plant-translated PVP complex and the ERSP portion is composed of about 3 to 60 amino acids. In some embodiments it is 5 to 50 amino acids. In some embodiments it is 10 to 40 amino acids but most often is composed of 15 to 20; 20 to 25; or 25 to 30 amino acids. The ERSP is a signal peptide so called because it directs the transportation of a protein. Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals. The signal peptides for ER trafficking are often 15 to 30 amino acid residues in length and have a tripartite organization, comprised of a core of hydrophobic residues flanked by a positively charged amino terminal and a polar, but uncharged carboxyterminal region. (Zimmermann, et al, “Protein translocation across the ER membrane,” Biochimica et Biohysica Acta, 2011, 1808: 912-924).

[0142] Many ERSPs are known. It is NOT required that the ERSP be derived from a plant ERSP, non-plant ERSPs will work with the procedures described herein. Many plant ERSPs are however well known and we describe some plant derived ERSPs here. For example, ins some embodiments, the ERSP can be a barley alpha-amylase signal peptide (BAAS), which is derived from the plant, Hordeum vulgare, and has an amino acid sequence as follows: “MANKHLSLSLFLVLLGLSASLASG” (SEQ ID NO: 29)

[0143] Plant ERSPs, which are selected from the genomic sequence for proteins that are known to be expressed and released into the apoplastic space of plants, include examples such as BAAS, carrot extensin, and tobacco PR1. The following references provide further descriptions, and are incorporated by reference herein in their entirety: De Loose, M. et al. “The extensin signal peptide allows secretion of a heterologous protein from protoplasts” Gene, 99 (1991) 95- 100; De Loose, M. et al. described the structural analysis of an extension—encoding gene from Nicotiana plumbaginifolia, the sequence of which contains a typical signal peptide for translocation of the protein to the endoplasmic reticulum; Chen, M.H. et al. “Signal peptide- dependent targeting of a rice alpha-amylase and cargo proteins to plastids and extracellular compartments of plant cells” Plant Physiology, 2004 Jul; 135(3): 1367-77. Epub 2004 Jul 2. Chen, M.H. et al. studied the subcellular localization of α-amylases in plant cells by analyzing the expression of α-amylase, with and without its signal peptide, in transgenic tobacco. These references and others teach and disclose the signal peptide that can be used in the methods, procedures and peptide, protein and nucleotide complexes and constructs described herein.

[0144] In some embodiments, the ERSP can include, but is not limited to, one of the following: a BAAS; a tobacco extensin signal peptide; a modified tobacco extensin signal peptide; or a Jun a 3 signal peptide from Juniperus ashei. For example, in some embodiments, a56330291.3- 95 -Docket No. 277702-548911 plant can be transformed with a nucleotide that encodes any of the peptides that are described herein as Endoplasmic Reticulum Signal Peptides (ERSP), and a PVP.

[0145] The tobacco extensin signal peptide motif is another exemplary type of ERSP. See Memelink et al, the Plant Journal, 1993, V4: 1011-1022; Pogue GP et al, Plant Biotechnology Journal, 2010, V8: 638-654, the disclosures of which are incorporated herein by reference in their entireties.

[0146] In some embodiments, a PVP ORF can have a nucleotide sequence operable to encode a tobacco extensin signal peptide motif (UniProt accession no Q40402). In one embodiment, the PVP ORF can encode an tobacco extensin motif according to SEQ ID NO: 30. In another embodiment, the PVP ORF can encode an a modified tobacco extensin signal peptide according to SEQ ID NO: 31.

[0147] An illustrative example of how to generate an embodiment with an extensin signal motif is as follows: A DNA sequence encoding an extensin motif is designed (for example, the DNA sequence shown in SEQ ID NO: 32 or SEQ ID NO: 33) using oligo extension PCR with four synthetic DNA primers; ends sites such as a restriction site, for example, a Pac I restriction site at the 5’-end, and a 5’-end of a GFP sequence at the 3’-end, can be added using PCR with the extensin DNA sequence serving as a template, and resulting in a fragment; the fragment is used as the forward PCR primer to amplify the DNA sequence encoding a PVP ORF , for example “gfp-l-PVP” contained in a pFECT vector, thus producing a PVP ORF encoding (from N’ to C’ terminal) “ERSP-GFP-L-PVP” wherein the ERSP is extensin. The resulting DNA sequence can then be cloned into Pac I and Avr II restriction sites of a FECT vector to generate the pFECT-PVP vector for transient plant expression of GFP fused PVP.

[0148] In some embodiments, an illustrative expression system can include the FECT expression vectors containing PVP ORF is transformed into Agrobacterium, GV3101, and the transformed GV3101 is injected into tobacco leaves for transient expression of PVP ORF.

[0149] Translational stabilizing protein (STA)

[0150] A Translational stabilizing protein (STA) can increase the amount of PVP in plant tissues. One of the PVP ORFs, ERSP-PVP, is sufficient to express a properly folded PVP in the transfected plant, but in some embodiments, effective protection of a plant from pest damage may require that the plant expressed PVP accumulate. With transfection of a properly constructed PVP ORF, a transgenic plant can express and accumulate greater amounts of the correctly folded PVP. When a plant accumulates greater amounts of properly folded PVP, it can more easily resist, inhibit, and / or kill the pests that attack and eat the plants. One method of increasing the accumulation of a polypeptide in transgenic tissues is through the use of a translational stabilizing protein (STA). The translational stabilizing protein can be used to56330291.3- 96 -Docket No. 277702-548911 significantly increase the accumulation of PVP in plant tissue, and thus increase the efficacy of a plant transfected with PVP with regard to pest resistance. The translational stabilizing protein is a protein with sufficient tertiary structure that it can accumulate in a cell without being targeted by the cellular process of protein degradation.

[0151] In some embodiments, the translational stabilizing protein can be a domain of another protein, or it can comprise an entire protein sequence. In some embodiments, the translational stabilizing protein can be between 5 and 50 amino acids, 50 to 250 amino acids (e.g., GNA), 250 to 750 amino acids (e.g., chitinase) and 750 to 1500 amino acids (e.g., enhancin).

[0152] One embodiment of the translational stabilizing protein can be a polymer of fusion proteins comprising at least one PVP. A specific example of a translational stabilizing protein is provided here to illustrate the use of a translational stabilizing protein. The example is not intended to limit the disclosure or claims in any way. Useful translational stabilizing proteins are well known in the art, and any proteins of this type could be used as disclosed herein. Procedures for evaluating and testing production of peptides are both known in the art and described herein. One example of one translational stabilizing protein is Green-Fluorescent Protein (GFP) (SEQ ID NO:24; UniProt / Swiss-Prot Accession No. P42212).

[0153] In some embodiments, a protein comprising an Endoplasmic Reticulum Signal Peptide (ERSP) can be operably linked to a PVP, which is in turn operably linked to a Translational Stabilizing Protein (STA). Here, this configuration is designated as ERSP-STA- PVP or ERSP-PVP-STA, wherein said ERSP is the N-terminal of said protein and said STA may be either on the N-terminal side (upstream) of the PVP, or of the C-terminal side (downstream) of the PVP. In some embodiments, a protein designated as ERSP-STA-PVP or ERSP-PVP-STA, comprising any of the ERSPs or PVPs described herein, can be operably linked to a STA, for example, any of the translational stabilizing proteins described, or taught by this document including GFP (Green Fluorescent Protein; SEQ ID NO: 24; UniProtKB / Swiss-Prot: P42212.1), or Jun a 3, (Juniperus ashei; SEQ ID NO:25; GenBank: AAF31759.1 also UniProtKB / Swiss- Prot: P81295.1).

[0154] Additional examples of translational stabilizing proteins can be found in the following references, the disclosures of which are incorporated herein by reference in their entirety: Kramer, K.J. et al. “Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta” Insect Biochemistry and Molecular Biology, Vol. 23, Issue 6, September 1993, pp.691-701. Kramer, K.J. et al. isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta. Hashimoto, Y. et al.56330291.3- 97 -Docket No. 277702-548911 “Location and nucleotide sequence of the gene encoding the viral enhancing factor of the Trichoplusia ni granulosis virus” Journal of General Virology, (1991), 72, 2645-2651. These references and others teach and disclose translational stabilizing proteins that can be used in the methods, procedures and peptide, protein and nucleotide complexes and constructs described herein.

[0155] In some embodiments, a PVP ORF can be transformed into a plant, for example, in the tobacco plant, Nicotiana benthamiana, using a PVP ORF that contains a STA. For example, in some embodiments, the STA can be Jun a 3. The mature Jun a 3 is a ~30 kDa plant defending protein that is also an allergen for some people. Jun a 3 is produced by Juniperus ashei trees and can be used in some embodiments as a translational stabilizing protein (STA). In some embodiments, the Jun a 3 amino acid sequence can be the sequence shown in SEQ ID NO: 25.

[0156] LINKERS

[0157] Linker proteins assist in the proper folding of the different motifs composing a PVP ORF. The PVP ORF described in this invention also incorporates polynucleotide sequences encoding intervening linker peptides between the polynucleotide sequences encoding the PVP (PVP) and the translational stabilizing protein (sta), or between polynucleotide sequence encoding multiple polynucleotide sequences encoding PVP, i.e., (l-PVP)N or (PVP-l)N, if the expression ORF involves multiple PVP domain expression. The intervening linker peptides (LINKERS or L) separate the different parts of the expressed PVP construct, and help proper folding of the different parts of the complex during the expression process. In the expressed PVP construct, different intervening linker peptides can be involved to separate different functional domains. In some embodiments, the LINKER is attached to a PVP and this bivalent group can be repeated up to 10 (N=1-10) and possibly even more than 10 times (e.g., N = 200) in order to facilitate the accumulation of properly folded PVP in the plant that is to be protected.

[0158] In some embodiments the intervening linker peptide can be between 1 and 30 amino acids in length. However, it is not necessarily an essential component in the expressed PVP in plants.

[0159] In some embodiments, the PVP-fungicidal proteincomprises at least one PVP operably linked to a cleavable peptide. In other embodiments, the PVP-fungicidal proteincomprises at least one PVP operably linked to a non-cleavable peptide.

[0160] A cleavable linker peptide can be designed to the PVP ORF to release the properly PVP from the expressed PVP complex in the transformed plant to improve the protection the PVP affords the plant with regard to pest damage. One type of the intervening linker peptide is the plant cleavable linker peptide. This type of linker peptides can be56330291.3- 98 -Docket No. 277702-548911 completely removed from the expressed PVP ORF complex during plant post-translational modification. Therefore, in some embodiments, the properly folded PVP linked by this type of intervening linker peptides can be released in the plant cells from the expressed PVP ORF complex during post-translational modification in the plant.

[0161] Another type of the cleavable intervening linker peptide is not cleavable during the expression process in plants. However, it has a protease cleavage site specific to serine, threonine, cysteine, aspartate proteases or metalloproteases. The type of cleavable linker peptide can be digested by proteases found in the pathogenic microbe environment. Using the information taught by this disclosure it should be a matter of routine for one skilled in the art to make or find other examples of LINKERS that will be useful in this invention.

[0162] In some embodiments, the PVP ORF can contain a cleavable type of intervening linker, for example, the type listed in SEQ ID NO: 26, having the amino acid code of “IGER”. The molecular weight of this intervening linker or LINKER is 473.53 Daltons. In other embodiments, the intervening linker peptide (LINKER) can also be one without any type of protease cleavage site, i.e., an uncleavable intervening linker peptide, for example, the linker “ETMFKHGL” (SEQ ID NO: 28).

[0163] In some embodiments, the PVP-fungicidal protein can have two or more cleavable peptides, wherein the fungicidal protein comprises an fungal cleavable linker (L), the fungal cleavable linker being fused in frame with a construct comprising (PVP-L)n, wherein “n” is an integer ranging from 1 to 200, or from 1 to 100, or from 1 to 10. In another embodiment, the PVP-fungicidal protein or peptide, and described herein, comprises an endoplasmic reticulum signal peptide (ERSP) operably linked with a PVP, which is operably linked with an fungal cleavable linker (L) and / or a repeat construct (L-PVP)n or (PVP-L)n, wherein n is an integer ranging from 1 to 200, or from 1 to 100, or from 1 to 10.

[0164] In some embodiments, a protein comprising an Endoplasmic Reticulum Signal Peptide (ERSP) can be operably linked to a PVP and an intervening linker peptide (L or Linker); such a construct is designated as ERSP-L-PVP, or ERSP-PVP-L, wherein said ERSP is the N- terminal of said protein, and said L or Linker may be either on the N-terminal side (upstream) of the PVP, or the C-terminal side (downstream) of the PVP. A protein designated as ERSP-L-PVP, or ERSP-PVP-L, comprising any of the ERSPs or PVPs described herein, can have a Linker “L” that can be an uncleavable linker peptide, or a cleavable linker peptide, and which may be cleavable in a plant cells during protein expression process, or may be cleavable in an pathogenic microbe.

[0165] In some embodiments, a PVP-fungicidal protein can comprise any of the intervening linker peptides (LINKER or L) described herein, or taught by this document,56330291.3- 99 -Docket No. 277702-548911 including but not limited to following sequences: IGER (SEQ ID NO: 26), EEKKN, (SEQ ID NO: 27), and ETMFKHGL (SEQ ID NO: 28), or combinations thereof.

[0166] In various embodiments, an exemplary fungicidal protein can include a protein construct comprising: (ERSP)-(PVP-L)n; (ERSP)-(L)-(PVP-L)n; (ERSP)-(L-PVP)n; (ERSP)-(L- PVP)n-(L); wherein n is an integer ranging from 1 to 200 or from 1 to 100, or from 1 to 10. In various related embodiments described above, a PVP is the aforementioned PvD1 Variant Polypeptides, L is a non-cleavable or cleavable peptide, and n is an integer ranging from 1 to 200, preferably an integer ranging from 1 to 100, and more preferably an integer ranging from 1 to 10. In some embodiments, the PVP-fungicidal protein may contain PVP peptides that are the same or different, and fungal cleavable peptides that are the same or different. In some embodiments, the C-terminal PVP is operably linked at its C-terminus with a cleavable peptide that is operable to be cleaved in a pathogenic microbe, for example a fungus. In some embodiments, the N-terminal PVP is operably linked at its N-terminus with a cleavable peptide that is operable to be cleaved in a pathogenic microbe.

[0167] Generally, the presence of certain proteases and peptidases in the pathogenic microbe follow the pH of the microbes cell interior. Certain proteases and peptidases in the human gastrointestinal system may include: pepsin, trypsin, chymotrypsin, elastase, carboxypeptidase, aminopeptidase, and dipeptidase.

[0168] Some of the available proteases and peptidases found in a pathogenic microbe may include: (1) serine proteases; (2) cysteine proteases; (3) aspartic proteases, and (4) metalloproteases.

[0169] The papain family contains peptidases with a wide variety of activities, including endopeptidases with broad specificity (such as papain), endopeptidases with very narrow specificity (such as glycyl endopeptidases), aminopeptidases, dipeptidyl-peptidase, and peptidases with both endopeptidase and exopeptidase activities (such as cathepsins B and H). Other exemplary proteinases found in the various pathogenic microbes include trypsin-like enzymes, e.g. trypsin and chymotrypsin, pepsin, carboxypeptidase-B and aminotripeptidases.

[0170] Serine proteases are widely distributed in nearly all animals and microorganisms (Joanitti et al., 2006). In higher organisms, nearly 2% of genes code for these enzymes (Barrette- Ng et al., 2003). Being essentially indispensable to the maintenance and survival of their host organism, serine proteases play key roles in many biological processes. Serine proteases are classically categorized by their substrate specificity, notably by whether the residue at P1: trypsin-like (Lys / Arg preferred at P1), chymotrypsin-like (large hydrophobic residues such as Phe / Tyr / Leu at P1), or elastase-like (small hydrophobic residues such as Ala / Val at P1) (revised by Tyndall et. al.., 2005). Serine proteases are a class of proteolytic enzymes whose central56330291.3- 100 -Docket No. 277702-548911 catalytic machinery is composed of three invariant residues, an aspartic acid, a histidine and a uniquely reactive serine, the latter giving rise to their name, the “catalytic triad”. The Asp-His- Ser triad can be found in at least four different structural contexts (Hedstrom, 2002). These four clans of serine proteases are typified by chymotrypsin, subtilisin, carboxypeptidase Y, and Clp protease. The three serine proteases of the chymotrypsin-like clan that have been studied in greatest detail are chymotrypsin, trypsin, and elastase. More recently, serine proteases with novel catalytic triads and dyads have been discovered for their roles in digestion, including Ser-His- Glu, Ser-Lys / His, His-Ser-His, and N-terminal Ser.

[0171] One class of well-studied digestive enzymes found in the gut environment of pathogenic microbes is the class of cysteine proteases. The term “cysteine protease” is intended to describe a protease that possesses a highly reactive thiol group of a cysteine residue at the catalytic site of the enzyme. There is evidence that many phytophagous pathogenic microbes and plant parasitic nematodes rely, at least in part, on midgut cysteine proteases for protein digestion. These include but are not limited to Hemiptera, especially squash bugs (Anasa tristis); green stink bug (Acrosternum hilare); Riptortus clavatus; and almost all Coleoptera examined to date, especially, Colorado potato beetle (Leptinotarsa deaemlineata); three-lined potato beetle (Lema trilineata); asparagus beetle (Crioceris asparagi); Mexican bean beetle (Epilachna varivestis); red flour beetle (Triolium castaneum); confused flour beetle (Tribolium confusum); the flea beetles (Chaetocnema spp., Haltica spp., and Epitrix spp.); corn rootworm (Diabrotica Spp.); cowpea weevil (Callosobruchus aculatue); boll weevil (Antonomus grandis); rice weevil (Sitophilus oryza); maize weevil (Sitophilus zeamais); granary weevil (Sitophilus granarius); Egyptian alfalfa weevil (Hypera postica); bean weevil (Acanthoseelides obtectus); lesser grain borer (Rhyzopertha dominica); yellow meal worm (Tenebrio molitor); Thysanoptera, especially, western flower thrips (Franklini ella occidentalis); Diptera, especially, leafminer spp. (Liriomyza trifolii); plant parasitic nematodes especially the potato cyst nematodes (Globodera spp.), the beet cyst nematode (Heterodera schachtii) and root knot nematodes (Meloidogyne spp.).

[0172] Another class of digestive enzymes is the aspartic proteases. The term “aspartic protease” is intended to describe a protease that possesses two highly reactive aspartic acid residues at the catalytic site of the enzyme and which is most often characterized by its specific inhibition with pepstatin, a low molecular weight inhibitor of nearly all known aspartic proteases. There is evidence that many phytophagous pathogenic microbes rely, in part, on midgut aspartic proteases for protein digestion most often in conjunction with cysteine proteases. These include but are not limited to Hemiptera especially (Rhodnius prolixus) and bedbug (Cimex spp.) and members of the families Phymatidae, Pentatomidae, Lygaeidae and Belostomatidae; Coleoptera, in the families of the Meloidae, Chrysomelidae, Coccinelidae and56330291.3- 101 -Docket No. 277702-548911 Bruchidae all belonging to the series Cucujiformia, especially, Colorado potato beetle (Leptinotarsa decemlineata) three-lined potato beetle (Lematri lineata); southern and western corn rootworm (Diabrotica undecimpunctata and D. virgifera), boll weevil (Anthonomus grandis), squash bug (Anasatristis); flea beetle (Phyllotreta crucifera), bruchid beetle (Callosobruchus maculatus), Mexican bean beetle (Epilachna varivestis), soybean leafminer (Odontota horni), margined blister beetle (Epicauta pestifera) and the red flour beetle (Triolium castaneum); Diptera, especially housefly (Musca domestica). See Terra and Ferreira (1994) Comn. Biochem. Physiol.109B: 1-62; Wolfson and Murdock (1990) J. Chem. Ecol.16: 1089- 1102.

[0173] Other examples of intervening linker peptides can be found in the following references, which are incorporated by reference herein in their entirety: a plant expressed serine proteinase inhibitor precursor was found to contain five homogeneous protein inhibitors separated by six same linker peptides, as disclosed in Heath et al. “Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata” European Journal of Biochemistry, 1995; 230: 250-257. A comparison of the folding behavior of green fluorescent proteins through six different linkers is explored in Chang, H.C. et al. “De novo folding of GFP fusion proteins: high efficiency in eukaryotes but not in bacteria” Journal of Molecular Biology, 2005 Oct 21; 353(2): 397-409. An isoform of the human GalNAc-Ts family, GalNAc-T2, was shown to retain its localization and functionality upon expression in N. benthamiana plants by Daskalova, S.M. et al. “Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins” BMC Biotechnology, 2010 Aug 24; 10: 62. The ability of endogenous plastid proteins to travel through stromules was shown in Kwok, E.Y. et al. “GFP-labelled Rubisco and aspartate aminotransferase are present in plastid stromules and traffic between plastids” Journal of Experimental Botany, 2004 Mar; 55(397): 595-604. Epub 2004 Jan 30. A report on the engineering of the surface of the tobacco mosaic viru...

Claims

Docket No. 277702-548911 CLAIMS 1. A PvD1 variant polypeptide (PVP) having fungicidal activity against one or more pathogenic microbe species, said PVP comprising or consisting of: (i) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2- G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; or (ii) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to an amino acid sequence of any one of SEQ ID NOs: 4-23, or an agriculturally acceptable salt thereof.

2. The PVP of claim 1, wherein X1 is G or A, and X2 is A or K, and X3 is F or L.

3. The PVP of claim 1, comprising or consisting of an amino sequence as set forth in any one of SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof.

4. The PVP of any one of claims 1-3, wherein the PVP is a homopolymer or heteropolymer of two or more PVPs, wherein the amino acid sequence of each PVP is the same or different.

5. The PVP of any one of claims 1-3, wherein the PVP is a fused protein comprising two or more PVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each PVP may be the same or different.

6. The PVP or claim 5, wherein the linker is cleavable inside the pathogenic microbe.

7. A composition comprising or consisting of a PVP of any one of claims 1-6, or combinations thereof, and at least one excipient.

8. A polynucleotide operable to encode a PVP, said PVP comprising or consisting of, an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R- S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3is F, L, G, A, V, I or M; or an agriculturally acceptable56330291.3- 150 -Docket No. 277702-548911 salt thereof; or SEQ ID NOs: 4-23; or an agriculturally acceptable salt thereof, or a complementary nucleotide sequence thereof.

9. The polynucleotide of claim 8, wherein if the polynucleotide encodes a PVP wherein X2is A or K and X1 is G or A and X3 is F or L.

10. A polynucleotide operable to encode a PVP, the PVP comprising or consisting of an amino sequence as set forth in any one of SEQ ID NOs: 5-23, or an agriculturally acceptable salt thereof.

12. A plant, plant tissue, plant cell, plant seed, or part thereof, comprising one or more PVPs, or a polynucleotide encoding the same, said PVP comprising an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to an amino acid sequence, according to Formula (I): X1-K-T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N- K-E-H-L-R-S-G-R-C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2 is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an amino acid sequence of any one of SEQ ID NOs: 4-23, or an agriculturally acceptable salt thereof, and optionally, wherein the PVP comprises from one to three amino acid conservative substitutions relative to the sequence of a PVP as set forth in SEQ ID NO:1, with the proviso that the conservative amino acid substitution does not occur at X1, X2and X3.

13. The plant, plant tissue, plant cell, plant seed, or part thereof of claim 12, wherein the PVP comprises a peptide sequence according to Formula (I) wherein X2 is A or K and X1 is G, A, V, L, I, F, T, Y, N, Q or D.

14. The plant, plant tissue, plant cell, plant seed, or part thereof of any one of claims 12-13, wherein the PVP comprises or consists of an amino sequence as set forth in any one of SEQ ID NOs: 5-23.

15. The plant, plant tissue, plant cell, plant seed, or part thereof of claim 12, wherein the PVP further comprises a homopolymer or heteropolymer of two or more PVPs, wherein the amino acid sequence of each PVP is the same or different.56330291.3- 151 -Docket No. 277702-548911 16. The plant, plant tissue, plant cell, plant seed, or part thereof of claim 12, wherein the PVP is a fused protein comprising two or more PVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each PVP may be the same or different.

17. The plant, plant tissue, plant cell, plant seed, or part thereof of claim 16, wherein the linker is cleavable inside the pathogenic microbe.

18. A method of producing a PVP, the method comprising: a. preparing a vector comprising a first expression cassette comprising a polynucleotide operable to encode: (i) a PVP, or complementary nucleotide sequence thereof, said PVP comprising an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K- T-C-E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R- C-R-D-D-F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1 is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3is F, L, G, A, V, I or M; or (ii) a PVP comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 4-23; b. introducing the vector into a yeast strain; and c. growing the yeast strain in a growth medium under conditions operable to enable expression of the PVP and secretion into the growth medium.

19. The method of claim 18, wherein X1is G, A, V, L, I, F, T, Y, N, Q or D; X2is K or A and X3 is F or L.

20. The method of claim 18, wherein the PVP comprises an amino sequence as set forth in any one of SEQ ID NOs: 5-23.

21. The method of claim 18, wherein the PVP is a homopolymer or heteropolymer of two or more PVPs, wherein the amino acid sequence of each PVP is the same or different.

22. The method of claim 18, wherein the PVP is a fused protein comprising two or more PVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each PVP may be the same or different.

23. The method of claim 22, wherein the linker is cleavable inside the pathogenic microbe.56330291.3- 152 -Docket No. 277702-548911 24. The method of claim 18, wherein the vector is a plasmid comprising an alpha-MF signal.

25. The method of claim 18, wherein the vector is transformed into a yeast strain.

26. The method of claim 25, wherein the yeast strain is selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces.

27. The method of claim 26, wherein the yeast strain is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

28. The method of claim 27, wherein the yeast strain is Kluyveromyces lactis.

29. The method of claim 18, wherein the PVP is secreted into the growth medium.

30. The method of claim 18, wherein expression of the PVP provides a yield of: at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, at least 190 mg / L 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L of PVP per liter of yeast culture medium.

31. The method of claim 18, wherein expression of the PVP in the medium results in the expression of a single PVP in the medium.56330291.3- 153 -Docket No. 277702-548911 32. The method of claim 18, wherein expression of the PVP in the medium results in the expression of a PVP polymer comprising two or more PVP polypeptides in the medium.

33. The method of claim 18, wherein the vector comprises two or three expression cassettes, each expression cassette operable to encode the PVP of the first expression cassette.

34. The method of claim 18, wherein the vector comprises two or three expression cassettes, each expression cassette operable to encode the PVP of the first expression cassette, or a PVP of a different expression cassette.

35. The method of claim 18, wherein the expression cassette is operable to encode a PVP according to SEQ ID NO: 1, wherein X1 is the amino acid G or A, and X2 is an amino acid selected from: A or K; and X3is an amino acid F or L.

36. A method for protecting a plant, or plant part thereof, from a pathogenic microbe, the method comprising: (1) applying a composition comprising a PVP of claim 7 to at least a part of a plant, or plant seed, (2) providing a plant or plant seed that expresses a PVP, or a polynucleotide encoding the same, or (3) combinations thereof.

37. The method of claim 36, wherein said PVP comprises or consists of: (i) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C-E-N-L- A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D-F-R-C- W-C-T-K-N-C (SEQ ID NO: 1), wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3 is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; or (ii) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to any one of SEQ ID NOs: 4-23.

38. The method of claim 37, wherein X1 is G or A, X2 is A or K, and X3 is F or L.

39. The method of any one of claims 36 to 38, wherein the PVP comprises or consists of an amino sequence as set forth in any one of SEQ ID NOs: 5-23.56330291.3- 154 -Docket No. 277702-548911 40. The method of claim 37, wherein the PVP further comprises a homopolymer or heteropolymer of two or more PVPs, wherein the amino acid sequence of each PVP is the same or different.

41. The method of claim 37, wherein the PVP is a fused protein comprising two or more PVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each PVP may be the same or different.

42. The method of claim 41, wherein the linker is cleavable inside the pathogenic microbe 43. The method of claim 36, wherein the pathogenic microbe comprises a species selected from the genera: Monilinia, Botrytis, Fusarium, Venturia, Wilsonomyces, Botryosphaeria, Penicillium, Rhizopus, Aspergillus, Podosphaera, Erysiphe, Golovinomyces, Leveillula, Peronospora, Pseudoperonospora, Plasmopara, Bremia, Cladosporium, Neofabraea, Microdochium, Marssonina, Sclerotinia, Rhizopus, Didymella, Alternaria, Verticillium, Phytophthora, Colletotrichum, Cercospora, Phakopsora, Rhizoctonia, Sclerotinia, Pythium, Phoma, Gaeumannomces, Leptoshaeria, or Puccinia.

44. The method of claim 43, wherein the pathogenic microbe is a pathogenic microbe belonging to the genera: Monilinia, Botrytis, Fusarium, Phytophthora, Cercospora, or Aspergillus.

45. The method of claim 44, wherein the pathogenic microbe is Monilinia fructicola, Botrytis cinerea, Fusarium graminearum, Phytophthora infestans, Cercospora sojina, or Aspergillus niger.

46. A method for controlling pathogenic microbes comprising, providing to said pathogenic microbe a transgenic plant that comprises in its genome a stably incorporated expression cassette, wherein said stably incorporated expression cassette comprises a polynucleotide operable to encode a PVP.

47. A method of combating, controlling, or inhibiting a pest comprising, applying a pesticidally effective amount of the composition of claim 7 to the locus of the pest, or to a plant or animal susceptible to an attack by the pest.56330291.3- 155 -Docket No. 277702-548911 48. The method of claim 46, wherein the pest comprises a species selected from the genera: Monilinia, Botrytis, Fusarium, Venturia, Wilsonomyces, Botryosphaeria, Penicillium, Rhizopus, Aspergillus, Podosphaera, Erysiphe, Golovinomyces, Leveillula, Peronospora, Pseudoperonospora, Plasmopara, Bremia, Cladosporium, Neofabraea, Microdochium, Marssonina, Sclerotinia, Rhizopus, Didymella, Alternaria, Verticillium, Phytophthora, Colletotrichum, Cercospora, Phakopsora, Rhizoctonia, Sclerotinia, Pythium, Phoma, Gaeumannomces, Leptoshaeria, or Puccinia.

49. A vector comprising a polynucleotide operable to encode a PVP having an amino acid sequence with at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identity to a sequence as set forth in any one of SEQ ID NOs: 1, or 4-23, or SEQ ID NOs: 5-23.

50. A yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode a PVP, said PVP comprising: (i) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-K-T-C- E-N-L-A-D-T-Y-X2-G-P-C-X3-T-T-G-S-C-D-D-H-C-K-N-K-E-H-L-R-S-G-R-C-R-D-D- F-R-C-W-C-T-K-N-C (SEQ ID NO: 1), wherein X1is G, A, V, L, I, F, T, Y, N, Q or D, X2is A, K, V, G, L or I, and X3is F, L, G, A, V, I or M; or an agriculturally acceptable salt thereof; or (ii) an amino acid sequence that is at least 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to the amino acid sequence according to any one of SEQ ID NOs: 4-23.

51. The yeast strain of claim 50, wherein X1is G or A, X2is A or K, and X3is F or L.

52. The yeast strain of claim 50, wherein the PVP comprises or consists of an amino sequence as set forth in any one of SEQ ID NOs: 5-23.

53. The yeast strain of any one of claims 50-52, wherein the yeast cell is selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces.56330291.3- 156 -Docket No. 277702-548911 54. The yeast strain of claim 53, wherein the yeast cell is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

55. The yeast strain of claim 54, wherein the yeast cell is Kluyveromyces lactis or Kluyveromyces marxianus.56330291.3- 157 -

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