phytovaccines

Abstract

The present application relates to compositions comprising one or more elicitor proteins, polypeptides, or peptides that induce defense responses in plants, thereby enhancing resistance to biotic and abiotic stress and promoting growth. These compositions can be applied to seeds, plants, or soil to improve crop performance and yield. The invention further includes transgenic plants expressing nucleic acids encoding elicitor proteins. It further discloses methods for controlling plague infestation improving the yield of a plant by applying the composition comprising the elicitor protein.

Description

PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WOPHYTOVACCINESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 719,402, filed November 12, 2024, which is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] This application relates to new compositions comprising proteins, polypeptides and / or peptides that act as elicitors of plant defense responses. These compositions enhance plant resistance to biotic and abiotic stress, promote growth, and improve crop yield and quality. It is also about the application of a formulation, either to a plant or to seed inoculation and the generation of a transgenic plant.BACKGROUND OF THE INVENTION

[0003] In crop protection, there is an ongoing need of compositions that improve plant health, since such improvement results in increased yields and / or a higher plant or crop quality. In addition, healthier plants resist biotic and abiotic stress better. In turn, increased resistance against biotics stress enables farmers to reduce the quantity of chemical pesticides they apply.

[0004] Plants defend themselves against potential invading pathogens either through morphological structures that act as physical barriers that inhibit the entry and development of the pathogen, or through biochemical reactions that take place in different plant tissues and produce substances which are toxic for the pathogen or generate conditions that inhibit the entry and growth of the microorganism in the host. These defenses can be found in a constitutive form, establishing a non-specific protection against a wide range of microorganisms, or can be induced in response to the presence of the attacking pathogen. In the latter case, plants, like other living beings, activate their own defense systems when they recognize a molecule derived from a pathogenic organism or when they perceive a molecule generated during the pathogen invasion. Any such defense system is called a defense “inductor” or “elicitor”. These inductor molecules alert the presence of the invading pathogen when it is recognized by the host plant. When the plant recognizes the elicitors of the attacking pathogen early, it activates its defense mechanisms rapidly. Said mechanisms block the infection, thus stopping the progress of the pathogen. It this case, the plant / pathogen interaction is said to be incompatible, because it does not lead to disease, and the phytopathogenic strain is defined as avirulent This interaction triggers different associated systemic defense responsesPCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WOthat include the induction of several PR (Pathogenesis-related proteins) genes, the accumulation of phytoalexins, the induction of reactive oxygen species (ROSs) and the Hypersensitive response (HR). The elicitors trigger the rapid and transitory production of reactive oxygen species (ROS) in plants.

[0005] Few elicitors have been reported to effectively induce resistance. Harpin proteins have been registered as biopesticides that act as elicitors. Said proteins are an alternative to the use of methyl bromide, which are effective against the bacterial leaf spot, the bacterial halo blight and certain fungal diseases; chitosan, which is active against the downy and powdery mildew and the gray mold and phosphite that are active against different phytopathogenic fungi.

[0006] The identification and isolation of elicitor proteins are the result of several basic researches that seek to understand the interaction between phytopathogenic microorganisms and plants. A first line of defense is the hypersensitive response (HR), a plant cell death at the point of the infection. Cell death creates a physical barrier to the movement of the pathogen and some dead cells of plants can release compounds that are toxic for the invading pathogen. Research indicated that phytopathogenic microorganisms might have a simple factor that was responsible for activating the HR. One research in particular identified a specific bacterial protein responsible for the HR. The protein under study was a known protein, encoded by a group of bacterial genes termed hypersensitive response and pathogenicity gene cluster (hrp). The hrp cluster in the bacteria Erwinia amylovora (Ea), the causal agent of fire blight in pears and apples, was identified as a protein that caused HR in certain plants. This protein was named Harpin (and later, Harpin Ea). This was the first time that such protein and gene of any bacterial species was identified.

[0007] Since then, several different harpin proteins of the Erwinia, Pseudomonas, Ralstonia, Xanthomonas and Pantoea, species, among others, have been identified.

[0008] After their identification and isolation, Harpin proteins were described as having the ability to induce disease resistance in plants and increase plant yield. A notable finding was that the application of purified harpin protein strengthened the plant’s resistance to a subsequent attack by pathogens, including in parts of the plant distant from the injection site. This means that harpin proteins may activate a systemic acquire resistance (SAR), a plant’s defense mechanism that provides resistance to a range of viral, bacterial and fungal pathogens. The application of elicitors induces resistance to several viruses, bacteria, oomycetes andPCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WOpathogenic fungi in several crops, comprising wheat, rice, apple, pear, citrus, cucumber, tobacco, tomato, pepper and strawberry.

[0009] Elicitors have the capacity to increase crop growth and yields. They increase photosynthesis, nutrients absorption, seed germination, root development and plant growth. Elicitor proteins induce several responses that promote plant growth, activating the ethylene signaling system, increasing photosynthesis rates and the expression of the expansin gene (EXP). For example, they increase the production of peaches, basically increasing the size of the fruit. They also improve the development of the fruit and induce an earlier ripening of the fruit. It has been proved that the yield of Green Tea (Cammelia sinensis) increased by over 55% in comparison to the untreated control plants. In tobacco, the elicitors increased leaf yield by 30.4% in comparison to the control plants. In addition, they improved the quality of tobacco leaves, with an increase of 50.2% against the control plants. It has been demonstrated that the Harpin protein acts as a growth promoter in orchids. (Phalaenopsis).

[0010] In view of the aforesaid and having detected the lack of new solutions and tools related to the provision of new treatments and / or prevention of plant diseases, we have developed compositions comprising several proteins or polypeptides that act as elicitors, triggering several defense mechanisms that immunize the plant and render it resistant to disease, while promoting plant growth.

[0011] In this sense, the use of elicitor proteins (polypeptides and / or peptides) represents an advantageous alternative for the agricultural systems, due to their natural characteristics and significant biological activity.BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1: Example of the hypersensitive response (HR) of tobacco leaves infiltrated with different dilutions of the composition of SEQ ID NO: 2 (1).

[0013] Figure 2. Resistance trial to Ralstonia solanacearum and plant-growth in tomato plants treated with the composition of SEQ ID NO: 2. References: (-) negative control without protein, (1) protein extract based on SEQ ID NO: 2; the number between brackets is 1 % survival for each treatment.

[0014] Figure 3 : Biocontrol assay of soil fungi: (A) Fusarium graminearum, (B) Rhizoctonia solani, and (C) Macrophomina phaseolina, after treatment of soybean seeds with the composition of elicitor protein based on SEQ ID NO 2. Treatments: (1) Absolute control (non-infected), (2) Inoculated control, (3) Seeds treated with the SEQ ID NO: 2-based composition, (4) Chemical control, and (5) Combined treatment with SEQ ID NO: 2-basedPCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WOcomposition and chemical control. (D) Plant length, (E) Plant weight, (F) Germination Percentage.

[0015] Figure 4: Determination of the accumulation of Reactive Oxygen Species (ROS) in plants after the foliar application of the compositions of elicitor proteins. (-) negative control without protein, (1) compositions based on SEQ ID NO: 2, (2) compositions based on SEQ ID NO: 4.

[0016] Figure 5. Fluorescence microscopy images of tobacco leaves infiltrated with compositions of: A) SEQ ID NO: 2, B) SEQ ID NO: 4. Callose depositions are shown in blue.BRIEF DESCRIPTION OF THE SEQUENCES

[0017] The proteins of SEQ ID NO: 1-20 belong to a group of proteins that share the characteristic of eliciting the plant's defense response.

[0018] The proteins of SEQ ID NO: 21-120 are homologous to the protein of SEQ ID NO: 1, with which they share a sequence identity of over 45% and the presence of a Pilin-like domain. In addition, the proteins of SEQ ID NO: 22, 26, 37 and 58 have a sequence identity of over 80% with the protein of SEQ ID NO: 1.

[0019] The proteins of SEQ ID NO: 121-216 are homologous to the protein of SEQ ID NO: 2, with which they share a sequence identity of more than 48% and the presence of a Necrosis-inducing Phytophthora protein (NPP1) domain. In addition, the proteins of SEQ ID NO: 122, 137, 188 and 200 share a sequence identity of over 80% with the protein of SEQ ID NO: 2.

[0020] The proteins of SEQ ID NO: 217-315 are homologous to the protein of SEQ ID NO: 3, with which they share a sequence identity of over 60% and the presence of a domain of type Glycosyl hydrolase family 45. Additionally, the proteins of SEQ ID NO: 223, 238, 258, 279 and 293 share a sequence identity of over 80% with the protein of SEQ ID NO: 3.

[0021] The proteins of SEQ ID NO: 316-414 are homologous to the protein of SEQ ID NO: 4, with which they share a sequence identity of more than 42%, and the presence of a Necrosis-inducing Phytophthora protein (NPP1) domain. In addition, the proteins of SEQ ID NO: 326, 334, 363 and 388 share a sequence identity of over 80% with the protein SEQ ID NO: 4.DETAILED DESCRIPTION OF THE INVENTION

[0022] The present application relates to a liquid or solid composition of macromolecules which elicit plant defense, termed phytovaccines. It is further related to the method of producing, formulating said phytovaccines and applying them to a plant or seed in order toPCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WOinduce a response in the plant. The application relates to recombinant proteins, elicitor polypeptides or peptides that can have a different identity but perform a common function in plants. Specifically, it refers to an amino acid sequence, which may be a short sequence, like a peptide, and has an eliciting activity in plants. In particular, the proteins of the application relate to elicitor polypeptides or peptides recombinant proteins, identified as follows: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. These sequences can originate from phytopathogenic fungi and also from bacteria of the following genres: Botrytis spp, Cercospora spp, Corinespora spp, Fusarium spp, Microphomina spp, Phyllosticta spp, Phytophthora spp, Pythium spp, Magnaporthe spp, Rhizoctonia spp, Sclerotinia spp, Xanthomonas spp, Bacillus spp.

[0023] As used in the description and the set of claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0024] Additionally, as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated enumerated elements, as well as the lack of combinations when interpreted in the alternative (“or”).

[0025] The term “approximately”, whenever used herein, refers to a measurable value such as a quantity, concentration or similar, is deemed to comprise variations of ± 10%, ± 5%, ± 1%, ± 0.5% or even ± 0.1% of the specified value as well as the specified value. For example, “approximately X”, wherein X is the measurable value, is deemed to include X, as well as the variations of ± 10%, ± 5%, ± 1%, ± 0.5% and also variations of ± 0.1% of X. An interval provided herein for a measurable value can include any other interval and / or individual value therein.

[0026] As used herein, phrases such as “between X and Y” and “between approximately X and Y” must be understood to include X and Y. As used herein, phrases such as “between approximately X and Y” mean “between approximately X and approximately Y” and phrases such as “from approximately X to Y” mean “from approximately X to approximately Y”.

[0027] The mention of interval values herein is merely intended to be an abbreviated form to refer individually to each separate value that falls in the interval, unless otherwise indicated herein; and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the interval 10 to 15 is disclosed, then the intervals 11, 12, 13 and 14 are also disclosed.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0028] The terms “comprise” “it comprises” and “comprising”, as used herein, specify the presence of the features, whole numbers, steps, operations, elements and / or components indicated, but do not preclude the presence or addition of one or more features, whole numbers, steps, operations, elements and / or groups thereof.

[0029] As used herein, the transitional phrase “consisting essentially of’ means that the scope of the claim includes specified materials or steps mentioned in the claim, together with any additional materials or steps which do not materially affect the basic and novel features of the claimed subject matter. Therefore, the phrase “consisting essentially of’, whenever used in a claim of the present aplication, must not be construed as equivalent to “comprising”.

[0030] As used herein, the terms “increase”, “enhance”, “promote”, “improve” and “which improves” (and the grammatical variations thereof) describe an increase of at least 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more in comparison with a control.

[0031] As used herein, the terms “reduce”, “reduced”, “which reduces”, “reduction”, “diminish” and “decrease” (and grammatical variations thereof), describe, for instance, a decrease of at least 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% in comparison to a control. In specific embodiments, a reduction may result in nil or essentially nil (i.e. a negligible quantity, for example, less of approximately 10% or even 5%) in a detectable activity or quantity.

[0032] A “heterologous” or “recombinant” nucleotide sequence is a sequence not naturally associated with a host cell into which it is introduced, comprising non- naturally occurring multiple copies of a naturally occurring nucleotide sequence.

[0033] A “native” or “wild type” nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. Thus, for example, a “wild-type mRNA” is an mRNA which is naturally present in or is endogenous to the organism. A “homologous” nucleic acid sequence refers to a nucleotide sequence naturally associated with a host cell into which it is introduced.

[0034] As used herein, the terms “a nucleic acid”, “a nucleic acid molecule”, “nucleotide sequence” and “polynucleotide” mean linear or branched RNA or DNA that is single- or double stranded or a hybrid thereof. The terms also encompass RNA / ADN hybrids.

[0035] As used herein the term “nucleotide sequence” means a nucleotide heteropolymer or a sequence of said nucleotides from the 5’ to the 3’ end of a molecule of nucleic acid and includes molecules of DNA or RNA , comprising the cDNA, a fragment or portion of DNA,PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WOgenomic DNA, synthetic DNA (e.g., synthesized chemically), plasmid DNA, mRNA and antisense RNA, any of which can be single- stranded or double-stranded. The terms “sequence of nucleotides”, “a nucleic acid”, “a molecule of nucleic acid”, “construct of nucleic acid”, “oligonucleotide” and “polynucleotide” are also used interchangeably herein to refer to an heteropolymer of nucleotides. The molecules of nucleic acid and / or the sequences of nucleotides provided herein are represented in the 5’ to 3’ direction, from left to right and are represented using the standard code used to represent nucleotides characters.

[0036] As used herein, the term “gene” refers to a molecule of nucleic acid that can be used to produce mARN, antisense RNA, miRNA, antisense oligodeoxyribonucleotide (AMO) and similar. The genes may or may not be capable of producing a functional formula or a genetic product. The genes can include coding and noncoding regions (e.g.: introns, regulatory elements, promoters, enhancers, terminator sequences and / or 5’ and 3’ untranslated regions). A gene may be “isolated”, i.e., a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium of the recombinant production, and / or various chemicals used in chemically synthesizing the nucleic acid.

[0037] A “portion” or “fragment” of a nucleotide sequence shall be deemed to mean a nucleotide sequence of a reduced length (e.g. reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) relative to a reference nucleic acid or nucleotides sequence and comprising, consisting essentially of and / or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g.: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence.

[0038] The term “transformation”, as used herein, means that a heterologous nucleic acid is introduced into the cell. The transformation of a cell may be stable or transient. Thus, in some embodiments, a host cell or host organism may be stably transformed with a molecule of a polynucleotide / a nucleic acid of the application. In some embodiments, a host cell or host organism may transiently be transformed with a construct of a polynucleotide of the application.

[0039] The term “bio stimulation” means the capacity of a substance, molecule or microorganism to cause an improvement in the nutritional efficiency of the plant under a certain condition.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0040] An “emulsifier”, “emulsifying agent” or “emulgent” is a substance that helps mix other two substances that are typically immiscible or difficult to mix, enabling the formation of an emulsion over time.

[0041] A “surfactant” is a compound that reduces the surface tension of the liquid to which it is added. An adjuvant surfactant helps to improve the dispersion of the drop on the plant’s leaf.

[0042] The “additives” are substances added to industrial products for technical purposes, e.g.: to improve harmlessness, increase the conservation period, modify its sensorial properties, or enhance its general features.

[0043] “Preservatives” are products that prevent the deterioration caused by bacteria, fungi, mold and yeasts, thus increasing duration or delaying or preventing changes in color or texture and delaying the rancidity of the formulation.EXAMPLES

[0044] Example 1 : Effect of extracts of elicitor proteins

[0045] The application of compositions that comprise elicitor proteins triggers a complex set of metabolic responses in the treated plant, which is mainly manifested with the activation of the plant’s defense mechanism and the stimulation of plant growth. This was recorded through the following laboratory trials: determination of the hypersensitive response (HR), analysis of callose depositions, evaluation of the increase in reactive oxygen species (ROS), biostimulation trials in different crops, protein-pathogen challenge trials and determination of increases in gene expressions associated with defense and growth. In addition, field trials for elicitor proteins were conducted with crops of interest to each composition.

[0046] 1) Hypersensitive response (HR) induced by elicitors

[0047] HR is manifested as an injury or necrosis in the leaf. Said injury originates when the plant responds to an attack, activating its defense mechanism and causing a programmed cell death in the injured area.

[0048] The compositions based on elicitor proteins and peptides with different concentrations of the active principles were infiltrated into leaves of Nicotiana tabacum. The plant’s response is observed after at least 72 hours.

[0049] The results are shown in Figure 1.

[0050] (2) Growth promotion -Yield increase - biostimulation

[0051] A. Bio stimulation of crops with elicitor proteins. Trial on soybean crops under controlled conditions.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0052] The trial studied the incidence rate of a biostimulant based on compositions with SEQ ID NO: 2 in the growth of soybeans seedlings. Three seed treatment trials were conducted in pots with different varieties and under controlled conditions. The areal dry weight and the root dry weight were determined. The results are summarized in Table 1, the seeds treated with 150 and 300 cc / lOOkg of compositions of SEQ ID NO: 2 reached an increase of approximately 20% respectively, i.e., the results were statistically significant improvement to the results observed in absolute control and statistically similar to the results observed in the chemical control and the commercial biological control. No statistically significant differences were observed between applied doses. Similar results were obtained regarding root weight. Under the study’s conditions, the performance observed by the samples were equivalent or even superior to the behavior of the commercial control.

[0053] Table 1. Determination of the aerial and root dry weight in soybean crops made in plant pots under controlled conditions.

[0054] The values refer to the average of the different trials conducted. The results were evaluated by means of analyses of variance (ANOVA) and the comparison of means by the Duncan test, with a significance level of 5% (a: 0.05) and 10 % (a: 0.1). The averages that share the same letters are not significantly different.

[0055] B. Crop biostimulation with elicitor proteins in soybean field trials.

[0056] Field trials were conducted to evaluate the biostimulant effect of the composition with SEQ ID NO: 2 on the emergency, development and yield of the field cultivation of soybean. Four trials were conducted in different agro-ecological regions for this crop, for two doses of proteins (150 and 300 cc / lOOkg). Table 2 shows the averages of these results for the following variables: fresh weight, dry weight, NDVI (the Normalized Difference VegetationPCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WOIndex is a simple indicator of photosynthetically active biomass), yield and weight of 1000 grams. In relation to the conditions under which the trial was conducted, it can be highlighted that, regardless of the dose of the composition with SEQ ID NO: 2 used as treatment of seeds, the increment observed was statistically significant in relation to the absolute control in the ND VI, areal weight, yield and weight of the 1000 soybean seeds in relation to the absolute control and, in certain cases, in relation to the commercial controls.

[0057] Table 2. Average determination of fresh weight, dry weight, NDVI, yield and weight of 1000 grains of trials in soybean field trials.

[0058] The results were evaluated by means of ANOVA and the Duncan test with a significance level of 5% and 10 %. The averages that share the same letters are not significantly different.

[0059] C. Bio stimulation of crops elicitor proteins in barley and wheat field trials.

[0060] An evaluation was conducted on the efficiency of the composition comprising SEQ ID NO: 4 on the development of barley and wheat cultivation in plant pots under controlled conditions. The effect of the application of the biostimulants applied may be observed in the increase in the vigor and aerial dry weight of plants. Afterwards, the pertinent field trials were conducted. Three agronomic efficiency trials of the composition that comprises SEQ ID NO: 4 in barley were conducted in different agroecologic regions, with three doses 75, 150 and 300 cc / 100 kg. The results showed a statistically significant increase in the number, vigor, freshPCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WOweight and yield of the plants in relation to the absolute control (Tables 3 and 4 show the average of the three trials), comparable to the result observed with commercial controls. No statistically significant differences were observed with the commercial controls. No statistically significant differences were observed between the doses. However, 150 cc / 100 kg reached the largest increases. The field trials of wheat showed similar results as regards the number, vigor, fresh weight and yield of the plants in relation to the absolute control, showing a biostimulant effect with an efficiency at least similar to the one observed with the biological commercial control.

[0061] Table 3. Average determination of Vigor, ND Vic Fresh weight in barley crops field trials.

[0062] The results were evaluated by means of ANOVA and the Duncan test, with a level of significance of 5% and 10 %. The averages that share the same letters are not significantly different.

[0063] Table 4. Average determination of the number of ears and yield of barley crops field trials.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0064] The results were evaluated by means of ANOVA and the Duncan test, with a level of significance of 5% and 10 %. The averages that share the same letters are not significantly different.

[0065] 3) Plague resistance

[0066] A. Plague resistance of tomato plants to Ralstonia solanacearum generated by a treatment with elicitor proteins.

[0067] Tomato seeds were treated with the composition of SEQ ID NO: 2 and were germinated and grown in approximately 7 days. After this period, the seeds were submerged in cultures of Ralstonia solanacearum (3x109 UFC / mL) and were finally transplanted to soil. The survival and height of the plants were evaluated 14 days after treatment. Figure 2 shows that the treatment with the composition of SEQ ID NO: 2 resulted in an increase in the survival of tomato plants infected with Ralstonia solanacearum. Furthermore, the plants that survived showed an 88.6% increase in height and a 20% increase in dry weight. These results show that the compositions that comprise SEQ ID NO: 2 foster activities that promote growth and protect the plant against pathogens.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0068] B. Biocontrol assay of soil fungi: (A) Fusarium graminearum, (B) Rhizoctonia solani, and (C) Macrophomina phaseolina, following treatment of soybean seeds with an elicitor protein composition.

[0069] Three-liter pots were filled with substrate, and three planting holes were prepared per pot. Each hole was inoculated with 3 g of infective inoculum of Macrophomina phaseolina, Rhizoctonia solani, and Fusarium graminearum, and subsequently sown with 10 soybean (Glycine max) seeds. A chemical control (CC) (Fludioxonil + Metalaxyl) was applied at a dose of 100 mL of active ingredient per 100 kg of seed. In the absolute control (AC), untreated and non-inoculated seeds were evaluated, while in the infection control (IC), untreated seeds inoculated with the pathogen were used. The protein composition based on SEQ ID NO: 2 was applied at a dose of 300 mL per 100 kg of seed. 21 days after sowing, the number of emerged seedlings, the length, as well as the fresh weight of roots and aerial parts, were evaluated (Figure 3).

[0070] C. The results shown in Figure 3d-f indicate an increase in germination percentage, as well as greater shoot and root length, and higher fresh weight in seeds treated with the composition based on SEQ ID NO: 2 compared with the infection control. These results correspond to Macrophomina phaseolina. Similar results were observed for Rhizoctonia solani and Fusarium graminearum. Bacterial canker (Clavibacter michiganensis subesp. michiganensis) in tomato crops (Solanum Lycopersicum).

[0071] The bacterial canker is vascular disease produced by a bacterium named Clavibacter michiganensis subesp. michiganensis, whose only host is the tomato plant. It is one of the main diseases which affect the crop and may lead to yield losses of between 20 % and 80 %. Table 5 shows that the foliar application of a composition that comprises SEQ ID NO: 1 in the two trials conducted resulted in a significant reduction of the bacterial infection intensity in the tomato plant. The average control efficacy of the 4 dates evaluated was 85.4, 93 and 94.7% for the doses tried. Independently of the doses, the composition with SEQ ID NO: 1 was effective in the reduction of the incidence, severity and intensity of the bacterial canker on the tomato plant. Similar results are obtained with the foliar application of the composition that comprises SEQ ID NO: 3 (Table 6).

[0072] Table 5. Bacterial infection intensity and efficacy in the control in tomato plants treated with the composition that comprises SEQ ID NO: 1.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0073] The table shows the average of the two independent trials in tomato plants treated with 1000, 2000 and 5000 cc / ha. The results were evaluated by means of ANOVA and a Duncan test, with a level of significance of 5% and 10 %. The averages that share the same letters are not significantly different.

[0074] Table 6. Bacterial infection intensity and efficiency control in tomato plants treated with a composition that comprises SEQ ID NO: 3.

[0075] Table 6 shows the average of independent trials in tomato plants treated with 1000, 2000 and 5000 cc / ha. The results were evaluated by means of ANOVA and the Duncan test, with a significance level of 5% and 10 %. The averages that share the same letter are not significantly different. Oidium control (Oidium neolycoperisici) in tomato crops (Solanum lycopersicun).PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0076] An evaluation was performed on the efficacy of the composition that comprises SEQ ID NO: 1 in the control of Oidium (Oidium neolycoperisici) in the tomato crop (Solarium lycopersicun) (Table 8. A). This is a disease that causes damages in different parts of the plant and that may affect the whole plant, causing a significant reduction in yield. The foliar application was performed in three doses: 1000, 2000 and 5000 cc / ha. The number of affected leaves and the percentage damage in each leaf were evaluated 7, 14 and 21 days after the application (DDA) and the intensity of the disease was determined. The evaluated doses of the compositions of SEQ ID NO: 1 reduced the intensity of Oidium on the leaves of tomato plants in a statistically significant manner against the absolute control. It was observed that efficacy increased when the doses of the composition of SEQ ID NO: 1 were increased, and the efficacy achieved by applying of the composition of SEQ ID NO: 2 at a rate of 2000 cc / ha was statistically significant and greater than the efficacy observed by applying a composition of SEQ ID NO: 1 at a rate of 1000 cc / ha. On the other hand, the concentration of 5000 cc / ha was not statistically significant. Similar results were observed with the composition of SEQ ID NO: 3 (Table 7. B).

[0077] Table 7. Average intensity of the Oidium (powdery mildew) infection in trials and efficacy in the control of the oidium infection in the middle third of the plant, average in trials

[0078] A) Trials using the composition with SEQ ID NO :1.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0079] The data were submitted to a variance analysis (ANOVA) and the Duncan test was used to evaluate the differences between the means of the treatments, reaching a significance level of 5%.

[0080] B) Trials using the composition with SEQ ID NO: 3.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0081] The data were submitted to a variance analysis (ANOVA) and the Duncan test was used to evaluate the differences between the means of the treatments, reaching a significance level of 5%.

[0082] 4) Increase in the production of reactive oxygen species (ROS).

[0083] The foliar application of formulations based on elicitor proteins generates an induction of ROS. The induction of these compounds at low concentrations is essential for cell signaling and are involved in numerous biological processes, such as the plant defense response, among others.

[0084] Figure 4 shows the result of spraying tomato leaves with the compositions that comprise SEQ ID NO: 2 and SEQ ID NO: 4. Two hours after spraying, they were incubated with marker compounds of ROS: 3,3’-diaminobenzidine (DAB), as a histological marker of the production of oxygen peroxide (H2O2) nitro blue tetrazolium chloride (NBT) as a hydrogen superoxide marker (O2-).

[0085] 5) Physiological defense response of plans: Callose depositions.

[0086] Callose is a -1,3-glucan polysaccharide present in plants that performs a defense role. As a response to biotic or abiotic stress, callose depositions are generated at plasmodesmata which diminish the communication between cells.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0087] Tobacco plants were infiltrated with formulations of elicitor proteins. Afterwards, the fluorescent staining technique with KOH-aniline blue was applied and a fluorescence microscope was used to observe said formulations. Figure 5 shows fluorescence microscopy images where the callose depositions in the leaves infiltrated with proteins of SEQ ID NO: 2 and SEQ ID NO: 4 are shown in blue.

[0088] 6) Increase in defense-related gene expressions.

[0089] Real Time PCR assays were performed with samples of tobacco leaves infiltrated with elicitor proteins compositions.

[0090] The Table 8 shows the expression levels of plant defense-related genes in tabacco infiltrated with two elicitor compositions: SEQ ID NO 2 and SEQ ID NO 3. In the infiltration of both compositions induces the expression of different genes related to the defense-response pathways: with SED ID NO 2, ACS1 (1 -aminocyclopropane- 1 -carboxylic acid synthase 1, key enzyme in the ethylene biosynthesis pathway), PR1 AC (Pathogenesis-Related Protein 1), PR3 (Pathogenesis-Related Protein 3, chitinase), and HMG (3-hydroxy-3-methylglutaryl-CoA reductase, key enzyme in the mevalonate pathway, leading to the synthesis of isoprenoids, phytoalexins, and other defense-related secondary metabolite) were significantly upregulated, indicating activation of general defense pathways. In the case of SEQ ID NO3, HSR203 (Hypersensitive Response Protein) and HMG expression increased significantly.

[0091] Also, defense-related and growth genes were induced in soybean after seed inoculation.

[0092] Table 8. Differential expression of tobacco defense-related genes in response to elicitor treatments.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO

[0093] Example 2: Production of proteins of interest

[0094] This protein, polypeptide, and / or sequence may be produced by means of a recombinant technology in a bacterial strain of non-pathogenic Escherichia coli. Alternatively, it can be produced in other microorganisms such as Saccharomyces cerevisiae and Pichia pastoris. It also involves the de novo solid phase peptide synthesis technology.

[0095] The pertinent sequence is cloned in an inducible protein expression vector, e.g.: Isopropyl-P-D-1 -thiogalactopyranoside (IPTG), lactose, arabinose or methanol, with a concentration which may range from 0.1 to 100 pM. The expression may be carried out in Erlenmeyer flasks, a pilot-plant reactor or larger, with a supplemented fermentation medium appropriate for the system. This medium may be rich, such as Luria-Bertani (LB), or a minimum medium with the appropriate concentrations of compounds essential for bacterial growth.

[0096] The specific expression conditions will vary with each protein. Basically, after the inducer has been added, they are incubated from 4 to a maximum of 16 hours, within a temperature range of 25 °C and 37° C.

[0097] Finally, the cell rupture of the obtained crude extract is carried out, which can be done through a chemical or physical process, e.g., using ultrasound waves or high-pressure homogenization. The remains of cellular components and membrane can be separated by centrifugation at 5000 g in cold conditions. It is also possible to continue using the homogenized crude extract directly without separation. Finally, various additives that enhance its stability and function can be incorporated.

[0098] Example 3: Formulation of proteins of interest

[0099] The homogenate obtained in the production process is formulated with components that make it possible to maintain its properties intact for at least 24 months. For this purpose, an appropriate formulation is used for the preparation of the final product.[000100] The final composition will depend on its subsequent use or method of application. For example, for foliar application, a standardized amount of crude extract measured by optical density is combined with additives to enhance and maintain its activity over time and to ensure the product adheres to the plant leaves. In this case, the components would be as follows:[000101] Crude extract or pure proteins solutions in a regulator solution to check optimal pH values, from 7 and 8.8, such as PIPES, Tricine, Tris-HCl, buffer phosphate, MOPS, or HEPES.[000102] Non-ionic emulsifier agents and / or surfactants, for example: polyethylene glycol, polyalkylene glycol ethers, polysorbates, octoxynol, glycerol and the ethers thereof.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO[000103] Additives which improve the stability of the active principle and enhance the defense-eliciting and / or biostimulant activity. As illustrative examples, we cite peptides, polysaccharides, sugar alcohols, EDTA, bovine serum albumin (BSA), chitosan, acetic acid, among other.[000104] Appropriate preservatives with biocide properties such as benzisothiazolinone, metilisotiazolinone, iodopropynyl butylcarbamate, sodium azide, bronopol, methylparaben, potassium sorbate or sodium benzoate.[000105] EMBODIMENTS[000106] The first embodiment discloses a composition comprising a defense-eliciting and biostimulant protein or fragment thereof, which may be a polypeptide or even a small elicitor peptide, wherein the defense elicitor protein or fragment thereof comprises an amino acid sequence of SEQ ID NO: 1-414.[000107] The second embodiment discloses a composition wherein the elicitor protein confers an improvement in plant yield.[000108] The third embodiment discloses a composition wherein the elicitor protein confers plague resistance in plants.[000109] The fourth embodiment discloses a composition wherein the elicitor protein comprises SEQ ID NO: 1 and 21-120.[000110] The fifth embodiment discloses a composition wherein the elicitor protein comprises SEQ ID NO: 2 and 121-216.[000111] The sixth embodiment discloses a composition wherein the elicitor protein comprises SEQ ID NO: 3 and 217-315.[000112] The seventh embodiment discloses a composition wherein the elicitor protein comprises SEQ ID NO: 4 and 316-414.[000113] The eighth embodiment discloses a composition wherein the elicitor protein selected from the group consisting of SEQ ID NO: 5-20.[000114] The ninth embodiment discloses a composition that also comprises a buffer solution.[000115] The tenth embodiment discloses a composition wherein the buffer solution is selected from a group consisting of: PIPES, Tricine, Tris-HCI, Buffer phosphate, MOPS or HEPES.[000116] The eleventh embodiment discloses a composition that also comprises emulsifiers and / or surfactants.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO[000117] The twelfth embodiment discloses a composition wherein the emulsifiers and / or surfactants are selected from a group consisting of: polyethylene glycol, polyalkylene glycol ethers, polysorbates, octoxynol, glycerol and the ethers thereof.[000118] The thirteenth embodiment discloses a composition that also comprises additives.[000119] The fourteenth embodiment discloses a composition wherein the additive is selected from a group consisting of: EDTA, peptides, polysaccharides such maltodextrin, sugar alcohols such as mannitol, bovine serum albumin (BSA), chitosan, acetic acid.[000120] The fifteenth embodiment discloses a composition that also comprises a preservative.[000121] The sixteenth embodiment discloses a composition wherein the preservative has a biocide or bacteriostatic effect.[000122] The seventeenth embodiment discloses a composition wherein the preservative is selected from a group consisting of: benzisothiazolinone, metilisotiazolinone, iodopropynyl butylcarbamate, sodium azide, bronopol, methylparaben, potassium sorbate or sodium benzoate.[000123] The eighteenth embodiment discloses the composition wherein the composition is liquid or solid.[000124] The nineteenth embodiment discloses a method of controlling plague infestation, wherein said method comprises a. contacting a seed or a plant with an effective quantity of one or more elicitor proteins or fragments thereof, having an amino acid sequence of SEQ ID NO: 1-414, b. cultivating a plant mentioned in point a, c. placing the plant in contact with the plague.[000125] The twentieth embodiment discloses a method wherein the plant belongs to an intensive and / or extensive crop selected from a group comprising maize, soybean, sorghum, sunflower, sugar cane, tobacco, barley, wheat, tomato, potato, pepper, cotton, eggplant, rice, lettuce, lentils, chickpea, lemon, orange, pear, apple, plum, peach, apricot, strawberry, blueberries.[000126] The twenty-first embodiment discloses a for improving the yield of a plant which comprises: a. contacting a seed or a plant with an effective quantity of one or more elicitor proteins or fragments thereof comprising an amino acid sequence of SEQ ID NO: 1-414, b. cultivating the plant mentioned in point a, c. harvesting the plant’s products.[000127] The twenty-second embodiment discloses a method wherein the plant belongs to an intensive and / or extensive crop selective from the group comprising maize, soybean, sorghum, sunflower, sugar cane, tobacco, barley, wheat, tomato, potato, pepper, cotton, eggplant, rice,PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WOlettuce, lentils, chickpea, lemon, orange, pear, apple, plum, peach, apricot, strawberry, blueberries.[000128] The twenty-third embodiment discloses a method for improving a plant’s yield which comprises: a. inoculating a plant or seed with the composition of the first embodiment, b. cultivating and growing the plant mentioned in point a, c. harvesting the plant’s product.[000129] The twenty-fourth embodiment discloses the use of a defense-elicitor protein or fragment thereof to improve a plant’s resistance to plagues, wherein the defense- elicitor protein or fragment thereof comprises an amino-acid sequence of de SEQ ID NO 1-414.[000130] The twenty-fifth embodiment discloses the use of a defense-elicitor protein or fragment thereof to improve a plant’s resistance to plagues, wherein the defense- elicitor protein or fragment thereof comprises an amino acid sequence of SEQ ID NO: 1-414.[000131] The twenty-sixth embodiment discloses a transgenic plant comprising a nucleic acid encoding the elicitor protein of claim 1, wherein expression of said nucleic acid induces a defense response in the plant.

Claims

PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WOCLAIMSWe claim:

1. A composition comprising at least one elicitor and bio-stimulating protein or a fragment thereof capable of inducing a defense response in a plant, wherein said defense response enhances resistance to biotic and / or abiotic stress and promotes plant growth.

2. The composition of claim 1, wherein the elicitor protein confers an improvement in the yield of plants.

3. The composition of claim 1, wherein the elicitor protein confers resistance to plagues in plants.

4. The composition of claim 1, wherein the elicitor protein comprises a sequence selected from the group consisting of SEQ ID NO: 1 and 21-120.

5. The composition of claim 1, wherein the elicitor protein comprises a sequence selected from the group consisting of SEQ ID NO: 2 and 121-216.

6. The composition of claim 1, wherein the elicitor protein comprises a sequence selected from the group consisting of SEQ ID NO: 3 and 217-315.

7. The composition of claim 1, wherein the elicitor protein comprises a sequence selected from the group consisting of SEQ ID NO: 4 and 316-414.

8. The composition of claim 1, wherein the elicitor protein comprises a sequence selected from the group consisting of SEQ ID NO: 5-20.

9. The composition of claim 1, wherein the composition also comprises a buffer solution.

10. The composition of claim 9, wherein the buffer solution is selected from a group consisting of: PIPES, Tricine, Tris-HCI, buffer phosphate, MOPS or HEPES.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO11. The composition of claim 1, wherein the composition also comprises a sequence of emulsifiers and / or surfactants.

12. The composition of claim 11, wherein the emulsifiers and / or surfactants are selected from a group consisting of: polyethylene glycol, ethylene glycol, polyalkylene glycol ethers, ethoxylated alcohols, methylated vegetable oils, polysorbates, octoxynol, glycerol and the ethers thereof.

13. The composition of claim 1, wherein the composition also comprises additives.

14. The composition of claim 13, wherein the additive is selected from a group consisting of: EDTA, peptides, polysaccharides such as maltodextrin, sugars alcohols such as mannitol, sorbitol, bovine serum albumin (BSA), chitosan, and acetic acid.

15. The composition of claim 1, wherein the composition also comprises a preservative.

16. The composition of claim 15, wherein the preservative has a biocide or bacteriostatic effect.

17. The composition of claim 16, wherein the preservative is selected from a group comprising: benzisothiazolinone, metilisotiazolinone, iodopropynyl butylcarbamate, sodium azide, bronopol, methylparaben, potassium sorbate or sodium benzoate.

18. The composition of claim 1, wherein the composition is liquid, lyophilized, dried or solid.

19. A method of controlling plague infestation, wherein said method comprises:a. contacting a seed or a plant with an effective amount of one or more elicitor proteins or fragments thereof, having an amino acid sequence of SEQ ID NO: 1-414, b. cultivating the plant of a,c. contacting the plant with the plague.PCT / US25 / 54972 11 November 2025 (11.11.2025)Attorney Docket No. 124047.PH826WO20. The method of claim 19, wherein the plant belongs to an intensive and / or extensive crop selected from a group comprising maize, soybean, sorghum, peanut, sunflower, sugar cane, tobacco, barley, wheat, tomato, potato, pepper, cotton, eggplant, rice, lettuce, lentils, chickpea, lemon, orange, pear, apple, plum, peach, apricot, cherries, strawberry, blueberries, grape, olives.

21. A method for improving the yield of a plant comprising:a. contacting a seed or a plant with an effective amount of one or more elicitor proteins or fragments thereof comprising an amino acid sequence of SEQ ID NO: 1- 414,b. cultivating the plant of a,c. harvesting the plant’s products.

22. The method of claim 21, wherein the plant belongs to an intensive and / or extensive crop selected from a group comprising maize, soybean, sorghum, sunflower, sugar cane, tobacco, barley, wheat, tomato, potato, pepper, cotton, eggplant, rice, lettuce, lentils, chickpea, lemon, orange, pear, apple, plum, peach, apricot, strawberry, blueberries.

23. A method for improving the yield of a plant comprising:a. inoculating a plant or seed with the composition of claim 1,b. cultivating and growing the plant of a,c. harvesting the plant’s product.

24. Use of a elicitor protein or fragment thereof to improve a plant’s resistance to plagues, wherein the elicitor protein or fragment thereof comprises an amino acid sequence of SEQ ID NO 1-414.

25. Use of a elicitor protein or fragment thereof to improve the yield of a plant exposed to plagues, wherein the elicitor protein or fragment thereof comprises an amino acid sequence of SEQ ID NO: 1-414.

26. A transgenic plant comprising a nucleic acid encoding the elicitor protein of claim 1, wherein expression of said nucleic acid induces a defense response in the plant.

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