# Patent

## Title
Plant defense signal peptides

## Abstract

A 23 amino acid peptide, AtPep1, plays an important role as a signaling component of the innate immune system of Arabidopsis. The peptide precursor gene is transcribed in response to elicitors generated by pathogens, and AtPep1 is produced to amplify the signaling pathways. Seven paralogs of the AtproPep1 gene have been identified in the Arabidopsis genome, and orthologs have been identified in species of several agriculturally important families. AtPep1 and its paralogs and orthologs play important roles as endogenous signals to amplify innate immunity. The sequences of two AtPep1 receptors from Arabidopsis are also provided.

## Background
RELATED APPLICATIONS
This application is a continuation of and claims benefit of co-pending U.S. patent application Ser. No. 13/592,971, filed Aug. 23, 2012, which claims priority to U.S. Provisional Patent Application No. 61/124,199 entitled Plant Defense Signal Peptides, filed Apr. 14, 2008, and U.S. patent application Ser. No. 11/795,733, filed Jun. 4, 2008, which is a national stage entry of WO 2006/08130 filed Jan. 24, 2006, which claims priority to U.S. Provisional Patent Application No. 60/647,708 filed Jan. 26, 2005.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
The invention was supported, at least in part, by a grant from the Government of the United States of America (grant no. IBN 0090766 from the National Science Foundation). The United States Government has certain rights to the invention.


FIELD
The present invention relates to materials and methods for enhancing plant disease resistance.
BACKGROUND
Plants are exposed to numerous denizens of their environment, including bacteria, viruses, fungi, and nematodes. Although many of the interactions between these organisms and plants, particularly via the roots of the plants, are beneficial, many of the interactions are harmful to the plants. The decimation of agricultural crops, ornamental plants, and other plants by diseases caused by plant pathogens is a worldwide problem that has enormous economic impact.
Damage to plants is caused by pathogens of multiple genera. These genera include Alternaria, Ascochyta, Aspergillus, Botrytis, Cercospora, Colletotrichum, Diplodia, Erwinia, Erysiphe, Fusarium, Gaeumanomyces, Helminthosporium, Macrophomina, Magnaporthe, Mycosphaerella, Nectria, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Pseudomonas, Puccinia, Puthium, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, and Xanthomonas. 
Macrophomina, Magnaporthe, Mycosphaerella, Nectria, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Pseudomonas, Puccinia, Puthium, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, and Xanthomonas. 
Many chemical compounds have been developed to combat these various pathogens. The activity of these compounds is typically limited to several species. As a consequence of the large number and diversity of plant pathogens, these compounds have not provided an effective solution to limiting infections in plants.
An alternative approach to controlling pathogenic infections in plants involves exploiting the natural defense mechanisms of plants to confer resistance. Many plants have developed natural resistance to some pathogens. However, resistance may be limited to certain genera of pathogens, or crops of agronomic interest may not exhibit sufficient resistance. Thus, natural plant defenses often do not provide sufficient protection against pathogens. By broadening the spectrum of pathogen defense or strengthening the defense response, it may be possible to enhance existing resistance mechanisms and promote pathogen defense in otherwise susceptible plants.
When present and active, the natural defense mechanisms of plants can be highly effective in preventing pathogen colonization and disease. Resistance is multi-tiered, with passive and active, constitutive and inducible elements.
Following the invasion of a plant by a potential pathogen, the pathogen either successfully proliferates in the host, causing associated disease symptoms, or its growth is halted by the defenses of the host plant. One such defense is the hypersensitive response (HR), rapid apoptotic cell death near the site of the infection that correlates with the generation of activated oxygen species, production of antimicrobial compounds, and reinforcement of host cell walls (Dixon and Lamb, Annu. Rev. Plant Physiol. Plant Mol. Biol. 41:339-367, 1990). Other defenses include systemic acquired resistance, which effectively protects the plant against subsequent attack by a broad range of pathogens (Ryals et al., Proc. Natl. Acad. Sci. USA 92:4202-4205, 1995).
Pathogens that elicit an HR on a given host are “avirulent” on that host, the host is “resistant,” and the plant-pathogen interaction is “incompatible.” If a pathogen proliferates and causes disease to the host, the pathogen is “virulent,” the host is “susceptible,” and the plant-pathogen interaction is “compatible.”
In many cases in which strains (“races”) of a particular fungal or bacterial pathogen differ regarding virulence on various cultivars (or wild accessions) of a particular host species, avirulent strains of the pathogen, but not virulent strains, possess one or more avirulence (avr) genes corresponding to “resistance” genes in the host. Resistance gene products are activated in response to pathogen signal molecules termed elicitors, production of which is controlled by pathogen avirulence genes. This observation is the basis for the “gene-for-gene” model of plant disease resistance (Crute et al., pp. 197-309 in Mechanisms of Resistance to Plant Disease, Fraser, ed., 1985; Ellingboe, Annu. Rev. Phytopathol. 19:125-143, 1981; Flor, Annu. Rev. Phytopathol. 9:275-296, 1971; and Keen et al., in Application of Biotechnology to Plant Pathogen Control, Chet, ed., John Wiley & Sons, 1993, pp. 65-88).
Normally avirulence and resistance genes are organized in functional pairs. A given resistance gene is generally effective only against pathogen strains that express a specific cognate avirulence gene (Flor, Annu. Rev. Phytopathol. 9:275-296, 1971; Keen, Annu. Rev. Genet. 24:447-463, 1990). However, exceptions to this rule exist. For example the Arabidopsis RPM1 gene product (Grant et al., Science 269:843-846, 1995) is involved in the recognition of elicitors produced by P. syringae expressing the avirulence genes avrRpm1 or avrB (Bisgrove et al., Plant Cell 6:927-933, 1994), suggesting that resistance gene products may function as common points in transduction of distinct pathogen signals.
A number of avirulence genes have been cloned. Many cloned avirulence genes have been shown to correspond to individual resistance genes in the cognate host plants and confer an avirulent phenotype when transferred to an otherwise virulent strain. A number of plant disease resistance genes have also been cloned. Similar features have been discovered among many of these resistance genes, in spite of the diversity of pathogens against which they act. These features include a leucine-rich-repeat (LRR), a motif found in a multitude of eukaryotic proteins with roles in signal transduction (Kobe and Deisenhofer, Trends Biochem. Sci. 19:415-421, 1994). The LRR motif is thought to be involved in protein-protein interactions and may allow interaction with other proteins that are involved in plant disease resistance. In addition, sequences predicted to encode nucleotide binding sites and leucine zippers are shared among many resistance genes (Dang), Cell 80:383-386, 1995; Staskawicz et al., Science 268:661-667, 1995). These motifs are present and similarly organized among resistance gene products from plants as diverse as tobacco, tomato, rice, flax, and Arabidopsis, suggesting a common mechanism underlying disease resistance signal transduction throughout the plant kingdom.
The local perception of pathogen attack is conveyed to distant tissues via a transmissible signal that involves salicylic acid (SA), further activating gene expression and conditioning a state known as systemic acquired resistance (SAR). It has subsequently been found that resistance can be expressed near the region of pathogen attack, as local acquired resistance, or can be induced systemically, depending on triggering signal and plant species. Thus, the systemic and local responses collectively are referred to as acquired resistance (AR). Establishment of AR is a powerful line of plant defense because it can provide broad-spectrum resistance against viral, bacterial, and fungal challenges that would otherwise cause disease. The AR response triggers the transcriptional activation of a suite of genes encoding pathogenesis-related (PR) proteins. Included among these are hydrolases, cell-wall strengthening proteins, proteins involved in oxidative burst, the combination of which are believed to promote heightened resistance. Biochemical and genetic analyses have identified genes and molecular signals associated with acquired resistance. The Npr1/Nim1 gene plays a key regulatory role in the AR defense in Arabidopsis against a broad spectrum of fungal and bacterial pathogens (WO 98/06748; WO 94/16077; WO 98/26082). The central importance of Npr1 in dicots was further substantiated by transgenic overexpression of the cloned gene, which led to heightened disease resistance in Arabidopsis against both fungal and bacterial pathogens (WO 98/06748).
Although the bulk of AR research has defined the pathway in dicotyledonous plants, monocotyledonous plants, such as wheat, rice, and barley, have an inducible pathway that protects against pathogen attack. Acquired resistance can be conditioned by different external stimuli, including avirulent pathogen challenge, pathogen elicitor exposure, and chemical treatments, including application of SA or SA analogs, such as 2,6-dichloroisonicotinic acid (INA) or benzo(1,2,3) thiodiazole-7-carbothioic acid S-methyl ester (BTH). Given the inducibility of the AR pathway by the same classes of activating compounds in monocot and dicot plants, there is likely to be partial conservation of signaling pathways, as subsets of PR genes appear to be induced in both groups. In monocots, induced acquired resistance is broad-spectrum, extending to fungal and bacterial pests, irrespective of pathogen race, with activated resistance persisting for weeks to months. Thus, manipulation of the AR pathway in plants may promote resistance to pathogens for which there exists no genetic source of resistance.
Thus, there is a need to identify genes that may play key roles in disease defense. Expression of these genes in transgenic plants may enhance the level of disease resistance against certain pathogens.
Within the past decade, the mechanisms by which plants activate innate immunity have been found to share a number of similarities with the innate immune responses of animals (Nimchuk et al., Annu. Rev. Gen. 37:579-609, 2003; Jones and Takemoto, Curr. Opin. Immun. 16:48-62, 2004; Nürnberger and Scheel, Trends Plant Sci. 8:372-379, 2001; Nümberger et al., Immun. Rev. 198:249-266, 2004; Guttman, Biotech. Adv. 22:363-382, 2004; Staskawicz et al., Science 292:2285-2289, 2001; Nürnberger and Brunner, Curr. Opin. Plant Biol. 5:1-7, 2002). Innate immunity is initiated in animals and plants through the recognition of a variety of pathogen associated molecules that in animals are called “pathogen-associated molecular patterns,” or PAMPS, and in plants are called elicitors. Peptides derived from pathogens can be powerful elicitors of plant defense responses (Hahlbrock et al., Proc. Natl. Acad. Sci. USA 92:4150-4157, 1995; van den Askerveken et al., Plant Physiol. 103:91-96, 1993; Kammpren, Curr. Opin. Plant Biol. 4:295-300, 2001; Kunze et al., Plant Cell, 16:3496-3507, 2004; Navarro et al., Plant Physiol. 135:1113-1128, 2004; Fellbrich et al., Plant J. 32:375-390, 2002); He et al., Cell 73:1255-1266, 1993).
We previously identified a number of novel defense signal peptides from dicot and monocot plant species that are useful for enhancing plant resistance against various biotic or abiotic stresses, including, but not limited to, disease resistance. See U.S. provisional patent application Ser. No. 06/647,708, filed Jan. 26, 2005, and PCT/US2006/002661, filed Jan. 24, 2006.
BRIEF SUMMARY
An embodiment provides for an isolated polynucleotide that encodes a polypeptide comprising a 10 amino acid peptide motif, where the 10 amino acid peptide motif consists of a glycine residue at position C-7 of the peptide, and the polypeptide causes a change of at least 0.2 pH units at a concentration of 25 pM/ml in a plant cell suspension. The polynucleotide may be isolated from genomic DNA, or synthesized based on genomic DNA, where the genomic DNA encodes three or more structural elements including a first EDKR repeat, a 1-5-10 helix motif, a serine repeat, a second EDKR repeat, and a K/R positive charge region, where the structural elements are positioned 5′ upstream of the polynucleotide. An additional embodiment provides for a transgenic plant comprising the isolated polynucleotide.
According to another embodiment, the isolated polynucleotide may encode one more defense signal peptides including TcPep1, GhPep2, SePep1, PtPep2, VvPep2, VvPep3, HaPep1, HaPep2, OsPep3b, OsPep8, SoPep1a, SoPep2a, PvPep1, OsPep3c, OsPep4b, OsPep4c, OsPep5b, OsPep5c, OsPep6b, ZmPep4b, ZmPep4c, TaPep3b, TaPep3c, HvPep1b, HvPep1c, SoPep1b, SoPep1c, SoPep1d, SoPep1e, SoPep1f, SoPep2b, SoPep2c, SoPep2d, and SoPep2e. An additional embodiment provides for a transgenic plant comprising one or more of the defense signal peptides.
According to another aspect, an isolated polypeptide is provided that comprises a 10 amino acid peptide motif, where the 10 amino acid peptide motif consists of a glycine residue at position C-7 of the peptide, and the polypeptide causes a change of at least 0.2 pH units at a concentration of 25 pM/ml in a plant cell suspension. An additional embodiment provides for a transgenic plant comprising the isolated polypeptide.
According to another embodiment, the isolated polypeptide may encode one more defense signal peptides including TcPep1, GhPep2, SePep1, PtPep2, VvPep2, VvPep3, HaPep1, HaPep2, OsPep3b, OsPep8, SoPep1a, SoPep2a, PvPep1, OsPep3c, OsPep4b, OsPep4c, OsPep5b, OsPep5c, OsPep6b, ZmPep4b, ZmPep4c, TaPep3b, TaPep3c, HvPep1b, HvPep1c, SoPep1b, SoPep1c, SoPep1d, SoPep1e, SoPep1f, SoPep2b, SoPep2c, SoPep2d, and SoPep2e. An additional embodiment provides for a transgenic plant comprising one or more of the defense signal peptides.
According to a further embodiment, the polypeptide is a propeptide. In a preferred embodiment, the propeptide is between 75 and 154 amino acid residues in length and may comprise one or more defense signal peptides including TcPep1, GhPep2, SePep1, PtPep2, VvPep2, VvPep3, HaPep1, HaPep2, OsPep3b, OsPep8, SoPep1a, SoPep2a, PvPep1, OsPep3c, OsPep4b, OsPep4c, OsPep5b, OsPep5c, OsPep6b, ZmPep4b, ZmPep4c, TaPep3b, TaPep3c, HvPep1b, HvPep1c, SoPep1b, SoPep1c, SoPep1d, SoPep1e, SoPep1f, SoPep2b, SoPep2c, SoPep2d, and SoPep2e. An additional embodiment provides for a transgenic plant comprising the propeptide.
According to another embodiment, the propeptide may be processed in a plant to produced one or more defense signal peptides including TcPep1, GhPep2, SePep1, PtPep2, VvPep2, VvPep3, HaPep1, HaPep2, OsPep3b, OsPep8, SoPep1a, SoPep2a, PvPep1, OsPep3c, OsPep4b, OsPep4c, OsPep5b, OsPep5c, OsPep6b, ZmPep4b, ZmPep4c, TaPep3b, TaPep3c, HvPep1b, HvPep1c, SoPep1b, SoPep1c, SoPep1d, SoPep1e, SoPep1f, SoPep2b, SoPep2c, SoPep2d, and SoPep2e.
According to another aspect, an isolated polynucleotide is provided that encodes a polypeptide comprising a defense signal peptide receptor protein, where the defense signal peptide receptor protein may include AtPepR1, AtPepR2, GmPepR1a, GmPepR1b, GmPepR2, P93194 (In), 803073 (Pt), 765043 (Pt), CAO23192 (Vv), OsPepR1, OsPepR2, GRMZM2G011806 (Zm), GRMZM2G128602 (Zm), and Sb07g021950 (Sb). An additional embodiment provides for a transgenic plant comprising the isolated polynucleotide.
According to an additional embodiment, an isolated polypeptide is provided that comprises a defense signal peptide receptor protein, where the defense signal peptide receptor protein may include AtPepR1, AtPepR2, GmPepR1a, GmPepR1b, GmPepR2, P93194 (In), 803073 (Pt), 765043 (Pt), CAO23192 (Vv), OsPepR1, OsPepR2, GRMZM2G011806 (Zm), GRMZM2G128602 (Zm), and Sb07g021950 (Sb). An additional embodiment provides for a transgenic plant comprising the isolated polypeptide.
According to a further embodiment, the transgenic plant may further comprise one or more defense signal peptides including TcPep1, GhPep2, SePep1, PtPep2, VvPep2, VvPep3, HaPep1, HaPep2, OsPep3b, OsPep8, SoPep1a, SoPep2a, PvPep1, OsPep3c, OsPep4b, OsPep4c, OsPep5b, OsPep5c, OsPep6b, ZmPep4b, ZmPep4c, TaPep3b, TaPep3c, HvPep1b, HvPep1c, SoPep1b, SoPep1c, SoPep1d, SoPep1e, SoPep1f, SoPep2b, SoPep2c, SoPep2d, and SoPep2e.
According to another embodiment, an isolated polynucleotide is provided that encodes a polypeptide comprising a 10 amino acid peptide motif, where the 10 amino acid peptide motif consists of a glycine residue at position C-7 of the peptide, and the polypeptide causes a change of at least 0.2 pH units at a concentration of 25 pM/ml in a plant cell suspension, where the isolated polynucleotide further comprises a promoter, where the expression of the isolated polynucleotide in a cell of a plant causes the plant to exhibit an improvement compared to a control plant lacking the polynucleotide. The improvement may be an improved yield of plant product, reduced disease symptoms, or enhanced resistance to disease infestation.
According to an additional embodiment, a composition is provided comprising one or more defense signal peptides and a biologically acceptable carrier. The one or more defense signal peptides may include TcPep1, GhPep2, SePep1, PtPep2, VvPep2, VvPep3, HaPep1, HaPep2, OsPep3b, OsPep8, SoPep1a, SoPep2a, PvPep1, OsPep3c, OsPep4b, OsPep4c, OsPep5b, OsPep5c, OsPep6b. ZmPep4b, ZmPep4c, TaPep3b, TaPep3c, HvPep1b, HvPep1c, SoPep1b, SoPep1c, SoPep1d, SoPep1e, SoPep1f, SoPep2b, SoPep2c, SoPep2d, and SoPep2e. In a further embodiment, a plant is provided where a surface of the plant has been treated with the composition. In yet a further embodiment, a seed is provided where the surface of the seed has been treated with the composition.
According to another aspect, compositions are provided that comprise one or more isolated defense signal peptides that are 10 or more amino acid residues in length and that have substantial defense signal peptide activity. Such defense signal peptides may be longer, e.g., 15, 20, 23 or more amino acid residues in length. For example, they are more easily synthesized. Accordingly, according to various embodiments, the defense signal peptide is between about 10 and about 50 amino acid residues in length, or between about 15 and about 50 amino acid residues in length. However, longer defense signal peptides may be made and used in the practice.
According to another embodiment, compositions are provided that comprise one or more polypeptides that are processed in a plant cell to produce defense signal peptides, for example, a defense signal peptide that comprises a sequence having at least 75 percent homology, or 80 percent homology, or 85 percent homology, or 90 percent homology, or complete homology with a polypeptide selected from the group consisting of TcPep1, GhPep2, SePep1, PtPep2, VvPep2, VvPep3, HaPep1, HaPep2, OsPep3b, OsPep8, SoPep1a, SoPep2a, PvPep1, OsPep3c, OsPep4b, OsPep4c, OsPep5b, OsPep5c, OsPep6b, ZmPep4b, ZmPep4c, TaPep3b, TaPep3c, HvPep1b, HvPep1c, SoPep1b, SoPep1c, SoPep1d, SoPep1e, SoPep1f, SoPep2b, SoPep2c, SoPep2d, and SoPep2e, that is, any of the plant defense peptides provided in Tables 2 and 3. Alternatively, the defense signal peptide comprises a sequence having at least 90 percent homology, or complete homology, with a dicot or monocot defense signal peptide consensus sequence, as discussed herein.
Such compositions comprising peptide or polypeptide compositions may further comprise biologically acceptable carriers and/or other substances used in formulating peptides and polypeptides. For example, such compositions may be agricultural formulations that are suitable for application to plants. Accordingly, in another embodiment, plants or seeds of plants are provided that comprise such a composition applied to a plant or seed surface, respectively. When applied to plants under suitable conditions, such compositions induce the plants' innate immunity and enhance their defense against attack by pathogens.
According to another aspect, polynucleotides that express defense signal peptides (or polypeptides, including, for example, pro-forms of defense signal peptides that are processed in plant cells to produce defense signal peptides) in plants are provided. Transgenic expression of such defense signal peptides induces the plants' innate immunity. Accordingly, one embodiment is an isolated polynucleotide comprising a sequence that encodes a defense signal peptide (as described above) operably linked to a plant promoter. Expression of the polynucleotide in a cell of a plant causes the plant to exhibit an improvement compared to a control plant lacking the polynucleotide that is selected from the group consisting of improved yield of plant product, reduced disease symptoms, and enhanced resistance to disease infestation. The encoded defense signal peptide is 10 or more, or 15 or more, or 20 or more, 23 or more amino acid residues in length. Alternatively, such a polynucleotide comprises a sequence that encodes a polypeptide that is processed in a plant cell to produce the defense signal peptide.
According to one embodiment, such polynucleotides encoding defense signal peptides have at least 80 percent, or at least 90 percent, or at least 95 percent, or complete sequence similarity to a polynucleotide sequence that encodes a plant defense signal peptide selected from the group consisting of TcPep1, GhPep2, SePep1, PtPep2, VvPep2, VvPep3, HaPep1, HaPep2, OsPep3b, OsPep8, SoPep1a, SoPep2a, PvPep1, OsPep3c, OsPep4b, OsPep4c, OsPep5b, OsPep5c, OsPep6b, ZmPep4b, ZmPep4c, TaPep3b, TaPep3c, HvPep1b, HvPep1c, SoPep1b, SoPep1c, SoPep1d, SoPep1e, SoPep1f, SoPep2b, SoPep2c, SoPep2d, and SoPep2e, that is, any of the plant defense peptides provided in Tables 2 and 3.
According to another embodiment, an isolated polynucleotide is provided that comprises a sequence that encodes a defense signal peptide operably linked to a heterologous promoter. Polynucleotides for expression in plant, bacterial, fungal (including yeast), insect, and other types of cells are contemplated. In one embodiment, expression of the polynucleotide in a cell of a plant causes the plant to exhibit an improvement compared to a control plant lacking the polynucleotide that is selected from the group consisting of improved yield of plant product, reduced disease symptoms, and enhanced resistance to disease infestation. The heterologous promoter may, for example, be a constitutive promoter or a non-constitutive promoter, including, but not limited to, an organ- or tissue-specific promoter or an inducible promoter.
According to another embodiment, cells are provided that comprise one or more of the above-mentioned polynucleotides, including, but not limited to, plant, bacterial, fungal (including yeast), and insect cells. According to another embodiment, plants that comprise such cells are provided, including, but not limited to, plants such as: acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, cantaloupe, carrot, cassava, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, clementines, clover, coconut, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon, mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticalc, turf grass, turnip, a vine, watermelon, wheat, yams, and zucchini. According to another embodiment, such a plant exhibits reduced symptoms from, or enhanced resistance to, a disease caused by an organism of a genus selected from the group consisting of Alternaria, Ascochyta, Aspergillus, Botrytis, Cercospora, Colletotrichum, Diplodia, Erwinia, Erysiphe, Fusarium, Gaeumanomyces, Helminthosporium, Macrophomina, Magnaporthe, Mycosphaerella, Nectria, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Pseudomonas, Puccinia, Puthium, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, and Xanthomonas. An embodiment further encompasses parts of such plants, including, but not limited to, seeds, seed pods, flowers, fruit, tubers, stems, cuttings, and pollen. Products resulting from processing of such plants or parts thereof are also encompassed.
Formulations of such polynucleotides are also provided. Therefore, according to another aspect, a composition is provided that comprises one or more of the above-described polynucleotides and a biologically acceptable carrier.
According to another embodiment, methods are provided for making a defense signal peptide comprising expressing in a cell a polynucleotide as described above. Included are, for example, plant cells, bacterial cells, fungal cells, and insect cells. Such methods may further comprise purifying the defense signal peptide.
According to an additional embodiment, methods are provided for making a transgenic plant, comprising introducing into a cell of a plant one or more of the above-described polynucleotides, thereby producing a transformed cell, and regenerating a transgenic plant from the transformed cell, wherein, compared to a control plant lacking the polynucleotide, the transgenic plant exhibits a characteristic selected from the group consisting of substantially improved yield of plant product, substantially reduced disease symptoms, and substantially enhanced resistance to disease infestation.
According to another embodiment, methods are provided for making a plant that comprises a transgene comprising a sequence that encodes a defense signal peptide operably linked to a plant promoter, such methods comprising sexually crossing a plant that comprises the transgene with a plant that lacks the transgene, thereby producing a plurality of progeny plants, and selecting a progeny plant comprising the transgene.
According to another embodiment, methods are provided for making a plant that comprises a transgene comprising a sequence that encodes a defense signal peptide operably linked to a plant promoter, the method comprising asexually reproducing a plant that comprises the transgene, thereby producing a plurality of progeny plants, and selecting a progeny plant comprising the transgene.
According to an additional embodiment, methods are provided for growing a plant comprising planting a seed that comprises one or more of the above-mentioned polynucleotides, and growing the seed to produce a plant, wherein, compared to a control plant lacking said polynucleotide sequence, the plant grown from the seed exhibits a characteristic selected from the group consisting of substantially improved yield of plant product, substantially reduced disease symptoms, and substantially enhanced resistance to disease infestation.
According to another embodiment, methods are provided for detecting a plant cell comprising one or more of the above-mentioned polynucleotides of the disclosure in a biological sample, the method comprising contacting the biological sample with a probe that binds specifically to the polynucleotide, and detecting said binding. One such probe is a PCR primer, in which case the method comprises performing PCR on the sample and detecting said binding by detecting an amplification product diagnostic of the presence of the polynucleotide in the sample.
According to another embodiment, kits are provided for detecting a plant cell comprising a polynucleotide in a biological sample, the kit comprising one or more probes that bind specifically to the polynucleotide, or to the defense signal peptide encoded by the polynucleotide, and instructions for use.
According to another embodiment, methods are provided for detecting a plant cell comprising a polynucleotide in a biological sample, the method comprising contacting the biological sample with a probe that binds specifically to the polynucleotide, or with a probe that binds to the defense signal peptide encoded by the polypeptide (such as, for example, an antibody probe), and detecting said binding.
According to another embodiment, plant cells are provided that comprise an insertion of a foreign promoter upstream of a coding sequence for a defense signal protein, wherein the foreign promoter is operably linked to the coding sequence for the defense signal protein and the plant is characterized by a substantially enhanced resistance to a disease compared to a control plant lacking the insertion of the foreign promoter.
According to another embodiment, methods of making a transgenic plant are provided that comprise (a) introducing into cells of a plant a polynucleotide that comprises a heterologous promoter, thereby producing a cell comprising an insertion of the heterologous promoter upstream of a coding sequence for a defense signal protein, wherein expression of the defense signal protein is controlled by the foreign promoter, and (b) regenerating a transgenic plant from said cell comprising the insertion.
According to another embodiment, methods of identifying a defense signal peptide are provided, such methods comprising: (a) providing a plurality of candidate peptides having a length of at least 10 amino acids; (b) assaying said plurality of candidate peptides for defense signal peptide activity in an alkalinization assay; and (c) selecting a candidate peptide that has substantial defense signal peptide activity. The candidate peptides may be provided for such methods by, for example, chemically synthesizing the candidate peptides. Such methods may further comprise administering the candidate peptide to a plant by applying a composition comprising the candidate peptide to the plant. Alternatively, such methods may comprise administering the candidate peptide to a plant by expressing within a cell of the plant a polynucleotide that comprises a sequence that encodes the candidate peptide, thereby producing the candidate peptide within the cell of the plant.
According to another embodiment, methods are provided for identifying a substance that enhances defense of a plant against a disease comprising (a) contacting an isolated AtPep1 receptor with a plurality of candidate substances (e.g., peptides or non-peptide compounds); (b) selecting a candidate substance that has a detectable interaction with the isolated AtPep1 receptor, and (c) applying the selected candidate substance to a plant to determine whether the selected candidate substance enhances defense of the plant against a disease.
According to another embodiment, methods are provided for conferring on a plant cell a response to a plant defense signal peptide to which the plant cell would not normally respond, the method comprising expressing in the plant cell a polynucleotide comprising a sequence encoding a receptor for the plant defense signal peptide (including, for example, AtPepR1 or AtPepR2) operably linked to a promoter that is expressible in the plant cell. The plant cell response could include, for example, alkalinization of the plant cell in response to administration of the plant defense signal peptide, transcription of pathogen defense genes, enhanced resistance to a pathogen, etc.
According to another embodiment, plant cells are provided that comprise a polynucleotide comprising a sequence encoding a receptor for the plant defense signal peptide operably linked to a promoter that is expressible in the plant cell, wherein expression of the polynucleotide confers on the plant cell a response to a plant defense signal peptide to which the plant cell would otherwise be unresponsive.
The foregoing and other aspects of the preferred embodiments will become more apparent from the following detailed description, accompanying drawings, and the claims.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A-T provides the nucleotide and deduced amino acid sequences for the precursor protein for AtPep1, Atpropep1, and a number of paralogs and orthologs. Sequences that correspond to AtPep1 are underlined.
(a) AtproPep1 nucleotide (SEQ ID NO: 81) and amino acid (SEQ ID NO: 82) sequences (Locus tag At5g64900; Gene: GenBank GeneID: 836613, Arabidopsis thaliana Chromosome V [GenBank ID#AB019236], region 25954396-25955302; mRNA: NCBI RefSeq ID#NM—125888-499 bp; Protein: NCBI RefSeq ID#NP—569001-92 aa)
Paralogs (Arabidopsis thaliana):
(b) AtproPep3 nucleotide (SEQ ID NO: 83) and amino acid (SEQ ID NO: 84) sequences (Locus tag At5g64890; Gene: GenBank GeneID: 836612, Arabidopsis thaliana Chromosome V [GenBank ID#AB019236], region 25951798-25952735; mRNA: NCBI RefSeq ID#NM—125887-568 bp; Protein: NCBI RefSeq ID#NP—569000-109 aa)
(c) AtproPep4 nucleotide (SEQ ID NO: 85) and amino acid (SEQ ID NO: 86) sequences (Locus tag At5g64905; Gene: GenBank GeneID: 836614, Arabidopsis thaliana Chromosome V [GenBank ID#AB019236], region 25956800-25957285; mRNA: NCBI RefSeq ID#NM—125889-486 bp; Protein: NCBI RefSeq ID#NP—569002-96 aa)
(d) AtproPep5 nucleotide (SEQ ID NO: 87) and amino acid (SEQ ID NO: 88) sequences (Locus tag At5g09980; Gene: GenBank GeneID: 830859, Arabidopsis thaliana Chromosome V [GenBank ID#AB019236], region 3122757-3123909; mRNA: NCBI RefSeq ID#NM—121035-460 bp; Protein: NCBI RefSeq ID#NP 568223-81 aa)
(e) AtproPep2 nucleotide (SEQ ID NO: 89) and amino acid (SEQ ID NO: 90) sequences (Locus tag At5g09990; Gene: GenBank GeneID: 830860, Arabidopsis thaliana Chromosome V [GenBank ID#AB019236], region 3124569-3125073; mRNA: NCBI RefSeq ID#NM—121036-412 bp; Protein: NCBI RefSeq ID#NP—568224-86 aa)
(f) AtproPep6 nucleotide (SEQ ID NO: 91) and amino acid (SEQ ID NO: 92) sequences (Locus tag At2g22000; Gene: GenBank GeneID: 816736, Arabidopsis thaliana Chromosome II [GenBank ID#AC007019], region 9369406-9370082; mRNA: NCBI RefSeq ID#NM—127769-397 bp; Protein: NCBI RefSeq ID#NP—179791-104 aa)
(g) AtproPep7 nucleotide (SEQ ID NO: 93) and amino acid (SEQ ID NO: 94) sequences (Unannotated AtproPep; Gene: No GenBank GeneID, located on Arabidopsis thaliana Chromosome V [GenBank ID#AB019236], region 3121350-3121577; mRNA: No mRNA predicted, therefore no ID#; a 228 bp open reading frame (ORF) is encoded in genomic DNA as shown; Protein: Not predicted, therefore no ID #, Translation of ORF encoded on chromosome V yields the 75 as sequence shown.)
Orthologs:
(h) BnproPep1 from canola (Brassica napus) (SEQ ID NO: 95; GenBank ID#CD816645, Protein: 95 aa)
(i) StproPep1 from potato (Solanum tuberosum) (SEQ ID NO: 96; GenBank ID#CV505388, Protein: 116 aa)
(j) PbproPep1 from poplar (Populus balsamifera) (SEQ ID NO: 97; GenBank ID#CV230975, Protein: 121 aa)
(k) BeproPep1 from birch (SEQ ID NO: 98; GenBank ID#CD276952, Protein: 110 aa)
(l) GmproPep1 from soybean (Glycine max) (SEQ ID NO: 99; GenBank ID#CD401281, Protein: 115 aa)
(m) MsproPep1 from alfalfa (Medicago saliva) (SEQ ID NO: 100; GenBank ID#BI311441, Protein: 127 aa)
(n) VvproPep1 from grape (Vitis vinifera) (SEQ ID NO: 101; GenBank ID#CF604664, Protein: 83 aa)
(o) OsproPep1 from rice (Oryza saliva) (SEQ ID NO: 102; GenBank ID#CF333408; Locus tag:Os04g54590, Protein 154 aa)
(p) OsproPep2 from rice (Oryza saliva) (SEQ ID NO: 103; GenBank ID#AK111113; Locus tag:Os08g07600, Protein: 93 aa)
(q) TaproPep1 from wheat (Triticum aestivum) (SEQ ID NO: 104; GenBank ID#AL809059, Protein: 82 aa)
(r) TaproPep2 from wheat (Triticum aestivum) (SEQ ID NO: 105; GenBank ID#BF201609, Protein: 75 aa)
(s) ZmproPep1 from maize (Zea mays) (SEQ ID NO: 106; GenBank ID#DN214793, Protein: 142 aa)

    
    
        (t) HvproPep1 from barley (Hordeum vulgare) (SEQ ID NO: 107; GenBank ID#BQ763246, Protein: 93 aa)
    
    


FIG. 2A-D shows the sequence of an AtPep1 receptor gene (SEQ ID NO: 108; At1g73080), designated as AtPEPR1, and its deduced amino acid sequence (SEQ ID NO: 109; Also noted are several features of the receptor polypeptide: a signal sequence (residues 1-24); cysteine pairs (residues 64 and 71; and residues 836 and 854); leucine-rich repeats (residues 76-827); transmembrane domain (residues 870-892); kinase domain (residues 927-1208); and an intron (between residues 1099 and 1100).
FIG. 3A-D shows the structure of a second AtPep1 receptor gene (SEQ ID NO: 110; At1g17750) designated as AtPEPR2, and its deduced amino acid sequence (SEQ ID NO: 111). Also noted are several features of the receptor polypeptide: a signal sequence (residues 1-26); cysteine pairs (residues 62 and 71; and residues 709 and 727); leucine-rich repeats (residues 99-697); transmembrane domain (residues 738-760); kinase domain (residues 793-1079); and an intron (between residues 966 and 967).
FIG. 4 shows the concentration dependence of synthetic AtPep peptides deduced from the seven members of the AtproPep1 gene family in the alkalinization assay. Peptide concentrations (left to right for each peptide): 0.25 nM, 2.5 nM, 25 nM, 250 nM.
FIG. 5 shows activity of synthetic AtPep1 peptides from the C-terminus of AtPep1 in the alkalinization assay, from a 9-mer (SSGRPGQHN (SEQ ID NO: 75)) to a full-length 23-mer. The 15-mer (RGKEKVSSGRPGQHN (SEQ ID NO: 78)) is also shown. Ten microliter aliquots of each peptide solution were tested for activity at 0.25 nM (gray), 2.5 nM (dotted), and 25 nM (black).
FIG. 6 shows the activity of single alanine amino acid substitutions at every position in the 15-mer peptide at the carboxy terminus of AtPep1 (RGKEKVSSGRPGQHN (SEQ ID NO: 78)) in the alkalinization assay. The set of substituted 15-mer peptides was assayed using four-day-old Arabidopsis cells. Ten ml of each peptide (2.5 pmoles) was added to 1 ml of cells to make a final concentration of 2.5 nM. After 20 min, the pH of the media was recorded. The data is the average of three separate experiments.
FIG. 7A-F shows nucleotide sequence information for precursors of the novel plant signal defense peptides of Table 2: OsPep8 nucleotide (SEQ ID NO: 112) and amino acid (SEQ ID NO: 113) sequences; TcPep1 nucleotide (SEQ ID NO: 114) and amino acid (SEQ ID NO: 115) sequences; GhPep2 nucleotide (SEQ ID NO: 116) and amino acid (SEQ ID NO: 117) sequences; PtPep2 nucleotide (SEQ ID NO: 118) and amino acid (SEQ ID NO: 119) sequences; VvPep2 nucleotide (SEQ ID NO: 120) and amino acid (SEQ ID NO: 121) sequences; VvPep3 nucleotide (SEQ ID NO: 122) and amino acid (SEQ ID NO: 123) sequences; HaPep1 nucleotide (SEQ ID NO: 124) and amino acid (SEQ ID NO: 125) sequences; HaPep2 nucleotide (SEQ ID NO: 126) and amino acid (SEQ ID NO: 127) sequences; SoPep1a nucleotide (SEQ ID NO: 128) and amino acid (SEQ ID NO: 129) sequences; SoPep2a nucleotide (SEQ ID NO: 130) and amino acid (SEQ ID NO: 131) sequences; PvPep1 nucleotide (SEQ ID NO: 132) and amino acid (SEQ ID NO: 133) sequences. A (*) denotes a stop codon.
FIG. 8 shows representative monocot propeptide sequences containing multiple peptides. Nucleotide sequence information for precursors of the novel plant defense signal peptides of Table 3 are shown: Barley (SEQ ID NO: 134); Sugarcane1 (SEQ ID NO: 135); Sugarcane2 (SEQ ID NO: 136). Multiple peptides from the same precursor are named using letters starting from the carboxyl end of the precursor as shown.
FIG. 9 shows (A) an amino acid sequence alignment of predicted proAtPep1 orthologs (ProAtPep1 (SEQ ID NO: 82); ProAtPep2 (SEQ ID NO: 90); At2g22000 (SEQ ID NO: 137); Canola (SEQ ID NO: 138); Potato (SEQ ID NO: 139); Rice (SEQ ID NO: 140); Poplar (SEQ ID NO: 141); Medicago (SEQ ID NO: 142); Soybean (SEQ ID NO: 143) and (B) a gene domain model for proATPep1-like genes.
FIG. 10 shows a phylogenic tree of AtPEPRs homologues.
FIGS. 11A(1-4) and 11B(1-19) show amino acid alignment of AtPEPR homologues (A) and the DNA and amino acid sequences of identified AtPEPR homologues (B).
FIG. 12 shows a consensus amino acid sequence (SEQ ID NO: 172) of the protein kinase domain of AtPEPRs homologues.



DETAILED DESCRIPTION
We have identified defense signal peptides from Arabidopsis and a variety of other plants, including crop plants of commercial importance. In addition, we have identified genes that encode polypeptides, which are processed in plant cells to produce the shorter peptides, as well as receptors for the peptides. Transgenic plants in which these defense signal peptides are expressed under the control of a heterologous promoter, such as a constitutive promoter, exhibit improved yield of plant product, reduced disease symptoms, and/or enhanced resistance to disease infestation. Peptides that comprise as few as 10 amino acid residues from the carboxy-terminus of AtPep1, a 23 amino acid defense signal peptide from Arabidopsis thaliana, retain significant activity. In addition, individual amino acid residues that are important for activity have been defined by amino acid substitutions. Peptides and other substances that have defense signal peptide activity can be readily screened using an alkalinization assay that is described herein, and their identity can be confirmed by exogenous application to plants or transgenic expression in plants.
We have isolated novel defense peptides AtPep1 and AtPep2 from Arabidopsis. This is the first demonstration of the involvement of a plant-derived peptide signal in defense against pathogens. The proAtPep1 and 2 precursor genes have been identified and belong to a seven-member gene family. The DNA sequence of the gene from Arabidopsis thaliana that encodes AtproPep1 and the deduced amino acid sequence of AtproPep1 are provided in FIG. 1. Orthologs have been identified in such dicots as canola, potato, poplar, alfalfa, soybean, grape, and tomato, and in such monocots as rice, wheat, maize, and barley, and are likely commonly found across the plant kingdom. The sequences of several paralogs and orthologs of AtPep1 are also provided in FIG. 1.
The rapid, sensitive alkalinization assay that was used to identify and purify the AtPep1 and 2 peptides is useful for identifying defense signal peptides from any plant whose signaling pathways result from peptide-receptor interactions that initiate intracellular signaling through MAP kinases and proton pumps in the plasma membrane. Within minutes after adding systemin to cells, an ATP-driven proton pump is inhibited, causing the extracellular medium of the cells to become alkaline. When aliquots (e.g., 1-10 μL) from fractions from plant tissues that eluted from HPLC columns were added to 1 mL of suspension cultured plant cells grown at low pH (e.g., pH 5), some fractions caused the cell medium to increase in pH. The identification of a peptide as a defense signal peptide is confirmed by application of the peptide (e.g., as a plant fraction or isolated peptide) to plants or by expression of a transgene encoding the peptide, including, but not limited to, the gene encoding a pro-form of the peptide (such as, for example, AtproPep1, the pro-form of AtPep1) in plants and observation of detectable defense signal peptide activity, such as, for example, enhanced disease resistance.
We established a suspension cultured Arabidopsis cell line to be used in the alkalinization assay to seek novel peptides in Arabidopsis leaf extracts. Cells are grown unbuffered near pH 5 or less in order to record an alkalinization of about 1 pH unit in response to peptide signals. In order to establish an Arabidopsis suspension cell culture for use in the alkalinization assay, Arabidopsis cells were regularly transferred and maintained in the growth chamber room for several months, when they equilibrate at a pH of about 5.0 during exponential growth. Cell cultures that grow at low pH from several other plant species, including tomato (Lycopersicon esculentum), tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), maize (Zea mays), petunia (Petunia hybrida), nightshade (Solanum nigrunt), and sweet potato (Ipomoea batatus) have been developed for use in the alkalinization assay, and developing similar cultures from other plants is readily accomplished.
For the work described in Example 1, we used a typical purified peptide fraction from Arabidopsis leaves. Kilogram quantities of leaf material were extracted and peptides were separated on an HPLC column. A 10 microliter (μL) aliquot from each fraction eluting from the column was assayed with the alkalinization assay using suspension cultured Arabidopsis cells. Two novel peaks were identified that were called AtPep1 and AtPep2. The peptides were purified through several additional column separations until homogeneous, as verified by MALDI-MS and amino-terminal sequencing. The peptides were each 23 amino acids in length and the amino acids sequences of the two were identical at 10 residues. Neither peptide was post-translationally modified. The two peptides were chemically synthesized and, in alkalinization assays exhibited identical activities as the native peptides, in the sub-nanomolar concentration range. Searches of protein data bases revealed that the peptides were derived from the C-terminus of two members of a seven-member gene family. The deduced precursors were from 92 to more than 100 amino acids in length and did not have leader sequences at their N-termini.
These properties are similar to tomato systemin (Pearce et al., Science 253:895-897, 1991). Other similarities between proAtPep1 prosystemin were the absence of a leader sequence in the precursors, the low nanomolar concentrations needed to activate the alkalinization response, the processing of the peptides from the C-termini of their precursor proteins, and the presence of KEK motifs in the precursors that are commonly found in proteins that are involved in protein-protein interactions. The expression of the proAtPep1 gene in excised Arabidopsis leaves was induced by AtPep1, similar to the expression of the prosystemin gene in tomato plants being induced by systemin.
The tissue-specific expression of the proAtPep1 gene was analyzed using RT-PCR analyses. All tissues of the plant expressed the gene at low levels, which did not reveal a clue as to its function. To assess whether stresses to Arabidopsis plants might affect AtPep1 gene expression, the plants were subjected to cold and drought stresses and treatments with abscissic acid (ABA), methyl jasmonate (MeJA), methyl salicylate (MeSA), UV-B, and wounding. Only MeJA and wounding induced a strong expression of the gene. These results indicated that the gene was behaving in Arabidopsis in a similar manner as systemin in tomato plants (Ryan et al., Plant Cell. 14:251-264, 2002) and suggested that the peptide may be a defense signaling peptide.
Pathogen defense is well characterized in Arabidopsis, where two defense pathways have been identified in which jasmonate is a signaling component (Lorenzo and Solano, Curr. Opin. Plant Biol 8:532-540, 2005; Lorenzo et al., Plant Cell 16:1938-1950, 2004.). In one pathway, wounding and jasmonate activate defensive genes through the octadecanoid pathway, with COI1 and AMYC2 playing major roles in transcription of defensive genes that includes LOX2 and VSP2 (Lorenzo et al., Plant Cell 16:1938-1950, 2004). In the second pathway jasmonate, in concert with ethylene, activates PDF1.2, and several PR proteins, with active oxygen playing a key signaling role (Lorenzo et al., Plant Cell 15:165-178, 2003; Penninckx et al., Plant Cell 10:2103-2113, 1998; Penninckx et al., Plant Cell 8:2309-2323, 1996; Coego et al., Plant Cell 17:2123-2137, 2005; Mackerness et al., Plant Cell Environ. 22:1413-1423, 1999). To assess the possible involvement of AtPep1 with the known pathways, Arabidopsis plants were excised and supplied with the AtPep1 peptide at 10 nM concentrations, and several known wound-inducible genes and pathogen defense genes were assayed for expression levels two hours later. Only the pathogen defense genes were induced, with PDF2.2 and PR-1 being most strongly expressed, with PR-3, PR-4, and TAT expressed at lower levels. LOX2 and VSP2 genes were not induced by the peptide.
Arabidopsis plants were transformed with a 35S-AtPep1 fused gene, and many stable transformants that strongly expressed the gene were recovered. The overexpression of the gene did not visibly affect the growth of the transgenic plants compared to wild type plants. The progeny of a stable transformant that strongly expressed the gene was analyzed to determine if the overexpression of the gene would affect the expression of defense genes. Plants constitutively overexpressing the proAtPep1 gene also constitutively overexpressed the AtPep1-inducible genes.
To determine if the transgenic plants were more resistant to a pathogen, the soil of plants in which the transgenic line was grown was inoculated with the root pathogen Pythium irregulare and the plants were monitored with time to assess pathogenicity. The aerial parts of the transgenic plants infected with Pythium were visibly more robust than infected wild type plants. However, the roots of the infected transgenic plants were much denser and healthier than roots of infected wild type plants. The transgenic plants were growing almost as well as uninfected wild type plants. These experiments indicated that overexpression of the gene was enhancing resistance to the root pathogen.
Using photoaffinity labeling of AtPep1, a high affinity binding protein for the peptide was identified on the surface of Arabidopsis suspension cultured cells by photoaffinity labeling. The protein was purified to homogeneity. The binding of the photoaffinity label to the receptor is strongly competed by unlabeled AtPep1, but not by tomato systemin. The binding of AtPep1 is powerfully competed by suramin, a potent inhibitor of many ligand-receptor interactions, including the binding of systemin with its receptor. Amino acid sequence analysis has shown the protein to be a leucine-rich repeat (LRR) receptor kinase. The gene (At1G73080) contains 27 LRR motifs, a 23 amino acid membrane-spanning region, and an intracellular protein kinase domain. The protein is glycosylated, as evidenced by the loss of about 20% of its mass by enzymatic deglycosylation. An ortholog (At1G17750) is present in Arabidopsis, and LRR receptor kinase orthologs from rice and morning glory have been reported in GenBank that share a high percentage of amino acid identity with the AtPep1 receptor. The paralog in Arabidopsis shares more than 90% amino acid identity with AtPep1, but it has only 24 LRRs and does not have either a transmembrane domain or a kinase domain. The DNA and deduced amino acid sequences for the AtPep1 receptors are provided in FIG. 2 (Ag1g73080) and FIG. 3 (At1g17750).
The AtPep1 peptide, like systemin, is apparently a cytosol-derived peptide that involves the jasmonate/ethylene signaling pathway. How the peptide is processed from a precursor and arrives at the cell surface to activate a signaling pathway is unknown, but, like systemin, transport of either the precursor or the processed peptide to the cell surface may occur in order for the peptide to react with its receptor.
AtPep1 is a component of the defense signaling of Arabidopsis plants and is therefore the first plant peptide hormone to be associated with a known pathogen defense pathway in any plant.
Table 1 shows examples of the C-terminal sequences of paralogs and orthologs of AtproPep1 that we have identified in a wide variety of plants. In addition to the examples listed in Table 1, for example, two orthologs of the AtPep1 precursor gene (called preproLePep1) have been identified in tomato plants. The tomato cDNA has been isolated and shown to code for an AtPep1-like defense peptide. Other paralogs and orthologs of AtproPep1 in these and other plant species may be found by amino acid sequence homology. Additional defense signal peptides may be identified by screening plants for peptides having defense peptide activity, as described herein.







TABLE 1







AtPep1, Paralogs, Orthologs, and Dicot and Monocot Consensus Sequences










Peptide
Source
Alignment of C-terminal Sequences
SEQ ID NO:













AtPep1
Arabidopsis thaliana




1


Paralogs





AtPep2
Arabidopsis
-SLNVM RKGIR KQPVS SGKRG GVN
2



thaliana




AtPep3
Arabidopsis
-DNKAK SKKRD KEKPS SGRPG QTN
3



thaliana




AtPep4
Arabidopsis
-EIKAR GKNKT KPTPS SGKGG KHN
4



thaliana




AtPep5
Arabidopsis
-GLPGK KNVLK KSRES SGKPG GTN
5



thaliana




AtPep6
Arabidopsis
-ITAVL RRRPR PPPYS SGRPG QNN
6



thaliana




AtPep7
Arabidopsis
-VSGNV AARKG KQQTS SGKGG GTN
7



thaliana




Orthologs





BnPep1
Canola
-VARLT RRRPR PP-YS SGQPG QIN
8



(Brassica





napus)




StPep1
Potato
-PTERR GRPPS RPKVG SGPPP QNN
9



(Solanum





tuberosum)




PbPep1
Poplar
-DAAVS ALARR TPPVS RGGGG QTN
10



(Papulus





balsamifera)




BePep1
Birch
-DLVMA VNAPP RPSLT PGSGA QIN
11



(Betula spp.)




GmPep1
Soybean
-ASLMA TRGSR GSKIS DGSGP QHN
12



(Glycine





max)




MsPep1
Alfalfa
-LSSMG RGGPR RTPLT QGPPP QHN
13



(Medicago





saliva)




VvPep1
Grape (Vitis
-EKVRE KQKKG EDGES VGR PG KKN
14



vinifera)




Dicot consensus sequence





15


OsPep1
Rice (Oryza
-ARLRP KPPGN PREGS GGNGG HHH
16



sativa)




OsPep2
Rice (Oryza
-DDSKP TRPGA PAEGS GGNGG AIH
17



sativa)




TaPep1
Wheat
-AVRRP RPPTT PREGR GGGGG SHN
18



(Triticum





aestivum)




TaPep2
Wheat
-AAPAP QRPGA PAEGA GGQGG IIH
19



(Triticum





aestivum)




ZmPep1
Maize (Zea
-VRRRP TTPGR PREGS GGNGG NHH
20



mays)




HvPep1
Barley
-QLARP RPPGP PRQGH GGDGG AIH
21



(Hordeum





vulgare)




Monocot consensus sequence





22









Among the native defense signal peptides identified so far, the shortest deduced peptide sequence is 23 amino acids in length (for example, AtPep1) and the longest is 36 amino acids (AtPep3).

The dicot and monocot disease signal peptides in particular have a substantial degree of homology in the C-terminal region of the peptides, allowing us to define consensus sequences as shown in Table 1, which shows C-terminal alignments for AtPep paralogs and orthologs that correspond to the sequence of AtPep1 and dicot and monocot consensus sequences for defense signal peptides.
According to one aspect, defense signal peptides are provided that comprise a dicot or monocot consensus sequence or have a high degree of amino acid sequence homology to such a consensus sequence, e.g., 75 percent homology, or 80 percent homology, or 90 percent homology. In one embodiment, such defense signal peptides are 10 amino acid residues in length or longer and have defense signal peptide activity.
Analogs of AtPep1 were synthesized and assayed in the alkalinization assay. An analog of AtPep1 missing the carboxy-terminal amino acid was completely inactive, whereas deletions from the amino-terminus of the peptide resulted in a sequential reduction in activity, until peptides with 9 carboxy-terminal amino acids remaining (SSGRPGQHN (SEQ ID NO: 75)) were inactive. A peptide consisting of only the 15 C-terminal amino acids was nearly as active as the native peptide (at approximately 2.5 nM), but analogs of 14 amino acids and smaller were progressively less active.
AtPep1 is 23 amino acid residues in length. However, truncated forms of AtPep1 of 10 amino acid residues from the C-terminus of AtPep1 retain activity, and peptides having 15 residues retain substantially full defense signal peptide activity (Example 3). As a result of the foregoing studies of shorter defense signal peptides, according to another embodiment, defense signal peptides comprising at least 10 amino acid residues, particularly those including sequences from the C-terminus of native defense signal peptides or including consensus dicot or monocot sequences are provided. Such shorter peptides may be 11 or more, 12 or more, or 15 or more amino acid residues in length, provided that they retain substantial defense signal peptide activity.
The 15-mer was substituted with alanine in each position to assess which amino acids were necessary for the alkalinating activity. A Ser to Ala substitution at position 7, counting from the amino-terminus of the 15-mer, and a Gly to Ala substitution at position 9 exhibited little activity. Computer modeling predicted that these two amino acids would be involved in a hairpin-turn within the peptide region of -SSGR- (compare with residues 15-18 of the sequence of AtPep1 shown in Table 1). Substituting Ala for Ser (-ASGR-) abolished the predicted turn and severely reduced activity (half-maximal activity at approximately 25 nM), while substituting Ala for Gly was even less active (half-maximal activity of >250 nM). However, neither of these analogs were able to compete with the non-substituted 15-mer for receptor binding, indicating that the structural changes in this region may have severely modified the conformation without competing for the receptor binding site. Other Ala substitutions had no effect on activity. These results will guide the skilled artisan in making desired substitutions in other defense signal peptides.
Additional Plant Signal Defense Peptides
In addition to the plant signal defense peptides that are shown in Table 1 above, we have discovered additional plant signal defense peptides from a number of dicot and monocot plant species. These include the AtPep1 orthologs shown in







TABLE 2







AtPep1 and Additional Dicot and Monocot Orthologs










Peptide
Source
Alignment of C-terminal Sequences
SEQ ID NO:





AtPep1
Arabidopsis thaliana




23


Orthologs





TcPep1
Alpine
-VTKFK AKMKE REKVS TGRSG QHN
24



pennycress





(Thlaspi caerulescens)




GhPep2
Cotton
-LPMVS LFTPK RPGTS AGSGP QIN
25



(Gossypium hirsutum)




SePep1
Tomato
-ATDRR GRPPS RPKVG SGPPP QNN
26



(Solanum lycopersicum)




PtPep2
Poplar
-AVTVS ALARR TPPVS SGSGG QIN
27



(Populus balsamifera)




VvPep2
Grape (Vitis vinifera)
-LRRGP TRPPI SFESR PGGGS QIN
28


VvPep3
Grape (Vitis vinifera)
-KMSSD FRQPP RPPID PGQGG QIN
29


HaPep1
Sunflower
-RGLTR RPPPP RGPIS SGGGG QTN
30



(Helianthus annuus)




HaPep2
Sunflower
-RVNLV GYDYS GYGQS TGKPS ECN
31



(Helianthus annuus)




OsPep8
Rice (Oryza sativa)
-QRNEI RSRGV DPSVS GGKQP GIN
32


SoPep1a
Sugarcane
-ASVLM RGPAQ PVPPT EGAXG RGG
33



(Saccharum officinarum)




SoPep2a
Sugarcane
-ASVLM RGPAQ PGPPT EGAGR RGG
34



(Saccharum officinarum)




PvPep1
Switchgrass
-VVTRV WAVRR PREGS GGNGG VHH
35



(Panicum virgatum)









FIG. 7 shows nucleotide sequence information for precursors of the novel plant signal defense peptides of Table 2.
We have also identified additional plant signal defense peptides in precursor nucleotide sequences in which only one plant signal defense peptide had been identified previously. These are shown in Table 3.







TABLE 3







AtPep1 and Additional Monocot Orthologs from Precursor Genes


that Encode Multiple Peptides










Peptide
Source
Alignment of C-terminal Sequences
SEQ ID NO:





AtPep1
Arabidopsis thaliana




36


Orthologs





OsPep3b
Rice (Oryza
-RRPTP PGGAG PREGS GGRGG VIH
37



sativa)




OsPep3c
Rice (Oryza
-SLAGA NVLVR DAPPE TGGGP HHN
38



sativa)




OsPep4b
Rice (Oryza
-RRPTP PGGAG PREGS GGRGG VIH
39



sativa)




OsPep4c
Rice (Oryza
-LAGAN VLLRD DAPPE GGRGP HHN
40



sativa)




OsPep5b
Rice (Oryza
-RRPTP PGGAG PREGR GGRGG VIH
41



sativa)




OsPep5c
Rice (Oryza
-QLAGA KVLVR DAPPE TGGGP HHN
42



sativa)




OsPep6b
Rice (Oryza
-GGVRP TPPGN PREAQ KGGGV IHA
43



sativa)




ZmPep4b
Maize (Zea
-ALRGP APPAR PKEGS GGKVH VVS
44



mays)




ZmPep4c
Maize (Zea
-LWPAP SPKGR PGAPR QGSGG QVH
45



mays)




TaPep3b
Wheat
-DASSL APQLR RTSPG EGTSG RIH
46



(Triticum





aestivum)




TaPep3c
Wheat
-IAPTL QPSSA PVEGT GGQVM VLN
47



(Triticum





aestivum)




HvPep1b
Barley
-DASSL PLQLM RTPPG EGAGG RIH
48



(Hordeum





vulgare)




HyPep1c
Barley
-SVLPD QPPSA PAEGT GGQVM VLN
49



(Hordeum





vulgare)




SoPep1b
Sugarcane
-ASVLL RGPAP PGRPV EGSGG KVH
50



(Saccharum





officinarum)




SoPep1c
Sugarcane
-AHMVI RGPAR PGLPA QGSGG KVH
51



(Saccharum





officinarum)




SoPep1d
Sugarcane
-MATPM RRPTP PGPPA QGSGG KTN
52



(Saccharum





officinarum)




SoPep1e
Sugarcane
-SRAAP SPKGS PGAPR QGSGG HVH
53



(Saccharum





officinarum)




SoPep1f
Sugarcane
-APASP LRRQL LRYVS SGLVA ALH
54



(Saccharum





officinarum)




SoPep2b
Sugarcane
-AHMVI RGPAR PGLPA QGRGG KVH
55



(Saccharum





officinarum)




SoPep2c
Sugarcane
-MATPM RRPTS PGPPA QGSGG KTN
56



(Saccharum





officinarum)




SoPep2d
Sugarcane
-SRAVP SLKGR PGAPR QGSGG HVH
57



(Saccharum





officinarum)




SoPep2e
Sugarcane
-APASP LRRQL LRYVS SGLVA ALH
58



(Saccharum





officinarum)









FIG. 8 shows nucleotide sequence information for precursors of the novel plant defense signal peptides of Table 3.
DEFINITIONS AND METHODS
The following definitions and methods are provided to better define the preferred embodiments and to guide those of ordinary skill in the art in the practice of the disclosed embodiments. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may also be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. The nomenclature for DNA bases as set forth at 37 CFR 1.822 is used. The standard one- and three-letter nomenclature for amino acid residues is used.
Polynucleotides
“Polynucleotide.” The term “polynucleotide” refers to a polymer of nucleotide monomers, including but not limited to ribonucleotides or deoxyribonucleotides or nucleotide analogues. Polynucleotides include, for example, DNA and RNA molecules, including cDNA, genomic DNA, primers, probes, vectors, and so on, and include single- and double-stranded forms thereof. Polynucleotides may be chemically modified by well known methods by labeling, coupling to solid supports, etc.
“Defense signal peptide (or polypeptide)polynucleotide”. The term “defense signal peptide polynucleotide” refers to a polynucleotide that encodes a defense signal peptide, and a “defense signal polypeptide polynucleotide” refers to a polynucleotide that encodes a defense signal polypeptide (i.e., a polypeptide that, when processed in a plant cell, produces a defense signal peptide), whether a cDNA or genomic sequence or synthetic form thereof. Such polynucleotides may comprise wild type polynucleotides sequences encoding defense signal polypeptides, such as those listed in Table 1, operably linked to a heterologous promoter, i.e., a promoter not associated in nature with such native, or wild type, polynucleotide sequences. Alternatively, such polynucleotides may comprise non-naturally occurring recombinant polynucleotides that comprise a sequence that encodes a defense signal peptide operably linked to a suitable promoter. For expression of defense signal peptides for exogenous application to plants, a defense signal peptide polynucleotide may be operably linked to a promoter suitable for expression in a bacterial, fungal, insect, or other suitable cell. For transformation of plants or plant cells or tissues, a defense signal peptide may be operably linked to a promoter suitable for expression in a plant cell, i.e., a plant promoter. According to another embodiment, a heterologous promoter may be introduced into a plant or a plant cell or tissue for insertion into the genome, thereby producing an insertion of the promoter upstream of a sequence that encodes a defense signal peptide, operably linking the sequence encoding the defense signal peptide to the heterologous promoter. Such an expression unit, including the heterologous promoter and the sequence encoding the defense control peptide, is another embodiment of a defense signal peptide (or polypeptide) polynucleotide.
“Native.” The term “native” refers to a naturally-occurring (“wild type”) polynucleotide, polypeptide or peptide.
“Isolated.” An “isolated” polynucleotide is one that has been substantially separated or purified away from other polynucleotide sequences in the cell of the organism in which the polynucleotide naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, by conventional purification methods. The term also embraces recombinant polynucleotides (including promoter insertions operably linked to a defense signal peptide gene) and chemically synthesized polynucleotides.
“Heterologous.” A heterologous polynucleotide is one that is not normally present in a particular context. For example, with reference to a cell, tissue or organism, heterologous polynucleotide sequence is one that is not found in such a cell, tissue or organism in nature unless introduced into such cell, tissue or organism. As another example, a heterologous promoter is a promoter not associated in nature with a particular protein coding sequence.
Fragments, Probes, and Primers
A fragment of a polynucleotide is a portion of a polynucleotide that is less than full-length and comprises at least a minimum length capable of hybridizing specifically with a native polynucleotide sequence under stringent hybridization conditions. The length of such a fragment is preferably at least 15 nucleotides, more preferably at least 20 nucleotides, and most preferably at least 30 nucleotides of a native polynucleotide sequence.
A “probe” is an isolated polynucleotide to which is attached a conventional detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme. “Primers” are isolated polynucleotides that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase. Primer pairs can be used for amplification of a polynucleotide sequence, e.g., by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods.
Probes and primers are generally 15 nucleotides or more in length, preferably 20 nucleotides or more, more preferably 25 nucleotides, and most preferably 30 nucleotides or more. Such probes and primers hybridize specifically to the target polynucleotide sequence under high stringency hybridization conditions under at least moderately stringent conditions.
Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates) (hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR-primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such-as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).
Primers and probes based on the native defense signal polypeptide polynucleotide sequences that are disclosed herein can be used to confirm (and, if necessary, to correct) the disclosed polynucleotide sequences by conventional methods, e.g., by re-cloning and re-sequencing.
“Substantial similarity.” A first polynucleotide is “substantially similar” to a second polynucleotide if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other polynucleotide (or its complementary strand), there is at least about 75% nucleotide sequence identity, preferably at least about 80% identity, more preferably at least about 85% identity, and most preferably at least about 90% identity. Sequence similarity can be determined by comparing the nucleotide sequences of two polynucleotides using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wis.
Alternatively, two polynucleotides are substantially similar if they hybridize under stringent conditions.
“Operably Linked.” A first nucleic-acid sequence is “operably linked” with a second nucleic-acid sequence when the first nucleic-acid sequence is placed in a functional relationship with the second nucleic-acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
“Recombinant.” A “recombinant” polynucleotide is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques. Techniques for nucleic-acid manipulation are well-known (see, e.g., Sambrook et al., 1989, and Ausubel et al., 1992). Methods for chemical synthesis of polynucleotides are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of polynucleotides can be performed, for example, on commercial automated oligonucleotide synthesizers.
Preparation of Recombinant or Chemically Synthesized Polynucleotides: Vectors, Transformation, Host Cells
Natural or synthetic polynucleotides according to the present disclosure can be incorporated into recombinant nucleic-acid constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct preferably is a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
For the practice of the present embodiments, conventional compositions and methods for preparing and using vectors and host cells are employed, as discussed, inter alia, in Sambrook et al., 1989, or Ausubel et al., 1992.
A cell, tissue, organ, or organism into which has been introduced a foreign polynucleotide, such as a recombinant vector, is considered “transformed”, “transfected”, or “transgenic.” A “transgenic” or “transformed” cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing such a “transgenic” plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a recombinant polynucleotide construct.
A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987); Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
Examples of constitutive plant promoters include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odel et al., Nature 313:810, 1985), including monocots (see, e.g., Dekeyser et al., Plant Cell 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet. 220:389, 1990); the nopaline synthase promoter (An et al., Plant Physiol. 88:547, 1988) and the octopine synthase promoter (Fromm et al., Plant Cell 1:977, 1989).
A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals, also can be used for expression of defense signal peptides in plant cells, including promoters regulated by (1) heat (Callis et al., Plant Physiol. 88:965, 1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al., Plant Cell 1:471, 1989; maize rbcS promoter, Schaffner and Sheen, Plant Cell 3:997, 1991; or chlorophyll a/b-binding protein promoter, Simpson et al., EMBO J. 4:2723, 1985), (3) hormones, such as abscisic acid (Marcotte et al., Plant Cell 1:969, 1989), (4) wounding (e.g., wunl, Siebertz et al., Plant Cell 1:961, 1989); or (5) chemicals such as methyl jasminate, salicylic acid, or Safener. It may also be advantageous to employ (6) organ-specific promoters (e.g., Roshal et al., EMBO J. 6:1155, 1987; Schernthaner et al., EMBO J. 7:1249, 1988; Bustos et al., Plant Cell 1:839, 1989), including promoters that express specifically in the root, leaf, seed, etc.
Plant expression vectors optionally include RNA processing signals, e.g., introns, which may be positioned upstream or downstream of a polypeptide-encoding sequence in the transgene. In addition, the expression vectors may also include additional regulatory sequences from the 3′-untranslated region of plant genes (Thornburg et al., Proc. Natl. Acad. Sci. USA 84:744 (1987); An et al., Plant Cell 1:115 (1989), e.g., a 3′ terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3′ terminator regions.
Useful dominant selectable marker genes include genes encoding antibiotic resistance genes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin, or spectinomycin); and herbicide resistance genes (e.g., phosphinothricin acetyltransferase). A useful strategy for selection of transformants for herbicide resistance is described, e.g., in Vasil, Cell Culture and Somatic Cell Genetics of Plants, Vols. I-III, Laboratory Procedures and Their Applications Academic Press, New York, 1984.
An expression vector for expression of a defense signal peptide or polypeptide in a plant may also comprise a gene encoding another polypeptide, including a herbicide-tolerance gene (e.g., tolerance to glyphosate, glufosinate, etc.); a polypeptide conferring insect resistance (e.g., a Bacillus thuringensis insecticidal protein or a Xenorhabdus insecticidal protein); a pathogen protein (e.g., virus coat protein); a trait for improving yield, drought resistance, cold tolerance, etc.; a trait for modifying the oil, protein or starch composition of seeds; or another gene that has a desirable activity when expressed in a plant. For example, U.S. Pat. No. 5,571,706 describes the introduction of the N gene into tobacco to confer resistance to tobacco mosaic virus; WO 95/28423 describes the expression of the Rps2 gene from Arabidopsis thaliana in plants as a means of creating resistance to bacterial pathogens including Pseudomonaos syringae; WO 98/02545 describes the introduction of the Prf gene into plants to obtain broad-spectrum pathogen resistance; and U.S. Pat. No. 6,762,285 describes the expression of the Bs2 resistance proteins in plants to confer resistance to Xanthomonas compestris. Such plant defense genes may also be co-expressed on the same or a different expression vector with a defense signal polypeptide or peptide.
Nucleic-Acid Hybridization: “Stringent Conditions”; “Specific”
The term “stringent conditions” is functionally defined with regard to the hybridization of a nucleic-acid probe to a target polynucleotide (i.e., to a particular nucleic-acid sequence of interest) by the specific hybridization procedure discussed in Sambrook et al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52, 9.56-9.58; Kanehisa, Nucl. Acids Res. 12:203-213, 1984; and Wetmur and Davidson, J. Mol. Biol. 31:349-370, 1968.
Regarding the amplification of a target nucleic-acid sequence (e.g., by PCR) using a particular amplification primer pair, “stringent conditions” are conditions that permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild type sequence (or its complement) would bind and preferably to produce a unique amplification product.
The term “specific for (a target sequence)” indicates that a probe or primer hybridizes under given hybridization conditions only to the target sequence in a sample comprising the target sequence.
Nucleic-Acid Amplification
As used herein, “amplified DNA” refers to the product of nucleic-acid amplification of a target nucleic-acid sequence. Nucleic-acid amplification can be accomplished by any of the various nucleic-acid amplification methods known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990.
See also the examples below regarding RT-PCR, for example.
Nucleotide-Sequence Variants of Native Defense Signal Polypeptide Polynucleotides and Amino Acid Sequence Variants of Native Defense Signal Proteins and Peptides
Using the nucleotide and the amino-acid sequences disclosed herein, those skilled in the art can create DNA molecules, polypeptides, and peptides that have minor variations in their nucleotide or amino acid sequence, respectively.
“Variant” DNA molecules are DNA molecules containing minor changes in a native sequence, i.e., changes in which one or more nucleotides of a native sequence is deleted, added, and/or substituted, preferably while substantially maintaining a desired biological activity. Variant DNA molecules can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing the variant DNA molecule or a portion thereof. Such variants preferably do not change the reading frame of the protein-coding region of the polynucleotide and preferably encode a protein having no change, only a minor reduction, or an increase in a desired biological activity.
Amino-acid substitutions are preferably substitutions of single amino-acid residues. DNA insertions are preferably of about 1 to 10 contiguous nucleotides and deletions are preferably of about 1 to 30 contiguous nucleotides. Insertions and deletions are preferably insertions or deletions from an end of the protein-coding or non-coding sequence and are preferably made in adjacent base pairs. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a final construct.
Preferably, variant polynucleotides according are “silent” or “conservative” variants. “Silent” variants are variants of a native sequence or a homolog thereof in which there has been a substitution of one or more base pairs but no change in the amino-acid sequence of the polypeptide or peptide encoded by the sequence. “Conservative” variants are variants of a native (or consensus) sequence in which at least one codon in the protein-coding region of the gene has been changed, resulting in a conservative change in one or more amino acid residues of the encoded polypeptide encoded, i.e., an amino acid substitution. A number of conservative amino acid substitutions are listed below. In addition, one or more codons encoding cysteine residues can be substituted for, resulting in a loss of a cysteine residue and affecting disulfide linkages in the polypeptide.







TABLE 4







Conservative Amino-Acid Substitutions










Original Residue
Conservative Substitutions







Ala
Ser



Arg
Lys



Asn
Gln, His



Asp
Glu



Cys
Ser



Gln
Asn



Glu
Asp



Gly
Pro



His
Asn; Gln



Ile
Leu; Val



Leu
Ile; Val



Lys
Arg; Gln; Glu



Met
Leu; Ile



Phe
Met; Leu; Tyr



Ser
Thr



Thr
Ser



Trp
Tyr



Tyr
Trp; Phe



Val
Ile; Leu










Substantial changes in function are made by selecting substitutions that are less conservative than those listed above, e.g., causing changes in: (a) the structure of the polypeptide backbone in the area of the substitution; (b) the charge or hydrophobicity of the polypeptide at the target site; or (c) the bulk of an amino acid side chain. Substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
Polypeptides and Peptides
For the polypeptide and peptide sequences presented herein, either the three-letter code or the one-letter code may be used for representing amino acid residues, as provided in Table 5 below.







TABLE 5







Three-letter Code and One-letter Code for Amino Acids











Amino Acid
Three-Letter Code
One-Letter Code







Alanine
Ala
A



Cysteine
Cys
C



Aspartic acid
Asp
D



Glutamic acid
Glu
E



Phenylalanine
Phe
F



Glycine
Gly
G



Histidine
His
H



Isoleucine
Ile
I



Lysine
Lys
K



Leucine
Leu
L



Methionine
Met
M



Asparagine
Asn
N



Proline
Pro
P



Glutamine
Gln
Q



Arginine
Arg
R



Serine
Ser
S



Threonine
Thr
T



Valine
Val
V



Tryptophan
Trp
W



Tyrosine
Tyr
Y



Unknown or
Xaa
X



Unspecified










“Defense Signal Polypeptide”: “Defense Signal Peptide”

In general, a peptide is considered a short polypeptide. The term “defense signal polypeptide” (or protein) refers to a polypeptide encoded by a defense signal protein polynucleotide, including, but not limited to, the polynucleotides listed in Table 1, and other polynucleotides that encode orthologs, paralogs, homologs, and variants of a native defense signal polypeptide. Defense signal peptides result from the processing of a native defense signal polypeptide, such as AtproPep1, in a plant cell. As a result, a native defense signal polypeptide includes sequences in addition to defense signal peptide sequences. Recombinant polypeptides that are not processed intracellularly, but that have defense signal peptide activity, are also considered defense signal polypeptides or peptides.
Recombinant fusion polypeptides may be made that, when processed in a plant cell, result in the production of more than one defense signal peptide, or in the production of a defense signal peptide and another biologically active polypeptide or peptide.
The term “defense signal peptide” refers to a peptide about 10 or more amino acids in length that has substantial defense signal peptide activity. Such defense signal peptides may have a length of 11, 12, 13, 14, 15, or more amino acids. AtPep1 is a native defense signal peptide from Arabidopsis that is 23 amino acids in length, although sequences from the C-terminal end of AtPep1 as short as 10 amino acids retain substantial defense signal peptide activity, and such truncated peptides increase in activity with increasing length. Defense signal peptides longer than 23 amino acids retain defense signal peptide activity. The native defense signal polypeptides that we have identified range encodes propeptides of 75 to 154 amino acids that are processed intracellularly to produce the shorter defense signal peptides. Defense signal peptides up to about 160 amino acid residues, or 100, or 90, or 80, or 70, or 60, or 50, or 40, or 30, or 23, or 20 amino acid residues are included among the defense signal peptides disclosed herein.
Defense signal peptides may be produced by expression of a polynucleotide that encodes such a peptide intracellularly, e.g., in a plant cell, or in a non-plant cell, e.g., a bacterial, fungal, insect, or other cell used in recombinant production of polypeptides. Alternatively, defense signal peptides may be produced by chemical synthesis. Techniques for chemical synthesis of polypeptides are described, for example, in Merrifield, J. Amer. Chem. Soc. 85:2149-2156, 1963, and peptide synthesizers are commercially available. For chemical synthesis, shorter forms of the defense signal peptides are preferable to longer forms, including but not limited to, defense signal peptides between about 10 and about 30 amino acids in length.
Polypeptide Sequence Homology
Ordinarily, defense signal peptides encompassed by the present disclosure are at least about 75 percent homologous to a native defense signal peptide, including but not limited to any of the defense signal peptides listed in Tables 1, 2, or 3 or a dicot or monocot consensus defense signal peptide sequence, or at least about 80 percent, 85 percent, 90 percent, or 100 percent (complete) homology, and that has substantial defense signal peptide activity. Such homology is considered to be “substantial homology,” although more important than shared amino-acid sequence homology is the possession of characteristic structural features and highly conserved amino acid residues from the C-terminal region of native defense signal peptides or consensus sequences.
Polypeptide homology is typically analyzed using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wis.). Polypeptide sequence analysis software matches homologous sequences using measures of homology assigned to various substitutions, deletions, substitutions, and other modifications.
“Isolated,” “Purified,” “Homogeneous” Polypeptides and Peptides
An “isolated” polypeptide or peptide has been separated from the cellular components (polynucleotides, lipids, carbohydrates, and other polypeptides) that naturally accompany it. Such a polypeptide or peptide can also be referred to as “pure” or “homogeneous” or “substantially” pure or homogeneous. Thus, a polypeptide that is chemically synthesized is isolated. A defense signal peptide or polypeptide is also considered “isolated” if it is the product of the expression of a recombinant polynucleotide (even if expressed in a homologous cell type). Thus, if AtPep1, for example, is recombinantly expressed in an Arabidopsis plant, it is considered “isolated” if the polynucleotide that encodes it is under the control of a promoter that is different from the native AtproPep1 promoter, or if the polynucleotide encodes a polypeptide other than the wild type, or native, AtproPep1 polypeptide but, when processed in a plant cell produces a native AtPep1 peptide, or the AtPep1 peptide produced by expression of the polynucleotide and processing of the encoded polypeptide differs from that of the native AtPep1 peptide in any way, for example in length or sequence.
A monomeric polypeptide or peptide is isolated when at least 60% by weight of a sample is composed of the polypeptide or peptide, or 90% or more, or 95% or more, or more than 99%. Protein purity or homogeneity is indicated, for example, by polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band upon staining the polyacrylamide gel; high pressure liquid chromatography; or other conventional methods.
Protein Purification
The polypeptides and peptides of the present disclosure can be purified by any of the means known in the art. Various methods of protein purification are described, e.g., in Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982.
Variant and Modified Forms of Defense Signal Peptides and Polypeptides
Encompassed by the defense signal peptides and polypeptides of the present disclosure are variant peptides and polypeptides in which there have been substitutions, deletions, insertions or other modifications of a native (i.e., wild type) peptide or polypeptide. The variants substantially retain structural characteristics and biological activities of a corresponding native peptide or polypeptide and are preferably silent or conservative substitutions of one or a small number of contiguous amino acid residues.
Regarding the terms “paralog” and “ortholog”, homologous polynucleotide sequences and homologous polypeptide sequences may be paralogs or orthologs of the claimed polynucleotide or polypeptide sequence. Orthologs and paralogs are evolutionarily related genes that have similar sequence and similar functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event. Sequences that are sufficiently similar to one another will be appreciated by those of skill in the art and may be based upon percentage identity of the complete sequences, percentage identity of a conserved domain or sequence within the complete sequence, percentage similarity to the complete sequence, percentage similarity to a conserved domain or sequence within the complete sequence, and/or an arrangement of contiguous nucleotides or peptides particular to a conserved domain or complete sequence. Sequences that are sufficiently similar to one another will also bind in a similar manner to the same DNA binding sites of transcriptional regulatory elements using methods well known to those of skill in the art.
The term “equivalog” describes members of a set of homologous proteins that are conserved with respect to function since their last common ancestor. Related proteins are grouped into equivalog families, and otherwise into protein families with other hierarchically defined homology types. This definition is provided at the Institute for Genomic Research (TIGR) website, “tigr.org” under the heading “Terms associated with TIGRFAMs”.
“Allelic variant” or “polynucleotide allelic variant” refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations may be “silent” or may encode polypeptides having altered amino acid sequence. “Allelic variant” and “polypeptide allelic variant” may also be used with respect to polypeptides, and in this case the terms refer to a polypeptide encoded by an allelic variant of a gene.
“Splice variant” or “polynucleotide splice variant” as used herein refers to alternative forms of RNA transcribed from a gene. Splice variation naturally occurs as a result of alternative sites being spliced within a single transcribed RNA molecule or between separately transcribed RNA molecules, and may result in several different forms of mRNA transcribed from the same gene. Thus, splice variants may encode polypeptides having different amino acid sequences, which may or may not have similar functions in the organism. “Splice variant” or “polypeptide splice variant” may also refer to a polypeptide encoded by a splice variant of a transcribed mRNA.
As used herein, “polynucleotide variants” may also refer to polynucleotide sequences that encode paralogs and orthologs of the presently disclosed polypeptide sequences. “Polypeptide variants” may refer to polypeptide sequences that are paralogs and orthologs of the presently disclosed polypeptide sequences.
A native defense signal peptide or polypeptide sequence can be modified by conventional methods, e.g., by acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, and labeling, whether accomplished by in vivo or in vitro enzymatic treatment or by the synthesis of a defense signal peptide or polypeptide using modified amino acids.
Labeling
There are a variety of conventional methods and reagents for labeling polypeptides and fragments thereof. Typical labels include radioactive isotopes, ligands or ligand receptors, fluorophores, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al., 1989 and Ausubel et al., 1992.
Peptide Fragments
The present disclosure also encompasses fragments of a defense signal peptide that lacks at least one residue of a native full-length defense signal peptide. Preferably, such a fragment retains substantial defense signal peptide activity, including but not limited to substantial activity in an alkalinization assay and/or the ability to enhance disease resistance in a plant.
“Defense Signal Peptide Activity”: Biological Activity of Polypeptides or Peptides
The terms “biological activity”, “biologically active”, “activity” and “active” refer primarily to the characteristic biological activity or activities of a native defense signal peptide or polypeptide. Defense signal peptide activity includes activity in an alkalinization assay. Substantial defense signal peptide activity in an alkalinization assay includes a change of at least 0.2 pH units when a 10 microliter aliquot of a solution having a concentration of 25 nM of the peptide is added to 1 mL of plant cells in the assay. More substantial defense signal peptide activity in the assay is the observation of a change in pH of at least 0.2 pH units using a solution having concentrations of 2.5 nM or 0.25 nM of the peptide, or when a change of at least 0.5 pH units are observed at a given peptide concentration, or when the activity is at least 25 percent, or 50 percent, or 75 percent that of a native defense signal polypeptide.
For the alkalinization assay, cells suspensions are grown in a volume of 40 ml in a 125 ml flask. Tobacco and Arabidopsis cells are typically grown for 3-5 days before use. Tomato cells are typically grown for 4-7 days before use. Soybean cells are generally grown for 3-7 days before use. Preferably, cells are in mid to log or log phase when they are to be used for the alkalinization assay. A 1 ml pipette tip with the end cut off (to prevent clogging of the tip with the cells) is used to aliquot 1 ml of the cells from the suspension into a well in a 24-well culture cluster plate. The flask is swirled between aliquots to ensure that the cells remain evenly dispersed throughout the cell suspension and an roughly equivalent number of cells is provided to each well. The plate(s) are then shaken at 160 rpm for 1 hour. Small aliquots (1-10 ml) of extracted peptide fractions are then added to the wells. After 20 min, the pH of the cell media is measured and recorded.
Alternatively, a substantial defense signal peptide activity is the ability to enhance plant disease resistance and substantially improve yield of plant product, with enhancement of plant disease resistance evidenced by reduced disease symptoms, enhanced resistance to disease infestation, etc., when a defense signal peptide is applied to a plant exogenously or recombinantly expressed within a plant. A defense signal peptide substantially enhances disease resistance of a plant if it increases the resistance of a plant to a pathogen of at least 10 percent as compared to a control plant under similar conditions, or more substantially, of at least 25, or 50, or 75, or 100 percent, as measured by standard quantitative measures of plant disease resistance to a given pathogen, e.g., the number or size of lesions, increased growth, survival rate, rate of disease progression, higher root mass, better seed viability, seed quantity and quality, etc. Alternatively, a substantial defense signal peptide activity is present where the peptide, when applied to a plant exogenously or recombinantly expressed within a plant, confers a substantial change in any resistance to a biotic or abiotic stress that involves the jasmonate/ethylene or salicylic acid pathways, as measured by standard methods.
Fusion Polypeptides
The present disclosure also provides fusion polypeptides including, for example, heterologous fusion polypeptides in which a defense signal polypeptide coding sequence is joined to a heterologous promoter (i.e., a promoter from gene other than the promoter that is operably linked to that coding sequence in nature), or in which the coding sequence for the defense signal peptide is joined to a fusion partner, i.e., a protein-coding sequence other than sequences with which the coding sequence for the defense signal peptide is joined in nature. Such fusion polypeptides can exhibit biological properties (such as substrate or ligand binding, enzymatic activity, antigenic determinants, etc.) derived from each of the fused sequences.
Polypeptide Sequence Determination
The sequence of a polypeptide of the present disclosure can be determined by any of the various methods known in the art.
Polypeptide Coupling to a Solid-Phase Support
The polypeptides of the present disclosure can be free in solution or coupled to a solid-phase support, e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, or glass wool, by conventional methods.
Antibodies
The present disclosure also encompasses polyclonal and/or monoclonal antibodies capable of specifically binding to a particular defense signal peptide and/or fragments thereof. Such antibodies are raised against a defense signal peptide or fragment thereof and are capable of distinguishing a defense signal peptide from other polypeptides, i.e., are specific for the particular defense signal peptide.
For the preparation and use of antibodies according to the present disclosure, including various immunoassay techniques and applications, see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, 2d ed, Academic Press, New York, 1986; and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988. Defense signal peptide-specific antibodies are useful, for example in: purifying a defense signal peptide polypeptide from a biological sample, such as a host cell expressing a recombinant defense signal peptide; in cloning a paralog, ortholog, or homolog from an expression library; as antibody probes for protein blots and immunoassays; etc.
Antibodies can be labeled by any of a variety of conventional methods. Suitable labels include, but are not limited to, radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles, etc.
Obtaining Paralogs, Orthologs, and Homologs of Defense Signal Peptides
As discussed in the examples below, defense signal peptides homologous to AtPep1 and other defense signal peptides exist in many plant species. Based upon the availability of the defense signal peptide and polypeptide sequences and their corresponding gene sequences disclosed herein, paralogs and orthologs can be obtained by conventional methods, e.g., by screening a cDNA or genomic library with a probe that specifically hybridizes to a native defense signal peptide sequence under at least moderately stringent conditions, by PCR or another amplification method using a primer or primers that specifically hybridize to a native defense signal peptide or polypeptide sequence under at least moderately stringent conditions, or by screening an expression library using defense signal peptide-specific antibodies.
EDKR Repeat Regions
Highly negatively charged glutamate/aspartate repeat interrupted by lysine/arginine residues in the precursor region of the molecule.
1-5-10 Helix Motifs
The precursor genes encode a hydrophobic motif interrupted by a conserved lysine and cysteine, with a consensus sequence of xXxxxXCxxx, where x denotes some hydrophobic residue, X represents any nonhydrophobic amino acid, and x specifically designates F, I, L, or V. The protein secondary structure prediction programs SSpro version 2.0 (Pollastri et al. 2001) and SSpro8 (Baldi et al. 1999) predict that this region to be helical in gene family members. This consensus sequence coincides with the 1-5-10 calmodulin recognition domain motif, which requires an F, I, L, or V in the 1, 5 and 10 positions interspersed with cationic charges. This motif generates an amphipathic alpha helix that has the potential to interact with calmodulin in the presence of calcium (Rhoads and Friedberg 1997).
Serine Repeat
A stretch of multiple serines in a row in the precursor region. In some precursors, there are stretches of repeated alanines rather than serines. Note that this structural characteristic is common, but does not occur in every precursor.
K/R Positive Region
The region comprising the proposed amino end of the processed peptide, is positively charged, having in Arabidopsis between 3 and 7 lysine or arginine residues encoded in this area by each family member. Other plant species may have fewer lysines or arginines here (for instance poplar has just two), but all predicted peptides have at least on lysine or arginine in this region.
Core Peptide Motif
The carboxyl terminal of the predicted peptide region. All orthologs contain the conserved glycine residue at the C-7 position (7 amino acids prior to the predicted carboxy terminus of the peptide). Each precursor also encodes a conserved asparagine or histidine as the final amino acid in the predicted peptide followed by a stop codon. Most dicots encode a serine or threonine in the C-9 position, whereas this position is less conserved in the monocot sequences.
Plant Transformation and Regeneration: Transformed Plant Cells, Plants, and Parts and Products of Transformed Plants
Various polynucleotide constructs that include a sequence that encodes a defense signal polypeptide or a defense signal peptide are useful for producing plants having enhanced disease resistance or enhanced resistance to another biotic and abiotic stress that involves the jasmonate/ethylene or salicylic acid pathways.
Polynucleotides that comprise a sequence that encodes a defense related polypeptide or a defense related peptide can be expressed in plants or plant cells under the control of an operably linked promoter that is capable of expression in the plant or plant cell. Any well-known method can be employed for plant cell transformation, culture, and regeneration in the practice of the present disclosure with regard to a particular plant species. Conventional methods for introduction of foreign DNA into plant cells include, but are not limited to: (1) Agrobacterium-mediated transformation (Lichtenstein and Fuller In: Genetic Engineering, Vol 6, Rigby, ed., London, Academic Press, 1987; and Lichtenstein and Draper, in: DNA Cloning, Vol II, Glover, ed., Oxford, IRI Press, 1985); (2) particle delivery (see, e.g., Gordon-Kamm et al., Plant Cell 2:603, 1990; or BioRad Technical Bulletin 1687), (3) microinjection (see, e.g., Green et al., Plant Tissue and Cell Culture, Academic Press, New York, 1987), (4) polyethylene glycol (PEG) procedures (see, e.g., Draper et al., Plant Cell Physiol. 23:451, 1982); Zhang and Wu, Theor. Appl. Genet. 76:835, 1988), (5) liposome-mediated DNA uptake (see, e.g., Freeman et al., Plant Cell Physiol. 25:1353, 1984), (6) electroporation (see, e.g., Fromm et al., Nature 319:791, 1986); and (7) vortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci. USA 87:1228, 1990).
Once a transformed plant cell or tissue has been obtained, it is possible to regenerate a full-grown plant from it. Means for regeneration vary from species to species. In one approach a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable. Plant regeneration is described, for example, in Evans, et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. 1, 1984, and Vol. III, 1986). Practically all plants can be regenerated from cultured cells or tissues, including monocots, dicots, gymnosperms, etc.
After the DNA construct is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crosses or by asexual propagation. With respect to sexual crossing, any of a number of standard breeding techniques can be used depending upon the species to be crossed. Cultivars can be propagated in accord with common agricultural procedures known to those in the field.
The term “plant” encompasses any higher plant and progeny thereof, including monocots, dicots, gymnosperms, and other plants and includes parts of plants, including reproductive units of a plant (e.g., seeds), fruit, flowers, etc.
A “reproductive unit” of a plant is any totipotent part or tissue of the plant from which one can obtain a progeny of the plant, including, for example, seeds, cuttings, tubers, buds, bulbs, somatic embryos, cultured cells (e.g., callus or suspension cultures), etc.
According to one aspect of the disclosure, plant cells are provided that comprise a polynucleotide sequence that comprises a sequence that encodes a defense signal peptide or polypeptide operably linked to a plant promoter. Another aspect of the disclosure is directed to plants comprising such cells, i.e., transformed or transgenic plants. Another aspect is a part or product of such plants.
Agronomically and commercially important products and/or compositions of matter derived from transgenic plants according to the disclosure include, but are not limited to, animal feed, commodities, products and by-products that are intended for use as food for human consumption or for use in compositions and commodities that are intended for human consumption, including but not limited to plant parts, including but not limited to seeds, seed pods, flowers (including flower buds), fruit, tubers, stems, cuttings, pollen, and products derived from processing such plant parts, including but not limited to flour, meal, syrup, oil, starch, cakes, cereals, and the like. Such compositions may be defined as containing detectable amounts of a polynucleotide sequence as set forth herein, and thus are also diagnostic for any transgenic event containing such nucleotide sequences. These products are more likely to be derived from crops propagated with fewer pesticides and organophosphates as a result of their incorporation of the nucleotides of the present disclosure for controlling plant disease. For example, such commodities and commodity products can be produced from seed produced from a transgenic plant, wherein the transgenic plant comprises cells that express a defense signal protein of the present disclosure.
Identifying Transgenic Plants According to the Disclosure and Parts and Products Thereof
Transgenic plants according to the present disclosure, parts of such plants, and products derived from the processing of such plants, can be readily identified by using probes and primers to specifically identify the presence of a transgene that encodes a defense signal peptide or the presence of a specific defense signal peptide. In order to perform such an identification, a biological sample thought to contain such a plant, part or product is contacted with a probe that binds specifically to the transgene containing a defense signal peptide- or polypeptide-encoding polynucleotide (such as one or more PCR primers, cDNA probe, etc.), and detecting such binding (e.g., by identifying the production of an amplification product of a diagnostic size after gel electrophoresis, or by autoradiography). Alternatively, one may use a probe that binds specifically to the defense signal peptide or polypeptide itself, such as an antibody probe, wherein binding can be detected by an enzyme-linked immunosorbent assay (ELISA), etc.
Conferring Resistance to Biotic and Abiotic Stresses to Plants and Enhancing Plant Growth
As one aspect of the disclosure, resistance to disease resistance, or to another biotic and abiotic stress that involves the jasmonate/ethylene pathway or the salicylic acid pathway, is conferred on a plant, or resistance may be enhanced in the plant, or growth of the plant is enhanced, by expression of polynucleotides that encode one or more defense peptides in cells of the plant.
As another aspect of the disclosure, methods are provided that comprise growing a seed into a plant, wherein the plant comprises cells comprising a polynucleotide sequence comprising a sequence that encodes a defense signal protein or polypeptide, wherein the plant exhibits one or more of the following: improved yield of plant product, reduced disease symptoms, or enhanced resistance to disease infestation; compared to a control plant lacking the recombinant polynucleotide.
According to another aspect of the disclosure, improved yield, reduced disease symptoms, or enhanced resistance to disease infestation is conferred or enhanced, by application of compositions comprising one or more defense signal peptides to a plant.
Where absolute immunity against infection by a pathogen or detrimental affects or other stresses is not to be conferred, the severity of the disease is reduced and symptom development is delayed. This method of imparting resistance has the potential for enhancing plant resistance to a variety of diseases for which other approaches were ineffective in providing effective control.
The methods of the present disclosure are useful in imparting resistance to a wide variety of pathogens including viruses, bacteria, and fungi.
With regard to the use of the compositions and methods of the present disclosure to enhance plant growth, various forms of plant growth enhancement or promotion can be achieved. This can occur as early as when plant growth begins from seeds or later in the life of a plant. For example, plant growth according to the present disclosure encompasses greater yield, increased percentage of seeds germinated, increased plant size, greater biomass, more and bigger fruit, earlier fruit coloration, earlier flower opening, improved flower longevity (i.e., shelf-life), and earlier fruit and plant maturation. As a result, the present disclosure provides significant economic benefit to growers. For example, early germination and early maturation permit crops to be grown in areas where short growing seasons would otherwise preclude their growth in that locale. Increased percentage of seed germination results in improved crop stands and more efficient seed use. Greater yield, increased size, and enhanced biomass production allow greater revenue generation from a given plot of land.
To confer such enhanced resistance, one may express a single gene copy, or in order to express a defense signal peptide at high levels, e.g., expression of multiple copies of a transgene encoding such a defense signal peptide and/or the use of strong promoters to drive expression may be employed. Expression of a transgene encoding a defense signal peptide in plant cells at a sufficiently high level may initiate the plant defense response constitutively in the absence of signals from the pathogen. A constitutive plant promoter can be used. Alternatively, an inducible promoter, or an organ- or tissue-specific promoter, for example, can be used.
If a plant cell is selected to be transformed, it may be of any type capable of being transformed, preferably one with an agronomic, horticultural, ornamental, economic, or commercial value. Examples of such plant cells include, but are not limited to: acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, clementine, clover, coconut, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon, mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf grass, turnip, a vine, watermelon, wheat, yams, and zucchini.
Compositions Comprising Defense Signal Peptides for Application to Plants
According to one embodiment, compositions for application to plants comprise an oil flowable suspension, comprising purified defense signal peptides or unpurified forms of the peptides, including lysed or unlysed bacterial cells or fractions thereof that contain one or more of the defense signal peptides disclosed herein. Any such bacterial host cell expressing the novel polynucleotides disclosed herein and producing a defense signal peptide is contemplated to be useful, such as Bacillus spp., including B. thuringiensis, B. megaterium, B. subtilis, B. cereus, Escherichia spp. including E. coli, and/or Pseudomonas spp., including P. cepacta, P. aeruginosa, and P. fluorescens. 
In another embodiment, compositions for application to plants comprise a water dispersible granule or powder comprising purified or unpurified defense signal peptides.
In another embodiment, compositions for application to plants comprise a wettable powder, spray, emulsion, colloid, aqueous or organic solution, dust, pellet, or colloidal concentrate comprising purified or unpurified defense signal peptides. Such dry forms of the insecticidal compositions may be formulated to dissolve immediately upon wetting, or alternatively, dissolve in a controlled-release, sustained-release, or other time-dependent manner. Alternatively, such a composition may consist of a combination of one or more of the following compositions: lysed or unlysed bacterial cells, spores, crystals, and/or purified crystal proteins.
In another embodiment, compositions for application to plants comprise an aqueous solution or suspension comprising purified or unpurified defense signal peptides. Such aqueous solutions or suspensions may be provided as a concentrated stock solution which is diluted prior to application, or alternatively, as a diluted solution ready-to-apply.
Such compositions may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers. Detergents may be included to facilitate uptake of the defense signal peptides by plant tissues and cells.
Regardless of the method of application, the amount of the active component(s) are applied at an amount that is effective to confer enhanced disease resistance to plants, which will vary depending on such factors as, for example, the specific disease to be controlled, the specific plant or crop to be treated, the environmental conditions, and the method, rate, and quantity of application of the composition.
Such compositions may be made by formulating purified or unpurified defense signal peptides with the desired biologically-acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluents, such as saline or other buffer, for example. The formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art.
The term “biologically-acceptable carrier” refers to all carriers that are compatible with the growth and development of a cultured cell or tissue, an excised plant part, a seed, a plant grown under greenhouse or field conditions, or other biological entity, e.g., aqueous solutions, buffers, adjuvants, etc. that are ordinarily used in connection with the biological entity, including but not limited to any carrier used in bacterial or plant cell or tissue culture and agriculturally-acceptable carriers. The term “agriculturally-acceptable carrier” covers all adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in formulation technology for compositions used in agriculture to be applied to plants, soils, etc. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding.
Such compositions are applied to the environment of the plant for uptake into plant tissues and cells, typically onto the foliage of the plant or crop to be protected, by conventional methods, such as by spraying. The strength and duration of application will be set with regard to conditions specific to the particular pest(s), crop(s) to be treated and particular environmental conditions. The proportional ratio of active ingredient to carrier will naturally depend on the chemical nature, solubility, and stability of the defense signal protein(s), as well as the particular formulation contemplated.
Other application techniques, e.g., dusting, sprinkling, soaking, soil injection, seed coating, seedling coating, spraying, aerating, misting, atomizing, and the like, are also feasible and may be required under certain circumstances.
The defense signal peptides may be employed in such compositions singly, in a mixture of defense signal peptides, or in combination with other compounds, including and not limited to other proteins or chemical compounds used for treatment of plants, including but not limited to proteins or chemical compounds used to treat plants for pathogens, insect pests, etc. The method may also be used in conjunction with other treatments such as surfactants, detergents, polymers or time-release formulations.
The compositions of the present disclosure may be formulated for either systemic or topical use. The concentration of insecticidal composition which is used for environmental, systemic, or foliar application will vary widely depending upon the nature of the particular formulation, means of application, environmental conditions, and degree of biocidal activity. Typically, the composition will be present in the applied formulation at a concentration of at least about 0.1% by weight and may be up to and including about 99% by weight. Dry formulations of the compositions may be from about 0.1% to about 99% or more by weight of the composition, while liquid formulations may generally comprise from about 0.1% to about 99% or more of the active ingredient by weight.
The formulation may be administered to a particular plant or target area in one or more applications as needed, with a typical field application rate per hectare ranging on the order of from about 0.1 g to about 1 kg, 2 kg, 5, kg, or more of active ingredient.
Identifying Defense Signal Proteins
According to one aspect of the disclosure, methods are provided for identifying native defense signal peptides from plants and also for screening synthetic peptides for defense signal peptide activity.
A sensitive, rapid “alkalinization assay” (see Examples) is useful for isolate native defense signal peptides from plants or synthetic defense signal peptides produced by chemical synthesis or other means. Cultured plant cells, for example suspension cell cultures that grow at about pH5 are used. Several laboratories have developed such cell cultures for Arabidopsis, tomato (Lycopersicon esculentum), tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), maize (Zea mays), petunia (Petunia hybrida), nightshade (Solanum nigrum), and sweet potato (Ipomoea batatus), for example. Within minutes after adding systemin to cells, an ATP-driven proton pump is inhibited, causing the extracellular medium of the cells to become alkaline. When 1-10 μL aliquots from fractions from plant tissues, e.g., leaves, that have eluted from HPLC columns were added to 1 mL of suspension cultured cells, some fractions caused the cell medium to increase in pH. In order to confirm that a candidate peptide is a defense signal peptide, the peptide is applied to a plant as described in the Examples below or a polynucleotide sequence encoding the candidate peptide is expressed in a plant in order to observe whether disease resistance is enhanced in the plant. Confirmation of the identity of a candidate peptide may be obtained by determining whether the peptide induces defense gene expression (for example, of PDF1.2 and PR-1), e.g., by supplying a solution of the peptide to excised leaves through their cut petioles then analyzing transcript levels, e.g., by semi-quantitative RT-PCR.
Identifying Compounds that Interact with Receptors for Defense Signal Proteins and that Enhance Plant Disease Resistance
According to another aspect of the disclosure, substances other than peptides and polypeptides, for example, chemical compounds, are screened for their ability to enhance plant defense against diseases. In one approach, the alkalinization assay is used to screen such substances. Candidate substances are added to cultured plant cells and a rise in pH indicates that a candidate substance interacts with a receptor. In a second approach, candidate substances are assayed for binding by an AtPep1 receptor or another receptor for a defense signal peptide. A composition comprising one or more candidate substances that are selected after being screened in an alkalinization assay or receptor binding assay may then be administered to plants in a greenhouse or field trial to assess whether the candidate substance(s) confer enhanced plant defense against a disease. Substances that have activity in conferring enhanced plant defense may be formulated according to standard formulation approaches for application to plants, to seeds, to the soil, etc. The present disclosure also includes compositions comprising an amount of such substances that is effective to enhance plant defense against a disease and a biologically (including agriculturally) compatible carrier. Such compositions may also include other ingredients that are used in formulations for application to plants as detailed above.
Preferred embodiments will be better understood by reference to the following examples, which are intended to merely illustrate the best mode now known for practicing the embodiments. The scope of the disclosure is not to be considered limited thereto.
Example 1
Isolation and Analysis of AtPep1 and Paralogs and Orthologs Thereof
Innate immunity is initiated in animals and plants through the recognition of a variety of pathogen associated molecules that in animals are called “pathogen-associated molecular patterns,” or PAMPS, and in plants are called elicitors. Peptides derived from pathogens can be powerful elicitors of plant defense responses (Hahlbrock et al., Proc. Natl. Acad. Sci. USA 92:4150-4157, 1995; van den Askerveken et al, Plant Physiol. 103:91-96, 1993; Kammpren, Curr. Opin. Plant Biol. 4:295-300, 2001; Kunze et al., Plant Cell, 16:3496-3507, 2004; Navarro et al., Plant Physiol. 135:1113-1128, 2004; Fellbrich et al., Plant J. 32:375-390, 2002; He et al., Cell 73:1255-1266, 1993), but plant-derived peptides have not been identified previously that are elicitors of immune responses directed against pathogens.
We have isolated and characterized a 23 amino acid peptide, called AtPep1, that is a signaling component of the innate immune system of Arabidopsis. The peptide precursor gene is transcribed in response to elicitors generated by pathogens, and AtPep1 is produced to amplify the signaling pathways. Seven paralogs of the AtproPep1 gene are present in the Arabidopsis genome, and orthologs have been identified in species of several agriculturally important families including Solanaceae, Poaceac, Salicaceae, Vitaceae, and Fabaceae. AtPep1 and its paralogs and orthologs play important roles as endogenous signals to amplify innate immunity.
Materials and Methods
Plant Growth Conditions.
Arabidopsis thaliana ecotype Columbia seeds were grown in soil in four-inch square pots for six days under low light at approximately 8° C. Germinated seedlings were then grown under day lengths of 16 hours at 21° C. Mutant plants were grown in autoclaved soil.
Alkalinization Assay.
Arabidopsis suspension cells were grown with shaking in the dark in 125 mL flasks, using 40 mL NT media as previously described (Pearce et al., Proc. Natl. Acad. Sci. USA 98:12843-12847, 2001). The cells were transferred weekly (2.5 mL) and used for assays 3-5 days after transfer. One mL aliquots of cells were transferred to wells of 24-well culture plates and allowed to equilibrate for one hour while agitated on a rotary shaker at 160 rpm. Aliquots of 1-10 μL from extracts or fractions eluted from HPLC columns were added to cells and the pH of the media was monitored after 20 min.
Purification of AtPep1.
Arabidopsis thaliana (Columbia ecotype), 28 days after planting, consisting of rosettes, flowers, stems and seed pods, were harvested, frozen in liquid nitrogen, ground to a powder, and stored at −20° C. Peptides were extracted from 600 g of powder as previously described (Pearce et al., Nature 411:817-820, 2001; Pearce and Ryan, J. Biol. Chem. 278:30044-30050, 2003), using 1200 mL 1% trifluoroacetic acid (TFA). The clear extract was applied to a reversed-phase C18 flash column (Bondesil, Varian Analytical Instruments, Walnut Creek, Calif.) that was equilibrated with 0.1% TFA/H2O. After washing with equilibration buffer, the column was eluted with 50% methanol/0.1% TFA. The eluate was vacuum-evaporated and lyophilized to dryness. This material was dissolved in 0.1% TFA and chromatographed on a G-25 Sephadex column (2.5×33 cm), and the fractions were monitored with the alkalinization assay. The broad peak of activity that eluted between 1-1.5 void volumes was collected and lyophilized. The yield of dry powder was 109 mgs.
Two hundred forty mg of the powder was dissolved in 9 mL of 0.1% TFA/H2O, centrifuged, and the clarified solution was applied to a 5 micron, 10×250 mm semi-preparative C18 column (#218TP510, Vydac, Hesperia, Calif.) with a flow rate of 2 ml/min and monitored at 225 nm. After 2 min, a gradient from 0-40% acetonitrile/0.1% TFA was applied to the column and 1 min fractions were collected and assayed as above. A defined activity peak was identified in fractions 36-37 and designated Arabidopsis Peptide 1 (AtPep1). AtPep1 was further purified by strong cation exchange chromatography on a 5 μm, 4.6×200 mm PolySulfoethyl AspartamideTM column (The Nest Group, Southborough, Mass.) equilibrated with 5 mM potassium phosphate, pH 3, in 25% acetonitrile. Two min after applying AtPep1 to the column, a gradient of 0-100% elution buffer consisting of 5 mM potassium phosphate, 1 M potassium chloride, pH 3, in 25% acetonitrile was applied for 60 min. Absorbance was monitored at 214 nm and the flow rate was 1 mL/min. Fractions were collected at minute intervals and 10 μL aliquots were assayed for alkalinization activity. The fractions with activity, 58 and 59, were pooled and lyophilized. Further purification of AtPep1 was performed on a narrow-bore reversed-phase 218TP52, 5 μm, 2.1×250 mm C18 column (Vydac, Hesperia, Calif.) that had been equilibrated with 0.1% TFA/H20. The lyophilized material was dissolved in the equilibration buffer and applied to the column. After two min, a gradient of 0-50% acetonitrile in 0.1% TFA was applied over 90 min with a flow rate of 0.25 mL/min, and monitored at 214 nm. Fractions were collected at 1 min intervals and assayed as above. The activity was present in fractions 48-50, which were pooled and lyophilized. Further purification was obtained on the same narrow-bore column but using a 0-50% methanol/0.05% TFA gradient over 90 min for elution. The activity was found exclusively in fractions 63-64. These fractions were pooled and subjected to amino acid sequence analysis and MALDI-mass spectroscopy.
Peptide Sequence Analysis and Synthesis.
N-terminal sequence analysis was performed using Edman chemistry on an Applied Biosystems Procise Model 492 protein sequencer. MALDI-mass spectroscopy was performed on a PerSeptive Biosystems Voyager time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm) with α-cyano-4-hydroxycinnamic acid as the matrix. Peptide synthesis was performed using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry by solid phase techniques using an Applied Biosystems Model 431 synthesizer. Synthetic peptides were purified by reversed-phase C18 HPLC. Peptide stocks (250 μM) were assayed for purity and the mass verified with a Finnigan LC/Q mass spectrometer using direct injection.
Plant Stress and Hormone Treatments.
To examine effects of cold stress, plants were placed in a refrigerated growth chamber set to 2° C. To simulate drought stress conditions, plants grown under standard growth chamber conditions were grown without watering. Methyl jasmonate (Bedoukian Research Inc., Danbury Conn.) was applied as a 625 μM solution in 0.1% Triton X-100 to the upper surface of leaves and the plants were incubated in plexiglass boxes. Methyl salicylate (Sigma-Aldrich, St. Louis Mo.), was applied to leaf surfaces at 2 mM in a 0.1% Triton X-100 solution. Ethephon (Phytotechnology Laboratories, Shawnee Mission Kans.) was sprayed on plants as a 7 mM solution in 0.1% Triton X-100 (Sigma-Aldrich, St. Louis Mo.). ABA effects were analyzed by spraying plants with a 100 μM solution (mixed isomer, Sigma-Aldrich, St. Louis Mo.) in 0.1% Triton X-100 (Denekamp and Smeekens, Plant Physiol. 132, 1415-1423, 2003).
Excised-Leaf Assays.
AtPep1 peptide dissolved in double distilled water was supplied to excised leaves of 3 to 4 week old Arabidopsis plants. Leaves were excised and the petioles were immersed in 800 μL centrifuge tubes containing either the peptide solution or distilled water, and placed in a closed clear plexiglass box containing a thin layer of water for humidity, and a small opening to allow air to enter. Boxes were incubated in a growth chamber under the plant growth conditions described above and sprayed with a fine mist of distilled water every half hour to ensure humidity and prevent wilting. To determine variations in basal levels of the AtproPep1 transcript among assays, four different leaves from four different plants were used for each treatment, and leaves supplied with either water or AtPep1 were taken from the same plants. Assays were terminated by immersing the leaves in liquid nitrogen.
Hydrogen peroxide accumulation was visualized using diaminobenzidine (DAB) (Thordal-Christensen et al., Plant J. 11:1187-1194, 1997).
Semi-Quantitative RT-PCR Analysis of Relative Gene Expression Levels.
RNA was isolated using Trizol reagent and manufacturer's instructions (Invitrogen, Carlsbad Calif.), and 2 μg of RNA template was reverse transcribed with a RETROscript kit (Ambion, Austin Tex.). PCR reactions were carried out with ExTaq Hot Start polymerase and reagents (Fisher Scientific, Pittsburgh Pa.). The AtproPep1 forward and reverse primers with the respective sequences of 5′ CTT ATC AGA TCT CAA TGG AGA AAT C 3′ (SEQ ID NO: 59) and 5′ CAA TGT AAC TTA AAG TGC CTA ATT ATG 3′ (SEQ ID NO: 60) generated a 310 bp intron-spanning product. Primers to β-tubulin (At5g62690) of 5′ CAA CGC TAC TCT GTC TGT CC 3′ (SEQ ID NO: 73) and 5′ TCT GTG AAT TCC ATC TCG TC 3′ (SEQ ID NO: 74) generated a 681 base pair intron-spanning product. An initial denaturing/polymerase activating step of 5 minutes at 94° C. was followed by 31 repetitions of the following three steps: a thirty second denaturation phase at 94° C., a thirty second annealing period at 55.5° C., and a one minute elongation step at 72° C. The amplification program was terminated with a 10 minute final 72° C. elongation phase.
The products of each reaction were separated by electrophoresis and were visualized on a Bio Imaging System (SynGene, Frederick Md.) using GeneSnap version 6.00.26 software (SynGene, Frederick Md.). A high resolution image of the gel was analyzed using GeneTools analysis software version 3.02.00 (SynGene, Frederick Md.). Relative band intensities for each band were normalized to the 3-tubulin band. A numerical ratio of amplified AtproPep1 cDNA to amplified tubulin cDNA was obtained for every sample. To calculate average values, semi-quantitative RT-PCR assays were performed in duplicate, and RNA extractions were performed in triplicate.
Transformation of Arabidopsis with a CaMV 3SS:proAtPep1 Gene.
Genomic DNA was isolated from Arabidopsis leaves using the DNAzol reagent (Invitrogen, Carlsbad Calif.). The genomic sequence encoding AtproPep1 was amplified using a forward primer 5′ ATA AAG AGT CAC ACC CAA TAC CG 3′ (SEQ ID NO: 76) and a reverse primer 5′ TGA TAC TGG TTA TGA ACT TAT GAT GG 3′ (SEQ ID NO: 77) to generate a 1078 base pair product. A 5′ Xho 1 recognition site and a 3′ BamH 1 site were amplified onto the genomic fragment for ligation into the pART-7 vector (Gleave, Plant Mol. Biol. 20:1203-1207, 1992). Both the proAtPep1 genomic product and the pART-7 vector were digested with BamH 1 and Xho 1 enzymes (Promega Biosciences Inc., San Luis Obispo Calif.), and ligated using the LigaFast rapid DNA ligation system (Promega Biosciences Inc., San Luis Obispo Calif.). The construct was transformed into chemically-competent E. coli TOP10F′ cells (Invitrogen, Carlsbad Calif.) that were plated out on LB-ampicillin (50 μg/mL). A plasmid clone containing the full AtproPep1 genomic DNA insert with no nucleotide errors was used to generate an AtproPep1/pBART construct. Both pBART and AprotPep1/PART-7 plasmid were digested with Not 1 (Promega Biosciences Inc., San Luis Obispo Calif.) to enable ligation of the CaMV 35S/AtproPep1 expression cassette into the digested pBART plasmid using the Promega LigaFast kit (Promega Biosciences Inc., San Luis Obispo Calif.). An empty pART-7 vector was digested with Not 1 to generate a control pBART construct. TOP10F′ chemically competent cells were transformed with the constructs and grown in Luria-Berftani media containing 100 μg/mL spectinomycin (Sigma-Aldrich, St. Louis Mo.), 40 μL of a 40 mg/mL solution of X-Gal (Sigma-Aldrich, St. Louis Mo.) and 40 μL of a 100 mM IPTG (Sigma-Aldrich, St. Louis Mo.) stock. A pBART clone containing the CaMV 35S/proAtPep1 construct, and a second clone containing the empty CaMV 35S construct, were transformed into Agrobacterium tumefaciens strain AGLO cells (Lazo and Ludwig, Biotechnology (N Y) 9:963-967, 1991) by electroporation using a BioRad electroporator (BioRad Laboratories, Hercules, Calif.). The transformed cells were grown on 2XYT media (Lazo et al., Biotechnology (N Y) 9:963-967, 1991) containing 100 μg/mL spectinomycin, and viable colonies were screened using RT-PCR with the pART F and pART R primers.
Liquid cultures of Agrobacterium carrying the CaMV 35S:AtproPep1 or empty CaMV 35S constructs were grown in 2XYT media and used for floral dip transformation of Arabidopsis plants (Clough and Bent, Plant J. 16:735-743, 1998). Transformed plants were grown to maturity, and the seed was collected and planted. Newly germinated seedlings were treated with a 350 μM solution of the herbicide BASTA (glufosinate ammonium, brand name Finale; Farnam Companies Inc., Phoenix Ariz.) four times at three day intervals, and healthy plants were screened for the proAtPep1 transgene via PCR. Plants that were both glufosinate-resistant and that amplified products of the appropriate size were grown to maturity and the seeds planted to recover T2 progeny.
Growth and Inoculation of Plants with Pythium irregulare. 
Two strains of the oomycete root pathogen Pythium irregulare, strain 110305) were grown on water-agar (1%) plates for maintenance of stock cultures and, after growing at room temperature in the dark for one week, were stored at 4° C. Pythium stocks for infection assays were grown on 1× potato dextrose agar (Sigma-Aldrich, St. Louis Mo.) in the dark for one week at room temperature.
Week-old P. irregulare cultures were scraped from the plates into 20 mL of sterile distilled water, the mixture was lightly ground with a mortar and pestle to produce a uniform suspension. Aliquots (250 μL) of the suspension or water were pipetted into the soil of plants having a rosette diameter of 2-3 cm. Plants were grown for 25 days as described above and assayed. The experiments were repeated five times. After two and a half weeks, the plants were photographed to show rosette morphology, and at three and a half weeks were harvested and the roots examined. The day prior to harvest, plants were not watered, so that the soil would easily separate from the roots. Soil was gently rinsed from the roots of each plant with water, taking care to minimize damage, and each plant was trimmed at the base of the rosette to fully expose the root structure, and photographed.
Identification the AtproPep1 Gene and Homologous Genes.
The gene locus encoding the AtPep1 peptide precursor was identified using the National Center for Biotechnology Information (NCBI) TBLASTN version 2.2.7 algorithm (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997) to search genomic sequences from Arabidopsis thaliana. To determine possible localization of the protein in the cell, several predictive programs were employed, including pSORT (Nakai and Kanehisa, Proteins 11:95-110, 1991), ChloroP41 and MitoProt (Emanuelsson et al., J. Mol. Biol. 300:1005-1016, 2000; Emanuelsson et al., Prot. Sci. 8:978-984, 1999). Orthologs to the AtproPep1 gene were identified using the NCBI TBLASTN version 2.2.7 and Institute of Genomic Research (TIGR) TBLASTN 2.0 MP algorithms (Gish, TBLASTN 2.0 MP-WashU [27 Aug. 2000] [linux-i686 21:46:47 28 Aug. 2000] Copyright 1996-2000 Washington University, Saint Louis, Mo. USA. [http://blast.wustl.edu]). The predicted protein sequence for each was aligned using the program Clustal W version 1.8, available at the Baylor College of Medicine Search Launcher website.
Analysis of Gene Expression Using RT-PCR.
Gene expression was analyzed using RT-PCR (Nishimura et al., Plant Cell 16:1365-1377, 2004). Forward and reverse primers used for RT-PCR analysis are shown in Table 6.
Results
We have isolated a 23 amino acid peptide called AtPep1 from extracts of Arabidopsis leaves that exhibits characteristics of an elicitor of the innate immune response. Endogenous peptide elicitors of innate immunity have not been previously known. The identification and isolation of the peptide from soluble extracts of Arabidopsis leaves was facilitated by its ability, at sub-nanomolar concentrations, to cause an alkalinization response that is typical of elicitors (Moyen and Johannes, Plant Cell Environ. 19:464-470, 1996; Felix and Boiler, Plant J. 7:381-389, 1999; Pearce et al., Proc. Natl. Acad. Sci. USA 98:12843-12847, 2001; Pearce et al., Nature 411:817-820, 2001; Pearce and Ryan, J. Biol. Chem. 278:30044-30050, 2003).
A bioactive component, AtPep1, was identified and purified to homogeneity. Peptides present in a 1% TFA/water extract of Arabidopsis tissues were passed through a reverse phase semi-preparative C18 flash chromatography column and separated on a G-25 Sepharose column. The breakthrough peak was applied to a C18 HPLC column and 10 μL from 2 mL fractions from the column were assayed for alkalinization activity. The peak identified as AtPep1 was further purified through two additional chromatography steps and finally purified by narrow bore HPLC. Fractions were assayed for alkalinization activity and the active peak was analyzed by MALDI mass spectroscopy. The amino acid sequence of the purified peptide was determined by Edman degradation. Its identity as a peptide was established by its molecular mass (M/Z, 2492.65) and amino acid sequence (from amino terminus to carboxy terminus, ATKVKAKQRGKEKVSSGRPGQHN (SEQ ID NO: 1; see Table 1) with a calculated molecular mass of 2491.8). The kD determined by mass spectroscopy matched the kD calculated from the amino acid sequence, indicating that the peptide was not post-translationally modified. The chemically synthesized peptide was found to be as active as the native AtPep1, with a 1⁄2 maximal activity of 0.25 nM.
The sequence of AtPep1 was identified in GenBank as being derived from the accession At5g64900, which encodes a small protein of 92 amino acids, with its C-terminal 23 amino acids comprising AtPep1. FIG. 1 shows the amino acid sequence of the AtPep1 precursor protein, AtproPep1, deduced from the protein encoded by the gene At5g64900. The AtPep1 sequence at the carboxyl terminus of the precursor protein, is underlined. The amino acid sequence of the precursor protein is highly charged and lacks a leader sequence, indicating that it is not synthesized through the secretory pathway, but rather on cytoplasmic ribosomes.
Expression Analysis of AtroPep1 in Response to Abiotic and Biotic Cues.
As a first step in seeking a possible function for AtproPep1 and its encoded peptide, the basal expression level of the gene was assessed in leaves, stems, roots and flowers of Arabidopsis plants was studied using semi-quantitative RT-PCR analysis of AtproPep1 gene expression in response to treatment of leaves with MeJA, ethephon, MeSA, and AtPep1. The relative abundance of the proAtPep1 transcript was estimated from the expression of the β-tubulin gene as a control. Leaves were wounded by crushing once across the mid-vein with a hemostat. Plants were sprayed with a 250 μM solution of MeJA in 0.1% Triton X-100; with a 2 μM solution of MeSA in 0.1% Triton X-100 or with a 7 mM solution of ethephon in 0.1% Triton X-100. AtPep1 peptide (10 nM in water) was supplied through cut petioles of excised leaves. Total RNA was extracted and analyzed. The forward and reverse primers for real-time PCR(RT-PCR) analysis are shown in Table 6 below.







TABLE 6







RT-PCR primers









Gene
Primers
Product size





AtproPep1
SEQ ID NO: 59:
310 bp



5′ CTTATCAGATCTCAATGGAGAAATC 3′ F*)



(At5g64900)
SEQ ID NO: 60:




5′ CAATGTAACTTAAAGTGCCTAATTATG 3′ (R)



 


PDF1.2
SEQ ID NO: 61:
243 bp



5′ ATGGCTAAGTTTGCTTCCA 3′ (F)



(At5g44420)
SEQ ID NO: 62:




5′ TTAACATGGGACGTAACAGATAC 3′ (R)



 


PR-1
SEQ ID NO: 63:
306 bp



5′ GGAGCTACGCAGAACAACTA 3′ (F)



(At2g14610)
SEQ ID NO: 64:




5′ AGTATGGCTTCTCGTTCACA 3′ (R)



 


TAT3
SEQ ID NO: 65:
330 bp



5′ TACAGGGGTAGTTCAAGCAA 3′ (F)



(At2g24850)
SEQ ID NO: 66:




5′ CCTAGAGCCACTCCTGGTAT 3′ (R)



 


LOX2
SEQ ID NO: 67:
312 bp



5′ ACGGTAGAAGACTACGCACA 3′ (F)



(At3g45140)
SEQ ID NO: 68:




5′ TAAGGTCTCGAGCTCCTCTT 3′ (R)



 


VSP2
SEQ ID NO: 69:
317 bp



5′ CAAAATATGGATACGGGACA 3′ (F)



(At5g24770)
SEQ ID NO: 70:




5′ ATTGCCAACGATGTTGTATC 3′ (R)



 


ATT13
SEQ ID NO: 71:
231 bp



5′ TGGCAATGAAGTCAGTTTCT 3′ (F)



(At2g43530)
SEQ ID NO: 72:




5′AGAAGTCGCAGAAGCACTTA 3′ (R)



 


β-tubulin
SEQ ID NO: 73:
681 bp



5′CAACGCTACTCTGTCTGTCC 3′ (F)



(At5g62690)
SEQ ID NO: 74:




5′ TCTGTGAATTCCATCTCGTC 3′ (R)





*F = Forward primers;


R = Reverse primers.






The AtproPep1 gene was expressed at low levels in all tissues, giving no clues as to its possible function. Monitoring the expression of AtproPep1 in intact plants exposed to different environmental conditions and chemicals, including drought and cold stress, UV-B irradiation, wounding, methyl jasmonate (MeJA), methyl salicylate (MeSA), abscissic acid (ABA) and Ethephon®, provided more definitive clues. Whereas most treatments did not cause changes in expression of AtproPep, wounding, MeJA, ethephon, and AtPep1 all induced expression of AtproPep), indicating a possible relationship of the gene and its encoded peptide in plant defense. Transcription of the gene in response to wounding was detected within about 8 h, whereas spraying the plants with a 250 μM solution of MeJA or 7 mM ethephon induced a strong expression of the gene within an hour. Supplying 10 nM AtPep1 through cut petioles of excised leaves induced expression of the AtproPep1 gene within two hours.
AtPep1 Regulates Transcription of Pathogen Defense Genes.
The expression of AtproPep1 in response to MeJA and ethylene (Et) suggested that the encoded peptide might have a role in activating innate immunity in Arabidopsis, although an endogenous peptide had not previously been reported in the innate immune system of any plant. The jasmonic acid (JA)/Et signaling pathway in Arabidopsis activates the expression of defensive genes including PDF1.2 (defensin), while the salicylic acid (SA) pathway activates several pathogen-related (PR) genes (Penninckx et al., Plant Cell 8:2309-2323, 1996; Lorenzo et al., Plant Cell 15:165-178, 2003; Zimmerli et al., Plant J. 40:633-646, 2004; Penninckx et al., Plant Cell 10:2103-2113, 1998; Hammond-Kosack and Parker, Curr. Opin. Biotechnol. 14:177-193, 2003; Mauch-Mass and Matreau, Ann. Bot. 82:535-540, 1998).
In order to determine whether AtPep1 regulates defense gene expression, we determined the fold induction of defense related genes in excised Arabidopsis leaves in response to 10 nM AtPep1 supplied through their cut petioles. After 2 hr, transcript levels were analyzed for expression levels of PDF1.2 (defensin), PR-1 (pathogenesis-related 1) LOX2 (lipoxygenase 2), VSP2 (vegetative storage protein2) and ATTI3 (Arabidopsis thaliana trypsin inhibitor 3), relative to levels in untreated excised leaves. Expression was determined by semi-quantitative RT-PCR using a β-tubulin gene as a control. Supplying excised Arabidopsis leaves with solutions of AtPep1 through their cut petioles induced a strong expression of PDF1.2 and PR-1.
We also performed similar assays with an Arabidopsis triple mutant (fad3-2, fad7-2, fad8; McConn and Browse, Plant Cell 8:403-416, 1996) that is incapable of synthesizing jasmonic acid, and a mutant (ein2-1; Guzman and Ecker, Plant Cell 2:513-523, 1990) that is incapable of perceiving ethylene. AtPep1 was supplied at 10 nM for 2 hr, and RNA isolated and assayed by semi-quantitative RT-PCR for gene expression levels. AtPep1 did not induce the expression of AtproPep1, PDF1.2 or PR-1. These experiments suggested that AtPep1 acts upstream from the JA/Et and SA pathways to activate PDF1.2, PR-1 and AtproPep1.
We also studied the accumulation of H2O in leaves supplied for 2 hr with water, 10 nM AtPep1, or with 10 nM AtPep1, all containing 1 mg/mL of DAB to visualize H2O2 accumulation. Leaves treated with AtPep1 and DAB were also co-supplied with 100 μM DPI, an inhibitor of NADPH oxidase. We also studied the transcription of PDF1.2 and PR-1 in leaves of wild type plants in response to supplying with 10 nM AtPep1 in the presence or absence of DPI (diphenylene iodonium chloride), an inhibitor of NADPH oxidase in both plants and animals (O'Donnell et al., Biochem. J. 290:41-49, 1993). The expression of each gene was analyzed by RT-PCR and compared to expression in excised plants treated only with water. The results indicated that reactive oxygen species (ROS) generated in both the JA/Et and SA pathways is required for PDF1.2 and PR-1 transcription (Penninckx et al., Plant Cell 8:2309-2323, 1996; Hammond-Kosack and Parker, Curr. Opin. Biotechnol. 14:177-193, 2003; Mackerness et al., Plant Cell and Environ. 22:1413-1423, 1999).
AtproPep1 Over-Expression in Arabidopsis Enhances Innate Immunity.
Arabidopsis plants were transformed with a CaMV-3SS-AtproPep1 transgene in order to assess the effects of the constitutive synthesis of AtPep1 on the expression of defense genes using semi-quantitative RT-PCR. In previous studies, overexpression of the tomato prosystemin precursor gene (McGurl et al., Proc. Natl. Acad. Sci. USA 91:9799-9802, 1994) caused a constitutive over-expression of defense genes. This is apparently due to the constitutive synthesis of prosystemin in the cytoplasm of cells (prosystemin, like AtproPep1, lacks a leader sequence) where it is processed to systemin. Analysis of transgenic Arabidopsis plants overexpressing AtproPep1 behaved in a similar manner as the prosystemin gene in that it caused an over-expression of defense genes, in this case of PDF1.2 and PR-1. The fold expression of the various genes (compared to wild type) was found to be: AtproPep1, 12.7±6.4; PDF1.2, 4.4±0.5; PR-1, 2.1±0.4; LOX2, 1.0±0.2; VSP-2, 0.9±0.1; and ATTI3, 1.2±0.1. These results indicated that plants over-expressing AtproPep1 were synthesizing AtPep1 in the absence of pathogen attacks or elicitors, constitutively signaling the defense response.
Transgenic Arabidopsis plants constitutively over-expressing AtproPep1 were assayed for enhanced resistance against a root pathogen, Pythium irregulare, an oomycete that has been employed previously to demonstrate the effects of signaling mutants of Arabidopsis on disease resistance (Staswick et al., Plant J. 15:747-754, 1998; Vijayan et al., Proc. Natl. Acad. Sci. USA 95:7209-7214, 1998). The soils of young wild type (Columbia) and transgenic plants overexpressing a 35S:AtproPep1 gene (having rosette diameters of 2-3 cm) were inoculated with either a suspension of Pythium irregulare strain 110305 propagules, or with sterile water, and the plants were grown for 25 days post-inoculation. Five repetitions were performed with 16 plants of each genotype in each experiment. The aerial parts of the wild type plants inoculated with Pythium were slightly smaller than uninoculated wild type or transgenic plants. However, the roots of plants from duplicate experiments in which the root masses of uninoculated wild type and transgenic plants are compared to the roots of inoculated wild type and transgenic plants clearly showed that the over-expression of AtproPep1 had enhanced the resistance of the plants toward the root pathogen.
AtProPep1 Paralogs.
AtproPep1 belongs to a seven-member gene family in Arabidopsis of which one gene is unannotated. Three paralogs, At5g64890, At5g64900 (AtproPep1), and At5g64905, are sequentially encoded in a 5.5 kilobase region of chromosome V (NCBI Arabidopsis Genome Database). Paralogs At5g09980 and At5g09990 and the unannotated gene are also found on chromosome V, but in a 3.8 kb region at a distal region on the second arm of the chromosome. At2g22000 is found on chromosome 11. In comparing the amino acid sequences of the open reading frames of the paralogs, a low overall amino acid sequence identity was found, but within the C-terminal region of each gene where the putative AtPep1 sequences reside, the amino acid identities ranged from 35% to 65%. Four genes, At5g64905, At5g64900, At5g64890 and At5g09980, are expressed relatively strongly in excised Arabidopsis leaves in response to supplying either AtPep1 through cut petioles, or by spraying with MeJA. However, spraying plants with MeSA strongly induced only two of the genes that are induced by AtPep1 and MeJA, i.e. At5g64890 and At5g64905. This differential regulation of AtproPep1 paralogs suggests that a complex signaling network is at play in the leaves, and that cross-talk occurs between the JA/Et and SA pathways, regulating the expression levels of the paralogs. This differential expression of AtproPep1 paralogs was also found in the results from several recent microarray analyses of Arabidopsis genes transcribed in response to pathogens and elicitors. In these analyses the AtproPep1 paralogs were included without any knowledge of their possible signaling roles.
We performed a transcript analysis in order to determine the increases in transcription of AtproPep1 paralogs in Arabidopsis leaves in response to the pathogens P. infestans (oomycete), B. cineria (fungus), and Ps. syringae DC 3000 (bacteria) (Toufighi et al., Plant J. 43:153-163, 2005; Craigon et al., Nucleic Acids Res. 32:D575-577, 2004), and to the elicitors NPP1, HrpZ, flg22, and elf18, derived from oomycetes, and bacteria, respectively (Kammpren, Curr. Opin. Plant Biol. 4:295-300, 2001; Kunze et al., Plant Cell, 16:3496-3507, 2004; Navarro et al., Plant Physiol. 135:1113-1128, 2004; Fellbrich et al., Plant J. 32:375-390, 2002); He et al., Cell 73:1255-1266, 1993). All of these treatments strongly induce the transcription of At5g64890 and At5g64905, the two paralogs that are strongly induced by treating leaves with MeJA, MeSA and AtPep1. However, the lack of induction of At5g64900 by the pathogens, a gene induced by MeJA and AtPep1, and the induction of At5g64900, At5g64890 and At5g64905 by elicitors as well as by the pathogens, indicates that differential induction of the AtproPep family of genes may be governed by the types of elicitors related to individual pathogens. The data presented herein supports a model in which the paralogs At5g64900, At5g64905, and At5g64890 are transcribed in response to elicitors of the JA/Et signaling pathway, while the genes At5g64905, and At5g64890 are transcribed in response to elicitors of SA. The nascent proproteins or processed peptides are transported to the apoplast, where they interact with a cell surface receptor(s) to amplify the immune response. Thus, the AtproPep1 paralogs are components of an amplification system for a broad spectrum of elicitors that activate the innate immune response.
Discussion
Some fundamental similarities are found among signaling components of animal and plant innate immune systems, including the recognition of PAMPS and/or elicitors from pathogens, the involvement of LRR receptor kinases that monitor the signals, and the resulting activation of defense gene transcription of genes involved in early steps of innate immunity. Several peptides originating from plant pathogens can activate the plant innate immune response, including Pep13, AVR9, and elicitins derived from fungi (Hahlbrock et al., Proc. Natl. Acad. Sci. USA 92:4150-4157, 1995; van den Askerveken et al., Plant Physiol. 103:91-96, 1993; Kammpren, Curr. Opin. Plant Biol. 4:295-300, 2001), and the peptides hrpZ, NPP1, flg22 and elf13 from bacteria (Kunze et al., Plant Cell, 16:3496-3507, 2004; Navarro et al., Plant Physiol. 135:1113-1128, 2004; Fellbrich et al., Plant J. 32:375-390, 2002; He et al., Cell 73:1255-1266, 1993), as examples. However until this report endogenous plant peptides have not been reported that are involved with signaling roles directed against pathogen attacks. We have discovered a family of genes that encode small peptides are rapidly and are strongly transcribed along with defense genes in response to pathogens and their elicitors, appearing to assure a rapid, strong amplification of the innate immune response. The low expression or lack of expression of some of the AtproPep genes demonstrates that the paralogs are differentially expressed in response to pathogen infections and elicitors. Fusions of all paralogs with green fluorescent protein (GFP) and beta-glucuronidase (GUS) are used to investigate expression of the paralogs to determine their tissue-specific expression and their roles in defense responses.
The induction of the defense responses by AtPep1 is mediated by a binding protein on the cell surface of Arabidopsis suspension cultured cells that interacts with AtPep1 with the characteristics of a receptor, further supporting the fundamental concept proposed in the model described above in which the AtproPep1 paralogs serve as components of an amplification system for a broad spectrum of elicitors that activate the innate immune response.
Searches of plant genomic databases and EST collections identified AtproPep orthologs in species from several plant families. Table 1, supra, shows the C-terminal sequences of paralogs and orthologs of AtproPep1 aligned with the AtPep1 peptide sequence. Paralogs are grouped above and dicot and monocot orthologs are grouped below.
The regions with the highest amino acid identities among the deduced proteins occur within the C-terminal residues of each where the AtPep homologs are found. The deduced canola peptide exhibited the highest identity with the AtPep peptides, being a Brassicaceae species. All of the putative AtPep homologs have a conserved glycine at residue #17 (numbers aligned with AtPep1), and all but paralogs from the Poeceae family contain an asparagine at residue #23. Each peptide contains several proline, glycine, and serine residues within a 10 amino acid C-terminal region that may be important for receptor recognition.
The chemical and physiological properties of the AtPep1 family members, their precursor proteins, and their genes, are strikingly similar to the properties of the 18 amino acid peptide signal systemin, its precursor prosystemin, and its gene, that are components of the signaling pathway for defense against herbivorous pests of the Solanaceae family (Ryan and Pearce, “Peptide hormones/systemins,” in Encyclopedia of Biological Chemistry, ed. Lennarz and Lane, vol 3, pp. 381-384, Elsevier, 2004). Both AtPep1 homologs and tomato systemin homologs are cleaved from the carboxy (C)-termini of precursor proteins that lack leader peptides, both precursors are small, highly positively charged proteins, and each activates defense genes. The mechanism for processing AtPep1 is not known, nor is it known if it is the AtPep1 or its peptide precursor that is transported to the apoplast, or if more than one receptor is involved in recognizing the different peptides.
These results indicate that the major role for receptor-mediated defense-signaling peptides in plants is to amplify signaling that is initiated by wounding and elicitors to mount a rapid, strong defense against herbivores and pathogens. If AtproPep orthologs behave in the same manner when over-expressed in other plant species by constitutively expressing defense genes, they may provide an important new approach to enhance innate immunity in a broad spectrum of agriculturally important crops.
Example 2
An LRR Receptor Kinase is a Component of AtPep1 Amplification of Innate Immunity in Arabidopsis 
Over 200 LRR receptor kinases are present in the Arabidopsis genome. Only a few LRR receptor kinases have been identified that interact with the peptide signals in plants. This includes the receptor for the defense peptide signal, systemin (Scheer and Ryan, Proc. Natl. Acad. Sci. 99:9585-9590, 2002) and receptors for the developmental peptide signals CLAVATA1 (Clark and Mcyerowitz, Cell 89:575-585, 1997), phytosulfokine-alpha (Matsubayashi et al., Science 296:1470-1472, 2002) and the pathogen-derived peptide flg22 (Gomez-Gomez and Boiler, Trends Plant Sci. 7:251-256, 2000).
The discovery that the AtPep family of endogenous peptides from Arabidopsis leaf extracts cause an alkalinization of the medium of Arabidopsis suspension cultured cells indicates that the peptide plays a role in plant cells by interacting with a cell surface receptor. Investigations of the biological role of the peptide in Arabidopsis plants indicated that it activates the innate immune system of the plants and functions through an interaction with a cell surface receptor.
We have isolated from Arabidopsis a cell surface LRR Thr/Ser kinase receptor for AtPep1, a 23 amino acid signal that that amplifies defense genes for innate immunity. The interaction of AtPep1 with the receptor is saturable and exhibits a Kd of 0.25 nM. Two SALK mutant lines with T-DNA insertions in exons of At1g73080 do not express the receptor gene and are not labeled by an AtPep1 photoaffinity analog that was used to identify and isolate the receptor protein. However, in contrast to wild type plants, the SALK insertional lines constitutively expressed high levels of PDF1.2 and PR-1, indicting that the receptor was negatively regulating defense gene expression in the absence of AtPep1. The AtPep1 receptor plays a central role in amplifying innate immunity activating defense gene expression when interacting with AtPep peptides that are synthesized in response to elicitors.
Methods
Synthesis of AtPep1 Analogs.
AtPep1, Cys-AtPep1 and Tyr-AtPep1 were synthesized using solid-phase instrumentation, (peptide synthesizer Model 431A; Applied Biosystems, Foster City, Calif.). After synthesis, the polypeptides were purified using C18 reverse-phase, high-performance liquid chromatography (HPLC), as previously described (Pearce and Ryan, J. Biol. Chem. 278:30044-30050, 2003; Pearce et al., Proc. Natl. Acad. Sci. USA 98:12843-12847, 2001; Pearce et al., Nature 411:817-820, 2001; Scheer and Ryan, The Plant Cell 11:1525-1535, 1999; Shevchenko et al., Anal. Chem. 68:850-858, 1996). Stock solutions of the peptides (2.5 mM) were prepared in water and stored at −20° C. Iodination of Tyr-AtPep1 was performed using IODO-GEN Pre-Coated Iodination Tubes (Pierce, Rockford, Ill.). Two hundred μl of NaI (20 mM in 0.1 M phosphate buffer, pH 8.0) solution was oxidized in the IODO-GEN Pre-Coated Iodination Tube for 6 min. Oxidized NaI solution was transferred into a 1.5 ml tube containing 100 nmol Tyr-AtPep1, and maintained at room temperature for 6 min in the dark gently agitating the tube every 30 sec. Iodinated Tyr-AtPep1 was purified using HPLC as described previously (Scheer and Ryan, The Plant Cell 11:1525-1535, 1999), and quantified with bicinchoninic acid (Pierce, Rockford, Ill.). All analogs were analyzed by LCQ ion trap mass spectrometry (Finnigan, San Jose, Calif.). Cys-AtPep1 was coupled through a disulfide bond to the photoaffinity cross-linker, N-(4-[p-azidosalicylamido]butyl)-3′-(2′-pyridyldithio)propionamide (APDP) (Pierce, Rockford, Ill.) to produce azido-Cys-AtPep1, which was purified by HPLC by methods previously described (Scheer and Ryan, Proc. Natl. Acad. Sci. 99:9585-9590, 2002).
Radioactive iodinations of Tyr-AtPep1 and azido-Cys-AtPep1 were performed using 2 mCi of Na125I and 12.5 nmol and the products were purified by HPLC (Scheer and Ryan, Proc. Natl. Acad. Sci. 99:9585-9590, 2002). The specific activity of the purified mono-iodinated Tyr-AtPep1 and azido-Cys-AtPep1 were 2.58 mCi/nmol, while the specific activity of the diiodinated forms was 5.15 mCi/nmol. The mono-iodinated analog of azido-Cys-AtPep1 was found to comprise 90% of the iodinated analog and was employed for photoaffinity labeling.
Alkalinization Assay.
Medium alkalinization activity of Arabidopsis suspension cultured cells by AtPep1 analogs was analyzed as previously described for systemin (Pearce et al., Science 253:895-897, 1991), RALF (Pearce et al., Proc. Natl. Acad. Sci. USA 98:12843-12847, 2001), HypSys peptides (Pearce and Ryan, J. Biol. Chem. 278:30044-30050, 2003; Pearce et al., Nature 411:817-820, 2001), and AtPep1 (supra). The alkalinization activity of azido-Cys-AtPep were carried out after incubation the analog with cells in darkness for 10 min, when the pH was recorded.
Binding Assays.
Binding assays of Arabidopsis cells with 125I1-Tyr-AtPep1 were performed by the methods of Scheer and Ryan (Pearce et al., Nature 411:817-820, 2001) modified as follows: Arabidopsis suspension cultured cells were subcultured and grown for 4-5 days, washed with culture medium, and diluted with medium to a level of 0.2 mg fresh weight/ml. Two mL of cells were aliquoted into each well of 12-well culture plates and allowed to equilibrate on an orbital shaker (160 rpm) at room temperature for 1 hr. 125I1-Tyr-AtPep1 was added to the medium, and 500 μL of cells were removed at selected times and filtered through a 2.5 cm Type A/E Glass Fiber Filter (Pall Corporation, Ann Arbor, Mich.) using a 12-well vacuum filtration manifold (Millipore, Bedford, Mass.). The filtered cells were washed three times with 5 mL of cold MS medium containing 3% sucrose, suspended in 1 mL MS medium containing 3% sucrose in a glass test tube, and analyzed for total radioactivity in a gamma-ray counter (Isodata 2020; Isodata Inc., Palatine, Ill.). Specific binding was calculated by subtracting nonspecific binding (binding in the presence of 250-fold native AtPep1) from total binding. 125I1-Tyr-AtPep1 bound to the cell surface within a minute, and equilibrated within 4 min.
Photoaffinity Labeling.
To 1 mL of cultured cells in darkness was added 125I-azido-Cys-AtPep1 (0.25 nM final concentration as described above) and the cells were shaken for 10 min on an orbital shaker (160 rpm) at room temperature. The cells were transferred to a 1.5 mL glass tube, centrifuged at 10,000×g and the sedimented cells were dispersed in 1 mL cold MS medium containing 3% sucrose and centrifuged as above. This wash was repeated twice. The cells were resuspended in 1 mL MS medium and irradiated with UV-B for 10 min on ice to photoactivate the azido group to effect the crosslinking. The cells were washed with 1 mL MS medium, centrifuged as above, and resuspended in 400 μL of 5% SDS. The cells were disrupted by boiling for 30 min, and the insoluble debris was removed by centrifugation at 10,000×g. Proteins in the clear supernatant were precipitated by adding 1.25 volumes of methanol/chloroform (vol/vol). After centrifugation at 10,000×g, the pellet was recovered and dissolved in 100 μL of Laemmli sample buffer containing 5% SDS, boiled for 10 min, and separated using 8% SDS-PAGE. The gels were dried and exposed to X-ray film for 50 hr to visualize labeled proteins. In competition assays, unlabeled AtPep1 and suramin were added to the cells and incubated for 10 min before adding 125I-azido-Cys-AtPep1. The same procedures described above were employed to detect labeled proteins.
Purification of AtPep1-Binding Protein.
125I-azido-Cys-AtPep1 (0.25 nM) was added to 1 L of Arabidopsis suspension cultured cells and incubated for 10 min in the dark as described above, and collected on Miracloth (Calbiochem, San Diego, Calif.). After washing the cells with 1 L of cold water, the cells were suspended in 500 ml of MS medium containing 3% sucrose, and irradiated with UV-B for 15 min while being mixed on an orbital shaker at 160 rpm. The cells were again collected on Miracloth and washed with 1 L cold water. Microsomal fractions were prepared by differential centrifugation as previously described (Pearce et al., Proc. Natl. Acad. Sci. USA 98:12843-12847, 2001), and stored at −80° C. This process was repeated three times and the microsomal proteins were pooled. Protein was measured by Bio-Rad Protein Assay reagent (BIO-RAD, Hercules, Calif.) using bovine serum albumin as a standard.
Purification of AtPep1-binding protein from the membranes was performed as described previously with modifications (Pearce et al., Proc. Natl. Acad. Sci. USA 98:12843-12847, 2001). Briefly, 25 and 55 mg of radiolabeled and unlabeled microsomal proteins, respectively, were mixed and separated by 2 sets of 7.5% SDS-PAGE (0.6×14×9 cm) for 16 with 37 V at room temperature. The gels were sliced horizontally into 5 mm width and the radioactivity measured with a gamma-counter (Isodata 2020; Isodata Inc., Palatine, Ill.). The gel slices near 180 kD, containing the highest radioactivity were pooled. The gel slices were mascerated and the proteins were recovered by incubating the mascerate three times for 30 min with 25 mL of 20 mM Tris-HCl, 0.5 M NaCl, pH 7.5. The eluted proteins were pooled and incubated with 75 μL of Conconavalin A-Sepharose 4B (Amersham Bioscience, Piscataway, N.J.) for 6 h at room temperature to trap the Con A-protein complexes. The Con A-Sepharose was washed with 50 mL of 0.5 M alpha-methyl-D-glucoside three times, followed by 50 mL of H2O three times, to elute loosely bound proteins. Bound proteins were eluted by boiling the Con A-Sepharose in 500 μL of 5% SDS, and the eluted proteins were precipitated by adding 1.25 volumes of methanol/chloroform (2:1, vol/vol). Half of the eluted proteins were separated using 7.5% SDS-PAGE (0.15×14×8.5 cm) at 100 V for 8 h at room temperature. The gel was sliced horizontally into 1 mm widths, and the gel slice containing highest radioactivity was digested with trypsin (Promega, Madison, Wis.) according to Shevchenko et al. (Anal. Chem. 68:850-858, 1996). The other half of the eluted proteins were digested with Peptide-N-Glycosidase F (PNGase F) (Prozyme, San Leandro, Calif.), separated by 7.5% SDS-PAGE, recovered as above, and digested with trypsin. Peptides generated in the trypsin digests were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS), Voyager-DE RP Biospectrometry Workstation (Applied Biosystems, Framingham, Mass.). Protein was identified by searching in the National Center for Biotechnology Information database using Mascot (www.matrixscience.com).
Analysis of T-DNA Insertional Lines.
Arabidopsis thaliana T-DNA insertional lines, SALK 014538, SALK 059281 and SALK 064539, were obtained from ABRC (Ohio State University) through The Arabidopsis Information Resource. The plants were screened by RT-PCR using gene-specific primer pairs and a primer specific for the T-DNA left border. Total RNA was purified from rosette leaves.
Microsomal fractions were prepared from one-month-old plants by differential centrifugation as previously described (Scheer and Ryan, Proc. Natl. Acad. Sci. 99:9585-9590, 2002). The membranes were photoaffinity labeled (Takayama et al, Nature 413:534-538, 2001) using the radiolabeled azido analog used with suspension cultured cells. The membranes were incubated for 60 min at approximately 4° C., irradiated with UV-B for 15 min, separated by SDS-PAGE, dried and analyzed by radioautography to identify labeled proteins.
Results
An analog of AtPep1 was synthesized with a Tyr residue attached to its N-terminus and radiolabeled with 125I to quantify binding. The Tyr analog and mono- and di-iodo-Tyr-AtPep1 analogs were separated by HPLC, and the concentration-dependent activities of AtPep1 and these analogs where tested in the alkalinization assay. All were found to be as fully active as AtPep1 in the alkalinization assay. The mono-iodinated Tyr-AtPep1 comprised about 95% of the iodinated proteins and was employed for binding studies. Saturation kinetics of the binding of mono-125I-Tyr-AtPep1 with Arabidopsis suspension cultured cells (six repetitions using 106 cells/assay) showed that the 125I-labeled peptide was maximally bound to Arabidopsis cells within about 10 min, saturating the sites at about 0.1 nM peptide. A Kd of 0.25 nM was estimated from Scatchard analysis of the saturation data, which is typical of ligand-receptor interactions and indicates that AtPep1 was interacting with a cell surface binding protein with the characteristics of a receptor.
A photoaffinity labeled AtPep1 was prepared by synthesizing an analog with a Cys residue at its N-terminus so that an 125I-labeled azido adduct, APDP, could be crosslinked to the peptide through a disulfide bond. All procedures were in darkness unless otherwise specified. The azido-Cys-AtPep1 was purified by HPLC and assayed for its alkalinization response under red light to avoid photoactivating the azido group. The analog was as fully active as the native peptide in the alkalinization assay. The azido analog was iodinated with 125I and incubated with Arabidopsis suspension cultured cells for 10 min and subjected to UV-B irradiation to activate the azido group for crosslinking with binding proteins. SDS-PAGE analyses of the radiolabeled membrane proteins revealed a single labeled protein band of Mr approximately 180 kD. Pre-incubation of 2.5 nM AtPep1 to cells totally abolished labeling. Tomato systemin (LeSys), a nonhomologous 18 amino acid peptide signal from tomato plants (Pearce et al., Science 253:895-897, 1991) did not compete for binding with the labeled analog and had not effect on photoaffinity labeling. Suramin, a polycyclic non-specific inhibitor of peptide hormone-receptor interactions in both animals and plants (Stratmann et al., Proc. Natl. Acad. Sci. USA, 97:8862-8867, 2000), inhibited the labeling of the 180 kD protein by the photoaffinity AtPep1 analog at 100 nM, supporting a membrane association for the 180 kD labeled protein. The labeled protein band from SDS-PAGE gels was eluted and incubated with the carbohydrase PNGaseF to enzymatically remove covalently bound carbohydrates. This enzyme caused a decrease in the kD of the photoaffinity labeled protein from about 180 kD to about 150 kD, indicating that the binding protein was glycosylated.
Purification of the radiolabeled protein from 1 L of Arabidopsis cells in late log phase was achieved using final steps of ConA-Sepharose affinity chromatography followed by SDS-PAGE. After electrophoresis, the labeled 180 kD protein band was excised from the gel and the protein recovered. Half of the protein was digested with trypsin and the fragments were analyzed by MALDI-TOF mass spectroscopy. The other half of the eluted protein was treated with the enzyme PNGase F to remove carbohydrate and was subjected to gel electrophoresis. The protein in the 150 kD band was recovered, digested with trypsin, and was also analyzed by mass spectroscopy. The amino acid sequences of 18 tryptic fragments from the 180 kD peptide exactly matched sequences of the Arabidopsis LRR receptor kinase gene, At1g73080. The deglycosylated protein yielded three large fragments of from 12 to 18 amino acids in length that were also exact matches to sequences within the At1g73080 gene. The nucleotide sequence of the AtPep1 receptor gene (At1g73080) the deduced receptor polypeptide is provided in FIG. 2 shows the structure of the At1g73080 gene, which is comprised of an 646 amino acid extracellular domain containing 27 LRR motifs; a 22 amino acid transmembrane domain; and a 280 amino acid Ser/Thr receptor kinase domain.
The leaves of two SALK T-DNA insertional lines having insertions in the exons of the gene At1g73080, SALK 014538 and SALK 059281, did not express the gene when analyzed by RT-PCR, using wild type plants and a SALK 059281 insertional mutant of gene At5g55480 as controls. Microsomal membrane proteins from the two At1g73080 mutant lines, and from wild type plants and the control SALK 059281 line, were analyzed for proteins that were specifically photoaffinity labeled by 125I-azido-Cys-AtPep1. A protein was labeled in the membranes from wild type plants and the SALK 059281 plants, but not from the SALK mutants unable to express the receptor gene. The label was found in a 180 kD protein band and in a slightly lower doublet band that appears to be degradation products of the receptor protein, since they were labeled. The proteins labeled in the wild type and SALK 059281 microsomal membranes were absent when the membranes were preincubated with 2.5 nM AtPep1 and then photoaffinity labeled, indicating that the proteins labeled in the membranes of wild type and SALK 059281, and not in membranes from the lines with mutants in the At1g73080 gene, were the AtPep1 receptor and its biologically active fragments.
To further investigate the role of the 180 kD protein as the receptor of AtPep1, competition experiments were performed between the 125I1-Tyr-AtPep1 analog and synthetic peptide homologs derived from sequences at the N-termini of all seven of the AtPep family members that corresponded to the 23 amino acid AtPep1 (gene At5g684900). The peptides were purified after synthesis on HPLC and each assayed for biological activities at increasing concentrations in the alkalinization assay to determine the concentration of each that caused maximal activity. FIG. 4 shows the concentration dependence of synthetic AtPep peptides deduced from the seven members of the AtproPep1 gene family in the alkalinization assay. All peptides except those derived from At1g09980 gene and the unannotated gene were fully active at about 2.5 nM concentrations. The two genes with diminished activity were from Subfamily II, which reside relatively close together on Chromosome V. All seven synthetic peptides competed with the 125I1-Tyr-AtPep1 analog for binding with Arabidopsis suspension cultured cells, with a pattern similar to their biological activities in the alkalinization response. As in the alkalinization assay, the peptides derived from the At5g09980 gene and the unannotated gene were much weaker competitors than peptides derived from the other genes.
Example 3
Shorter Peptides from the C-Terminus of AtPep1 Possess Substantial Defense Signal Peptide Activity
Analogs of AtPep1 from the C-terminus of AtPep1 were synthesized and assayed in the alkalinization assay. One mL aliquots of 4 day old Arabidopsis cells were allowed to equilibrate on an orbital shaker at 180 rpm for one hour. A 10 μL aliquot of each peptide solution was added to the cells. Peptide concentrations of 0.25 nM, 2.5 nM, and 25 nM were tested. After 20 min, the pH of the media was recorded. The results are shown in FIG. 5. An analog of AtPep1 missing the carboxy-terminal amino acid was completely inactive, whereas deletions from the amino-terminus of the peptide resulted in a sequential reduction in activity, until peptides with 9 carboxy-terminal amino acids remaining (SSGRPGQHN (SEQ ID NO: 75)) were inactive. A peptide with 10 carboxy-terminal amino acids remaining had substantial defense signal peptide activity at the 25 nM peptide concentration, causing a change in pH of over 0.20 units, and longer analogs had progressively greater activity. A peptide consisting of only the 15 C-terminal amino acids was nearly as active as the native peptide at approximately 2.5 nM and had substantial activity even at the lowest concentration tested. It is expected that substantial defense signal peptide activity will be retained by analogs of other defense signal peptides that comprise sequences from the C-terminus of the peptides.
Example 4
Alanine Substitutions in Residues of a 15-mer Analogs from the C-Terminus of AtPep1
The 15-mer from the carboxy-terminus of AtPep1 (RGKEKVSSGRPGQHN (SEQ ID NO: 78)) was substituted with alanine at each position to assess which amino acids were necessary for the alkalinizing activity. FIG. 6 shows the effect of these single alanine amino acid substitutions on the activity of the 15-mer peptide in the alkalinization assay. The set of substituted 15-mer peptides was assayed using four-day-old Arabidopsis cells. Ten ml of each peptide (2.5 pmoles) was added to 1 ml of cells to make a final concentration of 2.5 nM. After 20 min, the pH of the media was recorded. The data is the average of three separate experiments.
A Ser to Ala substitution at position 7, counting from the amino-terminus of the 15-mer, and a Gly to Ala substitution at position 9, exhibited little activity. Computer modeling predicted that these two amino acids would be involved in a beta-turn within the peptide region of -SSGR- (SEQ ID NO: 79) (compare with residues 15-18 of the sequence of AtPep1 shown in Table 1). Substituting Ala for Ser (-ASGR-) (SEQ ID NO: 80) abolished the predicted turn and severely abolished activity (half-maximal activity at  ̃250 nM), while substituting Ala for Gly was even less active (half-maximal activity of >250 nM. However, neither of these analogs were able to compete with the non-substituted 15-mer for receptor binding, indicating that the structural changes in this region may have severely modified the conformation without competing for the receptor binding site. Peptides with alanine substitutions at all residues were synthesized and assayed, with most showing no differences in activity than the native AtPep1.
Example 5
Identification of Additional Plant Defense Signal Peptides from Various Dicot and Monocot Plant Species
Additional searching of nucleotide sequence databases has revealed additional plant defense signal peptides, shown in Table 2. In addition, it was discovered that a single precursor gene may encode multiple plant defense signal peptides. Additional plant defense signal peptides shown in Table 3 are encoded by such precursor genes.
Identification of Orthologs of the proAtPep1 Gene in Other Plant Species.
To search for orthologs to the proAtPep1 gene in other plants, the NCBI protein query versus translated database (TBLASTN version 2.2.7) search algorithm (Altschul et al. 1997) was employed to search both plant genomic sequence and EST databases. For these searches no search filter was used and the expect value was set for either 10,000 or 20,000. Additionally, each individual plant EST database maintained by the Institute of Genomic Research (TIGR) was searched using the algorithm TBLASTN 2.0 MP (Gish) with no filter and an expect value of 10,000. Each member of the Arabidopsis proAtPep1 family was used as the input sequence for the database searches, and as orthologs in other plants were identified, they were also used as input sequences to search for other orthologous genes.
In order for a gene found in database searches to be classified as a proAtPep1 ortholog, the sequence had to fulfill several criteria. First, the gene had to encode a small protein—the proAtPep1 orthologs identified encode proteins ranging in size from 95 amino acids (canola) to 151 amino acids (rice). Next, each ortholog had to have a region in the encoded precursor protein that was glutamate or aspartate and lysine or arginine rich (designated E/DK/R repeats). The precursor region of the protein was required to contain some variation of the amphipathic helical hydrophobic/cationic 1-5-10 motif. Finally, each predicted protein was required to have several conserved characteristics in the carboxyl region from which the active peptide would likely be cleaved. A high concentration of cationic charges in the amino end of the peptide region was a criterion, and in the carboxyl end a conserved glycine residue at position C-7 was required. In addition to these obligatory elements, several distinguishing features helped identify a gene as orthologous, but were not absolutely required. Most proteins encoded by the orthologs have repetitive serine residues in the precursor region. Additionally, in most predicted proteins a conserved asparagine is found as the carboxyl-terminal amino acid (C-1 position), immediately followed by a stop codon. Also, two residues prior to the conserved glycine in the peptide, all but one of the predicted proteins contain a serine or threonine residue.
Predicting Secondary Structure of Proteins Encoded by proAtPep1 and Homologs.
To predict secondary structure of the proAtPep1 protein and predicted proteins encoded by homologous genes, the SCRATCH server from the Institute for Genomics and Bioinformatics at the University of California at Irvine, Calif. was used. This server simultaneously integrates predictions from four different programs into a single output. One of these programs is SSpro version 2.0 (Pollastri et al. 2002a), which predicts three different classes of secondary structure; helix, strand, or neither. A second program SSpro8 (Baldi et al. 1999) further refines predictions into eight classifications, including three different types of helix, extended strand, β-bridge, turn, bend or none of the above. To predict whether the number of contacts each residue is involved in is above or below average for that residue, the program CONpro (Pollastri et al. 2001) was used. Finally, the ACCpro program (Pollastri et al. 2002b) was employed to estimate the relative solvent accessibility of each residue.
FIG. 7 shows nucleotide sequence information for precursors of the novel plant signal defense peptides of Table 2.
FIG. 8 shows nucleotide sequence information for precursors of the novel plant defense signal peptides of Table 3.
FIG. 9 shows an amino acid sequence alignment of predicted proAtPep1 orthologs (A) and a gene domain model for proATPep1-like genes (B).
Example 6
PROPEP Family Regulation of Pathogen Defense in Arabidopsis thaliana 
AtPep1 is a 23 amino acid peptide derived from the carboxyl terminus of a precursor protein encoded by the PROPEP1 gene of Arabidopsis thaliana. Expression of the PROPEP1 gene is induced by methyl jasmonate and wounding, and the AtPep1 peptide specifically interacts with an LRR-RK, PEPR1, to regulate expression of genes encoding the pathogen-defense proteins PDF1.2 (plant defensin) and PR-1 (pathogenesis response protein 1). Transgenic plants constitutively expressing the PROPEP1 gene also express PDF1.2 and PR-1 at levels higher than in wild type plants and are more resistant to the root pathogen Pythium irregulare. Arabidopsis encodes five other PROPEP genes, raising the question of whether these genes act redundantly, synergistically or differ functionally. Expression studies of the individual PROPEP gene family members reveal differential induction in response to pathogens, elicitors and the defense-related hormones methyl salicylate, methyl jasmonate and ethylene. The AtPep peptides encoded by these PROPEP genes differentially regulate expression of PDF1.2 and PR-1. To better understand the regulatory role of individual PROPEP genes, we are employing microarray analysis of global gene expression patterns in transgenic plants overexpressing the different PROPEP genes. Additionally, we are investigating the role of individual AtPeps PROPEP genes in salicylate-associated defense responses by studying resistance of PROPEP-overexpression plants to infection with Pseudomonas syringae pv. tomato DC3000. Based on our studies we hypothesize that peptides encoded by the PROPEP gene family enhance pathogen defense responses by acting as endogenous signals to amplify defense signaling initiated by pathogens through both the jasmonate/ethylene and salicylate pathways.
Example 7
Tobacco Transgenic Cells Expressing AtPEPR1
To further investigate the role of AtPEPR1 as the AtPep1 receptor, gain-of-function experiments were carried out with tobacco suspension-cultured cells that do not respond to AtPep1 by alkalinating the cell medium. Tobacco cells were transformed with a CaMV-35S-AtPEPR1 gene to determine whether the foreign receptor protein was targeted to the cell surface, where, in response to AtPep1, it would activate intracellular signaling to effect the alkalinization of the medium of the transgenic tobacco cells. Expression of the AtPEPR1 gene was confirmed by RT-PCR analysis of RNA from three independently transformed tobacco cell lines. All three of the tobacco lines responded to the addition of nanomolar concentrations of AtPep1 by producing a strong, reproducible alkalinization of the cell culture medium, whereas no alkalinization was detected in the culture medium of wild type cells challenged with AtPep1. These results not only confirm the identity of AtPEPR1 as the AtPep1 receptor, but they indicate that the receptor-mediated intracellular signaling pathway in tobacco cells that regulates alkalinization in response to peptide ligands can accommodate signaling by AtPEPR1 and its peptide ligand. AtPep2-7 was also supplied to the transgenic tobacco cells expressing AtPEPR1. At 2.5 nanomolar, AtPep2, AtPep3, Atpep5 and Atpep6 caused a change in pH of up to 0.6 units. But AtPep4 and AtPep7 did not increase medium pH. This is a similar pattern to their activities in the alkalinization response and in the competition with 125I1-Tyr-AtPep1 analog for binding with Arabidopsis suspension-cultured cells.
Expression Analysis of AtPEPR1 and AtPEPR2.
In the Arabidopsis genome, there is a gene encoding a receptor kinase, which is very similar to AtPEPR1 (At1g73080) with 72% amino acid similarity for the entire sequence and was designated as AtPEPR2 (At1g17750). In order to elucidate the role of AtPEPR2 in AtPeps signal transduction, the gene expression patterns were analyzed in comparison with AtPEPR1. Northern hybridization and quantitative RT-PCR analysis showed that the expression patterns of AtPEPR1 and AtPEPR2 were very similar. The transcripts of AtPEPR1 and AtPEPR2 were induced by wounding, methyl jasmonate and AtPep supplying within 30 min, but not by methyl salicylate or aminocyclopropanecarboxylic acid, a precursor of ethylene. Of the 6 AtPeps, AtPep1, AtPep2 and AtPep3 were strong inducer of AtPEPR1 and AtPEPR2 transcripts.
The induction of AtproPep1 and MPK3 Gene Expression Upon Supplying AtPep1 Using T-DNA Insertional Mutants.
In addition to T-DNA insertional mutants of AtPEPR1 (SALK—059281 and SALK—014538), T-DNA insertional mutants of AtPEPR2 (SALK—036564 and SALK—004447) were obtained from The Arabidopsis Information Resource (http://www.arabidopsis.org/index.jsp). In order to elucidate how much AtPEPR1 and AtPEPR2 contribute to the AtPeps perception, SALK—014538 and SALK—004447 were crossed to obtain a double mutant. Another double mutant of AtPEPR1 and AtPEPR2 (SALK—059281 and SALK—036564) was provided by Dr. Frans Tax (Arizona University). It was confirmed that full length mRNAs of AtPEPR1 and/or AtPERP2 are not transcribed in the mutant lines by RT-PCR.
Using AtproPep1 and MPK3 as marker genes, the response to AtPep1 of the T-DNA insertional mutants was analyzed. Two-week-old seedlings grown in liquid medium were supplied with 10 nM AtPep1 for 30 min. The AtPEPR1 mutants (SALK—059281 and SALK—014538) showed more than a 50% reduction of AtproPep1 and MPK3 induction when supplied with AtPep1, but still responded, indicating that AtPEPR1 is not a sole receptor for AtPep1. The AtPEPR2 mutants (SALK—036564 and SALK—004447) did not show the clear reduction of the response to AtPep1. However, the double mutants of AtPEPR1 and AtPEPR2 completely lost the response when supplied with AtPep1. These data indicated that AtPEPR1 is the primary receptor for AtPep1, and AtPEPR2 is also involved in AtPep1 perception and signal transduction in Arabidopsis. 
Example 8
Identification of Orthologs of the AtPEPR Gene in Other Plant Species
Proteins and putative proteins, which are highly homologous to AtPEPR, were searched from several databases including NCBI, PlantGDB, and Phytozome by the BLAST program. Secondly, a phylogenic tree was made using these proteins and members of Arabidopsis leucine-rich repeat receptor-like kinase (LRR-RLK) X1 subfamily. Then the proteins, which belong to same clade with AtPEPR1 and AtPEPR2, were selected as AtPEPR homologues.
FIG. 10 is a simplified version of the AtPEPRs homolog phylogenic tree. Among them, OsPEPR1 and OsPEPR2 were confirmed their ability for perception of monocot defense peptides. Amino acid sequences were compared by the Clustal W program and phylogenic tree was drawn by the Tree View program. AtPEPR homologues are indicated in color. The numbers indicate bootstrap values. Arabidopsis BRI1 was used as an out group. At, Arabidopsis thaliana. Pt, Populus trichocarpa. Vv, Vitis vinifera. Zm, Zea mays. Sb, Sorghum bicolor, Os, Oriza saliva. Gm, Glycine max, In, Ipomoea nill. 
The plant LRR-RLK family is a very large family, and more than 200 and 600 LRR-RLKs were predicted in the Arabidopsis and rice, respectively. All of them have an N-terminal signal sequence, an extracellular LRR region, two sets of cystein pairs around the LRR region, a transmembrane region, and an intracellular protein kinase domain. LRR regions are repeats of a leucine-rich motif consisted of 24 amino acids, and vary in length.
FIG. 11 is an amino acid alignment of AtPEPR homologues. Conserved amino acids are written in white letter with black background, and conserved similar amino acids are written with gray background. The intracellular protein kinase domain was indicated by red ruler.
FIG. 12 is a consensus amino acid sequence of the protein kinase domain of AtPEPRs homologues.
Example 9
AtPEPR1 is a Receptor for all AtPep Peptides and AtPEPR2 for AtPep1 and AtPep2
AtPEPR1 and AtPEPR2 mutants are unable to respond to AtPEP1. To further examine the importance of AtPEPR1 and AtPEPR2 in AtPep1 signaling, the expression level of WRKY transcription factor genes, WRKY22, WRKY29, WRKY33, WRKY53 and WRKY55, were monitored by qRT-PCR after supplying AtPep1 to wild type Arabidopsis and T-DNA insertional mutants of AtPEPR1 and AtPEPR2. These WRKY genes were reported to be induced by the fungal PAMP, chitin (Wan et al., Plant Cell 20: 471-481, 2008), and the bacterial PAMPs, flg22 and elf18 (Zipfel et al., Cell 125:749-760, 2006). Two-week-old seedlings grown in liquid medium were incubated with AtPep1 (10 nM) for 30 min and subjected to qRT-PCR analysis. WRKY33, WRKY53, WRKY22, WRKY29 and WRKY55 were highly induced by 10 nM AtPep1 within 30 min in wild type. In pepr2-1 and pepr2-2 (SALK—059281 and SALK—014538, respectively) seedlings, these genes were induced by AtPep1 to similar levels as wild type seedlings. A 50% lower induction of these genes by AtPep1 was observed with pepr1-1 and pepr1-2 (SALK—036564 and SALK—004447) seedlings. In the double mutants, pepr1-1/pepr2-1 and pepr1-2/pepr2-2, the responses were completely abolished. We also examined the effect of supplying AtPep1 to the double mutants on the expression of the antimicrobial protein genes, defensin (PDF1.2) and pathogenesis related protein (PR-1) genes, since AtPep peptides are known to induced these genes (Huffaker and Ryan, Proc. Natl. Acad. Sci. USA 104: 10732-10736, 2007). Since the expression levels of PDF1.2 and PR-1 gene in Arabidopsis seedlings grown in liquid medium were already high, the method of supplying AtPep1 was changed. Four-week-old Arabidopsis plants grown in soil were sprayed with 1 μM AtPep1 in 0.01% Silwet L-77, and subjected to qRT-PCR analysis after 6 hours. The PDF1.2 and PR-1 genes were induced by 30- and 60-fold, respectively in wild type plants, and were not induced by AtPep1 in the double mutants.
AtPEPR2 Binds to AtPep1.
The results shown above strongly suggested that AtPEPR2 was also a receptor for AtPep1 in addition to AtPEPR1, whose ability to bind AtPep peptides was confirmed by loss of function and gain of function experiments (Yamaguchi et al., Proc. Natl. Acad. Sci. USA 103: 10104-10109, 2006). In these experiments, photoaffinity labeling using microsomal fractions of the T-DNA insertional mutants of AtPEPR1, pepr1-1 and pepr1-2, did not show any AtPep1 binding proteins (Yamaguchi et al., Proc. Natl. Acad. Sci. USA 103: 10104-10109, 2006). However, if the protein level of AtPEPR2 or binding capacity of AtPEPR2 to AtPep1 is much lower than AtPEPR1, it would be difficult to detect binding between AtPEPR2 and AtPep1 using Arabidopsis plants grown under normal condition. To clarify whether AtPEPR2 binds to AtPep1, the AtPEPR2 coding region was fused to the CaMV 35S promoter and introduced into tobacco suspension-cultured cells by Agrobacterium. Tobacco cells expressing AtPEPR1 and GUS genes were also created as positive and negative controls, respectively. RT-PCR analysis revealed that transgenic cells selected on kanamycin-containing medium expressed each transgene. These transgenic lines were analyzed for alkalinizing activity as previously described (Pearce et al., Proc. Natl. Acad. Sci. USA 98, 12843-12847, 2001). The cells expressing AtPEPR1 and AtPEPR2 caused alkalinization of the media in response to AtPep1, whereas wild type tobacco and GUS expressing cells did not respond to AtPep1. The level of response to AtPep1 for the AtPEPR2-expressing cells was similar to that of AtPEPR1-expressing cells and to Arabidopsis cells (Yamaguchi et al., Proc. Natl. Acad. Sci. USA 103: 10104-10109, 2006, Huffaker et al., Proc. Natl. Acad. Sci. USA 103: 10098-10103, 2006), with a maximal change of 1-1.2 pH units and a half-maximal peptide activity of 0.25 nM.
For final confirmation, a photoaffinity-labeling experiment was performed using the transgenic tobacco cells. The transgenic cells were incubated with 0.25 nM 125I1-azido-Cys-AtPep1 (Yamaguchi et al., Proc. Natl. Acad. Sci. USA 103: 10104-10109, 2006) and irradiated with UV-B to cross-link the AtPep1-binding proteins. After separation of extracted proteins by SDS-PAGE, the labeled proteins were detected on X-ray film. A major protein band of 170 kDa, consistent with the size of AtPEPR1 in Arabidopsis (Yamaguchi et al., Proc. Natl. Acad. Sci. USA 103: 10104-10109, 2006), and 150 kDa were labeled in AtPEPR1 and AtPEPR2 expressing cells, respectively, but not in the cells incubated with 50 nM unlabeled AtPep1 as a competitor in addition to 125I1-azido-Cys-AtPep1. The intensity of the 170 kDa protein was greater than that of the 150 kDa protein, requiring a one-tenth dilution of extract from AtPEPR1 expressing cells for use in SDS-PAGE to give comparable intensities to undiluted AtPEPR2 expressing cell extract. Since no specific labeling was detected in wild type tobacco and GUS expressing cells, the proteins labeled specifically by 125I1-azido-Cys-AtPep1 in AtPEPR1 and AtPEPR2 expressing cells were products from the AtPEPR1 and AtPEPR2 genes.
AtPEPR2 Contributes to AtPep1 and AtPep2 Perception.
The response to all AtPep peptides in transgenic tobacco cells expressing either AtPEPR1 or AtPEPR2 was analyzed by the medium alkalinization assay. As reported previously, for AtPEPR1 expressing cells, AtPep1, AtPep2, AtPep3, AtPep5 and AtPep6 showed strong activity with a maximal change of 1.2 pH units and a half-maximal activity of ≈0.25 nM, and AtPep4 and AtPep7 showed weaker activity with a half-maximal activity of ≈2.5 nM. In contrast, only AtPep1 and AtPep2 showed strong activity to the AtPEPR2 expressing cells with a maximal change of 1.2 pH unit and a half-maximal activity at ≈0.25 nM with the other peptides showing very weak or no activity, indicating that AtPEPR2 contributes to AtPep1 and AtPep2 perception by itself.
Example 10
AtPep Peptides Pre-Infiltration Reduces Symptom Development by P. syringe 
Overexpression of AtPROPEP1 showed increased resistance to a root pathogen, Pythium irregulare (Huffaker et al., Proc. Natl. Acad. Sci. USA 103: 10098-10103, 2006). The resistance to P. irregulare requires the JA signaling pathway in Arabidopsis (Staswick et al, Plant J. 15: 747-754, 1998), and AtPep peptides induced PDF1.2, a marker gene for the JA response (Huffaker et al., Proc. Natl. Acad. Sci. USA 103: 10098-10103, Huffaker and Ryan, Proc. Natl. Acad. Sci. USA 104: 10732-10736, 2007). In addition to PDF1.2, supplying AtPep peptides also induced expression of the PR-1 gene, a marker gene for the SA response (Huffaker et al., Proc. Natl. Acad. Sci. USA 103: 10098-10103, Huffaker and Ryan, Proc. Natl. Acad. Sci. USA 104: 10732-10736, 2007), suggesting that AtPep peptides may be involved in the SA signaling pathway, which increases resistance to Pseudomonas syringae. In order to examine this possibility, Arabidopsis plants were inoculated with P. syringae pv. tomato DC3000 (Pst DC3000) with or without AtPep1 pre-infiltration. Pst DC3000 was extracted from the infected leaves 4 days after infection and colony forming units (cfu/cm2) were calculated. Pre-infiltration of a 10 nM solution of AtPep1 reduced Pst DC3000 growth in leaves and this reduction was concentration-dependent. AtPep1 pre-infiltration was more effective than flg22 pre-infiltration, which was reported to cause growth reduction of Pst DC3000 (Zipfel et al., Nature 428: 764-767, 2004). The reduction of Pst DC3000 growth was not observed when [A17]AtPep1 (9-23), an inactive derivative of AtPep1 (Pearce et al., Peptides. 29: 2083-2089, 2008) was pre-infiltrated. The reduction of Pst DC3000 growth was also not observed when the double mutant, pepr1-1/pepr2-1, was pre-infiltrated with AtPep1. These results indicated that reduction of Pst DC3000 growth by AtPep1 was specific. Pre-infiltration of AtPep2-6 also attenuated Pst DC3000 growth to different degrees. The attenuation of Pst DC3000 by AtPep1 pre-infiltration was differentially diminished in leaves of JA deficient (fad3, 7, 8), SA signaling (npr1-1) and SA biosynthesis (sid2-2) mutants, indicating that JA and SA signaling pathway are required for full activity of AtPep1.
To assess the importance of AtPEPR1 and AtPEPR2 in the resistance to Pst DC3000 after infiltration of AtPep1, T-DNA insertional mutants were inoculated. When 1 μM AtPep1 was infiltrated into leaves one day prior to Pst DC3000 inoculation, the size of the necrotic regions were reduced in both wild type and single mutant lines, pepr1-1 and pepr2-1. On the other hand, AtPep1 pre-infiltration did not affect symptom development by Pst DC3000 infection in double mutants, pepr1-1/pepr2-1. The bacterial growth was reduced to 1/100 in wild type and pepr2-1, and to 1/25 in pepr1-1 by AtPep1 pre-infiltration, but no decrease in colony growth was observed with the double mutants. Similar results were obtained when another set of mutant lines, pepr1-2, pepr2-2 and pepr1-2/pepr2-2, was used. These results were consistent with the results obtained in the analysis of defense gene induction by AtPep1 in the mutant lines, and indicated that AtPEPR1 contributed more to AtPep1 signaling than AtPEPR2.
Example 11
Rice Homologues of AtPEPR1 are Receptors for Rice Peptides
We identified rice homologues of AtPEPR1 in the Rice Annotation Project database (see the website located at www.rapdb.dna.affrc.go.ip). The rice homologues, OsPEPR1 (Os08g0446200) and OsPEPR2 (Os08g0446400), have 60% and 62% amino acid similarities, respectively. The OsPEPR1 has 26 LRRs in the extracellular region, which is the same as AtPEPR1. The OsPEPR2 has a shorter extracellular region with 22 LRRs. Transgenic tobacco cells expressing either OsPEPR1 or OsPEPR2 were created to examine the possibility of receptor binding to monocot peptides. OsPep3a, OsPep3b, OsPep3c, ZmPep1 and ZmPep4a were supplied to suspension cultured transgenic tobacco cells, and medium pH was measured. OsPep3a, OsPep3b, ZmPep1 and ZmPep4a caused medium alkalinization in OsPEPR1 expressing cells with a 1.2 pH unit increase and a half-maximal activity of 0.25 nM. OsPep3c did not show any activity to OsPEPR1 expressing cells. However, OsPep3c showed weak activity to OsPEPR2 expressing cell with a 0.5 pH unit increase when the cells were incubated with peptide at 250 nM. These results indicated that OsPEPR1 and OsPEPR2 are receptors for monocot peptides.
Example 12
Soybean Homologues of AtPep Peptide and AtPEPR
Precursor gene expression of soybean homologues of the AtPep peptide, GmPROPEP1, GmPROPEP2 and GmPROPEP3, were analyzed by semi-quantitative RT-PCR. GmPROPEP2 and GmPROPEP3 were expressed in leaves, stems and roots. GmPROPEP1 was expressed mainly in roots, and very weakly in leaves and stems. The transcripts of GmPROPEP1 increased 4-fold 1 hour after supplying MeJA and 3-fold 8 hours after wounding. The transcripts of GmPROPEP2 increased 5-fold 2 hours after supplying MeJA. GmPep1-3 also affected the precursor gene expression. The GmPROPEP1 was induced by infiltration of GmPep1-3 (50 nM), and GmPROPEP2 was induced by GmPep2 (50 nM) and GmPep3 (50 nM) infiltration.
To examine whether GmPep1-3 are involved in defense responses, expression of soybean defense-related genes were analyzed by semi-quantitative RT-PCR after GmPep1-3 infiltration. The transcripts of D6aH/CYP93a1, a biosynthetic enzyme of the soybean phytoalexin glyceollin (Schopfer et al., FEBS Lett. 432: 182-1860, 1998), were induced 2 to 3-fold by GmPep1-3 infiltration. The expression of three chitinase genes, Chib1-b, Chi 111 and Chi 1 (Watanabe et al., Biosci. Biotechnol. Bioche. 63: 251-256, 1999, Gijzen et al., J. Exp. Bot. 52: 2283-2289, 2001), was also induced 3 to 15-fold by GmPep1-3 infiltration.
We found soybean homologues of AtPEPR1 and AtPEPR2, GmPEPR1a (Glyma10g33970), GmPEPR b (Glyma20g33620) and GmPEPR2 (Glyma15g00360), which have more than 60% amino acid similarities with AtPEPR1, in the soybean genome database (see the website located at www.phytozome.net/soybean.php). The GmPEPR1a and GmPEPR1b have 26 leucine-rich repeats in the extracellular region, similar to the AtPEPR1. The GmPEPR2 has a shorter extracellular region with 25 leucine-rich repeats, similar to AtPEPR2. A phylogenetic analysis of amino acid sequences revealed that GmPEPRs are more closely related to AtPEPRs than OsPEPRs, indicating that soybean homologues of AtPEPR1 and AtPEPR2 are possible receptors for soybean defense peptides.

## Claims

1. A transgenic plant comprising a recombinant polypeptide consisting of
i) a consensus amino acid sequence





wherein
X is any amino acid;
residue 19 is Pro or Gly;
residue 20 is Pro or Gly;
and residues 1-14 include at least one Lys or Arg

or

ii) an amino acid carboxy terminal fragment of said consensus amino acid sequence comprising at least 10 amino acids; wherein the recombinant polypeptide has defense signal activity.

2. A method of treating a plant or a seed to increase plant defense signaling activity, comprising
applying to a surface of said plant or said seed a polypeptide consisting of
i) a consensus amino acid sequence





wherein
X is any amino acid;
residue 19 is Pro or Gly;
residue 20 is Pro or Gly;
and residues 1-14 include at least one Lys or Arg

or

ii) an amino acid carboxy terminal fragment of said consensus amino acid sequence comprising at least 10 amino acids; wherein the recombinant polypeptide has defense signal activity.

3. A plant comprising a polypeptide applied to a surface of said plant, said polypeptide consisting of
i) a consensus amino acid sequence





wherein
X is any amino acid;
residue 19 is Pro or Gly;
residue 20 is Pro or Gly;
and residues 1-14 include at least one Lys or Arg

or

ii) an amino acid carboxy terminal fragment of said consensus amino acid sequence comprising at least 10 amino acids.

4. A seed comprising a polypeptide applied to a surface of said seed, said polypeptide consisting of
i) a consensus amino acid sequence





wherein
X is any amino acid;
residue 19 is Pro or Gly;
residue 20 is Pro or Gly;
and residues 1-14 include at least one Lys or Arg

or

ii) an amino acid carboxy terminal fragment of said consensus amino acid sequence comprising at least 10 amino acids; wherein the recombinant polypeptide has defense signal activity.