WO2010087805A2 - Insult resistant plants and methods of producing and using the same - Google Patents

Abstract

A method of increasing resistance to an insult in a plant, particularly a pathogen.

Description

Insult Resistant Plants and Methods of Producing and Using the Same Inventors: Christian A. Voigt and Shauna Somerville

FIELD OF THE INVENTION

[0001] The present invention relates to nucleic acids encoding callose synthase and the expression of callose synthase. More particularly, the present invention relates to increased presence of callose synthase in a plant cell to provide improved resistance and response to an insult to the cell, such as the insult of a pathogen attack.

RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application 61/129,658, filed July 10, 2008, which is incorporated herein in its entirety.

BACKGROUND

[0003] As human population increases, space for growing plants becomes less to accommodate living space but demand also increases especially in terms of providing nutrients. Consequently, the crops that are grown must be better cared for to avoid ruin and destruction by pathogens.

[0004] For decades, reliance has been placed upon the use of chemicals such as organophosphates to ward of potential pathogens. However, studies have revealed detrimental long term effects from human exposure to these chemicals. Consequently, research has shifted to other means of protecting a plant from a pathogen.

[0005] One such method is the use of genetically modified plants. Plants can be modified to express proteins hazardous to a pathogen or to express oligonucleotides that are fatal to a pathogen through the process of RNAi (ribonucleic acid interference). Processes for transforming a plant with exogenous nucleotides are known in the art. However, much of the population is wary of consuming these plants on the basis that the proteins are potentially hazardous for human consumption, or that the RNAi will affect human cells as well. [0006] In spite of the need to protect plants, plants do typically possess an endogenous response to a wound or an insult from a pathogen. It has previously been reported that knock-out or a reduced level of expression of the callose synthase gene, GSL5 (also referred to as PMR4 (powdery mildew resistant)) will improve a plant's resistance to an insult, such as a pathogen attack. Callose synthase is an enzyme that aids in the production of the β-glucan, callose.

[0007] Callose is a polysaccharide of D-glucose. Glucose polysaccharides are abundant in all types of cells. Callose is marked by the 1,3 linkage of glucose molecules. Callose is traditionally laid down at plasmodesmata, at the dividing cell plate and during pollen development. Accordingly, strategies for improving plant resistance have been targeted toward reducing the expression of callose synthase.

SUMMARY OF THE INVENTION

[0008] The present invention is based on the unexpected finding that increasing expression of callose synthase improves the resistance of a plant cell to an insult, such as a pathogen attack or wound. The present invention provides that over-expression or constitutive expression of callose synthase improves plant pathogen resistance. In one embodiment, the callose synthase is derived from a plant. In one embodiment, the callose synthase is derived from Arabidopsis. The callose synthase may be GSL5 derived from Arabidopsis, or a homolog or a functional equivalent thereof derived from other plant species.

[0009] The present invention provides for over-expression of callose synthase in a plant cell. Callose synthase may be over-expressed by introducing to a plant cell a nucleic acid that encodes callose synthase operably linked to a promoter. The promoter may be constitutively active. The promoter may be tissue-specific. The callose synthase may be encoded by GSL5.

[0010] The present invention provides nucleic acids that encode callose synthase. The nucleotide sequences may include both the naturally occurring sequences as well as mutant (variant) forms. [0011] The present invention also provides for polypeptides comprising callose synthase and fragments thereof that retain or demonstrate the enzymatic activity of native, full- length or "wild-type" callose synthase.

[0012] The present invention provides for over-expressed callose synthase in a plant cell. The callose synthase may be derived from any plant. The callose synthase may be derived from Arabidopsis. In one embodiment, the callose synthase is encoded by the Arabidopsis gene GSL5, or the comparable equivalent thereof derived from other plant species.

[0013] The present invention provides transgenic and transformed plant cells and plants wherein the cells express callose synthase. Expression of callose synthase in a transformed plant cell may be under the control of a constitutively active promoter. The present invention provides for transforming a plant cell with expression vectors having a linear or circular nucleic acid molecule comprising a polynucleotide encoding a callose synthase protein operably linked to additional nucleotides that provide for its expression. In some embodiments, the nucleic acid encoding the callose synthase becomes integrated into the host cell's genome.

[0014] The present invention disclose a method for increasing the rate of callose deposit in response to an injury comprising increasing the presence of callose synthase in a cell. The present invention provides for methods of protecting a cell from an insult comprising over-expressing callose synthase in the cell. The methods of the present invention provide for improved immediate and early response to an insult to a plant cell. The present invention provides that by providing a cell with over-expressed or constitutively expressed callose synthase, the cell can demonstrate an improved immediate and early response to an insult to the cell. The callose synthase may be encoded by GSL5 or a homolog or functional equivalent thereof. The methods of the present invention comprise contacting a plant cell with a nucleotide that encodes callose synthase. The nucleic acid encoding callose synthase may be under the control of a promoter. The nucleic acid encoding callose synthase may be under the control of a constitutively active promoter. The promoter may be tissue-specific. In some embodiments the cell is a plant cell. The plant cell may be contained in a plant. [0015] The present invention provides a method of increasing disease-resistance of a plant cell to a pathogen. The pathogen may be a fungus, nematode or oomycete. In some embodiments, the pathogen is powdery mildew. In other embodiments, the pathogen is selected from Golovinomyces cichoracearum, Mycosphaerella musicola, Fusaήum graminearum, Blumeria graminis f. sp. hordei, Puccinia hordei, Puccinia graminis f. sp. tritici, Puccinia striiformis, Pyrenophora teres, Rhynchospoήum secalis, Erysiphe necator, Colletotrichum graminicola, Puccinia sorghi, Ustilago maydis, Blumeria graminis f. sp. avenae, Phytophthora infestans, Magnaporthe grisea, Oidium neolycopersici, Leveillula taurica, Phakopsora pachyrhizi, Phytophthora sojae, Puccinia melanocephala, Peronosclerospora sacchari, Ustilago scitaminea, Blumeria graminis f. sp. tritici, Puccinia graminis f. sp. tritici, Tilletia indica.

[0016] The present invention also provides methods to increase resistance of a plant cell to a pathogen following sustaining a wound. The present invention provides methods of limiting the formation of blemishes at the site of a wound to a plant. The present invention provides for enhancing the defensive mechanism of action of a plant against infection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows the modification of callose synthase in the model plant Arabidopsis thaliana.

[0018] Figure 2 shows the disease phenotype seven days post-inoculation with powdery mildew.

[0019] Figure 3 shows plant biomass ten days post-inoculation with powdery mildew.

[0020] Figure 4 shows callose synthase activity at early stages of infection with powdery mildew.

[0021] Figure 5 shows callose deposition six hours post-inoculation with powdery mildew.

[0022] Figure 6 shows callose deposits at early stages of infection with powdery mildew.

[0023] Figure 7 shows fungal penetration success at early stages of infection with powdery mildew. [0024] Figure 8 shows callose deposition and fungal growth seven days post-inoculation with powdery mildew.

[0025] Figure 9 shows regulation of callose synthesis by phosphorylation.

[0026] Figure 10 shows the impact on second generation biofuels, particularly with relation to biofuels from plant biomass.

[0027] Figure 11 shows a phylogenetic tree based on amino acid sequence to GSL5. As a result, GSL5-like genes can be easily identified in other plant species. Plants species are as follow: Arabidopsis thaliana: AtGSL, 12 sequences, Brachypodium distachyon: BdGSL, 11 sequences, Rice, Oryza sativa: OsGSL, 11 sequences, Polar, Popular trichocarpa: PtGSL, 7 sequences, Sorghum bicolor. SbGSL, 12 sequneces. the sequences contained in the boxed area further only have 1 intron in their genomic DNA.

DETAILED DESCRIPTION

[0028] The present invention provides novel means for plants to respond to an insult. The present inventors have discovered that operably linking a gene that produces callose synthase to a constitutively active promoter in a plant cell yields improved response to an insult to the plant cell. The callose synthase may be encoded by the GSL5 gene from Arabidopsis or a comparative gene from another species of plant.

[0029] The present invention provides methods of increasing the resistance and response to an insult. The insult may be a pathogen. In some instances, the present invention provides methods of increasing the early response to an insult and increases the rate and amount of callose deposited at the site of the insult.

[0030] As used herein with respect to nucleic acids, polypeptides and proteins, the term "isolated" may include a polypeptide or nucleic acid that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. For example, an isolated callose synthase may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.

[0031] As used herein, the term "variant" may include any polypeptide having an amino acid sequence substantially identical to a polypeptide, or peptide, of the invention, in which one or more residues have been conservatively substituted with a functionally similar residue, and further which displays substantially identical functional aspects of the polypeptides as described herein. Examples of conservative substitutions include substitution of one non-polar (hydrophobic) residue for another (e.g. isoleucine, valine, leucine or methionine) for another, substitution of one polar (hydrophilic) residue for another (e.g. between arginine and lysine, between glutamine and asparagine, between glycine and serine), substitution of one basic residue for another (e.g. lysine, arginine or histidine), or substitution of one acidic residue for another (e.g. aspartic acid or glutamic acid). A variant polypeptide also includes a polypeptide derived from the native protein by deletion (or truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, e.g., genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art. A variant may b a naturally occurring callose synthase polypeptide or a callose synthase polypeptide having amino acid substitutions and functions in the same manner as the wild type callose synthase polypeptide.

[0032] Thus, expressed polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of callose synthase can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art, for example, conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred, e.g., Aliphatic: Glycine (G), Alanine (A), Valine .(V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur- containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q).

[0033] As used herein, a "homolog" may include any polypeptide having a tertiary structure substantially identical to a GSL5 of Arabidopsis which also displays the functional properties of the polypeptides as described herein.

[0034] As used herein, "fusion" may refer to nucleic acids and polypeptides that comprise sequences that are not found naturally associated with each other in the order or context in which they are placed according to the present invention. A fusion nucleic acid or polypeptide does not necessarily comprise the natural sequence of the nucleic acid or polypeptide in its entirety. Fusion proteins may have two or more segments joined together through normal peptide bonds. Fusion nucleic acids may have two or more segments joined together through normal phosphodiester bonds. An example of a fusion protein may be callose synthase or a fragment thereof fused to another protein.

[0035] As used herein, "cleavage" may refer to the severing of an amino acid or nucleotide sequence. By way of example, cleavage may occur with the use of enzymes, such as trypsin and chymotrypsin. By way of further example, nucleotide sequences can be cleaved with the use of restriction endonucleases.

[0036] Callose synthase may be isolated or obtained in substantially pure form. Substantially pure means that the proteins and/or polypeptides and/or peptides are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. In particular, the polypeptides are sufficiently pure and are sufficiently free from other biological constituents of their host cells so as to be useful in, for example, generating antibodies, sequencing, or producing pharmaceutical preparations. By techniques well known in the art, substantially pure polypeptides may be produced in light of the nucleic acid and amino acid sequences disclosed herein. The polypeptide may comprise only a certain percentage by weight of the preparation. The polypeptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems.

[0037] The term "native" or "wild type" gene refers to a naturally occurring gene that may be present in a genome of an untransformed cell, i.e., a cell not having a known mutation.

[0038] A "marker gene" encodes a selectable or screcnable trait.

[0039] A "transgene" refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, e.g., genes that are either heterologous or homologous to the genes of a particular plant to be transformed. Transgenes may also comprise native genes inserted into a non-native organism, or chimeric genes. An "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" or "exogenous" gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.

[0040] Individual substitutions deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are "conservatively modified variations", where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservatively modified variations."

[0041] Callose Synthase

[0042] Callose synthase refers to an enzyme involved in the production of callose. Callose is a non-cellulosic polysaccharide, characterized as a chain of glucose molecules linked through b-1,3 linkages (Gibeaut et al. FASEB J 8:904-917, 1994). As used for the present invention callose synthase includes functional equivalents or homologs thereof capable of performing the enzymatic function of wild-type callose synthase. An example showing of a portion of the callose, β-glucan, is presented below:

[0043] Callose is traditionally generated in the space between the plasma membrane and cell wall of a plant cell. Callose is laid down at plasmodesmata, at the dividing cell plate and during pollen development. Callose is also produced in response to an insult to a plant or a plant cell. Insults include wounding, infection by pathogens, aluminum and abscisic acid. Pathogens may include, eukaryotes, prokaryotes or viruses. Callose, along with celluose (1,4 β D-glucan), are the only polysaccharides known to be made at the plasma membrane of plant cells.

[0044] Callose is typically synthesized in a plant by a callose synthase enzyme that assists in catalyzing the 1,3 β linkage {see, e.g., Verma et al. Plant MoI. Biol. 47: 693- 701, 2001). Twelve genes have been identified in Arabidopsis that encode for callose synthases, identified as GSLl -12 (Richmond et al. Plant Physiol. 124: 495-498 (2001). It has also previously been reported that GSL5 in particular is useful in forming callose in response to a wound such as slicing with a razor (Jacobs et al. The Plant Cell 15: 2503-2513, 2003). However, this same study, as well as others, reported that knocking out the callose synthase function from GSL5 results in improved resistance to pathogen attack, primarily through increased production of salicylic acid production (Jacobs et al. The Plant Cell 15: 2503-2513, 2003; Nishimura et al Science 301 : 969-972, 2003).

[0045] In contrast, the present invention provides that increasing expression of callose synthase improves the resistance of a plant cell to an insult, such as a pathogen attack or wound. The present invention provides that over-expression or constitutive expression of callose synthase improves plant resistance to an insult. The expressed callose synthase may be GSL5 callose synthase derived from Arabidopsis, or a homolog or a functional equivalent thereof derived from other plant species.

[0046] The present invention provides for over-expression of callose synthase in a plant cell. The callose synthase may be over-expressed by introducing into a plant cell a nucleic acid that encodes callose synthase operably linked to a promoter. The promoter may be constitutively active. The promoter may be tissue-specific. The callose synthase may be encoded by GSL5, also known as PMR4 (powdery mildew resistant 4) or a functional equivalent or homolog thereof. The GSL5 may be derived from Arabidopsis or another plant.

[0047] The present invention also provides for callose synthase or functional equivalents or homologs thereof and methods of expressing the same. The present invention also provides for plant cells transformed with callose synthase or functional equivalents or homologs thereof and methods for transforming plant cells with an expression vector for over-expression of callose synthase. The present invention provides methods for protecting plants from an insult and methods for providing increased resistance against an insult by the expression of callose synthase or functional equivalents or homologs thereof. The present invention also provides methods for defending plants from an attack by the expression of callose synthase or functional equivalents or homologs thereof.

[0048] Callose Synthase

[0049] The present invention also provides for polypeptides comprising callose synthase. The present invention also provides for fragments of callose synthase that retain or demonstrate the enzymatic activity of full-length or "wild-type" callose synthase.

[0050] The present invention provides for over-expressed callose synthase in a plant cell. The callose synthase may be derived from Arabidopsis. In other embodiments, the callose synthase is encoded by the Arabidopsis gene GSL5, or the comparable equivalent thereof derived from other plant species.

[0051] As used herein with respect to proteins and polypeptides, the term "recombinant" may include proteins and/or polypeptides and/or peptides that are produced or derived by genetic engineering, for example by translation in a cell of non-native nucleic acid or that are assembled by artificial means or mechanisms.

[0052] A polypeptide fragment or active fragment, as used herein, refers to a callose synthase polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of a full-length callose synthase enzyme polypeptide or a homologous sequence thereof, wherein the fragment retains enzymatic activity. For purposes of the present invention, the degree of identity between two amino acid sequences is determined by the Clustal method (Higgins, CABIOS 5: 151-153 (1989)).

[0053] An isolated or purified callose synthase protein, or enzymatically active fragment thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0054] A callose synthase protein that is substantially free of cellular material includes preparations of callose synthase protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of "contaminating protein." For example, when the callose synthase protein or active portion thereof is recombinantly produced, preferably, culture medium represents less than about 30%, 20%, 10%, or 5% of the volume of the protein preparation. When a callose synthase protein is produced by chemical synthesis, preferably the preparations has less than about 30%, 20%, 10%, or 5% (by dry weight) of the chemical precursors.

[0055] The polypeptides of the present invention have at least about 20%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the enzymatic activity of the polypeptide consisting of the amino acid sequence of a callose synthase. In one embodiment, the callose synthase is encoded by GSL5 or functional equivalents or homologs thereof.

[0056] Proteins and peptides of the invention may be prepared by any available means, including recombinant expression of the desired protein or peptide in eukaryotic or prokaryotic host cells (see U.S. Patent 5,696,238). Methods for producing proteins or polypeptides of the invention for purification may employ conventional molecular biology, microbiology, and recombinant DNA techniques within the ordinary skill level of the art. Such techniques are explained fully in the literature. See, for example, Maniatis et al., (1989) Molecular Cloning: A Laboratory Manual, 2d ed. Cold Spring Harbor Laboratory Press; Glover, (1985) DNA Cloning: A Practical Approach, VoIs. 1- 4, IRL Press; Gait, (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Hames & Higgins, (1985) Nucleic Acid Hybridisation: A Practical Approach, IRL Press; Perbal, (1984) A Practical Guide To Molecular Cloning, Wiley.

[0057] In its broadest sense, the term "substantially similar" when used herein with respect to polypeptide means that the polypeptide has substantially the same structure and function as the reference polypeptide. In addition, amino acid sequences that are substantially similar to a particular sequence are those wherein overall amino acid identity is at least 65% or greater to the instant sequences. Modifications that result in equivalent nucleotide or amino acid sequences are well within the routine skill in the art. The percentage of amino acid sequence identity between the substantially similar and the reference polypeptide is at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, wherein the reference polypeptide is an Arabidopsis callose synthase polypeptide encoded by a gene with a promoter, a nucleotide sequence comprising an open reading frame which encodes a callose synthase or a functionally active fragment thereof. One indication that two polypeptides are substantially similar to each other, besides having substantially the same function, is that an agent, e.g., an antibody, which specifically binds to one of the polypeptides, specifically binds to the other.

[0058] A polypeptide that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the polypeptides of the invention, or biologically active fragments thereof, are recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein of interest chemicals.

[0059] The present invention also provides for fusion proteins of the callose synthase proteins. As used herein, "fusion" may refer to nucleic acids and polypeptides that comprise sequences that are not found naturally associated with each other in the order or context in which they are placed according to the present invention. A fusion nucleic acid or polypeptide does not necessarily comprise the natural sequence of the nucleic acid or polypeptide in its entirety. Fusion proteins have the two or more segments joined together through normal peptide bonds. Fusion nucleic acids have the two or more segments joined together through normal phosphodiester bonds.

[0060] In some embodiments, the present invention provides a fusion polypeptide comprising a callose synthase polypeptide or a functionally active fragment thereof fused to additional polypeptides. In some embodiments, there are one, two, three, four, or more additional polypeptides fused to the callose synthase polypeptide. In some embodiments, the additional polypeptides are fused toward the amino terminus of the callose synthase polypeptide. In other embodiments, the additional polypeptides are fused toward the carboxyl terminus of the callose synthase polypeptide. In further embodiments, the additional polypeptides flank the callose synthase polypeptide.

[0061] In some embodiments, the additional polypeptides may comprise an epitope. In other embodiments, the additional polypeptides may. comprise an affinity tag. By way of example, fusion of a polypeptide comprising an epitope and/or an affinity tag to the callose synthase polypeptide may aid purification and/or identification of the protein. By way of example, the additional polypeptide may be a His-tag, a myc-tag, an S-peptide tag, a MBP tag (maltose binding protein), a GST tag (glutathione S -transferase), a FLAG tag, a thioredoxin tag, a GFP tag (green fluorescent protein), a BCCP (biotin carboxyl carrier protein), a calmodulin tag, a Strep tag, an HSV-epitope tag, a V5-epitope tag, and a CBP tag. The use of such epitopes and affinity tags is known to those skilled in the art.

[0062] In further embodiments, the additional polypeptides may provide sites for cleavage of the protein. As an example, a polypeptide may be cleaved by hydrolysis of the peptide bond. In some embodiments, the cleavage is performed by a protease enzyme. In some embodiments cleavage occurs in a cell. In other embodiments, cleavage occurs through artificial manipulation and/or artificial introduction of a cleaving enzyme. By way of example, protease enzymes may include aspartic proteases, serine proteases, metalloproteases and cysteine proteases.

[0063] The fusion polypeptides of the present invention may be prepared by any known techniques. For example, the polypeptides may be expressed through genetic engineering. By way of example, the translation of recombinant DNA. The polypeptides may also be prepared synthetically. By way of example, the polypeptide may be synthesized using the solid-phase synthetic technique initially described by Merrifield (J. Am. Chem. Soc. 85:2149-2154.), which is incorporated herein by reference. Other polypeptide synthesis techniques may be found, for example, Kent et al. Synthetic Peptides in Biology and Medicine, eds. Alitalo, Partanen, and Vakeri, Elsevier Science Publishers, pp. 295-358 (1985).

[0064] Fusion proteins and peptides of the invention may be prepared by any available means, including recombinant expression of the desired protein or peptide in eukaryotic or prokaryotic host cells {see U.S. Patent 5,696,238). Methods for producing proteins or polypeptides of the invention for purification may employ conventional molecular biology, microbiology, and recombinant DNA techniques within the ordinary skill level of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d ed. Cold Spring Harbor Laboratory Press; Glover (1989), (1985) DNA Cloning: A Practical Approach, VoIs. 1-4, IRL Press; Gait, (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Hames & Higgins,

(1985) Nucleic Acid Hybridisation: A Practical Approach, IRL Press; Perbal, (1984) A Practical Guide To Molecular Cloning, Wiley.

[0065] The present invention provides nucleic acids that encode callose synthase or functional equivalents or homologs thereof. The nucleic acids of the present invention include both the naturally occurring sequences as well as mutant (variant) forms thereof.

[0066] The nucleic acid encoding callose synthase or functional equivalents or homologs thereof may be derived from a plant, for example Arabidopsis. The callose synthase may be a wild type enzyme. A wild-type nucleic acid or polypeptide refers to a nucleic acid or polypeptide sequence found in nature without any known mutation. In other embodiments, the callose synthase may be an active fragment or a functional equivalent fragment thereof. An active fragment refers to a callose synthase polypeptide having one or more amino acids deleted from full-length callose synthase polypeptide or a homologous sequence thereof, wherein the fragment retains enzymatic activity for producing callose.

[0067] Examples of plant callose synthase genes include twelve callose synthase genes identified in Arabidposis. They are numbered sequentially GSLl to GSL12, derived and encoded by GenBank accession numbers AF237733 (GSLl), AC006223 (GSL2), AL353013 (GSL3), AB025605 (GSL4), AC006436 (GSL5), AL163527 (GSL6), AC007592 (GSL7), AB023038 (GSL8), ACOl 2395 (GSL9), AC006922(GSL10), AF162444 AL161502 AC012392 (all 3 GSLl 1), and AC005142 AF071527 AL161497 (all 3 GSL12). GSL5 and GSLl have less than 5 introns, while the remaining 10 GSL genes in Arabidopsis have greater than 20 introns. In one embodiment, the callose synthase may be encoded by GSLl or GSL5. In another embodiment, the callose synthase may be encoded by GSL5.

[0068] In other embodiments, the callose synthase is encoded by a callose synthase gene from another plant species. The presence of callose and of callose synthases in other plant species is known in the art. A callose synthase corresponding to GSL5 in Arabidopsis from another plant may be utilized. Methods for identifying a homologous gene or protein in another plant species are known in the art. For example, PCR or hybridization with probes can be used to identify the mRNA encoding comparable proteins in other plant species. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. Collins et al. (Nature 425: 973-977, 2003) demonstrate use of probes for identifying a comparable gene in barley to the Arabidops is gene penl. Similarly, the MIo function in tomatoes for powdery mildew resistance was initially identified based on homology based on comparison to tomato and to Arabidopsis genes (Bai et al. Molecular Plant-Microbe Interactions 21 : 30-39, 2008). Bioinformatic analysis can also be used to identify comparable genes and proteins in other plants, see, e.g., Nelson et al. PNAS 104: 16450-16455, 2007; see also Century et al. Plant Physiol. 147: 20-29, 2008). In rice, the sequence of Os01g55040 shares greater than 60% identity with GSL5 and Os01g48200 shares approximately 70% identity in amino acid sequence to the Arabidopsis GSL5. Furthermore, these two genes, like GSL5, have less than 5 introns. In barley, the encoded proteins MLO, ML06 and MLO 12 each share approximately 50% identity to GSL5, and all have been demonstrated to be functional homologs and all confer some level of disease resistance to powdery mildews when mutated.

[0069] A nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. The term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. A particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.

[0070] A "fragment" of a nucleic acid refers to a fraction of a given nucleic acid molecule. In higher plants, deoxyribonucleic acid (DNA) is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. "Nucleic acid", "oligonucleotide" or "nucleic acid sequence" may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.

[0071] With respect to double stranded nucleic acid molecules, sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (e.g., the strand having a sequence homologous to the mRNA). Transcriptional and translational control sequences are nucleic acid regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

[0072] The present invention provides for the introduction of callose synthase into a plant cell, wherein the nucleic acid encoding the callose synthase may be derived from the genomic DNA of the plant cell, or may be derived from the genomic DNA of another species of plant. The terms "heterologous DNA sequence", "exogenous DNA segment" or "heterologous nucleic acid", as used herein, refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Accordingly, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms may also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. A heterologous region of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. Accordingly, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.

[0073] A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced. DNA sequences are substantially homologous when at least about 85%, at least about 90%, or at least about 95% of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified, for example, in a Southern hybridization or northern hybridization experiment under stringent conditions as defined for that particular system. Stringent conditions refer to the temperature, salt, and/or pH conditions required for hybridization. Defining appropriate hybridization conditions is within the skill of the art. Homologous in the context of nucleotide sequence identity refers to the similarity between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under stringent conditions, as is well understood by those skilled in the art (e.g. as described in Haines and Higgins, eds., Nucleic Acid Hybridization, IRL Press, Oxford, U.K.), or by the comparison of sequence similarity between two nucleic acids or proteins.

[0074] A substantially similar sequence refers to nucleotide and amino acid sequences that represent functional and structural equivalents of the sequences disclosed herein. In its broadest sense, substantially similar, when used herein with respect to a nucleotide sequence, refers to the nucleotide sequence that is part of a gene which encodes a polypeptide having substantially the same structure and function as a polypeptide encoded by a gene for the reference nucleotide sequence, e.g., the nucleotide sequence comprises a promoter from a gene that is the ortholog of the gene corresponding to the reference nucleotide sequence. Substantially similar refers as well to promoter sequences that are structurally related the promoter sequences particularly exemplified herein, i.e., the substantially similar promoter sequences hybridize to the complement of the promoter sequences exemplified herein under high or very high stringency conditions. For example, altered nucleotide sequences which simply reflect the degeneracy of the genetic code but nonetheless encode amino acid sequences that are identical to a particular amino acid sequence are substantially similar to the particular sequences. Substantially similar sequences also include nucleotide sequences wherein the sequence has been modified, e.g., to optimize expression in particular cells, as well as nucleotide sequences encoding a variant polypeptide having one or more amino acid substitutions relative to the (unmodified) polypeptide encoded by the reference sequence, which substitution(s) does not alter the activity of the variant polypeptide relative to the unmodified polypeptide. Moreover, a nucleotide sequence that is substantially similar to a reference nucleotide sequence is said to be "equivalent" to the reference nucleotide sequence. One skilled in the art will recognize that equivalent nucleotide sequences encompassed by this invention can also be defined by their ability to hybridize, under low, moderate and/or stringent conditions (e.g., 0.1 x SSC, 0.1% SDS, at 65° C), with the nucleotide sequences that are within the literal scope of the instant claims.

[0075] What is meant by "substantially the same activity" when used in reference to a polynucleotide or polypeptide fragment is that the fragment has at least 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, ' 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to at least 99% of the activity of the full length polynucleotide or full length polypeptide. A functionally equivalent callose synthase may have substantially the same activity as a wild-type callose synthase.

[0076] An "isolated" or "purified" nucleic acid molecule or polypeptide is a nucleic acid molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic or transformed host cell. For example, an isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated" or "purified" nucleic acid may be free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic sequence of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

[0077] A variant with respect to a sequence (e.g., a polypeptide or nucleic acid sequence) refers to substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, e.g., with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site- directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein. Generally, nucleotide sequence variants of the invention will have at least about 40, 50, 60, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide sequence identity to the native (wild type or endogenous) nucleotide sequence. A nucleic acid encoding a functionally equivalent callose synthase may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide sequence identity to wild type callose synthase. A functionally equivalent callose synthase may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid sequence identity to the naturally occurring or wild type callose synthase.

[0078] A "conservative" variation of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic codes, a large number of functionally identical nucleic acids encode any given polypeptide. Thus, a codon can be altered without altering the encoded amino acid. Such nucleic acid variations are often referred to as "silent variations." One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques.

[0079] The nucleic acid molecules of the invention can be "optimized" for enhanced expression in plants of interest (see, for example, WO 91/16432; Perlak 1991; Murray 1989). In this manner, the open reading frames in genes or gene fragments can be synthesized utilizing plant-preferred codons (see, for example, Campbell & Gowri, 1990 for a discussion of host-preferred codon usage). Thus, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used. Variant nucleotide sequences and proteins also encompass, sequences and protein derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create a new polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Strategies for such DNA shuffling are known in the art (see, for example, Stemmer 1994; Stemmer 1994; Crameri 1997; Moore 1997; Zhang 1997; Crameri 1998; and U.S. Pat. Nos. 5,605,793 and 5,837,458).

[0080] A nucleic acid "coding sequence" is a double-stranded nucleic acid sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence. A nucleic acid may thereby "encode" the corresponding amino acid sequence.

[0081] The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) reference sequence, (b) comparison window, (c) sequence identity, (d) percentage of sequence identity, and (e) substantial identity, (a) A reference sequence is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence, (b) A comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches. Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Computer implementations of mathematical-algorithms can . be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters. The BLAST programs are based on the algorithm of Karlin and Altschul, publicly available through the National Center for Biotechnology Information and involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N penalty score for mismatching residues; always <0).

[0082] For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative- scoring residue alignments, or the end of either sequence is reached. In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g. BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989).

[0083] Alignment may also be performed manually by inspection. An "equivalent program" is intended to include any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program, (c) Sequence identity, or identity, in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have sequence similarity or similarity. Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non- conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.). Percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions {i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity, (e) (i) Substantial identity or substantial similarity of polynucleotide sequences for a protein encoding sequence means that a polynucleotide comprises a sequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. Substantial identity or similarity of polynucleotide sequences for promoter sequence means (as described above for variants) that a polynucleotide comprises a sequence that has at least about 40, 50, 60, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least about 70%, 80%, 90%, or 95%.

[0084] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C to about 20° C, depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, (ii) Substantial identity in the context of a peptide indicates that a peptide comprises a sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, p80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. Optimal alignment may be conducted using a homology alignment algorithm such as that of Needleman and Wunsch (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.

[0085] The present invention also provides a vector comprising a nucleic acid encoding callose synthase or functional equivalents or homologs thereof wherein the nucleic acid is in or part of a vector. A vector may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.

[0086] Vectors may further contain a promoter sequence. A promoter may include an untranslated nucleic acid sequence usually located upstream of the coding region that ^■ contains the site for initiating transcription of the nucleic acid. The promoter region may also include other elements that act as regulators of gene expression. In further embodiments of the invention, the expression vector contains an additional region to aid in selection of cells that have the expression vector incorporated. The promoter sequence is often bounded (inclusively) at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Commonly used promoters are derived from polyoma, bovine papilloma virus, CMV (cytomegalovirus, either murine or human), Rouse sarcoma virus, cauliflower mosaic virus, adenovirus, and simian virus 40 (SV40). Other control sequences (e.g., terminator, polyA, enhancer, or amplification sequences) can also be used.

[0087] Vectors may further contain one or more marker sequences suitable for use in the identification and selection of cells which have been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. The vectors may be those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.

[0088] The nucleic acid encoding callose synthase may be contained in an expression vector. An expression vector is one into which a desired nucleic acid sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Expression refers to the transcription and/or translation of an endogenous gene, transgene or coding region in a cell. An expression vector is constructed so that the polypeptide coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed and translated under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence). The control sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site. If the selected host cell is a plant cell, the control sequences can be heterologous or homologous to the coding sequence, and the coding sequence can either be genomic DNA containing introns or cDNA.

[0089] The nucleic acid encoding callose synthase may be operably linked to any regulatory regions. A coding sequence and regulatory sequences are operably joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.

[0090] The terms "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).

[0091] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C, and a wash in Ix to 2x SSC (2Ox SSC is 3.0 M NaCl/0.3 M tri-sodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C, and a wash in 0.5x to Ix SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C, and a wash in 0. Ix SSC at 60 to 65° C.

[0092] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA- DNA hybrids, the T_m can be approximated from the equation of Meinkoth and Wahl (Anal. Biochem., 138:267-284, 1984): T_m=81.5° C + 16.6 (log M)+0.41 (% GQ-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T_m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T_m is reduced by about 1 ° C for each 1% of mismatching; thus, T_m> hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with approximately 90% identity are sought, the T_m can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (T_m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize hybridization and/or wash at 1, 2, 3, or 4° C lower than the thermal melting point; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C lower than the thermal melting point; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C lower than the thermal melting point. Using the equation, hybridization and wash compositions, and desired T_m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T_m of less than 45° C (aqueous solution) or 32° C (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York (2000).

[0093) Thus, with the foregoing information, the skilled artisan can identify and isolate polynucleotides that are substantially similar to the present polynucleotides. In isolating such a polynucleotide, the polynucleotide can be used as the present polynucleotide

[0094] The coding sequence refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an uninterrupted coding sequence, i. e. , lacking an intron, such as in a cDNA, or it may include one or more introns bounded by splice junctions which are known in the art. An intron is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.

[0095] An open reading frame (ORF) refers to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. An initiation codon and a termination codon refer to a unit of three adjacent nucleotides (codon) in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).

[0096] A "functional RNA" refers to an antisense RNA, ribozyme, or other RNA that is not translated. [0097] The term "RNA transcript" refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA" (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA.

[0098] A transcription regulating nucleotide sequence, or regulatory sequence, refers to nucleotide sequences influencing the transcription, RNA processing or stability, or translation of the associated (or functionally linked) nucleotide sequence to be transcribed. The transcription regulating nucleotide sequence may have various localizations with the respect to the nucleotide sequences to be transcribed. The transcription regulating nucleotide sequence may be located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of the sequence to be transcribed (e.g., a coding sequence). The transcription regulating nucleotide sequences may be selected from the group comprising enhancers, promoters, translation leader sequences, introns, 5'-untranslated sequences, 3'_:untranslated sequences, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences, which may be a combination of synthetic and natural sequences. As is noted above, a transcription regulating nucleotide sequence is not limited to promoters. However, preferably a transcription regulating nucleotide sequence of the invention comprises at least one promoter sequence (e.g., a sequence localized upstream of the transcription start of a gene capable to induce transcription of the downstream sequences). In one embodiment, the transcription regulating nucleotide sequence of the invention comprises the promoter sequence of the corresponding gene and, optionally and preferably, the native 5 '-untranslated region of said callose synthase gene. Furthermore, the 3'-untranslated region and/or the polyadenylation region of the callose synthase gene may also be employed.

[0099) A 5' non-coding sequence refers to a nucleotide sequence located 5' (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.

[00100] A 3' non-coding sequence refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[00101] A translation leader sequence refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.

[00102] A signal peptide refers to the amino terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into, the secretory pathway. The term "signal sequence" refers to a nucleotide sequence that encodes the signal peptide. The term "transit peptide" as used herein refers part of a expressed polypeptide (preferably to the amino terminal extension of a polypeptide), which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into a cell organelle (such as the plastids (e.g., chloroplasts) or mitochondria). The term "transit sequence" refers to a nucleotide sequence that encodes the transit peptide.

[00103] Promoters

[00104] The present invention provides for nucleic acids encoding callose synthase under the control of a promoter. In some embodiments, the promoter is constitutively active. In other embodiments, the promoter is inducible or tissue-specific.

[00105] A promoter refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. A promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. A promoter also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an enhancer is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements, derived from different promoters found in nature, or even comprise synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[00106] The initiation site is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e , further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5¹ direction) are denominated negative.

[00107] Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters." In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A "minimal or core promoter" thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.

[00108] The promoter may be selected from the group consisting of a viral coat protein promoter, a tissue-specific promoter, a monocot promoter, a ubiquitin promoter such as UBQl, a stress inducible promoter, a CaMV 35S promoter, a CaMV 19S promoter, an actin promoter, a cab promoter, a sucrose synthase promoter, a tubulin promoter, a lectin promoter, a napin R gene complex promoter, a tomato E8 promoter, a BTH6 promoter, NOS promoter, ROLD promoter, CsVMV promoter, UBI-IL promoter, UBI-I S promoter, a patatin promoter, a mannopine synthase promoter, a soybean seed protein glycinin promoter, a soybean vegetative storage protein promoter, a bacteriophage SP6 promoter, a bacteriophage T3 promoter, a bacteriophage T7 promoter, a heat-shock promoter, a Ptac promoter, a root-cell promoter, an ABA-inducible promoter and a turgor-inducible promoter.

[00109] "Constitutive expression" refers to expression using a constitutive or regulated promoter. "Conditional" and "regulated expression" refer to expression controlled by a regulated promoter. A "constitutive promoter" refers to a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant. Each of the transcription-activating elements do not exhibit an absolute tissue-specificity, but mediate transcriptional activation in most plant parts at a level of at least 1% of the level reached in the part of the plant in which transcription is most active. By way of example, constitutive promoters include CaMV 35S, NOS, ubiquitin promoters, actin promoters, ROLD, UBI-IL, UBI-I S, and CsVMV.

[00110] A promoter may be a strong promoter, such as CaMV35S. Other examples of strong promoters include but are not limited to CoYMV (Commelina yellow mottle virus), CLCuV (Cotton leaf curl virus), Figwort Mosaic Virus promoter, Chlorella virus adenine methyltransferase gene promoter, Maize polyubiquitin promoter, and Rubi3 (Rice ubiquitin gene promoter) (Medberry et al. Plant Cell 4: 185-191, 1992; Sanger et al. Plant MoI. Biol. 14: 433-443, 1990; Mitra et al. Plant MoI. Biol. 26: 85-93, 1994; Science in China Series C-Life Sciences 44(1): 8-17, 2001 ; Sivamani et al. Plant MoI. Biol. 60: 225-239, 2006; and, Christensen et al. Plant MoI. Biol. 18: 675-689, 1992).

[00111] A regulated promoter refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and includes both tissue-specific and inducible promoters. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al. (1989). Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol- inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysone- inducible systems. Inducible promoter also include those regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, or a pathogen.

[00112] Tissue-specific promoter refers to regulated promoters that are not expressed in all plant cells but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence. By way of example and not as a limitation tissue-specific promoters include the Brassica derived napin gene promoter, the Arabidopsis derived 2S albumin gene promoter, the pr-la promoter derived from tobacco, the maize derived promoter from the zein gene, the rice derived promoter for glutelein, the potato derived pin2 promoter, the phaseolin promoter (U.S. Patent 5,504,200), the legumin promoter, the USP (unknown seed protein) promoter, the promoter of the sucrose binding protein (PCT Application WO 00/26388) and the LeB4 promoter.

[00113] Transgenic Plants

[00114] The present invention provides transgenic and transformed plant cells and plants wherein the cells express callose synthase. The expression of callose synthase in a transformed plant cell may be under the control of a constitutively active promoter. The present invention provides for transforming a plant cell with an expression vectors having a linear or circular nucleic acid molecule comprising a polynucleotide encoding a callose synthase protein operably linked to additional nucleotides that provide for its expression. In some embodiments, the nucleic acid encoding the callose synthase becomes integrated with the host cell's genome. [00115] The word "plant" refers to any plant, particularly to agronomically useful plants (e.g., seed plants), and "plant cell" is a structural and physiological unit of the plant, which comprises a cell wall but may also refer to a protoplast. The plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ differentiated into a structure that is present at any stage of a plant's development. Such structures include one or more plant organs including, but are not limited to, fruit, shoot, stem, leaf, flower petal, etc. The term "plant" includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seeds (including embryo, endosperm, and seed coat) and fruits (the mature ovary), plant tissues (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same.

[00116] The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous. Included within the scope of the invention are all genera and species of higher and lower plants of the plant kingdom. Included are furthermore the mature plants, seed, shoots and seedlings, and parts, propagation material (for example seeds and fruit) and cultures, for example cell cultures, derived therefrom. Examples of plants include those from the following plant families: Amaranthaceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Tetragoniaceae. Plant materials may be derived from plants of these plant families.

[00117] Annual, perennial, monocotyledonous and dicotyledonous plants are useful as host organisms for the generation of transgenic plants. The use of the recombination system, or method according to the invention is furthermore advantageous in all ornamental plants, forestry, fruit, or ornamental trees, flowers, cut flowers, shrubs or turf. Said plant may include, but shall not be limited to, bryophytes such as, for example, Hepaticae (hepaticas) and Musci (mosses); pteridophytes such as ferns, horsetail and clubmosses; gymnosperms such as conifers, cycads, ginkgo and Gnetaeae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms) and Euglenophyceae.

[00118] Plants for the purposes of the invention may comprise the families of the Rosaceae such as rose, Ericaceae such as rhododendrons and azaleas, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such as pinks, Solanaceae such as petunias, Gesneriaceae such as African violet, Balsaminaceae such as touch-me-not, Orchidaceae such as orchids, Iridaceae such as gladioli, iris, freesia and crocus, Compositae such as marigold, Geraniaceae such as geraniums, Liliaceae such as Drachaena, Moraceae such as ficus, Araceae such as philodendron and many others.

[00119] The transgenic plants according to the invention are furthermore selected in particular from among dicotyledonous crop plants such as, for example, from the families of the Leguminosae such as pea, alfalfa and soybean; the family of the Umbelliferae, particularly the genus Daucus (very particularly the species carota (carrot)) and Apium (very particularly the species graveolens var. dulce (celery)) and many others; the family of the Solanaceae, particularly the genus Lycopersicon, very particularly the species esculentum (tomato) and the genus Solarium, very particularly the species tuberosum (potato) and melongena (aubergine), tobacco and many others; and the genus Capsicum, very particularly the species annum (pepper) and many others; the family of the Leguminosae, particularly the genus Glycine, very particularly the species max (soybean) and many others; and the family of the Cruciferae, particularly the genus Brassica, very particularly the species napus (oilseed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli); and the genus Arabidopsis, very particularly the species thaliana and many others; the family of the Compositae, particularly the genus Lactuca, very particularly the species sativa (lettuce) and many others.

[00120] The transgenic plants according to the invention may be selected among monocotyledonous crop plants, such as, for example, cereals such as wheat, barley, sorghum and millet, rye, triticale, maize, rice or oats, and sugarcane. Further of use are trees such as apple, pear, quince, plum, cherry, peach, nectarine, apricot, papaya, mango, and other woody species including coniferous and deciduous trees such as poplar, pine, sequoia, cedar, oak, etc. Further of use are Arabidopsis thaliana, Nicotiana tabacum, oilseed rape, soybean, corn (maize), wheat, linseed, potato and tagetes.

[00121] The present invention provides for the plasmid(s) or vector(s) described herein to be contained in a host cell, a plant cell, or a transgenic plant. The plant may be Arabidopsis thaliania or selected from the group consisting of wheat, corn, peanut, cotton, oat, and soybean plant.

[00122] Methods, vectors, and compositions for transforming plants and plant cells in accordance with the invention are well-known to those skilled in the art, and are not particularly limited. For a descriptive example see Karimi et al., TRENDS in Plant Science, 7: 193-195, 2002, incorporated herein by reference.

[00123] "Chromosomally-integrated" refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes are not chromosomally integrated, they may be stably expressed/transformed or transiently expressed/transformed. Transiently transformed refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium- mediated transformation or biolistic bombardment), but not selected for stable maintenance. Stably transformed refers to cells that have been selected and regenerated on a selection media following transformation.

[00124] Genetically stable and heritable refer to chromosomally-integrated genetic elements that are stably maintained in the plant and stably inherited by progeny through successive generations.

[00125] A primary transformant and To generation refer to transgenic plants that are of the same genetic generation as the tissue which was initially transformed (/. e. , not having gone through meiosis and fertilization since transformation).

[00126] Secondary transformants and the Ti, T₂, T₃, etc. generations refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants. [00127] An altered plant trait refers to any phenotypic or genotypic change in a transgenic plant relative to the wild-type or non-transgenic plant host.

[00128] Transformation refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as transgenic cells, and organisms comprising transgenic cells are referred to as transgenic organisms. Examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation and particle bombardment technology (e.g. U.S. Patent No. 4,945,050). Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan.

[00129] Transformed, transgenic, and recombinant refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome generally known in the art. For example, transformed, transformant, and transgenic plants or calli have been through the transformation process and contain a foreign gene integrated into their chromosome. Untransformed or native refers to normal plants that have not been through the transformation process.

[00130] Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et αl. Meth. Enzymol. 143, 277 (1987)) and particle- accelerated or "gene gun" transformation technology (Klein et αl. Nature 327, 70-73 (1987); U.S. Pat. No. 4,945,050, incorporated herein by reference). Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be 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. 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 αl., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et αl., 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.

[00131] Higher eukaryotic cell cultures, such as those dervied from mammals, may also be used to express the callose synthase or homologs or functional equivalents thereof of the present invention, whether from vertebrate or invertebrate cells, including insects, and the procedures of propagation thereof are known. See, for example, Kruse and Patterson (1973) Tissue Culture, Academic Press. Suitable host cells for expressing the polypeptides of the present invention in higher eukaryotes include: 293 (human embryonic kidney) (ATCC CRL- 1573); 293F (Invitrogen, Carlsbad CA); 293T and derivative 293T/17(293tsA1609neo and derivative ATCC CRL-1 1268) (human embryonic kidney transformed by SV40 T antigen); COS-7 (monkey kidney CVI line transformed by SV40)(ATCC CRL1651); BHK (baby hamster kidney cells) (ATCC CRLlO); CHO (Chinese hamster ovary cells); mouse Sertoli cells; CVI (monkey kidney cells) (ATCC CCL70); VERO76 (African green monkey kidney cells) (ATCC CRLl 587); HeLa (human cervical carcinoma cells) (ATCC CCL2); MDCK (canine kidney cells) (ATCC CCL34); BRL3A (buffalo rat liver cells) (ATCC CRLl 442); W 138 (human lung cells) (ATCC CCL75); HepG2 (human liver cells) (HB8065); and MMT 060652 (mouse mammary tumor) (ATCC CCL51).

[00132] The present invention also provides methods for using transgenic plants. For example, transgenic plants may be used as a crop, wherein the transgenic expression of callose synthase allows for the crop to have an increased resistance to a wound or a pathogen attack.

[00133] Methods of protecting plants

[00134] The present invention provides methods for increasing the rate of callose deposit in response to an injury comprising increasing the presence of callose synthase in a cell. The cell or a plant comprising the cell may have enhanced pathogen and/or insult resistance. The cell and/or plant with increased callose synthase or functional equivalents or homologs thereof expression may be resistant to insects, bacterium, fungus, or other pathogens and abiotic stimuli and mechanical wounding. The present invention provides methods of protecting a cell against an insult comprising over- expressing callose synthase in the cell. The present invention provides for methods of increasing the defense of a cell against an attack comprising over-expressing callose synthase in the cell. The various methods of the present invention comprise introducing a nucleic acid encoding callose synthase or a homolog or a functional equivalent thereof into a cell, such as a plant cell. The nucleic acid encoding callose synthase may be under the control of a promoter. The nucleic acid encoding callose synthase may be under the control of a constitutively active promoter. The promoter may be tissue-specific. In some embodiments the cell is a plant cell. The plant cell may be contained in a plant.

[00135] The present invention provides a method of increasing disease-resistance of a plant cell to a pathogen by increasing the level of callose synthase or a homolog or a functional equivalent thereof within the plant cell. Callose synthase, or a homolog or functional equivalent thereof may be increased by introducing a nucleic acid encoding the same into a plant cell of a plant. The nucleic acid encoding the callose synthase or homolog or functional equivalent thereof may be under the control of a promoter. The promoter may be constitutively active or tissue specific. The promoter may be reactive to an agent, such as when the agent contacts the plant cell and/or the promoter, the agent induces transcription.

[00136] The pathogen may be a fungus, nematode or oomycete. The pathogen may be powdery mildew. The pathogen may be selected from Golovinomyces cichoracearum, Mycosphaerella musicola, Fusarium graminearum, Blumeria graminis f. sp. hordei, Puccinia hordei, Puccinia graminis f. sp. tritici, Puccinia striiformis, Pyrenophora teres, Rhynchosproium secalis, Erysiphe necator, Colletotrichum graminicola, Puccinia sorghi, Ustilago maydis, Blumeria graminis f. sp. avenae, Phytophthora infestans, Magnaporthe grisea, Oidium neolycopersici, Leveillula taurica, Phakopsora pachyrhizi, Phytophthora sojae, Puccinia melanocephala, Peronosclerospora sacchari, Ustilago scitaminea, Blumeria graminis f. sp. tritici, Puccinia graminis f. sp. tritici, Tilletia indica.

[00137] The present invention provides a method of regulating and/or modulating a plant's resistance to a pathogen and/or insult comprising introducing callose synthase into a plant. This allows the growth of a healthy crop and/or enhances the crop yield. The pathogen may be an insect, bacterium, fungus, or other pathogens. The insult may be a pathogen, or other abiotic stimuli or mechanical wounding.

[00138] The present invention provides plants with resistance to insults and/or pathogen attacks. The present invention also provides for breeding of plants with increased pathogen resistance and/or insult resistance. The present invention provides for breeding of transgenic plants that deomstrate increased callose synthase or homologs or functional equivalents therof expression. The plants of the present invention have higher or enhanced resistance to insults and/or pathogen attacks as compared to plants that do not have the endogenous machinery for increased expression of callose synthase. The present invention provides methods for producing transgenic plants by the incorporation of a nucleotide sequence encoding callose synthase in the plant to be modified. By introducing callose synthase, or a homolog or functional equivalent thereof, into a plant, the pathogen resistance can be influenced. No pleiotrophic gene effects are to expected, and accordingly, the crop yield of the stock material may remain undisturbed. The resulting transgenic plants show a higher resistance with respect to pathogens, such as, for example and not by way of limitation, against fungal pathogens including Plasmodiophoromycetes, Oomycetes, Ascomycetes, Chytridiomycetes, Zygomycetes, Basidiomycetes, and Deuteromycetes varieties.

[00139] In one embodiment, the callose synthase may be derived from the Arabidopsis gene GSL5. In another embodiment, the callose synthase may be derived from another species of plant and corresponds to GSL5. Methods of identifying the corresponding genes in other species of plant are known in the art, e.g., as performed between the dicot Arabidopsis and the moncot barley by Collins et al. Nature 425: 973-977, 2003 and Consonni et al. Nature Genet. 38: 716-720, 2006, incorporated herein by reference. Accordingly, the callose synthase expressed in a plant cell can be derived from any species of plant. The callose synthase over-expressed may correspond to GSL5 in Arabidopsis. The species of plant may include Amaranthaceae; Brassicaceae; Carophyllaceae; Chenopodiaceae; Compositae; Cucurbitaceae; Labiatae; Papilionoideae; Liliaceae; Linaceae; Malvaceae; Rosaceae; Saxifragaceae; Scrophulariaceae; Solanaceae; Tetragoniaceae; bryophytes such as, for example, Hepaticae (hepaticas) and Musci (mosses); pteridophytes such as ferns, horsetail and clubmosses; gymnosperms such as conifers, cycads, ginkgo and Gnetaeae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms) and Euglenophyceae; Rosaceae such as rose; Ericaceae such as rhododendrons and azaleas; Euphorbiaceae such as poinsettias and croton; Caryophyllaceae such as pinks; Solanaceae such as petunias; Gesneriaceae such as African violet; Balsaminaceae such as touch-me-not; Orchidaceae such as orchids; Iridaceae such as gladioli, iris, freesia and crocus; Compositae such as marigold; Geraniaceae such as geraniums; Liliaceae such as Drachaena; Moraceae such as ficus, Araceae such as philodendron; Leguminosae such as pea, alfalfa and soybean; Umbelliferae, particularly the genus Daucus (particularly the species carota (carrot)) and Apium (particularly the species graveolens var. dulce (celery)) and many others; Solanaceae, particularly the genus Ly coper sicon, particularly the species esculentum (tomato) and the genus Solarium, particularly the species tuberosum (potato) and melongena (aubergine), tobacco and many others; Capsicum, very particularly the species annum (pepper) and many others; Glycine, particularly species max (soybean) and many others; Cruciferae, particularly the genus Brassica, particularly the species napus (oilseed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea- cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli); Arabidopsis, particularly the species thaliana and many others; Compositae, particularly the genus Lactuca, particularly the species sativa (lettuce) and many others; monocotyledonous crop plants, such as, e.g., cereals such as wheat, barley, sorghum and millet, rye, triticale, maize, rice or oats, and sugarcane; trees such as apple, pear, quince, plum, cherry, peach, nectarine, apricot, papaya, mango, and other woody species including coniferous and deciduous trees such as poplar, pine, sequoia, cedar, oak, etc; Nicotiana tabacum; oilseed rape; soybean; corn (maize); wheat; linseed; potato; and, tagetes. Suitable plants in the sense of the invention are plants which provide nutrients and raw materials, for example carbohydrate providing plants (such as wheat, corn, rice, rye, potatoes, barley, oat and millet), oil and fat producing plants (such as peanut, palm oil, olive, grape and sunflower), sugar producing plants (such as sugar beets, sugar cane, sugar millet), protein producing plants (such as strawberries, beans, peas, lentils and soy beans), fiber producing plants (such as cotton, flack, hemp, jute), pleasure substance providing plants (such as tobacco, tea and cocoa), wood producing plants (such as birch, fig, fir, Douglas, pine, larch, Limba, mahogany, beech, oak, cedar), feed material providing plants (such as Lucerne and feed beets), vegetables (such as cucumbers, types of cabbage, pumpkin, carrot, paprika, lettuce, spinach, radish and tomato), fruits (such as apples, pears, cherries, melons, grapes, citrus, pineapple and bananas).

[00140] In some embodiments, the over-expression of callose synthase does not lead to increased callose until an insult is inflicted on the cell. In some instances, the over- expressed callose synthase increases only the mRNA for callose synthase, but not the expressed protein. In other instances, the over-expression of callose synthase provides for over-expressed callose synthase protein that is not functionally active in producing callose until an insult is inflicted on the cell.

[00141] In some embodiments, the callose synthase is GSL5 derived from Arabidopsis and is expressed in the cell of a plant. It is known in the art that plants can express genes derived from Arabidopsis.

[00142] In other embodiments, the callose synthase may be a functional equivalent or a homolog of wild type callose synthase.

[00143] The present invention also provides methods of increasing broad-spectrum resistance to bacterial pathogens that utilize pilli or similar structures to interact with a plant cell by expressing callose synthase in a plant cell. In some embodiments, the present invention provides for improved callose deposit at the site of an insult, more particularly the rate of callose deposit is increased so that the rate of penetration of the insult is impeded as compared to a wild-type cell not over-expressing callose synthase. In some embodiments, the callose synthase is produced by introducing a nucleic acid encoding the callose synthase into the cell. The nucleic acid may be under the control of a promoter. In some further embodiments, expression of callose synthase may directed to a specific site or organ or organelle within a plant through the use of tissue-specific promoters.

[00144] In some embodiments, expression of callose synthase may be induced through the use of an inducible promoter. Methods of the present invention further include contacting a cell comprising a nucleic acid comprised of an inducible promoter and a nucleic acid encoding callose synthase or a homolog or a functional equivalent thereof with an agent that induces the promoter to transcribe the nucleic acid encoding callose synthase or a homolog or a functional equivalent thereof.

[00145] The present invention also provides for increasing the rate of callose deposited at the site of an insult by increasing callose synthase within the cell. In one embodiment, the methods of the present invention provide for increased immediate and early response to an insult to a plant cell. The present invention provides that by providing a cell with over-expressed or constitutively expressed callose synthase, the cell can demonstrate an improved immediate and early response to an insult to the cell. In other embodiments, the callose synthase may be encoded by GSL5 or a functional equivalent thereof.

[00146] The present invention also provides methods to increase resistance of a plant cell to a pathogen following sustaining a wound by introducing callose synthase to the cell. The present invention provides methods of limiting the formation of blemishes at the site of a wound to a plant. In some embodiments the pathogen is powdery mildew.

[00147] The present invention also provides methods to increase resistance of a plant to a pathogen by introducing to the plant callose synthase. The methods may comprise making a transgenic plant that introduces and/or over-expresses callose synthase as compared to a non-transgenic form of the plant.

[00148] The present invention also provides for methods for identifying agents that modulate callose synthase activity. The agent may increase callose synthase enzymatic activity or may increase expression of callose synthase. Conversely the agent may inhibit callose synthase expression or inhibit callose synthase enzyme activity. The method comprises contacting a test cell expressing callose synthase or a nucleic acid encoding callose synthase with a test agent. Those skilled in the art will appreciate that positive and/or negative controls will be useful in determining the efficacy of the test agent. For example, inhibition or stimulation of callose synthase expression may be determined by comparison of callose synthase protein or RNA expression as compared to a non-treated cell. Inhibition or stimulation of callose synthase may be determined by assaying callose synthase activity and comparing to a non-treated cell. The callose synthase may be endogenous to the cell or may be introduced into the cell.

[00149] The present invention also provides methods fore identifying agents that modulate a promoter for callose synthase. Those skilled in the art will recognize that endogenous callose synthase production is regulated by a promoter region upstream of the coding region. The methods involve contact a cell with a test agent and comparing callose synthase levels with a control, such as a cell not treated with the test agent or with a known agent. The level of callose synthase may increase in response to the test agent. The level of callose synthase may decrease in response to the test agent.

[00150] By way of example, the present invention provides methods of providing in the following plant species an increased resistance to diseases caused by the listed pathogens:

EXAMPLES

[00151] Fungal diseases are a serious threat to plant life, and methods to combat plant fungi have generated substantial financial support. Of the total budget into plant protection research BASF AG dedicates almost 40% (1,170 million Euros) and Bayer CropScience dedicates over one quarter ( 1 ,200 million Euros) to fungicide development.

[00152] An exemplary fungal pathogen is Fusarium graminearum, which is a filamentous, necrotrophic pathogen to cereals. The fungus produces cob rot in maize and Fusarium head blight in wheat. The fungus exerts its effects through production of mycotoxins, deoxynivalenol, and zearalenone.

[00153] Fusarium graminearum has been demonstrated to function or rely on its secreted lipase FGLl. Inoculation of maize with a mutant Fusarium graminearum that has a disrupted fgll gene demonstrated markedly reduced virulence than the wild-type or ectl (ectopic) strains.

[00154] These studies were also repeated in wheat, and again reduced virulence was noted in comparison to wild-type or ect inoculation.

[00155] The fungal Fusarium graminearum infection acts somewhat similarly to cereal spikes as powdery mildew does on plant leaves, wherein both adhere, germinate, penetrate and propagate, and both generate plant defense responses which lead to callose deposition, and the production of jasmonate and ethylene or salicylic acid.

[00156] Callose deposits are typically in between the cell wall and the plasma membrane of a plant cell. The extracellular presence of a conidium triggers the response of a receptor to create a response to the penetration peg of the appressorium, thereby providing a callosic papilla to combat the penetration into the cell.

[00157] Studies with wheat plants infected with a mutated fgll (Δfgll) Fusarium graminearum that measured the concentration of callose present after inoculation demonstrated levels comparable with the control, whereas wild-type Fusarium graminearum resulted in markedly decreased callose presence, declining from the basal levels of -0.45 μg/mg plant tissue to nearing zero around 14 days after inoculation. Callose synthase activity was then measured during this same 2 week course post- Fusarium graminearum inoculation. Again the wheat plants inoculated with Δfgll showed similar if not improved callose synthase activity over the control, whereas wheat plants inoculated with wild-type Fusarium graminearum showed markedly diminished callose synthase activity. [00158] It was next examined what fatty acids, which along with glycerol are a byproduct of lipase activity on triacylglycerol. Saturated and unsaturated (18:0, 18: 1, 18:2, 18:3) free fatty acids were administered to plant cells and callose synthase activity was measured. All forms of fatty acids resulted in inhibiting callose synthase activity, all especially at concentrations over 10 μM. Addition of exogenous free fatty acids to wheat plants suppressed callose accumulation and rendered the plants susceptible to the Δfgll mutant (see Voigt et al, Plant Journal 42: 364-375, 2005).

[00159] Given the previously reported results concerning disruption of the callose synthase GSL5 providing improved resistance to pathogen attack, it was next examined what the effect of over-expressed GSL5 would be on a plant's resistance. The GSL5 gene was placed under the control of the constitutive promoter, CaMV35S (Cauliflower Mosaic Virus 35S), and transformed into plant cells. See Figure 1. The previously reported pmr4 gene (deficient in callose synthase activity) as utilized along with wild- type plant cells as a control. Plants were then inoculated with powdery mildew. Powdery mildew typically has a life cycle of first presenting as a conidium to the cell wall of an epidermal plant cell, followed by presentation of the appressorium to penetrate the cell wall, resulting in the penetration peg. The haustorium then forms and followed by the formation of conidiospores.

[00160] Cells over-expressing wild-type GSL5 showed a remarkable and unexpectedly improved resistance to powdery mildew. See Figure 2. While the expression oϊpmr4 showed improved resistance over wild-type plants, over-expressed GSL5 did not result in a weakened or more prone plant, but instead surpassed the resistance as compared to pmr4. The mass of plants over-expressing GSL5 was near baseline 10 days post- inoculation with powdery mildew, whereas wild-type and pmr4 plants showed significant decrease in plant mass 10 days following inoculation. See Figure 3.

[00161] It was next examined what the extent of callose synthase activity was at certain point before and during inoculation, namely 6, 12, and 24 hours. See Figure 4. Plants over-expressing GSL5 showed a markedly increased level of activity at 6 hours, well above wild-type or pmr4 plants. At 12 days, the wild-type activity had increased, as well as slightly thepmr4. By 24 hours, the 35S:GSL5 plants showed diminished activity, comparable to wild-type and the now increased pmr4 plants. These data indicate that GSL5 is an early responding callose synthase, and further that other callose synthase genes respond later to aid the plant's resistance.

[00162] The deposition of callose in these plants was next examined. Following 6 hours, the over-expressed GSL5 showed a significantly higher deposition of callose as compared to wild-type plants. In corroboration with the callose synthase activity data, pmr4 plants did not show significant deposits of callose.. See Figure 5. The diameter of callose deposits was next determined, and two different 35S:GSL5 transformants were assessed, along with wild-type plants. Both plants over-expressing GSL5 showed markedly higher initial callose deposition. See Figure 6. Furthermore, 24 hours after inoculation, the transformed plants continued to demonstrate a moderately well maintained callose deposit, whereas wild-type plant deposits had dwindled to a diffuse deposit.

[00163] It was then assessed how well powdery mildew could penetrate plants over- expressing GSL5. See Figure 7. At 12 and 24 hours post-powdery mildew inoculation, it was determined how well the cells of the plants were penetrated by the pathogen. Both wild-type and pmr4 plants demonstrated over 80% penetration at 12 hours post- inoculation, whereas both lines of 35S:GSL5 demonstrated no detectable penetration by powdery mildew at 12 or 24 hours after inoculation. The fungal growth and callose deposition were then determined a week after powdery mildew inoculation. See Figure 8. Over-expressed GSL5 plants showed less wide-spread callose deposition, but also showed significantly less fungal growth, indicating less areas for callose deposition were required. Plants expressing pmr4 showed a reduced callose deposits, but also reduced fungal growth, confirming earlier results.

[00164] It was then assessed what the role of phosphorylation of callose synthase is for plant resistance. The serine at amino acid 1053 was mutated either to an alanine to inhibit signaling or to a glutamate to mimic constitutive phosphorylation. Plants expressing the S1053A mutant showed reduced callose deposition in response to pathogen attack or wounding, whereas S1053D demonstrated improved callose deposition and disease resistance as compared to native plants. See Figure 9.

[00165] These data are summarized in Figure 10. The over-expression of callose synthase through coupling GSL5 to the constitutively active promoter CaMV35S, yields improved fungal resistance as well as providing for conservation of the mass of the pathogen attacked plant, and provides for increased levels of callose deposits.

[00166] It should be understood that the foregoing discussion and examples merely present a detailed description of certain preferred embodiments. It therefore should be apparent to those of ordinary skill in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention. All journal articles, other references, patents, and patent applications that are identified in this patent application are incorporated by reference in their entirety.

Claims

We claim:

Claim 1 : A transgenic plant genetically engineered to comprise a nucleic acid that encodes callose synthase a functional equivalent thereof, or a homolog thereof.

Claim 2: The transgenic plant of claim 1, wherein the nucleic acid comprises a promoter and a gene.

Claim 3: The transgenic plant of claim 2, wherein the gene is GSL5.

Claim 4: The transgenic plant of claim 2, wherein the promoter is constitutively active.

Claim 5: The transgenic plant of claim 2, wherein the promoter is Cauliflower Mosaic Virus 35S (CaMV35S).

Claim 6: The transgenic plant of claim 1, wherein the plant is a cereal plant.

Claim 7: The transgenic plant of claim 1, wherein the plant is a fruit bearing plant.

Claim 8: The transgenic plant of claim 1, wherein the plant is a vegetable.

Claim 9: The transgenic plant of claim 1, wherein the nucleic acid comprises an expression vector.

Claim 10: A method for producing a transgenic plant with increased pathogen resistance, comprising: introducing a nucleic acid encoding a callose synthase or a functional equivalent or homolog thereof under the control of a constitutive promoter into a plant cell, and culturing the plant cell under conditions to allow the plant cell to grow into a transgenic plant. Claim 11 : A transgenic plant produced by the method of claim 10.

Claim 12: A method of improving the response of a plant to an insult comprising introducing into the plant or part thereof a nucleic acid encoding callose synthase.

Claim 13: The method of claim 12, wherein the nucleic acid integrates into the plant's genome.

Claim 14: The method of claim 12, wherein the nucleic acid comprises a constitutively active promoter and a gene encoding callose synthase.

Claim 15: The method of claim 14, wherein the promoter is Cauliflower Mosaic Virus 35S.

Claim 16: The method of claim 14, wherein the gene is GSL5.

Claim 17: The method of claim 12, wherein the insult is a pathogen attack.

Claim 18: The method of claim 17, wherein the pathogen is Golovinomyces cichoraceanim .

Claim 19: The method of claim 12, wherein the insult is a wound.

Claim 20: The method of claim 12, wherein the plant is a cereal plant.

Claim 21 : The method of claim 12, wherein the plant is a fruit bearing plant.

Claim 22: The method of claim 12, wherein the plant is a vegetable.

Claim 23 : A method of increasing disease-resistance of a plant cell to a pathogen that directly penetrates the plant cell, comprising introducing into the plant cell a nucleic acid sequence encoding a callose synthase, a functional equivalent, or a homolog thereof. Claim 24: The method of claim 23, wherein the nucleic acid is under the control of a constitutively active promoter.

Claim 25: The method of claim 23, wherein the plant cell is derived from a plant wherein the plant is selected from the group consisting of Arabidopsis thaliana, banana, Brachypodium distachyon, barley, grape, maize, oat, potato, tomato, soybean, sugarcane, and wheat.

Claim 26: The method of claim 23, wherein the pathogen is bacterium, insect, fungus, nematode or oomycete.

Claim 27: The method of claim 23, wherein the pathogen is powdery mildew.

Claim 28: The method of claim 24, wherein the pathogen is selected from the group consisting of Golovinomyces cichoracearum, Mycosphaerella musicola, Fusarium graminearum, Blumeria graminis f. sp. hordei, Puccinia hordei, Puccinia graminis f. sp. tritici, Puccinia striiformis, Pyrenophora teres, Rhynchosproium secalis, Erysiphe necator, Colletotrichum graminicola, Puccinia sorghi, Ustilago maydis, Blumeria graminis f. sp. avenae, Phytophthora infestans, Magnaporthe grisea, Oidium neolycopersici, Leveillula taurica, Phakopsora pachyrhizi, Phytophthora sojae, Puccinia melanocephala, Peronosclerospora sacchari, Ustilago scitaminea, Blumeria graminis f. sp. tritici, Puccinia graminis f. sp. tritici, and Tilletia indica.

Claim 29: A method to increase broad-spectrum resistance of a plant to bacterial pathogens that utilize pilli or similar structures to interact with a host plant cell comprising introducing to the host plant cell a nucleic acid encoding a callose synthase.

Claim 30: The method of claim 29, wherein the nucleic acid is regulated by a constitutively active promoter. Claim 31 : A method to increase broad-spectrum disease resistance of a plant to pathogens that gain entry via wounds comprising introducing the nucleic acid of claim 1 to a cell of the plant.

Claim 32: A method to reduce the formation of blemishes at wound sites of a plant comprising introducing to a cell of the plant a nucleic acid comprising a promoter operably linked to a gene, wherein the gene encodes callose synthase .

Claim 33: A method for increasing resistance of a plant to a pathogen comprising: a) stably transforming a plant cell with an expression vector, wherein the expression vector comprises a nucleic acid operably linked to a promoter that drives expression in a cell of the plant, and further wherein the nucleic acid encodes a callose synthase, a functional equivalent, or a homolog thereof; and b) regenerating a transformed plant from the plant cell, wherein the level of resistance to the pathogen in the plant cell is increased in comparison to a plant that does not comprise the expression vector.

Claim 34: The method of claim 33, wherein the promoter is constitutively active.

Claim 35: A transgenic plant produced by the method of claim 33.

Claim 36: A method for producing a plant crop resistant to pathogen, comprising introducing to a cell of a plant of the crop a nucleic acid encoding a callose synthase, a functional equivalent, or a homolog thereof.

Claim 37: A method for identifying an agent that modulates callose synthase expression comprising contacting a cell comprising expressing callose synthase, a functional equivalent, or a homolog thereof with a test agent, wherein the agent modulates the level of callose synthase expression as compared to a control cell. Claim 38: The method of claim 37, wherein callose synthase expression is increased by the test agent.

Claim 39: The method of claim 37, wherein callose synthase expression is decreased by the test agent.

Claim 40: A method for identifying an agent that modulates callose synthase activity comprising contacting a cell comprising expressing a callose synthase, a functional equivalent, or a homolog thereof with a test agent, wherein the agent modulates the level of callose synthase activity as compared to a control cell.

Claim 41 : The method of claim 40, wherein callose synthase activity is increased by the test agent.

Claim 42: The method of claim 40, wherein callose synthase activity is decreased by the test agent.