WO2012040851A1 - Horsetail silicon transporter genes and uses thereof - Google Patents

Abstract

The present invention features polynucleotides encoding horsetail silicon transporters, vectors, cells, and plants including such polynucleotides, and methods for making such plants. The invention also features horsetail silicon transporter polypeptides and fragments thereof. Plants expressing this horsetail transporter exhibit both increased silicon uptake and increased resistance to biotic and abiotic stresses. In particular, plants such as soybean expressing this horsetail silicon transporter may exhibit increased resistance to pathogens such as rust.

Description

HORSETAIL SILICON TRANSPORTER GENES

AND USES THEREOF

Cross-reference to related application

[0001] This application claims priority from US provisional application 61/388,003 filed on September 30, 2010, the content of which is hereby incorporated by reference in its entirety.

Background of the Invention

[0002] The invention relates to compositions and methods which may be useful for increasing silicon uptake and increasing resistance to biotic and abiotic stresses in plants (such as for example soybean). Biotic and abiotic stresses on plants cause billions of dollars worth of damage to crops each year. For example, Soybean rust, a disease caused by the Phakopsora pachyrhizi iungus, resulted in approximately $1 billion worth of damage in Brazil in 2003. This disease has now begun to spread into the United States, the largest producer of soybean worldwide. While the rust can be treated using chemical fungicides, doing so is expensive, potentially damaging to the environment, and may only be partially effective. Accordingly, there is a need for additional or improved methods for protecting plants against biotic as well as abiotic stresses. Prevention or control of soybean rust is an important application in this regard.

Summary of the Invention

[0003] We have discovered silicon influx transporter gene in horsetail (Equisetum arvense), a plant known to take up silicon efficiently. The encoded transporter protein increase resistance to biotic and abiotic stresses when expressed in a plant (such as for example Aribidopsis or soybean). The present invention thus features polynucleotides encoding a silicon transporter; vectors, cells, and plants including such polynucleotides; and methods for making such plants. The invention also features silicon transporter polypeptides and fragments thereof. Particularly useful are plants transformed with the silicon transporter described herein, where expression of the silicon transporter results in increased resistance to biotic or abiotic stresses. More particularly useful are soybean plants transformed with the silicon transporter described herein, where expression of the silicon transporter results in increased resistance to soybean rust.

[0004] Accordingly, in a first aspect, the invention features an isolated or a substantially pure polynucleotide including a nucleic acid sequence substantially identical (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical) to a sequence selected from the group consisting of SEQ ID NOs: 1 , 3 and 5 or a fragment thereof. The invention also features a polynucleotide including a nucleic acid sequence that encodes a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 7, or a fragment thereof. In some embodiments, expression of the polypeptide encoded by the polynucleotide of the first aspect in a cell increases or is capable of increasing silicon transport into the cell. The polynucleotide may be less than 1 ,000, 500, 100, 50, 30, 20, 15, 10, 8, 6, 5, 4, 3, or 2 kb in length.

[0005] In a second aspect, the polynucleotide may be operably linked to a promoter, for example, a promoter capable of expression in a plant cell. The promoter may be time- dependent, cell specific (e.g., root cells), or tissue specific (e.g., in any tissue described herein). The promoter may be constitutive or inducible, for example, under environmental conditions such any abiotic or biotic stress (e.g., those described herein).

[0006] In a third aspect, the invention also features a vector including a polynucleotide of the invention.

[0007] The invention also features a cell such as a plant cell (e.g., a soybean cell or a cell from any plant described herein), a bacterial cell, or any cell described herein including the vector. The cell may, in some embodiments, be part of a plant seed or a tissue from a plant (e.g., any described herein). The invention also features a polypeptide, or fragment thereof, encoded by any of the polynucleotides described herein. The polypeptide may be substantially pure or may be expressed in a cell recombinantly.

[0008] In another aspect, the invention features a plant (e.g., soybean or any plant described herein), plant tissue, or seed including one or more heterologous

polynucleotides including a nucleic acid sequence substantially identical to a nucleic acid sequence encoding a silicon influx transporter or a fragment thereof (e.g., a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs:1 , 3 and 5 or a fragment thereof) or a nucleic acid sequence encoding a polypeptide substantially identical to an amino acid sequence SEQ ID NOs: 2, 4, 6 and 7, or a fragment thereof. The polypeptide encoded by the heterologous polynucleotide may increase or be capable of increasing the transport of silicon into at least one tissue or cell (e.g., root cells) within the plant upon expression. The plant may exhibit increased resistance to one or more biotic or abiotic stress (e.g., those described herein). In one embodiment, the plant is a soybean plant exhibiting increased resistance to soybean rust, or a tissue or seed from such a plant. [0009] In yet another aspect, the invention features a plant (e.g., soybean or any plant described herein), plant tissue, or seed including a heterologous polynucleotide substantially identical to a nucleic acid sequence encoding a silicon influx transporter or a fragment thereof. The polynucleotide may include a nucleic acid sequence substantially identical (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical) to (a) a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1 , 3 and 5; (b) a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6 and 7, or (c) a fragment thereof. The polypeptide encoded by the heterologous polynucleotide may increase or be capable of increasing the transport of silicon into at least one tissue or cell (e.g., root, stem, or leaf cells) within the plant upon expression.

[0010] The invention also features methods for generating any of the plants, plant tissues, or seeds described above. In one aspect, the method includes (a) providing a first vector including a polynucleotide substantially identical to a nucleic acid sequence encoding a silicon influx transporter or a fragment thereof (e.g., any of those described above); (b) transforming a plant cell (e.g., a soybean cell or a cell from any plant described herein) with the vector; and (c) growing a plant from the cell, where the plant expresses the polynucleotide, thereby generating a plant with increased silicon uptake. The transformation may be performed using any method known in the art (e.g., any method described herein).

[0011] In another aspect, the invention also features a method of generating a plant, plant tissues, or plant seeds with increased silicon transport. The method includes (a) providing a first vector including a polynucleotide substantially identical to a nucleic acid sequence encoding a silicon transporter, or a fragment thereof (e.g., an influx transporter such as any of those described above); (b) transforming a plant cell (e.g., a soybean cell or cell from any plant) with the vector; and (c) growing a plant from the cell, where the plant expresses the polynucleotide, thereby generating a plant with increased silicon transport.

[0012] In either of the previous two methods, the method may further include step (d) generating seeds from the plant or harvesting at least one tissue from the plant. In the aspects directed to plants, plant tissues, and seeds or related methods, the plant tissue may be, for example, root, fruit, ovule, male tissue, seed, integument, tuber, stalk, pericarp, leaf, stigma, pollen, anther, petal, sepal, pedicel, silique, and stem. Seed tissues include embryo, endosperm, and seed coat. [0013] Other features and advantages of the invention will be apparent from the following Detailed Description, the drawings, and the claims.

Detailed Decription of the invention

Brief Description of the Drawings

[0014] Figure 1 is an alignment of rice and wheat Lsi1 (a), NIP B, NIP C and NIP D (b) and with all Lsi1 (c) nucleic acid sequences. In these alignments, the first sequence is the reference. Identical nucleic acids in other sequences are marked by a dot

[0015] Figure 2a is an alignment of rice and wheat Lsi1 , and horsetail NIP B, NIP C and NIP D predicted amino acid sequences. NPA loops and pore residues are boxed and transmembrane domains are indicated above the sequence. Figure 2b is a Kyte and Doolittle mean hydrophobicity profile of rice and wheat Lsi1 , and horsetail NIP B, NIP C and NIP D aligned predicted amino acid sequences. Regions above zero indicate hydrophobic zones of the proteins.

[0016] Figure 3 is a quantification of silicon concentration in oocytes after 0, 30 or 60 min of incubation in a solution without or with 1.7 mM Si. Control oocytes were injected with water (negative control). NIP B, C and D oocytes were injected with horsetail silicon transporters and Lsi1 wheat oocytes were injected with wheat Lsi1 silicon transporter (positive control).

[0017] Figure 4 is a microscopic observation of GPF fluorescence in Tobacco leaves when GFP alone or C-terminally fused to TaLsil (TaLsi1 -GFP) or OsLsil (OsLsi1-GFP) was expressed. GFP-derived fluorescence (a-c) and fluorescence super-imposed over the transmission image (d-f). (a, d) leaves expressing GFP alone, (b, e) leaves expressing TaLsil -GFP and (c, f) leaves expressing OsLsil -GFP. Images are representative of the results seen on five samples.

[0018] Figure 5 is a microscopic observation of GFP fluorescence when TaLsil -GFP or OsLsil -GFP fusion proteins are expressed in Arabidopsis. As similar observations are noticed both in rice and wheat, fluorescence super-imposed over the transmission image of wild-type plants (a, c and e) are compared to representative Lsi1-GFP lines (b, d and f).

[0019] Figure 6 is a OsLsil and TaLsil relative expression measurement in roots or leaves of Arabidopsis by quantitative real-time PCR analysis. Mean values ± SD from two different lines by transgenic plant are shown (n=3). [0020] Figure 7 is X-ray microanalysis mapping images super-imposed over SEM images showing Si deposition on leaves harvested from WT plants (a, b) and transgenic plants expressing Lsi1 transporter (c, d) treated without (a) or in presence of 1.7 mM Si (b, c and d). Si concentration is indicated by color, where red represents the highest concentration of Si and black indicates no Si. Observations are representative of analyses on five samples.

[0021] Figure 8 is a measurement of Si concentration in aerial part of 35S-OsLsi1 or 35S- TaLsil transgenic plants and corresponding WT plants. Plants were grown hydroponically without Si or in presence of 1.7 mM Si and Si concentrations within leaves was determined by ICP-MS. Mean values ± SD from a pool of ten plants by lines are shown (n=3).

[0022] Figure 9 is a measurement of Si concentration by ICP-OES in aerial parts of WT, 35S::TaLsi1 and pNIP5.:TaLsi1 transgenic plants grown with or without 1.7 mM Si for 7 days. [0023] Figure 10 is a photographic observation of Arabidopsis thaliana infected with powdery mildew (Erysiphe cichoracearum) seven days after inoculation. Red arrows show leaf spots infected.

[0024] Figure 11 is a quantification of silicon concentration in soybean plants grown in 1.7 mM Si. "Variety Jack" represents the control variety that was also transformed with the wheat silicon transporter (TaLsil ) subsequently named SYDC04U.

Definitions

[0025] By "isolated" or "substantially pure" polynucleotide is meant a nucleic acid (e.g., a DNA or an RNA molecule) that is free of the genes which, in the naturally- occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. [0026] By "isolated" or "substantially pure" polypeptide is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 30%, 50%, 60%, 70%, 80%, 90% 95%, or even 99%, by weight, free from the proteins and naturally- occurring organic molecules with which it is naturally associated. A substantially pure polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis.

[0027] By "transformed cell" is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule, for example, a DNA molecule encoding a silicon influx transporter or any of the nucleic acids described herein.

[0028] By "fragment" of a polynucleotide or amino acid sequence is meant at least 10, 15, 20, 25, 30, 50, 75, 100, 250, 300, 400, or 500 contiguous nucleic acids or amino acids of any of a longer sequence (e.g., a sequence described herein).

[0029] The term "substantial identity" as applied to nucleic acid sequences denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 50 percent, preferably 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical as compared to a reference (e.g., any of the sequences described herein).

[0030] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C to about 20°C, usually about 10°C to about 15°C, lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60°C. For instance in a standard Southern hybridization procedure, stringent conditions will include an initial wash in 6x SSC at 42°C followed by one or more additional washes in 0.2x SSC at a temperature of at least about 55°C, typically about 60°C, and often about 65°C. [0031] Nucleotide sequences are also substantially identical for purposes of this invention when said nucleotide sequences encode polypeptides and/or proteins which are substantially identical. Thus, where one nucleic acid sequence encodes essentially the same polypeptide as a second nucleic acid sequence, the two nucleic acid sequences are substantially identical even if they would not hybridize under stringent conditions due to degeneracy permitted by the genetic code (see, Darnell et al., Molecular Cell Biology, Second Edition Scientific American Books W. H. Freeman and Company New York, 1990 for an explanation of codon degeneracy and the genetic code). Protein purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization upon staining. For certain purposes high resolution may be needed and HPLC or a similar means for purification may be used.

[0032] The term "substantial identity" as applied to amino acid sequences denotes a characteristic of a polypeptide, wherein the peptide comprises a sequence that has at least 60 % 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to another sequence (e.g., any of the sequences of Figure 1 , or a fragment thereof).

[0033] A "silicon influx transporter" is a polypeptide that is able to increase silicon transport into a cell.

[0034] By a polypeptide which "increases silicon transport" into or from a cell is meant a polypeptide whose expression in that cell results in increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 300%, 500%, 1 ,000%, 5,000%, or 10,000%) in the rate of silicon or germanium transport through the cell membrane (e.g., into or out of the cell) as compared to a cell lacking the polypeptide, but does not substantially disrupt the cell membrane or increase transport of other molecules (e.g., glycerol) in a non-specific manner.

Description of particular embodiments

[0035] Horsetail and grasses such as wheat, oat, sorghum, and barley are known to be high accumulators of silicon. Horsetail, in particular, is known to accumulate silicon very efficiently, and silicon compounds can make up to 15% of horsetail dry weight. We therefore hypothesized that these plants, due to their high silicon content, would likely transport silicon efficiently, i.e.. able to cause accumulation of high concentrations of silicon in the plant, able to transport silicon rapidly, or both. These transporters may therefore be used to increase silicon uptake in a heterologous cell (e.g.. in a plant that normally has lower silicon uptake or transport) by expressing a transporter described herein. Because increased silicon content in plants is associated with increased resistance to both biotic and abiotic stresses, expression of these transporters in a plant may increase resistance to stress. Such an approach may be particularly useful in soybean, where soybean rust caused by Phakopsora pachyrhizi fungus causes significant damage to the soybean crop. Accordingly, the present invention features polynucleotides and polypeptides having sequence identity to the silicon transporters identified herein, vectors, cells, and plants (e.g., soybean) containing such polynucleotides, and methods for making such plants. Plants expressing silicon transports may exhibit increased resistance to fungus such as rust.

Silicon in plants

[0036] Silicon (Si) is absorbed by the root system in the form of silicic acid where it can eventually accumulate in the form of polymerized silicon in the shoots and leaves of plants. However, plants vary greatly in their ability to absorb silicon, thereby causing variability in their ability to benefit from Si feeding. In a survey of nearly 500 plant species, plants were ranked into three groups according to their Si accumulation: 1 ) high Si accumulators including Gramineae (grasses); 2) intermediate accumulators including Cucurbitaceae; and 3) low accumulators including most other plant species (for a summary see Ma and Takahashi, Soil, Fertilizer, and Plant Silicon Research in Japan,

Amsterdam Elsevier Science, 2002). For example, grasses such as oat, rye, and ryegrass, contained 2.04, 2.41 , and 2.34 % Si0₂, when grown in soil containing 45 ppm Si0₂ in solution at pH 6.0. By contrast, crimson clover, peas, and mustard, in the same soil, contained 0.12, 0.25, and 0.15% Si0₂, respectively (Jones et al, Advances in Agronomy, 107- 149, 1967). Differences in Si accumulation have been attributed to the ability of the roots to take up Si whereby plants would possess one of three modes of absorption: active, passive, or rejective uptake.

[0037] Silicon is one of the most abundant elements on the surface of the earth, but its essentiality in plant growth has not been clearly established (Epstein, Silicon in Agriculture. Datnoff et al., eds. New York: Elsevier Science; 2001 :1- 15; Epstein, Proc Natl Acad Sci USA 91:11-17, 1994; Epstein, Annu Rev Plant Physiol Plant Mol Biol 50:641-664, 1999). While its nutritional role in plants appears limited, there is accumulating evidence that Si absorption plays an important function in protection against biotic and abiotic stresses. Many reports have implicated Si with improved plant growth in situations of nutrient deficiency or excess. Si fertilization has also been linked to increased resistance of plants to diseases, including powdery mildew pathogens on wheat, barley, rose, cucumber, muskmelon, zucchini squash, grape, and dandelion and for other diseases such as blast (Pyricularia grisea) and brown spot (Bipolaris oryzae) on rice, Botrytis cinerea, Didymella bryoniae, Fusarium wilt, and root rot caused by Pythium ultimum and P. aphanidermatum on cucumber.

[0038] Three silicon transporters have been identified in rice (Ma et al., Nature 440:688-691 , 2006; Ma et al., Nature 448:209-212, 2007; Yamaji et al, The Plant Cell 20: 1381-1389, 2008), including two Si influx transporters (SIITI, also referred to as Lsil; and SIIT2, also referred to as Lsi6) and a Si efflux transporter (SIETI, also referred to as Lsi2). The influx transporters SIITI and SIIT2 are predicted to be membrane proteins similar to water channel proteins, aquaporins. These proteins belong to the NIP subfamily (Nod26-like intrinsic protein). The channel is formed from six transmembrane segments (TM), two hydrophilic loops (HL3 between TM3 and TM4; HL4 between TM4 and TM5) and two Asn- Pro-Ala (NPA) motifs, an arrangement that is conserved in aquaporins. A pore structure and constrictions that may determine selective water permeability are assembled with HL3 and the second NPA domain (NPA2) in the extracellular side and with HL4 and the first NPA domain (NPAI) in the cytoplasmic membrane (Wallace and Roberts, 2004, Plant Physiology 135(2) 1059-1068).. The NPA boxes may be important for correct assembly of the three-dimensional structures of aquaporins, because such proteins with mutations near NPA boxes can be folded improperly. Some aquaporins of the NIP subfamily have an alternate NPV motif, but the substitution of Ala for Val does not appear to

experimentally affect the transport properties of several plant NIPs (Wallace and Roberts, 2005, Biochemistry 44: 16826-16834). The expression of the SIITI transporter appears to be localized in roots with a constitutive expression regulated by Si level. The transporter SIIT2 appears to be expressed in the root tips and in the xylem parenchyma cells of leaf sheaths and blades.

Polynucleotides

[0039] We have identified and cloned 3 silicon transporter genes from horsetail

(Equisetum arvense). Accordingly, the invention features polynucleotides having substantial identity to any of the polynucleotides described herein, or fragments of such polynucleotides. In certain embodiments, the polynucleotides may encode functional silicon transporter polypeptides (e.g., polypeptides, that when expressed in a cell, are capable of increasing silicon influx). Identification of exemplary polynucleotides of the invention is described in greater detail herein. [0040] The invention also features fragments of the polynucleotides described herein. Such fragments may also encode functional silicon transporter polypeptides. Shorter fragments may be useful as primers, or may encode antigenic polypeptide sequences. Fragments may include the transmembrane segments, or the hydrophilic loops of the transporter.

Identification of silicon influx transporters in plants

[0041] We identified silicon transporter sequences in horsetail through a transc ptomic approach. BLAST searches on the database created, allowed the identification of three transporter sequences in horsetail and termed these sequences NIP B, NIP C and NIP D (SEQ ID NO: 1 , 3 and 5 respectively). Comparison of the horsetail cDNA, coded sequences (SEQ ID NO:2, 4 and 6), to the 296 amino acid rice polypeptide sequence (SEQ ID NO:15) and the corresponding wheat sequence (SEQ ID NO:17) revealed from 35 to 39% identity only.

Consensus sequence based on NIP B. NIP C and NIP D sequences

[0042] We have generated a consensus sequence based on the polypeptide sequences encoded by the NIP B, NIP C and NIP D nucleic acid sequences identified above. This consensus sequence is represented by SEQ ID NO.7

Promoters

[0043] Any polynucleotide described herein can be operatively linked to an appropriate promoter to confer gene expression (e.g., in a cell or in an in vitro system such as a cell extract). Promoters can regulate expression in a time- dependent, cell specific (e.g., root cells), or tissue specific manner. Promoters useful in the expression cassettes of the invention include any promoter that is capable of initiating transcription in a plant cell. Such promoters include those that can be obtained from plants, plant viruses, and bacteria that contain genes that are expressed in plants, such as Agrobacterium and

Rhizobium. In one embodiment, the promoter is constitutively active in root cells (e.g., the Atl 7.1 promoter). In another embodiment, the promoter is induced by a biotic or abiotic stress.

[0044] The promoter may be constitutive, inducible, developmental stage- preferred, cell type-preferred, tissue-preferred, or organ-preferred. Constitutive promoters are active under most conditions. Examples of constitutive promoters include the CaMV 19S and 35S promoters (Odell et al., Nature 313:810-812,1985), the sX CaMV 35S promoter (Kay et al., Science 236:1299- 1302, 1987), the Sepl promoter, the rice actin promoter (McElroy et al., Plant Cell 2: 163-171 , 1990), the Arabidopsis actin promoter, the ubiquitin promoter

(Christensen et al., Plant Mol. Biol. 18:675-689, 1989), pEmu (Last et al., Theor. Appl. Genet. 81:581-588, 1991), the figwort mosaic virus 35S promoter, the Smas promoter (Velten et al., E BO J 3:2723-2730, 1984), the GRP 1-8 promoter, the cinnamyl alcohol dehydrogenase promoter (US 5,683,439), promoters from the T-DNA of Agrobacterium, such as mannopine synthase, nopaline synthase, and octopine synthase, and the small subunit of ribulose biphosphate carboxylase (ssuRUBISCO) promoter.

[0045] In other embodiments, an inducible promoter is used. Such promoters are preferentially active under certain environmental conditions, such as the presence or absence of a nutrient or metabolite, heat or cold, light, pathogen attack, anaerobic conditions, or under any abiotic or biotic stress (e.g., those described herein). For example, the hsp80 promoter from Brassica is induced by heat shock; the PPDK promoter is induced by light; the PR-I promoters from tobacco, Arabidopsis, and maize are inducible by infection with a pathogen; and the Adhl promoter is induced by hypoxia and cold stress. Plant gene expression can also be facilitated via an inducible promoter (for review, see Gatz, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108, 1997). Chemically inducible promoters are especially suitable if gene expression is wanted to occur in a time specific manner. Examples of such promoters are a salicylic acid inducible promoter (WO

95/19443), a tetracycline inducible promoter (Gatz et al. Plant J. 2:397-404, 1992), and an ethanol inducible promoter (WO 93/21334). An inducible promoter is a stress-inducible promoter. Such promoters may be activated based on sub-optimal conditions associated with salinity, drought, temperature, metal, chemical, pathogenic, and oxidative stresses. Stress inducible promoters include, but are not limited to, Cor78 (Chak et al., Planta 210:875-883, 2000; Hovath et al., Plant Physiol. 103:1047-1053, 1993), Corl5a (Artus et al., Proc Natl Acad Sci USA 93: 13404-09, 1996), Rci2A (Medin et al., Plant Physiol. 125:1655-66, 2001 ; Nylander et al., Plant Mol. Biol. 45:341-52, 2001 ; Navarre et al., EMBO J. 19:2515-24, 2000; Capel et al., Plant Physiol. 115:569-76, 1997), Rd22 (Xiong et al., Plant Cell 13:2063-83, 2001 ; Abe et al., Plant Cell 9:1859-68, 1997; Iwasaki et al, Mol. Gen. Genet. 247:391-8, 1995), cDeto (Lang et al., Plant Mol. Biol. 20:951-62, 1992), ADHI (Hoeren et al., Genetics 149:479-90, 1998), KATI (Nakamura et al. Plant Physiol. 109:371-4, 1995), KSTI (Muller-Rober et al, EMBO 14:2409-16, 1995), Rhal (Terryn et al, Plant Cell 5:1761-9, 1993; Terryn et al, FEBS Lett. 299:287-90, 1992), ARSK I (Atkinson et al, 1997, GenBank Accession # L22302, and WO 97/20057), PtxA (Plesch et al, GenBank Accession # X67427), SbHRGP3 (Ahn et al. Plant Cell 8:1477-90, 1996), GH3 (Liu et al. Plant Cell 6:645-57, 1994), the pathogen inducible PRPI -gene promoter (Ward et al. Plant. Mol. Biol. 22:361-366, 1993), the heat inducible hsp80-promoter from tomato (US 5,187,267), cold inducible a-amylase promoter from potato (WO 96/12814), or the wound- inducible pinll- promoter (EP 375091 ). Other examples of drought, cold, and salt-inducible promoters, such as the RD29A promoter, are described by Yamaguchi-Shinozalei et al. (Mol. Gen. Genet. 236:331 -340, 1993).

[0046] Developmental stage-preferred promoters are preferentially expressed at certain stages of development. Tissue and organ preferred promoters include those that are preferentially expressed in certain tissues or organs, such as roots, xylem, leaves, or seeds. An example of an organ-preferred and stress upregulated promoter is the Atl 7.1 promoter, which drives gene expression in the roots and vascular system of soybean plants (Mazarei et al., Mol Plant Pathol 5:409-423, 2004). Other examples of tissue-preferred and organ- preferred promoters include, root-preferred, fruit-preferred, ovule-preferred, male tissue-preferred, seed -preferred, integument-preferred, tuber-preferred, stalk- preferred, pericarp-preferred, and leaf-preferred, stigma-preferred, pollen- preferred, anther-preferred, petal-preferred, sepal-preferred, pedicel-preferred, silique-preferred, and stem-preferred. Seed-preferred promoters are preferentially expressed during seed development and/or germination. For example, seed-preferred promoters can be embryo- preferred, endosperm- preferred, and seed coat-preferred (see Thompson et al., BioEssays 10:108, 1989). Examples of seed preferred promoters include cellulose synthase (celA), Ciml, gamma-zein, globulin- 1 , and maize 19 kD zein (cZ19BI).

[0047] Other suitable tissue-preferred or organ-preferred promoters include the napin- gene promoter from rapeseed (US 5,608,152), the USP- promoter from Vicia faba

(Baeumlein et al., Mol. Gen. Genet. 225:459-67, 1991 ), the oleosin-promoter from Arabidopsis (WO 98/45461 ), the phaseolin-promoter from Phaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoter from Brassica (WO 91/13980), or the legumin B4 promoter (LeB4;

Baeumlein et al., Plant Journal, 2:233-9, 1992), as well as promoters conferring seed-specific expression in monocot plants including maize, barley, wheat, rye, and rice. Suitable promoters are the Ipt2 or Iptl-gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890 (promoters from the barley hordein-gene, rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oat glutelin gene, Sorghum kasirin-gene, and rye secalin gene). Other promoters useful in the invention include the major chlorophyll a/b binding protein promoter, histone promoters, the Ap3 promoter, the β-conglycin promoter, the napin promoter, the soybean lectin promoter, the maize 15 kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter, the γ-zein promoter, the waxy, shrunken 1 , shrunken 2, and bronze promoters, the Zml 3 promoter (US 5,086,169), the maize polygalacturonase promoters (PG) (US 5,412,085 and US 5,545,546), and the SGB6 promoter (US 5,470,359), as well as synthetic or other natural promoters.

Vectors

[0048] A polynucleotide encoding a silicon transporter (e.g., any of those described herein such as a polynucleotide operably linked to a promoter) may be part of an expression vector. Any suitable vector known in the art may be used. The vector may be an autonomously replicating vector, i.e., a vector existing as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated simultaneously with the chromosomes into which it has been integrated.

[0049] Plant expression vectors can include (1 ) a cloned plant gene (e.g., a silicon transporter gene) under the transcriptional control of 5' and optionally 3' regulatory sequences (e.g., a promoter such as a promoter described herein). The vector may also include a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region (for example, one conferring inducible or constitutive, pathogen- or wound- induced, 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.

[0050] Plant expression vectors may also optionally include RNA processing signals, e.g., introns, which have been shown to be important for efficient RNA synthesis and accumulation. The location of the RNA splice sequences can dramatically influence the level of transgene expression in plants. An intron may therefore be positioned upstream or downstream of a silicon transporter coding sequence in the transgene to alter levels of gene expression.

[0051] In addition to the aforementioned 5' regulatory control sequences, the expression vectors may also include regulatory control regions which are generally present in the 3' regions of plant genes. For example, the 3' terminator region may be included in the expression vector to increase stability of the mRNA. One such terminator region may be

J 3. derived from the Pl-ll terminator region of potato. In addition, other commonly used terminators are derived from the octopine or nopaline synthase signals.

[0052] The plant expression vector also typically contains a dominant selectable marker gene used to identify those cells that have become transformed. Useful selectable genes for plant systems include the aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II), genes encoding antibiotic resistance genes, for example, those encoding resistance to hygromycin, kanamycin, bleomycin, neomycin, G418, streptomycin, or spectinomycin. Genes required for photosynthesis may also be used as selectable markers in photosynthetic-deficient strains. Finally, genes encoding herbicide resistance may be used as selectable markers; useful herbicide resistance genes include the bar gene encoding the enzyme phosphinothricin acetyltransferase and conferring resistance to the broad-spectrum herbicide Basta® (Bayer Cropscience Deutschland GmbH, Langenfeld, Germany). Other selectable markers include genes that provide resistance to other such herbicides such as glyphosate and the like, and imidazolinones, sulfonylureas, triazolopyrimidine herbicides, such as chlorosulfron, bromoxynil, dalapon, and the like. Furthermore, genes encoding dihydrofolate reductase may be used in combination with molecules such as methatrexate.

[0053] Efficient use of selectable markers is facilitated by a determination of the susceptibility of a plant cell to a particular selectable agent and a determination of the concentration of this agent which effectively kills most, if not all, of the transformed cells. Some useful concentrations of antibiotics for tobacco transformation include, for example, 20-100 pg/ml (kanamycin), 20-50 g/ml (hygromycin), or 5-10 pg/ml (bleomycin). A useful strategy for selection of transformants for herbicide resistance is described, for example, by Vasil (Cell Culture and Somatic Cell Genetics of Plants, Vol I, II, III Laboratory Procedures and Their Applications Academic Press, New York, 1984). In addition to a selectable marker, it may be desirable to use a reporter gene. In some instances, a reporter gene may be used without a selectable marker. Reporter genes are genes which are typically not present or expressed in the recipient organism or tissue. The reporter gene typically encodes for a protein which provide for some phenotypic change or enzymatic property. Examples of such genes are provided in Weising et al. (Ann. Rev. Genetics 22:421-478, 1988). Preferred reporter genes include without limitation glucuronidase (GUS) gene and GFP genes.

J4. Genetic Transformations of Plants

[0054] Any method for genetic transformation can be used to insert a polynucleotide encoding a silicon transporter into a plant. In some cases, it may be desirable to transform a plant with a silicon influx transporter (e.g., any of the transporters described herein). Methods for the transformation of many plants, including soybeans, are well known to those of skill in the art. For example, techniques which may be employed for the genetic transformation of soybeans include electroporation, microprojectile bombardment,

transformation and direct DNA uptake by protoplasts. To effect transformation by electroporation, one can employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In this technique, one can partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wound tissues in a controlled manner.

[0055] Protoplasts can also be employed for electroporation transformation of plants (Bates, Mol. Biotechnol., 2:135-145, 1994; Lazzeri, Methods Mol. Biol., 49:95-106, 1995). For example, the generation of transgenic soybean plants by electroporation of cotyledon- derived protoplasts is described in WO 92/17598. A particularly efficient method for delivering transforming DNA segments to plant cells is microprojectile bombardment. Here, particles are coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells can be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.

[0056] An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a surface covered with target soybean cells. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. The smaller aggregates are believed to reduce the damage inflicted on cells by larger projectiles, thus resulting in higher transformation efficiency. Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species (e.g., soybean or any plant described herein). The application of microprojectile

JS bombardment for the transformation of soybeans is described, for example, in US

5,322,783, hereby incorporated by reference.

[0057] >4grobacfe/7um-mediated transfer is another widely used system for introducing gene loci into plant cells. An advantage of the technique is that DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations (Klee et al., Bio. Tech., 3:637-642, 1985). Recent technological advances in vectors for Agrobacterium- mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes. Additionally, Agrobacterium containing both armed and disarmed Ti genes can be used for transformation. Agrobacterium-mediated

transformation is described in US 6,384,301 and US 6,037,522.

[0058] In those plant strains where >4gro6acfe/7um-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene locus transfer. The use of Agrobacterium-mediaied plant integrating vectors to introduce DNA into plant cells is well known in the art (Fraley et al., Bio. Tech., 3:629-635, 1985; US

5,563,055). Use of Agrobacterium in the context of soybean transformation has been described, for example, by Chee et al. (Methods Mol. Biol., 44:101-119, 1995) and in US 5,569,834.

[0059] Transformation of plant protoplasts also can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., Mol. Gen. Genet., 199:169-177, 1985; Omirulleh et al., Plant Mol. Biol., 21 :415-428, 1993; Fromm et al., Nature, 319(6056):791- 793., 1986; Uchimiya et al., Mol. Gen. Genet., 204:204-207, 1986; Marcotte et al., Nature, 335:454- 457, 1988). The demonstrated ability to regenerate soybean plants from protoplasts makes each of these techniques applicable to soybean (Dhir et al., Plant Cell Rep., 10:97-101 , 1991 ). Plants

[0060] Any plant may be used in the present invention. In certain embodiments, a plant that naturally does not accumulate high levels of silicon is used. Many plants do not efficiently accumulate silicon including soybean. In other embodiments, it may be desirable to increase silicon uptake in a plant that efficiently accumulates silicon (e.g., rice or a grassy plant such as wheat, oat, sorghum, or barley). Plants that may be used in the invention include a monocotyledonous crop plant such as barley, maize, oats, rice, rye, sorghum, and wheat; and a member of the grass family of Poaceae, such as Phleum spp., Dactylis spp., Lolium spp., Festulolium spp., Festuca spp., Poa spp., Bromus spp.,

Agrostis spp., Arrhenatherum spp., Phalaris spp., and Trisetum spp., for example, Phleum pratense, Phleum bertolonii, Dactylis glomerata, Lolium perenne, Lolium multiflorum, Lolium multiflorum westervoldicum, Festulolium braunii, Festulolium loliaceum,

Festulolium holmbergii, Festulolium pabulare, Festuca pratensis, Festuca rubra, Festuca rubra rubra, Festuca rubra commutata, Festuca rubra trichophylla, Festuca duriuscula, Festuca ovina, Festuca arundinacea, Poa trivialis, Poa pratensis, Poa palustris, Bromus catharticus, Bromus sitchensis, Bromus inermis, Deschampsia caespitosa, Agrostis capilaris, Agrostis stolonifera, Arrhenatherum elatius, Phalaris arundinacea, and Trisetum flavescen.; and a dicotyledonous plant, such as alfalfa, carrot, cotton, potato, sweet potato, oilseed rape, radish, soybean, sugarbeet, sugar cane, sunflower, tobacco, and turnip; vegetables such as asparagus, bean, carrot, chicory coffee, celery, cucumber, eggplant, fennel, leek, lettuce, garlic, onion, papaya, pea, pepper, spinach, squash, pumpkin, and tomato; vegetable brassicas such as brussel sprouts, broccoli, cabbage, and cauliflower; fruits, such as avocado, banana, blackberry, blueberry, grapes, mango, melon, nectarine, orange, papaya, pineapple, raspberry, strawberry; rosaceous fruits such as apple, apricot, peach, pear, cherry, plum, and quince; herbs such as anise, basil, bay laurel, caper, caraway, cayenne pepper, celery, chervil, chives, coriander, dill, horseradish, lemon balm, liquorice, marjoram, mint, oregano, parsley, rosemary, sesame, tarragon, and thyme; woody species, such as eucalyptus, oak, pine, and poplar.

Screening of transformed plants

[0061] Once a plant is transformed with a Si-transport gene, screening can be

accomplished by any means known in the art. In some cases, screening is performed using the silicon detection techniques described below. Other screening techniques may involve screening for uptake, transport, or efflux of germanium (Ge). As described above, Ge has been used to evaluate silicon uptake in Xenopus oocytes. Such an approach can also be used to evaluate silicon uptake in higher plants, as molar ratios between Ge and silicon have been observed to remain constant following uptake in different plant tissues (Nikolic et al., Plant Physiol 143:495-503, 2007).

J 7- [0062] In other embodiments, the plants can be screened for resistance to one or more biotic stresses, one or more abiotic stresses, or any combination thereof. In one example, soybean plants transformed with a silicon influx transport, a silicon efflux transporter, or both are screened for resistance to soybean rust {Phakopsora pachyrhyzi). In general, untransformed plants and transformed plants are grown in the presence of a stress (e.g., any described herein), and the effect of silicon transporter expression on stress resistance is determined by measuring a phenotypic response to the stress (e.g., growth, survival, weight, yield), where an improvement in the phenotypic response (e.g., increased growth, higher rate of survival) in the transformed plant as compared to the non-transformed plants indicates that the transformation with the silicon transporter is beneficial.

[0063] Any appropriate abiotic stress may be used to evaluate the effect of transforming a cell or plant with a silicon transporter. Exemplary abiotic stresses include salinity, temperature (e.g., heat or cold), oxidative stress, insufficient or excess water

(waterlogging or drought), insufficient or excessive minerals (e.g., mineral toxicity), physical stress (e.g., wind). Health or growth parameters, such as height, weight, yield, or survival are recorded and compared to untransformed control plants subjected to the same stress.

[0064] Alternatively, plants may be subjected to biotic stresses, such as bacteria, fungus, or an insect. Any biotic stress known in the art may be used to screen plants. Other pathogens affecting soybean include Phytophthora megasperma fsp. glycinea,

Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthephaseolorum var. sojae (Phomopsis sojae), Diaporthephaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines, and Fusarium solani.

[0065] In general, exemplary fungi include Alternaria (Alternaria brassicola; Alternaria solani), Ascochyta (Ascochyta pisi); Botrytis (Botrytis cinerea); Cercospora (Cercospora kikuchii; Cercospora zeae-maydis); Colletotrichum (Colletotrichum lindemuthianum);

US- Diplodia (Diplodia maydis); Erysiphe (Erysiphe graminis f. sp. graminis; Erysiphe graminis f. sp. hordei); Fusarium (Fusarium nivale; Fusarium oxysporum; Fusarium graminearum; Fusarium culmorum; Fusarium solani; Fusarium moniliforme; Fusarium roseum);

Gaeumanomyces (Gaeumanomyces graminis f, sp. tritici); Helminthosporium

(Helminthosporium turcicum; Helminthosporium carbonum; Helminthosporium maydis); Macrophomina (Macrophomina phaseolina); Magnaporthe (Magnaporthe grisea); Nectria (Nectria haematococca); Peronospora (Peronospora manshurica; Peronospora tabacina); Phoma (Phoma betae); Phymatotrichum (Phymatotrichum omnivorum); Phytophthora (Phytophthora cinnamomi; Phytophthora cactorum; Phytophthora phaseoli; Phytophthora parasitica; Phytophthora citrophthora; Phytophthora megasperma f. sp. sojae;

Phytophthora infestans); Plasmopara (Plasmopara viticola); Podosphaera (Podosphaera leucotricha); Puccinia (Puccinia sorghi; Puccinia striiformis; Puccinia graminis f. sp. tritici; Puccinia asparagi; Puccinia recondita; Puccinia arachidis); Pyrenophora (Pyrenophora tritici-repentis); Pyricularia (Pyricularia oryzae); Pythium (Pythium aphanidermatum;

Pythium ultimum); Rhizoctonia (Rhizoctonia solani; Rhizoctonia cerealis); Sclerotium (Sclerotium rolfsii); Sclerotinia (Sclerotinia sclerotiorum); Septoria (Septoria lycopersici; Septoria glycines; Septoria nodorum; septoria tritici); Thielaviopsis (Thielaviopsis basicola); Uncinula (Uncinula necator); Venturia (Venturia inaequalis); and Verticillium (Verticillium dahliae; Verticillium albo-atrum).

[0066] Examples of rusts include rust caused by Basidiomycetes of the order Uredinales;

Puccinia (P. graminis, P. stiiformis, P. recondita, P. hordei, P. coronata, P. sorghi., P. polysora, P. purpurea, P. sacchari P. kuehnii, P. stakmanii, P. asparagi, P. chrysanthemi,

P. malvacearum, and P. antirrhini); Gymnosporangium (G. juniperi-virginianae, G.

globosum); Hemileia (H. vastatrix); Phragmidium; Uromyces (U. caryophyllinus);

Cronartium (C. ribicola, C. quercuum f. sp. fusiforme, C. quercuum f. sp. virginianae, C. comptoniae, C. comandrae, C. strobilinum); Melampsora (M. lini); Coleosporium (C. asterinum); Gymnoconia; Phakopsora (P. pahyrhizi) and Tranzschelia.

[0067] In one example, transformed plants and untransformed controls are grown hydroponically in a nutritive solution containing 1.7 mM Si, the maximum solubility of Si in solution. Plant roots and aerial parts are harvested, and their Si content is measured by techniques described below.

JO- Si detection, localization, and quantification

[0068] Once a cell or a plant (e.g., a Xenopus oocyte or a plant described herein) has been transformed to express a silicon transporter, it may be desirable to measure the amount of silicon in the cell or plant. Several methods exist to determine a Si content in different substrates. Typically, when measuring silicon in biological sample, Si quantification is performed through a spectrometric analysis, which, in some cases, may result in destruction of the sample.

[0069] One non-destructive analytical method is X-ray fluorescence spectroscopy. This technique allows detection and quantification of Si in biological material, for example, by measuring and analyzing the secondary radiation emitted from a substrate excited with a X-ray source. Prior to visualization, samples are frozen to -80°C and then lyophilized. Once completely dry, they are attached to carbon SEM stubs and coated with gold.

Samples are then submitted to X-rays and the secondary radiation is recorded and quantified.

[0070] Other spectrometric analyses are performed on treated samples, for which Si is solubilized, usually in an acid solution. Samples can be prepared by autoclave-induced digestion, acid digestion, microwave assisted acid digestion, or NaOH fusion. The resulting solution can then be analyzed using a colorimetric method (either yellow silicomolybdic acid or blue silicomolybdous acid procedure), atomic absorption spectrometry, or inductively coupled plasma (ICP).

[0071] ICP is reported to have the lowest detection limit (3 ppb) and the greatest precision. Thus, it may be the method of choice when dealing with Si quantification, where solubilization is possible. Once a sample is prepared for an ICP analysis, it is converted into aerosol with a nebulizer. A desolvation/volatilization phase occurs, in which water is driven off while solid and liquid fractions are converted into gases. Then an atomization phase takes place where gas phase bonds are broken. This step produces a plasma which requires a high temperature (5000 to 8000°C) to maintain an inert chemical environment, usually provided by Argon. The plasma is then excited by X-rays and releases electromagnetic radiation (hv) in an element-specific wavelength. For instance, Si emits at 251 ,611 nm. A detector then measures the light emitted and quantifies it. ICP can thus be used to assess Si-transport efficiency following oocyte transformation and also to measure Si absorption in plants (e.g., transformed or untransformed). Indirect, analytical methods for measuring silicon uptake using a germanium tracer may be used. This approach is described using oocytes above but can also be applied to higher plants. Using small amounts of radioactive germanium (⁶⁸Ge) as a tracer can be used as a means for measuring silicon uptake (see, e.g., Nikolic et al., Plant Physiol 143:495-503, 2007). Finally, silicon influx or efflux can be measured by another method following oocyte transformation and incubation in a solution containing silicon: oocytes were washed, solubilized in HN0₃ and the silicon content was directly quantified by atomic-absorption (AA) spectrometry. Atomic-absorption spectroscopy uses the absorption of light to measure the concentration of gas- phase atoms. Because samples are usually liquids or solids, the analyte atoms or ions must be vaporized in a flame or graphite furnace. The atoms absorb ultraviolet or visible light and make transitions to higher electronic energy levels. The analyte concentration is determined from the amount of absorption. Silicon concentration measurements were determined from a working curve after calibrating the instrument with standards of known concentration.

Synthesis of silicon transporter polypeptides

[0072] Nucleic acids that encode silicon transporter polypeptides or fragments thereof may be introduced into various cell types or cell-free systems for expression, thereby allowing purification of these polypeptides for biochemical characterization, large-scale production, antibody production, and patient therapy.

[0073] Eukaryotic and prokaryotic silicon transporter expression systems may be generated in which a silicon transporter gene sequence is introduced into a plasmid or other vector, which is then used to transform living cells. Constructs in which the silicon transporter cDNA contains the entire open reading frame inserted in the correct orientation into an expression plasmid may be used for protein expression. Alternatively, portions of the silicon transporter gene sequences, including wild-type or mutant silicon transporter sequences, may be inserted. Prokaryotic (e.g., E. coli) and eukaryotic expression systems allow various important functional domains of the silicon transporter proteins to be recovered, if desired, as fusion proteins, and then used for binding, structural, and functional studies and also for the generation of appropriate antibodies.

[0074] Typical expression vectors contain promoters that direct the synthesis of large amounts of mRNA corresponding to the inserted silicon transporter nucleic acid in the plasmid-bearing cells. They may also include a eukaryotic or prokaryotic origin of replication sequence allowing for their autonomous replication within the host organism, sequences that encode genetic traits that allow vector-containing cells to be selected for

2L in the presence of otherwise toxic drugs, and sequences that increase the efficiency with which the synthesized mRNA is translated. Stable long-term vectors may be maintained as freely replicating entities by using regulatory elements of, for example, viruses (e.g., the OriP sequences from the Epstein Barr Virus genome). Cell lines may also be produced that have integrated the vector into the genomic DNA, and in this manner the gene product is produced on a continuous basis.

[0075] Expression of foreign sequences in bacteria, such as Escherichia coli, requires the insertion of the silicon transporter nucleic acid sequence into a bacterial expression vector. Such plasmid vectors contain several elements required for the propagation of the plasmid in bacteria, and for expression of the DNA inserted into the plasmid. Propagation of only plasmid-bearing bacteria is achieved by introducing, into the plasmid, selectable marker- encoding sequences that allow plasmid-bearing bacteria to grow in the presence of otherwise toxic drugs. The plasmid also contains a transcriptional promoter capable of producing large amounts of mRNA from the cloned gene. Such promoters may be (but are not necessarily) inducible promoters that initiate transcription upon induction. The plasmid also preferably contains a polylinker to simplify insertion of the gene in the correct orientation within the vector.

[0076] Once the appropriate expression vectors containing a silicon transporter gene, fragment, fusion, or mutant are constructed, they are introduced into an appropriate host cell by transformation techniques, such as, but not limited to, calcium phosphate transfection, DEAE-dextran transfection, electroporation, microinjection, protoplast fusion, or liposome-mediated transfection. The host cells that are transfected with the vectors of this invention may include (but are not limited to) E. coli or other bacteria, yeast, fungi, plant cells, insect cells (using, for example, baculoviral vectors for expression in SF9 insect cells), or cells derived from mice, humans, or other animals. In vitro expression of silicon transporter proteins, fusions, polypeptide fragments, or mutants encoded by cloned DNA may also be used. Those skilled in the art of molecular biology will understand that a wide variety of expression systems and purification systems may be used to produce recombinant silicon transporter proteins and fragments thereof.

[0077] Once a recombinant protein is expressed, it can be isolated from cell lysates using protein purification techniques such as affinity chromatography. Once isolated, the recombinant protein can, if desired, be purified further, e.g., by high performance liquid

XL chromatography (HPLC; e.g., see Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, Eds., Elsevier, 1980).

[0078] Polypeptides of the invention, particularly short silicon transporter fragments can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, IL).

[0079] A particular aspect of the invention provides an isolated polypeptide sequence having at least 67% identity to a polypeptide encoded by the isolated nucleic acid selected from the group consisting of SEQ ID NOS: 1 , 3 and 5. In particular, the isolated polypeptide sequence is selected from the group consisting of SEQ ID NOs: 2, 4, and 6. Method of increasing resistance of a plant to biotic and abiotic stress

[0080] The present invention also provides a method of increasing the resistance of a plant to biotic or abiotic stresses, comprising: a) providing a vector comprising a nucleic acid sequence having at least 50% identity to a sequence selected from the group consisting of SEQ ID NO: 1 , 3 and 5; b) transforming a plant cell with said vector; and c) growing a plant from said cell, wherein said plant expresses a protein encoded by said nucleic acid sequence; whereby the plant exhibits an increased resistance to biotic or abiotic stresses. Particularly, the plant cell is a dicotyledonous plant cell, more particularly, a soybean cell.

[0081] In accordance with a particular embodiment, the method of inscreasing the resistance of a plant to a biotic or abiotic stress is carried out with a nucleic acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to a sequence selected from the group consisting of SEQ ID NOs: 1 , 3, and 5.

[0082] Particularly, the nucleic acid sequence is operatively linked to a promoter capable of expression in a plant cell. More particularly, the promoter is a tissue-specific or a tissue- preferred promoter. Still, more particularly, the promoter is a root-preferred promoter or a leaf-preferred promoter.

[0083] In accordance with a particular aspect of the imethod of the invention, the promoter is an inducible promoter. Alternatively, the promoter is a constitutive promoter.

[0084] In a particualry aspect of the invention, the biotic stressor is fungi. Particularly, the fungi is selected from the group consisting of Alternaria (Alternaria brassicola; Alternaria solani), Ascochyta {Ascochyta pisi); Botrytis {Botrytis cinerea); Cercospora {Cercospora kikuchii; Cercospora zeae-maydis); Colletotrichum {Colletotrichum lindemuthianum); Diplodia {Diplodia maydis); Erysiphe {Erysiphe graminis f. sp. graminis; Erysiphe graminis f. sp. hordei); Fusarium {Fusarium nivale; Fusarium oxysporum; Fusarium graminearum; Fusarium culmorum; Fusarium solani; Fusarium moniliforme; Fusarium roseum); Gaeumanomyces {Gaeumanomyces graminis f, sp. tritici); Helminthosporium {Helminthosporium turcicum; Helminthosporium carbonum; Helminthosporium maydis); Macrophomina {Macrophomina phaseolina); Magnaporthe {Magnaporthe grisea); Nectria {Nectria haematococca); Peronospora {Peronospora manshurica; Peronospora tabacina); Phoma {Phoma betae); Phymatotrichum {Phymatotrichum omnivorum); Phytophthora {Phytophthora cinnamomi; Phytophthora cactorum; Phytophthora phaseoli; Phytophthora parasitica; Phytophthora citrophthora; Phytophthora megasperma f. sp. sojae; Phytophthora infestans); Plasmopara {Plasmopara viticola); Podosphaera {Podosphaera leucotricha); Puccinia {Puccinia sorghi; Puccinia striiformis; Puccinia graminis f. sp. tritici; Puccinia asparagi; Puccinia recondita; Puccinia arachidis); Pyrenophora {Pyrenophora tritici-repentis); Pyricularia {Pyricularia oryzae); Pythium {Pythium aphanidermatum; Pythium ultimum); Rhizoctonia {Rhizoctonia solani; Rhizoctonia cerealis); Sclerotium {Sclerotium rolfsii); Sclerotinia {Sclerotica sclerotiorum); Septoria {Septoria lycopersici; Septoria glycines; Septoria nodorum; septoria tritici); Thielaviopsis {Thielaviopsis basicola); Uncinula {Uncinula necator); Venturia {Venturia inaequalis); Verticillium {Verticillium dahliae; Verticillium albo-atrum); Basidiomycetes of the order Uredinales; Puccinia (P. graminis, P. stiiformis, P. recondita, P. hordei, P. coronata, P. sorghi., P. polysora, P. purpurea, P. sacchari P. kuehnii, P. stakmanii, P. asparagi, P. chrysanthemi, P. malvacearum, and P. antirrhini); Gymnosporangium (G. juniperi-virginianae, G. globosum); Hemileia (H. vastatrix); Phragmidium; Uromyces (U. caryophyllinus); Cronartium (C. ribicola, C. quercuum f. sp. fusi forme, C. quercuum f. sp. virginianae, C. comptoniae, C. comandrae, C. strobilinum); Melampsora (M. lini); Coleosporium (C. asterinum); Gymnoconia; Phakopsora (P. pahyrhizi) and Tranzschelia.

[0085] The following examples are intended to illustrate, rather than limit, the invention. EXAMPLE 1 - Identification of horsetail (Equisetum arvense) Lsi1 silicon

transporters

Previous attempts to identify horsetail silicon transporters.

[0086] The first attempts to identify horsetail silicon transporters were performed using a PC approach with primers designed from known monocots homologues. Neither PCR with classic primers or degenerate primers were successful. In order to assess the level of homology between horsetail and cereal transporters, we then performed Southern blot analysis on genomic DNA from horsetail and several cereals (rice, wheat, barley) that have Lsi1 homolog genes. Using probes from wheat Lsi1 , no signal was detected from horsetail. This led us to believe that the horsetail transporters could have only distant homology to the known sequences from cereals.

Transcriptomic approach

[0087] We successfully used a transcriptomic approach to identify horsetail silicon transporters. This approach has a significant advantage over PCR on genomic DNA: transcriptome analysis confirms that the genes identified in this study are expressed in the plant organ that absorbs silicon: the root.

RNA extraction conditions

[0088] Total root RNA was extracted from actively growing horsetail plants using the TRIzol reagent. Silicon is known to be an essential element for horsetail (Chen and Lewin, 1969, Canadian Journal of Botany 47(1 )125-131). Actively growing horsetail plants abundantly accumulate this element, while silicon-deprived horsetail plants die. Horsetail silicon transporters need to be expressed in the roots, since this element comes from the soil solution. Active growth was thus the best stage to perform RNA extraction on roots in order to identify the expressed transporters. Transcriptome sequencing and assembly

[0089] Horsetail root messenger RNAs were reverse transcribed into complementary DNA (cDNA) and sequenced using the lllumina Genome Analyser 2 platform. The 60 million of 72 bp sequences obtained were assembled using the CLC bio software to reduce the number of sequences at ca. 6000 contigs. Identification of horsetail silicon transporter homologues

[0090] Using the BLAST tool from CLC bio software, three Lsi1 homologues were identified from the horsetail transcriptome. The three Lsi1 homologues belong to the NIP (Nodulin-26-like intrinsic proteins) family of aquaporins, and were named NIP B, NIP C and NIP D.

EXAMPLE 2 - Cloning of horsetail silicon transporter homologues

[0091] PCR primers with EcoRI and Xbal restriction sites were designed from the sequences identified in the horsetail transcriptome to clone the silicon transporters. PCR was performed on 20 ng cDNA reverse-transcribed from the same RNA used for lllumina sequencing. The Superscript III reverse transcriptase (Invitrogen) was used for the synthesis of cDNA according to the manufacturer's instructions.

[0092] Primers NIP B Forward EcoRI (SEQ ID NO: 8) and NIPB Reverse Xbal (SEQ ID NO:9) were used to amplify the NIP B ORF (SEQ ID NO:1 ) using the following PCR conditions using a Pfu DNA polymerase: a first round at 98°C for 30 seconds, followed by 20 cycles of 98°C for 10 seconds; 65°C for 15 seconds; and 72°C for 15 seconds; followed by a final extension of 72°C for 5 minutes.

[0093] Primers NIP C Forward EcoRI (SEQ ID NO:10 ) and NIPC Reverse Xbal (SEQ ID NO: 11 ) were used to amplify the NIP C ORF (SEQ ID NO:3) using the following PCR conditions using a Pfu DNA polymerase: a first round at 98°C for 30 seconds, followed by 35 two-step cycles of 98°C for 10 seconds and 72°C for 45 seconds;; followed by a final extension of 72°C for 5 minutes.

[0094] Primers NIP D Forward EcoRI (SEQ ID NO: 12) and NIPD Reverse Xbal (SEQ ID NO: 13) were used to amplify the NIP D ORF (SEQ ID NO:5) using the following PCR conditions using a Pfu DNA polymerase: a first round at 98°C for 30 seconds, followed by 35 two-step cycles of 98°C for 10 seconds and 72°C for 45 seconds;; followed by a final extension of 72°C for 5 minutes.

[0095] All PCR products were then sub-cloned in a pUC18 vector and transformed into E. coli competent cells strain Top10. The presence of the proper insert was screened by colony PCR with 0.2 μΜ each M13 Forward and M13 Reverse universal primers using the following conditions: 94°C for 2 minutes, followed by 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 68°C for 1 minute, followed by a final extension of 68°C for 5 minutes. A plasmid extraction was performed on positive clones using the Miniprep kit from QIAgen. Several clones were sequenced from each PCR product to confirm the sequences. EXAMPLE3 - Analysis of horsetail silicon transporters

[0096] The horsetail sequences were compared with known homologues from rice and wheat. The identity and similarity percentages are detailed in Tables 1 and 2. Sequences were aligned with the ClustalW program of the BioEdit software. For the percentage similarity between amino acids, the BLOSUM62 substitution matrix was used. The horsetail genes and proteins show distant homology with previously identified silicon transporters. Alignments of Lsi1 homologues from rice, wheat and horsetail are shown in Figure 1c.

21. Table 1: Identity percentages ofnucieic acid sequences of LsH homologues from rice, wheat and horsetail

% ID acides nucleiques ORFs

Lsi1 rice Lsi1 wheat NIPBhorsetail NIPChorsetail NIPDhorsetail

SEQIDNO:14 SEQIDNO:16 SEQIDNO:1 SEQIDNO:3 SEQIDNO:5

Lsi1 rice

SEQIDNO:14 100 84,2 43,0 47,6 43,7

Lsi1 wheat

SEQIDNO:16 100 43,4 48,6 44,7

NIPBhorsetail

SEQIDNO:1 100 70,6 68,7

NIPChorsetail

SEQIDNO:3 100 68,9

NIPDhorsetail

SEQIDNO:5 100

Table 2: Identity and similarity percentages of amino acid sequences of LsH homologues from rice, wheat and horsetail

% ID et SIM

proteines

Lsi1 wheat NIPB horsetail NIPC horsetail NIPD horsetail

Lsi1 rice SEQIDNO:15 SEQIDNO:17 SEQIDNO:2 SEQIDNO:4 SEQIDNO:6

CD

<z %ID %SIM %ID %SIM %ID %SIM %ID %SIM %ID %SIM CD Lsi1 rice

CD

SEQIDNO:15 100 100 81 ,9 93,3 35,8 50,5 37,3 51 ,3 36,6 50,7

Lsi1 wheat

SEQIDN0:17 100 100 36,7 51 ,2 39,5 55,4 37,8 51 ,9 m IPB horsetail

CD SEQIDN0:2 100 100 67,2 779 68,3 76,6 I

m NIPC horsetail

m SEQIDN0:4 ^' ·^'.- ί 100 100 70,9 78,1

NIPD horsetail

73

<Z SEQIDN0:6 100 100

I

m

EXAMPLE 4 - Analysis of horsetail Lsi1 homologues (NIPs) overall structure and pore structure

[0097] The four amino acid residues forming the pore seem to be critical for aquaporin selectivity and transport activity. The horsetail NIPs reported herein show a novel pore structure that is different from previously reported Lsi1 homologues, and from any known plant aquaporins. The pore for horsetail NIP B, C and D is composed of the residues S T A R (Serine, Threonine, Alanine and Arginine) while the pore for known Lsi1 homologues from wheat and rice is composed of the residues G S G R (Glycine, Serine, Glycine, Arginine). An alignment of Lsi1 proteins from rice, wheat, and the three horsetail NIPs along with the position of the four pore residues, is shown at Figure 2a. The alignment was generated using the ClustalW program from the CLC bio software. Pore residues and NPA loops were identified by homology with the rice Lsi1. (For examples of authors using this methodology to identify NIP pore residues, please see for example Wallace and Roberts, 2005, Biochemistry 44:16825- 16834 or Forrest and Bhave, 2997, Functional & Integrative Genomics, 7:263-289). Putative transmembrane domains were identified using the Transmembrane Helix Prediction tool at http://www.cbs.dtu.dk/services/TMHMM/. Figure 2b shows an hydrophobicity profile of aligned predicted protein sequences from rice and wheat Lsi1 , and horsetail NIP B, NIP C and NIP D. Different from the other NIP sequences, horsetail NIP B has a second NPV box instead of NPA, but as mentioned above, this does not appear to affect the transport function or selectivity of the protein.

EXAMPLE 5 - Characterization of horsetail Lsi1 transporters - oocyte assay

[0098] To assess and compare the efficiency of Si-transporters encoded by the polynucleotides described herein, transformed oocytes from Xenopus laevis can be used. Si-transporter cRNA can be generated using any method known in the art and can be injected into the oocytes, resulting in production of functional Si-transport proteins. Using this system, the rate of silicon uptake for different transporters can be evaluated. Such a system allows the characterization of Si-transporter(s) and selection of transporters with desirable traits, including more rapid rate of silicon uptake or a greater total silicon uptake.

a [0099] Oocytes have been widely used to study proteins through transient expression of the corresponding genes. Oocytes are particularly well suited for studies of receptors, channels, and ion pumps because these proteins often display normal electrophysiological characteristics in oocytes. It is therefore possible to study assembly, membrane insertion, and function of such proteins. In addition, because oocytes are mammalian cells, complex proteins that require post-translational modification can be produced and retain their functionality.

[00100] As noted above, such oocytes can be injected with cRNA to produce transient production of the encoded protein. A gene of interest can be cloned into an expression vector capable of producing cRNA containing the gene. The production of a functional cRNA can be obtained by in vitro transcription of the DNA sequence of the gene of interest to produce a pre-cRNA. The pre-cRNA is then capped with a 7- methylguanosine, which mimics most eukaryotic mRNAs found in vivo. Capping of RNA improves its stability and therefore the yield of translation. Purified, capped cRNA can then be microinjected in prepared oocytes. Such a process is described by Hildebrand et al. (Nature 385:688-689, 1997) to study a silicon transporter found in diatoms. Following oocyte transformation, the rate of silicon influx or efflux can be measured using any method known in the art (e.g., those described herein). Exemplary approaches are also described in Ma et al. (Nature 440:688-691 , 2006). Ma et al. use germanium 68 (⁶⁸Ge) as a tracer for silicon to assay uptake into Xenopus oocytes. Because germanium is toxic, it is used at relatively low concentrations in assays where viability of the cell is required. By measuring the radioactivity of low germanium concentrations in the oocytes in the presence or absence of a putative silicon transporter, it is possible to determine whether expression of a particular protein lead to increased silicon transport. To determine whether silicon is specifically transported upon expression of the protein, transport of a molecule such as glycerol can be used as a negative control.

Cloning of silicon transporters in an expression vector

[00101] An expression vector, Poll (SEQ ID NO: 18), was used for the in vitro transcription of horsetail Lsi1 coding sequences. These sequences were inserted into the Poll vector using restriction sites that were added by the primers used for PCR amplification: an EcoRI/Xbal fragment containing the coding sequence of Lsi1

3L was inserted from pUC18 to PoH vector using an excision/ligation procedure known in the art. New vectors were transformed in E. coli DH5a strains and kept frozen at - 80 °C.

Production of cRNAs for oocytes microinjection

[00102] Plasmids were recovered from a fresh bacteria culture using a miniprep plasmid extraction kit from QIAgen. Plasmids were digested with Nhe1 restriction enzyme (Roche) allowing the linearization of the plasmid. The digestion was purified using a PCR purification kit (QIAgen) and 1 g of DNA was used for the in vitro transcription using the mMessage m achine 17 Ultra kit (Ambion). cRNA were recovered, solubilized in DEPC-treated water and kept frozen at -80 °C until use.

Si detection, localization, and quantification

[00103] Once a cell or a plant (e.g., a Xenopus oocyte or a plant described herein) has been transformed to express a silicon transporter, it may be desirable to measure the amount of silicon in the cell or plant. Several methods exist to determine a Si content in different substrates. Typically, when measuring silicon in biological sample, Si quantification is performed through a spectrometric analysis, which, in some cases, may result in destruction of the sample.

[00104] One non-destructive analytical method is X-ray fluorescence spectroscopy. This technique allows detection and quantification of Si in biological material, for example, by measuring and analyzing the secondary radiation emitted from a substrate excited with a X-ray source. Prior to visualization, samples are frozen to -80 °C and then lyophilized. Once completely dry, they are attached to carbon SEM stubs and coated with gold. Samples are then submitted to X-rays and the secondary radiation is recorded and quantified.

[00105] Other spectrometric analyses are performed on treated samples, for which Si is solubilized, usually in an acid solution. Samples can be prepared by autoclave-induced digestion, acid digestion, microwave assisted acid digestion, or NaOH fusion. The resulting solution can then be analyzed using a colorimetric method (either yellow silicomolybdic acid or blue silicomolybdous acid procedure), atomic absorption spectrometry, or inductively coupled plasma (ICP).

[00106] ICP is reported to have the lowest detection limit (3 ppb) and the greatest precision. Thus, it may be the method of choice when dealing with Si quantification, where solubilization is possible. Once a sample is prepared for an ICP analysis, it is converted into aerosol with a nebulizer. A desolvation/volatilization phase occurs, in which water is driven off while solid and liquid fractions are converted into gases. Then an atomization phase takes place where gas phase bonds are broken. This step produces a plasma which requires a high temperature (5000 to 8000 °C) to maintain and an inert chemical environment, usually provided by Argon. The plasma is then excited by X-rays and releases electromagnetic radiation (hv) in an element-specific wavelength. For instance, Si emits at 251 ,611 nm. A detector then measures the light emitted and quantifies it. ICP can thus be used to assess Si-transport efficiency following oocyte transformation and also to measure Si absorption in plants (e.g., transformed or untransformed).

[00107] Indirect, analytical methods for measuring silicon uptake using a germanium tracer may be used. This approach is described using oocytes above but can also be applied to higher plants. Using small amounts of radioactive germanium (⁶⁸Ge) as a tracer can be used as a means for measuring silicon uptake (see, e.g., Nikolic et al, Plant Physiol 143:495-503, 2007).

[00108] Finally, silicon influx or efflux can be measured by another method following oocyte transformation and incubation in a solution containing silicon: oocytes were washed, solubilized in HN0₃ and the silicon content was directly quantified by atomic-absorption (AA) spectrometry. Atomic-absorption spectroscopy uses the absorption of light to measure the concentration of gas-phase atoms. Because samples are usually liquids or solids, the analyte atoms or ions must be vaporized in a flame or graphite furnace. The atoms absorb ultraviolet or visible light and make transitions to higher electronic energy levels. The analyte concentration is determined from the amount of absorption. Silicon concentration measurements were determined from a working curve after calibrating the instrument with standards of known concentration. EXAMPLE 6 - Consensus sequence based on the amino acid sequences of NIP B, NIP C and NIP D

[00109] The amino acid horsetail NIP B, C and D sequences were aligned and a consensus sequence was generated by the CLC bio software. This sequence allowed to highlight conserved domains between the 3 Lsi1 homologues found in horsetail and to achieve a BLAST search against other Si transporters. The horsetail NIP consensus sequence was compared with known homologues from rice, wheat and barley. E-values and percentage identities are detailed in Table 3. Sequences were aligned with the CLC bio software. For the percentage identity between amino acids, the BLOSUM62 substitution matrix was used. Horsetail proteins show distant homology with previously identified silicon transporters.

Table 3: Percentage of identity of Horsetail NIP consensus sequence with know sequences from Barley, Wheat and Rice silicon transporters

EXAMPLE 7 - Expression of cRNAs in Xenopus oocytes and quantification of Si content

[00110] Oocytes were taken from Xenopus laevis females. After dissection, a set of oocytes was transferred to a physiological medium with antibiotics. They were kept at 18 °C until use for up to 72 h. cRNA was dissolved in RNase-free water. Twenty to 50 nl of the injection fluid (500 ng /μΙ cRNA) was injected into each prepared oocyte using a micromanipulator. The oocytes were then incubated at 18°C for about 48 h to allow protein synthesis and membrane integration. Si uptake measurement was then performed. Si was added to the physiological medium to reach a 1.7 mM concentration. After 0, 30, or 60 min, oocytes were rinsed to remove external Si. Si content in oocytes was then measured by atomic absorption spectrometry, as described above.

[00111] Whereas in water injected oocytes, the concentration remained constant over the experimental period, in all other transformed oocytes, a higher concentration of silicon was detected over time (Figure 3). Interestingly, at 60 min, NIP B and NIP D exhibited a 2-fold better activity than NIP C or wheat Lsi1 Si transporter. These results indicate that the sequences identified in horsetail are functional homologues of the wheat Si transporter and that two out of the three seem to be more efficient.

EXAMPLE 8 - Transformation of Arabidopsis plants with Si-transport gene

Localization of OsLsM or TaLsi1 -GFP fusion proteins in N. benthamiana and A. thaliana

[00112] The OsLsi1 -GFP and Tal_si1 -GFP fusion constructs were obtained as follows. The OsLsH and TaLsil coding regions (excluding the stop codon) were amplified using gene-specific primers (N-OsLsi1 and S-OsLsi1 bis or N-Tal_si1 and S-TaLsi1 bis, Table 4) with cDNA obtained by reverse transcription (Superscript II, Invitrogen) of rice or wheat root RNA, respectively. Amplicons were cloned into the pGEM-T Easy vector (Promega). An Nco\-Spe\ fragment carrying either the OsLsH or TaLsil coding sequence was inserted into the corresponding sites in pCAMBIA1302 (CAMBIA, Canberra, Australia), thereby producing an in-frame fusion with the GFP coding region under the control of the CaMV35S promoter (constructs 35S::OsLsi1::GFP and 35S:: TaLsi1::GFP). These constructs were then transformed into Agrobacterium tumefaciens GV3101/pMP90. [00113] Transient expression of OsLsi1-GFP or TaLsi-GFP in N. benthamiana leaves was performed as described by (Johansen ei al. 2001 ). As a control, pCAMBIA1302 vector carrying the GFP gene under the control of the 35S promoter was used. Every agroinfiltrations included bacteria containing pGD-p19 plasmid, which expresses the Tomato Bushy Stunt Virus (TBSV) p19 coding region to minimize host RNA silencing (Bragg ef al. 2004). A. tumefaciens cultures containing 35S::GFP, 35S::OsLsi1::GFP or 35S::TaLsi1::GFP and pGD-p19 were mixed in equal proportions and co-infiltrated on the abaxial surface of the leaves. Transgenic leaves were taken five days post agroinfiltration and observed by confocal laser scanning microscopy (Olympus FV1000).

[00114] For stable expression, A. thaliana plants were transformed using the floral dip method (Ciough er a/. 1998). Transformants (T1 ) were selected on hygromycin (30mg/L) and the presence of the OsLsH or TaLsH transgene was verified by PCR using OsLsil- Fow and OsLsi1-Rev or Tal_si1-Fow and Tal_si1-Rev (Table 4) for rice and wheat, respectively. T2 seeds were harvested and sown on MS Medium containing the necessary antibiobic to select single locus lines. The homozygous transgenic plants T3 were grown and the OsLsi1-GFP or Tal_si1-GFP fusion proteins were located using confocal microscopy. Expression of OsLsH or TaLsH genes in A. thaliana

[00115] The 35S::OsLsi1 and 35S::TaLsi1 constructs were prepared in similar fashion to the fusion protein constructs except that the OsLsH and TaLsH amplicons were obtained using reverse primers that included the stop codon (S-OsLsi1 or S-TaLsi1 , Table 4). The resulting binary plasmids were individually introduced into A. tumefaciens strain GV3101/pMP90. Transformation and selection of transgenic lines were performed as described above.

-3Z Table 4. List of primers used.

Quantitative Real-Time RT-PCR

[00116] Expression of the OsLsH and TaLsil genes in A. thaliana was assessed by qRT-PCR using a Mastercycler EP Realplex 2S (Eppendorf, Hamburg, Germany). Total RNA was extracted as described above from 100 10-day-old seedlings (roots and aerial parts) for each of three transformants per construct and quantified using a Nanovue spectrophotometer (GE Healthcare, Chalfont St Giles, UK). For each of three biological replicates, 1 g of total RNA was reverse transcribed into first-strand cDNA. Primer pairs for selected genes were designed with the Oligo Explorer software (Genelink, Hawthorne, NY, USA) and reported in Table 4 (Atactin2-Fow and Atactin2-Rev for reference gene; Lsi1-Fow and Lsi1- Rev for target gene).

[00117] PCR amplifications were performed in a 20 μΙ reaction volume using the Quantitect SYBR green PCR kit (QIAgen) with 2 μΙ of sample cDNA. PCR was performed as follows: 15 min at 95°C followed by 45 cycles of 20 s at 95°C, 20 s at 58°C and 20 s at 72°C. Each sample from each biological replicate was analyzed twice. The specificity of primer pairs was verified both visually on 2% agarose gels and by melting curve analysis. Amplification efficiencies for all primer pairs were determined using the Realplex software (Eppendorf) and the quantification of the relative changes in gene expression was performed using the Pfaffl method (Pfaffl 2001).

Light microscopy, X-ray microanalysis mapping and scanning electron microscopy (SEM)

[00118] For microscopy, 10 leaf samples were taken from A. thaliana plants grown during seven days in hydroponic solution supplemented with 0.4 or 1.7mM of Si (Si+) and without Si (Si-). For light microscopy, samples were examined with an Olympus S261 bifocal microscope (Shinjuku-KV, Tokyo, Japan) and images were analyzed using Photoshop (Adobe, San Jose, USA). X-ray microanalysis and SEM analyses were used to measure the accumulation of Si within the leaves of A. thaliana. Samples were analyzed as described in Guevel et al. (2007) using a CAMECA SX-100 Universal EPMT microscope (Cameca instruments Inc., Trumbull, USA).

Si content in A. thaliana shoots

[00119] Si content in A. thaliana plants was measured by inductively coupled plasma optical emission spectrometry (ICP-OES, Jobin-Yvon Horiba JY2000-2, Longjumeau, France). Aerial parts of 10 plants from each treatment (three replicates) were collected and freeze-dried one week following the beginning of Si amendment. Samples were ground to a powder and total Si analysis was carried out at 251.611 nm by ICP-OES as described previously by Cdte-Beaulieu ef al. (2009).

TaLsil localizes to the plasma membrane

[00120] To investigate the sub-cellular localization of wheat Lsi1 , we prepared a C- terminal fusion between the green fluorescent protein (GFP) and TaLsil (TaLsi -GFP) as well as GFP fused to OsLsil (OsLsi1-GFP) or GFP alone. To ensure that the constructs were functional, they were first expressed transiently in Nicotiana benthamiana leaves by agro-infiltration under the control of the CaMV35S promoter (Figure 4). When expressed alone, GFP fluorescence was observed in the nucleus, in the cytosol and at the periphery of the cell around a large central vacuole (Figure 4a, d). In contrast, the OsLsi1-GFP or TaLsi1-GFP fusion proteins were both restricted to the membrane lining the cell, suggesting a plasma membrane localization of the TaLsil protein similar to that previously described for OsLsil (Figure 4b, c, e and f).

[00121] To verify the cellular localization of wheat and rice Lsi1 transporters and their expression profile during plant development, 35S::OsLsi1::GFP and 35S::TaLsi1::GFP constructs were used to obtain stable expression in A. thaliana by

transformation. Among 20 OsLsi1::GFP and 19 TaLsi1::GFP primary transformants, ten of each were chosen randomly. Of these, three transformants with a single T-DNA integration locus (3:1 segregation in T2) and presenting a strong GFP fluorescence were chosen. T3 lines homozygous for the T-DNA were identified and examined for GFP fluorescence 7 days post-germination and during flowering by confocal microscopy. The distribution of fluorescence was similar for both fusion proteins and representative images are shown in Figure 5. In seedlings, proteins were expressed in primary and lateral roots (Figure 5b, d). Fluorescence was stronger in the central root cylinder within the differentiation zone and was weaker in the basal zone (Figure 5b). No expression was observed in root hairs (Figure 5d). In primary root tips, GFP fusion proteins were expressed in the cap and in the root meristem (Figure 5b) but weakly expressed in the differentiated cortex and endodermis. In flowering plants, only weak fluorescence was observed at the base and inside trichomes of young leaves (Figure 5f). No fluorescence was observed in flowers (data not shown).

Expression of Lsi1 transporters in A. thaliana

[00122] To produce Si accumulating plants, stable transformants of A. thaliana harboring either the 35S::OsLsi1 or Z5S..TaLsi1 construct were obtained. As described previously, a first set of ten transformants with a single T-DNA insertion was selected from each group of primary transformants (33 for 35S::OsLsi1 and 17 for 35S::TaLsi1). Two homozygous T3 plants transformed with rice or wheat transporters were selected and OsLsil or TaLsil relative expression in leaves and roots was measured by qRT-PCR (Figure 6). In all transgenic constructs, the expression of each gene was highly abundant compared to the wild-type (WT) and similar among a given transformant both in roots and

.AO- shoots except in the first 35S::OsLsi1 line where the leaf expression was approximately 20% higher than in roots.

A. thaliana plants expressing Lsi1 transporters allows Si uptake and deposition

[00123] In order to evaluate Si deposition in leaves of 35S::TaLsi1 or 35S::OsLsi1 plants grown with 0, 0.2 and 1.7 mM Si, X-ray microanalysis mapping were performed. Si accumulation was similar for both transgenic plants and representative images are shown in Figure 7. In absence of Si, no signal was detected within transgenic or WT leaves (Figure 7a). By contrast, leaves of transgenic plants amended with 1.7 mM Si showed a constant pattern of Si deposition at the base of trichomes, in guard cells of stomata and at the periphery of a necrotic zone (Figure 7c, d). A very weak accumulation was also noticed at the base of trichomes in Si-amended WT plants (Figure 7b). At 0.2 mM, a less intense but comparable Si deposition pattern was observed (data not shown).

[00124] We subsequently measured Si concentration by ICP-OES in aerial parts of WT, 35S::OsLsi1 and 35S:: TaLsi1 transgenic plants grown with or without 1.7 mM Si for 7 days (Figure 8). In absence of Si, there was no statistic difference in Si accumulation between transgenic and WT. By contrast, in 35S..OsLsi1 amended plants, Si concentration was 3-4 fold higher compared to WT. Similar results were obtained with transgenic plants expressing the TaLsil transporter in which Si concentration was 4-5 fold higher than in WT. These results indicate that either wheat or rice Lsi1 transporters could be used to promote an efficient Si uptake and deposition in plants with low innate Si transport activity.

[00125] Considering that it is possible to express a silicon transporter gene in a foreign plant, it is soundly predicted that expression of the horsetail silicon transporters NIP

B, C or D as defined herein as well a homologues thereof can be expressed in plants such as Arabidopsis. EXAMPLE 9. Expression of TaLsi^'1 gene under root promoter in A. thaliana and Si content.

[00126] The 2.5-kb region upstream of the initiation codon of NIP5;1 was amplified by PCR from BAC clone F24G24 (obtained from the ABRC) using primers 5'- GAGAAAGCTTGAAAGCAAGCATTCCCTG-3' (SEQ ID N0.19) and 5'- GAGCCATGGCCAACGTTTTTTTTTTTGGT-3' (SEQ ID NO.20). Using Hind\\\ and Λ/col, the amplified fragment was subcloned into 35S-TaLsi1 construct without the 35S promoter. The resulting pNIP5::TaLsi1 plasmids (SEQ ID N0.21 ) were individually introduced into A.

L tumefaciens strain GV3101/pMP90. Transformation and selection of transgenic lines were performed as described above.

[00127] Si concentrations were measured by ICP-OES (see above) in aerial parts of WT, 35S::TaLsi1 and pNIP5 :TaLsi1 transgenic plants grown with or without 1.7 mM Si for 7 days (Figure 9). In amended pNIP5::TaLsi1 plants, Si concentration was 1.5 fold higher compared to WT. Similar results were obtained with transgenic plants expressing the TaLsil transporter under 35S promoter. These results confirm that a root specific promoter is as efficient as a constitutive promoter to drive silicon absorption using the heterologous transporter TaLsil . EXAMPLE 10 - Effect of silicon feeding on control and transformed plants of

Arabidopsis thaliana infected with Powdery mildew (Erysiphe cichoracearum)

Materials and methods

Inoculum

[00128] Arabidopsis thaliana, accession PAD4, was used to produce the powdery mildew (Erisyphe cichoracearum) inoculum. The accession PAD4 is a mutant with increased susceptibility to powdery mildew (Reuber, ef a/. 1998. Correlation of defense gene induction defects with powdery mildew susceptibility in Arabidopsis enhanced disease susceptibility mutants. Plant Journal. 16 :473-485).

[00129] Powdery mildew inoculum was collected from heavily infected PAD4 plants by brushing the leaves with a small paintbrush over a petri dish.

Plant material

[00130] A. thaliana accession Col-0 was used as a control throughout the experiment. Mutants were Col-0 plants previously transformed by Agrobacterium tumefasciens containing the pNIP5:TaLsi1 construct where pNIP5 is a root specific promoter from A. thaliana and TaLsil is a gene coding for a wheat silicon transporter.

[00131] Four-week-old plants, grown in soilless medium (Connaisseur Mix, Fafard, Riviere-du-Loup, Qc) in a growth chamber under a 6h-photoperiod, with a maximum temperature of 22°C during the day and a minimum of 19°C during the night, were used in this experiment. Silicon amendment (Si+) (1 ,7 mM Si in Hoagland solution 1/10) was started 7 days prior to inoculation with powdery mildew following a 3 times a week schedule. The control treatment (Si-) consisted of a Hoagland solution 1/10.

Inoculation.

[00132] Plants (5 plants/pot), control and transgenic, were brushed gently with a paintbrush contaminated with conidia on ca. 3 - 4 rosette leaves per pot. After inoculation, plants were placed in the growth chamber with a plastic cover to minimize air convection during infection. Plastic covers were removed 3 days after inoculation. Growth chamber conditions were kept as described above.

Results

[00133] Four days after inoculation, white colonies appeared on Col-0 and on some mutants Si-. Seven days after inoculation, disease severity was evaluated (Table 5, Figure 10). Transformants fed with silicon amended solution showed less powdery mildew infection spots than control plants (Si- or Si+) and transformants Si-.

Tables

Seven days after inoculation, disease severity was estimated with a visual scale where (-) means no powdery mildew symptom, (+) at least one leaf where infection has started, (++) at least one leaf infected on more than 50% of its surface, and (+++) more than two leaves infected on 50% of the total leaf area. Each treatment was represented by 3 pots containing 5 plants each.

EXAMPLE 11- Transformation of soybean plants with TaLsil (silicon transporter from wheat)

Material and methods - Si deposition and content within soybean leaves.

[00134] Fungal and plant material. Cultivars Jack (wild-type) and SYDC04U (Jack with TaLsil insert with the CMP constitutive promoter, which is described in US 7,166,770, incorporated herein by reference) were used. Plants were grown in a greenhouse maintained at 25/20°C with a relative humidity of 65% and a photoperiod of 14h.

[00135] Hydroponic system and silicon amendment. Plant were grown in peatmoss or in hydroponic systems. Plants were fed with soluble silicon two weeks after seedling emergence by amending the Hoagland solution or the peat-moss with 1.7 mM Si in the form of potassium silicate (Kasil #6, 23,6 % Si02, National Silicates, Toronto).

[00136] Si deposition and content within leaves. Soybean leaves were collected three weeks after Si amendment. Scanning electron microscopy and X-ray microanalysis mapping were used to determine Si deposition for the different treatments. Silicon content was quantified by inductively coupled-plasma optical emission spectrometry (ICP-OES) (Figure 11 ).

Results

[00137] In CMP::Tal_si1 Si amended plants, Si concentration was ca, 25% higher compared to WT. These results indicate that wheat Lsi1 transporter could be used to promote an efficient Si uptake and deposition in plants with low innate Si transport activity.

[00138] These plants, once grown, can be tested for increased resistance to various biotic and abiotic stresses, including soybean rust. Desirably, such transformed plants will have increased resistance to such stresses, including increased resistance to soybean rust.

[00139] Based on these results, it can be soundly predicted that soybean, once transformed with the horsetail Si promoter of the present invention, can express the Si transporter and demonstrate increased Si uptake to fight-off biotic and abiotic stress.

EXAMPLE 12- Transformation of soybean plants with horsetail Si-transport gene

[00140] Several genes can be introduced into a plant during a single transformation event. For the present invention, one example of a DNA construct consisting of an Agrobacterium p-CAMBIA plasmid containing the following sequence can be introduced in the plant genome using antibiotic resistance as a selection marker: CaMV 35 S promoter - antibiotic resistance gene - terminator - CaMV 35S promoter - SIITI gene - terminator - CaMV 35 S promoter - SIIT2 gene - terminator -CaMV 35 S promoter - SIETI gene - terminator, (see, e.g., Dans and Wei Plant Science 173:381-389, 2007 for an example of soybean transformation with two insect resistance genes). In another example, a DNA construct consists of a plasmid comprising a root-preferred promoter (e.g. AR6, described in US 7,615,624, which is incorporated herein by reference) - NIP D silicon transporter gene - terminator - actin promoter - herbicide tolerance gene - terminator. The DNA construct is introduced in Agrobacterium tumefasciens bacteria. [00141] Soybean calluses are co-cultured with the Agrobacterium. The plant cells are then transferred to a culture medium containing the selection marker, for example an antibiotic or a herbicide. Only the plant cells that have integrated the DNA construction and expressed the antibiotic-resistance gene or the herbicide resistance gene will grow.

[00142] Additional controls can be performed using PCR. To verify that the NIP B, NIP C and NIP D genes are integrated in the plant genome, total plant DNA is extracted, and PCR is performed using primers specific for either the NIP B, NIP C and NIP D genes. To verify that NIP B, NIP C and NIP D genes are expressed in the plant, root RNA is extracted and reverse-transcribed in complementary DNA (cDNA, as described in Example 1 ). PCR is then performed on the cDNA using primers specific for the NIP B, NIP C and NIP D genes using standard methods.

[00143] These plants, once grown up, can be tested for increased resistance to various biotic and abiotic stresses, including soybean rust. Desirably, such transformed plants will have increased resistance to such stresses, including increased resistance to soybean rust. [00144] All patents, patent applications and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent, patent application, or publication was specifically and individually indicated to be incorporated by reference.

AS.

Claims

I . An isolated or substantially pure polynucleotide comprising a nucleic acid sequence with at least 50% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 , 3 and 5.

2. The polynucleotide of claim 1 , wherein expression of the polypeptide encoded by said polynucleotide in a cell is capable of increasing silicon transport into said cell.

3. The polynucleotide of claim 1 , wherein said identity is selected from the group consisting of at least: 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% and 100%.

4. The polynucleotide of claim 1 , wherein said polynucleotide is less than 20 kB in length.

5. The polynucleotide of any one of claims 1 to 4, operably linked to a promoter.

6. The polynucleotide of claim 5, wherein said promoter is capable of expression in a plant cell.

7. The polynucleotide of claim 6, wherein said plant cell is a root cell.

8. A vector comprising the polynucleotide of claim 5.

9. A cell comprising the vector of claim 8.

10. The cell of claim 9, wherein said cell is a plant cell.

I I. The cell of claim 10, wherein said plant is a dicotyledonous plant.

12. The cell of claim 11 , wherein said plant cell is a soybean plant cell.

13. A seed comprising the cell of claim 9.

14. A substantially pure polypeptide encoded by the polynucleotide of any one of claims 1 to 7.

15. A plant comprising a heterologous polynucleotide comprising a nucleic acid sequence according to any one of claims 1 to 7.

16. The plant of claim 15, wherein said plant is a dicotyledonous plant.

17. The plant of claim 16, wherein said plant is a soybean plant.

18. A method of generating a plant with increased silicon uptake, said method comprising:

(a) providing a first vector comprising the polynucleotide comprising a nucleic acid sequence having at least 50% identity to a sequence selected from the group consisting of SEQ ID NO: 1 , 3 and 5;

(b) transforming a plant cell with said vector; and

(c) growing a plant from said cell, wherein said plant expresses said polynucleotide, thereby generating a plant with increased silicon uptake.

19. The method of claim 18, wherein said plant cell is a dicotyledonous plant cell.

20. The method of claim 19, wherein said plant cell is a soybean cell.

21. A polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 50% identity to an amino acid sequence of SEQ ID NO.7.

22. The polynucleotide of claim 21 , wherein expression of the polypeptide encoded by said polynucleotide in a cell is capable of increasing silicon transport into said cell.

23. The polynucleotide of claim 21 , wherein said identity is selected from the group consisting of at least: 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% and 100%.

24. The polynucleotide of claim 21 , wherein said polynucleotide is less than 20 kB in length.

25. The polynucleotide of any one of claims 21 to 24, operably linked to a promoter.

26. The polynucleotide of claim 25, wherein said promoter is capable of expression in a plant cell.

-4Z

27. The polynucleotide of claim 26, wherein said plant cell is a root cell.

28. The polynucleotide of claim 27, wherein said plant cell is a dicotyledonous plant cell.

29. The polynucleotide of claim 28, wherein said plant cell is a soybean plant cell.

30. A method of increasing the resistance of a plant to biotic or abiotic stresses, comprising:

a) providing a vector comprising a nucleic acid sequence having at least 50% identity to a sequence selected from the group consisting of SEQ ID NO: 1 , 3 and 5; b) transforming a plant cell with said vector; and

c) growing a plant from said cell, wherein said plant expresses a protein encoded by said nucleic acid sequence;

whereby the plant exhibits an increased resistance to biotic or abiotic stresses.

31. The method of claim 30, wherein said plant cell is a dicotyledonous plant cell.

32. The method of claim 31 , wherein said plant cell is a soybean cell.

33. The method of claim 30, wherein said nucleic acid sequence has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to a sequence selected from the group consisting of SEQ ID NOs: 1 , 3, and 5.

34. The method of claim 30, wherein said nucleic acid sequence is operatively linked to a promoter capable of expression in a plant cell.

35. The method of claim 34, wherein said promoter is a tissue-specific or a tissue- preferred promoter.

36. The method of claim 35, wherein said promoter is a root-preferred promoter.

37. The method of claim 36, wherein said promoter is a leaf-preferred promoter.

38. The method of claim 34, wherein said promoter is an inducible promoter.

39. The method of claim 34, wherein said promoter is a constitutive promoter.

40. The method of claim 30, wherein the biotic stressor is fungi.

41. The method of claim 40, wherein the fungi is selected from the group consisting of Alternaria (Alternaria brassicola; Alternaria solani), Ascochyta (Ascochyta pisi); Botrytis (Botrytis cinerea); Cercospora (Cercospora kikuchii; Cercospora zeae-maydis);

Colletotrichum (Colletotrichum lindemuthianum); Diplodia {Diplodia maydis); Erysiphe (Erysiphe graminis f. sp. graminis; Erysiphe graminis f. sp. hordei); Fusarium {Fusarium nivale; Fusarium oxysporum; Fusarium graminearum; Fusarium culmorum; Fusarium solani; Fusarium moniliforme; Fusarium roseum); Gaeumanomyces (Gaeumanomyces graminis f, sp. tritici); Helminthosporium (Helminthosporium turcicum; Helminthosporium carbonum; Helminthosporium maydis); Macrophomina (Macrophomina phaseolina);

Magnaporthe (Magnaporthe grisea); Nectria (Nectria haematococca); Peronospora

(Peronospora manshurica; Peronospora tabacina); Phoma (Phoma betae); Phymatotrichum (Phymatotrichum omnivorum); Phytophthora (Phytophthora cinnamomi; Phytophthora cactorum; Phytophthora phaseoli; Phytophthora parasitica; Phytophthora citrophthora; Phytophthora megasperma f. sp. sojae; Phytophthora infestans); Plasmopara (Plasmopara viticola); Podosphaera (Podosphaera leucotricha); Puccinia (Puccinia sorghi; Puccinia striiformis; Puccinia graminis f. sp. tritici; Puccinia asparagi; Puccinia recondita; Puccinia arachidis); Pyrenophora (Pyrenophora tritici-repentis); Pyricularia (Pyricularia oryzae); Pythium (Pythium aphanidermatum; Pythium ultimum); Rhizoctonia (Rhizoctonia solani; Rhizoctonia cerealis); Sclerotium (Sclerotium rolfsii); Sclerotinia (Sclerotinia sclerotiorum); Septoria (Septoria lycopersici; Septoria glycines; Septoria nodorum; septoria tritici);

Thielaviopsis (Thielaviopsis basicola); Uncinula (Uncinula necator); Venturia (Venturia inaequalis); Verticillium (Verticillium dahliae; Verticillium albo-atrum); Basidiomycetes of the order Uredinales; Puccinia (P. graminis, P. stiiformis, P. recondita, P. hordei, P. coronata, P. sorghi., P. polysora, P. purpurea, P. sacchari P. kuehnii, P. stakmanii, P. asparagi, P. chrysanthemi, P. malvacearum, and P. antirrhini); Gymnosporangium (G. juniperi- virginianae, G. globosum); Hemileia (H. vastatrix); Phragmidium; Uromyces (U.

caryophyllinus); Cronartium (C. ribicola, C. quercuum f. sp. fusi forme, C. quercuum f. sp. virginianae, C. comptoniae, C. comandrae, C. strobilinum); Melampsora (M. lini);

Coleosporium (C. asterinum); Gymnoconia; Phakopsora (P. pahyrhizi) and Tranzschelia.

42. An isolated polypeptide sequence having at least 67% identity to a polypeptide encoded by the isolated nucleic acid of claim 1.

43. The isolated polypeptide sequence of claim 42, wherein the polypeptide sequence is selected from the group consisting of SEQ ID NOS: 2, 4, and 6.