US20060005271A1

US20060005271A1 - Transgenic plants expressing L3 delta proteins are resistant to trichothecene fungal toxins

Info

Publication number: US20060005271A1
Application number: US11/010,795
Authority: US
Inventors: Nilgun Tumer; Rong Di
Original assignee: Rutgers State University of New Jersey
Current assignee: Rutgers State University of New Jersey
Priority date: 2003-12-12
Filing date: 2004-12-13
Publication date: 2006-01-05
Also published as: US20120042415A1; US20100146667A1; US8026410B2

Abstract

Disclosed are specific mutants of L3 and transgenic plants that produce them. The plants exhibit increased resistance to fungal toxins that target ribosomal L3 protein. Also disclosed are transgenic plants that co-produce L3 mutant and an RIP protein, and exhibit increased resistance to various fungal toxins and viruses, while reducing toxicity normally associated with production of the RIP. Uses of the L3 mutants in animals are further disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 60/529,348 filed Dec. 12, 2003, the disclosure of which is hereby incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The development of this invention was supported in part by the United States Department of Agriculture grant USDA-RS-58-5325-758. Therefore, the Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

The subject of plant protection against pathogens remains the area of utmost importance in agriculture. Many commercially valuable agricultural crops are prone to infection by plant viruses and fungi capable of inflicting significant damage to a crop in a given season, and drastically reducing its economic value. The reduction in economic value to the farmer in turn results in a higher cost of goods to ultimate purchasers.
Fungal pathogens contribute significantly to the most severe pathogen outbreaks in plants. Plants have developed a natural defense system, including morphological modifications in their cell walls, and synthesis of various anti-pathogenic compounds. See, e.g. Boller, et al., Plant Physiol. 74:442-444 (1984); Bowles, Annu. Rev. Biochem. 59:873-907 (1990); Joosten, et al., Plant Physiol. 89:945-951 (1989); Legrand, et al., Proc. Natl. Acad. Sci. USA 84:6750-6754 (1987); and Roby, et al., Plant Cell 2:999-1007 (1990). Several pathogenesis-related (PR) proteins have been shown to have anti-fungal properties and are induced following pathogen infection. These are different forms of hydrolytic enzymes, such as chitinases and β-1,3-glucanases that inhibit fungal growth in vitro by destroying fungal cell walls. See, e.g. Boller, et al., supra; Grenier, et al., Plant Physiol. 103:1277-123 (1993); Leah, et al., J. Biol. Chem. 266:1464-1573 (1991); Mauch, et al., Plant Physiol. 87:325-333 (1988); and Sela-Buurlage Buurlage, et al., Plant Physiol. 101:857-863 (1993).
Several attempts have been made to enhance the pathogen resistance of plants via recombinant methodologies using genes encoding pathogenesis-related proteins (such as chitinases and β-1,3-glucanases) with distinct lytic activities against fungal cell walls. See, e.g., Broglie, et al., Science 254:1194-1197 (1991); Vierheilig, et al., Mol. Plant-Microbe Interact. 6:261-264 (1993); and Zhu, et al., Bio/Technology 12:807-812 (1994). Recently, two other classes of genes have been shown to have potential in conferring disease resistance in plants. Wu, et al., Plant Cell 7:1357-1368 (1995), reports that a transgenic potato expressing the Aspergillus niger glucose oxidase gene exhibited increased resistance to Erwinia carotovora and Phytophthora infestans. The hypothesis is that the glucose oxidase-catalyzed oxidation of glucose produces hydrogen peroxide, which when accumulates in plant tissues, leads to the accumulation of active oxygen species, which in turn, triggers production of various anti-pathogen and anti-fungal mechanisms such as phytoalexins (see Apostol, et al., Plant Physiol. 90:109-116 (1989) and Degousee, Plant Physiol. 104:945-952 (1994)), pathogenesis-related proteins (Klessig, et al., Plant Mol. Biol. 26:1439-1458 (1994)), strengthening of the plant cell wall (Brisson, et al., Plant Cell 6:1703-1712 (1994)), induction of systemic acquired resistance by salicylic acid (Chen, et al., Science 162:1883-1886 (1993)), and hypersensitive defense response (Levine, et al., Cell 79:583-593 (1994)).
In addition to the studies on virus resistance in plants, ribosome inactivating proteins (RIPs) have been studied in conjunction with fungal resistance. For example, Logeman, et al., Bio/Technology 10:305-308 (1992), report that an RIP isolated from barley endosperm provided protection against fungal infection to transgenic tobacco plants. The combination of barley endosperm RIP and barley class-II chitinase has provided synergistic enhancement of resistance to Rhizoctonia solani in tobacco, both in vitro and in vivo. See, e.g., Lea, et al., supra; Mauch, et al., supra; Zhu, et al., supra; and Jach, et al., The Plant Journal 8:97-109 (1995). PAP, however, has not shown antifungal activity in vitro. See Chen, et al., Plant Pathol. 40:612-620 (1991), which reports that PAP has no effect on the growth of the fungi Phytophthora infestans, Colletotrichum coccodes, fusarium solani, fusarium sulphureum, Phoma foreata and Rhizoctonia solani in vitro.
Lodge, et al., Proc. Natl. Acad. Sci. USA 90:7089-7093 (1993), report the Agrobacterium tumefaciens-mediated transformation of tobacco with a cDNA encoding wild-type pokeweed antiviral protein (PAP) and the resistance of the transgenic tobacco plants to unrelated viruses. Pokeweed antiviral protein (PAP) is a 29-kDa ribosome inactivating protein that catalytically removes two adenines and a guanine from the sarcin/ricin (S/R) loop of the large rRNA (Endo et al., J. Biol. Chem. 263:8735-8739 (1988); Hudak et al., J. Biol. Chem. 274:3859-3864(2000) and disrupts binding of elongation factors to the ribosome (Montanaro et al., Biochemical J. 146:127-131(1975); Osborn et al., European J. of Biocham. 193:401-407 (1990)). Aside from this demonstration of broad spectrum resistance to viruses, it has been demonstrated that when expressed in transgenic plants, PAP also confers broad spectrum antifungal (Zoubenko et al., Nature Biotechnol. 15:922-996 (1997); Zoubenko et al., Plant Mol. Biol. 44:219-229 (2000)) activity. It has also been shown that PAP recognizes its ribosomal substrate by binding to L3 (Hudak et al., J. Biol. Chem. 274:3858-3864 (1999)).
Lodge also reports, however, that the PAP-expressing tobacco plants (i.e., above 10 ng/mg protein) tended to have a stunted, mottled phenotype, and that other transgenic tobacco plants that accumulated the highest levels of PAP were sterile. U.S. Pat. Nos. 5,756,322 and 5,880,322 teach PAP mutants that when produced in plants exhibit less toxicity than wild-type PAP and exhibit biological activities (e.g., resistance to viruses, fungi and other pests) akin to wild-type PAP. It has also been reported that PAP II and PAP II mutants exhibit reduced phytotoxicity compared to wild-type PAP. See Wang, et al., Plant Mol. Biol. 38:957-964 (1998).
The trichothecenes are a family of low molecular weight sesquiterpenoid mycotoxins synthesized by various Fusarium species of fungi. Deoxynivalenol (DON) produced by F. graminearum or F. culmorum that causes fusarium head scab of wheat is a worldwide problem for human health concern and poses a major impact on animal production if present in feeds (Miller et al., Nat. Toxins 5:234-237 (1997)). Other trichothecenes include fusarenon X, trichothecin, verrucarin A, nivalenol, trichodermin, T-2 toxin and diacetoxyscirpenol (DAS). Trichothecenes inhibit peptidyl transferase reaction of protein synthesis by binding to the 60S ribosomal subunit. In addition, they cause membrane damage (Feinberg et al., C. S. 1989. Biochemical mechanism of actions of trichothecene mycotoxins. Pages 27-36 in: Trichothecene mycotoxixosis: Pathophysiological effects, Vol. 1. V. R. Beasley, ed. Boca Raton, Fla., CRC Press. Khachatourians, Canad. J. Physiol. Pharm. 68:1004-1008 (1990); Miller et al., Nat. Toxins 5:234-237 (1997)). Mitterbauer et al., 7^thInternational Congress of Plant Pathology, Edinburgh, Scotland, 5.4.6. (1998) demonstrate that trichothecene resistance in the yeast, Saccharomyces cerevisiae, could result from either alterations in the target of trichothecenes, the ribosomal protein L3 or the increased drug efflux due to over-expression of a membrane transporter protein encoded by the PDR5 gene.
L3 is a highly conserved ribosomal protein that participates in the formation of the peptidyltransferase center that in turn allows elongation of the ribosome along the messenger RNA (mRNA). Hampl, et al., J. Biol. Chem. 256:2284-2288 (1981); Noller, J. Bacteriol. 175:5297-5300 (1993). L3 also plays an essential role in the catalysis of peptide bond formation. See, Green, et al., Annu. Rev. Biochem. 66:679-716 (1997). This is an essential step in protein synthesis in yeast, animals and higher plants. L3 is encoded by the rpl3 gene. Trichodermin, a substituted 12,13-epoxytrichothecene, is known to inhibit peptide bond formation by binding to the peptidyl transferase center. A mutation in the Rpl3 gene, designated tcm-1, which contains a single amino acid substitution of tryptophan to cysteine at position 255 (i.e., W255C) was initially identified in yeast by conferring resistance to trichodermin (Fried, et al., Proc. Natl. Acad. Sci. USA 78:238-242 (1981)). U.S. Pat. No. 6,060,646 to Harris, et al., teaches modified peptidyl transferase (L3) genes that provide resistance to trichothecene mycotoxins, such as the tcm-1 gene. Transgenic plants transformed with genes encoding L3 proteins are disclosed in WO 00/39291. The L3 proteins include wild-type L3, spontaneously occurring mutants and other non-naturally occurring mutants. It also teaches plants transformed with L3 genes and genes encoding ribosome inactivating proteins such as PAP.
Studies by Muhitch et al., Plant Science 157:201-207 (2000) demonstrated that tobacco plants transformed with either the Saccharomyces cerevisiae gene PDR5, which encodes a multi-drug transporter, or with the Fusarium sporotrichioides gene TRI101, which encodes a trichothecene 3-O-acetyltransferase, showed increased tolerance to the trichothecene 4,15-diacetoxyscirpenol (DAS). Even more recently, Harris et al., Physiol. Mol. Plant Path. 58:173-181 (2001), showed that transgenic tobacco tissues transformed with a modified Rpl3 gene from rice displayed resistance to DON.

SUMMARY OF THE INVENTION

U.S. Pat. No. 6,060,646 to Harris, et al., teaches that the entire area between amino acid residues 240-263 of the L3 gene (which Harris refers to as the peptidyl transferase gene) is highly conserved in rice, Arabidopsis, yeast, bovines, humans, mice and rats, and is critical from the standpoint of conferring resistance to trichothecenes. It also teaches an L3 mutant, tcm-1, which results in an amino acid change at position 255 (W255C) in L3 that confers resistance to the trichothecene mycotoxin, thus substantiating this belief.
Applicants have discovered that N-terminal fragments of L3 that do not contain this region, when produced in plants, provide increased resistance to fungi, especially Fusarium, that produce trichothecenes. The N-terminal fragments of L3 do not contain the tcm-1 mutation (resulting in the amino acid change, W255C) in L3 that confers resistance to the trichothecene mycotoxin. Applicants have also discovered that expression of the N-terminal L3 fragments in transgenic plants confers better resistance to trichothecene mycotoxins than the full length L3 gene, and that co-expression of these fragments and a ribosome inactivating protein (RIP) such as pokeweed antiviral protein (PAP) serves to reduce or eliminate the toxicity associated with expression of the RIP. As a result, RIPs such as wild type PAP protein can be expressed at much higher levels in plants containing the N-terminal fragments of L3 than in plants containing the wild type PAP gene alone. Applicants discovered in the presence of the L3 N-terminal polypeptides, PAP does not auto-regulate i.e., degrade, its own mRNA, which results in higher expression levels and thus greater resistance to diseases caused by fungi, and that PAP does not depurinate the RNA of the cell, resulting in less toxicity to the cell.
Accordingly, a first aspect of the present invention is directed to a transgenic plant comprising an exogenous nucleic acid (i.e., a nucleic acid in addition to the native genome of the host) comprising a transgene functional therein and that encodes a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein, or an analog of said polypeptide, wherein said plant exhibits increased resistance to toxins that target eucaryotic ribosomal L3 protein compared to a non-transgenic control plant. Such toxins include trichothecene mycotoxins, e.g., deoxynivalenol (DON) and 4,15-diacetoxyscirpenol (DAS). In some embodiments, the plant is cereal crop plant, e.g., maize, wheat, barley, rice and oat. In some embodiments, the polypeptide contains from at least the first 21 to 99 N-terminal amino acids, and in other embodiments, the polypeptide contains the first 100 N-terminal amino acids of a eurcaryotic ribosomal L3 protein. In some embodiments, the polypeptide has an amino acid sequence that corresponds to the yeast, rice, Arabidopsis or a tobacco L3 protein. In some embodiments, the exogenous nucleic acid further contains another transgene that encodes a RIP that targets a euraryotic ribosomal L3 protein, such as PAP, PAP-v, PAP II, ricin or a Shiga toxin. Seed generated from the transgenic plants are also provided.
A second aspect of the present invention is directed to a protoplast transformed with an exogenous nucleic acid having a transgene encoding a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein or an analog thereof, wherein expression of the transgene in a transgenic plant generated from the transformed protoplast provides greater resistance to toxins that target a eucaryotic ribosomal L3 protein compared to a non-transgenic control plant. In some embodiments, the exogenous nucleic acid further comprises a transgene encoding a RIP protein that targets a eurcaryotic L3 ribosomal protein. The two transgenes can be introduced into the protoplast together by way of a single vector, or separately. Compositions containing the protoplasts and a suitable (e.g., culture or regeneration) medium, and callus derived from the protoplasts are also provided.
A third aspect of the present invention is directed to plant tissue transformed with an exogenous nucleic acid having a transgene encoding a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein or an analog thereof, wherein expression of the nucleic acid in a transgenic plant generated from the transformed plant tissue provides greater resistance to toxins that target a eucaryotic ribosomal L3 protein compared to a non-transgenic control plant. In some embodiments, the exogenous nucleic acid further comprises a transgene encoding a RIP protein that targets a eurcaryotic L3 ribosomal protein. Compositions containing the plant tissue and a suitable (e.g., culture or regeneration) medium are also provided.
A fourth aspect of the present invention is directed to a vector functional in plant cells (e.g., suitable for use in transforming plants, or parts thereof such as protoplasts and plant tissue and which is replicable and viable therein), comprising a first nucleic acid fragment comprising a first promoter functional in a plant cell in operable association with a first nucleic acid encoding a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein or an analog thereof. In some embodiments, the vector further contains a second nucleic acid fragment encoding an RIP protein e.g., PAP, that targets a eurcaryotic ribosomal L3 protein, in operable association with a second promoter functional in a plant cell, wherein the first and second promoters may be the same or different.
A fifth aspect of the present invention is directed to a method of making a transgenic plant having increased resistance to infestation by fungi that produce toxins that target a eurcaryotic L3 protein, e.g., trichothecene-producing fungi that produce deoxynivalenol (DON) or 4,15-diacetoxyscirpenol (DAS), comprising preparing a transgenic plant having a genome that contains an exogenous nucleic acid comprising a transgene encoding a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein or an analog thereof, wherein expression of the transgene in the transgenic plant confers increased resistance to toxins that target a eucaryotic ribosomal L3 protein compared to a non-transgenic control plant.
A sixth aspect of the present invention is directed to a method of making a transgenic plant having resistance to infestation by fungi that produce toxins that target a eurcaryotic L3 protein, e.g., trichothecene mycotoxins deoxynivalenol (DON) and 4,15-diacetoxyscirpenol (DAS), comprising preparing a transgenic plant having a genome that contains a first exogenous nucleic acid having a first transgene encoding a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein or an analog thereof, and a second exogenous nucleic acid having a second transgene encoding an RIP protein that targets a eucaryotic ribosomal L3 protein, wherein expression of the first and second transgenes in the transgenic plant confers increased resistance to the fungi, and with less toxicity to the plant compared to a transgenic control plant that contains the second transgene but does not contain the first transgene.
A further aspect of the present invention is directed to a transgenic non-human animal comprising an exogenous nucleic acid having a transgene encoding a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein or an analog thereof, wherein expression of the transgene in the animal provides increased resistance toxins that target a eucaryotic ribosomal L3 protein e.g., trichothecene mycotoxins deoxynivalenol (DON) and 4,15-diacetoxyscirpenol (DAS), compared to a non-transgenic control non-human animal.
A further aspect of the present invention is directed to a pharmaceutical composition for treating fungal infections caused or mediated by a fungal toxin that targets a eucaryotic L3 ribosomal protein, comprising an anti-fungal effective amount of a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein or an analog thereof, and a pharmaceutically acceptable carrier.
A further aspect of the present invention is directed to a method of reducing toxicity associated with a ribosome inactivating protein (RIP) that targets a eucaryotic ribosomal L3 protein, in an animal in need thereof, comprising administering to an animal in need thereof, a composition comprising an effective amount of a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein, or an analog thereof. In some embodiments, the RIP is PAP, e.g., wild type PAP. The L3 polypeptide or analog thereof can be administered prior to, simultaneous with or after administration of the RIP, such that it is present in animal or human to exert its anti-cytotoxic effect of or on the RIP. In some embodiments, the RIP is conjugated to a ligand that binds a receptor present on or in the target cell. The methods are particularly useful in the treatment of cancer and viral infections (e.g., HIV) in mammals, preferably humans.
A further aspect of the present invention is directed to a polypeptide or analog thereof, comprising at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein.
A further aspect of the present invention is directed to a polynucleotide having a sequence encoding a polypeptide having at least the first 21 to 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein. Constructs containing the polynucleotides, e.g., vectors such as plasmids functional (e.g., replicable and viable) in a host cell such as a bacterial, yeast or animal cell, wherein the polynucleotide is operably associated with a promoter functional in the cell or non-cellular system in which the polynucleotide is intended to be expressed, and host cells transformed with the polynucleotide, are further provided. The phrase “targets a eucaryotic ribosomal L3 protein”, as used herein, includes interaction between the L3 protein and a toxin such as DON or DAS, or a RIP such as PAP, that results in depurination of ribosomes and toxicity to the cell. By the term “about 99”, it is meant to include polypeptides having the first 100 N-terminal amino acid residues of a eucaryotic L3 ribosomal protein.
These and other aspects of the present invention are more fully described in the sections that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows constructs used to generate transgenic tobacco plants described in working examples.
FIGS. 2A, B and C show integration of L3, L3(1-100) and PAP genes in transgenic plants analyzed by PCR or Southern blot. PCR reactions were performed by L3, L3(1-100) and PAP-specific primers. Southern blot was performed on the L3(1-100) PCR products from transgenic plants using ³²P-labeled L3(1-100) fragment as the probe. A. PCR analysis for L3 gene in NT243 and NT250 plants. Lanes 1-2: NT243-6, 8. Lanes 3-4: NT250-1, 4. Lane 5: wt NN. Lane 6: 1 kb MW standard. B. PCR analysis of NT243 and NT245 transgenic tobacco plants for PAP gene. Lane 1: wt NN. Lanes 2-5: NT243-6, 7, 8, 9. Lanes 6-9: NT245-1, 2, 3, 4. Lane 10: wt nn. Lane 11: 1 kb MW standard. C. Southern blot analysis of L3(1-100) PCR products of NT245 and NT252 transgenic plants. Lane 1: L3(1-100) fragment released by restriction enzymes contained in a plasmid. Lane 2-3: NT245-1, 2. Lane 4: wt nn. Lane 5-6: NT252-1, 4.
FIGS. 3A, B and C show Western blot analysis on PAP expression level in transgenic tobacco plants. 10 μg of protein for each sample was electrophoresed on 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane and probed with PAP-specific polyclonal antibody. A. NT243 R0 transgenic plants. B. NT243 R2 transgenic plants. C. NT245 R0 transgenic plants.
FIG. 4 shows results of a depurination assay by primer extension of NT243 and NT245 R2 transgenic plants. Ribosomal RNAs were isolated as described and incubated with ³²P end-labeled oligonucleotide complementary to the 3′-end of the plant large rRNA. Primer extension was performed by reverse transcriptase. Lane1: wt rRNA treated with PAP as in vitro positive control. Lane2: wt rRNA not treated as in vitro negative control. Lane3: PAPv (less toxic PAP variant) rRNA as in vivo positive control. Lane4: PAPx (active site mutant) rRNA as in vivo negative control. Lane5: NT245-12. Lane6: NT245-21. Lane7: NT243-64. Lane8: no rRNA plus probe control. Lane9: NT245-12 treated with PAP. Lane10: NT245-21 treated with PAP.
FIGS. 5A and B show results of a DAS fungal toxin resistance test. Tobacco seeds were surface sterilized and germinated on MS medium containing 1 μM of DAS. The root length of 10 plants for each transgenic line was measured and averaged six weeks after as graphed in A. Pictures of the root growth of the wild type tobacco and transgenic plants are shown in B.
FIGS. 6A and B show results of a DON fungal toxin resistance test. Tobacco seeds were surface sterilized and germinated on MS medium containing 10 μM of DON. The root length of 10 plants from each construct was measured and averaged six weeks after as graphed in A. Pictures of the root growth of the wild type tobacco and transgenic plants are shown in B.
FIG. 7. shows results of a virus resistance test on the R1 transgenic plants of NT243. Two leaves of each plant were inoculated with TMV of 2 μg/ml. The local lesion numbers on the upper leaves of ten plants were counted and averaged and compared to the wild type plants.
FIG. 8 shows a Northern blot analysis to detect tobacco basic chitinase in R1 plants of NT243 inoculated with TMV (I) or with H₂O (U). Total RNA was isolated and electrophoresed-in denaturing agarose gel and transferred onto Duralose UV-membrane (Stratagene). The membrane was hybridized with ³²P-labeled basic chitinase cDNA.
FIGS. 9A, B and C show gene expression of tobacco ribosomal protein L3A and L3B analyzed by real-time quantitative PCR and Western blot. In real-time PCR (A. and B.), Oligo d(T) was used to prime the total RNA to synthesize the first-strand CDNA with SuperScript RT. Real-time PCR was performed with specific primers for tobacco L3A and L3B. The gene expression level was calculated as ddCT using tobacco tubulin gene as an internal control. The experiment was repeated three times. In Western blot (C.), 10 μg protein of each cytosolic sample was electrophoresed on 10% SDS-PAGE gel and transferred to nitrocellulose membrane. The blot was probed with L3 polyclonal antibody and PEPc to show equal loading. Lanes 1-12: wt nn, wt NN, NT243-64, NT243-81, NT245-12, NT245-21, NT250-11, NT250-41, NT252-11, NT252-41, PAPx and PAPv.
FIGS. 10A, B and C show gene expression of yeast L3 or L3(1-100) and PAP was analyzed by real-time quantitative and Western blot. In real-time PCR (A. and B.), oligo d(T) was used to prime the total RNA to synthesize the first-strand cDNA with SuperScript RT. Real-time PCR was performed with L3Δ- and PAP-specific primers. The gene expression level was calculated as ddCT using tobacco tubulin as an internal control. In Western blot (C.), 10 μg protein of each ribosomal sample was electrophoresed on 10% SDS-PAGE gel and transferred to nitrocellulose membrane. The blot was probed with PAP polyclonal antibody. Lanes 1-8: wt nn, PAPx, PAPv, NT243-64, NT243-81, NT245-12, NT245-21 and PAP standard.
FIGS. 11A and B show alignments of the amino acid sequences of full-length L3 proteins from Arabidopsis (i.e., AtRPL3A and AthRPL3B), Nicotiana tabacum (i.e., NtRPL3-8d and NtRPL3-10d), yeast (i.e., YRPL3), and rice (i.e., HvRPL3) various L3 proteins, and their first 100 amino acid residues, respectively.
FIGS. 12, 13 and 14 show nucleotide and corresponding amino acids sequences of the yeast wild-type L3 gene (rpl3), the tobacco “8d” L3 and “10d) proteins, and the mutant tcm1 gene.
FIGS. 15 and 16 show polynucleotide and corresponding amino acid sequences of a wild-type PAP and PAP II, respectively.
FIGS. 17, 18 and 19 show the polynucleotide and corresponding amino acid sequences of ricin and two different Shiga toxins, respectively.
FIGS. 20 and 21 show results of cytotoxicity experiments conducted in yeast transformed with PAP and L3(1-99) or L3 (1-100).
FIGS. 22A and B show results of ribosome depurination experiments conducted in yeast transformed with PAP and L3(1-99) or L3(1-100).
FIG. 23 shows results of a ribosome depurination assay conducted in vitro.
FIG. 24 shows results of a real time PCR analysis of production of PAP mRNA in yeast cells transformed with L3 N-terminal polypeptides of the present invention.
FIG. 25 schematically shows the stem loop structure of the 5′ end of L3 mRNA that encodes amino acid residues 1-21.
FIG. 26 shows results of a growth assay of yeast cells transformed with PAP and a polynucleotide encoding L3 polypeptides of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

A primary aspect of the present invention is directed to DNA sequence that encodes a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein (hereinafter “L3 N-terminal polypeptides”, or “L3 N-terminal polypeptide fragments,” or an analog of the L3 polypeptide. Eucaryotic L3 proteins include, but are not limited to human, yeast, bovine, mice, rat and higher plant (e.g., rice wheat, barley, and tobacco) and Arabidopsis L3 proteins. An alignment of the amino acid sequences of full-length L3 proteins from Arabidopsis (i.e., AtRPL3A and AthRPL3B), Nicotiana tabacum (i.e., NtRPL3-8d and NtRPL3-10d), yeast (i.e., YRPL3), and rice (i.e., HvRPL3) various L3 proteins, and their first 100 amino acid residues, are illustrated in FIGS. 11A and B. Nucleotide and corresponding amino acids sequences of the yeast wild-type L3 gene (rpl3), the tobacco “8d” L3 and “10d) proteins, and the mutant tcml gene, are illustrated in FIGS. 12, 13 and 14.
The polypeptides of the present invention may include the first 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 N-terminal amino acid residues of a eurcaryotic L3 protein. These polypeptides are referred to herein as L3(1-21), L3(1-22), L3(1-23), L3(1-24), L3(1-25), L3(1-26), L3(1-27), L3(1-28), L3(1-29), L3(1-30), L3(1-31), L3(1-32), L3(1-33), L3(1-34), L3(1-35), L3(1-36), L3(1-37), L3(1-38), L3(1-39), L3(1-40), L3(1-41), L3(1-42), L3(1-43), L3(1-44), L3(1-45), L3(1-46), L3(1-47), L3(1-48), L3(1-49), L3(1-50), L3(1-51), L3(1-52), L3(1-53), L3(1-54), L3(1-55), L3(1-56), L3(1-57), L3(1-58), L3(1-59), L3(1-60), L3(1-61), L3(1-62), L3(1-63), L3(1-64), L3(1-65), L3(1-66), L3(1-67), L3(1-68), L3(1-69), L3(1-70), L3(1-71), L3(1-72), L3(1-73), L3(1-74), L3(1-75), L3(1-76), L3(1-77), L3(1-78), L3(1-79), L3(1-80), L3(1-81), L3(1-82), L3(1-83), L3(1-84), L3(1-85), L3(1-86), L3(1-87), L3(1-88), L3(1-89), L3(1-90), L3(1-91), L3(1-92), L3(1-93), L3(1-94), L3(1-95), L3(1-96), L3(1-97), L3(1-98) and L3(1-99), respectively. L3(1-99) is also referred to herein, particularly in the working examples, as “L3Δ1-99” or L3Δ99”. By way of specific example, as shown in FIG. 11B, L3(1-99) in yeast has an amino acid (and corresponding nucleotide) sequence as set forth below.
Yeast L3(1-99):

- +1
- MSHRKYEAPRHGHLGFLPRKRAASIRARVKAFPKDDRSKPVALTSFLGYKAGMTTI VRDLDRPGSKFHKREVVEAVTVVDTPPVVVVGVVGYVETPRGL+99)
- Yeast L3 (1-99) nucleotide
  +1
- ATGTCTCACAGAAAGTACGAAGCACCACGTCACGGTCATTTAGGTTTCTTGCCAAGA AAG
- AGAGCTGCCTCCATCAGAGCTAGAGTTAAGGCTTTTCCAAAGGATGACAGATCCAA GCCA
- GTTGCTCTAACTTCCTTCTTGGGTTACAAGGCTGGTATGACCACCATTGTCAGAGA TTTG
- GACAGACCAGGTTCTAAGTTCCACAAGCGTGAAGTTGTCGAAGCTGTCACCGTTGT TGAC
- ACTCCACCAGTTGTCGTTGTTGGTGTTGTCGGTTACGTCGAAACCCCAAGAGGTTTG A+298. Thus, the amino acid sequences corresponding to yeast L3(1-21) to L3(1-99) may be easily ascertained, as follows: L3(1-21) MSHRKYEAPRHGHLGFLPRKR; L(1-22) MSHRKYEAPRHGHLGFLPRKRA; L3(1-23) MSHRKYEAPRHGHLGFLPRKRAA; L3(1-24 MSHRKYEAPRHGHLGFLPRKRAAS; L3(1-25) MSHRKYEAPRHGHLGFLPRKRAASI, etc.

It is readily apparent that although the L3 proteins illustrated in FIGS. 11A and B possess a high level of sequence similarity, there are differences in various first 99 residues. Such differences occur at positions 6 (F or Y), 8 (H or A), 11 (H or T), 13 (S or H), 23 (N, S or A), 24 (R or S), 25 (H or I), 27 (G or A), 28 (K or R), 29 (V or C), 31 (A or S), 37(Q, P, T, R or K), 38 (T, N, or S), 41 (C or V), 42 (K, R, A, or H), 43 (F or L), 45 (A or S), 47 (M or L), 55 (H or T), 60 (V or L), 61 (E or D), 62 (K or R), 67, (L, F or M), 70 (K or R), 72 (T or V), 73 (C or V), 75 (A or L), 78 (I or V), 79 (I or V), 80 (E or D), 83 (A or P), 84 (M, V or I), 85 (V or I), 86 (V or I), 91 (A or G) and 94 (K or E). Thus, L3(1-21)-L3(1-99) from yeast, as well as from rice, Arabidopsis, and tobacco L3 proteins illustrated in FIG. 11B constitute specific examples of polypeptides of the present invention. Yet other polypeptides of the present invention may be based on amino acid sequences of L3 proteins not specifically disclosed herein in accordance by resort to the literature or standard techniques (e.g., probing genomic or cDNA libraries with probes corresponding to conserved regions of L3 proteins as shown in FIGS. 11A and B.
In certain embodiments, depending on the nature of the restriction enzyme and the vector, use of L3(1-99) will result in expression of L3 (1-100). This would occur, for instance, when L3 DNA starting material is produced by treating yeast L3 DNA with BglII, inserting the DNA encoding L3(1-99) into a vector with a BamHI or BglII site, and then transforming a cell with the vector. In this case, an “R” codon would be added. Since native yeast L3 contains an R at residue 100, the corresponding expression product would be L3 (1-100). Thus, the polypeptides of the present invention include L3 (1-100). L3(1-100). is also referred to herein, particularly in the working examples, as “L3Δ100” or L3Δ1-100”.
The present invention also includes analogs of the L3 N-terminal polypeptides. In general, analogs differ from the native sequences of the L3 N-terminal polypeptides In general, analogs of the polypeptides in terms of amino acid alterations or modifications, substitutions, insertions or deletions, and preferably in terms of one or more conservative or non-conservative amino acid substitutions. In preferred embodiments, the analogs differ in terms of one or more conservative amino acid substitutions, particularly in any of amino acids 1-21, which as illustrated in FIG. 25, the mRNA of which forms a secondary stem loop structure. Referring again to FIG. 11B, L3(1-21) from yeast may have an “H” residue substituted for the “A” residue at position 8. There is relatively more latitude for analogs of L3 N-terminal polypeptides that contain additional amino acids, i.e., having from at least the first 22 to about 99 amino acids, and amino acids substitutions may be conservative or non-conservative in nature. Analogs of the present invention also possess the desired properties, e.g., providing increased resistance to toxins (e.g., trichodermin toxins) that target eurcaryotic L3 ribosomal proteins when present in a given host, and when present along with an RIP that targets a eurcaryotic L3 ribosomal protein, serves to reduce toxicity associated with the RIP.
It is also well understood by the skilled artisan that there is a limit to the number of changes that may be made within a portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity of function. There are several general guidelines to consider in determining whether a given change in an amino acid sequence will result in an unacceptable change in the desired activity. First, tolerance to change increases with the length of the peptide or protein. It is also well understood that where certain residues are shown to be particularly important to the biological or structural properties of a polyamino acid, such residues may not generally be exchanged. Amino acid substitutions are generally based on the relative similarity of the various types of amino acid side-chains, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents. To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, which are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). The importance of the hydropathic amino acid index in conferring interactive biological function on a protein, and correspondingly a polyamino acid, is generally understood in the art. It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within 2 is preferred, those which are within approximately 1 are particularly preferred, and those within approximately 0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. As disclosed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±I); serine 5 (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. In some embodiments, analogs of the polypeptides contain amino acid substitutions in the positions where as shown in FIGS. 11A and B, variability exists.
Expression of a transgene encoding the polypeptide or analog in a plant (transformed with the transgene) confers increased resistance to toxins and antibiotics that target eurcaryotic ribosomal protein L3, and which are toxic to and cause disease in plants. Such toxins include trichothecene mycotoxins (also referred to as sesquiterpene antibiotics) such as fusarenon X, trichothecin, verrucarin A, nivalenol, trichodermin, T-2 toxin, diacetoxyscirpenol (DAS) and deoxynivalenol (DON). Trichothecenes are a class of toxic, sesquiterpenoid secondary metabolites that are produced mainly by plant pathogenic fungi (Fernandez-Lobato et al., Biochem. J. 267:709-713 (1990)). Fusarium graminearum and F. culmorum produce the trichothecene mycotoxins deoxynivalenol (DON), also known as vomitoxin, which contaminates a substantial. portion of agricultural crops such as wheat, barley and maize, and 4,15-diacetoxyscirpenol (DAS). The resistance to these toxins and the diseases they cause will be greater than the level of resistance exhibited by a non-transgenic control plant. Transgenic plants of the present invention particularly show greater resistance to at least two trichothecene mycotoxins, DON and DAS (and thus the diseases they cause), than a non-transgenic control plant of the same species. Resistance may also be about equal to or greater than a transgenic control plant that expresses an exogenous transgene that encodes wild type yeast L3 (as shown in FIG. 12). This determination can be made in accordance with the protocols described in the working examples.
Thus, plants transformed with nucleic acids encoding L3 N-terminal polypeptide or an analog thereof exhibit increased resistance to diseases and infections or infestations caused or mediated by trichothecene mycotoxins, particularly DON and DAS. Thus, in general, transgenic plants of the present invention will exhibit resistance against diseases caused by Fusarium infection (e.g., root rot of bean, dry rot of potatoes, head blight (scab) in wheat), Pythium (one of the causes of seed rot, seedling damping off and root rot), Phytophthora (the cause of late blight of potato and of root rots, and blights of many other plants), Bremia, Peronospora, Plasmopara, Pseudoperonospora and Sclerospora (causing downy mildews), Erysiphe graminis (causing powdery mildew of cereals and grasses), Verticillium (causing vascular wilts of vegetables, flowers, crop plants and trees), Rhizoctonia (causing damping off disease of many plants and brown patch disease of turfgrasses), Cochliobolus (causing root and foot rot, and also blight of cereals and grasses), Giberella (causing seedling blight and foot or stalk rot of corn and small grains), Gaeumannomyces (causing the take-all and whiteheads disease of cereals), Schlerotinia (causing crown rots and blights of flowers and vegetables and dollar spot disease of turfgrasses), Puccinia (causing the stem rust of wheat and other small grains), Ustilago (causing corn smut), Magnaporthae (causing summer patch of turfgrasses), and Schlerotium (causing southern blight of turfgrasses). Other important fungal diseases include those caused by Cercospora, Septoria, Mycosphoerella, Glomerella, Colletotrichum, Helminthosporium, Alterneria, Botrytis, Cladosporium and Aspergillus. Since the L3 N-terminal polypeptides also affect viral frameshifting, the plants might also exhibit resistance to certain plant viruses e.g., barley yellow dwarf virus, potato leafroll virus, citrus tristeza virus and beet western yellows virus.
Nucleic acids encoding the L3 N-terminal polypeptides and analogs thereof of the present invention can be prepared in accordance with standard procedures such as cloning or synthetic synthesis. In addition to the nucleotide sequence for L3(1-99) from yeast that is set forth above, representative nucleic acid sequences are contained in FIGS. 11A and B, 12, 13 and 14. That is, the portion of the full-length polynucleotide that encodes the L3 N-terminal polypeptide may be easily designed by introducing a “stop” codon immediately after the C-terminal amino acid residue of the desired N-terminal polypeptide. For example, a representative polynucleotide encoding yeast L3(1-21) may have a sequence as follows: 5′ atgtctcaca gaaagtacga agcaccacgt cacggtcatt taggtttctt gccaagaaag agataa 3′. Allelic versions of the sequences and homologous sequences encoding L3 proteins may be found in eurcaryotic cell types other than plants such as humans and rodents (e.g., rats and mice). For example, nucleic acid sequences encoding L3 proteins are obtainable from a variety of publicly accessible web sites related to various genome projects. Yet other nucleic acids having sequences encoding the L3 N-terminal polypeptides and analogs thereof may be prepared based on considerations of the degeneracy of the code and the codon preference of a given host cell, e.g., plant or other eucaryotic cell such as an animal or human cell, in which the polynucleotide is to be expressed.
In other embodiments of the present invention, transgenic plants containing nucleic acids encoding L3 N-terminal polypeptides and analogs also contain exogenous nucleic acids encoding a ribosome inactivating protein (RIP) such as a Pokeweed Anti-viral Protein (PAP) protein. PAP proteins include wild-type PAP, variant PAP (i.e. PAP-v, which differs from wild-type PAP in terms of the double amino acid substitutions, Leu20Arg and Tyr49His), PAP mutants having reduced toxicity (e.g., phytotoxicity) compared to wild-type PAP or PAP-v, and which have intact catalytic active site amino acid residues (Glu176 and Arg179), and PAP II proteins. Wild-type PAP, PAP-v and various PAP mutants are described in U.S. Pat. Nos. 5,756,322 and 5,880,322. Aside from the differences in the codons resulting in the two amino acid changes, the third change in the PAP-v nucleotide sequence (i.e., TCG →TCA for the first occurring Ser in the signal sequence) has no effect on the amino acid sequence. PAP II is reported in Poyet, et al., FEBS Letters 347:268-272 (1994). The term “PAP-II,” is inclusive of the 310 amino acid polypeptide disclosed in Poyet, et al., the 285-amino acid polypeptide containing amino acid residues 26-310 of said polypeptide (i.e., “PAP II (1-285)”) and which excludes the N-terminal twenty-five (25)-amino acid signal sequence and analogs of PAP II (1-285) such as fragments and mutants (e.g., amino acid additions, deletions and substitutions) that substantially retain PAP II anti-viral and anti-fungal properties and exhibit reduced phytotoxicity compared to PAP. PAP II and PAP II mutants are described in WO 99/60843, published Dec. 2, 1999. Polynucleotide and corresponding amino acid sequences of a wild-type PAP and PAP II are illustrated in FIGS. 15 and 16, respectively. Other RIPs useful in the present invention include ricin toxin and Shiga toxin. Nucleotide and corresponding amino acid sequences of ricin and two different Shiga toxins are illustrated in FIGS. 17, 18 and 19, respectively. Other RIPs include but are not limited to trichosanthin, saporin, mirabilis antiviral protein, momordin, dianthin and gelonin.
Transgenic plants expressing exogenous nucleic acids encoding a RIP protein will exhibit increased resistance to plant fungi that produce toxins that target eucaryotic L3 ribosomal proteins. Thus, expression of an RIP may provide increased resistance to diseases caused by fungi such as Fusarium infection (e.g., root rot of bean, dry rot of potatoes, head blight (scab) in wheat), Pythium (one of the causes of seed rot, seedling damping off and root rot), Phytophthora (the cause of late blight of potato and of root rots, and blights of many other plants), Bremia, Peronospora, Plasmopara, Pseudoperonospora and Sclerospora (causing downy mildews), Erysiphe graminis (causing powdery mildew of cereals and grasses), Verticillium (causing vascular wilts of vegetables, flowers, crop plants and trees), Rhizoctonia (causing damping off disease of many plants and brown patch disease of turfgrasses), Cochliobolus (causing root and foot rot, and also blight of cereals and grasses), Giberella (causing seedling blight and foot or stalk rot of corn and small grains), Gaeumannomyces (causing the take-all and whiteheads disease of cereals), Schlerotinia (causing crown rots and blights of flowers and vegetables and dollar spot disease of turfgrasses), Puccinia (causing the stem rust of wheat and other small grains), Ustilago (causing corn smut), Magnaporthae (causing summer patch of turfgrasses), and Schlerotium (causing southern blight of turfgrasses). Other important fungal diseases include those caused by Cercospora, Septoria, Mycosphoerella, Glomerella, Colletotrichum, Helminthosporium, Alterneria, Botrytis, Cladosporium and Aspergillus.
RIPs might also provide increased resistance to viruses including but not limited to RNA viruses e.g., citrus tristeza virus, potexviruses such as (PVX, potato virus X), potyvirus (PVY), cucumber mosaic virus (CMV), tobacco mosaic viruses (TMV), barley yellow dwarf virus (BYDV), wheat streak mosaic virus, potato leaf roll virus (PLRV), plumpox virus, watermelon mosaic virus, zucchini yellow mosaic virus, papaya ringspot virus, beet western yellow virus, soybean dwarf virus, carrot read leaf virus and DNA plant viruses such as tomato yellow leaf curl virus. See also Lodge, et al., PNAS USA 90:7089-7093 (1993); Tomlinson, et al., J. Gen. Virol. 22:225-232 (1974); and Chen, et al., Plant Pathol. 40:612-620 (1991).
Since the RIPs of the present invention target L3 ribosomal proteins and as a result, are toxic to eucaryotic cells, the co-expression or transcription of a nucleic acid encoding a L3 N-terminal polypeptide will reduce such toxicity, relative to a control plant expressing an RIP transgene but which does not contain the L3 N-terminal polypeptide-encoding transgene.
Nucleic acids encoding L3 N-terminal polypeptides and analogs thereof, and in some embodiments, a PAP protein, can be introduced and expressed in a variety of plants including higher plants such as flowering plants, including both monocots and dicots, and preferably crop plants and cereal crop plants, in accordance with standard transformation techniques for the plant type of interest. See U.S. Pat. No. 5,675,322 (and references cited therein), Horsch, et al., Science 227:1229-1231 (1985); and Hartman, et al., Bio/technology 12:919-923 (1994). Preparation of expression cassettes and vectors for the introduction of the L3 nucleic acid into plant cells, protoplasts, whole plants and plant parts are also well known in the art. In general, any cloning vector can be used; the choice will reflect the host in which the final transformation is made and the manner in which transformation is accomplished. Vectors suitable for Agrobacterium transformation typically carry at least one T-DNA border sequence. These include vectors such as pBIN19 (Bevan, Nucleic Acids Research 12:8711-8721 (1984)) and pCIB200 (EP 0 332 104). Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors that contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. For example, pCIB3064 is a pUC-derived vector suitable for the direct gene transfer technique in combination with selection by the herbicide basta (or phosphinothricin), as described, for example, in WO 93/07278 and Koziel et al., Biotechnology 11:194-200 (1993).
For the transformation of plants, the cloning vector can further comprise a 3′ untranslated region. A 3′ untranslated region refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by directing the addition of polyadenylic acid tracks to the 3′ end of the mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5′ AATAAA-3′ although variations are not uncommon. Examples of suitable 3′ regions are the 3′ transcribed non-translated regions containing a polyadenylation signal of Agrobacterium tumor inducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene) and plant genes such as the soybean storage protein genes and the small subunit of the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene. The 3′ untranslated region from the modified peptidyl transferase gene of the present construct can be used for expression in plants, without any additional region. The vectors of the present invention can also contain a suitable promoter functional in the host. In the case of monocot transformation, for example, preferred promoters include the CaMV 35S promoter, ubiquitin promoter, and the actin promoter. For dicots, mention may be made of the CaMV 35S promoter, the enhanced CaMV 35S promoter, the L3 promoter or the FMV (figwort mosaic virus) promoter. In the embodiments of the present invention that entail transformation of a plant with a nucleic acid encoding a PAP protein, it is preferred to place the nucleic acids encoding L3Δ or analog thereof, and PAP under the control of separate regulatory units and polyadenylation sites (i.e., to prepare polycistronic rather than monocistronic expression cassettes. An expression cassette containing the nucleic acid(s) of the present invention may be inserted into a plant transformation vector by standard recombinant DNA methods. Alternatively, some or all of the elements of the expression cassette may be present in the vector, and any remaining elements may be added to the vector as necessary.
Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques which do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described in Paszkowski et al., EMBO J 3:2717-2722 (1984), Potrykis et al., Mol. Gen. Genet. 199:169-177 (1985), Reich et al., Biotechnology 4:1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques.
Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons (dicots) because of its high efficiency of transformation and its broad utility with many different species. The many crop species which are routinely transformable by Agrobacterium include tobacco, tomato, sunflower, cotton, oilseed rape, potato, soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432 (tomato), WO 87/07299 (Brassica), U.S. Pat. No. 4,795,855 (poplar)). Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident plasmid or chromosomally (e.g. strain CIB542 for pCIB200 (Uknes et al. Plant Cell 5:159-169 (1993)). The transfer of the recombinant binary vector, to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Höfgen & Willmitzer, Nucl. Acids Res. 16:9877 (1988)). Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols known in the art. Transformed tissue is regenerated on selectable medium carrying an antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
Preferred transformation techniques for monocots include direct gene transfer into protoplasts using PEG or electroporation techniques and particle bombardment into callus tissue. Transformation can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with this invention. Co-transformation may have the advantages of avoiding complex vector construction and generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al., Biotechnology 4:1093-1096 (1986)).
Published European Patent Applications EP O 292 435 and EP O 392 225, and PCT application WO 93/07278 describe techniques for the preparation of callus and protoplasts of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordeon-Kamm et al., Plant Cell 2:603-618 (1990), and Fromm et al., Biotechnology 11:194-200 (1993), describe techniques for the transformation of elite inbred lines of maize by particle bombardment.
Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-types (Zhange et al., Plant Cell Rep. 7:739-384 (1988); Shimamoto et al. Nature 338:274-277 (1989); Datta et al. Biotechnology 8:736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9:957-962 (1991)).
Patent Application EP 0 332 581 described techniques for the generation, transformation and regeneration of Pooideae protoplasts. Furthermore, wheat transformation has been described by Vasil et al., Biotechnology 10:667-674 (1992), using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al., Biotechnology 11:1553-1558 (1993), and Weeks et al., Plant Physiol. 102:1077-1084 (1993), using particle bombardment of immature embryos and immature embryo-derived callus.
Transformation of monocot cells such as Zea mays can be achieved by bringing the monocot cells into contact with a multiplicity of needle-like bodies on which these cells may be impaled, causing a rupture in the cell wall thereby allowing entry of transforming DNA into the cells. See U.S. Pat. No. 5,302,523. Transformation techniques applicable to both monocots and dicots are also disclosed in the following U.S. Pat. Nos.: 5,240,855 (particle gun); 5,204,253 (cold gas shock accelerated microprojectiles); 5,179,022 (biolistic apparatus); 4,743,548 and 5,114,854 (microinjection); and 5,149,655 5,120,657 (accelerated particle mediated transformation); 5,066,587 (gas driven microprojectile accelerator); 5,015,580 (particle-mediated transformation of soy bean plants); 5,013,660 (laser beam-mediated transformation); and 4,849,355 and 4,663,292. See also section 6.2.7 of U.S. Pat. No. 6,720,014, which describes transformation of monocots.
To aid in identification of transformed cells, the vectors may further contain a selectable marker (e.g., a reporter gene). For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformations include the nptII gene which confers resistance to kanamycin (Messing and Vierra, Gene 19:259-268 (1982); Bevan et al., Nature 304: 184-187 (1983)), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res. 18:1062 (1990); Spencer et al., Theor. Appl. Genet. 79:625-631 (1990)), the hph gene which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol. Cell Biol. 4:2929-2931)), and the dhfr gene, which confers resistance to methotrexate). Selection of successful transformation events may also be accomplished using a CGS gene as a reporter. See, e.g., WO 00/55303, published Sep. 21, 2000, to Tumer, et al.
The thus-transformed plant cells or plant tissue are then grown into full plants in accordance with standard techniques. Transgenic seed can be obtained from transgenic flowering plants in accordance with standard techniques. Likewise, non-flowering plants such as potato and sugar beets can be propagated by a variety of known procedures. See, e.g., Newell, et al. Plant Cell Rep. 10:30-34 (1991) (disclosing potato transformation by stem culture).
Techniques for transforming plants and regenerating plants are also disclosed in Lanfranco, Riv. Biol. 96(1):31-54 (2003); Job, Biochimie 84(11):1105-10 (2002); Taylor, et al., DNA Cell Biol. 21(12):963-77 (2002); Rakoczy-Trojanowska, Cell Mol. Biol. Lett. 7(3):849-58 (2002); Ow, Plant Mol. Biol. 48(1-2):183-200 (2002); Boch, J. Mol. Biol. 312(3):425-38 (2001); Casas, et al., Plant Breed Rev. 13:235-64 (1995); Newell, Mol. Biotechnol. 16(1):53-65 (2000); Bogorad, Trends Biotechnol. 18(6):257-63 (2000); Komari, et al., Curr. Opin. Plant Biol. 1(2):161-5 (1998); Dempsey, et al., Trends Microbiol. 6(2):54-61 (1998); Oard, Biotechnol. Adv. 9(1):1-11 (1991); and Holm, et al., Transgenic Res. 9(1):21-32 (2000). Specific examples of transformation in potato, rice, corn, barley and wheat are disclosed in Garg, et al., PNAS 99(25):15898-15903 (2002); Cheng, et al., PNAS 95:2767-2772 (1998); Wakita, et al., Genes Genet. Syst. 73:219-226 (1998); Lin, et al., PNAS 100(10):5962-5967 (2003); Breitler, et al., Theor. Appl. Genet. 104(4):709-719 (2002); Miller, et al., Transgenic Res. 11(4):381-96 (2002); Aulinger, et al., Plant Cell Rep. 21(6):585-91 (2003); Romano, et al., Transgenic Res. 12(4):461-73 (2003); de Vetten, et al., Nat. Biotechnol. 21(4):439-42 (2003); Park, et al., Protein Expr. Purif. 25(1):160-5 (2002); Sawahel, Cell Mol. Biol. Lett. 7(1):19-29 (2002); Frame, et al., Plant Physiol. 129:13-22 (2002); Hansen, et al., PNAS 93:14978-14983 (1996); Grosset, et al., Plant Mol. Biol. 34(2):331-8 (1997); Patnaik, et al., BMC Plant Biol. 3:1-11 (2003); Rasco-Gaunt, et al., J. Exp. Botany 52(357):865-874 (2001); Amoah, et al., J. Exp. Botany 52 (358):1135-1142 (2001); and Cheng, et al., Plant Physiol. 115:971-80 (1997).
Representative examples of transgenic plants of the present invention include maize, tomato, turfgrass, asparagus, papaya, sunflower, rye, oats, millet, beans, ginger, lotus, bamboo, potato, rice, peanut, barley, malt, wheat, alfalfa, soybean, oat, eggplant, squash, onion, broccoli, sugarcane, sugar beet, beets, apples, oranges, grapefruit, pear, plum, peach, pineapple, grape, rose, carnation, daisy, tulip, Douglas fir, cedar, white pine, scotch pine, spruce, peas, cotton, flax, canola, ornamentals and coffee.
In less preferred embodiments, the L3 polypeptides (with or without a RIP) may be applied directly to a plant or part thereof, in order to achieve increased resistance to fungal diseases.
The L3 N-terminal polypeptides or analogs thereof of the present invention also have pharmaceutical uses. For example, they may be introduced into other eukaryotic cells e.g., human or animal cells, such as by way of administration to an animal or human, to reduce the cytotoxic effect of various pharmaceutical and therapeutic agents that contain ribosome inhibitory proteins (RIP) such as PAP proteins, and particularly wild-type PAP. They are also useful in providing increased resistance to fungal infection, e.g., infections caused or medited by trichothecene mycotoxins, particularly DON and DAS, which are also toxic to human and animal cells. RIPs such as PAP are used to treat viral infections such as HIV (which tends to cause toxicity to host cells), and as targeted cytotoxic agents e.g., to treat cancers (in which case, there is some undesirable non-specific cytotoxicity). In the latter and/or former cases, the RIP may be administered in conjugated form to a ligand that recognizes a receptor on a target cell surface. See, e.g., U.S. Pat. Nos. 5,919,457 and 6,146,628. While not intending to be bound by any particular theory of operation, Applicants believe that the cytotoxic effect of these RIPs such as PAP proteins is mediated by binding to endogenous L3 proteins in the cell. Embodiments of the present invention include co-administration of a composition containing an L3 N-terminal polypeptide or analog thereof along with the RIP, or administration of separate compositions containing the L3 N-terminal polypeptide or analog thereof and the RIP, to an animal (e.g., a mammal such as a human) in need thereof. By co-administration, it is meant administration of the L3 N-terminal polypeptide or analog thereof suitably prior to, simultaneously with or after the administration of the RIP such that the L3 will be present in the cell to reduce to cytotoxic effect of the RIP on various cells, particlularly non-diseased cells. The compositions may include a pharmaceutically or veterinary acceptable carrier and at least one other pharmaceutical or veterinary acceptable excipient. Dosage amounts and modes of administration may be determined and optimized based on a consideration of factors such as the weight, age and overall health of the human or animal and severity of the infection, and in accordance with standard procedures in the field.
The L3 N-terminal polypeptides may be produced recombinantly or synthetically, preferably recombinantly, by standard techniques. Aside from production and isolation of the polypeptides from transformed plant hosts (as described above), the polypeptides may be recombinantly produced in bacterial cells, e.g., E. coli, Streptomyces, Bacillus subtilis, fungal cells such as yeast, insect cells, and animal cells. As in the case of plants, choice of appropriate vectors, promoters and other 5′ and 3′ regulatory flanking sequences, e.g., origin of replication, translation initiation and termination, leader sequence, marker genes, methods of introducing the DNAs encoding the polypeptides into the host cell, culturing, isolation and purification techniques, are all well known in the art. Cell-free translation systems may also be employed.
In embodiments of the present invention intended to provide a greater degree of resistance to trichothecene mycotoxins, the human or animal may be administered a composition comprising an effective amount of the L3 N-terminal polypeptide or analog thereof. Here again, dosage amounts and modes of administration may be determined and optimized based on a consideration of factors such as the weight, age and overall health of the human or animal and severity of the infection, and in accordance with standard procedures in the field. This effect may also be achieved by generation of a transformed human or animal (e.g., a non-human animal) containing a nucleic acid encoding L3 N-terminal polypeptide or an analog thereof. The transformed animals are more tolerant to at least the two trichothecene mycotoxins DON and DAS relative to the same species of animal that is not transformed with the nucleic acid. Techniques for generating transgenic animals have been developed and optimized in mice (Hogan et al., 1986, Manipulation of the mouse embryo: a laboratory manual. Cold Spring Harbour Laboratory Press: New York), sheep (Wright et al., 1991, Bio-technology NY 9: 831-834), goats (Ebert and Schindler, 1993, Teriogenology, 39: 121-135) and pigs (Rexroad and Purcel, 1988, Proc. 11th Int. Congress of Animal Reproduction and Artificial Insem. 5: 29-35)). In general, such methods are based upon pronuclear micro-injection of fertilized zygotes taken from super-ovulated female animals. Zygote pronuclei are micro-injected with several hundred copies of the novel gene construct, and then transferred to recipient females for the remainder of the gestation period. Confirmation of transgene integration is by Southern hybridization of somatic tissues taken from the offspring, and analysis of gene product or gene function. Suitable animal hosts include any non-human animal that has, at least as a part of its diet, the food grains obtained from plants suspectible to infection by fungi that produce DON and DAS. These animals would include but are not limited to livestock animals, bovines and equines. Examples of specific animals are cows, sheep, goats, pigs, horses, poultry, and rodents such as rats and mice. Methods of introducing transgenes into animal cells and the preparation of transgenic non-human animals are described in section 6.3.17 of U.S. Pat. No. 6,720,014, that specific disclosure,of which is hereby incorporated herein by reference.
The present invention is further:described in terms of the following non-limiting examples. Unless otherwise indicated, all parts and percentages are on a weight-by-weight basis.
In the first few examples that follow, Applicants have demonstrated, by a sensitive seed germination assay, that over-expression of L3Δ100 in transgenic tobacco plants conferred resistance to the trichothecene fungal toxins DON and DAS, and that in another embodiment of the present invention, namely transgenic tobacco plants transformed with both the yeast L3Δ100 gene and wild type PAP, showed the greatest resistance to DON, compared to prior art plants expressing wild-type L3 and PAP.
Specifically, Applicants generated transgenic tobacco plants expressing either the wild type yeast L3 gene or its N-terminal 100 amino acids (L3Δ100) alone or together with pokeweed antiviral protein (PAP) gene. In these first examples, gene expression of L3Δ99 resulted in the polypeptide L3Δ100. The results indicated that expression of L3Δ100 in transgenic tobacco plants conferred greater resistance to trichothecene mycotoxins, DON and DAS (4,15-diacetoxyscirpenol) than did wild type yeast L3. The combination of L3 and PAP in transgenic plants not only rendered most of the plants normal looking by reducing the toxicity of PAP, but also conferred significant resistance to both fungal toxins and tobacco mosaic virus. The combination of L3Δ100 and PAP resulted in all plants looking normal and high level of resistance to the fungal toxins. Analysis of ribosomes of the transgenic plants indicated that although the toxicity of PAP was reduced in plants containing wild type yeast L3 together with PAP, ribosomes were depurinated. In contrast, ribosome depurination was abolished in transgenic plants containing L3Δ100 and PAP, leading to elimination of PAP toxicity and regeneration of normal looking plants that expressed high levels of PAP. Expression of the endogenous tobacco ribosomal protein genes, L3A and L3B, was up regulated in all the transgenic tobacco lines.
Expression of the Yeast Wild Type L3, and the L3Δ Alone or Combined with PAP in Transgenic Tobacco Plants
Yeast wild type L3 gene, and the L3Δ100 (the N-terminal 100 amino acids of L3) were cloned singly into a plant expression vector resulting in constructs NT250, and NT252 (FIG. 1). Yeast L3, and L3Δ100 combined with wild type PAP were cloned separately into the plant expression vector generating constructs NT243, and NT245. The expression of the L3 genes and PAP gene were driven by CaMV 35S promoter individually. Kanamycin resistance gene (NPTII) served as the selection marker. All constructs were transformed into tobacco, Nicotiana tabacum cv Samsun NN or nn via Agrobacterium-mediated transformation (Lodge et al. Proc. Natl. Acad. Sci. USA 90:7089-7093 (1993)).
ELISA assays were performed on the regenerated plants to identify NPTII-positive transgenic plants (Agdia). PCR reactions with L3 and PAP specific primers were carried out to determine the presence of these genes in the transgenic plants. To confirm the identities of L3Δ100 PCR products, they were electrophoresed in agarose gel and transferred to Stratagene Duralose-UV membrane by capillary action overnight. The membrane was probed with ³²P-labeled L3Δ cDNA and Southern blot analysis was performed. Western blot analysis with PAP specific antiserum or L3 specific monoclonal antibody (kindly provided by J. Warner) was used to determine the expression levels of these two genes in the transgenic plants.
Ribosomal RNA Depurination Assay by Primer Extension
To test the viability of PAP transgene in NT243 (L3+PAP) and NT245 (L3Δ100+PAP) transgenic tobacco plants, ribosomes from the leaves were isolated as described (Hudak et al., J. Biol. Chem. 274:8359-3864 (1999)). Ribosomal RNAs were isolated from the -ribosomes and used as the substrate in primer extension analysis. Purified ribosomal RNAs were incubated with a 5′ ³²P end-labelled oligonucleotide (5′-AGGCGTTCAGTCATAATCC-3′) complementary to the 3′-end of the plant large rRNA. Primer extension was performed by reverse transcription with the GibcoBRL SuperscriptII reverse transcriptase and the reaction was precipitated in 100% ethanol and resuspended in formamide loading buffer. The primer extension products were separated on a 6% polyacrylamide/urea denaturing gel and visualized by autoradiography.
Fungal Toxin Resistance Assay
To determine the resistance of transgenic tobacco plants to fungal toxins, a sensitive seed germination assay was adopted. MS medium (cat. # 11117-066 GibcoBRL) with 30 g/l sucrose containing DAS or DON was solidified in petri dishes (100×15 mm, VWR) with agar. Tobacco seeds were surface sterilized with 50% bleach containing 0.1% Triton-X-100 for 10 min., rinsed with sterile water three times and distributed onto the petri dishes with MS medium. Dishes were incubated under 16/8 hr light/dark cycle at 25° C. The germination of the seeds and the growth of the seedlings were recorded, and the root length was measured after six weeks.
Virus Resistance Test
To assess the resistance of transgenic tobacco plants to plant viruses, two leaves were sprayed with carborandum and mechanically inoculated with 2 μg/ml tobacco mosaic virus (TMV) in phosphate buffered saline (PBS) solution. The plants were grown in the same conditions as above. Subsequently the local lesion numbers were recorded 5 days after the inoculation.
Northern Blot to Detect the Expression of PR Proteins
Pathogenesis related (PR) proteins have been associated with plant defense mechanisms (Ryals et al., Plant Cell 8:1809-1819 (1996); and Zoubenko et al., Nature Biotechnol. 15:992-996 (1997)). Total RNAs were extracted from tobacco leaves by TRIzol® Reagent (Invitrogen) according to the manufacturer. They were electrophoresed in denaturing formamide agarose gel and transferred to Stratagene Duralose-UV membrane by capillary action overnight. Tobacco basic chitinase cDNA was labelled with ³²P-dCTP by Pharmacia Ready-To-Go™ (#27-9240-01). DNA Labelling Beads were used to probe the expression of basic chitinase.
Real-Time Quantitative PCR
Total RNA was isolated from tobacco leaves using TRIzol® Reagent (Invitrogen) according to the manufacturer. SuperScript™ reverse transcriptase (Invitrogen) and oligo d(T) were used to produce the first-strand cDNA which was then applied to real-time PCR using gene-specific primers with ABI PRISM 7000SDS (Applied Biosystems). The relative expression levels of genes were calculated as ddCT (Livak and Schmitgen, 2001) using the expression of tobacco tubulin as an internal control.
Results
Integration and Expression of L3 and PAP in Transgenic Tobacco Plants
Several NPTII positive transgenic tobacco (N. tabacum NN) lines, containing NT250 (L3), and NT252 (L3Δ100) were identified by ELISA for the NPTII gene expression. The integration of L3 was confirmed by PCR (FIG. 2A.). The integration of L3Δ100 was confirmed by Southern blotting of the PCR products with L3Δ100-specific primers (FIG. 2C.). All of these transgenic plants were phenotypically normal and indistinguishable from wild type plants based on their appearance and growth characteristics. However, immunoblot analysis with yeast L3 specific monoclonal antibody revealed undetectable levels of L3 and L3L100 genes.
A total of 12 transgenic tobacco plants (N. tabacum NN) transformed with both wild type (wt) L3 and wt PAP (NT243) were identified by ELISA for NPTII. The transformation frequency, defined as the number of transgenic plants obtained per initial leaf disk times 100, was approximately 24%. The presence of both L3 and PAP genes were confirmed by PCR analysis (FIGS. 2A. and B.). However, immunoblot assay using monoclonal antibodies against yeast L3 did not detect expression of the yeast L3 in transgenic tobacco plants. The immunoblot analysis of the primary (R0) transgenic plants containing NT243 showed varied levels of PAP expression, with NT243-7 and NT243-9 as the highest expressers (FIG. 3A.). Only these two plants showed mottled symptoms on their leaves similar to transgenic plants expressing the toxic variant form of PAP (PAPv, 26139-19) (Lodge et al., Proc. Natl. Acad. Sci. USA 90:7089-7093 (1993)). NT243-7 and NT243-9, however, did not produce any viable seeds. The other plants appeared normal compared to untransformed plants. The PAP expression level in NT243-6 and NT243-8 plants was much lower compared to NT243-7 and NT243-9. None of the other transgenic plants tested showed detectable PAP expression. Immunoblot analysis performed on R2 generation plants from NT243-6 and NT243-8 demonstrated a considerable amount of PAP expression (FIG. 3B.), yet these plants appeared normal.
Several transgenic tobacco plants (N. tabacum nn) were generated with NT245, which contained both L3Δ100 and PAP genes. These plants were all phenotypically indistinquishable from the wild type plants. PCR and Southern blot analysis confirmed the presence of both genes in the transgenic plants (FIGS. 2B. and C.). While Western blot analysis did not reveal detectable levels of L3Δ100, PAP was expressed at high levels in all the plants tested, in contrast to the PAP expression in NT243 plants (FIG. 3C.).
Depurination Assay
Ribosomal RNA depurination assay by primer extension clearly showed that the rRNAs from the R2 plants of NT243 (L3+PAP) were depurinated by the constitutively expressed wild type PAP (FIG. 4, lane 7, NT243-64). However, depurination by wt PAP in these plants did not seem to affect the morphology of the plants or the viability of the seeds. PAP was expressed at very high levels in NT243-7 and NT243-9, which showed mosaic symptoms and did not produce seeds. The symptoms observed on these plants and their inability to produce seed may have been due to the higher level of depurination in these two plants. In contrast to the NT243 lines, rRNA in the R2 plants of NT245 (L3Δ100+PAP) was not depurinated (FIG. 4, lanes 5 and 6). However, when the ribosomes of NT245 R2 plants were isolated and treated with purified PAP in vitro (Hudak et al., J. Biol. Chem. 274:3859-3864 (1999)), the rRNAs were depurinated. These results indicated that the S/R loop was not resistant to depurination by PAP in NT245 lines. While not intending to be bound by any particular theory of operation, Applicants believe that the yeast L3Δ100 gene interacts with PAP, rendering it inactive in terms of depurination, resulting in healthy plants.
Fungal Toxin Resistance Assay
With the sensitive seed germination assay, the optimum concentrations of DON and DAS were determined by plating wild type tobacco seeds on the MS medium containing different concentrations of DON or DAS. Based on this analysis, 1 μM of DAS and 10 μM of DON were selected as the lowest concentrations that would give the best inhibition of wild type tobacco seed growth. Muhitch et al., Plant Science 157:201-207 (2000) have shown that DON is far more inhibitory than DAS toward wheat. When transgenic tobacco seeds were plated on the MS medium containing 1 μM of DAS, NT250 plants transformed with only yeast L3 were highly resistant to this trichothecene fungal toxin (FIG. 5). The resistance level as measured by the average root length of 10 plants, was almost as high as 4-fold compared to wild type tobacco plants. NT243 plants transformed with both L3 and PAP were equally resistant to DAS as NT245 (L3Δ100+PAP) plants, while transgenic tobacco plants expressing PAPv (Lodge et al., Proc. Natl. Acad. Sci. USA 90:7089-7093 (1993) and NT252 expressing L3Δ100 alone were also resistant to DAS but at a relatively lower level.
When transgenic tobacco seeds were plated on the MS medium containing 10 μM of DON, all transgenic plants including PAPv, NT250 (L3), NT252 (L3Δ100), NT243 (L3+PAP) and NT245 (L3Δ100+PAP) exhibited resistance to DON compared to wild type plants (FIG. 6). L3Δ100 plus PAP plants (NT245) showed the greatest resistance to DON, almost as high as 4-fold compared to wild type tobacco plants.
These data showed that yeast L3 or L3Δ100 alone in transgenic tobacco plants conferred considerable resistance to trichothecene Fusarium toxins DAS and DON. The combination of L3Δ100 with PAP conferred better resistance to DON than either gene alone.
Virus Resistance Assay
Five days after inoculation with TMV, the local lesion numbers on the leaves of NT250 (L3), and NT252 (L3Δ100) transgenic plants were similar to the untransformed plants (data not shown) indicating that L3 and L3Δ100 genes alone in transgenic plants did not confer resistance to plant virus. However, FIG. 7 shows that the local lesion numbers of TMV on the R1 plants of NT243 (L3+PAP)-6 and NT243-8 were significantly lower compared to the wild type tobacco. This indicates that the interaction between L3 and PAP significantly reduced the toxicity of PAP in NT243 plants, yet retained the antiviral characteristic of PAP.
ELISA results (data not shown) showed that NT245 (L3Δ100+PAP) R2 plants inoculated with PVY contained almost same amount of virus as the inoculated wild type plants. This seems to suggest that the interaction between the 99 amino acids at N-terminus of yeast L3 and PAP completely abolished the toxicity of PAP as shown by all normal-looking transgenic plants, but did not retain the antiviral activity of PAP as in NT243 (L3+PAP) plants.
Expression of PR Proteins in Transgenic NT243 (L3+PAP) Plants
Northern blot analysis (FIG. 8) revealed that prior to inoculation of TMV, the R1 plants of NT243-6 and NT243-8 did not show any accumulation of tobacco basic chitinase. The basic chitinase level in these transgenic plants was detectable only after they were inoculated with TMV, same as wild type plants. This is contrary to a previous finding in that the expression of PAPv (26139-19 line) constitutively induced the expression of the basic isoform of tobacco PR proteins (Zoubenko et al., Nature Biotechnol. 15:992-996 (1997)). Again, without intending to be bound by theory, this result suggests that the interaction between L3 and PAP seemed to have disrupted the induced disease resistance pathway as in PAPv (26139-19) transgenic plants, and that the viral resistance of NT243 plants may have come from the direct depurination of infecting viruses.
Expression of Tobacco L3A and L3B by Real-Time PCR
The gene expression level of tubulin was assessed and appeared to be relatively constant in all the plants (data not shown). Therefore it was used as an internal control for the calculation of the “fold expression” of transgenic plants compared to wild type tobacco. The real-time quantitative PCR results (FIGS. 9A. and B.) showed that the gene expression levels of L3A and L3B were enhanced in all the transgenic plants including PAPv, NT250 (L3), NT252 (L3Δ100), NT243 (L3+PAP) and NT245 (L3Δ100+PAP) compared to wt plants. PAPv plants displayed the highest level of gene expression in both L3A and L3B. The NT252 lines showed relatively higher elevation of both L3A and L3B gene expression compared to the rest of the transgenic lines. Western blot result of the cytosolic samples with tobacco L3 polyclonal antibody (FIG. 9C.) confirmed the real-time PCR data. These seemed to correlate slightly with the resistance level of these transgenic plants to fungal toxins. As in FIG. 6, PAPv demonstrated higher resistance to DON compared to the other transgenic lines and wild type plants, except NT245 lines. These results suggested that the elevated levels of L3A and L3B in transgenic tobacco plants might provide excessive targets for the fungal toxins, resulting in the resistance to DON and DAS tested.
Discussion
The observation that 12 transgenic NT243 (L3+PAP) tobacco plants were regenerated with a transformation frequency of 24% and the majority of plants appeared normal indicated an amazing difference from previously reported data in which the transformation frequency with wild type PAP (pMON8443) was only 0.7% (Lodge et al., Proc. Natl. Acad. Sci. USA 90:7089-7093 (1993)). This clearly demonstrated that the interaction between L3 and PAP existed at the transgene level in transgenic tobacco plants. In addition, this interaction was exhibited not only between the wild type L3 gene, but also between the truncated L3 with the N-terminal 100 amino acids, because all the transgenic plants containing NT245 (L3Δ100+PAP) appeared indistinguishable from the non-transgenic plants.
The gene expression level of both L3 and L3Δ100 was undetectable at the protein level in transgenic plants expressing L3 alone or together with PAP (NT243/L3+PAP, NT245/L3Δ100+PAP, NT250/L3 and NT252/L3Δ100). However, the expression level of either L3 or L3Δ100 could be detected by real-time PCR (FIG. 10A.). L3Δ100 in NT252. plants showed more than 1000-fold higher level of gene expression relative to the wild type plant. L3 expression levels in NT250 were 100- to 200-fold higher relative to that in wild type plant. Comparatively, the gene expression of L3 and L3Δ100 in NT243 (L3+PAP) and NT245 (L3Δ100+PAP) was much lower, from 25- to 38-fold. The expression level of PAP in NT243 (L3+PAP) coincided with the severity of the mottling symptoms just as PAPv (26139-19) plants, resulting in sterility of the highest expressers (NT243-7 and NT243-9). Depurination assay showed that the low level expression of PAP in NT243-6 and NT243-8 plants still resulted in the disruption of some tobacco ribosomes, but it did not seem to have much effect on the growth of these plants and the production of their seeds. The PAP expression level in NT245 (L3Δ100+PAP) plants, however, was very high in every plant. The PAP gene expression level in NT245-12 was even higher than PAPx, the non-depurinating active site mutant. And all NT245 plants were normal looking and fertile. Depurination assay indicated that the rRNAs of NT245 plants were not depurinated (FIG. 4). It demonstrated that the full-length yeast L3 in NT243 plants at undetectable protein level greatly reduced the toxicity of PAP, while L3Δ100 in NT245 also at undetectable protein level completely abolished the toxicity of PAP. This seemed to indicate the difference between the interaction of L3 and L3Δ100 with PAP and show that L3Δ100 works better than the full length L3 gene. In addition, the effect of L3 and L3Δ100 on PAP seems to be at the transcription level for the L3 and L3Δ100 genes verses the translation level for PAP. L3Δ100 comprises of the first 100 amino acids of yeast ribosomal protein L3. It has been shown to exert a trans-dominant effect on promoting the programmed −1 ribosomal frameshifting of the L-A double-stranded RNA virus and reducing the translation fidelity in yeast (Peltz et al., Mol. Cell Biol. 19(1):384-91 (1999)). A previous study in yeast showed that wild type L3 is required for PAP to bind to ribosomes and depurinate the 25S rRNA (Hudak et al., J. Biol. Chem. 274:3859-3864 (1999)). By studying PAP mutants, domains of PAP that are involved in toxicity to yeast cells have been identified. These domains can be separated from the depurination property of PAP (manuscript submitted). Again, without intending to be bound by theory, Applicants believe that L3 counter-interacts with the toxicity domains of PAP, and this counter-interaction is more specific for L3Δ100 than for L3.
The interaction between L3 and PAP in NT243 also disrupted the induction of one of the tobacco PR proteins, basic chitinase, as was previously observed in PAPv (26139-19) plants (Zoubenko et al., Nature Biotechnol. 15:992-996 (1997)). Therefore, the resistance to TMV of these plants might have resulted from the direct depurination or inhibition of infecting virus by the low level of PAP. The interaction between L3Δ100 and PAP in NT245 resulted in non-depurinating PAP and susceptibility of plants to PVY, although the PAP expression level was very high in these plants as demonstrated by Western blot of the cytosolic extracts (FIG. 3C.), real-time PCR analysis (FIG. 10B.) and Western blot on the ribosomal samples (FIG. 10C.).
The results have shown that all transgenic tobacco plants were resistant to trichothecene fungal toxins DAS and DON, with NT245 (L3Δ100+PAP) as the most resistant lines to DON. To investigate the mechanism of fungal toxin resistance in the transgenic plants, the levels of tobacco ribosomal proteins L3A and L3B, which are the targets for fungal toxins, were analyzed by real-time PCR (FIGS. 9A. and B.). The levels of L3A and L3B in transgenic tobacco plants expressing PAPv were both elevated by 2- to 3-fold compared to wild type plants. The L3A and L3B in NT250 (L3), NT252 (L3Δ100) and NT245 (L3Δ100+PAP) were elevated at much lesser levels, ranging from 0.5- to 2-fold. Again, without intending to be bound by theory, Applicants postulate that the fungal toxin resistance in 26139-19, NT250, NT252, and NT245 may have resulted from the elevated levels of L3A and L3B, which provided excessive targets for toxins and henceforth overcame the toxic effects. The levels of L3A and L3B might have been elevated by wild type L3, L3Δ100 and PAPv genes. This elevation might have been at the post-transcriptional level because these three genes were hardly detectable at the protein level in NT250 (L3), NT252 (L3Δ100), NT243 (L3+PAP), NT245 (L3Δ100+PAP) and PAPv plants. In addition, NT245 plants demonstrated the highest fungal toxin resistance although the L3A and L3B levels were not as high as they were in PAPv or in NT252-11 (FIGS. 9A. and B.). Again, without intending to be bound by theory, Applicants hypothesize that in these plants, both yeast L3Δ100 and wt PAP bound to the tobacco ribosomes in a way that the ribosomes were shielded from the fungal toxins.
This study has shown that NT243 expressing both low levels of wild type yeast L3 and wild type PAP conferred resistance to fungal toxins DAS and DON and TMV. NT245 expressing yeast L3Δ100 and PAP, NT250 expressing L3 and NT252 expressing L3Δ100 provided great resistance to fungal toxins. The results of this study demonstrate that by altering ribosomal protein L3, the target of fungal toxins and combining it with PAP, relatively high levels of resistance to trichothecene mycotoxins was obtained.
Interaction of Yeast L3 with Pokeweed Antiviral Protein (PAP) in Yeast Cells
Expression of the Yeast L3Δ100 and L3Δ99 Reduces the Cytotoxicity of PAP in Yeast
In the following examples, we show here that co-expression of a truncated form of yeast L3 (L3Δ100) which encodes the first 100 amino acids of L3 with wild type PAP in transgenic tobacco plants eliminates the autoregulation of PAP expression, ribosome depurination and cytotoxicity of PAP. Expression of the endogenous tobacco ribosomal protein L3 is upregulated in the transgenic lines and they are resistant to the Fusarium mycotoxins, DON and DAS. The L3Δ100 is much more effective in conferring resistance to PAP and the trichothecene mycotoxins than the full length L3 gene because expression of the full length L3 gene from yeast in transgenic tobacco plants does not completely eliminate the autoregulation of PAP expression, ribosome depurination and the cytotoxicity of PAP, but reduces it. Although L3Δ100 expressed in plants does not contain the tcm-1 mutation (W255C) or the (P257T) mutation found in mak8-1, which protect ribosomes from depurination by PAP, it is highly effective in preventing ribosome depurination, mRNA autoregulation and cytotoxicity of PAP. Co-expression of the first 99 amino acids of L3 with wild type PAP in yeast eliminates the autoregulation of PAP expression, ribosome depurination and cytotoxicity of PAP.
We also show that L3Δ99, that includes the first 99 amino acids of L3, works better in yeast than the L3Δ100. These results demonstrate that expression of an N-terminal fragment of L3 leads to high level of resistance to PAP and DON, providing evidence that both toxins target L3.by a common mechanism.
The polynucleotides encoding that first 100 or 99 amino acids at the N-terminus of yeast L3 gene were cloned in the yeast expression vector pAC55 under GAL1 promoter on a URA3 plasmid, resulting in NT760 (L3Δ100) and NT771 (L3Δ99), respectively. Both NT760 and NT771 were co-transformed into yeast cells together with NT188 containing the wild type PAP gene in a yeast expression vector under the GAL1 promoter with LEU2 marker. Transformants were selected on SD -leu-ura media. The presence of both L3A genes and PAP gene in each transformant was confirmed by isolating the plasmids from yeast cells and re-transforming into E. coli cells.
The cytotoxicity of PAP in yeast containing NT760 and NT188 was not affected since they did not grow on plates containing galactose (FIG. 20). However, the cytotoxicity of PAP in yeast containing NT771 and NT188 was abolished in three out of four transformants (FIG. 20). Immunoblot analysis showed that there was an equivalent amount of PAP produced in cells containing NT760 and NT188 cells as the cells containing NT188 alone (FIG. 21). In contrast, there was significantly more PAP protein produced in cells containing NT771 and NT188 (transformants 5 and 6) (FIG. 21). These results indicated that L3Δ99 could reduce the toxicity of PAP much better than L3Δ100 in yeast.
To assess the level of rRNA depurination by PAP in yeast cells, total RNAs from cells containing NT760/NT188 and NT771/NT188 were extracted and subjected to the dual-primer extension analysis. As shown in FIG. 22A, ribosome depurination was reduced by approximately 80% in cells containing NT760 and NT188 compared to cells containing NT188 alone. As shown in FIG. 22B, the depurination of rRNA was greatly reduced (transformants 3 and 4) or eliminated (transformants 5 and 6) in cells containing NT771 and NT188. These results showed that co-expression of L3Δ99 with PAP eliminated the cytotoxicity of PAP.
Since there was a large amount of PAP produced in the non-toxic NT771/ NT188 transformants 5 and 6, compared to the transformants 3 and 4 (FIG. 21), PAP was extracted from each transformant using SuperFine Sephadex G25 columns. The PAP proteins were then used in an in vitro depurination assay by incubating PAP with ribosomes from the wild type cells. The rRNAs were then extracted and ribosome depurination was analyzed by the dual-primer extension assay. As shown in FIG. 23, PAP protein extracted from transformants 3 and 4 could still depurinate ribosomes in vitro. In contrast, PAP protein isolated from transformants 5 and 6 could not depurinate the ribosomes in vitro.
Yeast L3Δ99 Abolishes the Autoregulation of PAP Gene Expression and Enhances the Stability of PAP mRNA.
To determine if PAP destabilizes its own mRNA in cells containing NT771 and NT188, we used real-time PCR analysis with ABI PRISM 7000 Sequence Detection System (Applied Biosystems) to examine the mRNA levels in transformants 3, 4, 5 and 6 after induction on galactose for six hours. Reverse transcription reaction was carried out using the total yeast RNAs as templates and oligo d(T) as the primer. The single stranded cDNAs produced were used in real-time PCR analysis using two PAP-specific primers (PAP690F, 5′-GGGTAAGATTTCAACAGCAATTCA-3′ and PAP769R, 5′-CACCACTGGCATCCACTAGCT-3′). The PAP gene expression level was normalized against the yeast G6PD mRNA as an internal control using the ddCT method. It is shown in FIG. 24 that PAP mRNA in transformants 3.and 4 accumulated to the highest level at 4 hr after induction and then gradually declined. This PAP gene expression pattern was exactly the same as in yeast expressing NT188, which destabilizes its own mRNA. In contrast, the PAP mRNA level in transformants 5 and 6 increased up to six hours and did not decrease after 6 hours of galactose induction. This expression pattern was similar to what was observed with the active site mutant NT224 (PAPX) which does not autoregulate its own mRNA, i.e., the stability of PAPx mRNA is not affected. These results indicate that L3Δ99 diminishes the effect of PAP on its own mRNA, resulting in stabilization of PAP mRNA.
Effect of the First 21 Amino Acids of L3.
The 5′ end of L3 mRNA contains a stem loop structure highly similar to the SRL of rRNA (FIG. 25). The sequence “14AGUACGA20” in the L3 mRNA stem loop is identical to the sequence of the sarcin ricin loop (SRL) “AGUACGAGAGGA”, which is the longest conserved sequence found in all large rRNAs. Since PAP binds to the SRL, this sequence in L3 may act as an SRL mimic and PAP may bind to it and destabilize the L3Δ mRNA instead of its own mRNA. To test this, a polynucleotide encoding the first 21 amino acids of yeast L3 was cloned into pTKB175 under the GAL1 promoter (NT803). NT803 was co-transformed into yeast cells with NT188. As shown in FIG. 26, all four transformants containing NT803 and NT188 grew better on galactose. This indicates that the first 21 amino acids may have similar effect on PAP's toxicity as L3Δ99.
All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A transgenic plant comprising an exogenous nucleic acid comprising a transgene functional therein and that encodes a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein, or an analog of said polypeptide, wherein said plant exhibits increased resistance to toxins that target eucaryotic ribosomal L3 protein compared to a non-transgenic control plant.

2. The transgenic plant of claim 1, wherein the transgene encodes a polypeptide having from the first 21 to 99 N-terminal amino acid residues of a full-length ribosomal L3 protein.

3. The transgenic plant of 1, the transgene encodes a polypeptide having amino acids 1-99 of a eucaryotic ribosomal L3 protein.

4. The transgenic plant of claim 1, wherein the transgene encodes a polypeptide having amino acids 1-100 of a eucaryotic ribosomal L3 protein.

5. The transgenic plant of claim 1, wherein the transgene encodes a polypeptide having amino acids 1-21 of a eucaryotic ribosomal L3 protein.

6. The transgenic plant of claim 1, wherein the transgene is a first transgene, and wherein said exogenous nucleic acid further comprises a second transgene that encodes a ribosome inactivating protein (RIP) that targets a eurcaroytic L3 ribosomal protein.

7. The transgenic plant of claim 6 wherein said second transgene encodes Pokeweed Antiviral Protein (PAP)-v.

8. The transgenic plant of claim 6, wherein the second transgene encodes wild-type PAP.

9. The transgenic plant of claim 6, wherein the second transgene encodes PAP-II.

10. The transgenic plant of claim 6, wherein the second transgene encodes ricin or a Shiga toxin.

11. The transgenic plant of claim 1, which is a monocot plant.

12. The transgenic plant of claim 1, which is a cereal crop plant.

13. The transgenic plant of claim 12, wherein the cereal crop plant is maize, rice, wheat, oat, barley or wheat.

14. The transgenic plant of claim 1, which is a dicot plant.

15. Seed derived from-the transgenic plant of claim 1.

16. Seed derived from the transgenic plant of claim 6.

17. A method of making a transgenic plant having increased resistance to infestation by fungi that produce toxins that target a eurcaryotic L3 protein, comprising preparing a transgenic plant having a genome that contains an exogenous nucleic acid comprising a transgene encoding a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein or an analog thereof, wherein expression of the transgene in the transgenic plant confers increased resistance to toxins that target a eucaryotic ribosomal L3 protein compared to a non-transgenic control plant.

18. The method of claim 17, wherein said preparing a transgenic plant comprises transforming a protoplast from a cell of a plant with the exogenous nucleic acid to produce a transformed protoplast, and generating the transgenic plant from the transformed protoplast.

19. The method of claim 17, wherein said preparing a transgenic plant comprises introducing the exogenous nucleic acid into tissue of a plant to produce transformed plant tissue, and regenerating the transgenic plant from the transformed plant tissue.

20. A method of making a transgenic plant having resistance to infestation by fungi that produce toxins that target a eurcaryotic L3 protein, comprising preparing a transgenic plant having a genome that contains a first exogenous nucleic acid having a first transgene encoding a polypeptide having at least the first 21 to about 99 N-terminal amino acid residues of a full-length eucaryotic L3 protein or an analog thereof, and a second exogenous nucleic acid having a second transgene encoding an RIP protein that targets a eucaryotic ribosomal L3 protein, wherein expression of the first and second transgenes in the transgenic plant confers increased resistance to the fungi, and with less toxicity to the plant compared to a transgenic control plant that contains the second transgene but does not contain the first transgene.