MXPA98000333A

MXPA98000333A - Pap mutants that exhibit antiviral activity and / or antifungica in plan

Info

Publication number: MXPA98000333A
Application number: MXPA/A/1998/000333A
Authority: MX
Inventors: E Tumer Nilgun
Original assignee: Rutgers The State University; E Tumer Nilgun
Priority date: 1995-07-11
Filing date: 1998-01-09
Publication date: 1998-11-09

Abstract

PAP mutants having reduced phytotoxicity compared to wild-type PAP and conferring broad spectrum resistance to viruses and / or fungi in plants are described. A group of PAP mutants is characterized by at least one amino acid substitution at the N terminus of mature PAP, such as glycine residue 75 or glutamic acid residue 97 two additional PAP mutant groups are characterized by truncations in the N-terminus region of mature PAP and truncations or substitutions of amino acids in the mature PAP terminus C region respectively and an additional group is enzymatically inactive which still exhibits antigenic properties. Also described are DNA molecules that encode PAP mutants, mutant PAP DNA constructs, and transgenic seeds and plants that contain the DNA. In addition, methods for identifying PAP mutants that have reduced phytotoxicity, as well as purified and isolated PAP mutants identified by the method are described.

Description

MUTANTS OF PAP THAT EXHIBITS ANTIVIRAL AND / OR ANTIFUNGAL ACTIVITY IN PLANTS TECHNICAL FIELD This invention relates generally to agricultural biotechnology and more specifically to methods and genetic materials for conferring resistance to fungi and / or viruses in plants. BACKGROUND TECHNIQUE The issue of plant protection against pathogens remains the most important area in agriculture. Many commercially valuable agricultural crops are prone to invention by plant and fungal viruses capable of inflicting significant crop damage in a given season and drastically reducing their economic value. The reduction in economic value for the farmer in turn results in a higher cost of goods for the last buyers. Several published studies have addressed the expression of capsid proteins from plant viruses in a plant in an effort to confer virus resistance. See, e.g., Abel et al., Science 232: 738-43 (1986); Cuozzo et al., Bio / Technology 6: 549-57 (1988); Hemenway et al., EMBO J. 7: 1273-80 (1988); Stark et al., Bio / Technology 7: 1257-62 (1989); and Lawson et al., Bio / Technology 8: 127-34 (1990). However, the transgenic plants exhibited resistance only to the homologous virus and related viruses, but not to unrelated viruses. Kawchuk and others, Mol. Plant-Microbe Interactins 3 (5): 301-07 (1990), describe the expression of the coated wild type virus (VHEP) leaf protein virus in potato plants. Although the infected plants exhibited resistance to the VHEP, all the transgenic plants that were inoculated with VHEP were infected with the virus and therefore the continuous transmission of the virus was unfavorably allowed so that high levels of resistance could not be expected. See US Pat. No. 5,304,730 Lodge et al., Proc Nati Acad Sci USA 907089-93 (1993) reports the transformation mediated by tobacco Agrobacterium tumefaciens with a cDNA encoding wild-type anti-viral I protein (PAP psi) and the resistance of transgenic tobacco plants to unrelated viruses, PAP, a protein that inhibits pbosomes (PIR) of type I found in the cell walls of Phytolacca americana (grana), is a single chain of polypeptides that catalytically removes a residue of adenine specific to a highly conserved stem-loop structure in 28S RNA of eukaryotic pbosomes and interferes with the synthesis of cellular binding protein and blocker of the elongation factor Ver, vgr Irving et al., Pharmac Ther 55. 279-302 (1992), Endo et al., Biophys Res. Comm., 150 1032-36 (1988), and Hartley et al., FEBS Lett. 290: 65-68 (1991). The observations by Lodge had a sharp contrast with previous studies, supra, which reported that transgenic plants expressing a viral gene were resistant only to that virus and to closely related viruses. See also Beachy and others, Ann. Rev. Phytopathol. 28.:45-74 (1990); and Golemboski et al., Proc. Nati Acad. Sci. USA 876311-15 (1990).

Lodge also reports, however, that tobacco plants that express PAP (ie, above 10 ng / mg protein) had tended to have an atrophied, mottled phenotype, and that other transgenic tobacco plants that accumulated levels Higher PAPs were sterile PIRs have proven to be unpredictable in other aspects such as white specificity Unlike PAPs (as shown in Lodge, supra), Castor bean seed castor isolate is 1000 times more active than in pbosomes of mammals that in plant pbosomes See V gr, Harley et al., Proc Nati Acad Sci USA 79.5935-5938 (1982) The PIR of barley endosperms also shows very little activity against plant pbosomes See, v gr, Endo and others , Biochem Biophys Acta 994224-226 (1988) and Taylor et al., Plant J 5827-835 (1984) Fungal pathogens contribute significantly to outbreaks of more severe pathogens in plants Plants have developed a system of natural defense, including morphological modifications in its cell walls and the synthesis of several antipathogenic compounds Ver, v gr, Boller et al., Plant Physiol 74.442-444 (1984), Bowles, Annu Rev Biochem 59.873-907 (1990), Joosten and others , Plant Physiol. 89: 945-951 (1989); Legrand et al., Proc. Nati Acad. Sci. USA 846750-6754 (1987); and Roby et al., Plant Cell 2: 999-1007 (1990). Several proteins related to patojeaseis (RP) have been shown to have antifungal properties and are induced after infection by pathogens. These are different forms of hydrolytic enzymes, such as quitmases and β-1,3-glucanases that inhibit fungal development in vitro by destroying fungal cell walls Ver, v gr, 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 improve resistance to plant pathogens via recombinant methodologies by using genes that encode pathogenesis-related proteins (such as quymatase and β-1,3-glucanases) with different activities against fungal cell walls. Ver, v gr, Broglie and others, Science 254 1194-1197 (1991) Vierheilig et al. Mol Plant-Microbe Interact 6261-264 (1993), and Zhu et al., Bio / Technology 12.807-812 (1994) Recently, two other classes of genes have been shown that have the potential to confer resistance The disease in plants Wu and others, Plant Cell 7 1357-1368 (1995) report that the transgenic potato expressing the glucose oxidase gene of Aspergillus niger exhibited increased resistance to Erwinia carotovora and Phytophthora infestans. The hypothesis is that oxidation catalyzed by glucose oxidase produces hydrogen peroxide, which, when accumulated in plant tissues, leads to the accumulation of active oxygen species, which, in turn, triggers the production of various antipathogenic and antifungal mechanisms such as phytoalexins (see Apostle et al., Plant Physiol 90.109-116 (1989) and Degousee, Plant Physiol 104945-952 (1994)), pathogenesis-related proteins (Klessig et al. Plant Mol Biol 26.1439-1458 (1994)), hardening of the cell wall of the plant (Bpsson 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 response to defense sa hypersensitive (Levine et al., Cell 79.583-593 (1994)) In addition to studies on virus resistance in plants, PIR has been studied along with fungal resistance. For example, Logeman et al., Bio / Technology 1JD 305-308 (1992) report that an isolated PIR of barley endosperm provided protection against fungal infection to transgenic tobacco plants The combination of tobacco endosperm PIR and barley class II chitinase provided synergistic increase in resistance to Rhizoctonia solani in tobacco, both in vitro and in vitro See, v. Lea and others above, Mauch et al., Supra Zhu et al., Supra, and Jach et al., The Plant Journal 8 97-109 (1995). However, PAP did not show in vitro antifungal activity. See Chen et al. Plant Pathol 40612-620 (1991), which reports that PAP has no effect on the development of the fungus Phytophthora infestans, Colletotrichum coccodes, fusarium solani, fusarium sulphureum, Phoma roeata and Rhizoctonia solani, in. Thus, there is still a need for a medium whereby broad-spectrum resistance of viruses and / or fungus is confined to plants without causing cell death or sterility and which requires a minimum number of transgenes. COMPENDIUM OF THE INVENTION present invention is directed to PAP mutants having reduced phytotoxicity and exhibiting biological activity of PAP in plants. By "biological activity of PAP" is meant PAP antiviral activity and / or PAP antifungal activity A preferred group of PAP mutants is characterized by at least one amino acid substitution at the N terminus of mature PAP, such as a substitution for the Glycine 7 residue or 97 glutamic acid residue Another group of PAP mutants is characterized by a truncating as many as 38 amino acids at the N terminus of mature PAP. Yet another preferred group of PAP mutants is characterized by mutations such as truncations in the C-terminal region of mature PAP. More preferred are PAP mutants truncated at their C terminus by at least about 26 to 76 mature PAP amino acids (not counting the C-terminal extension of 29 wild type PAP amino acids). An additional group of PAP mutants are enzymatically inactive and do not exhibit PAG antiviral activity in vitro or in plant:, exhibit PAP antifungal activity in plants. The PAP mutants of the present invention may also include the N-terminal signal sequence of 22 amino acids and / or the C-terminal extension of wild-type PAP. The present invention also provides DNA molecules encoding PAP mutants, which may or may not also encode the N-terminal signal sequence of 22 amino acids from mature PAG and / or the C-terminal extension of 29 amino acids of wild type PAP. they can be operably linked to a functional promoter in procapotic cells (v gr, E coli), or eukaryotic cells such as plants and are stably transformed into a functional vector in said cells. The hosts, v gr, are also provided with prokaryotic or eukaryotic cells (v gr , yeasts or plants), stably transformed with a DNA that encodes mutant PAP, as well as protoplasts stably transformed with the DNA. Transgenic plants and seeds containing the DNA are also provided. The expression of the DNA in the transgenic plants confers resistance to viruses and / or high-spectrum plants without being as phytotoxic to the plant as wild-type PAP Plants in Within the scope of the present invention are monocotyledons, such as cereal crops and dicotyledonous plants. The present invention further provides a method for identifying a PAP mutant having reduced phytotoxicity and exhibiting biological activity of PAP in plants. The method involves the steps of providing a transformed eukaryotic cell such as yeast containing a DNA molecule encoding mature PAP operably linked to a functional inducible promoter in the ecuariotic cell. The DNA encoding PAP is mutagenized before transformation, or the transformed cell is mutagenized (i.e., mutagenesis is performed after the cell is transformed with the PAG construct). The cells thus transformed are cultured in a suitable medium and after a predetermined time, an inducer is added to the medium to cause the expression of the DNA molecule. A determination is then made as to whether the survival of cultured cells is due to the expression of a mutant PAP. Such PAP mutants exhibiting a Substantial lack of toxicity to the host could be considered as PAP mutants exhibiting reduced phytotoxicity The PAP mutants thus identified that also exhibit resistance to broad spectrum viruses and / or fungi, determined m vitro (v gr, by exogenous application of the virus u fungus), or in vitro (v gr, by the expression in transgenic plants), could also be considered as PAP mutants that have biological activity of PAP in plants. The present invention also provides isolated and purified PAP mutants identified by the aforementioned method. BEST MODE FOR CARRYING OUT THE INVENTION Transgenic plants expressing DNA encoding the PAP mutants of the present invention exhibit phytotoxicity reduced compared to transgenic plants that produce wild type, mature PAP ("PAP") or variant PAP, ie PAP-v, but also exhibit antiviral and / or antifungal activities. By the term "reduced phytotoxicity" is meant that a transgenic plant expressing a DNA encoding PAP exhibits a normal and fertile phenotype and does not exhibit the characteristics of atrophied, mottled phenotypes of transgenic plants that produce mature PAP (as described in Lodge, supra, for example) By "wild type PAP" is meant the amino acid sequence? PAP 1-262, the 22-amino acid N-terminal signal peptide ("the N-terminal signal sequence of wild-type PAP") and the C-terminal extension of 29 amino acids (amino acid numbered 263-291), all illustrated in the following Table 1 as SEQ ID NO: 2. The corresponding nucleotide sequence is therefore displayed as SEQ ID NO: 1. Therefore, by the terms "mature wild type PAP", or "mature PAP", is understood as the amino acid sequence of PAP 1-262 shown in Table I.

Table I S'CTATGAAGTCGCGTCAAAGCATATACAsGCTATCCATTGTTAGAAACATrGATGCCT CTGATCCCGATAAACAATACAAATTAGACAATAAGATGACATACAAGTACCTAA CTG TGTATGGGGGAGTGAAACCTCAGCTGCTAAAAAAACCTTGTAAGAAAAAAAGAAAGT TGTGAGTTAACTACAGGGCGAAAGTATTGGAACT A AGCTAGTAGGAAGOGAAG ATG AAG TCG ATG CTT GTG GTG ACA ATA TCA ATA Mel Lyt Ser Mel Leu Vil VaJ Thr IJe Ser He (67) TGG CTC ATT CTT GCA CCA ACT TCA ACT TGG GCT GTG AAT ATÁ ATC TAC Tf Leu lie Leu AJa Pro Thx Ser Tl Ttp Ali Vil Aso Thr He He Tyr (I) (100) G AAT GTT GGA ACT ACC ACC ATC AGC AAA TAC GCC ACT TTT CTG AAT GAT CTT Handle VaJ Gly Ser Th / Thr He Ser Lyt Tyr AJa Thr Pbe Leu Aso Asp Leu (10) (20) CGT AAT GAA GCG AAA GAT CCA AGT TTA AAA TGC TAT GGA ATA CCA ATG CTG Arj Asa Glu AJa Lyi Asp Pro Ser Leu Lyi Cyi Tyr Gly He Pro Mel Leu (30) (40) C CCC AAT ACÁ AAT ACÁ AAT CCA AAG TAC CTG TTC GTT GAG CTC CAA GGT TCA Pro Asn Thr Handle Thr Handle Pro Lys Tyr Va] Leu Val Glu Leu Gln Gly Sa (50) AAT AAA AAA? CC.ATC ATÁ ATG CTG AGA CGA AAC AAT TTG TAT CTG ATG Aso Lys Lys Thr He Thr Leu Met Leu Arj Arj Asn Asn Leu Tyr Val Mel (60) (70) GGT TAT TCT GAT CCC TTT GA ACC AAT AAA TGT CGT TAC CAT ATC TTT AAT i Gly Tyr Be Asp Pro Pbe Glu Thr Asn Lys Cys Ar Tyr Hit He Pbe Asn (80 ) (90) / GAT ATC TCA GGT ACT GAA CGC CAA GAT GTA ACT ACT CTT TGC CCA AAT Asp ile Ser Gly Thr Glu Arj Gln Asp Val Glu Thr Thu Leu Cyt Pro Asa (100) GCC AAT TCT GTT AGT AAA AAC ATA AAC TTT GAT AGT CGA TAT CCA ACA Wing Asn Ser Arj Val Ser Lys Asn He Asn Pbe Asp Ser Arj Tyr Pro Thr (1 10) (120) TTG GAA TCA AAA GCG GGA GTA AAA TCA AGA AGT CAO GTC CA CTG GGA ATT Leu Glu Ser Lys Wing Gly Val Lys Ser Ar Ser Gln Val Gln Leu Gly He (130) (140) CAA ATA CTC GAC AGT AAT ATT GGA AAG ATT TCT CGA GTG ATG TCA TTC ACT Gla He Leu Asp Ser Asn He Gly Lyt He Ser Gly VaJ Met Ser Pbe Thr (150) GAO AAA ACC GAA OCC GAA TTC CTA TTO OTA OCC ATA CAA ATO OTA TCA GAO Glu Lys Thr Glu Wing Glu Pbe Leu Leu Val Wing Ue Gln Mel Val Ser Glu (160) (170) GCA GCA AGA TTC AAG TAC ATA GAG AAT CAO GTO AAA ACT AAT TTT AAC AGA Wing Wing Arg Pbe Lyt Tyr De Giu Asa Gln Val Lys T? R Asa Phe Asa? Rg (180) (190) GCA TTC AAC CCT AAT CCC AAA GTA CTT AAT TTO CAA GAG ATA TGG GGT AAG Ala Pbe Asa Pro Asn Pro Lys Val Leu Asa Leu Gln Glu Thr Tf Gly Lyt (200) (210) ATT TCA ACA GCA ATT CAT GAT GCC AAG AAT GOA GTT TTA CCC AAA CCT CTC Ue Ser Thr Ala Lie His Asp Ala Lyt Asa Gly Val Leu Pro Lys Pro Leu (220) GAG CTA GTG GAT GCC ACT GGT GCC AAG TGG ATA GTG TTO ACÁ GTG GAT CAÁ Glu Leu Vil Asp AJa Ser Gly Ala Lyt Tf lie Val Leu Arj Val Asp Glu (230) (240) ATC AAG CCT GAT GTA GCA CTC TTA AAC TAC sTT GGT GGO AGC TGT CAG ACÁ He Lys Pro Asp Val Ala Leu Leu Asa Tyr Val Gly Gly Ser Cyt Gto Thr (230) (260) ACT TAT AAC CAA AAT GCC ATG TTT CCT CAA CTT ATA ATG TCT ACT TAT TAT Thr Tyr Asa Gla Asa Wing Met Phe Pro Gla Leu lie Met Ser Thr Tyr Tyr (262) (270) AAT TAC ATO GTT AAT CTT GGT GAT CTA TTT GAA GGA TTC TGATCATAAACA Asn Tyr Mel Val Asa Leu Gly Asp Leu Phe Glu Gly Pbe (280) (290) TAATAAGGAGTATATATATATTACTCCAACTATATTATAAAGCTTAAATAAsAsGCCG TGT AATrAGTACTTsTTsccTrrrGCTTTATssTGTTsTrrATTATGccrrsTATGCTT GTAATATTATCTAGAGAACAAGATGTACTsTGTAATAsTCTTGTTTGAAATAAAACTr CCAATT? TGATGC ?? AAAAAAAA? AAAAr Table I further shows amino acids .3 JAP-y and corresponding nucleotides in appropriate alignment with wild-type PAG Basically, the amino acid sequence of PAP-v differs from that of wild-type PAP in terms of a Leu20Arg (i.e. an arginine residue in position 20 of mature PAP opposed to a leucine residue) and a substitution of Tyr 49H? s The third change in the nucleotide sequence of PAP-v (codon of TCG? TCA for the first Ser present in the sequence of signals) has no effect on the amino acid sequence Table 1 also shows flanking sequences without 5 'and 3' coding When expressed in eukaryotic cells, the N-terminal 22 amino acid sequence of wild type PAP is separated as translationally, producing a polypeptide that has a molecular weight of approximately 32 kD, which is then processed by separating the 29 terminal C amino acids ("the C-terminal extension of PAP type si lvestre "or" PAP (263-292) "), yielding mature wild-type PAP (hereinafter" PAP (1-262) ") (ie, one that is isolated from Phvtolacca americana leaves), which have a molecular weight of approximately 29 kD See Irving et al., Pharmac. Ther. 55: 279-302 (1992); Dore and others, Nuc. Acids Res. 21 (181: 4200-05 * 1993); Monzingo et al., J. Mol. Biol. 233: 705-15 (1993); Tumer et al., Proc. Nati Acad. Sci. USA 92: 8448-8452 (1995). By the phrase "PAP antiviral activity", it is understood that the expression of a mutant PAP of the present invention in a transgenic plant confers resistance to broad spectrum viruses, ie, resistance to or ability of its first infection by a number of unrelated viruses, including, but not limited to, AR N v. gr. , potexviruses such as (PVX, potato X virus), potyvirus (PVY), cucumber mosaic virus (VMP), tobacco mosaic virus (VMT), barley yellow dwarf virus (VEAC), stellate mosaic virus of wheat, potato leaf curl virus (VEHP), plum poxvirus, melon mosaic virus, zucchini yellow mosaic virus, papaya rings stains, yellow beet rust, soybean dwarf virus, carrot red leaf virus and DNA plant viruses such as tomato yellow leaf winding virus. See also Lodge et al., Supra, Tomlinson et al., J. Gen Virol. 22: 25-32 (1974); and Chen and others, Plant Pathol. 40..612-20 (1991). By the phrase "PAP antifungal activity" it is understood that the PAP mutants of the present invention confer broad spectrum fungal resistance to plants. The mutant PAPs of the present invention provide increased resistance to diseases caused by plant fungi, including those caused by Pythium (one of the causes of root rot), Phytophthora (the cause of late potato pest and root rot and pests). many other plants), Bremia, Peronospora, Plasmopara, Pseudoperonospora and Sclerospora (causing velvety molds), Erysiphe graminis (causing powdery mildew of cereals and grasses), Verticillium (causing rot disease on the stem of many plants and coffee patch disease) of lawns), Fusarium (causing root rot of beans, dry rot of potatoes), Cochiliobolus (causing rot of roots and feet and also pests of cereals and grasses), Giberella (causing pests of plant seeds and foot rot or petiole) of maize and small grams), Gaeumannomyces (causing podredu crown mers flower and vegetable pests is and disease of grassland dollar spots), Puccinia (causing oxidation of the wheat stem and other small grains), Ustilago (causing dirt of corn), Magnaporthae (causing summer patch of grassland), and Schelerotium (causing southern pests of grassland). Other important fungal diseases include those caused by Cercospora, Septoria. Mycosphoerella, Glomerella, Colletotrichum, Helminthosporium, Alterneria, Botryris, Cladosporium and Aspergillus. The Applicant also thinks that the mutant PAPs of the present invention confer increased resistance to insects, bacteria and nematodes in plants. Important bacterial diseases include those caused by Pseudomonas, Xanthomonas, Erwinia, Clavibacter and Streptomyces. The PAP mutants of the present invention differ from wild-type PAP in substantially the following manner: (1) those that exhibit altered compartmentalization in vitro: (2) C-terminal mutants, including but not limited to deletion mutants framework; (3) N-terminal mutants; and (4) active site mutants. The first category of PAP muta.ites may have altered compartmentalization properties in. vitro; that is, they can not be located in the same subcellular compartment as wild type PAP. While not intended to be attached to any particular theory of operation, the Applicant thinks that these PAP mutants are not capable of undergoing cotraslational processing (to remove the signal peptide of 22 amino acids) and / or post-translational processing (for remove the C-terminal fragment of 29 amino acids) in yeast, which results in decreased substantiality or insignificant cytotoxicity. These mutants are also phytotoxic. What is particularly surprising or unexpected about the function of these in vitro mutant PAPs is that the mutations are located within the sequence encoding the mature PAP (1-262) and not within the N-terminal signaling peptide or extension. C-terminal of 29 amino acids. In addition, the mutant PAPs are enzymatically active to inhibit in vitro translation, indicating that phytotoxicity is not only a function of enzymatic activity. Preferred PAP mutants include a conservative point mutation such that glycine 75 (Gly75) was changed from the amino acid residue of wild-type PAP to valine, alanine, isoleucine or leucine, or (2) a mutation of the conservative point or non-conservative in the glutamic acid 97 (Glu97) residue of amino acids of wild-type PAP. The most preferred PAP mutants are PAP (1-262, Gly75Val) and PAP (1-262, Gly97Lys), the respective DNs of which can be prepared by simply changing the codon of wild-type GGT by glycine 75 to GTT ( valine) and the GAA codon for glutamic acid 97 to AAA (lysine). Other PAP mutants that have altered compartment capacity properties can be identified by the screening method described below. Dore and others, supra, describes a PA P Arg67Gly mutant (numbered in Dore as A rg 68Gly due to the presence of an N-terminal methionine residue), but which is tonic for eukaryotic cells and non-toxic for prokaryotic cells such as E. coli . This mutant is not included within the scope of the present invention. The second category of PA P mutants of the present invention have deletions or substitutions of amino acids in the C-terminal region of PAP. The applicant has unexpectedly discovered that these mutants are also non-toxic i n vitro (i.e., non-phytotoxic) although they inhibit in vitro translation. Preferred mutants are the PAP, PAP (1-184) mutants, inclusive. Therefore, mutations starting approximately at the amino acid residue 237 of mature wild-type PAP, e.g. , PAP (1-236), PAP (1-235), PAP (1-234), PAP (1-233), PAP (1-232), PAP (1-231). PAP (1-230), PAP (1-229), PAP (1-228), PAP (1-227), PAP (1-226), PAP (1-225), PAP (1-224), PAP (1-223), PAP (1-222), PAP (1-221), PAP (1- 220). PAP (1-219), PAP (1-218), PAP (1-217), PAP (1-216), PAP (1-215), PAP (1-214), PAP (1-213). PAP (1-212), PAP (1-211), PAP (1-210), PAP (1- 209), PAP (1-208). PAP (1-207). PAP (1-206), PAP (1-205), PAP (1-204), PAP (1-203). PAP (1-202). PAP (1-201). PAP (1-200), PAP (1-199). PAP (1- 198). PAP (1-197), PAP (1-196). PAP (1-195). PAP (1-194), PAP (1-193). PAP (1-192). PAP (1-191). PAP (1-190). PAP (1-189). PAP (1-188). PAP (1-187). PAP (1-186), PAP (1-185). V PAP 111'18) are encompassed by the present invention. The most preferred mutants include PA P (1-184G lu), PA P (1-199Lys), PAP (1-206Glu), PAP (1-209) and PA P (1-236Lys). Suppressions shorter than about 26 (ie, between 1 and 25 amino acids, inclusive) or longer 76 amino acids of mature PAP are included within the scope of the present invention as long as they are not toxic to plant cells, which can be determined by the selection method described in detail below and confer resistance to the fungus and / or virus in plant. The latest properties can be determined in vitro, v. gr. , inoculating parts of plants, v. gr. , leaves, with the PA P mutant in the presence of a virus or fungus, or by separate in vitro analysis where a transgenic plant transformed with a DNA encoding mutant PAP is inoculated with a fungus or virus. A preferred C-terminal substitution mutant is PAP (1-262, Leu202Phe). Again, as long as it is not intended to be linked to any particular theory of operation, the Applicant thinks that the amino acid sequence of PAP 244Gly-259Cys (shown in Table I), which is homologous to the consensual sequence for the binding site of Prokaryotic membrane lipoprotein lipids (Hayashi et al., J Bioenerg, Biomem. 22: 451-71 (1990)) and which is absent from each of the PA P mutants described above, is involved in the binding of PAP to phospholipids in membranes of endoplasmic reticulum (ER) which facilitates the translocation of PAP into the cell's cytosol where it inhibits protein synthesis. Disarming this function, v. gr. , by deletion or by mutation of frame change, results in mutants of PAP having the properties described instantaneously. Dore et al., Supra, also disclose a Phe 195Tyr, Lys21 1 Arg PAP mutant (whose numeration is + 1 out of phase with the numbering used herein due to the N-terminal Met residue required for expression in E. coli). ) that is toxic to eukaryotic cells (such as plants) but not toxic to prokaryotes such as E. coli. Accordingly, this PA P morator described in the Dore publication is not included within the scope of the present invention. The third category of PAP mutants is characterized by truncations of 1 to at least about 38 N-terminal amino acid residues of mature PAP. These mutants include PAP (2-262), PAP (3-262), PAP (4-262), PAP (5-262), PAP (6-262). PAP (7-262). PAP (8-262). PAP (9-262), PAP (10-262). PAP (11-262). PAP (12-262). PAP (13-262), PAP (14-262), PAP (15-262), PAP (16-262), PAP (17-262), PAP (18-262). PAP (19-262), PAP (20-262), PAP (21-262). PAP (22-262). PAP (23-262), PAP (24-262), PAP (25-262), PAP (26-262). PAP (27-262). PAP (28-262). PAP (29-262), PAP (30-262), PAP (31-262), PAP (32-262), PAP (33-262), PAP (34-262), PAP (35-262), PAP (36-262), PAP (37-262), PAP (38-262) and PAP (39-262).

Truncations greater than 38 N-terminal amino acid residues of mature PAP are included within the scope of the present invention to the extent that they exhibit biological activity of PAP and reduced phytotoxicity in vitro. These properties can be determined according to the procedures exhibited in the following working examples. The fourth category of PAP mutants contains active site mutations that render the enzyme molecule inactive (measured by its inability to inhibit in vitro translation and / or eukaryotic ribosomes). The Applicant has surprisingly and unexpectedly found that these mutants exhibit broad spectrum antifungal activity when expressed in plants, although they exhibit insignificant PAP antiviral activity. The putative active site of PAP includes amino acid residues Tyr72, Tyr 123, Glu176, Arg179 and Trp208. Accordingly, PAP active site mutants, e.g., that contain a conservative or even non-conservative substitution at the active site of PAP or where at least one active site amino acid is deleted or replaced by another amino acid, where PAP becomes enzymatically inactive but retains antifungal activity, they are encompassed within the present invention. PAP mutants can be tested for enzymatic and antifungal activities using the methods of analysis described in the following working examples. A preferred PAP active site mutant is PAP (1-262, Glu176Val).

With respect to the PAP mutants described the phrase "which differs from wild type PAP substantially" means that except for the amino acid changes (described above), which are necessary to confer reduced phytotoxicity and antiviral and / or antifungal activity, the amino acid sequences of the mutant PAPs are substantially identical to those of mature PAP. By the term "substantially identical" it is understood that the PAP mutants of the present invention can be further modified in the form of additional substitutions, additions or deletions as long as the resulting PAP mutant retains the reduced phytotoxicity and biological activity of PAP as defined herein. For example, the N terminus of the mutant PAP can be changed to a methionine residue, either by substitution addition, to allow expression of a DNA encoding the mutant PAP in several host cells particularly E coli The PAP d mutants The present invention may also include the wild-type PAP N-terminal 22-amino acid signaling peptide and / or the C-terminal extension of 29 amino acids, both of which are shown in the following Table I. The DNAs encoding the PAP mutants of the present invention can be prepared by manipulation of known PAP genes. See Ausubel et al. (Eds.), Vol. 1, Chap. 8 in Current Protocols in Molecular Biology. Wíley, NY (1990). DNA can also be prepared via PCR techniques. See PCR Protocols. Innis et al., (Eds.), Academic Press, San Diego, CA (1990) DNA encoding mutant PAP (v. G, a cDNA) is inserted into a plant transformation vector in the form of a cassette. expression containing all the elements necessary for the transformation of plant cells The expression cassette normally contains, in an appropriate reading frame, a functional promoter in plant cells, a 5 'untranslated leader sequence, the mutant PAP DNA and a functional 3 'untranslated region in plants to cause the addition of polyadenylated nucleotides to the 3' end of the RNA sequence. Functional promoters in plant cells can be obtained from a variety of sources such as plants or plant DNA viruses. of a promoter used in the expression cassettes will determine the spatial and temporal expression pattern of the construction in the transgenic plant. The selected promoters can have const activity. itutiva and these include the CaMV 35S promoter, the actin promoter (McEIroy et al., Plant Cell 2 163-71 (1990), McEIroy et al. Mol. Gen Genet 231: 150-160 (1991), Chibbar et al., Plant Cell Rep. 12: 506-509 (1993), and the ubiquitme promoter (Binet et al., Plant Science 79: 87-94 (1991), Christensen. and others, Plant Mol. Biol. 12: 619-632 (1989); Taylor et al., Plant Cell Rep. 12: 491-495 (1993)). Alternatively, they can be induced by wounds (Xu et al., Plant, Mol Biol 22: 783-792 (1993), Firek et al, Plant, Mol. Biol. 22: 129-142 (1993), Warener and others Plant J. 3: 191-201 (1993)) and therefore directs the expression of the mutant PAP gene to the wound sites or pathogenic infection. Other useful promoters are expressed in specific cell types (such as leaf epidermal cells, meosphile, root bark cells) or in specific tissues and organs (roots, leaves or weeps, for example) Patent application WO 93/07278, for example, describes the isolation of maize trpA gene which is preferentially expressed in Hudspeth medulla cells & Gruía, Plant Mol Biol 12.579-589 (1989), has described a promoter derivative of the maize gene encoding phosphoenolpyruvate carboxylase (PEPC) with direct expression in a specific manner for the leaf. Alternatively, the selected promoter can direct the expression of the gene under a light-induced or other temporarily regulated promoter An additional alternative is that the selected promoter can be chemically regulated. DNA can also encode the N-terminal signal sequence and / or the C-terminal extension of wild type PAP. a variety of trans-decoupling and polyadenylation sites for use in expression cassettes These are responsible for the correct processing (formation) of the 3'-end of mRNA The appropriate transclassal separation and polyadenylation sites known to function in plants include the separation sites and polyadenylation CaMV 35S, the sites of separation and polyadenilacion of tml, the cleavage and polyadenylation sites of nopaline synthase, the separation sites and polyadenylation of rbcS E9. These can be used in both monocotyledons and dicotyledons. Numerous sequences have been found to increase the expression of the gene from within the transcptional unit and these sequences can be used together with the genes of this invention to increase their expression in transgenic plants. shown that several sequence sequences increase expression particularly in monocotyledonous cells. For example, it has been found that the Adhl gene of maize vortices significantly increase the expression of the wild-type gene under its analogous promoter when introduced into cells. It was found that intron 1 is particularly effective and increases expression in fusion constructs with chloramphenicol acetyl transferase gel ((Callis et al., Genes Develop 1 183-1200 (1987)) In the same experimental system, the intron of the bronze-l gene had a similar effect to increase expression (Calhs et al. sup ra) Sequences of introns have been routinely incorporated into plant transformation vectors, usually within the untranslated leader A number of untranslated sequences derived from viruses are also known to increase expression and these are particularly effective in cells dicotyledons Specifically, the leader sequences of the Tobacco Mosaic Virus (VMT, the "O sequence"), Corn Chlorotic Mottle Virus (VMCM), and Alfalfa Mosaic Virus (VMA) have been shown to be effective in increasing expression (Fig. gr, Galhe and others, Nucí.

Acids Res 15 8693-8711 (1987), Skuzeski and others Plant Mol. Biol. 1_565-79 (1990)) Numerous transformation vectors are available for the transformation of plants and the genes of this invention can be used together with any of said vectors. The selection of vector to be used will depend on the preferred transformation technique and the target species for transformation For certain target species, different antibiotics or herbicide section markers may be preferred. Selection markers routinely used in transformations include the nptll gene which confers resistance to kanamycin (Messing &Vierra, Gene 19.259-268 (1982 ), Bevan others, Nature 304 184-187 (1983)), the bar gene which confers resistance to herbicide fosfmotpcin (White et al., Nucí, Acids Res. 18.1062 (1990), Sener et al., Theor Appl Genet 79 .. 625-631 (1990)), the hph gene that confers resistance to antibiotic hygromycin (Blochinger &Diggelmann, Mol Cell Biol 4 2929-2931)) and the dhfr gene, which fers resistance to methotrexate (Fl ing & Elwell, 1980)) Suitable vectors for Agrobacterium transformation usually have at least one T-DNA borderline sequence. These include vectors such as pBIN19 (Bevan, Nucí Acids, Res. (1984) and pC1B200 (EP 0 322 104) Transformation without the use of Agrobacterium tumefaciens surrounds the requirement for T-DNA sequences in the selected transformation vector and consequently vectors lacking these sequences can be used in addition to vectors such as those described above which contain T-DNA sequences.Transformation techniques that are not based on Agrobacterium include transformation via particle bombardment, absorption of protoplasts (eg, PEG and electroporation) and microinjection.The choice of vector depends largely on the preferred selection for the species being transformed.For example, pCI B3064 is a vector derived from P UC suitable for direct gene transfer technique in combination with the selection by herbicidal phloem (or phosphinothricin) is described in WO 93/07278 and Koziel et al. ros (Biotech nology 1 _: 194-200 (1993)). An expression cassette containing the A DNA gene of PA P mutant containing the various elements described above can be inserted into a plant transformation vector by normal recombinant DNA methods. Alternatively, some or all of the elements of the expression cassette may be present in the veto and any remaining elements may be added to the vector as necessary. Transformation techniques for dicotyledons are well known in the art and include techniques based on Agrobacterium and techniques that do not require Agrobacterium. The techniques without Agrobacterium imply the absorption of exogenous genetic material directly by protoplasts or cells. This can be achieved by PEG or absorption mediated by electroporation, mediated delivery by particle bombardment or microinjection.

Examples of these techniques are described by Paszkowski et al., EMBO J 3: 2717-27222 (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 normal techniques. The transformation mediated by Agrobacerium is a preferred technique for the transformation of dicotyledons due to its high transformation efficiency and its wide utility with many different species. Species of many crops that are routinely transformed by Agrobacterium include tobacco, tomato, sunflower, cotton, rapeseed, potato, soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0249 432 (tomato), WO 87/07299 (Brassico), US 4,795,855 (poplar)). Agrobacterium transformation usually involves transfer to the binary vector carrying the foreign DNA of interest (eg, pCIB200 or pCIB2001) to an appropriate strain of Agrobacterium that may depend on the complement of vir genes carried by the host strain of Agrobacterium and either in a co-resident or chromosomally plasmid (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 achieved by a method of triparenteral mating using E. coli carrying the recombinant binary vector, an E. coli helper strain carrying a plasmid such as pRK2013 which is capable of mobilizing the recombinant binary vector to the white strain of Agrobacterum Alternatively, the binary vector recombinant can be transferred to Agrobacterium by transformation of A DN (Hofgen &; Willmitzer, N ucí Acids Res 16 9877 (1988)) Transformation of white plant species by recombinant Agrobacterium or suely involves the co-cultivation of Agrobacterium with plant explants and follows well-known protocols in the material The transformed weave is regenerated in selectable medium carrying a marker of resistance to the antibiotic or herbicide present between the limits of T-DNA of binary plasmid. The preferred transformation techniques for monocotyledons include the direct transfer of genes into protoplasts used P EG or electrophoresis and bombardment techniques. particles in callous tissue The transformation can be carried out with a single species of A DN or multiple species of A DN (ie, co-transformation) and both of these techniques are suitable for use with this invention. Co-transformation can have the advantage of avoiding the complex construction of the vector and of generating transgenic plants with lugare s unbound for the gene of interest and the selectable marker, allowing the removal of the selectable marker in subsequent generations, which should be considered desirable. However, a disadvantage of the use of cotransformation is the frequency less than 100% with which separate species of DNA are integrated into the genome (Schocher et al., Biotechnology 4 1003-1096 (1986)) Published Patent Applications EP 0292435 EP 0392 225 and WO 93/07278 describe techniques for the preparation of corn protoplasts and callus, transformation of protoplasts using PEG or electroporation and regeneration of transformed protoplast corn plants. Gordeon-Kamm et al., Plant Cell 2603-618 (1990) Fromm et al., Biotechnology H 194-200 (1993), describes techniques for the transformation of selected descent lines of corn by particle bombardment. Rice transformation can also be carried out by direct gene transfer techniques using protoplasts or particle bombardment. Protoplast-mediated transformation has been described for types of Japonica and Indica types (Zhange et al., Plant Cell Rep 7739-384 (1988), Shimamoto et al. Nature 338274-277 (1989), Datta et al., Biotechnology 8736-740 (1990)) Both types can also be routinely transformed using particle bombardment (Chpstou et al. Biotechnology 9957-962 (1991)) . Patent application E 0 332 581 described techniques for the generation, transformation and regeneration of Pooideae protoplasts. In addition, wheat transformation by Vasil et al. (Biotechnology 10: 667-674 (1992)) has been described using particle bombardment in type C long-term regenerable callus cells and also by Vasil et al. (Biotechnology 11 : 1553-1558 (19993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and aepvate callus of immature embryos Transformation of monocotyledonous cells such as Zea mays can be achieved by in contact the cells of monocotyledons with a multiplicity of needle-like bodies on which they can impale, causing a break in the cell wall thus allowing the entry of transformation DNA into the cells. See U.S. Patent No. 5,302,523. Transformation techniques applicable to both monocotyledons and dicotyledons are also described in the following patents from E U to 5,240,855 (particle gun), 5,204,253 (accelerated cold gas shock microprojectiles), 5,179,022 (biolistic apparatus), 4,743,548 and 5,114,854 (microinjection); and 5,149,655, 5,120,657 (mediated transformation of accelerated particles), 5,066,587 (gas-driven microprojectile accelerator), 5,015,580 (particle-mediated transformation of soybean plants), 5,013,660 (laser-mediated transformation); and 4,849,355 and 4,663,292. Cells from such transformed plants or plant tissue are then grown in whole plants according to normal techniques. The transgenic seed can be obtained from transgenic flowering plants according to normal techniques. Likewise, non-flowering plants such as potatoes and sugarcane can be propagated by a variety of known processes See, v. Gr., Newell et al. Plant Cell Rep 10 3C-.4 (1991) (describing the transformation of potato by stem culture) The DNAs encoding mutant PAP of the present invention confer resistance to fungi and broad spectrum viruses for any plant capable of expressing the DNAs, including monocots (v. gr, cereal crops) and dicotyledons. Specific examples include corn, tomato , grass, asparagus, papaya, sunflower, rye, beans, ginger, lotus, bamboo, potato, rice, peanut, barley, malt, wheat, alfalfa, soy, oats, eggplant, pumpkin, onion, broccoli, sugar cane, beet sugar, beets, apples, oranges, grapes, pear, plum, peach, pineapple, grapes, rose, carnation, sunflower, tulip, Douglas fir, cedar, white pine, amber pine, spruce, peas, cotton, linen and coffee PA mutants P different from those specifically described above can be identified by a selection system in eukaryotic cells. In a preferred embodiment, a DNA molecule encoding PAP, operably linked to a functional inducible promoter in the eukaryotic cell, is randomly mutagenized according to normal. The cell is then transformed to the mutagenized PAP construct. The cell thus transformed is then cultured in a suitable medium for a predetermined amount of time, e.g., sufficient to cause some growth of the cells, at which time an inducer is added to the medium to cause the expression of the DNA molecule. mutagenized If the cultured cell survives the induction of expression of the mutagenized PAP DNA molecule which indicates that the mutagenesis resulted in the expression of a non-toxic PAP mutant, the PAP mutant can then be analyzed m vitro or vitro to determine if it retains the biological activity of PAP Preferred m vitro analyzes include eukaryotic translation systems such as reticulocyte lysate systems where the degree of inhibition of protein synthesis in the system caused by the PAP mutant was determined. Preferred host cells are yeast cells such as Saccharomvces cerevisiae, as described in more detail in the following Example 1 This method can also be carried out with a plurality of DNA molecules encoding randomly mutagenized PAP PAP mutants identified as having reduced phytotoxicity and which retain antifungal and antiviral activity of PAP, determined After further analysis, they can be isolated, purified and sequenced according to normal techniques. In another embodiment, the mutagenesis is carried out after the transformation of the eukaryotic cell. The disadvantage with DNA mutagenization after transformation is that the chromosomal DNA of the The host can also be mutagenized. To determine whether mutations of the surviving cells are chromosomal or have plasmid by nature, this embodiment requires the step of replacing the DNA encoding transforming PAP with DNA encoding wild-type PAP under the control of an inducible promoter and developing the cells in the presence of the inductor. Mutants that retain the ability to grow are chromosomal mutants, whereas mutants that do not grow are mutants that have plasmids (ie, PAP). The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only and are not intended to be limited unless otherwise specified. EXAMPLE 1 A. Construction of yeast expression vectors and analysis of PAP expression in yeast. The full length cDNAs corresponding to PAP and PAP-v described in Lodge and others and shown in Table 1, were cloned into yeast expression vectors, under the control of the galactose-inducible promoter, GAL1. S. cerevisiae was chosen as the expression system since yeast has the advantage of providing post-translational modifications specific for eukaryotic cells. Since the yeast ribosomes are sensitive to PAP, a regulated promoter was used to direct PAP expression. The cDNAs encoding PAP and PAP-v were cloned into the yeast expression vector pAC55, containing the selectable marker, URA3, as fragments from BglII / Smal under the control of the galactose inducible promoter pGaH. The vectors containing PAP (NT123) and PAP-v (NT124) were transformed into the yeast strain W303 (Mat a, ade2-1 trp1-1 ura3-1 leu2-3, 112 his3-1, 15can1-100) ( Bossie et al., Mol. Biol. Cell, 3: 875-93 (1992)), according to the procedure described in Ito et al., J. Bacteriol. 153: 163-68 (1983) and transformants were selected in medium minus uracil with glucose at 30 ° C. Yeast cells containing NT123 (wild type PAP) or NT124 (PAP-v) were grown in non-uracil medium with 2% raffinose at 30 ° C for 48 hours at a density of 5x107 cells / ml. The expression of PAP proteins was induced by the addition of 2% galactose to half the culture, while the other half of the culture was used as an uninduced control. The cells were allowed to develop for an additional 4 h and then collected by centrifugation at 10,000 g for 5 min. The cells were resuspended in 9150 mM pH regulating solution RIPA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8) with protease inhibitors (0.1 μg) / ml of each of antipain, aprotinin, chemostatin, leupeptin, pepstatin) and was smooth using glass beads (0.5 mm). The extracts were loaded on a 10% SDS-PAGE gel according to the procedures described in Tomlinson, J. Gen. Virol., Supra. The immunoblot analysis was performed by the increased chemiluminescence (ECL) method (Amersham), using antibodies against purified PAP. PAP as PAP-v were expressed in yeast after galactose induction. Based on the comparison with PAP protein standards, yeast cells containing the PAP plasmid (NT123) expressed both the mature form of PA P and a larger form, whereas the cells containing PAP-v (NT 124) They expressed predominantly the largest form and very low levels of the mature form PA P was not detected in the medium of the content While it is not intended to be bound by any particular theory of oppression, the sunlit think that these results suggest the following (1) PA P is expressed as a precursor and processed to mature form in levad ura; and (2) PAP undergoes additional processing in addition to the cotraslational division of the peptide of N-terminal amino acid signal (Lodge and others su pra) since the size of the PA P uranium and the PAP expressed in yeast are found that it is smaller than the expected size after the removal of the signal sequence. B. In vitro translation and processing of PA P and PA Pv To examine the process of PAP m vitro, the constructs described in Example 1 A were transcribed and in vitro translators using the reticulocyte lysate translation system coupled of T7 in the presence of 35S-methionine with or without canine microsomal membranes (Promega). The PA P and PAP-v cDNAs were cloned into the pGem 3Z vector (Promega) downstream of the T7 promoter. An equal amount of DNA (1 μg) of each construct was transcribed and moved in vitro in the presence of 35S-methionine, using the translational system of T7-coupled reticulocyte lysate (promega) with or without canine pancreatic microsomal membranes (Promega). The translational products were incubated with 0.2 mg / ml proteinase K in the presence of 5 mM EDTA and 125 mM sucrose for 90 minutes. Proteinase K was activated by the addition of 4 mM PMSF and incubation at room temperature for 2 hours. The translation products were then treated with Endo-H (endo-N-acetylglucosaminidase) (1 mU / 10μl) in the presence of 0. 1% SDS and 0. 1 M sodium citrate at pH 5.5, at 37 ° C. C for 12 hours. Equal amounts of protein (3.5 μl) were analyzed in 10% SDS-PAGE E according to the procedure described in Laem m li et al., Natu re (London) 227: 680-5 (1970). PA P and PAP-v encode 33 and 34 kD precursor proteins respectively and both precursors were processed in a 32 kD form after membrane incubation. The processed proteins are even larger than the mature form (29k D) indicating that the precursor from PAP on additional post-translational processing. PAP does not contain any N-linked glycosylation site and the size of the proteins transferred in vitro did not change after treatment with endo-N-acetylglucosaminidase (Endo H), which removes carbohydrates. These results indicated that PAP precursors contain a sequence of N-terminal signals that are cotranslationally processed and another sequence, which was removed post-translationally. Additional evidence was obtained to process C-terminal from the analysis of X-ray structure, which showed that the mature PAP is 29 amino acids shorter in its C-terminus than the predicted sequence from cDNA. See Monzingo et al., J Mol Biol 233 705-15 (1993) C Transformed yeast growth In the presence of 2% raffinose, a non-repressor, non-inducing source of carbon in relation to the expression "of the GAL gene. yeast transformants containing NT 123 or NT124 were indistinguishable from the transformants containing the vector alone. The growth of the transformed yeast containing NT123 was subtracted by the addition of the inducer, galactose, to the medium. The cells containing NT123 or NT124 did not grow on galactose-containing plates. However, in the liquid medium, the degree of inhibition was higher with NT123 than with NT124, possibly due to lower levels of mature PAP produced in yeast containing NT124. PAP expression was detected within 2 h of the addition of galactose to the medium Maximum levels were reached from 6 to 8 h. Immunoblot analysis using antibodies against PAP detected a maximum PAP level of 1 μg / mg protein. a yeast in transformants of NT123 and 250 ng / mg of yeast protein in NT124 transformants. These results were in agreement with the production of active PAP in yeast. D. Mutagenesis of PAP plasmids. To isolate non-toxic PAP mutants for yeast, the expression plasmids containing PAP (NT123) or PAP-v (NT124) were mutagenized using hydroxylamine according to the methods set forth in (5), transformed into yeasts and the cells were seeded in plates in glucose containing medium and a replica was plated in galactose-containing plates. Approximately 10 μg of the purified plasmid DNA was added to 500 μl of freshly prepared hydroxylamine solution (0.35 g of hydroxylamine-HCl and 0.09 g NaOH in 5 ml of water) and incubated at 37 ° C for 20 h. To stop mutagenesis, 10 μl of 5M NaCl, 50 μl of 1 mg / ml BSA and 1 ml of 100% ethanol were added and the mutagenized DNA was precipitated by incubation at -70 ° C for 10 minutes. The DNA was resuspended in TE and precipitated again. The DNA was then transformed into yeast and plated in medium minus uracil containing 2% glucose and plated on a replica in medium containing 2% galactose. Colonies that grew in galactose were analyzed for PAP expression by ELISA described in Lodge et al., Supra, and by immunoblot analysis to identify mutants that expressed mutant PAP generated from hydroxylamine. E. Mutant Yeast Growth: Growths of NT123-derived mutants in galactose-containing medium were indistinguishable from growth in raffinose containing medium. Similar results were obtained with mutants derived from NT124. Analysis of protein accumulation in yeast indicated that wild-type PAP expression, but not mutant PAP generated from hydroxylamine, resulted in decreased protein accumulation in yeast (data not shown). After mutagenesis, the colonies growing on uracil deficient galactose plates were analyzed for PAP expression by ELISA using PAP antibodies and the positive ones were further analyzed by immunographic analyzes. Of a total of 28 mutants of NT123 mutants, six isolated Different expressed proteins that cross-reacted with PAP antibodies. Of the 44 mutant mutants isolated from NT124, 24 different isolates produced proteins that cross-reacted with PAP antibodies. Four mutants (HMNT123-1, 124-6, 124-7, and 124). -1) produced proteins that were larger than the mature form of PAP (29 kD), suggesting in the processing of PAP the mature form was blocked in these mutants Two mutants (HMNT123-2 and 123-3) produced proteins that co -migrated with the mature form of PAP, while many others (HMTN123-4, 123-5, 123-6, 124-2 and 124-3) produced smaller proteins. Roteins in the mutants ranged from 0005 to 008% of the total soluble protein F Nucleotide sequence analysis of PAP mutants The positions of the amino acid alterations in the PAP mutants were identified by sequence analysis of the plasmids rescued from yeast plasmids were isolated from the mutants, transformed into £ coli according to the procedure set forth in Rose et al., supra, and sequenced using the Sequenase 20 (USB) DNA sequencing kit. See Robzyk et al., Nucí Acids Res. 20 , 3790 (1992) The sequence analysis of HMNT123-2 revealed that it contains a single point mutation, changing the glutamic acid at position 176 to vahna (E176V) in the putative active site (Table II). HMNT123-2 produced a protein of the same size as the wild-type PAP. The glutamic acid at position 176 (E176) is highly conserved between the PIRs sequenced to date and it is proposed that it has a cleavage at the active site of PAP ( 4) See Stevens others, Expepentia 37257-9 (1981) HNT123-6, HMNT124-2 and HMNT124-3 all had a point of mutation close to termination C that introduced a stop codon instead of a tpptofan at position 237 (W237) (Table II) As a result of this mutation, 26 amino acids were deleted from the C terminus of the mutant PAP and a truncated protein HMNT123-5 was produced containing a frame shift mutation, which suppressed two nucleotides (GA) approximately in the codon for Glu184 (GAG), so that the reading frame was altered and the Asn190 codon became TAA, since the reading frame changed to position 1, resulting in the expression of a truncated protein, a mutation point in HMNT124-1 changed glutamic acid at position 97 to lysine (E97K) (Table 1) HMNT123-1 also contained a mutation at a single point, at position 75, changing glycine to valine (G75V). Both mutants expressed a larger protein than the purified mature potato, suggesting that PAP processing was inhibited in these mutants to confirm that the phenotypes of mutants observed were due to the mutations identified in the PAP sequence and not due to a chromosomal mutation , each mutant PAP plasmid was isolated and retransformed in the host strain, W303 and URA + transformants were selected. These transformants grew in wild-type regimes in medium containing galacosa, indicating that the ability of the transformants to survive the induction of PAP expression are linked to plasmids. Table II. Mutations which abolish the toxicity of PAP to eukaryotic cells. HMNT123-1 Gly-75 (GGT)? Val (GGT) HMNT123-2 Glu-176 (GAG)? Val (GTG) HMNT123-4 Trp-208 (TGG)? Stop (TAG) HMNT123-5 Glu-184 (GAG)? Glu (GAA) HMNT123-6 Trp-237 (TGG)? Stop (TAG) HMNT123-1 Glu-97 (GAA)? Lys (AAA) HMNT123-2 Trp-237 (TGG)? Stop (TAG) HMNT123-3 Trp-237 (TGG)? Stop (TAG) HMNT123-13 Leu-202 (CTT)? Phe (TTT) G. Enzymatic activity of PAP mutants: In vitro translation analysis was used to compare the enzymatic activity of PAP mutants. The bromine mosaic virus RNA (VMB) was transferred into the rabbit reticulocyte lysate system (Promega) in the presence of yeast extracts containing different amounts of PAP, as described in Lodge et al., Supra. The levels of PAP in yeast were quantified by ELISA. { Lodge and others, supra). The inhibition curves were linear in the scale from 0.1 to 1 ng PAP / ml. Table III shows the results of inhibition analysis of protein synthesis in the presence of 0.2 ng / ml PAP of yeast. The amount of total protein and PAP was adjusted to 87 ng / ml and 0.2 mg / ml, respectively, in each extract by adding wild- yeast extract or pH-regulating solution of RIPA. In previous experiments, when performed in vitro in the presence of 0.2 ng / ml of BSA, translation inhibition was not observed. When 0.2 ng / ml of untransformed yeast protein (TS) was added, slight translation inhibition was observed. Translation was inhibited in the presence of 0.2 ng / ml of: (1) purified PAP added to wild- yeast extract (TS + PAP); (2) extracts of yeast protein containing NT123 or NT124; Y (3) extracts of yeast protein containing the mutants HMNT123-3, HMNT124-1, HMNT124-3 and HMNT124-13 generated from hiroxylamine. In contrast, protein extracted from HMNT123-2 does not inhibit protein synthesis in the reticulocyte lysate system. Results were obtained when the in vitro translation experiments were performed using 0.1 ng / ml PAP. Table III. Inhibition of protein synthesis by PAP mutants Incorporation of Protein Synthesis Protein (incorporated average translation cpm) Without RNA 2,246 +/- 204 BSA 244,956 TS 176,723 ± 713 PAP + TS 146,660 ± 2474 NT123 110,007 ± 445 HMNT123-2 213,952 ± 767 HMNT123-3 134.202 ± 5522 HMNT124 84.959 ± 661 HMNT124-1 119.529 ± 2094 HMNT124-3 132.955 ± 3739 HMNT124-13 145.899 ± 4457 Example 2 Expression of PAP mutants in transgenic tobacco Mutant PAPs were treated for constitutive expression in plants in order to conclude whether they could be non-toxic to plants and whether they could retain antiviral properties of PAP. In order to insert the PAP genes into the plant expression vectors, the plasmid DNA encoding the mutant PAPs was isolated from yeast, transformed into E. coli as described in Example 1. The plasmid DNA that encodes the mutant PAPs was isolated from E. coli digested with HindIII, the Hind site was filled with Klenow DNA polymerase and the plasmid was digested with Sacl to isolate the 772 bp Sacl / Hind lll fragment encoding the mutant PAP . The Sacl / HindIII fragments encoding the mutant PAPs were cloned into pMON8443 (Lodge et al., Proc.Nat.Acid.Sci.A. 90: 7089-7093 (1993)) after digestion with Sacl and Sma1 to remove the inserts of wild- PAP cDNA. The SACI / Hindl l l fragment of HMNT123-2 was cloned into digested pMON8443 from SACI / Smal to generate NT144 and the Sacl / Hindl fragment from HMNT124-3 was cloned into digested Sacl / Smal pMON8443 to generate NT145. NT146 and NT147 were generated by replacing the 772 bp Sacl / H i nd lll fragment of the wild- PAP DNA insert in pMO N 8443 with the 772 bp Sacl / Hind lll fragment of HMN T123-2 and HM NT 124 -3 respectively. The plasmids NT144, NT 145, NT146 and NT147 were mobilized in the ABI strain of Agrobacterium tumefaciens for transformation into tobacco and potato (Lodge et al., 1993). For the transformation of N. tabacum, young leaves of one-month-old tobacco plants were covered with water for 20-30 min. The water was drained and the leaves were covered with 10% chlorox and 0.04% Tween 20 for 1 5 minutes and after rinsing three times with water. Leaf discs were cut with a single perforation and placed downward in the medium of MS 104 (4.4 g / l MS salts)., 30 g / l of sucrose, vitamins B5, 0. 1 mg / 1 NAA and 1.0 mb / 1 BA) for 1 day for pre-culture. The recultivated discs were placed in a 50 ml tube and inoculated with overnight culture of Agrobacterium which was diluted 1: 5. The tube was inverted several times. The discs were removed and plotted on sterile filter paper and placed down on MS104 feeding plates with a filter disc to co-culture for 2 days. The leaf discs were then transferred to MS104 medium with selection (100 μg / ml kanamycin and 300 μg / ml cefataxime) and placed in an incubator. Sprouts and calluses appeared in approximately three weeks. The shoots are transferred to plant cones with MSO medium (4.4 g / l of MS salts, 30 g / l sucrose and vitamins B5) with selection. Roots formed in two weeks, the pots with roots were transferred to soil and kept in an environment with high humidity. The regenerated plants were then screened by ELISA for the presence of neomycin phosphotransferase (NPTII) to identify the expressors. The transformation frequencies of N. tabacum cv Samsun normally range from 10 to 12% (number of transgenic plants obtained per leaf disk). As previously reported, the frequency of transformation of N. tabacum was significantly reduced when vectors containing wild type PAP (pMON8443) or variant PAP (pMON8442) were used in the transformation (Lodge et al., 1993). In contrast, as shown in Table IV below, no decrease in the frequency of transformation was observed when the vectors containing the non-toxic mutant PAPs were used. TABLE IV Plasmid Transformation frequency NT144 13% NT145 11% NT147 12% As previously reported, transgenic plants expressing wild-type PAP or variant PAP showed reduced growth, chlorosis and speckling on their leaves (Lodg e et al. 1993). You found, the transgenic plants expressing the mutant PAPs were phenotypically normal. They grow at the same rate as the wild type plants and did not show chlorosis or mottle on their leaves, indicating that the expression of the PA P mutants is not toxic for transgenic plants. The mutant PAPs were also expressed in E. coli and their expression does not affect the growth rate of E cells. coli, indicating that they are not toxic to E. coli Example 3 Antiviral activity of PA P mutant expressed in transgenic tobacco. Transgenic tobacco plants (N. tabacum cv Samsun) were analyzed by ELISA (Lodge et al., 1993) to determine the level of expression of the mutant PAPs. In Table V, the level of expression of the mutant PA P was compared to the expression level of the variant PAP (pMON8442) (Lodge et al., 199) expressed in transgenic plants. Table V: Level of PAP expression in transgenic tobacco Plant Number Expression level NT144-12 1.5 g / mg NT144-13 0 9 μg / mg NT145-13 4.4 ng / mg pMON8442 (26139-11) 96 ng / mg As shown in Table V, the transgenic plant containing the C-terminal deletion mutant (NT145-13) expressed similar levels of mutant PAP as the plant expressing the PAP variant (pMON8442) (Lodge et al., 1993). In contrast, the transgenic plants containing the active site mutant (NT144) expressed significantly higher levels of the mutant PAP. To test if the mutants of PAP (constructions of NT144 and NT145) had antiviral activity m vitro, wild-type tobacco plants were inoculated with potato X virus (VPX) in the presence of plant protein extracts expressing the mutant PAP, v-PAP (pMON8442) and tobacco ( wild type) not transformed. The levels of PAP in the transgenic plants were quantified by ELISA. The level of PAP expression in line 145-13 was 4.4 ng / mg and the level of PAP expression in line 144-12 was 1.5 μg / ml. The plants were inoculated with extracts of transgenic plants containing 5 ng of PAP per leaf and 1.1 mg of total protein. The protein extract was inoculated into tobacco leaves in the presence of different amounts of total protein from untransformed tobacco, ranging from 6.7 μg to 1 1 mg. Twenty tobacco plants were labeled with 50 μl of 1 μg / ml of VPX in the presence of of 6.7 μg-1 1 mg of total protein from non-transformed plants. As shown in Table VI, all TS plants were infected with VPX and showed local lesions, systemic symptoms and viru accumulation in the leaves above the leaves if nocudas (systemic leaves) these results show that the protein extracts of non-transformed tobacco plants have no effect on VPX infection. When the protein extract from untransformed tobacco plants was used in the presence of 5 and 10 ng of purified PAP (TS + PA P), lower numbers of VPX lesions were observed in inoculated leaves, indicating that the plants were protected. of tobacco infection with VPX in the presence of purified PAP However, although minor lesions were obtained in the inoculated leaves of these plants, they did show systemic symptoms and similar levels of VPX antigen as the plants were inoculated with VPX in the presence of of non-transformed tobacco extracts (TS).

Table VI Effects of PAP mutants on VPX infection of tobacco leaves1 PAP Extract na / 0 no. Of lesions PVX antiqeno plantas3 Level nq / ml) c WTd 0 66.6 ± 10.1 4.4 ± 1.4 TS + PAPe 5 9.0 ± 2.0 3.1 ± 1.9 1.5 ± 2.0 2.8 ± 2.6 26139 5 1.8 ± 2.9 NA 145-13 5 12.5 ± 7.4 0.2 ± 0.3 144-12 5 57.8 ± 7.4 3.2 ± 2.5 10 56.1 ± 4.9 2.5 ± 1.4 20 55.5 ± 13.8 4.3 ± 1.4 50 53.3 ± 14.9 2.8 ± 0.3 100 68.0 ± 11.7 3.0 ± 0.9 a The plant extract was prepared from tobacco leaves not transformed or transformed with plant expression vector (pMON8442, NT145, and NT144). b The number of injuries was counted 9 days after inoculation. c Three discs of leaves in a tube were taken from the 1st, 2nd. And 3a systemic leaves at 12 days after inoculation and then homogenized in pH ELISA buffer. The average levels of VPX antigen were graded by ELISA. The amount of total proteins in each extract was quantified by ELISA. The amount of total proteins in each extract was quantified by BCA reagent (Pierce). d The protein extract was made from untransformed tobacco leaves. ß PAP (Calbiochem) was added to an extract of proteins from untransformed tobacco leaves. 1 Twenty plants were used for wild type (ts), ten plants for ts + PAP and five plants for each transgenic protein extract. Two leaves of each plant were inoculated with 50 μl of VPX (μg / ml) in the presence of different amounts of PAP or PA-P mutants. When VPX was inoculated in the presence of 5 ng of protein from the transgenic plant (26139) expressing the PAP variant (pMON8442), there were significantly lower numbers of lesions on the leaves and noculated leaves and these plants escaped systemic infection. Similarly, when VPX was inoculated in the presence of 5 ng of protein of the transgenic plant (145-13) expressing the C-terminal deletion mutant, significantly lower lesions were obtained. These plants show no systemic symptoms and VPX antigen levels were significantly reduced in the inoculated leaves. In contrast, when VPX was inoculated in the presence of 5 to 100 ng of transgenic plant protein expressing the active site mutant (144-12), the numbers of lesions observed in the inoculated leaves were similar to the numbers of lesions observed in plants inoculated in the presence of protein from non-transformed tobacco plants (TS). Systemic symptoms were observed in these plants and VPX antigen levels in the systemic leaves were comparable with antigen levels in plants and noxicated with VPX in the presence of extracts from nontransformed tobacco plants. These results demonstrate that the C-terminal deletion mutant that is enzymatically active in vitro retains its antiviral activity in vitro. In contrast, the active site mutant that is enzymatically inactive in vitro does not retain its antiviral activity in vitro, suggesting that the PA P enzyme activity is critical for antiviral activity in vitro. EXAMPLE 4 Expression of PAP mutants in transgenic potato. Stems of potato were cut into 3mm pieces and placed in sterile water. Agrobacterium containing NT144, NT145, NT 146 and NT147 was developed overnight. The cells were centrifuged and resuspended in 10 ml of water, the Agrobacterium was diluted again to 1: 10 in water. The water was removed from the explants of the potato stem and the diluted Agrobacterium was added. Stem explants were incubated with Agrobacterum for 15 min. The bacteria were removed and the explants were placed in 1/10 MSO plates that were covered with sterile Whatman # 1 filters. MSO contains 4.4 g of MS salts, 30 g of sucrose and 1 ml of vitamin B5 (500X) in a volume of 1 liter, pH 5.7. After a two-day co-cultivation period in the dark, the explants were placed in pC medium. containing MSO plus 0 5 mg / l of zeatin pboside (RZ), 5 mg / l of AgNO3 and 0.1 mg / 1 NAA (naphthalene acetic acid) 100 mg of kanamycin and 300 mg of cefataxime per liter, for four weeks After 4 weeks, the explants were placed in the middle of PS containing MSO plus 5 mg / l of RZ, 0.3 mg / l of cefataxime per liter. The shoots were then removed and placed in culture cones containing PM medium (4.4 grams of MS salts, 30 g of sucrose, 0.17 g of NaH2PO4H2O, 2 ml of thiamine HCl and 0 1 g of Inositol in a volume of 1 liter. , pH 6 0 and Gelpte agar) The plants were then placed in the soil, hardened and analyzed by nPTil ELISA to identify the transgenic plants. The transgenic PAP plants were then analyzed by ELISA for PAP expression. Transgenic potato plants expressing NT144, NT145 and NT146 were identified by ELISA. Transformation frequencies were not affected when constructs containing PAP mutants were used and transgenic plants expressing PAP mutants were phenotypically normal, indicating that the expression of the mutant PAPs is not toxic to potato. Example 5 Expression of mutants in Transgenic Turfgrasses The mutant PAPs were treated for constitutive expression in monocotyledons. Perennial greasy grass (Agrostis palustris, Huds.) Which a lawn used in fields, slopes, starting point and golf courses, was used as the monocityl species for transformation In order to constitute an expression vector for monocotyledons, it was created NT168 cloning the promoter and the first codon of the maize ubiquitin gene (Toki et al., Plant Physiol., 100: 1503-1507, 1992) in pMON969. PMON969 was digested with Hindlll and Bgill to remove the promoter region CaMV 35S The plasmid pAHC20, containing the ubiquitin promoter and the first intron (Toke et al., 1992) was digested with Hmd lll and BamHl to isolate the 2016bp h? Ndlll / BamH1 fragment that chose the Hmdlll / BglII fragment from pMON969 to generate NT168. The monocot expression vectors containing the mutant PAP cDNAs were used. then in the transformation together with pSL12011, which contains the selectable marker, the bar gene (Hartmann et al., 1994 Biotechnology 12 919-923) Turf transformation was carried out using two different methods, transformation of biolistics using the particle gun and by protoplast transformation as described below. Embryogenic callus cultures were initiated from the sterilized seeds on the surface of 7 perennial grasses. 'Cobra', 'Emerald', 'PennLmks', 'Providence', 'Putter', 'Southshore' and 'SR1020' and were used in the biolistic transformation, as described in Hartmann and Bulls, Biotechnology 12: 919-923 ( 1994). The means of initiation of calluses were basal medium of MS and vitamins of MS, supplemented with 100 mg L "1 myo-inositol, 3% of sucrose and 150 mg I" 1 of asparagine and 2 mg of L "1 of casein hydrolyzate, 6.6 mg L'1 of dicamba and O 5 mg L'1 of b-BA for MMS Media solidified in 0 2% of Phytagel (Sigma) After 4 to 6 weeks in the dark at 25 ° C, Embryogenic callus lines were selected and transferred to fresh medium. Embryogenic callus culture suspensions were established by adding 1 to 2 g of callus to 250 ml flasks with 50 ml of liquid medium, incubated in the dark at 25 ° C with agitation at 120 rpm and subcultured twice a week. The plates were prepared for particle bombardment by placing 1 ml of cells in suspension in 5 5 cm filter discs in plates containing 2D MSA medium with the addition of 04M mannitol Plates were prepared 20 h before bombardment and kept in the dark Gold particles were prepared by heating at 95 ° C in 100% ethanol for 30 minutes, centrifuged briefly and resuspended in fresh ethanol. The particles were treated with sodium for 10-30 minutes, centrifuged briefly and resuspended in fresh ethanol. The particles were treated with sound for 10-30 minutes in a water bath, were washed 3 times in distilled sterile water and resuspended in water DNA samples consisting of 50 μl (5 mg) of gold suspensions, 10 μg of white DNA, 50 μl of 25M CaCl2 and 20 μl of 0.1M of spermidine, they were stirred, centrifuged and resuspended in ethanol. The washing with ethanol was repeated for a total of 3 times. The final pellet was resuspended in 30 μl of ethanol and 5 μl of DNA solution were used per shot. The bombardment was carried out using the HE Biolistic Delivery System, Bio-Rad PDS-1000 at 77 33 kg / cm3 The calluses of the bombardment experiments were seeded on plates in MSA2D medium containing 2 to 4 mg / l for bialaphos for selection 3 to 4 days after bombardment and continued for 8 weeks without transfer. After 8 weeks in plaque selection, calluses were transferred to MS medium without hormones for regeneration. The regenerations appeared within 2-8 weeks. The shoots were transferred to culture cones containing MS medium and the roots appeared within 2-4 weeks. For the protoplast transformation, protoplast isolation was carried out four days after the subculture Cells were incubated with filter-sterilized enzyme solution containing 1% (w / v) of Cel ulosa Onozuka RA (Yakult Pharmaceutical Co. LTD), 0 1% of Pectohasa Y-23 (Seishm Pharmaceutical Co. LTD) and 0 1% of M ES (2-N-morpholinoethane sulfonic acid) (Sigma) in culture medium (MSA2D or MMS with 5% mannitol) for 4 hours at 28 ° C with stirring at 50 rpm. Approximately 1 g of fresh weight suspension cultures were treated with 10 ml of enzyme solution. The protoplasts were filtered through Mirechoth and washed twice with culture medium containing 5 mannitol. Mannitol was used as an osmotic stabilizing agent. Protoplasts were cultured using a layered feeding system (Rhodes et al., 1988). Filtered, washed protoplasts were pipetted onto a black nitrocellulose membrane (Lee et al., 1989) were placed on a feeding layer of the suspension cells that were diffused onto the 5% manila culture medium. Then, the membranes with protoplasts were transferred to a fresh feeder layer on 3% mannitol culture plates. The protoplasts were removed from the feed layer 2 weeks after isolation. The efficiency for seed plates was determined by dividing the number of colonies. 3 weeks after isolation by the total number of protoplasts seeded on plates The plants were regenerated by placing callus derived from protoplasts on the MS medium without hormone or with 1 mg L "1 of orquinetma 6-BA After 4 to 5 weeks, the shoots are transferred to Plantcon® with MS medium without containing hormone to form roots. The protoplasts were transformed using PEG following the protocol of Negrutiu et al. (1987), or 1 70 volt cm-1 electroporation using a Gene-Pulster (BioRad) In P EG experiments, the newly isolated protoplasts were resuspended at a density of 1 x 107 protoplasts per ml in mannitol at 5% containing 15 mM MgCl 2 and 0 1% M ES Approximately 0 3 ml of protoplasts were incubated with 20 to 40 μg of plasmid DNA and 13% of PEG for 10 to 15 min, diluted in steps and resuspended in culture medium with 5% mannitol (pH 5 8) after centrifugation. In electroporation experiments, protoplasts were resuspended at a density of 5 x 10 6 protoplasts per ml in electroporation pH-regulating solution sterilized with cold filter containing 52 g L 1 KCl, or 835 g L -1 CaCl, 0 976 g L "1 of MES and 5% of mannitol at pH 58 Approximately 08 ml of protoplasts were mixed with 20 μg of DNA per inversion, electorporated at 170 volts cm" 1 and placed on ice for 15 minutes, then diluted to a total of 3 ml with culture medium containing 5% mannitol The selection with 4 mg L "1 of bialaphos was started 16 days after isolation and transformation of protoplasts Resistant colonies in MS medium without hormone, with 6-BA or kinetin was described earlier The shoots were transferred to PlantconsR for root formation A commercial formulation of bialaphos under the trade name Herb? aceR (Meiji Seika Kaishya, LTD) was used in greenhouse herbicide trials Herbicide regimens for Herb? aceR they were established using control plants and were based on the commercial regime of 084 kg Al / hectare (1x the field regime) The herbicide was applied to all the shoots on the ground with an artistic brush at a rate of 120 I per floor The floor dimension is 0 1431 m2 and contains 96 or 24 plants EXAMPLE EXPRESSION OF POT MUTANTS IN TRANSGENIC TOBACCO PLANTS AND VIRAL INFECTION RESISTANCE A. Expression of PAP mutants in transgenic tobacco To determine if the enzymatic activity of PAP is required for its antiviral activity, the cDNA encoding the active site mutant NT123-2 was cloned into the plant expression vector pMON8443 after removing the wild-type PAP insert, to generate NT144, as described in Example 2 Similarly, the A DNc encoding the C-terminal deletion mutant, NT124-3 was cloned into pMON8443 to generate NT 145 and NT 147, as described in Example 2 The expression of the PA P mutants were directed by the promoter of CaMV35S increased NT 144, NT 145 and NT 147 were mobilized in Agrobacterium tumefaciens for transformation into tobacco The frequencies of transformation of Nicotiana tabacum cv Samsun normally varied between 10 to 12% based on the number of transgenic plants obtained by leaf disk (Lodge et al., 1993) The transformation frequency was 13% using NT144 and 11% using NT 145 Transgenic plants expressing the mutant active site mutant C-terminal deletion were phenotypically normal They grew at the same rate as wild type plants and did not show chlorosis or speckled on their leaves, indicating that the expression of PAP mutants was non-toxic for transgenic tobacco. These results are in contrast with previously reported results (Lodge et al., 1993) in which the transformation frequencies of N Tabacum were reduced to 0 7% when they use a vector that contains PAP (pMON8443) and to 3.7% when a vector containing PAP-v (pMON8442) was used, both of which are enziomatically active (Lodge et al., 1993). Lodge and others did not recover any transgenic plant expressing high levels of PA P and the transgenic plants expressing high levels of PAP-v showed reduction of growth, chlorosis and mottling in their leaves.

The regenerated transgenic plants are first analyzed for the expression of neomycin phosphotransferase (NPTII) by ELISA. The NPTII positive plants were then analyzed for PAP expression by ELISA and immunoblot analysis. Eleven different transgenic plants expressed detectable levels of the active site mutant by ELISA. Plants expressing the PAP active site mutant produced a 29 kDa protein that comigrated with mature PAP, indicating that active site mutant PAP is completely processed to the mature form in transgenic plants (data not shown). The transgenic plants expressed significantly higher levels of the active site mutant than the plants expressing PAP or PAP-v. No bands corresponding to PAP were detected in wild-type work or in transgenic tobacco expressing β-glucuronidase. The C-terminal deletion mutant was expressed at significantly lower levels than the active site mutant. B. Antiviral activity of the active site mutant in transgenic tobacco. In order to determine if the transgenic lines expressing the active site mutant PAP are resistant to virus infection, progenies from plant lines transformed with VPX were inoculated. Autofertilized progenies were sifted for the presence of PAP by ELISA. The levels of PAP in the progeny of the transgenic lines varied depending on the age of the plants and growth conditions. The degree of variability in PAP levels was similar to that previously reported for transgenic lines expressing PAP or v-PAP (Lodge et al., 1993) Ten progenies of each transgenic line expressing the active site mutant PAP (144-1 and 144- 7), PAP-v (26139-19), PAP (33617-11) and 10 untransformed tobacco plants were inoculated with 1 μb / ml The development of symptoms on inoculated and systemic leaves was monitored visually each day up to 21 days after Inoculation In addition, the discs of the inoculum and the first, second and third systemic leaves of each plant were sampled 12 days after the inoculation in order to quantify the rephcation and diffusion of the virus. As shown in Table VII, the transgenic plants expressing PAP-v or wild-type PAP did not develop lesions in the leaves inoculated at nine days after inoculation In contrast, the transgenic plants expressing the active site mutant had so much s lesions on their inoculated leaves as the control plants. ELISA analysis of systemic leaves showed that 9% of wild type tobacco plants were systemically infected by VPX at 12 days after inoculation, whereas only 30 and 40% of transgenic plants expressed PAP and PAP-v , respectively, showed systemic VPX infection. In contrast, 100% of the plants expressing the active site mutant were infected with VPX (Table VII). Similar results were obtained when the plants were classified again at 21 days after inoculation Table VII PAP line PAP level Number of% of plants that Plant expressed ((nngg // mmgg)) aa lleessiioonneess "" mmuueessttrraann infec. Systemic TS 0 77 +/- 12 90 26139-19 PAP-v 5.6 +/- 2.6 0 ** 30 ** 33617-11 PAP 0.6 +/- 0.02 0 ** 40 * 144-1 E176V 43.8 +/- 4.8 78 +/- 16 100 144-7 E176V 46.2 +/- 5.6 72 +/- 11 100 a PAP levels were quantified by ELISA after taking four leaf discs from twenty plants per line. Average values of SD +/- are shown. "Ten plants of each line were inoculated with 50 μl of 1 μg / ml VPX in two leaves per plant, the number of lesions was counted 9 days after inoculation, mean values of +/- SD were shown c Three were taken Leaf discs of the 1st, 2nd and 3rd leaves were infected systemically at 12 days after inoculation and viral antigen levels were quantified by ELISA The amount of total protein in each extract was quantified using the BCA (Pierce). ** Significantly different from the wild type at the 1% level * Significantly different from the wild type at the 5% level. To determine whether transgenic plants expressing higher levels of the active site mutant are also susceptible to VPX infection, the homozygous progeny (generation R2) of the transgenic link 144-12, which expressed higher levels of the site mutant PAP were inoculated with 0 5 μg / ml of VPX As shown in the following Table VI II, the trangenic lines producing high levels of the active site mutant had the same numbers of lesions as the wild type tobacco plants in their inoculated leaves, while the progeny of transgenic plants that expressed PAP-v or PAP had significantly lower numbers of lesions The ELISA analysis of the systemic leaves showed that at 21 days after inoculation, 90% of tobacco plants type wild and 100% of the transgenic plants expressing the active site mutant were infected with VPX Found, the plants expressing P AP or PAP-v had minor lesions on the inoculated leaves and lower percentages of these plants were systemically infected with VPX. In additional experiments, the progeny of seven different transgenic lines expressing the active site mutant were analyzed for their susceptibility to VPX infection none of these lines showed resistance to VPX (data not shown) Table VI II Susceptibility of transgenic tobacco plants expressing the C-terminal deletion mutant (W237Stop) to VPX infection Table VI II PAP line Number level of plants% what Plant expressed PAP (ng / mg) to lleessiioonneess show systemic infection 12 dpi 21 dpi TS 24 +/- 15 90 90 26139-19 PAP-v 9.6 1 +/- 2 ** 10 ** 30 ** 33617-11 PAP 1.6 11 +/- 4 ** 10 * 40 * 144-12 E176V 1500 23 +/- 13 100 100 147-19 W237Stop 4.5 12 +/- 10 ** 20 60 145-13 W237Stop 4.4 6 +/- 4 ** 30 ** 60 a PAP levels were quantified by ELISA in the primary transgenic plants. b From eight to ten plants of the homozygous progeny (R2 generation) of each transgenic line were inoculated with 50 μl of 0. 5 μg / ml of VPX in two leaves per plant. The number of lesions was counted 12 days after inoculation. The mean values of +/- SD were shown. c Two discs of leaves of the first and second leaves infected systemically of each plant were taken 12 days after inoculation and two discs of leaves were taken from the third and fourth infected leaves systemically at 21 days after inoculation. Levels of viral antigens were quantified by ELISA. The amount of total protein in each extract was quantified using the BCA equipment (Pierce) ** Significantly different from the wild type at the 1% level * Significantly different from the wild type at the 5% level. C. Antiviral activity of C-terminal deletion mutant in transgenic tobacco. In order to determine if the transgenic lines expressing the C-term inal deletion mutant are resistant to virus infection, homozygous progeny (R2 generation) of transgenic lines 145-12 and 147-19 expressing the deletion mutant C -terminal (W237Stop) were inoculated with 0.5 μg / ml VPX and the number of lesions were counted at 12 days after i noculation. As shown in Table VI II above, plants of transgenic lines 145-13 and 147-19 had significantly lower numbers of lesions on their inoculated leaves compared to wild type plants. At 12 days after inoculum, only from 20 to 30% of the plants of the transgenic lines 147-19 and 145-2, respectively, showed systemic symptoms and contained VPX antigen by ELISA, while 90% of the control plants were infected with VPX. At 21 days after inoculation, there was an increase in the percentage of plants of lines 147-19 and 145-13 that showed systemic symptoms. As observed in previous tests, the progeny of transgenic lines expressing PAP-v and PAP were protected from VPX infection. Infected plants expressing the C-terminal deletion mutant (W237Stop), PAP or PAP-v showed more moderate symptoms compared to infected wild type plants or transgenic plants expressing the active site mutant (E176V) ELISA analysis was used for quantify levels of viril antigen in transgenic plants and wild type plants at 21 days after inoculation. The levels of VPX antigens were lower in plants of lines 147-19, 145-13 and 33617-11 compared to levels of antigens of wild type plants. The percentages of infected plants did not change when they were classified again at 4 weeks after inoculation. In additional experiments, a total of these different transgenic lines expressing the C-terminal deletion mutant were analyzed for their susceptibility to infection by VPX and four of these lines showed resistance to VPX infection (data not shown) Example 7 ANALYSIS OF FUNGAL RESISTANCE IN TRANSGENIC PLANTS EXPRESSING PAP AND PAP MUTANTS Seeds from plants of tobacco lines expressing PAP, PAP mutants and seeds were used. wild type tobacco plants. Four weeks later the seeds of germination were transferred to the growth chamber and developed in the sterile soil at 25 ° C, 80% relative humidity and photopepod of 16 hours. Recombinant constructs with chimeric PAP genes were introduced into Agrobacterium tumefaciens via tpaparenteral equalization. The Agrobacterium containing the modified PAP genes was used to transform Nicotiana tabacum cv. Samsun R2 transgenic plants resistant to kanamycin were self-pollinated and the seeds of R3 were used in the experiments Transgenic plants of lines 33617 (expressing wild type PAP), NT144 (expressing active site mutant PAP), NT145 were used and NT147 (both expressing C-terminal deletion mutant PAP) Four-week-old transgenic and control seeds were transplanted in sterile soil and inoculated with fungal pathogenic fungal Rhizoctoma solani. The development of disease symptoms was observed for two weeks. Weeks and seed mortality regimes were calculated Plants that survived the fungal infection were transplanted into individual containers and tissue samples were taken for further analysis After inoculation with R solani, the control tobacco seeds were separated very quickly by fungal pathogen The disease progressed rapidly, affecting more than 30% of Control seeds in six days after inoculation. In contrast, the susceptibility of transgenic lines to the infection was significantly lower. Six days after the inoculation, only 9.5% of the seeds of the lines with wild-type PAP, about 20% of seeds of the C-terminal truncated PAP line and 23% of the seeds of the active site mutant line they were affected. The number of seeds that survived at different time points is shown in the following Table IX. All transgenic lines exhibited a delay in appearance of disease symptoms and a lower mortality regimen. TABLE IX Progression of disease in PA P lines of transgenic tobacco with Rhizoctonia solani.

In a separate experiment, with a different strain of Rhizoctonia solani, the disease progressed very rapidly, essentially killing most seeds in five days. The survival of seeds after two weeks of growth in the infected soil is shown in the following Table X. Notably, the control plants, although they do not die at the time point of classification, atrophied extremely and exhibited symptoms of very severe disease. severe. In contrast, seeds in transgenic lines with truncated PAP showed much less tissue damage.

TABLE X Survival of transgenic tobacco lines with different PA P genes in Rhizoctonia solani resistance test Analysis of surviving plants Cell line protein analysis of transgenic lines was performed by separating proteins on 10% SDS-PAGE using a mini-PROTEA NII electrophoresis cells (Bio-Rad) and the proteins were transferred into nitrocellulose membrane using apparatus. Bio-Rad Trans-Blot semi-dry electrophoretic transfer according to the manufacturer's instructions. Western blot analysis was performed using PAP IgG or PR 1 a monoclonal antibodies. The detection was done by increasing the chemoluminescence using DuPont renaissance equipment.

Western blot analysis of cell extracts from trapsgenic plants showed that the PAP gene was expressed in all the plants that survived the fungal infection. The amount of PAP produced differed between individual plants. further, the apoplastic fluid was isolated from the same plants and extracellular proteins were analyzed by staining the native gel with silver nitrate. The expression of proteins related to pathogenesis (RP) was detected in plants expressing the antiviral protein genes of grana. Western graph analysis also showed elevated levels of PR 1 a in surviving plants. Significant reduction of fungal disease symptoms in transgenic tobacco lines was observed by expressing grana antiviral protein. As shown in tables IX and X, the transgenic lines with PAP exhibited a higher percentage of seed survival after infection by R. solani. In addition, the progression of the disease represented by the seed mortality regimen was also slower in transgenic PA P lines. Transgenic line 33617, which expressed the wild-type PAP, as well as transgenic tobacco lines that contained mutant forms of PAP, NT144-12 (which expresses the mutant PAP of active site), NT145-15 and NT147-19 (which expressed a truncated form of PAP, lacking 25 C-terminal amino acids) showed resistance to fungal infection. The expression of mutant PAP genes in tobacco proved to have no detectable phenotypic effect but surprisingly led to the constitutive expression of several pathogenesis-related proteins. Some of the induced genes are known for their antifungal activity. In view of this observation and while not intending to be limited to any particular theory of operation, the Applicant thinks that the resistance to infection of Rhizoctonia solani by tobacco lines expressing PA P mutant genes of the present invention can be explained by the action of host defense genes and that resistance to fungal infection in plants expressing PA P can be conferred by the double action of transgene of PAP and a number of host genes, constitutively expressed in transgenic tobacco. The applicant further believes that the induction of these plant defense genes also serves to protect the transgenic plants against other pathogens such as bacterial pathogens. EXAMPLE 8 AIS LAMINATION OF MULTIPLE MUES OF PA PA BY MUTAGEN IS IS C ROMOSOMA L AND IS LESSON IN DU RAS CAMERA A. Isolation of PAP Mutants Chromosomal mutagenesis and selection were used to isolate yeast mutants which allows the cells develop in the presence of PAP. The constitutive expression of PAP in S. cerevisiae is usually lethal. Therefore, the PAP gene was placed under the control of GaL1 promoter that induces galactose. This allows the cells carrying the plasmid with the PAP gene to develop normally in glucose when the expression of PAP is repressed, but it kills the cells developed in galactose when PAP is expressed. We had the advantage of having a PAP expression system and the toxicity of PAP to normal yeast cells, to isolate mutants that can grow in the presence of PAP The yeast cells carrying a plasmid with the wild-type PAP gene (NT123) were developed to the early log phase, pH 7 0, at a density of 1 x 108 cells / ml Three aliquots of 1 ml were removed and used for mutagenesis. Mutagenesis was performed using 5 μl or 25 μl of ethyl methanesulfonate (MSE). A non-mutagenized aliquot was maintained as the control to examine the frequency of spontaneous mutants. After the addition of EMS, the cells were incubated at 30 ° C for 1 hour, with gentle shaking. The mutagenesis was terminated by the addition of sodium thiosulfate at 55. The cells were then seeded on plates of uracil deficient plates with glucose 2% and incubated at 30 ° C. Based on the number of colonies that grew in the plates of the mutagenized cells against the unmutagenized control, 35% and 98% of the cells were killed with 5 μl and 25 μl of EMS, respectively. These colonies were plated in double plates for deficient medium of uracil with 2% galactose and sifted for colonies capable of growing in the presence of PAP. Approximately 13, 5000 colonies were sifted and 9 colonies were obtained, which were able to grow in galactose. The mutants were tested to see if the mutations were chromosomal or ligated plasmid. Plasmid segregation was performed in the mutants by cell growth for approximately 50 generations in non-selective medium (YEPD), planted in YEPD, followed by plating in duplicate, the colonies, having plasmid loss, not can develop in uracil deficient medium Cells secreted in plasmid were transformed with fresh NT 123 and examined for their ability to develop in uracil deficient medium with 2% galactose. Mutants that retained the ability to grow in galactose are chromosomal mutants which did not grow in mutations that have plasmids carrying galactose. Mutants that have plasmid were further characterized by performing immunoblot analyzes on whole cell extracts of the cells expressing these plasmids. This analysis revealed that 2 of the 7 plasmid mutants were expressed in a truncated form of PAP. The other 5 mutants did not express a truncated form of PAP. The 2 mutants that expressed truncated PAPs were examined by sequence analysis to determine the sites of the mutations. One mutant, NT 185, had a mutation site at the C terminus, changing Lys210 (AAG) to a stop codon (TAG), resulting in a deletion of approximately 3.5 kDa. The other mutant, NT 187 had a change in the N terminus, changing Tyr 16 (TAC) to a stop codon (TAA) and then was able to restart in Met39, resulting in a 24.8 kDa protein. B. The construction of the expression vector of E. coli To express the PA P madu suppressed N-terminal cells in £. coli, the A DN of plasmid NT187 was digested with restriction enzymes BstY1 and H ind l l l and the fragment around 830 bp was purified using the Gene Cleaning equipment (bio 101). The purified fragment was ligated to the expression vector of £ coli, pQE31 (QIAGEN Inc.), which was digested with BamH and Hindlll and then treated with alkaline phosphatase. The resulting plasmid, NT190, contained the N-terminal deletion mutant PAP in the expression vector of £ coli pQE31 C Expression of PAP mutants in £ coli NT 190 was isolated from DH 5a cells of £. coli and was transformed into the expression host, M15 col l (pR EP4) M19 cells containing NT190 were cultured in 50 ml of LB medium containing 2% glucose, 100 pg / ml ampicilm and 50 pg / ml of kanamycin at 37 ° C overnight with vigorous shaking The following day, a large culture was inoculated (500 ml of LB medium, containing 2% glucose, 100 μg / ml ampicillin, and 50 μg / ml kanamycin) and it was developed at 37 ° C with vigorous agitation until A6oo reached 0.9. I PTG was added to a final concentration of 2 mM and the culture was incubated at 37 ° C for 5 hours. Cells were collected by centrifugation at 4,000xg for 10 min and stored at -70 ° C. D. Purification of N-terminal suppressed PAP One gram of cells of £. coli was thawed and resuspended in 5 ml of pH A buffer (6M guanidmium hydrochloride, 0 1 M sodium phosphate and 0 01 M Tps-HCl, pH 8.0) and stirred for 1 hour at room temperature. The lisato of £. coli was centrifuged at 10,000 xg for 15 minutes at 4 ° C and the supernatant was collected. Two ml of a slurry of 50% Ni-agarose resin (QIAGEN Inc.), previously equilibrated in pH A buffer was added. After stirring at room temperature for 45 min, the resin was carefully loaded onto a chromatography column. 'poly-prep' (Bio-Rad) The column was loaded with 20 column volumes of buffer solution of pH A and 10 column volumes of buffer solution of pH B (8 M urea, 0.1 M sodium phosphate and 001M Tps-HCl, pH 8.0) Proteins that did not bind to the resin were washed with 20 column volumes of buffer solution of pH C (8M urea, 0 1M sodium phosphate and 0.01 M Tp-HCl, pH 63). Finally, the bound protein was eluted with 5 ml of pH C buffer containing 250 mM of imidazole and analyzed by SDS-PAGE and Western graph analysis. E. N-terminal suppressed PAP antiviral activity To determine if N-thermal suppressed PAP had antiviral activity, wild-type tobacco plants were inoculated with 1 μg / ml of VPX in the presence or absence of N-terminal deletion mutant. purified from £. coli The concentration of PAP was determined by ELISA and by SDS-PAGE. 15 ng / μl of mutant PAP were applied to tobacco leaves in the presence or absence of 1 μg / ml of VPX. As shown in Table XI, tobacco plants inoculated with VPX in the presence of 1.5 or 15 ng / μl of N-terminal suppressed PAP showed minor lesions in their inoculated leaves compared to plants inoculated with VPX in the absence of mutant PAP. In addition, as shown in Table XII, none of the plants inoculated with VPX in the presence of 15 ng / μl mutant PAP and only 13% of plants inoculated with VPX in the presence of 1.5 mg / μl of mutant PAP showed systemic VPX symptoms , whereas 100% of the plants were inoculated with VPX in the presence of buffer alone showed symptoms of systemic VPX. These results indicate that the exogenously applied N-terminal suppressed PAP protects tobacco against VPX infection and is therefore antiviral. Table XI Susceptibility of tobacco plants to VPX in the presence of PAP suppressed N-terminal applied exogenously Applied protein * VPX # mean of lesions (ng / μl) (ng / μl) none 20 +/- 16 PAP (1.5) 2 +/- 2 PAP (15) 2 +/- 2"Two leaves of each plant were inoculated with 50 μl of VPX (1 μg / ml) in the presence of 1.5 or 15 ng / μl of N-terminal suppressed PAP, Twelve plants were inoculated with VPX in the presence of buffer pH alone ( "none") and 8 plants were inoculated with VPX in the presence of 50 μl of 1.5 or 15 ng / μl of mutant PAP.

EBI number of injuries counted 7 days after inoculation. The mean values +/- SD are shown. Table XII Percentage of plants showing systemic symptoms in the presence of N-terminally applied PAP suppressed exogenously. Applied protein3 VPX% of plants that (ng / μl) (ng / μl) show systemic symptoms "none 1 100 PAP (1.5) 1 13 PAP (15) 1 0 Two leaves of each plant were inoculated with 50 μl of VPX (1 μg / ml) in the presence of 1.5 or 15 ng / μl of N-terminal suppressed PAP. Twelve plants were inoculated with VPX in the presence of buffer solution alone ("none") and 8 plants were inoculated with VPX in the presence of 50 μl of 1.5 or 15 ng / μl of mutant PAP. "Systemic symptoms were classified 11 days after inoculation, co-pending patent application Serial Nos. 08 / 500,611 500,694, of the applicant of July 11, 1995, are hereby incorporated by reference in their entirety." INDUSTRIAL APPLICABILITY The mutant PAP of the present invention they confer broad spectrum resistance to viruses and fungi when they are expressed in plants.

They provide this resistance efficiently in that a minimum number of transgenes is required. The mutant PAPs are also substantially non-phytotoxic and non-cytotoxic and therefore provide a distinct and unexpected advantage over the use of wild-type PA P Transgenic plants capable of expressing the mutant PAPs of the present invention are substantially more resistant to variety of pathogens, including viruses, fungi, bacteria, insect nematodes and therefore enjoy comparatively high yields of crops. All publications and patent applications mentioned in this specification indicate the level of experience of those experts in the field. All of these publications and patent applications are hereby incorporated herein by reference to the same description as if each individual publication or patent application was specified individually to be incorporated by reference. Vanas modifications of the invention described herein will be evident to those experts in The matter . Such modifications are intended to fall within the scope of the appended claims LIST OF SEQUENCES (1) GENERAL INFORMATION, (i) APPLICANT: Rutgers, The State University Piscataway, NJ 08855 (ii) TITLE OF THE INVENTION: Antiviral Protein Mutants of Grana (iii) NUMBER OF SEQUENCES: 2 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: Lerner, David, Littenberg, Krumholz & Mentlik (B) STREET: 600 South Avenue West (C) CITY: Westfield (D) STATE: NJ (E) COUNTRY: USA (F) ZP: 07090-1497 (v) LEADABLE COMPUTER FORM: (A) MIDDLE TYPE: Flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.25 (vi) CURRENT REQUEST DATA: (A) NUMBER OF APPLICATION: PCT (B) DATE OF SUBMISSION: 11-JUL-1995 (vii) PREVIOUS APPLICATION DATA: (A) NUMBER OF APPLICATION: US 08 / 500,611 (B) DATE OF SUBMISSION: 11-JULY-1995 (vii) DATA OF PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 08 / 500,694 (B) SUBMISSION DATE: JULY 11, 1995 (viii) APPORTER / AGENT INFORMATION: (A) NAME: Foley, Shawn P. (C) NUMBER REFERENCE / DOCUMENT: OCIRS 3.4-034CIP (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 908-5000 (B) TELEFAX: 908-654-7866 (C) TELEX: 139-125 (2) INFORMATION FOR SEC ID NO : 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1379 pairs d e base (B) TYPE: nucleic acid (C) HILATURE: double (D) TOPOLOGY: linear (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 225..1163 (ix) FEATURE: (A) ) NAME / KEY: mutation (B) LOCATION: replacement (233, "a") (ix) CHARACTERISTICS: (A) NAME / KEY: mutation (B) LOCATION: replacement (349, "g") (ix) FEATURE: (A) NAME / KEY: mutation (B) U BICATION: replacement (435, "c") (xi) SEQUENCE DESCRITION: SEQ ID NO: 1 CTATGAAGTC GCCTCAAAGC ATATACAGGC TATGCATTGT TAGAAACATT GATGCCTCTG 60 ATCCCGATAA ACAATACAAA TTACACAATA AGATGAC? TA CAAGTACCTA AACTGTGTAT 120 GGGGGGAGTGA AACCTCACCT CCTAAAAAAA CG7TGTAAGA AAAAAAGAAA GTTGTCAG7T 180 AACTACAGGG CCAAACTATT GGAACTACC? AGTAGGAAGG GAAG ATG AAG TCG ATG 236 Ket Lys Ser Ket 1 C7T CTG CTC ACÁ ATA TCA ATA TCG CTC ATT CTT CCA CCA ACT TCA. ACT 284 Leu Val Val Thr lie Ser Trp Leu lie Leu Wing Pro Thr Ser Tbr 5 10 15 20 TGC GCT ATA AT AT ATC ATC AAT CTT CGA AGT ACC ACC ATT AGC 332 Trp Ala Val Asn Thr lie lie Tyr Asn Vßl Cly Be Thr Thr lie Ser 25 30 35 AAA TAC CCC ACT TTT CTG AAT CAT CTT CCT AAT CAA GCC AAA CAT CCA 380 Lys Tyr Aa Thr Phe Leu Asp Asp Leu Arg Asn Clu Ala Lys Asp Pro 0 45 50 ACT TTA AAA TCC TAT CCA ATA CCA ATC CTC CCC AAT ACA AAT ATA AAT 28 Ser Leu Lys Cys Tyr Cly lie Pro Ket Leu Pro Asn Thr Asn Thr Asn 55 60 65 CCA AAC TAC GTC TTC CTT CAC CTC CA CCT TCA AAT AAA AAA ACC ATC -7 Pro Lv§ Tyr Val Leu Vai Clu Leu Clr »Cly Ser ASn Lys Lys Thr le 7C 75 80 ACÁ CTA ATC CTC ACÁ CGA AAC AAT TTG TAT GTG ATC CCT TAT TCT CAT 524 Thr Leu Ket Leu Arg Arg Asn Asn Leu Tyr Val Met Cly Tyr Ser Asp 85 90 95 100 CCC TTT CAA ACC AAT AAA TCT CCT TAC CAT ATC TTT AAT CAT ATC TCA 572 Pro Phe c: u Thr Ain Lys Cys Arg Tyr His lie Pha Asa Asp lie Ser 105 no iij "tirst ,, CCT ACT CAÁ CCC CAÁ GAT« A CAG ACT ACT CTT TCC CCA AAT CCC AAT 62C Gly Thr Glu Axg Gln Asp Val Glu Thr Thr Ltu Cys Pro Asn Wing Asn 120 125 130 TCT CGT CTT AGT AAA A? C ATA AAC TTT GAT ACT CGA TAT CCA ACA TTC 668 Ser Are Val Ser Lys Asn lie? Sn Phe Asp Str Arg Tyr Pro Thr Lau 135 1 * 0 1 * 5 GAA TCA AAA GCG GGA GTA AAA TCA ACA AGT CAC CTC CATC CTC GGA ATT 716 Glu Ser Lys Wing Gly Val Lys Ser Arg Ser Gln Val Gln Leu Gly lie 150 155 160 CAA ATA CTC GAC ACT AAT ATT GGA AAG ATT TCT GGA GTG ATG TCA TTC 76 ¿Gln lie Leu Asp Ser Asn He Gly Lys He Ser Gly Val Met Ser Phe 165 170 175 180 ACT GAG AAA ACC CAA GCC GAA TTC CTA TTC CTA CCC ATTA CTA ATC CTA 812 Thr Clu Lys Thr Clu Wing Clu Phe Leu Leu Val Wing Gln Ket Val 185 190 195 TCA CAC CCA CCA AC TTC AAC TAC ATA CAG AAT CAG GTG AAA ACT AAT 860 Ser Clu? Wing Arg Phe Lyß Tyr He Clu Asn Cln Val Lys Thr Asn 200 205 210 TTT AAC ACÁ CCA TTC AAC CCT AAT CCC AAA CTA CTT AAT TTG CAA GAG 908 Phe Asn Arg Ala Phe Asn Pro Aso Pro Lys Val Leu Asn Leu Cln Clu 215 220 223 ACÁ TCC CCT AAC ATT TCA ACÁ CCA ATT CAT CAT CCC AAC AAT CCA CTT 956 Thr Trp Cly Lys He Ser Thr Ala He His Asp Ala Lys Asn Cly Val 230 235 240 TTA CCC AAA CCT CTC CAC CTA CTC CAT GCC ACT CCT GCC AAC TCG ATA 1004 Leu Pro Lys Pro Leu Clu Leu Val? Sp? The Ser Cly Wing Lys Trp He 245 250 255 260 CTC TTC ACÁ CTC GAT CAA ATC AAC CCT CAT CTA CCA CTC TTA AAC TAC 1052 Val Leu Arg Val Asp Clu He Lys Pro Asp Val Wing Leu Leu Asn Tyr 265 270 275 TT CCT CCC CC TCT CAC ACA ACT TAT AAC AÁ AAT CC ATC TT CCT 1100 VaJ C'.and Cly Ser Cys Cln Thr Thr Tyr Asn Cln Asn Wing Met P? T Pro 280 285 290 CAÁ CTT ATA ATC TCT ACT TAT TAT AAT TAC ATC CTT AAT CTT CCT CAT 1148 Cln Leu He Ket Ser Thr Tyr Tyr Asn Tyr Ket Val Asn Leu Gly Asp 295 300 305 CTA TTT CAA CCA TTC TCATCATAAA CAT ATAACC ACTATATATA TATTACTCCA 1203 Leu Phe Clu Cly Phe 31C ACTATATTAT AAACCTTAAA TAACACCCCC TCTTAATTAC TACTTCTTGC CT7TTCCTTT 1263 ATCCTSTTCT TTATTATCCC TTCTATGCTT CTAATATTAT CTACACAACA AGATCTACTC 1323 TC ??? TACTC TTCTTTCAAA T? A ?? CTTCC AATTATCATG CA ?? AAAAAA AAAAAA 1379 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 313 amino acids (B) TYPE : amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Lys Ser Met Leu Val Val Thr He Ser He Trp Leu He Leu Ala 5 10 15 Pro Thr Ser Thr Trp? Val Asn Thr He He Tyr? Sn Val Gly Ser 20 25 30 Thr Thr He Ser Lys Tyr? Thr Pbe Leu? Sn? Sp Leu? Rg? Sn Glu 35 40 45 Wing Lys Asp Pro Ser Leu Lys Cys Tyr Cly He Pro Ket Leu Pro Asn 50 55 60 Thr Asn Thr? Sn Pro Lyß Tyr Val Leu Val Glu Leu Gln Cly Ser? Sn 65 70"7» 5 '80 Lys Lys Thr He Thr Leu Met Leu? Rg Arg Asn Asn Leu Tyr Val Met 85 90 95 Gly Tyr Ser Asp Pro Phe Clu Thr Asn Lys Cys Arg Tyr His He Phe 100 IOS 110 Asr. Asp He Ser Cly Thr Clu Arg Cln Asp Val Clu Thr Thr Leu Cys 115 120 125 Pro Asn Ala Asn Ser Arg Val Ser Lys Asn lie Asn Phe Asp Ser Arg 130 135 140 Tvr Pro Thr Leu Ciu Ser Lys Wing Cly Val Lys Ser Arg Ser Cln Val KS 15C 155 160 Cln Leu Cly He Cln He Leu Asp Ser AS? lie Cly Lys He Ser Cly 165 170 175 Val f.et Ser Phe Thr Clu Lys Thr Clu Wing Clu Phe Leu Leu Val Wing 180 185 19C le Cln f.et Val Ser Clu Aia Aiß Arg Phe Lys Tyr lie Clu Asn Cln 195 200 205 Val Lys Thr Asn Phe Asn Are Ala Pne Asn Pro Asn Pro Lys Val Leu 210 215 220 A «n Leu Cin Clu Thr Trp Cly Lys lie Ser Thr Ala He His ASp Ala 225 230 233 240 l-ys. sn Cly Val Leu Pro Lys Pro Leu Clu Leu Va l Asp Ala Ser Cly 2 * 5 250 255 ? the Lys Trp He Val Leu? rg Val? sp Cl? He Lys Pro? Sp Val? Lß 260 265 270 Leu Leu? Sn Tyr Val Gly Gly Ser Cys Gln Thr Thr Tyr Asn Gln Asn 275 280 285 Wing Met Phe Pro Cln Leu lie Met Ser Thr Tyr Tyr? Sn Tyr Met Val 290 295 300? Sn Leu Gly? Sp Leu Phe Clu Gly Pbe 305 310

Claims

CLAIMS 1 A PAP mutant having reduced phytotoxicity compared to mature wild type PAP or variant PAP and exhibiting antiviral or antifungal activity in plants 2 The PAP mutant of claim 1 which differs from Wild-type PAP substantially in that it has altered compartmentalization in vivo 3 The PAP mutant of claim 2, which has a conservative amino acid substitution in Gly75 4 The PAP mutant of claim 3, comprising PAP (1-262, Gly75Bal) 5 The PAP mutant of claim 2, having a non-conservative amino acid substitution in Glu97 6 The PAP mutant of claim 5, which is PAP (1-262, Glu97Lys) 7 The mutant of PAP of claim 1 which differs from wild-type PAP substantially in that it is truncated at its C-terminus by at least about 26 to about 76 mature PAP amino acids 8 The PAP mutant of claim 7, which is selected from group of PAP mutants that consist of PAP (1-236), PAP (1-235), PAP (1-234), PAP (1- 233), PAP (1-232), PAP (1-231), PAP (1-230), PAP (1-229), PAP (1-228), PAP (1-227), PAP (1-226), PAP (1-225), PAP (1-224), PAP (1-223), PAP ( 1-222), PAP (1-221), PAP (1-220), PAP (1-219), PAP (1-218), PAP (1-217), PAP (1- 216), PAP (1 -215), PAP (1-214), PAP (1-213), P.AP (1-212), PAP (1-211), PAP (1-210), PAP (1-209), P? P (1-208), PAP (1-207), PAP (1-206), PAP (1-205), PAP (1-204), PAP (1-2031 PAP (1-202), PAP (1 -201), PAP (1-200), PAP (1-199), PAP (1-198), PAP (1-197), PAP (1-196), PAP (1-195), PAP (1-198). 194), PAP (1-193), PAP (1-192), AP (1-191), PAP (1-190), PAP (1-189), PAP (1-188), PAP (1 -187), PAP (1-186), PAP (1-185), and PAP (1-184). 9. The PAP mutant of claim 8, comprising PAP (1-206GIU). 10. The PAP mutant of claim 8, comprising PAP (1-236Lys). 11. The PAP mutant of claim 8, comprising PAP (1-184GIU). 12. The PAP mutant of claim 8, comprising PAP (1-188Lys). 13. The PAP mutant of claim 1, which differs from Wild-type PAP substantially in that it is truncated at its N-terminus by 1 to about 38 amino acid residues. 14. The PAP mutant of claim 13, which is selected from the group of PAP mutants consisting of PAP (2-262), PAP (3-262), PAP (4-262), PAP (5-262), PAP (6-262), PAP (7-262), PAP (8-262), PAP (9-262), PAP (10-262), PAP (11-262), PAP (12-262), PAP (13-262), PAP (14-262), PAP (15-262), PAP (16-262), PAP (17-262), PAP (18-262), PAP ( 19-262), PAP (20-262), PAP (21-262), PAP (22-262), PAP (23-262), PAP (24-262), PAP (25-262). PAP (26-262), PAP (27-262), PAP (28-262), PAP (29-262), PAP (30-262). PAP (31-262). PAP (32-262), PAP (33-262), PAP (34-262), PAP (35-262). PAP (36-262), PAP (37-262), PAP (38-262) and i PAP (39-262). The PAP mutant of claim 1, which exhibits antifungal activity in plants 16 The PAP mutant of claim 15, which is enzymatically inactive The PAP mutant of claim 16, comprising PAP (1-262, Gly176Val) The PAP mutant of claim 1, which includes the N-terminal signal sequence of wild-type PAP 19 The PAP mutant of claim 1, which includes the C-terminal extension of wild-type PAP 20 A DNA molecule that encodes the PAP mutant of any of claims 1-19 A recombinant DNA molecule comprising the DNA molecule of claim 20 operably linked to a promoter capable of functioning in cells 22 The recombinant DNA molecule of claim 21, in wherein said promoter is capable of functioning in a yeast cell 23. The recombinant DNA molecule of claim 21, wherein said promoter is capable of functioning in a cell. that of plants 24. The recombinant DNA molecule of claim 21, wherein said promoter is inducible or constitutively regulatable. A vector stably transformed with the recombinant DNA molecule of claim 24. A protoplast stably transformed with the recombinant DNA molecule. of claim 24 A host stably transformed with the recombinant DNA molecule of claim 24, and which is capable of expressing said recombinant DNA molecule The host of claim 27, which is an E coli cell 29 The host of claim 27, which is a yeast cell 30 The host of claim 29, wherein said yeast cell is a cell of Saccharomvces cerevisiae 31 The host of claim 27, wherein said recombinant DNA molecule is operably linked to a functional inducible promoter in said host of claim 27, wherein said host It is a plant cell. A seed containing the recombinant DNA molecule of claim 27, wherein said seed is capable of developing a plant whose cells are capable of expressing said DNA molecule. 34. A transgenic plant produced by the seed of claim 33. A transgenic plant produced from the protoplast of claim 26 A transgenic plant comprising the DNA molecule of claim 20 and which is capable of expressing said DNA molecule. The transgenic plant of claim 36, which is a monocotyledonous plant. The transgenic plant of claim 37, wherein said monocotyledon is a cereal culture plant. The transgenic plant of claim 36 which is a dicotyledonous silver. A method for identifying a PAP mutant having reduced phytotoxicity compared to mature wild type PAP. or variant PAP, comprising the steps of (a) providing a stably transformed eukaryotic cell with a DNA molecule encoding mutagenized PAP operably linked to a functional inducible promoter in eukaryotic cells or with a DNA molecule encoding non-mutagenized PAP followed by the step of mutagenizing the cell thus transformed- ib) cul tivar said transformed cell in suitable medium; (c) adding an inducer to the medium to cause expression of the DNA molecule; and (d) determining whether said cultured cell survives the induction of expression of said DNA molecule encoding PAP so that the biological activity of PAP encoded by the mutagenized DNA molecule can be determined. The method of claim 40, wherein said step of provision comprises transforming a plurality of eucalypt cells with DNA molecules encoding randomly mutagenized PAP and said determining step comprises determining which transformed cells survive the induction of expression of said DNA molecules so that the biological activity of each of the PAPs encoded by the DNA molecule can be determined The method of claim 40. further comprising the step of analyzing the PAP encoded by the DNA molecule to determine whether it retains the antifungal or antiviral activity of PAP. The method of claim 42, wherein said step of analyzing comprises transferring nucleic acid into a eukaryotic translation system in the presence of predetermined amounts of the PAP encoded by the DNA molecule. 44. The method of claim 43, wherein said translation system comprises a reticulocyte lysate system. 45. The method of claim 44, wherein said translational system comprises a rabbit reticulocyte lysate system. 46. The method of claim 40, wherein said eukaryotic cell is a yeast cell. The method of claim 46 wherein said mvces cerevisiae yeast A purified and isolated PAP mutant identified by e claim 40