TRANSFORMATION OF CUCUMBER BY AGROBACTERIUM TUMEFACIENS AND THE REGENERATION OF TRANSFORMED CUCUMBER PLANTS BACKGROUND OF THE INVENTION Dicotyledonous plants are susceptible to infection from two pathogenic species of Gram-negative soil bacteria, Agrobacterium tumefaciens and Agrobacterium rhizogenes. see review by Bevan and Chilton, 1982, Ann.. R V. genet.. 16:357-384 which results in the diseases known as Crown Gall and Hairy Root, respectively. In each of these diseases the causative agent has been identified as resulting from the transfer and integration of a Segment of the large pTi or pRi DNA plasmid, known as the T-DNA regions Chilton et al. , 1977, Cell 11:263-271; Chilton et al., 1982, Nature. 295:432-434. The identification of several common steps in the pTi and pRi T-DNA transfer mechanism and the genes contained within these T-DNA regions which are responsible for the disease has resulted in the engineering of avirulent Agrobacterium strains, Hepburn et al., 1985, 2. General Microbio.. 131: 2961-2969; Hood et al., 1986, J_. Bacteriology. 168:1291-1301; Vilaine and Casse-Delbart, 1987, Mai. Sen. Genet. 206: 17-23. This information has also led to the construction of smaller, wide-hos -range, plasmids which are capable of replication in both £. coli and Agrobacterium strains. Such plasmids contain, the A ^g robacterium-derived pTi or pRi DNA signals necessary for the transfer of an engineered T-DNA region. These plasmids are referred to as binary plasmids, Bevan, 1984, Nucl. Acids. Res.. 12:8711-8721; An et al., 1985).

Many of the species belonging to the family Cucurbitaceae are known to be susceptible to infection by these Agrobacterj.um pathogens (see review by Anderson and Moore, 1979, Fhvtopath. 69:320-323; Smarrelli et al., 1986, Plant. Phvsiol.. 82:622-624). In addition, procedures for regenerating many of these species have already beeπ established (Malepszy and Nadolska-Orczyk, 1983, 2- Pflanzenphvsiol.. 111:273-276, Nadolska-Orczyk and Malepszy, 1984, Bulletin Pol. Acad. ≤c ., 32:423-428; Jelaska, 1986, Biotechnology in Agriculture and Forestry (ed. by Y.P.S. Bajai) 2:371-386). The combination of these two facts suggests that Agrobacterium-nediated tansfer of genetic information into the genome of cucurbit species should be readily achievable. The development of a system for the transfer and stable integration of genetic material into the genome of cucurbit species

will be useful for the transfer-of genetic traits which are difficult or impossible to transfer via conventional plant breeding techniques. The development of gene transfer technology for cucurbit species can be used to transfer engineered genes which will; confer resistance to viral infection, Powell-Able et al. , 1986, Science. 232:738-743; Cuozzo et al., 1988, Bio/tech.. 5:549-557, herbicide resistance Comai et al., 1985, Nature. 317:741-744; della-Cioppa et al. , 1987, Bio/tech.. 5:579-584; Lee et al., 1988, EMBO. J., 7:1241-1248; Stalker et al. , 1985, J. Biol. Chem. 260:4724-4728), or to transfer any other useful traits as they become available. *

INFORMATION DISCLOSURE The transformation and regeneration of transformed cucumber plants which express the NPT II gene has been reported by Trulson et al. (1986, Apol. Genet. 73:11-15). This transformation was achieved using Agrobacterium rhizogenes to transfer the T-DNA region of the binary plasmid pARC8, which contains a NOS-NPT II gene, by infecting cucumber hypocotyl sections. The resulting transformed tissues produced roots which were regenerated via an embryogenesis procedure.

S. Malepepsy, K. Niemirowicz-Szczytt, and J. Wiezniewska (1982), "Cucumber (cucumis sativus L.) somatic embryogenesis in vitro". Acta Biologica 10:218-220.

P. Chee and D. Tricoli (1988) "Somatic embryogenesis and plant regeneration from cell suspensions of Cucumis sativus ___" . , Plant Cell Reports 7:274-277. Definitions

As used herein the following terms mean:

"Plantlet" means a plant sufficiently developed to have a shoot and a root that is asexually reproduced by cell culture.

"Explaht" means a section or a piece of tissue from any part of a plant or plantlet for culturing, whether wild or cultivated, hybrid (somatic or sexual) , or genetically variant or transformed.

"Hormone" means a plant growth regulator that affects the growth or differentiation of plants, and is exogenous as used in reference to the various media herein. "Callus", and its plural "calli" refer to an unorganized group of cells formed in response to a cut, severing or injury of a plant, and herein refers to the unorganized cell growth which may form on explant tissues during culturing.

"Embryoid" means a structure similar in appearance to plant zygotic embryo

"Medium" means culturing media known in the art to be useful for culturing plant tissue "Kinetin" is a known hormone

2,4-D - 2,4-dichlorophenoxyacetic acid NAA - alpha-naphthaleneacetic acid

MS Medium - Murashige and Skoog Medium (Murashige & Skoog, 1962) SUMMARY OF THE INVENTION This invention provides: *

A method of generation of Cucumis sativus embryoids, from

^• Cucumis sativus explants obtained from cotyledon or hypocotyl tissue which comprises culturing the explants in an induction medium consisting of MS medium and exogenous hormones 2,4-D and kinetin for four to five weeks sufficient to induce competent embryoids. The embryoids provided by this method may be either transgenic or non- transgenic. Transgenic embryoids can be prepared by introducing foreign DNA into the plant material by either using agrobacterium as a vector or by microprojectile bombardment. Also provided is:

A method for producing Cucumis sativus transgenic plants which comprises subjecting embryoids generated by the method of claim 18 to conversion to obtain whole tranformed plants. Further provided Is: A composition consisting of Cucumis sativus plant tissue, an induction media, 2,4-D and kinetin.

EMBODIMENTS OF THE INVENTION In general the process of this invention involves the steps schematically shown in Chart 1. Exnlant Source and Preparation

Seeds of cucumber (Cucumis sativus L cv. Poinsett 76, Asgrow Seed Co.) were soaked in tap water for approximately 15 minutes. The seed coats were removed manually. The decoated seeds were surface sterilized with 70% ethyl alcohol for one minute. A twenty-five minute treatment with 25% (v/v) solution of commercial bleach (5.25% sodium hypochlorite) followed. The seeds were then rinsed four times with sterile distilled water. Sterilized seeds were germinated at 28 ^* C on 0.8% water agar (Difco Laboratories) in darkness. Unless

otherwise stated, all media were supplemented with 3% sucrose and solidified with 0.8 % phytagar (Gibco) . The pH of all media was adjusted to 5.8 before autoclaving. All media were autoclaved at 121 ^β C for 20 minutes. Eftft-πrpTe 1 Embryo Induction and Plant Regeneration

Three to five day old in vitro grown seedlings were used as a source of tissue. Tissue sections from cotyledons, 5x5 mm in size, and segments of hypocotyl, 0.6 to 1 mm long, were used as explants. The explants, about 100, were cultured on induction media supplemented with 2,4-D (1.0, 2.0 mg 1) + kineέin (0.5, 1.0, 2.0 mg/1) for four weeks at 26'C in darkness. After four weeks the cultures were evaluated for frequency and type of tissue produced. The cultures were then transferred to MS medium + 1.0 mg/1 NAA + 0.5 mg/1 kinetin. These cultures were incubated for an additional two weeks at 26 ^β C under diffuse cool white fluorescent lamps (4Klx) with a 16-hour photoperiod. The tissues were then transferred to MS medium with no growth regulators.

When plantlets developed an extensive roo.t system on the latter medium, they were transplanted to professional planting mix and covered with Ziploc ^® storage bags for hardening off. Subsequently, the regenerated plants were potted in soil and grown in a greenhouse. The concentration of 2,4-D added to the induction media range from 1.0 mg/1 to 2.0 mg/1 when explants from cotyledons and hypocotyl are used. The concentration of kinetin added is about 0.5 mg/1 for explants from cotyledons and hypotocol. Transformation Procedures Example g The Use of Agrobacteria for Transformation

Transformation of tissue was done according to the procedure described *by Horsch et al, Science. (1985), 227:1229-1231. Cotyledons from three-day old cucumber (Cucumis sativus .. cv. Poinsett 76) seedlings were excised and cut into small pieces. The pieces were submerged in an overnight liquid culture of either the avirulent agrobacterium strain C58Z707 (Hepburn et al. , 1985) or virulent strain C58 (Depicker et al. , 1980) . Both Agrobacterium strains contain the binary plasmid pGA481 (An et al. , 1985; An, 1986). After gentle shaking to ensure that all edges were infected, the pieces were blotted dry and prepared for regeneration. Example The Use of Microprojectiles for Transformation

Cucumber somatic embryos. derived using the tissue sources described in the embodiment of the invention are placed on MS medium containing 2.0 mg/1 of 2,4,D and 0.5 mg/1 kinetin. These tissues were bombarded with microprojectiles which have been coated with DNA containing a plasmid encoding a beneficial gene(s), as described by the manufacturer. After bombardment these tissues were transferred to plates containing fresh media. If the DNA construction used contained the plant expressible NPT II gene, the selection drug, kanamycin 100 to 200 mg/ml was added to the fresh plates. Cucumber tissues bombarded with microprojectiles were regenerated using the procedure described above in the embodiment of the invention.

It should also be recognized that transformation with microprojectiles can also be achieved by bombarding the embryogenlc callus. Example 4 Regeneration of Transformed Embryoids

Regeneration of potentially transformed cucumber callus tissues was done as follows: The cotyledon pieces from 3-6 days old germinating seedlings were cultured on MS basal medium supplemented with 2.0 mg/1 2.4D and 0.5 mg/1 kinetin. After four days, the Agrobacterium-infected cucumber cotyledon pieces were transferred to the same medium supplemented with 500 mg/ml carbinicillin and 100-200 mg/ml kanamycin and cultured for six additional weeks in the dark at 26'C.

After incubation for four weeks, characteristic gel like callus formed on the surface of the explants and at the site of contact with the medium. After six weeks cotyledon pieces were transferred to MS basal medium supplemented with 1 mg/1 NAA and 0.5 mg/1 kinetin. Within two weeks, embroyids began to appear on the surface of .gel like callus*. Plantlets were obtained by transferring the embroyoids to MS basal medium containing 50-100 mg/1 kanamycin and 500 mg/1 carbenicillin.

All of the callus tissue obtained from medium supplemented with kanamycin (100-200 mg/ml) were transformed. The Transfer of Genetic Material Example 5_ The transfer of virus coat protein genes

The purpose of this example is to generate a construction for the expression of a plant virus coat protein gene which, when expressed in a plant, results in reduced symptoms or resistance to

later infections by that virus. In general, after the identification of a coat protein gene by nucleotide sequencing, its sequences can be modified by using specific oligomers and the technique referred to as polymerase chain reaction (PCR) , to attach specific restriction enzyme sites to any coat protein gene. These restriction enzyme sites can be used to clone the coat protein gene into a plant expression vector which contains the necessary gene regulatory elements needed for controlling expression of the gene after transfer into the genome of various plants. A scheme describing the use of the plant expression vector plδUCcpexp (described in attachment B) for the cloning of various coat protein genes is shown in Chart 2. To facilitate Agrobacterium- mediated gene transfer the plant expressible coat protein is cloned into a binary vector and to facilitate micropro ectile-mediated gene transfers a marker gene such as the 3-glucuronidase gene (Jefferson, et al, 1987) is attached to the plasmid (see Chart 2).

Plants that are resistant to virus diseases and methods for producing them are described in EP 223,452, and in U. S. Patent application SN 07/234,404, filed August 19,1988, entitled "Cucumber Mosaic Virus Coat Protein Gene", and U.S. application SN 07/368,710 filed June 19, 1989, entitled "Potyvirus Coat Protein Genes and Plants Transformed Therewith". The use of viral coat protein gene to obtain resistance in transgenic plants has been described by Powell- Abel et al, (1986), Viral coat protein genes are isolated from any number of plant virus classes (tobamo, cucumo, poty, tobra, AMV, etc) and expressed constitu ively in plants after the attachment of the CaMV promoter and downstream 3' plant polyadenylation signals.

The construction of plant expressible coat protein genes is described in Examples 3 and 4 of U.S. Application SN 07/234,404 appended hereto as Appendix A and in Examples 8-11 and 15 of U.S. application SN 07/368,710 appended hereto as Appendix B. Example 6. Transfer of Herbicide Resistance Gene

This example illustrates how to generate plant expressible genes which allow a plant to be resistant to specific classes of herbicides. Such plants are useful for several reasons, (1) herbicides normally lethal can be used, and (2) different crops can be used in close rotations on soil which may contain residual amounts of a previously used herbicide that is normally lethal to the second

crop. Two genes of interest are mutant derivatives (derived from plant or bacterial sources) of the acetolactate synthase (ALS) gene which are not sensitive to chlorsulfuron and sulfσmeturon ethyll herbicides (Falco et al, 1985) and mutants of the gene encoding enolpyruvylshikimate-3-phosphate synthase (EPSPS) (Stalker et al, 1985) which are not sensitive to the herbicide glyphosate.

A gene which encodes an important enzyme which is either resistant to or detoxifies a specific herbicide is cloned downstream from a plant promoter, such as CaMV 35S (Pietrzak et al, 1986), ribulose-l,5-biphosphate carboxylase small subunit gene (Mazur and Chui, 1985) or other strong plant gene promoter and upstream from a plant gene poly (A) signal sequence (see Chart 3) .

This gene is then cloned into an Agrobacterium-derived vector (either binary or cis) and using the above described plant transformation methods this genetic material can be transferred and integrated into the genome (Chart 3) . This gene can also be cloned into a vector containing a plant expressible marker gene (Chart 3) and transferred into cucumber plants using the microprojectile- mediated gene transfer method (see Example 3) . Example 7 Transfer of Insect-Resistance Genes

In nature, numerous polypeptides exist which are toxic to insects and nematodes. The best known protein toxins are those associated with different strains Bacillus thuringiensis: for example, B_. israelenis is active against Diptera (mosquitoes and blackflies), B_. Thuringinensis is active against Lepidoptera, and £. san diego is active against Colioptera. The toxic proteins found in each of these bacteria is highly specific to insect pests; they are not toxic to other to other organisms. Thus the transfer and expression tof genes encoding such toxic proteins in plants are beneficial in reducing insect damage without using chemical insecticides thereby avoiding risk to other organisms. The genes encoding many of these toxic proteins have been isolated and sequenced (Schmeph et al, 1985; Waalwijk et al, 1985; Sekar et al, 1987) . The transfer of the B_. thuringiensis toxin gene into tobacco and its usefulness in protecting the plant from insect damage has been reported by Vaeck et al (1987). Thus, the combination of using the regeneration and transformation systems described here and plant expressible Bacillus toxin genes (Chart 4) allows for the transfer of

a useful genetic trait to cucumber (see Examples 2 and 3 for gene transfer methods) .

Example 8. Transfer of Antimicrobial Gene

This examples is to illustrate how to generate plant expressible genes which allow a plant to be resistant to infections by bacterial and fungi. In nature, several classes of polypeptides have been isolated and found to convey broad-spectrum antimicrobial activity; for example, magainins (Zasloff, 1987), deffensins (Daher et al, 1988), lysozymes (Boman et al, 1985) , Cercorpins (Boman et al, 1989), attacins (Halt ark et al, 1983), thionins (Bohlmann et al, 1988), and the like. Genes encoding these antimicrobial peptides or their more active modified forms can be synthesized and engineered for expression in plants. Promoters for the expression of these antimicrobial polypeptide genes can include the constitutive type or others which have tissue specificity. The use of tissue specific promoters, or wound inducible promoters (Sanchez-Serrano et al, 1987) would be useful so as to produce the antimicrobial peptide only when it is needed or in tissues which are more vulnerable to attack by bacterial or fungi, or both pests. Thus, the engineering of genes encoding antimicrobial polypeptide genes combined with the plant transformation and regeneration schemes described in and Examples 2 and 3 would allow for the transfer of a useful trait to squash plants. Chart 5 summarizes the construction of plasmids which could be used with the Agrobacterium-mediated and microprojectile-mediated gene transfer systems.

-9- CHART 1

Germinate Seed To Obtain A Young Plant

Excise Cotyledons of 3 Day-old Plants

Inoculate Plant Tissue by Dipping in Agrobacterium or By Bombarding With DNA Coated Microprojectiles

Transfer Tissue To Ms Medium Supplemented With 2,4D and Kinetin and Culture For 4 Days

Transfer To Fresh Medium Supplemented With 2,4=-D, Kinetin, Carbenicillin and Kanamycin ' Culture Six Weeks In Darkness

(Can See Callus Growing On Side)

Transfer To Fresh Medium Supplemented With NAA And Kinetin

Transfer Embroyids And Put In Regeneration Media

To Produce Plant

CHART 2

Coat Protein Gene Vector

BR

PGA482 Nos-Notll I

355 or coat protein promoter from other plant viruses

CHART 3

Herbicide Resistance Gene Vector

n -1\

BR Plant Mutant Plant BL

PCA482 ] I Nps-Notll | Promoter ALS or EPSPS gene Polv(A) f] or other herbicide resistant or detoxgene

CHART 4

Insect Resistance Gene Vector

5' 3' >

BR Plant Bacillus or Plant BR pG -82 Pi Nos-Notll \ Promoter other toxgene polvfA) | ] | for insect pest

GHART 5

Antimicrobial Gene Vector

•->

BR

APPENDIX A Examples 3 and 4 of U.S. Application SN 07/234,404 3_ Construction of a pUC19 Clone containing the CMV-WL coat protein gene The EcoRI fragments from lambda clone WL3Z8 were transferred to the plasmid vector, pUC19 (available from Bethesda Research, P.O. Box 6009, Gaithersbur , Md 20877), using standard methods to obtain clone pWL3Z8.1. The EcoRI cloned fragments in pUC19 were then sequenced by the technique described by Maxam and Gilbert (Methods in Enzvmologv 65:499, 1980). Based on this information the complete sequence of the CMV-WL coat protein gene was determined and this is shown in Chart 1. Additional sequencing showed that clone pWL3Z8.1 contains all but the 5'193 bp of the CMV-WL RNA3 molecule, as determined by comparison with the complete sequence of CMV-Q RNA3 (Davis and Symons, 1988, Virology 164:In press). The nucleotide and amino acid sequence of GMV-WL and CMV-C differ by 22.7% (Chart 2A) and 16% (Chart 3B) , respectively.

Example 4 Construction of a micro T-DNA plasmid containing a plant- expressible CMV-WL coat protein gene with the CaMV 35S polyadenylation signal

In order to attach the CaMV 35S promoter and polyadenylation signal, a fragment extending from an Apal site (located within the intergenic region of RNA3) to an EcoRI site (attached during the cloning experiment) was removed from lambda clone WL3Z8 and ligated into the multiple cloning site of the vector pDH51 (Pietrzak et al. , 1986) (available from Thomas Hohn, Friedrich Miescher Institute, P.O. Box 2543, CH-4002, Basel, Switzerland). This was accomplished by complete digestion with Apal and a partial digest with EcoRI of WL3Z8, creating a blunt-ended molecule out of the appropriate Apal to £co.RI 1090 bp fragment (using aung bean nuclease) , followed by ligating it into the Smal site of pDH51 (see Chart 4A) . This clone, designated pDH51/CPWL, was sequenced by the Maxa -Gilbert technique to confirm its suitability for expression in plants.

The plant expressible coat protein gene was then moved into a vector suitable for Agrobacterium-mediated gene transfer. Following partial digestion with EcoRI. the EcoRI to EcoRI fragment of about 1.9kb was removed from pDH51/CPWL and placed into the EcoRI site of the plasmid, pUC1813 (available from Robert Kay, Dept. of Chemistry,

Washington State University, -Pullman, Washington), creating the plasmid pUC1813/CPWL. A 1.9 kb fragment containing this plant expressible CMV-WL coat protein gene was removed by partial Hindlll digestion and ligated into the Hindlll site of the vector, pGA482 (An, 1986) (available from Gynehung An, Institute of Biological Chemistry. Washington State University). The plasmid pGA482 was previously modified to contain the plant expressible 9-glucuronide gene as described in WO 89/05858, incorporated above, and the modified plasmid is referred to as pGA482/G. After cloning the expression cassette the plasmid was designated *pGA482/CPWL/G (see Chart 5A) .

Chart 2A

Comparison of CMV-WL and CMV-C Coat Protein Gene

5 CMV-WL ATGGACAAATCTGGATCTCCCAATGCTAGTAGAACCTCCCGGCGTCGTCG 50

CMV-C A iTiGiGiAiCiAmAATnCTiGiAA MTCIAAC iCnAGT MGCMTGG MTCGMTA. . .AC ICIATC IGIAICIGMTCMG 47

. . . . C

CCCGCGTAGAGGTTCTCGGTCCG . . . CTTCTGGTGCGGATGCAGGGTTGC 97 o TC lCiGnCGnTCG iTG iGnTTnCCC nGCT MCCMGCCCC ICT ICICTCCG ICIGIGIAITIGICITAACT ITITA 97 GTGCTTTGACTCAGCAGATGCTGAAACTCAATAGAACCCTCGCCATTGGT 147

15 GiAGiTCTiTlGlTCiGCmAGCmAGCTiTTCGiCGAiCnTTAmATAiAGACGTTiAGCAG CTMGGMT 147

CGTCCCACTCTTAACCACCCAACCTTCGTGGGTAGTGAAAGCTGTAAACC 197

CiGnTCnCAAiCnTAT1T1A1A1C1C1A1C1C1C1A1A1C1CiTiTiTGnTAGnGGA mGTGmAACGmCTGmTAGAiCnC 197 20 . . . . .

CGGTTACACTTTCACATCTATTACCCTGAAACCGCCTGAAATTGAGAAAG 247

TG iGiGT iAnCAnCGT mTCAiCiAmTCTmATTiAmCCCiTiAA nAGC nCACCAAA mAATiAGA nCCGTG 247 25 GTTCATATTTTGGTAGAAGGTTGTCTTTGCCAGATTCAGTCACGGACTAT 297

AGTiCiTTmATTiACGmGTAiAAIAIGIGITITIGITITACTIACICITGIAITITI CIAIGITICIAICIGIGIAIATiAnT 297 GATAAGAAGCTTGTTTCGCGCATTCAAATCAGGGTTAATCCTTTGCCGAA 347

30

G mATAmAGAmAGCiTmTGTmTTCiGiCmGCAmTTCiAiAiAiTiTCG iAG mTTAmATCiCmTTTiGmCCGmAA 347

ATTTGATTCTACCGTGTGGGTTACAGTTCGGAAAGTACCTTCATCATCCG 397

35 A iTiTiTiGmATTiCiTiAiCiCiGiTiGiTiGmGGTiGA iCnAGnTCC nGTA iAnAGnTTC MCTIGC ICT ICICT ICIGG I 397

ATCTTTCCGTCGCCGCCATCTCTGCTATGTTTGGCGATGGTAATTCACCG 447

A iCTT iAT iCnCGnTTG iCiCmGCCiAiTiCiTmCTGmCTAiTiGmTTCG iCGG nACG nGAGCCT mCACmCG 447 40 . . . . .

^• GTTTTGGTTTATCAGTATGCTGCGTCCGGAGTTCAGGCCAACAATAAGTT 497

I I ! I M 1 1 1 1 1 ! 1 1 1 i 1 i I I I I l l l l l I I I I I I I I I I I I I

GTACTGGTTTATCAGTATGCCGCATCTGGAGTCCAAGCCAACAACAAACT 497

45 ACTTTATGACCTGTCCGAGATGCGTGCTGATATCGGCGACATGCGTAAGT 547

GTT iGT iTT iGlAlTC ITITT ICIGG iCG mATGmCGCG ICITIGIAITIAITIAG IGITG mACAmTGAG iAA MAGMT 547

• • • • •

ACGCCGTCCTGGTTTACTCGAAAGACGATAAACTAGAGAAGGACGAGATT 597 r 50 A iCiGiCiCmGTCmCTCG ITIGT IAITT ICIAA iAiAiGiAiCiGmATGCGC ITICG mAGAiCG mGACiGnAGiCT iA 597

GCACTTCATGTCGACGTCGAGCATCAACGAATTCCTATCTCACGGATGCT 647

55 G iTA iCiTiTiCiAiTmGTTG iAnCAT mCGAiGnCAiCC mAACiGlCA iTnTCnCCA iCAT nCTGG iAGT mGCTi

CCCGACTTAG 657 III I CCCAGTCTGA 657

SUBSTITUTESHEET

-17- Chart 3A

Comparison of CMV-WL and CMV-C Coat Protein Gene CMV-WL MDKSGSPNASRTSRRRRPRRGSRSA.SGADAGLRALTQQMLKLNRTLAIG 49 CMV-C M mDKSiES iTSA iGR i.NHR nRRnPRiRiGiSiRmSAPS MSADMANFR IVL ISQ IQILSRL INIKT ILIAIAG I 49

RPTLNHPTFVGSESCKPGYTFTSITLKPPEIEKGSYFGRRLSLPDSVTDY 99 R iPmTINiHiPiTiFiViGiSiEiRC iRmPGYnTFmTSInTLiKmPPKI nDRES nYYG iKB nT.T.T m.PDSmVTEY i

• • • • •

DKKLVSRIQIRVNPIJPKFDSTVWVTVRKYPSSSDLSVAAISAMFGDGNSP 149 D mKKLmVSRmIQIiRmWPmIJKmFDSmTVWmVTViRiKiViPiAS iSmDLSiViAnAInSAmMFAmDGAS nP

VLVYQYAASGVQANNKI YDLSEMRADIGDMRKYAVLVYSKDDKLEKDEI 149 . V mLVYiQiYmAASiGiViQmANNiKiLiLiFiDmLSAM nRAmDIGmDMRiKmYAViLmVYSiKmDDAL nETD iEnL

ALHVDVEHQRIPISRMLPT. 219 VLlHlVlDlIlElHlQlRlIlPlTSIGVILIPIV. 219

Chart 4A

Construction of RDH 51/cpWL

CMV-WL

WL3Z8 coat protein gene t sidues

CaMV 35S poly-A signal

CMV-WL 4. pDH51/CPWL

CaMV coat protein gene 4*

35S* A residues promoter

δhart 5A

Construction of pGA482/CPWL/G

CaMV 35S poly-A signal 4-

CMV-WL ρDH51/CPWL

CaMV coat protein gene t

35S A residues promoter

pGA482/CPWL/G NOS- CaMV 35S poly-A signal NPTII CMV-WL i

B II coat protein t gus gene B gene A residues

AEPENDIX B Examples 8, 9, 10, 11 and 15 of U.S. Application SN 07/368,710 Example 8, Construction of a Plant-expression Cassette for Expression of Various Genes in Transgenic Plants. In the preferred embodiment of the present invention, the following expression cassette was constructed to provide the necessary plant regulatory signals (which include the addition of a promoter, 5' untranslated region, translation initiation codon, and polyadenylation signal) to the gene inserts in order to achieve high level expression of the inserts. The expression cassette may be used to express any genes inserted therein. Accordingly, the applicability of the expression cassette goes beyond its use in expressing coat protein genes. Rather, the expression cassette may be used to express any desired protein in transgenic plants. The expression cassette is the preferred expression system for expressing viral coat protein genes in plants.

The expression cassette of the preferred embodiment contains: a constitutive promoter; a 5' untranslated region which enhances gene expression; an initiation codon which comprise Kozak's element; a cloning site where the gene to be expressed may be inserted to produce a functional expression unit; and a 3' untranslated region which comprises a poly(A) addition signal and untranslated flanking regions which result in a higher level of expression.

More specifically, the expression cassette which is the preferred embodiment of the present invention consists of the cauliflower mosaic virus (CaMV) 35S transcript promoter, the 5'- untranslated region of cucumber mosaic virus (CMV) , the CMV translation initiation codon, and the CaMV polyadenylation signal. The construction of this expression cassette utilized the Polymerase Chain Reaction (PCR) technique to obtain correct position of the plant regulatory signals and the addition of convenient restriction enzyme sites which allow for the easy addition of a coat protein gene and the excision of the completed cassette so it can be transferred to other plasmids. To accomplish the construction of this expression cassette the following oligomers were synthesized:

1. 5'-GAAGCTTCCGGAAACCTCCTCGGATTCC-3', contains a Hindlll site at its 5'-end and contains 21 bases which are identical to 21 bases

in the 5'-flanking region of CaMV.

2. 5'-GCCATGGTTGACTCGACTCAATTCTACGAC-3', contains a Ncol site at its 5' -end which contains a translation initiation codon which conforms to Kozak's rules and has 21 bases which are identical to 21 bases in the antisense strand of the CMV 5'-untranslated region.

3. 5'-GCCATGGTTGCGCTGAAATCACCAGTCTC-3', contains a Ncol site at its 5' -end (which contains the same translation initiation codon as oligomer 2) and has 20 bases which are identical to 20 bases in the 3' -untranslated region of CaMV. 4. 5'-GAAGCTTGGTACCACTGGATTTTGGTT-3' , contains a Hindlll site at its 3'-end and has a 20 base match with the flanking DNA region 3' of the CaMV polyadenylation site (on the antisense strand) .

These oligomers were used to amplify sequences contained within the CMV expression clone referred to as pUC1813/CP19, shown in Chart 6B, and referred to above. As depicted in Chart 8, the PCR technique was used to amplify the gene regulatory regions in pUC1813/CP19.

Amplification of the 5' -flanking, CMV 5' -untranslated region, and CMV initiation codon (which was modified to conform to Kozak's rule

AAXXATGG) resulted in a fragment of about 400 base pairs in length and amplification of the CaMV 3-untranslated and flanking regions resulted in a fragment of about 200 base pairs in length. These fragments were digested with Ncol and Hindlll, isolated from a polyacrylamlde gel, and then ligated into Hindlll digested and phosphatase treated pUC18. The resulting clone is referred to as pl8CaMV/CMV-exp and is shown in Chart 8B.

Example £ Identification of the WMVII Coat Protein Gene

The cloned WMVII cDNA insert from clone pWMVII-41-3.2 which was produced as described above, was sequenced by using both the chemical (Maxam and Gilbert, Methods of Enzymology 65:499, 1980) and enzymatic (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463, 1977) methods. Based on this information and comparative analysis with other potyviruses, the nucleotide sequence of clone pWMVII-41-3.2 was found to contain a complete copy of the WMVII coat protein gene. The N- ter inus of the coat protein was suggested by the location of the dipeptide sequence Gln-Ser. The length of the WMVII coat protein gene coding region (281 amino acids) is consistent with a gene encoding a protein of about 33 kD. The sequences of this WMVII coat protein gene and protein are shown in Chart 2B. In addition,

comparison of this sequence with that obtained from the related virus Soybean Mosaic Virus (SMV) strain N described by Eggeriberger et al. shows that they share overall about 88% identity and excluding the N- ter inal length differences they share about 92.5% identity, see Chart 5B. Because these two virus coat proteins share extensive amino acid identities, expression of the coat protein gene from WMVII is expected to yield plants resistant to WMVII infection and could yield plants resistant to SMV infection.

Example 10 Construction of a Plant-expressible WMVII Coat Protein Gene Cassette with CaMV 35S Promoter and Polyadenylation Signal and CMV Intergenic Region and Translation Initiator ATG.

As depicted in Chart 9B, attachment of the necessary plant regulatory signals to the WMVII coat protein gene was accomplished by using the PCR technique to amplify the WMVII coat protein gene using oligomers which would add the necessary sites to its 5' and 3' sequences. Following this amplification the resulting fragment Is digested with the appropriate restriction enzyme and cloned into the Ncol site of the above described expression cassette containing plasmid, pl8CaMV/CMV-exp. Clones containing the WMVII coat protein gene insert need only be checked to determine correct orientation with respect with the CaMV promoter. However, to ensure that no artifacts have been incorporated during the PCR amplification the entire coat protein gene region is checked by nucleotide sequence analysis. To obtain the amplified WMVII coat protein gene with Ncol restriction enzyme sites on both ends the following two oligomers were synthesized:

1. 5'-ACCATGGTGTCTTTACAATCAGGAAAAG-3', which adds a Ncol site to the 5'-eιid of the WMVII coat protein gene and retains the same ATG translation start codon which is present in the expression cassette, pl8CaMV/CMV-exp.

2. 5'-ACCATGGCGACCCGAAATGCTAACTGTG-3', which adds a Ncol site to the 3'-end of the WMVII coat protein gene, this site can be ligated into the expression cassette, pl8CaMV/CMV-exp. The cloning of this PCR WMVII coat protein gene, using these two oligomers, into pl8CaMV/CMV-exp yields a plant expressible WMVII gene (referred to as pl8WMVII-exp) which, following transcription and translation, will generate a WMVII coat protein which is identical to

that derived from the WMVII coat protein gene nucleotide sequence, see Chart 2B. However, this coat protein will differ, because of necessary genetic engineering to add the ATG initiation codon and by including the last four amino acids of the 54 kD nuclear inclusion protein (which is adjacent to the Glu-Ser protease cleavage site); the amino acids added are Val-Ser-Leu-Glu-N-ter WMVII. The addition of these four amino acid residues should not affect the ability of this coat protein to yield plants which are resistant to WMVII infections, because the N-terminal region of potyvirus coat proteins appear not to be well conserved for either length or amino acid identity. However, if this is found to be a problem its replacement would involve the use of a different oligoaer to obtain N-terminal variations of the WMVII coat protein gene. The cloned construction of the plant expressible WMVII coat protein gene is referred to as pl8WMVII-exp, and is shown in Chart 9B.

Example 11 Construction of a Micro T-DNA Plasmid Containing the Plant-expressible WMVII Coat Protein Gene Construction.

As depicted in Chart 10B, the plant expression cassette for the WMVII coat protein gene was transferred into a suitable micro-T-DNA vector which contains the necessary Agrobacterium T-DNA transfer signals (to mediated transfer from an Agrobacterium and integration into a plant genome) and wide-host range origin of replication (for replication in Agrobacterium) to form plasmid pGA482/G/CPWMVII-exp. To construct this plasmid, plasmid pl8WMVII-exp was digested with Hind III (which cuts within the polycloning sites of pUC18, well outside of the expression cassette), and an 1.8 kb fragment containing the plant-expressible cassette was removed and ligated into the Hind III site of the modified Agrobacterium-derived microvector' pGA482 (modification included the addition of the β- glucuronidase gene). The micro T-DNA vector, pGA482, is shown in Chart 7B and available from G. An, Institute of Biological Chemistry, Washington State University, Pullman, WA. The resulting plasmid was designated, pGA482/G/CPWMVII-exp is shown in Chart 10B. This plasmid (or derivatives thereof) was transferred into virulent or avirulent strains of Agrobacterium tumefaciens or rhizogenes, such as A208, C58, LBA4404, C58Z707, A4RS, A4RS(pRiB278b) , and others. Strains A208 C58, LBA4404, and A4RS are available from American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, MD. Bacteria

A4RS(pRiB278b)) is available from Dr. F. Casse-Delbart, C.N.R.A., Routede Saint Cyr. F78000, Versailles, France. Bacteria C58Z707 is available from Dr. A.G.Hepburn, Dept. of Agronomy, University of Illinois, Urbana, IL. After transfer of the engineered plasmid pGA482/G/CFWMVII-exp into any of the above listed Agrobacterium strains, these Agrobacterium strains can be used to transfer and integrate within a plant genome the plant-expressible WMVII coat protein gene contained within its T-DNA region. This transfer can be accomplished vising the standard methods for T-DNA transfers which are knoVm to those skilled in the art, or this transfer can be accomplished using the methods described in U.S. Patent application SN 07/135,655 filed December 21, 1987 entitled "Agrobacterium Mediated Transformation of Germinating Plant Seeds". In addition, it has recently been shown that such Agrobacteria are capable of transferring and integrating their T-DNA regions into the genome of soybean plants. Thus these strains could be used to transfer the plant expressible WMVII coat protein gene into the genome of soybean to develop a soybean plant line which is

"resistant to infection from soybean mosaic virus strains. (Examples 12 to 14 omitted)

Example 15 Construction of a Micro T-DNA Plasmid Containing the Plant-expressible ZYMV Coat Protein Gene Construction.

Following the teachings of Example 11 with appropriate modifications, the construction of a micro T-DNA plasmid containing a plant-expressible ZYMV coat protein was constructed. Plasmid pUC18cpZYMV (Chart 12B) was digested with Hind III (which cuts within the polycloning sites of pUC18, well outside of the expression cassette), and a 1.6 kb fragment containing the plant-expressible cassette vάs removed and ligated into the Hind III site of the microvector pGA482 (Chart 7B). The resulting plasmid was designated, pGA482GG/cpZYMV is shown in Chart 13B.

After transfer of the engineered plasmid pGA482GG/cpZYMV into Agrobacterium strains, the Agrobacterium strains can be used to transfer and integrate within a plant genome the plant-expressible ZYMV coat protein gene contained within its T-DNA region.

CHART 2B GTGTCTTTACAATCAGGAAAAGAAACAGTTGAAAATTTGGACGCAGGGAAAGAATCAAAG

1 . 60

ValSerLeuGlnSerGlyLysGluThrValGluAsnLetiAspAlaGlyLysGluSe rLys t AAAGATGCCAGTGACAAAGGGAATAAGCCGCAGAACTCGCAAGTTGGTCAGGGTAGCAAG 51 120

LysAspAlaSerAspLysGlyAsnLysProGlnAsnSerGiriValGlyGlnGlySe rLys

GAACCAAC^ΔAAACCGGCACAGTCAGCAAGGATGTAAATGTTGGATGGAAAGGAAA AGAA . 180 121 GluProThrLysThrGlyThrValSerLysAspValAsriValGlySerLysGlyLysGl u

GTCCCACGACTACAAAAGATAACAAAGAAAATGAATCTTCCAACAGTTGGTGGGAAA ATC

181 . 240

ValProArgLeuGlnLysIleThrLysLysMetAsnLeuProThrValGlyGlyLys Ile

ATTCTTAGCTTAGACCATTTGCTTGAGTACAAACCTAGTCAAGTTGATTTGTTTAAC ACT

241 300

IleLeuSerLeuAspHisLeuLeuGlyTyrLysProSerGlnValAspLeuPheAsn Thr CGAGCAACAAAAACACAATTTGAATCATGGTACAGCGCAGTCAAAGTTGAGTATGATCTT

301 360

Ar AlaThrLysThrGlnPheGluSerTrpTyrSerAlaValLysValGluTyrAspLeu

AATGATGAGCAAATGGGTGTGATTATGAATGGTTTTATGGTTTGGTGTATCGATAAC GGT 361 --. 420

AsnAspGluGlnMetGlyVallleMetAsnGlyPheMetValTrpCysIleAspAsn Gly

ACATCTCCAGATGTCAATGGAGTGTGGGTAATGATGGATGGGGAAGAGCAAGTTGAG TAC 421 480 ThrSerProAspValAsnGlyValTrpValMetMetAspGlyGluGluGlnValGluTyr

CCACTAAAGCCAATTGTTGAAAATGCAAAGCCAACTTTGAGACAAATCATGCACCAT TTC 481 . 540

ProLeuLysProIleValGluAsnAlaLysProThrLeuArgGlnlleMetHisHis Phe

TCAGATGCAGCGGAAGCATATATTGAAATGAGAAACTCTGAAAGTCCGTATATGCCT AGA 541 600

SerAspAlaAlaGluAlaTyrlleGluMetArgAsnSerGluSerProTyrMetPro Arg TACGGATTACTGAGAAATTTGAGAGACAGGGAATTAGCACGCTATGCTTTTGACTTCTAT

601 660

TyrGlyLeuLeuArgAsnLeuArgAspArgGluLeuAlaArgTyrAlaPheAspPhe Tyr

GAGGTTACTTCTAAAACGCCAAATAGGGCAAGAGAAGCAATAGCTCAAATGAAGGCC GCG 661 720

GluValThrSerLysThrProAsnArgAlaArgGluAlalleAlaGlnMetLysAla Ala

GCTCTCGCGGGAGTTAACAGCAGGTTATTTGGACTTGATGGTAATATCTCGACCAAT TCC

721 . 780 AlaLeuAlaGlyValAsnSerArgLeuPheGlyLeuAspGlyAsnlleSerThrAsnSer

CHART 2B (CONTINUED)

GAAAATACTGGGAGGCACACTGCAAGGGACGTAAATCAGAATATGCATACTTTGTTG GGT

781 . 840

Gl ^* uAsnThrGlyArhHisThrAlaArgAspValAsnGinAsnMetHisThrLe-uL euGly

ATGGGTCCACCGCAGTAAAGACTAGGTAAACTGGTCACAGTTAGCATTTCGGGTCGT TAT

841 . 900

MetGlyProProGlnEnd

AAGTTTTCTATAATATAACATGTCGCACTTTATTTTAGTATAGTGTGATTTCATCAC CT 901 960

TGTACTGTTTATGTTAGCGTGGTTTAACCACCTTTGTGTGTGCTTTATATTATAGTT TAT 961 .1020

GCGTAGCAGGGAGAACCATTACAATGCCGGAGTTGTTTGTAGTGTGATTTCATCACG GTT .1080

1081 AATAGCCGAGGTACGGTAATGTTTGTTGCCTAAAAAAAAAAAAAAAAAAAAAAAA 1135

CHART 5B

SMV SGKEKEGDMDADKDPKKSTSSSKG AGTSSKDVNV 34

WMVII S mGKEiTVE INILIDIAIGIKIEISK nKDAS iDKGNKPQNSQVGQGSKEPTKTG nTVS mKDVmNV 50

GSKGK PRIQKITRKMNIJ'MVEGKIILSLDHIJ-EYKPNQVDLFNTRATR 84

G iSnKGnKEW iiRmIjQKmITKiKiMiNmIJTV iGmGKImILSiLiDmHIJi-EiYmKPSQV iDiIiJiTmrRAmTK 100

TQFEAWYNAVKDEYELDDEQMGWMNGFMVWCIDNGTSPDANGVWVMMDG 134 T mGFEiSW iYiSA iVnKVE iYmDLNmDEQiMmGVImMNGmFMViWmCIDmNGTiSmPDVN iGiViWiYiMiMmDG 150

SMV EEQIEYT'LKPIVENAKPTLRQIMHHFSDAAEAYIEMRNSESPYMPRYGLL 184

WMVII ElElQlVlElYlPlIll^lIlVlElNlAlKlPlTlLlRlQlIlMlHlHlFlSlDlAlAlE lAlYlIlElMlRlNlSlElSlPlYlMlPlRlYlGlLlLl 200

SMV RNIi^RELARYAFDlTYEVTSKTPNRMlEAIAQMKAAALSGVNNKLFGLDG 234

WMVII R m1HImU)REiLmARYmAFDmFYEmVTSiKmTPNiRiAiRiEiAiIiAmQMKiAiAiAnLA G iVnNSR lLiFmGLDnG 250

SMV NISTNSENTERHTARDVNQNMHTLLGMGPPQ 285

WMVII NlIlSlTlNlSlElNlTlGlRlHlTlAlRlDlVlNlQlNlMlHlTlLlLlGlMlGlPlPl Ql 281

SUBSTITUTESHEET

-28- CHART 6B

pPRV117

I PRV-p Coat Protein Gene ]

pUC1813/Cpl9

Hindlll Ncol Tthllll BstXI Ncol Hindlll 4 4- 4* 4* 4- 4

* I I I I *

I ^p ca I ^τ c I I ^{017 Coat} *P«tein Gene| 'ca

pUC1813/Cpl9-PRVexp

Hindlll Ncol Tthllll BstXI Ncol Hindlll 4* 4* 4 I 4* il

I P.ca CMV I PRV-p Coat | I sca Coat Protein (16AA) gene

-29-

GHART 7B

pGA482

Hindlll

B _τ Nos | CaMV| B _T Gus Gene

pGA482/G/CP19-PRVexp

Hindlll Tthllll BstXI Hindlll

4 4 4 4

B,. Nos ^■ cai CMV I PRV-p Coat | |S _oa | |CaMV| |B _L | Coat Protein Gus Protein Gene Gene (16AA)

-30- GHART 8B

Hindlll Ncol Ncol Hindlll 1 4 4

J L l_l

^■ ca ca

pl8CaMV/CMV-exp

Hindlll Ncol Hindlll 4 4 4

ca I I sca

CHART 9B

pWMVII-41-3.2

I WMVII Coat Protein |

Gene

PCR Generated Gene

Ncol Ncol

4 4

WMVII Coat Protein Gene|

CHART 10B

pGA482/G/CPWMVII-exp

Hindlll Ncol Ncol Hindlll

4 4 I 4* * I I I U_ I I u IU U_i l-B-rl I Nos II P _ca I I _c I WWII Coat | | S _ca | | CaMV | |B _L |

Protein Gus

Gene Gene

-33- CHART 12B

pZYMV-15

IZYMV Coat ProteinI Gene

ZYMV Coat Protein Gene

Ncol Ncol 4 4*

ZYMV Coat Protein Gene

pUClδCpZYMV

Hindlll Ncol Ncol Hindlll BamHI Smal 4 4 4* 4 -4 4

I ^p ca I ^τ c I ² ™ ⁷ Coat Protein| | S _ca

Gene

CHART 13B

pGA482/GG/cpZYMV

Hindlll Ncol Ncol Hindlll 4 4 4 4*

* — l_l U I I U U U_L

I B _r I I Nos I P _ca | I _c I ZYMV Coat || S _ca H CaMV | |B _L |

Protein Gus

Gene Gene