CA2229168A1 - Methods and materials for producing pathogen-resistant plants - Google Patents

Abstract

This invention comprises a mutant plant virus gene which confers resistance on tobacco and tomato plants against tobacco mosaic tobamovirus and tomato mottle geminivirus infections and infection by other related geminiviruses. A gene was initially isolated from the known BC1 gene, between nucleotides 1278 and 2311 of the B component of tomato mottle geminivirus. Upon subcloning of this DNA fragment into an appropriate expression vector and transformation of the gene into tobacco plants, a truncated gene product was produced which confers resistance against viral infection to the recombinant plant in which it is expressed.

Description

CA 02229l68 l998-02-lO
W O 97/07217 PCT~US96/13097 I:)ESCRIPTION

METHODS AND MATERIALS FOR PRODUCING
PATHOGEN-RESISTANT PLANTS
S
Cross Referenee to Related ApplieAtion This applirAtinn elairns the benefit of U.S. Provisional ApplirAtinn No. 60/015,051, filed April 9, 1996, and U.S. Pn~vlalO~al ApplicAfion No. 60/002,158, filed August 11, 1995.

A~u~ e~ m~nt of Gu.. ll~u~ t Su~port This ul.,~Lioll was made with go~ support under USDA/DSRS CBAG Speeial Grants Prograrn, grant Nos. 93-34135-8607; 92-34135-7456; and the Florida Tomato C.
Grant No. 90153-C. The gU~.llllle.lL has eertain rights in this .lLùn.

Bacl~vulld of the Invention (i). Field of the L~"lLiul~
This illVCllLlUII pertains to the field of c.~,.L .;.. g pAthog~n l~ to plants. More palL-,ukulv, the hl~wlLioll is direeted to vi~us-resistant ll~ CV. ~;ç plants.

(ii) BacL~lound of the Invention Tomato ~JlUdUC~l~7 suffer ci.,";r;~ losses due to tomato mottle ~uiuivilua inf~rti~n Currently farmers must ~UII,LI~. rh~miçAlc in order to control tomato mottle virus in their tomato fields. Similarly, losses are ~ ;c ~cc;l by farmers produeing tobaceo as a result oftobaceo crop infection by tobaceo mosaic lob.ulluv~ s. Aceordingly, there is a need for a solution to this problem which is less eostly and less d-----S~g;-~g to the e.lvhol~ , -L than the Ch~ controls currently employed.
Pludu~Liull of L,A..cgç,~ plants with ell~ ~d phelluLyyic~hA~h~ iaLicsis a lelaLi-,~ly recent dev~l~mlont in the arsenal available to farrners. NC~.LIlCIe55, the value ofthis te(hnt~lo~
has been ~ t~ ,pc..t~ly in recent vears. However, what is required is the idrntifi~ Atif~n of appropriate genes to confer the desired ph&luLylJc, in this case, paLLo~
Tl A .1~ rh. . .-7.~ n of plants with portions of v iral ~ neC may result in plants with virus (Beachy, 1993). This pl.. l~.. \-~ is known as '~pilth~gton-derived l~ (Sanford W O 97/07217 PCT~US96/13097 and Johns-Jn, 1985) The level of ,~ obtained is variable This variability has been ~t~ t~d to the random nature of the LL~ r(J~ . process (T~ O..O~..rr, 1995) T l ~ l ,( Iines of plants g~ Yt~,d from a single L" ~rv ~ r~lJ ; ~ I mav contain different Llal sg~ e copy numbers inserted in valious cL ~c.~, ~ s PL~ u~y~ic Lrr~ c-,S have been noted among plant lines c . li; ; g a single copy of the l ,~ g ~ Some of the va iability in Llauag~C ~AI -~, ,a;u,. has also been d~ t ,1 to tissue culture induced changes (Phillips et al, 1994) This variability in tll phe,~o~y~c is also ol~sc. ~,1 in al bse~l ~ progeny derived from the Ro plants LLlu~ n of a. IA1;. (d.,rc~,Li~,)inonemotifofa", Il;~..l;rproteinhasbeen ~.~osel as a strategy for; l~ f~ g with viral reFIirAtion This ..IL~.f~nce with the function of wild-type genes has been referred to as a ~ negative mntAtif n Max~ell and his co-workers have cùuaLLu.l~d ~ C~ plants ~ Sail g a mo~lified tomato mottle ~ iVilUa replirAtion-A~soci~t--d protein (RAP), mutated in a NTP-binding motif, which appears to interfere with viral l.1.li~A~ ~ (Hanson, etal, 1991) This d~ ~ r ~ ,1 negative mutant for the tomato mottle g~. ~vuua RAP gene has been tested for tomato mottle g~vh~la ,~ , in Lo l ~t ,e~ Noris et al (1994, First Tntr~Ati~mAl Sy~yualuln on G~lilPivi~ua~,s, Almeria, Spain) found ;.. h;l.;l;.. - of Tomato Yellow Leaf Curl Virus (TYLCV) DNA replirAtinn in tobacco l,.u~upl~ ~; co~ rf- ,~1 with TYLCV and a CuuaLIu~,L of a L u..~,..t~,d RAP c.~.eascd under control of a CaMV 3~S ,uruluvt~r~
This ccm~rol strategy is lilcely to be verv virus specific since the RAP binding sites essential for function have been shown to re~uire a ic.~ ~ ~-specific ..lt~ ,Lio.. between RAP and the origin of replirAtinn (Fontes et al, 1994) This enables the Lli. r~; g factors of RAP to .1;~ ; ; ~1.;
beh~een the rrplirAtion origins of closely related ~,C l~uuvh u.,CS Because of C~WIfUflV1L ua ~ ~ a;Ly and ~lArtAbility, virus-specific control aL~..tC~,iCs are of limited value under field ~ 1;l ;- ~
There have been several reports in recent years relating to the inhihiti-n of ; f~ ;.. of certain plants bv specific viral p<.11 ~, c For cYAmrle Von Arnim and Stanley (1992) reportcd on 2~ the inhibitinn of systemic infaetinn by African Casava Mosaic Virus (ACMV) by a .. w~.. ,.. L
protein from the related G, luuY-lua, Tomato Golden Mosaic Virus (TGMV) T_is wasA~ l ~ by ",l,k.~ Jl-g the ACMV coat protein cod.~g L.e~ c with the BL l or BRl ~-W ~ ~.I.~.L
genesc~l~ r~fr~nTGMVandthentestingtheabilitvofthe.~o h; A I ACMVtoinfectitshost,Nicotzana benthamiana (which is also the host of TGMV) The authors found that the TGMV gene did not cf~mrllom~nt the ACMV ~cùU~ulduL, and h~ol~ that direct genomic expression of a (1~ I negative mutant might produce plants resistant against g~ivi~uacS.
Cooperetal (1995) ~ cl~edthatL,~ v, ;l~tobaccoplantse,.y.~saulgadefectivetobaccomosaic virus (TMV) mu~ L protein were resistant to infçctilm by multiple viruses, while W O 97/07217 PCTnUS96/13097 J. . ~, plants ~.~y~ i--g the natural ...u~ protein had i,.~ s~i ~ s~l;bility to ;.~f~ ~ l ;o. .
by TMV and other viruses.
Nejidat and Beachy (1990) ~ .sed that ~ . . ;c tobacco plants e.~ s~illg a TMV eoat protein have ill~,r~,~c,1 lc~ c against several of the toL~uv.. us~,s. Gilbertson et al. (1993) .~ 1 the reduced PA II .C ~ ;.; Iy of pseudol4.,~:.. l~;.. A .I ~ of two bipartite g4.11illiVil UsCs, tomato mottle (ToMoV) and TGV-MXl.

Brief Summar~ of the L1~ L;O11 We have dia~4 ~ ~ 4d a mutated plant virus gene which protects tobacco plants against tomato mottle gc.llilliV-l~ and tobacco mosaic tobu.l.~,v~u . infertions This .~ c gene has been ~lluducod into tobacco C111~ 5U~ DNA by genetie f...v;..c~ v- The LIA~C~ ;C tobaeeo plants C~,yl~ lg this gene sho w .~ to tomato mottle g~,.l~Vil u, and tobaceo mosaic tobal loVil~
;- ~f~ (lacli of or ~ v 1. ~~ ~ of disease ~ylll~ wherl inoclllAted with the viruses). The mutated gene can be ~.. LLoduced into cL.. ~.~s.. ~.c of desirable tomato and tobacco lines to develop ew~cl.,.ally i~ cd tomato and tobacco eultivars/hybrids.
Acc~,.dill~;ly, this invention c~....p. ;~e, a mutant plant virus gene which eonfers l.~ r, on tobaeeo and tomato plants against tobaeeo mosaic lob~ vi-us and tomato mottle ~ uuvuu5 ;. ~f~ 1 ;....~ as well as l~ A .. c to ;.. rC 1 ;~ of other related ~.lJilliVil us.,s. The know.n BC 1 gene, between ..~ s 1278 and 2311 of the B cu~ of tomato mottle g~v--us, was ~ C~ PCIintoana~ ieciApl~,a~ veetorand1.A.. ~r~ dintotobaeeoplants. Amutaf~ed gene product was ~luduf~i which confers -~ e against viral inf~ctif n to the lcccjl.Lill~t plant in which it is ~iA~.le;,~.
One object of this l.l. ~Liu.. is to provide a method for c~ " l il g viral l ~ c on a plant.
Another object of this ill~ ~lLiUll is to prc vide a mutated BC I gene and any L a ~,L thereof which confers viral ~ c on a plant.
Another object of this invention is to provide novel L-A~.sgf .;~ plants with ~ CC~1 viral 1 ~ ~; ~I A~ IC,C, Other objects and adva ltagcs of this hl~l~Li(lllwill become ~al~l~ from a review of the cnmrhPtP invention ~IiQrIr~c~re and the Al~ .flrd claims.
~ 30 Brief Description of the FiA~ures Figure 1 is the se~ e of the single stranded mutated tomato mottle g~llilliVil U ~ BC I
gene except for positions 1742-1766 which initiallv were not i~ i rir-~ wild-type mlrleofifl~!c which are ~ifferent in the mutant gene are sho~ in lower case text above the mutant gene S~ If-...~f WO 97/07217 PCT~US96/13097 Figure 2 is the ~c~1s~ c shown in Figure 1 along with its cnmpl~ n~y strand; theLIA~ AI start and stop codons are 1III-1. .. 1;~. .f1- the termini are HindlII l-~' ;. l ;O.1 sites.
Figure 3 is the deduced amino acid se~ c of the mutated gene product encoded by the mlrl~oti~1~ sc-lu- ~t~e of Figure 1, except for positions 1~1-159 which in initial ae~lu ~; e efforts S were not i~ ntifil ~l Figure 4 shows a cu. . ~f~A. ;C~ of the mutant and wild-type gene products (the mutant protein is the lower ac.~ ,.lce).
Figure ~ shows phc~lù~y~ic c~ -A- ;c.~.. of hAl~v~ ~;c Rl tobacco plants e~ aa~lg BCl protein of TMoV. T1A"'_I ~ plants were derived from a Ro plant which c-~ f ~s two copies of BCl gene (see Fig. 6) and which did not show any stunting. (A) Plants f~om left to right: a h....'_~...;.' plant (BC1-3-11-5) ~iA~ ,âa~llg ay~ n~ BC1 protein, shotnng stlmtine mr~ttlinv-, and curling on the leaves. Symptoms are more sever than those induced bv TMoV infi rtion b g., .;c plant (BC1-3-11-2) which contains one copy of the non-sy..~ ;r BC1 and the ay. . .l~ - BC l h a~g~e, showing mottling with no sl ~ ~ .l ;. .v c. ~ plant (BC 1-3-11-6 which contains one copv of non-s~lu~L~,llla~ic BC1 Lln~-~g. ~P, and d. non-L A .c~ . tobacco. (B) Plant on the left as in b, Fig. SA and on the right as in c, Fig. ~A. The plants in A were phuLu~al~hcd 45 days after trAn~plAnfinv-~ and in B 90 days.
Figure 6 shows S~lth~n blot analysis ofthe R, h- ~ plant with different ph.,~lu~ylJ~,s Segregation of the BC1 Lla lS~, in R, ~ ....,iOll of l,~ c~ tobacco plants which displayed diLr~.. t phe.luLy~es in Fig. S (BC1-3-11-1 and -2, mottling only, -4 and -~, severe stunting and mnttlinv~ -6 and -7, no visible ay~ LOll~). Blots from BC1-3-16-2 showing stunting and mottline and BC1-3-6-3 and -4, no visible svmptoms are shown for COlll~.. .i~., ~ul~u3CS; NT-I.. l.. ~r.. Aplant;andpKYsBCl,vectorcullaL u~LusedforLlA.. ~r~...... ;tm G~nomi~DNAof the ha~c~ ;c plants was e~tr~rted and digested with XbaI. Southem blots were sl~hject~A to lvb~ n tvith 32p-labeled Bcl DNA La~,lll.
Flgure 7 shows Westem blot analysis of the P3 0 fraction of tissue extracts from 1. ,...cg, .. ;c R~ tobacco plants CA~l~aalllg the BC I gene. Lanes l~,pl.,ae.ll e~tracts from plants dea~;liSJcd in Fig.
6 except for TMoV-infect. extract ~om TMoV infected tissue). The s..hr~ll..l~r fractions, P1, P30 and S30 were l)l~,d (Pasca's et al., 1993) and subjected to SDS-PAGE (S~h~g~r) with some .,.~1.~ .. and ;.. ~ ; using the polyclonal antsserum against e~!~leaa.,d BCl protein. The resu'sts of the P 1 and S30 fractions are not shown here.
Figure 8 shows Northern blût analysis of hc...~,.;c plants which express the BCl gene, probed with labeled-BCl DNA. Two BCl related h~a~ Ls were found in the hallag~c plants which t,.~ aicd the fi~'sl-length BCl gene, while only one Llanacli~t was found in the Llallsg~ic CA 02229168 1998-02-lO

plant which V-A~J~VS~ a 3 '-Llullcalbd forrn of the BC1 gene (BC1-3-11-6). The samples inAir~t~?A
are as in Fig. 6.
Figures 9A-1 thru 9A-5 and 9B show mlcit?4tiAç Sr~ f S (A) and ~l~livlvd amino acid Sc~ (B) of the TMoV BC l and its L. ,~ .~g, ~e mutants. The nllrl~otiAf sfe~lu ~re of TMoV BC l genefrom~nRi~nl ~rrfcQinn U14461. Thes~ r~ofthePCRi~mplifif,i BCl ORF uas veri_ed before and afier cloning into pGEM-T ~ ector. BC lA seqnt nre ~ f d from an aSymrtnmi~tir., m~ iropyLla~ vlnc plant which e.~lv~sed full length BCl protein. The se~ e was analyzed from the PCR product derived from genomic DNA (BC 1 -3-6-3A). BC lAt/r :,c~ e dt~
from the cDNA, the RT-PCR plulluvL~Tnrlified from the total RNA (BCl-3-1 1-6A). The S ~ f ~, was also verified by ~ - .. ;.. D the PCR product from the genomic DNA and from cloned PCR product. BClS SC~ f, ~ L ~ i from a SV~ CDf ~ plant which e,.~ ,;,sed full length BC 1 protei~ The s~ f, w as analvzed after RT-PCR of total RNA (BC 1-3-1 1-5S), aff~r PCR ~mrlifir~tinn of genomic DNA (BC 1-3-1 l-5S) and after PCR i~mplifiri~ti~m from 3 different lines with a similar phenotype. Note that identical nnrleQtiAt?s and amino acid residues are inAir~f.-A
by (.) Detailed necrTiptinn of the Invention The subject iu~v~,on ~n~r...... ~ a mutated plant virus gene that uhen e~lv~d in a plant confers on that plant a ~ rf~, to infection from plant p~thngf nc In one rTnhorlim~nt the mutated ~irusgeneisaBClgeneof~v~us. Themufatedgeneofthepresentu,.,~"lLiullcanbepl~,pal~dbv inserting the wild-tvpe gene into the genome of a plant and identifying those plants tri~ncform~-A
~ith the gene that exhibit h~l.,as.?d l~ -.re to viral inf~ctirm The subject invention also cl...~- ...c a method for cnnfrTring le~ .r~ on a plant to infection by plant pi~thngenq The subject method f~l~ ,es inserting a wild-tvpe viral llW ~ ~,.u~t gene, such as BCl, into the genome of a plant and then identifying those plants that do not e?;hibit pi~thogf nir svmptoms when the inserted gene is C~ ,sS~ but which have rnhi~nred l~ ic~ re to infectinn by p7~ll.n~...c The subject im~bntion also concerns ~ Dr-l;('. plants and plant tissue having a mutated gene of the present invention hlcull,ul~led into their genome.
The follo~hing is a specific example of the subject invention, a method for creating a virus-resistant plant7 using the BC 1 gene of tomato mottle g~,.llilliVU lls to illustrate the invention. The method is generally and broadly applicable to other plant viruses.
The c. l ' e se~ re of the BC l gene of tomato mottle g~,nLillivil u~ is l;no~n (Abouzid etal., 1992~hereinillcu~l~u~ bv~bfc~lcc) TheBClgeneoftomatomottleg~.~fvilu~ofthe SlJ~ 111 ~JTE SHEET (RULE 26) B ~ of the genome is isolated in ~- - r~...." quantity for ,~ c~ g in an Ci~.~L~S~;OLI vector.
This may be ~ulll~ by any of several methods well-known in the art. A simple method is to use a pair of speciiic primers to amplify the desired segment accc,.diug to the well known pol~.,l.,.~e chain reaction (PCR) t~hniql~e For this purpose, a useful primer pair such as:
5'-CCCAAGCTTCGAGTTCGAAACTGC-3'(SEQ ~ NO.l) and 5~-CCCAAGCTTAACGAAGTGTGTTTGAC-3~(SEQ ID NO. 2) mav be used. All or portions of the BC 1 gene may be used for this purpose.
Once suffirient qll~ntiti.-c of tne gene are obtained, the gene is cloned into a vector for ~L~lu~Lion of a stable source for mass produchon of the gene. Any vector known in the art can be used for this purpose, and mass .~ ;f~ of the vector may be cultured, for ~Y~mple7 by h,...~;r...~ l;O,. of c- ....~ .I bacterial cells such as E. coli followed by lh~ L~g of the plasmid DNA. Plef .ably, the gene is inserted into the multiple cloning site of a vector, such as the cul.u~c~,lally available pUC ~ectors or the pGEM vectors, which allow for excision of the gene having restrirtinn termini adapted for i~ iOu into any dcsuablc plant c,.~ loll or ;l't'.~
vector. For this purpose any vector in which a strong ~lOlllOt~, such as a viral gene ~r~lu~ is ù~C~aLi~ f linked to the coding ~e~u~ ~- c of the mutant gene of this iu.,~,.lLioll could be used. For ~mpl~ the powerfill 35S plUlJlUt~. of cauliflower mosaic virus could be used for this purpose. In one ~ ofthis iu~iol~, this ~ llU~ is ~h-~ f~ ~1 in a vector known in the art as pKYLX
71:35S2 (Morgan ef al., 1990). However, other plant e~l.,;,;,iull vectors could be used for this purpose.
Once the gene is excised and re-s~ on~ into a desirable e"~ ,;u.l vector, the gene is transforrned into â b~ - ;.--.. or other vector which is able to ~uLlù~luCC the gene into a plant cell.
Al~uaLi~,ly, the gene mav be iuLl~luccd into plant cells by a biolistic method (Carrer, 1995).
Preferably, c ~ A~,oba.,h, i~,. c~s are used for this purpose, and plant sections are exposed to the Agrobacterium ~ bUli~lg the BC1 gcne. 1~~ D, .. ~ of the plant cells in a scl~-,e medium to ensure the efficient uptake of the gene is ~lefe.l~,1, following which the l~ ..~d plants are grou~ under o~ ;,- d c-...~ ;....c for survival.
As a result of this process, it has been diacO~ d that a large l,lupulLiu.l of l~g_ne~aL~i tobacco plants which were h a~a~ uC for the BC1 gene had a ..p.,..~ ly mutated gene which GA~IGssed a mutated gene product. U- ~ ly, the plants harboring the mutated gene had hlClG&.iCd 1~ .i;Cli~.--'f, to viral inf~çtir n by both DNA and RNA plant viruses, without any ObSG. ~.~d d~ . effects resulting from t"~lG~:~;Oll of the mutated BC 1 gene (in contrast, ~ o~ . of the wild-type gene prù.luc.,;~ disease ~ l~ u~).
-While those skilled in ., ~I~lr~ biology are able to clone the known BC 1 gene into a plant C.,.,iOll vector to obtAin the mutated gene of the present invention, the mutant gene of this invention has also been d~osilcd prior to filing the instant patent ~rplir~tinn with the ,~m~rir~n Type Culture Coll~çtinn (ATCC), 12301 Parklawn Dri~ e, Rockville, Maryland 20852 USA. The mutant gene was cloned in a bacterial vector (pGEM-T) and the CuuaL- u.,~ is narned TMBC lm. The deposit has been ~ign~d accr ~ ," number ATCC ~To. 97244 by the ~~,~u~;L- y.
The subject deposit was d~u~iL~ under C~ ll .c that assure that access to the deposit will be available dunng the pedl~,...,y of this patent ~rpli(-~ti~1n to one dvt~ by the Cù~
of Patents and Tl ~ L ~ to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposit will be available as required by foreign patent laws in co~ .; s wherein C~uu~ of the subject applir~tion or its progeny, are filed. IIo.._~,., it should be ~m~hor.~tood that the availability of a deposit does not c~ l ;lul~ a license to practice the subject invention in d~.lugaLiull of patent rights granted by gu~v .. li l action.
Further, the subject deposit will be stored and made available to the public in accord with the ~.uv~;o~ of the Rl~larç~t Treaty for the Deposit of Mi.,.~ . . .c i.e., it will be stored with all the care l~c~;, .~y to keep it viable and ~ . r.. .l~ .1 for a period of at least five years after the most recent request for the f, .. . ~;'1-;--P of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the c,.~fo.~able life of any patent which may issue ~ the culture. The depositor aclinowledges the duty to replace the deposit should the ~ .~,o~;l . .. y be unable to furnish a sarnple when .~ u~ A due to the contlition of the deposit. All l\contheavailabilitvtothepublicofthesubiectculturedepositwillbeillc~ocàblyl~o.~.d upon the granting of a patent ~licrlncing it.
To use the biological materials dc~,o~ ,d, all that is ncccssa y is for the DNA to be solubilized in an &~ u~Jlialt:trAn~f~ ;V~ buffer for the cell type into which the gene is to be 1,.. ~f .. , ~A ForE. COIi~ CVI ~ , tc 1I cells are ~ al~d and L.~ .~fo.. ~1 a~.~li.. g to methods well h~wn in the art (see Maniatis et aL, 1982), and Ll A ~ ~ r(~ F'd cells sdected in an ~mri.-illin growth medium. The plasmid is then isolated from the E coli and excised from the pGEM-T vector using, for ~ mrl~ Hindm r~st~icti-n enyme. The excised gene La~e.lL has a size of about 1100 bp.
The Hindm fr~rn~nt is then cloned into the HindIII site of an a~JlUIJLi~.t; e~ ,.on vector as df ~ ~1 below. In addition to the above, Figure 1 provides the scq~ ~ of the mutant gene of this .lLioll, except for a stretch of 25 n~rleoti~l~s corresponding to positions 1742-1766, which were not ~ ntifi~d in initial se~ g efforts. There are several mnt~ti~nc in the pol~ of Figure 1. Those mllt~tionc are part of the instant ~ ,.lLion. Further, Fig~ure 2 provides the compl~ A~V strand of the mutant polvml~l~oti~to. and shows the Hindm termini. Figure 3 W O 97/07217 PCTf~S96/13097 pravides the dedlued amino acid ~v~l~.f .l ~, ofthe mutated gene product, except for arnino acids 151-159 which were not ir1~ntifi~rl in the initial w~lu .~ ;..g The ~li~c..-,e s in amino acid ~c~ n~4 between the wild-type BC 1 product and mutant BC 1 ~ U~L~, are shown in Figure 9B. Figure 4 shows a C~ IJA ;~ between the wild-type (upper ~C~1U .~ c) and mutant protein (lower SV~
based on initial seq~ v ~ ;.. g e forts.
While this description pluvidc, a specific gene and La~u~La thereof which conferI~L~cG on plants to ~vi ua and lob~ùvil ua inff rtion~ those s~lled in the art will ~~ro~ v that .. , .~ other than or in additifon to the specific .. u: ;.. c shown herein could achieve similar results. Tn fact, the method taught herein, bv which the mutant gene ~ ed herein was obtAin~yi is broadlv applicable to the ol,~t~ of similarlY useful mutated mu~ L genes of anv virus.
Furthermore, it is ~ Lable, based on the instant ~ lo~ that the instant genes and pGIv~ , "~ ,5 r1~r.rihed herein, as well as likewise-derived genes, can confer ,~ r, on a plant against ;- - ~; ~ l ;.... by a wide variety of plant p~lthOgf n~ which depend on .,.ù . _...c..l gene or other gene products for their pAth-. ,f .,r ;~ Q both DNA and RNA viruses.
1~
~mple 1 - D",~ of Ll .~v~ .;c tobacco plants A. G~u~L~u~ion of BCl ~ene into an ~,,.y~ siull vector. The BCl gene (""~
between 1278 and2311 oftheB ,-- ..~-o~ oftomatomottle~j.,,...nlvuua,Abouzidetal., 1992) was A- ~ ~p~ I from the e~racts of tomato mottle g~v,- ua infected tomato plants by poly chain reaction (PCR) t~rhnol-~gy. The primers used to amplif~r viral BC 1 were 5'-CCCAAGCTTCGAGTTCGAAACTGC-3' (SEQ ID NO. 1~ and ~ '-CCCAAGCTTAACGAAGTGTGTTTGAC-3 ' (SEQ ID NO. 2).
The . .~ ~ BCl segment was cloned into a pGEM-T vector and then digested with Hind m. The excised BCl segment was ligatedinto theunique Hind m site of the bina~y pKYLX 71:3~ s2 vector.
B. A~u~ t -;~ dt;~ C~ IcellsofAgrobacteriumrumefaciencesLBA
4404 were prepared as d~s~ ~ ;hed by An, et al., (1985). The BCl gene in the pKYLX 71:35 s2 vectorwas d~y ~r~ d into theAgrobaclerium. The clone was kept in a -80~ C frxzer for further use.
C. Plant ~ r-- ~ iO~ TheAgrobacterfum car~vingtheBC1 geneinthepKYLX71:35 S2 vector was used to ~ '.. the leaf discs of Niconana fobacum cv. Xanthi. The Agroba.,-~er. u".
cells were cult~ed in YEP broth ~ n;~ g 50 ,ug/ml ~ v.,.,- and 10 ~g/ml tetracycline and 25 ~/mls~ v~ for 24-30 hours. Agrobacterium cells were collected and ~ r .,,lr~ in YEP

WO 97/07217 PCTrUS96/13097 broth. Leaf discs cut from ~ d young sterile see~l1ingc were dipped into theAgrobacterium ..c;o" and then placed on a sele~iLi~,-, medium C~ P 200 ~g/ml Mt?fnYin and 100 ~g/ml kanamvcirL Res~ aLion and s~lç~tion were carried out with the media, and took 6-8 weeks. The Lallduly~il .. resistant plants were individually gro ~n in soil under sterile c- ...~l ;l i.... for a weeL, and then tr~ncpl~nt~d to pots in a growth room and/or gl... hv .c~

FxAmple 2 - PCR Southern blot and ELISA analvsis Tr~ncformAtinn of the tobacco plants was c- .. . r;. . . ~d by PCR analysis for BC 1 gene in cLo,..ns~ l DNA extracts, by Southern blotting with a BCl probe, and by ELISA analysis for NPTII(Ne~ v~ Phncrh-~~~~f~.. seII). Twenty-threeplantswere~ Cg~ forBC1.

F,~ATllrl(~ 3 - Western blot analvsis Infected leaves of tomato plants uere powdered after freezing in liquid nitrogen snd eAtc~i~,ly ground with a mortar and pestle in t~o volumes of ice-cold grinding bu~fer (GB: 100 rnMTris-HC1,pH8.0, l0mMEDTAand~ml liLLiuLl............. ~,.Lol)(Deom,etal., 1990). M~blal~eand cell-wall fractions were prepared as ~1~ ,- ;1 e~l by Pascal, et al., (1993). The blotting ~luc~ was col-d~.~le~l ecs~ontiAlly as ~c. jl-~ by Towbin, et al., (1979) using a Bio-Rad Mini-Protein E1C.;L uphc~l~,;.is Cell and Bio-Rad Trans-Blot Ele~;LI~holeLic Transfer Cell. The s~dLOl gel for small proteins was prepared with 12.5% polv_wyl~ide in gel buffer (T .A~.mmli, 1970). The protein gels were ~l~L.l~l to nitroc~ -lnse ~lbl~c (Bio-Rad Trans-Blot, 0.4,L4m). The ~ n of ~ ul'~,i,sed BC1 protein in L1~.~.C~. I;C tobacco plants was cnn~lncted with Western-Light~Chlomil.. ;,.F ~ ,I Detection Systern (TROPIX, Inc.). The BC1 protein was detected at a high level and extracts from about 50% of the plants showed a s aller (~ lcat~ ) BC1 protein (28k Da) than the wild-type (33k Da).
F,YATllrl~? 4 - Ev,~ t;u" of 1-,- ,~o.. ~ tobacco plants for svrnptorns due to the e~ ,;on of the BC1 gÇ~
The Bc l gene has been ;" l~ as a ~yll~ inducing elernent of a bipartite g~ vi us during inft~ctinn Eleven L~ Cg~ ;r tobacco plants which ~ se1 the full length BC1 protein showed disease ay~ n~C Twelve plants e,.~ h~g the truncated BC1 protein did not show disease svrnptorns.

W O 97/07217 PCT~US96/13097 Example j - R ~ e to tomato mottle ~.~.llllLlVLl U~ and tobacco mosaic Lob_l-uYil ua T~ (."..~tobaccoplants(R, ~n~r~tinn)tA~L~,aaillgBClweretestedfora~ G~ hilify to tomato mottle ~ fllhvLlus infrctit~n by natural ~.~.,c...;cc;~... with the white~y vector and by "~ TA1;. . with e~acts from infected plants. The innc~ ted plallts were evaluated for ~ to tomato motLle g~llilllVil ua by aylll~Lo n develnpmenf and by en~-me linked immuno-asssys (ELISA) using aLlLL ~WLIIll reactive to tomato mottle gCL~iluYll ua coat protein. The ~ . . ,;r plants c;.Y~ r~sa~lg the LrUL~.~,aL~l BC1 protein were free of aylll~LoLus and had very low ELISA
readings. T1A ~C~ ;G tobacco plants aulJic~hd to ~ inoclllAtinn with tobacco mosaic LcJ~ùvilua showed reduced disease aylll~lullls cunl~ed to inoclllAtpd non-L~allsgw.ic plsnts.

--I,k6-AnalvsisoftheBCl ~Yenee~ ul L uncAL~,dprotein The BCl gene from the tobacco plants e~ ,s~iug the L.ullc.~t~d BC1 protein was PCR-Amplified and ~e~ .1 This data ;..~ Atr~ that the BCl gene has ulld~gul~e ~v~
mIltAti~m(s) in about 50% of the L.,..-~. .;c BCl tobacco plants During the tissue culture phase, plant cells c~ the mutated BCl gene may have a selective &lv~Lagc over the wild-type BCl Jl ~,aa Ulg cells.

FY~ 1C 7 - Plu.~ ;0~1 of L~ P~ .ir tomato ~lan~s The mutated BCl gene in the pKYLX 71:35 s2 vector is suitable for the pro~11ctinn of I~ t~r forthe gene viaAgrobacterium trA~ncfnnnAtinn as ~ 5 ;~ above for tobacco.The mutated BC1 gene provides similam~ c to tomato mottle p~e.lfL~I-vil ua in tomato as seen in ~ C~ ~1;1 tobacco. The illllu lu~Liûu of this mutsted BCl gene into the .,Lr...-os~ - of desirable tomato lines leads to tomato mottle g~lfillivuua l~ ,r. in cc~ ..;ally ar~ Ahk tornato cultivars/hybrids. In addition, it is pl~ Ab1e that this .. ~ e is active against other E;~.~lfillivuua inf~ctio~c F~ li ,rr! to tobacco mosaic virus was also detected in the L-~s jwuC tobacco aaillg the mutated BC 1 gene, i. ..1;- AI ;1 Ig that l. ;~I Al 1~ C to RNA viruses also is possible with the .resaion of this mutated gene from a DNA plant virus. The mutated gene in tomato offers c to tomato mosaic tobanluv.. us, a virus related to tobacco mosaic ~ùballluvilua.

Example 8 - Production of BC1 ene fi~ S useful for cu~ u-~ virus l~ c to plants.Fla~wlla ofthe mutant BCl gene which are useful for c~ virus 1U~ rC to plants can be ,UlU~lUCell by use of BAL3 1 ~ om~ Ace for time-controlled limited ~ii~sti~n of the mutant BC 1 gene. Methods of using BAL3 1 e ~ c for this purpose are well known in the art, and havebeenwidelyusedforoveradecade(WeietaL, 1983). ByusingBAL31 r~ rIF~cc~onecan W O 97/07217 PCT~US96/13097 easily remove ....r.~ J~ f s from either or both ends of the mutant BC 1 gene to ay~ .Ally and certainly generate a wide a~e~LIu~u of DNA fragments which have controlled lengths and are from controlled ]nç~Atinnc along the entire length of the mutant BCl gene. IIuud l,ds of such Lla~wl~
from various points along the entire mutant BC1 gene DNA se~ c can be ay~t~ ;c~lly generated in one ~Aft~rn-~on These gene La~u~ La are then cloned into ~upliaLe vectors and n1tim,Atf~y L,.",.-.L,.l~d into plant cells according to the methods l1icr1Osed above. Plant cells r ,.. 1 with these L .. ~ll~ are luuLulclv cultured and l~ " al~,d into plants, which are then tested for l.~;~l7~ c to viruses. In this manner, fragments of the mutant BC1 gene which are a..r~. ~..1 toconfervirall~ ~;o ni~rG are l-)ulillely and~l~di~L7~lyi-lfnti~0 F,Y7~mrtlfe 9--Production of ~ 1;1 ;o~l mutants co.~.. IllD virus ~~ c to Dlants.
Tobacco was tr~Ancfnnnf~d with the 1~l0~ L protein (p7~thoglonirity) gene (BC13 from tomato mottle g~vuua (TMoV) using Agrobacterium~ d l.~ rl.. :;nn Different 1,. .cg, ., r tobacco lines ~,~l~a~g the BCl protein had phc.l~,~yl,es ranging from plants with severe st~mting and leaf mottling to plants with no visible sy~p~c~ c The se~ .rf, data for the BCI
1.~ ICg~lf forthediff=tpL~ly~s; ..I;~t~d....~ 7~ (s)~ AmutatedBCl l.~S
au~l~acd the p~ulv~ nl~ ofthe ay---l~ AI;- BCl gene in tobacco lines c- ...IA;~ g both copies of the BC l gene. The present iu~ h~n shows ay~ f'Ou~ .. I ,~1 ;n.~ in the haus~ e to be common inA~,~)ba~ m-l l lf ~ r~ l lc~ and this ph~ .- - - can be utilized in the creation and selection of pAIhn~;l - -resistant plants using p~thogf ni--ity genes during ~ r~.. " .~, ;n.~
The e.~yl~ ~a;Ou of tomato mottle gemini virus (TMoV) (Abouzid et al., 1992) movement protein gene (BCl) was ~ rcl in Llauag~, liC tobacco plants for evaluation of function and for possible 1l*1i7A*nn in p,.~ derived l~ f The BC 1 gene has been imr1irAtsd as a s~mptom induring element of a bipartite g lllluvilua during expression in trAncgf~nir plants (Pascal et al., 1993; vonAmim and Stanlev, 1992). T. .c~ . ~ tc)bacco e.~y~ aiug the BCl gene was CCl aL~u~kd utilizing standard Agrobac~c,-u", m~i~t~i L~ ~rO~ n Surprisingly, a number of plants e~ aiug TMoV BCl protein based on Westem blot analysis, did not show the t,~l.c~,l d virus-symptom phe.lolvl.e. Only 11 of the 19 Lla~S~ C Ro tobacco plants which e~yle;.a~ the BCl protein showed disease ayul~lulu~ ranging from mild to severe. The ùbs~. vaLion that eight plants e~ylei~aiugtheBC 1 proteindidnotshow~vlul~LC~u s was .~ e~
From further analysis, the three phc~uLv~,s were observed in the R, generation derived from a Ro plant which did not show arlya~l)a~ lL stunting (Fig. 5). The three obsen ed ph,.luLy~cs were:
I) Severe stunting and mr~ttling, more severe than the tvpical ayluyLwll5 "~o~ l~1 with TMoV

, W O 97/07217 PCT~US96/13097 inf~tion~ in tobacco; 2) Mottling with no stunting of growth; and 3) No visib}e by~ , plants in~ ;"g~ l.Aklt? from n~, lhA~ro~ d plants, These ~ plants were analvzed bv Southern blots to identify gene copy number (Fig 6). The ~, d~ plant showing slight mottling with no stunting had two copies of the BCl gene Other progeny from this line which had a severe ayl~lulll ph~lu~y~c or a non-ay~pl~ùLylJc orllv had one copy~ Progenv from three other lines ~h ~ one with ay~ A~ ;~ and two with non-avlll~lùlllaLic ph~uLvyca, had 3, 3, and ~ copies of the BCl gene, ~c;~,u~iLi~ ly.
High levels of the BCI protein ~ were i...l;-.~t.,~l in the young tissues in all ("..nv.... c plants by Western blot anal,vsis except for one non-ayll~lo~aLic line which showed low levels of a h u~ ~l BC 1 protein. Non-av.~ ol,laLic plant (BC I -3-6 1; ph~uly~C not shown) had asimilarlevelofBClproteinasthes~ plant(BCl-3-~ ). Extractsfromthe ~ AtA-ayl~Lull~ plant (Fig. 5A and B; BCl-3-l 1-2) showed both full-length and Llull~,a~ BCl proteins The low level of the h ulll,alC;I BCI protein ~t~tinn may be due to the loss of epitopes since 12l amino acid residues were lost at the carboxy end (see below). BCI proteins (full-length or hU~ .t~
form) fr~n the non-ay.. ~ ;, L _ ' plants were not detected in older tissue, unlil;e that sc~n for the L1AI~Cg~ ;C plants e.~le~a..lg the severe aylll~Lum type BCl protein. This ;...I; ~tl-i that certain .. .~ C in the BC1 protein may affect its stability in planta.
Northernblots;.~ t~dahighha,ls~ LnumberforalltheLlala~ ..clines(Fig~8)~ The non-svmptomatic plant shown in Fig. ~A had a smaller than ~ ~l e ~t~ d ha~ . This a~a~
delction in the ~n~ lis~ nth the hu~aL~d BCl protein seen in West~n blots (Fig. 7).
The ~ L level for the plants e.~ sai lg the Llull~àL~l BCI protein was high and LL C~lC the low level of L, u.l~ ~l BC l protein detected in Westem blots (Fig. 7) is not due to h a~ activit~ .
The larger than expected ~ l is the result of a l t~adLlll uu~ll of Bc l l.. .. , ~ ; . . A : ;n. , signals into the vector rbcS I.. ;.. ; ;on s~ of the pKYLX vector.
The BCI gene from the hanS~ iC tobacco plants showing the ~lifferent ph~ ~uLy~J~,s was Amrlifi~d bv pOly~aa~, chain reaction (PCR) and a~ rl The se~ , . .e data revealed mntAtinnc (amino acidresidue 215 G-S, 219 S-L, and 247 E-G) near the carboxyl terlIunus of the BCl protein (Fig. 9) for the severe stunting phenutv~ (Fig. 5A). T-YO mutants were A~o.~iAIf~ with non-ay~ ~L;~" LLa~La~f~LLLC tobacco. One mutant (resolved from BC 1-3-6-3) showed several changes near the arnino l~ mino acid residue 6 V-F, 7 N-S, and 35 F-L) while the other (resolved from BC 1-3-11-6, Fig 5A) shoued a cnange in amino acid residue 12 F-C, a deletion of amino acid residues 174-293, and an ~ i ri~ fusion sequence of 26 Amino acid residues starhng after arnino acid residue 173 (Fig. 9). This was C~ with the fletecti~n of a l~ caL~d BCl protein (~ 10 kDa smaller in size cu...~_~ to ~e wild t,vpe) in Western blots from e~acts from these Ll~uLagw~ic _ plants. TheL,~s.;..yLforthetruncated,BC1proteinwasse~ P~lafterreverse~ c~ t;~ of e~ll.._Led total RNA using oligo dT primer followed bv PCR Ampl;r;~ ;n~ using a BCl specific primer.
The non-aylllyLvlllaLic7 L A ~g, .;r Ro tobacco plants revealed se~e~aLivll in the R~
S generation as inrlirAtf d by the ayy_dl auCC of several s-ymptomatic plants in this gr ~ P1 ;OI) Some lines with aymyLolll n;U ~ (Fig. 5B) ~ ;l to se~ ~,gd~e in the R~ generation but the non-aylu~Lv~_L;C plants did not. Southern blot analysis (Fig. 6) ;...~ AI. d multiple copies of the BCl gene in the Ro tobacco. AYP~W1LIV some of the R" tobacco lines c~ Fd copies of both the symptomatic and non-~y~ ;c fomls of BC1. This was CO.~r;...--Ji by Southem blot and Western blot analyses of selected R, tobacco plants which t~ere a~ coci ~ ~ with the ~ erent yL~uLy~J~, (Fig. 5). The mottling phw~uLyye with no stunting ~lFcrrihed above (Fig. S) had one copy eachoftheay~ v~A~;~ andnon-sy~ A~ fomlsofBC1. TA~g~ tobaccoc.~ gcopies of both av...,.L..."~1ir and non-ay...l.lo..~ ;c fomls of the BC1 gene (,Fig. 5B) resulted in a n.~..~....l;.lv ~vith mild mottling ph,~uLyye. This in-iirAteC~ that the non-sy...l.L~ ;r BCl gene ~u~y~sacd (trans~ ~- ~;--~ ~I negative iut~f~wlce) the ayll.l.LO.Il ;---1~. ;-~o element(s) of ay~
BC 1 gene in l .. cg, .. ;~ plants C~ g both fomls. Tl .~ .. C_~ .. F cilFnrin~ (Meins Jr. and Ku~z, l99'i) was not evident in these plants since both protei~s were detected in Western blots (Fig. 7, BC1-3-11-2). F...~h.-II--J-G, the~ ~s.unofthe~.~ fi~yh~,~lvLyycins..I.se~l~v~generations ,1 that the ayluyLvluaLic BC1 gene was not in an inactive forrn in the phc,lvLyyc :~uyylG~ cd Ro tobacco. The ayuuylvll~ Suyyl~aiull was also effective against virus ;. . r~ . . since tobacco plants with the mntAt~A non-ay~ ",.AI i~ BCl Ll~ag~,ne .e...a~.cd free of TMoV syrnptoms under high disease pressure from viruliferous whiteflies over a 3-month period.
All of the BC1 L- ~5gf ~ic plants that were analy-zed revealed ~ OI~C/~ Ye~
mntatinnc in the BC1 gene. Point III~ IIC were found in all L~ analyzed, and onetransgene sho~ved a major deletion at the 3' end and with a fusion of an unitllontifi~ sc~ e of ~250 . .. ,~ s ~no dose l~ h;l . with â~ u~,~lCCS in GenBank using BLAST). This may have occurred bv a C1,.V~ S~ A1 cross-over event during plant cell division after the BCl gene was illLc~laL~;d into the tobacco .,l. .l. os-~ ..f, In the latter case, a Llull~,aL~,d BC1 protein (~ 10 kDa smaller in size ~-~.~1 to the wild type) was detected in Western blots and a smaller Llalls~ was detected in Northern blots. This in~lirAt~d that a deletion in the L~ a~ lC as well as point mntAtionc scrihecl previously) are sources of variation in Ll~l5~ 1C e.~lcaaion.
Other studies with the expression of foreign genes in LIAI~Cg~ ~ic plants show varying levels of e.~pll,ssi-i~y in the different g~e~aLC;i lines or in siblings in a Ll....cge..;c line (Hull, 1994).
Var,vinglevelsofl~,;.iaL~ul~indifferentL~alL,gc...~,plantlinestr~ncfo~n~dwiththesamegeneappear WO 97/07217 PCT~US96/13097 to be the norm in ,~nll ~(~gf ~-derived l~L~,cc studies. These variations are not ~lf~ ely f-YrlAinf~A
bv P~.~;l ;f~..A1 effects due to the random h1LC~ dLiul~ in the plant C 11l. ,.. ~f~ ....r. during t.~ n~ r. ,.., .nl ;n., .'~ik~n~infJ of gcnes in Ll ~"~v, ..;~ plants is considered a general phr..c ....~ . ~f~ when multiple copies of hA~X.~C are ~.-Lludu.,cd into plant cells (Meins Jr. and Kunz, 1995). All of our Ro lla~ V. .~;f plants analyzed c~ n;.. r~l multiple copies of the BC1 Llf ~ISg~lc with no a~ar~lL ~u~ a;OIl of Llf~,-c~ e e,~lf ;.alf~
Because the ~ h;..gc herein used the classical methods of Agrobacterium-~ A;~f -l r~ f ... c.~.... ~ .n. ~l,v used bv others in the art, some of the variation in the ~Yre(~.tf-d phenotype reported in the li~.fiLulc can be eYrlAin~d by Sp~ l;..,r~ lnl;f~ occ~ n-v during Agrob.~. ~f, f u ,. m~iAt~ tr~Ancffnrm~tif~n and dunng C111~ .. ~5~ AI r~Arrtmgem~ntc as reported here for TMoV BC1. Thus it is shown that s~u.~ln~ uc point ...~ C in the lf n.~7~C~I~. during Agrobacterf;um-m-~AiAtf~ I . n~ ~ r~ .. n 1 ;UI l and other mf flifirAtionf in the L f lag.,.lC by cL,o.. ~cn.. AI
rearrAngemf~.ntc affect gene function and regnlAtic.n ~ith L,n..~ge..r s The subject invention also cfn. .. r - - ~~ the pOlv--~f~l~OI ;f1~ mf l~f~ c shown in Figure 9A and the poly~cp~id~,s encoded therebv shown in Fifgure 9B, f~S well as other mutated poly. .. ~ c~ c c~.. . r~ . . ;. .g viral lc71~ c that can be ~-ùduccl using the ~f ~h;l~PC ofthe present illV~tiOll.
The ~7~ f ;~11 IC that can be ~ludu~l in viral l~lu . ~ genes using the methods and m~At~oriAl$ of the present hl~ltioll during Agrobacterium-. . . f~l;nl~ d hn~ r... ~.Af ;. .. provide a simple wav to develop pAth~v-~n-resistant plants. For gC~ iviluacS, the illhuJ~ l;r~n of the pnlh~g~ ;lv gene (BCl for the bipartite g~Lliv~ Ua~.7~ AC4 for the ~--~ .. .n~ ; like tomato yellow leafcurlvirus)intoplantcellsbyAgrobflcterium-m~ At~Lln~ru~ nl;u~willresultinse ;u~
since l~ ;r~ 1 cells which express the non-mutated p~thogtonirity genes will not grow as well as those cells which e~press the mutated p~th~g~irity gene. After Ll n~ ~ rU....~1 ;nn, visual evaluation for the non-~yl~LulllaLic phenuly~e and Western blot analysis for p~thngl~nicity gene protein C~y~ ,;ull iS âll that is needed to select g~llilliYilus-resistant plants for further 5.,l~ Lillg and cvalLldLiùlL All 1. ~ g. .;r tobacco ~hith a non-av~Lulllalic ph~,~luLy~e and with TMoV BC 1 protein showed virus 1 ~;~I; "rc Similarly, certain pathngtonirity genes from bacterial or fungal plant pathogl7nq can be i,lL,ùdu-,ed into ll~g~,. ic plants according to these t~chinSJ~ and the sel~ctinn pressure will result in pathogen-resistant plants.
The amino acid s~ iqrl~sod heran are based on standard single letter abbreviations for amino acid residues.
While the fole~uillg de~ ,Lioll and ~ nnrles provide details r~audillg the met_ods of making and using the invention, inrhl~linP its best mode, it is to be ulldc.~Lood that obvious W O 97/07217 PCT~US96/13097 idtiUll:i and r.,. . ~ uivdl~ theleof are to be considered par~ of this ill~ ~lio. . and l~ f fall u~thin ~e scope of the claims uhich follow.

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Claims (12)

Claims

1. A mutated plant virus BC1 gene which confers enhanced viral resistance to plants harboring said mutated gene.

2. The gene of claim 1 wherein the viral resistance is against a virus selected from the group consisting of tobamovirus and geminivirus.

3. The mutated gene of claim 1 prepared by the process of isolating the natural gene, inserting the natural gene into the genome of a plant, and identifying plants that have increased resistance to viral infection.

4. The mutated gene of claim 1 which encodes a gene product of about 28 kDa.

5. A method for conferring on a plant enhanced resistance against plant viral infection which comprises insertion of a viral movement gene into said plant and identifying a plant spontaneously expressing a mutant of said gene which confers said enhanced resistance against plant viral infection on said plant while at the same time not inducing pathogenic symptoms in the plant.

6. The method of claim 5 in which the viral movement gene is a plant virus BC1 gene.

7. A transgenic plant having increased resistance to viral infection, said plant being transgenic for a mutated plant virus BC1 gene.

8. The plant of claim 7 which is a transgenic tomato or tobacco plant.

9. The plant of claim 8 in which the plant has enhanced resistance against infection by tomato mottle geminivirus or tobacco mosaic tobamovirus.

10. The mutated gene of claim 1 which comprises any or all of the mutations as compared to the wild-type gene, shown in Figure 1 or Figure 9A.

11. The mutated gene of claim 1 comprising all or a portion of the sequence shown in Figure 1.

12. A mutant BC1 protein comprising any or all of the wild-type to mutant amino acid substitutions shown in Figure 4 or Figure 9B.