Note: Descriptions are shown in the official language in which they were submitted.
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
CA 02229168 1998-02-10
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
CA 02229168 1998-02-10
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
CA 02229168 1998-02-10
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
W O 97/07217 PCTAUS96/13097
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)
CA 02229168 1998-02-10
W O 97/07217 PCTAUS96/13097
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~).
-
CA 02229168 1998-02-10
W O 97/07217 PCTAUS96/13097
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
CA 02229168 1998-02-10
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
CA 02229168 1998-02-10
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.
CA 02229168 1998-02-10
W O 97/07217 PCT~US96/13097
I0
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
CA 02229168 1998-02-10
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
,
CA 02229168 1998-02-10
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
_
CA 02229168 1998-02-10
WO 97/07217 PCTAUS96/13097
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
CA 02229168 1998-02-10
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
CA 02229168 1998-02-10
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.
CA 02229168 1998-02-10
W O 97/07217 PCTrUS96/13097
Rerw~ cc;.
Hanson, S.F., RL. Gilbertson7 P.G. Ahlquist, D.R Russell, D.P. Maxwell (1991) "Site-Specific ..~
in colons of the positive NTP-bin&g motif of the ALl gene of bea~ gold mosaic g~il~ivilu7
abolish i~c~Livil~,''Pi~,vtopathology 81:124-127.
Noris, E., A.M. Vaira, G.P. Accotto (1994) "T~ ;l;.... of TYLCV DNA repl;rAti~n in ~ ~cc of a
L~ ;u.~,d Cl gene," First Internanonal Symposium on Geminiviruses, Elejido/Almeria, Spa~n,
Septemi~-r 14-17.
Fontes, E.P.B., H.J. GlA~lfelt~r~ RL. SrhAff~r7 I.T.D. Petty L. Hanlev-Bowdoin~ (1994) "G~livi
replirAti~ n origins have a modular ~ e Plant Cell 6:405-416.
Von Arnim7 A., J Stanley (1992) "T..l.;l.il;~.. of Africian Cassava Mosaic Virus systemic ;..r~ ... by a
nlu . ~..~L protein from the related g~llL~iVi~ u~ Tomato Golden Mosaic Virus," Yirology 187:555-
564.
Cooper, B., M. Lapidot, J.A. Heick, J.A. Dodds, and RN. Beachy, RN (1995) "A d~,f~ii~ U~
protein of TMV in I ~ g~ ~ -ir plarltS COrlferS 1~ Ae to multiple viruses whereas the r. - .- -~ ;n- ~nl
analog ill~,lcases susceptibilit~r," Yirology 206: 307-313.
Nejidat, Ali, Roger N. Beach~ (1990) "Tl~.g~,.. c Tobacco Plants E~ .g a Cûat Protein Gene of
Tobacco Mosaic Virus Are Resistant to Some Other Toba~..ov.lu 7cs," A~olt~cv~7r PlantMicrobe
In~erac ion 3:247-251.
GiL~1~l7 RL., E.J. PAI,l.. : Ac~ p.D~ Grieco~ v.p~ pateL and D~p~ Maxwe~ 993) ''A~ r~ n of Bean~
Tobacco, and Tomato with Two Bipartite Gc~ ..ivL.u .es Isolated from Tomato," P*ytopa~hology
83:1350.
Abouzid, Ahmed M., J.E. Polston, E. Hiebert (1992) "The m~rl~oti~le S~ e of tomato mottle virus, a neu
g~v - - .~ . isolated from ~ O~ 5 in Florida," J. General Yirology 73 :3225-3229.
Carrer, H., P. Maliga (1995) "Targeted Insertion of Foreign Genes into the Tobacco Plastid Genome vwithout
Physical Linkage to the S~l~~çtAhl~ Marl;er Gene,"Bio~echnolo,~ 13:791-794.
Maniatis, T., E.F. Fritsch, J. S~.blool; (1982) "Mol~r~ Cloning: A Labu~aL~ y Manual," Cold Spring
Harbor LaboratoIv.
An, G., B.D. Watson7 S. Stachel, M.P. Gordon7 E.W. Nester (1985) "New cloning vehicles for
LLA..~r;..,...AI;-~n of higherplants,"EA~BOJ. 4:277.
Deom, C.M.7 KR Schubert, C.A. Holt7 W.J. Lucas, RN. Beachy (1990) ''MO~ 1IAr C1 IAI 0~ A~ and
biological function of the m~ l protein of tobacco mosaic virus in ~ Sg~, liC plants," Proc
~atl. Acad Sci USA 87:3284-3288.
Pascal, E.J.7 P.E. Goodlove7 E.C. Wu, S.G. La~uwi~ (1993) "Transgenic tobacco C;~ g the
gC ~illliYi~ ~L protein BLl exhibit ~y~--yLo~ of viral disease7 Plant Cell 5:795-807.
Towbin, H., T. Staehelin, J. Gordon (1979) "Ele~iL~ uuh~ ic transfer of proteins from polyacrylamide gels
to rrutrocell~lose sheets: ~luccd-u~s and some appli~ati~.n 77 Proc Nafl. Acad. Sci USA 76:4350-
4354.
CA 02229168 1998-02-10
W O 97/07217 PCT~US96/13097
Groning, B.R, RJ. Hayes, KW. Buc~i (1994) ' S;~ u~ reglll~tinn of tomato golden mosaic viras coat
protein and ALl gene .,.~ ,;.ai.~ of the AL4 gene may col ll l ;l ~ to .u~ ;.sion of the
ALl gene," Journal of General Yirology 7~, 72:1-726.
Hanson, S.F., RA. Hoogstraten, P.G. Ahlquist, RL. Gilbertson, D.R Russell, D.P. Maxwell (1995)
"~nt~tion~l analysis of a putati-~e NTP-binding domain in the repli.~tinn .1 ~G~ .n,;l protein (AC 1)
of bean golden mosaic gw,lu,ivi, us," Virology 211: 1-9.
Ingham, D.J., E. Pascal, S.G. L~ ,wiL~ (1995) "Both bipartite ~ vhu. m~ ~w~ L proteins define viral
host range, but only BL1 d~ t~,, . ,;.~ s viral pc ~I~..g, ,;- ;Iy," Yirology 207: 199-204.
Morgan, B.D., M.H. MacDonald, R Gla~lJoswh, A.G. Hunt (1990) "Upstream 5~ other than
AAUAAAarerequiredforefficient.~ . ,. RNA3-endfnnn~tinn inplants," PlantCeU 2:1261-
1272.
Sanford, J.C. (1990) "The biolistic plant L~ r~ ;nn,"Physiol. Plant 79:206.
T aemmli U.K (1970)"Clea~ageof 7LI Ul:t,Ul al proteinsduringtheassemblyoftheheadofba~l ;n~ D~
T4,"Nan~re 227:680-685.
Wei, et al., (1983) J. Biol. Chem. 2~8: 13506-13512.
