U.S. patent application number 08/838151 was filed with the patent office on 2001-08-02 for transgenic plants expressing mutant geminivirus ac1 or c1 genes.
Invention is credited to AHLQUIST, PAUL G., GILBERTSON, ROBERT L., HANSON, STEVEN F., LUU, HANG T., MAXWELL, DOUGLAS P., STOUT, JOHN T..
Application Number | 20010011379 08/838151 |
Document ID | / |
Family ID | 21771857 |
Filed Date | 2001-08-02 |
United States Patent
Application |
20010011379 |
Kind Code |
A1 |
STOUT, JOHN T. ; et
al. |
August 2, 2001 |
TRANSGENIC PLANTS EXPRESSING MUTANT GEMINIVIRUS AC1 OR C1 GENES
Abstract
The invention involves production of transgenic plants
containing DNA encoding AC1/C1 wildtype and mutant sequences that
negatively interfere in trans with geminiviral replication during
infection. The transgenic plants produced by the invention are
resistant to viral infection.
Inventors: |
STOUT, JOHN T.; (KALAMAZOO,
MI) ; LUU, HANG T.; (KALAMAZOO, MI) ; HANSON,
STEVEN F.; (MADISON, WI) ; MAXWELL, DOUGLAS P.;
(VERONA, WI) ; AHLQUIST, PAUL G.; (MADISON,
WI) ; GILBERTSON, ROBERT L.; (DAVIS, CA) |
Correspondence
Address: |
ROCKEY MILNAMOW & KATZ LTD
TWO PRUDENTIAL PLAZA SUITE 4700
180 NORTH STETSON AVENUE
CHICAGO
IL
60601
US
|
Family ID: |
21771857 |
Appl. No.: |
08/838151 |
Filed: |
April 15, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60015517 |
Apr 16, 1996 |
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Current U.S.
Class: |
800/279 ;
800/280 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 15/8283 20130101; C12N 2750/12022 20130101; C07K 14/005
20130101 |
Class at
Publication: |
800/279 ;
800/280 |
International
Class: |
A01H 005/00 |
Claims
We claim:
1. A transgenic plant comprising chromosomal DNA, the plant
harboring geminivirus DNA integrated into the chromosomal DNA, the
geminivirus DNA encoding a protein required for geminivirus
replication, and the geminivirus DNA conferring resistance to viral
infection.
2. Transgenic plants according to claim 1 in which the geminivirus
DNA is wild type DNA.
3. The transgenic plant according to claim 1 in which the
geminivirus DNA comprises an ORF selected from the group consisting
of AC1 and C1.
4. The transgenic plant according to claim 3 in which the
geminivirus DNA comprises DNA encoding an amino acid sequence
selected from the group consisting of FLTYpxC; pHlHvliQ; vKxYxdKd;
FHPNlQxak; EGx.sub.2RTGKt; and NviDDi.
5. The transgenic plant according to claim 1 in which the
geminivirus DNA is a transdominant interference mutant of a
geminivirus gene.
6. The transgenic plant according to claim 5 in which the
geminivirus DNA comprises an ORF selected from the group consisting
of AC1 and C1.
7. The transgenic plant according to claim 5 in which the
geminivirus DNA encodes a sequence motif selected from the group
consisting of a DNA-nicking domain and a NTP-binding domain.
8. The transgenic plant according to claim 7 in which at least one
mutation region of the transdominant interference mutant of the
geminivirus DNA encodes an amino acid sequence comprising FLTYpxC;
pHlHvliQ; vKxYxdKd; FHPNlQxak; EGx.sub.2RTGKt; and NviDDi.
9. The transgenic plant according to claims 7 or 8 in which the
geminivirus DNA consists of at least one mutation of FLTYpxC;
pHlHvliQ; vKxYxdKd; FHPNlQxak; EGx.sub.2RTGKt; and NviDDi in the
AC1 ORF.
10. The transgenic plant according to claims 7 or 8 in which the
geminivirus DNA consists of at least one mutation of FLTYpxC;
pHlHvliQ; vKxYxdKd; FHPNlQxak; EGx2RTGKt; and NviDDi.
11. A transgenic plant according to claim 8 in which the
geminivirus DNA is a ToMoV AC1 mutant selected from the group
consisting of Sequence ID Nos. 3, 5, 7, 13, 14 and 15.
12. A transgenic plant according to claim 8 in which the
geminivirus DNA is a TYLCV C1 mutant selected from the group
consisting of Sequence ID Nos. 23, 26, and 29.
13. A transgenic plant according to claim 8 in which the
geminivirus DNA is a BGMV AC1 mutant selected from the group
consisting of Sequence ID Nos. 45, 48, 51, and 54.
14. A method of interfering with geminivirus infection of a
transgenic plant comprising: selecting a transgenic plant according
to claim 1; and growing said transgenic plant.
Description
DESCRIPTION OF INVENTION
[0001] A variety of geminivirus genes and mutant derivatives were
generated and transferred to plant cells. Transgenic plants
containing these genes were produced. Transgenic plants containing
trans-dominant mutations developed resistance to geminivirus
infection.
BACKGROUND
[0002] Geminiviruses present the most serious disease problem in
many vegetable crops in tropical and subtropical regions. For
example, major epidemics of geminivirus infections of beans and
tomatoes have occurred in Florida, the Caribbean Basin, Mexico, and
Central America. In the past, traditional breeding methods failed
to produce cultivars with significant levels of resistance to
geminiviruses. An alternative approach lies in producing
virus-resistant transgenic plants according to the present
invention.
[0003] The geminivirus group are single stranded DNA viruses that
infect both monocotyledonous (monocot) and dicotyledonous (dicot)
plants. A common feature among all gemini viruses is the mode of
genomic replication, which involves a rolling circle mechanism.
[0004] Tomato mottle virus (ToMoV) is one example of a geminivirus.
It has a two component (bipartite) genome, an ability to infect
dicot plants and is transmitted by whitefly. The DNA of its two
genomic components, DNA-A and DNA-B, has previously been cloned and
sequenced. Isolated clones of DNA-A and DNA-B of ToMoV are
themselves infectious when mechanically inoculated into tomato and
N. benthamiana, or when delivered to either host by
agroinoculation. An invariant geminiviral DNA sequence required for
replication is present in an intergenic, common region (CR) in each
genomic component.
[0005] The ToMoV DNA-A genomic component has four ORF, one of
which, AC1, must be expressed for efficient replication of both A
and B components. The AC1 ORF encodes a protein having several
functional activities: a DNA binding site specific to the DNA-A CR;
a DNA nicking activity; and a NTP binding activity. The DNA binding
region mediates an initiator protein function during rolling circle
replication.
[0006] AC3 protein is a second ToMoV-coded function involved in DNA
replication and production of single-stranded circular DNA.
[0007] Tomato yellow leaf curl virus (TYLCV) is another example of
a geminivirus. TYLCV has a monopartite genome organization, infects
monocot plants, and is leaf-hopper transmitted. The TYLCV C1
protein is required for replication, encoded by the C1 ORF.
[0008] Being DNA viruses, geminiviruses offer advantages for
antiviral strategies. Several geminiviruses have been cloned and
sequenced. Transgenic plants having mutant viral genes can be
produced, e.g., by introducing expression cassettes comprising
mututated virus genes directly into plants with a particle gun, or
into plant suspension cells or protoplasts by electroporation, or
by Agrobacterium transfection.
SUMMARY OF THE INVENTION
[0009] The invention involves production of transgenic plants
containing DNA encoding AC1/C1 wildtype and mutant sequences that
negatively interfere in trans with geminiviral replication during
infection. The resulting transgenic plants are resistant to viral
infection.
DESCRIPTION OF THE FIGURE
[0010] FIG. 1 shows the results of a transient assay for
trans-dominance done with BGMV-GA in NT-1 cells.
DETAILED DESCRIPTION OF THE INVENTION
[0011] A. Production of infectious clones
[0012] Infectious clones of geminiviruses are produced by methods
known to the skilled worker. Geminivirus DNA is extracted from
tissue as follows. Young tissue is collected from infected plants,
frozen in liquid nitrogen and ground in a mortar in the presence of
extraction buffer (10 mM Tris-Cl, pH 7.5, 10 mM EDTA, and 1% SDS,
1:4 wt/vol ratio) and centrifuged for about 10.sup.5 g minutes. The
supernatant is adjusted to about 1 M NaCl and stored at about
4.degree. C. for about 12 hr, then centrifuged for about 10.sup.7 g
minutes. After phenol extraction, the solution is adjusted to 0.3 M
sodium acetate, and the DNA is precipitated in alcohol. Viral
nucleic acids are isolated by agarose gel electrophoresis.
[0013] These viral nucleic acid fractions are digested with
restriction enzymes and isolated by agarose gel electrophoresis.
The DNA is cloned in a suitable cloning vector, e.g., pBluescript
KS+, and its identity is confirmed by sequencing.
[0014] Full-length clones of the geminivirus genome are
constructed, e.g., by a PCR-based cloning strategy. Primers are
synthesized that will amplify the entire ORF plus about ten
nucleotides on each side of the ORF. The primers should include
mismatched bases to create restriction sites before and after the
C1 or AC1 ORF which will allow convenient cloning without altering
initiation and termination codons of C1 or AC1 ORFs.
[0015] Primer 1 is complementary to and anneals with the viral
sense strand of the geminiviral genome. The 5' end of the primer is
located 40-50 base pairs 3' of the translation start, and the 3'
end is located 10-20 base pairs 3' of the translation start site.
Translation start is defined by CAT on the viral sense strand; AC1
or C1 ORFs are located on the complementary strand of the viral
genome and sequence coordinates are given for the viral sense
polarity strands.
[0016] Primer 2 is complementary to and anneals with the strand
(complementary sense polarity) of the geminiviral genome. The 5'
end of the primer is located 40-50 base pairs 3' of the AC1 or C1
termination condon, and the 3' end of the primer is located 10-20
base pairs 3' of the translation stop as determined on the
complementary sense polarity strand.
[0017] The primers are used in a PCR reaction to amplify the C1 or
AC1 ORF from cloned viral DNA or purified geminivirus DNA. The
amplified DNA is digested with appropriate restriction enzymes to
cut sites engineered in the ends of the PCR fragment and the
resulting fragment is cloned into a suitable vector. C1- or
AC1-containing clones are identified and sequenced to confirm the
presence and integrity of the cloned C1 or AC1 ORF.
[0018] The sequence of the AC1/C1 ORF is used for designing the
primers for amplification of the PCR fragment of AC1/C1 ORF. For
example, these primers are designed so that this ORF is cloned into
the BamHI and HindIII sites of pBluescript KS+. The BamHI site is
located at the 5' end of the complementary sense primer, which
amplifies the amino terminal end of the ORF. A HindIII site is
located at the 5' end of the viral sense primer which anneals to
the carboxy end of the ORF.
[0019] Infectious clones preferably are selected. The infectivity
of the clones are determined by construction of Agrobacterium
having greater-than-full-length viral genes with at least two
common regions of DNA-A and DNA-B. Infectivity is determined by
microparticle inoculation. Seeds are germinated on moist filter
paper to produce 1-3 cm long radicles of a host, and this tissue is
bombarded by DNA-coated particles with a particle gun. Inoculated
plants are placed in a growth chamber at about 26.degree. C. with
about 14 hour photoperiods. Infectivity is confirmed by PCR with
primers specific for geminiviruses or Southern blot analysis. For
example, 1.3-kb PCR products are expected when primers PAL1v1978
and PAV1c715 are used.
[0020] Cloned viral DNA is digested with restriction enzymes and
analyzed on agarose gels to produce a unique 2.5 to 2.7-kb
fragment. The DNA bands are removed from the gel and cloned into an
appropriate vector. For monopartite geminiviruses, the insert
preferably includes the entire genome. For bipartite geminiviruses,
entire inserts of both genomic components are preferable. The
single insert of the monopartite geminivirus or both cloned
components of bipartite geminiviruses are introduced into a host
plant and tested for infectivity by biolistic delivery or
agroinoculation.
[0021] The cloned C1 (monopartite viruses) or AC1 (bipartite
viruses) ORF are isolated by selecting for the following
characteristics:
[0022] A. The AC1 or C1 ORF encodes a protein product of about 42
Kd.
[0023] B. The nucleotide sequence of the C1 or AC1 ORF is at least
60% homologous to the AC1 ORF of a previously identified
geminiviruses (e.g., BDMV, ToMoV, or TYLCV). The deduced amino acid
sequence of the ORF will contain several characteristic sequences
which are similar in sequence and relative position within the ORF
i.e., motifs within the C1 or AC1 sequences.
[0024] B. Introducing Mutations
[0025] Mutations are introduced by site-directed mutagenesis of
cloned C1 or AC1 ORF by methods known in the art, e.g., using the
method of Kunkel et al. (Recombinant DNA Methodology, 1989, pp.
587-601) (herein, "Kunkel mutagenesis").
[0026] In particular, mutations are introduced into amino acid
sequence motifs in C1 or AC1 ORF that are highly conserved among
all gemini viruses. Four motifs are preferred in the DNA-nicking
domain of the protein. These include (capital letters denote high
conservation of amino acid, lower case denotes some conservation,
and "x" denotes a variable position in the motif):
[0027] (1) FLTYpxC
[0028] (2) HlHvliQ
[0029] (3) vKxYxdKd; and
[0030] (4) FHPNIQxak.
[0031] Additionally two motifs are preferred in the NTP-binding
domain of the protein. These include:
[0032] (5) EGx.sub.2RTGKt; and
[0033] (6) NviDDi.
[0034] The individual codons specifying the most highly conserved
amino acids within these motifs are mututated. For example, one or
more of the following mutations introduced to the C1 or AC1
ORF:
[0035] (1) vKxYxdKd to
[0036] (a) vKxFxdKd;
[0037] (b) vKxAxdKd;
[0038] (c) vKxYxdRd;
[0039] (2) EGx2RTGKt to
[0040] (a) EGx2RTGHt;
[0041] (b) EGx.sub.2RTGAt;
[0042] (c) RGx.sub.2RTGKht;
[0043] (3) NviDDi to
[0044] (a) NviRDi;
[0045] (b) NviKDi; or
[0046] (c) NviDYi,
[0047] (herein mutations 1(a), 1(b), 1(c), 2(a), 2(b), 2(c), and
3(a), 3(b), 3(c), respectively). Acidic or basic amino acids are
changed to the opposite charge, to alanine (alanine scanning) or to
other neutral amino acids. Combinations of mutants are also made.
For example, a single C1 or AC1 ORF containing codon changes
corresponding to vKxFxdKd and EGx2RTGHt (double mutations 1(a) and
2(a), above) are constructed and tested. Other mutants in motifs
within AC1/C1 are possible and are used. The presence of the codon
change is confirmed by DNA sequencing. Agrobacterium-mediated
transfer of the plant expressible mutated AC1/C1 ORF is done using
procedures known to those skilled in the art.
[0048] If an infectious clone of the geminivirus is available,
effects of mutations on replication can be tested. The mutation is
introduced into the C1 or AC1 ORF of an infectious clone. Mutant
DNA is transferred to plant cells. Replication of wild type viruses
is tested for infection as a positive control. Mutations which
create transdominant molecules generally abolish replication when
engineered into infectious clones. A number of mutations which
change codons for conserved amino acids within these motifs will be
lethal and potentially transdominant. Other mutations in C1 or AC1
which abolish replication should also be considered potentially
transdominant. Any non-functional C1 or AC1 molecule has the
potential to be transdominant.
[0049] Mutated C1 or AC1 ORFs are installed into a suitable plant
transformation vector in the sense orientation and under the
control of a strong constitutive promoter sequence and suitable
terminator for high level expression in the target plant species.
This step is performed for each of the C1/AC1 mutants created.
[0050] C. Assays
[0051] A transient assay is useful to screen candidate constructs
for transdominant interference activity. This is done by first
coinoculating protoplasts or a plant cell suspension culture with
the infectious geminivirus clone and a plasmid containing mutant C1
or AC1 ORF under control of a strong constitutive plant promoter.
Control treatments are inoculated with an infectious clone. Total
DNA is harvested from inoculated cells, and is assayed for viral
replication. Transominant C1 or AC1 mutants are identified as those
which suppress geminiviral replication relative to control
treatments after coinoculation.
[0052] In vitro assays for transdominance correlate lethal
mutations and transdominant activity in transient assays. This is
exemplified in a BGMV-GA model system. These results are readily
applicable to produce a transdominant C1 or AC1 ORFs from other
geminiviruses. Transgenic plants resistant to ToMoV were created by
transforming them with an AC1 ORF derived from ToMoV and engineered
to contain similar mutations.
[0053] Expression cassettes constructed above are installed into
binary plasmids and transformed into Agrobacterium strains for
plant transformation protocols. Plants are transformed by methods
tailored to the specific variety or line.
[0054] Transgenic status of R.sub.0 and later generation plants and
their segregating progeny is verified by routine methods,
including: ELISA assays for NPTII protein detection; DNA assays
such as PCR amplification with the AC1/C1 primers of plants and
Southern blot hybridization for detection of transgenes using
AC1/C1 as viral probes; and Southern blot hybridization to detect
AC1 or C1 transgenes. Demonstration that R.sub.1 plants transformed
with geminivirus gene constructions express NPTII protein is done
by ELISA. Protein in leaf tissue samples taken from R.sub.1
transgenic plant seedlings is extracted and analyzed for NPTII
protein by ELISA.
[0055] Geminivirus transgene expression is also measured by
Northern blot analysis. Transgene expression in a number of R.sub.0
and R.sub.1 plants was done by Northern blot hybridization. Total
RNA extracted from leaves of transgenic plants is separated by
agarose gel electrophoresis. After electrophoresis, RNA is pressure
blotted onto membrane. Membranes are hybridized with radiolabeled
probes, washed, and autoradiographed.
[0056] D. Identification of gemini-resistant transgenic plants
[0057] Geminivirus-resistant transgenic plants are identified by
challenging transgenic plants and progeny. R.sub.1 plants from self
pollinated R.sub.0 primary regenerants are agroinoculated about 3
weeks after sowing. Alternative methods include biolistic
inoculation, sap transmission from infected tissue (if the isolate
is mechanically transmissible), insect transmission, or grafting.
For bipartite geminiviruses, agroinoculation preferably involves
delivery of greater-that-full-length (i.e., at least 2 common
regions) DNA-A and DNA-B components into the seedlings using
Agrobacterium strains, e.g., containing a binary vector having in
its T-DNA a partial or full tandem duplication of infectious
geminivirus DNA. Geminivirus-resistant plants are incorporated into
traditional breeding programs to develop elite breeding lines that
include the resistance-conferring transgene. These changes produce
C1 or AC1 molecules when made alone or in combination with a
mutant.
[0058] Plants showing the highest steady state levels of transgene
RNA are challenged by Agrobacterium-mediated inoculation.
Resistance is determined by lack or delay of symptom expression and
low levels of viral DNA in plants as determined by squash blot
hybridization tests with viral probes (Gilbertson et al., 1991.
Plant Disease 75:336-342.). Resistance is also determined by
inoculation with viruliferous Bemisia tabaci as described. It is
expected that plants with low levels of mRNA accumulation for the
mutated AC1/C1 ORF have symptoms and those with high levels have no
symptoms.
[0059] Since the AC1/C1 proteins have domains required for
DNA-nicking and NTP-binding that are conserved among all
geminiviruses, an antiviral strategy involving mutated AC1/C1
protein is applicable to plant-geminivirus systems in general.
[0060] Other viruses include:
1 Virus Genes/Regions SEQ ID NO. TGV-GA1 AC1 ORF DNA-A
(complementary seq) 57 TGV-GA1 Common & Intergenic Region
(viral) 58 TLCV-IND Full Length Seguence (stemloop begin) 59
"Chino" Partial AC1, Common region, AV1 60 PHV AC1, Common region,
Intergenic, AV1 61 PHV BV1 ORF 62 PHV BC1, Hypervar., Common, &
Interg. Reg 63
[0061] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLE 1
[0062] Gene Expression Vectors
[0063] All E. coli culture and plasmid DNA isolation methods were
carried out according to standard methods. Restriction digestions,
filling in of 5' end overhangs, calf intestinal alkaline
phosphatase treatments of DNA and ligation of DNA fragments and gel
electrophoretic separation of DNA fragments and their isolation
from gels were done according to manufacturers' recommendations and
methods. Agrobacterium large plasmid DNA was isolated.
Agrobacterium transformation and culture is performed according to
general methods known to those skilled in the art.
[0064] Tables 1A and 1B list geminiviral transcribed sequences,
expression vectors, binary plasmids, and Agrobacterium strains
described in the Examples.
2TABLE 1A Constructs used to Create Transgenic Plants Transcribed
Seq. Expression Binary Agrobacterium (open reading frame Vector
Vector tumefaciens or antisense seq.) Used Used designation
ToMoV-AC1as pRT101e pJTS246.DELTA. RTAC ToMoV-AC1 pRT101e
pJTS246.DELTA. RTSC ToMoV-AC1as DH51 pJTS235 DHAC ToMoV-AC1 DH51
pJTS235 DHSC ToMoV-AC1-AC2-AC3as pRT101e pJTS246.DELTA. RT3AA
ToMoV-AC1as pRT101e pJTS246.DELTA. RTFS ToMoV-AC1 pLAT
pJTS246.DELTA. LASD, LASU ToMoV-AC1d1m pRT101e pJTS246.DELTA. MEU
ToMoV-AC1d1m pRT101e pJTS246.DELTA. MEU2 ToMoV-AC1d1m1 pRT101e
pJTS246.DELTA.- MUA ToMoV-AC1d1m23 pRT101e pJTS246.DELTA. MUB
ToMoV-AC1 p.DELTA.1CO35 pJTS246.DELTA. COALS ToMoV-AC1d1m
p.DELTA.1CO35 pJTS246.DELTA. CODLM ToMoV-AC1d1m1 pRTIN
pJTS246.DELTA. MUAIN ToMoV-AC1d1m23 pRTIN pJTS246.DELTA. MUBIN
TYLCV-C1as pRT101e pJTS246.DELTA. LCA TYLCV-.DELTA.C2as pRT101e
pJTS246.DELTA. LCR' TYLCV-C1as TYLCV-V1as pRT101e pJTS246.DELTA.
LCR" TYLCV-C1-.DELTA.C2-.DELTA.C3as pRT101e pJTS246.DELTA. RT3CA
TYLCV-C1-.DELTA.C2-.DELTA.C3as p.DELTA.1CO35 pJTS246.DELTA. CO3CA
TYLCV-C104 eP.sub.max-T.sub.plus pGA482.DELTA. + C104 HYG.sup.R
TYLCV-C225 eP.sub.max-T.sub.plus pGA482.DELTA. + C225 HYG.sup.R
TYLCV-C259 eP.sub.max-T.sub.plus pGA482.DELTA. + C259 HYG.sup.R
TYLCV-C1-.DELTA.C2-.DELTA.C3 pRT101e pJTS246.DELTA. RT3CS
[0065]
3TABLE 1B Constructs for Agrobacterium Inoculation Geminivirus
strain and Binary Vector Agrobacterium Sequence Used Designation
ToMaV-A dimer pJTS222 A.t. ToMoV-A ToMoV-B 1.67mer pJTS222 A.t.
ToMoV-B TYLCV-EG dimer pJTS222 A.t. TYLC-EG
EXAMPLE 1.1
[0066] Synthesis of expression vector pRT101e
[0067] The pRT101e expression vector listed in Table 1A was made by
removing a 325-bp HindIII-EcoRV fragment from pUC8-CaMVCAT
(Pharmacia) and inserting it into HincII-HindIII-digested pRT101
(Dr. Topfer, Max Planck-Institut fur Zuchtungsforschung, 5000 Koln
30, Germany), thereby adding a segment of the 35S promoter
containing the upstream enhancer (Kay et al., Science, 1987,
236:1299-1302) to the 5' end of the 35S promoter sequence of
PRT101.
EXAMPLE 1.2
[0068] Expression vector pDH51
[0069] The pDH51 expression vector of Table 1A (T. Hohn, Friedrich
Miescher Institute, P.O. Box 2543, CH-4002, Basel, Switzerland) is
comprised of a CaMV 35S promoter-35S terminator expression
cassette.
EXAMPLE 1.3
[0070] Synthesis of expression vector p.DELTA.1CO35
[0071] The p.DELTA.1CO35 expression vector of Table 1A was derived
from pCO1bam (Dr. Neil Olszewski, University of Minnesota-Twin
Cities, College of Biological Sciences). The 1.0-kb EcoR1-SalI
fragment of pCO1bam, containing the promoter controlling expression
of the commelina yellow mottle virus (CoYMV) transcript (Medberry
et al., Plant Cell, 1992, 4:185-92), was inserted into
EcoR1-SalI-digested pSL1180 (Pharmacia). A 1.1-kb EcoRI-DraI
fragment of the resulting construct was inserted into
EcoRI-HincII-digested pRT101, thereby replacing the CaMV 35S
promoter of pRT101 with the CoYMV promoter. Some restriction sites,
including BamHI and BglII, were removed by partially digesting this
plasmid with HindIII and recircularizing it with T4 DNA ligase to
produce p.DELTA.1CO35.
EXAMPLE 1.4
[0072] Synthesis of expression vector pRTIN
[0073] The pRTIN expression vector of Table 1A is a derivative of
pRT101e and pCOIN, in which the 35S terminator of pRT101e was
replaced with the protease inhibitor gene
terminator/polyadenylation site (T.sub.INH) of pCOIN. To produce
pCOIN, the 760-bp HindIII-XbaI fragment of pTPI-1 (Dr. C. Ryan,
Washington State University, Pullman, Wash.) containing T.sub.INH
was inserted into HindIII-XbaI-digested Bluescript II
KS+(Stratagene). The 770-bp XbaI-KpnI fragment of the resulting
construct was inserted into XbaI-KpnI-digested pUC19. The 800-bp
PstI-KpnI terminator fragment of the resulting plasmid was ligated
with the KpnI-PstI fragment of p.DELTA.1CO35 to produce pCOIN. The
805-bp SphI-SspI fragment of pCOIN was inserted into
SphI-SmaI-digested pRT101e, thereby replacing the 35S terminator of
pRT101e with T.sub.INH. The resulting plasmid was further modified
by inserting into a EcoRI site a DNA fragment with EcoRI ends and
internal restriction sites including BamHI to produce pRTIN.
EXAMPLE 1.5
[0074] Synthesis of expression vector eP.sub.mas-T.sub.phas
[0075] The expression vector eP.sub.mas-T.sub.phas of Table 1A was
assembled by combining an octopine synthase upstream activating
sequence (ocs UAS) and a mannopine synthase promoter (mas2').
[0076] The ocs UAS was excised from pAL1050 (Dr. Paul J. J.
Hooykaas, State Univ. of Leiden, 2333 AL Leiden, The Netherlands),
which was isolated from Agrobacterium tumefaciens strain LBA4404
(Dr. P. J. J. Hooykaas). A 2.8-kb EcoRI fragment of pAL1050,
containing nt 13362-16202 of ocs UAS was inserted into the EcoRI
site of pSL1180 (Pharmacia). A 311-bp SacI-BamHI fragment of the
resulting plasmid, containing nt 13774-14085 of the ocs UAS, was
ligated into SacI-BamHI-digested pBluescript II KS+. A 285-bp
XhoI-MfeI fragment containing the ocs UAS was ligated with the
EcoRI-XhoI fragment of pBluescript II KS+ with ocs UAS to produce a
plasmids having tandemly repeated ocs UAS element structure. A
EcoRI-XhoI fragment of the recombinant plasmid was ligated with
another MfeI-XhoI fragment to produce a recombinant plasmid,
pBluescript+UAS.sup.3, having three tandemly repeated ocs UAS
elements.
[0077] The mas2' promoter element was isolated as follows. Plasmid
pE93 (Dr. Stan Gelvin, Purdue University) is derived from pRK290
(Ditta et al., 1980). EcoRI fragment #13 of pE93 contains nt
16202-21634 of the octopine Ti plasmid, and lacks an internal ClaI
fragment at nt 8672-20128 (Eco13.DELTA.Cla). A 4-kb EcoRI-XhoI
fragment was ligated with the SalI-EcoRI fragment of pBR322, to
produce pJTS213. This plasmid was introduced into E. coli GM119
(Dr. Gurnam Gill, Pharmacia & Upjohn, Kalamazoo, Mich.), which
is deficient in DNA adenine methylation. Thus, normally
undigestible ClaI site beginning at nt 20128 in Eco13.DELTA.Cla is
cleavable by ClaI. A 951-bp ClaI-NcoI fragment of pJTS213
containing nt 21079-20128 was isolated and ligated with the
ClaI-NcoI fragment of pSL1180 to produce pSL1180+P.sub.mas.
[0078] A ocs UAS-enhanced mannopine synthase promoter cassette
(Ep.sub.mas) was assembled as follows. A 365-bp ClaI-FspI mas2'
fragment from pSL1180+P.sub.mas was ligated with the ClaI-EcoRV
fragment of pBluescript +VAS.sup.3. Clones in which the mas2' was
inserted downstream of the ocs UAS repeat were identified by
restriction digestion. To facilitate the addition of the phaseolin
transcription terminator, a 250-bp multiple cloning site (mcs)
XhoI-SalI fragment from pSL1180 was ligated into the XhoI-digested
recombinant plasmid. Two plasmids,
pBluescript+UAS.sup.3+P.sub.mas+mcs (orientations I and II),
containing a construct with the mcs inserted in the two possible
orientations were isolated.
[0079] The phaseolin terminator was added to
pBluescript+UAS.sup.3+P.sub.m- as+mcs, completing the assembly of
Ep.sub.mas-mcs-T.sub.phas, as follows. A 1.1-kb PstI-EcoRI fragment
of pUC19-hph-T.sub.phas (described below in the assembly of
pGA482.DELTA.+HYG.sup.R), which contains the phaseolin
transcription terminator (Tag), was ligated with
PstI-EcoRI-digested pBluescript II KS+. A 1.2-kb SacII-ClaI
fragment of the resulting plasmid was ligated with the SacII-ClaI
fragment of pBluescript+UAS.sup.3+P.sub.m- as+mcs (orientation I)
to produce a plasmid having the eP.sub.mas-T.sub.phas insert.
EXAMPLE 1.6
[0080] Synthesis of expression vector pLAT
[0081] The expression vector pLAT of Table 1A was produced as
follows. The promoter of the LAT52 gene (Twell et al., Development,
1990, 109:705-13) was used to construct an AC1 gene construct in
sense orientation that does not express in vegetative tissue. A
600-bp NcoI-SalI fragment of pLAT52-7a (Dr. S. McCormick, Plant
Gene Expression Center, USDA ARS, Albany, Calif.), which contains
the LAT52 promoter, was ligated with NcoI-SalI-digested pSL1180 to
produce pLAT.
EXAMPLE 2.1
[0082] Synthesis of binary vector pJTS246.DELTA.
[0083] The binary vector pJTS246.DELTA. of Table 1A was produced as
as a derivative of pGA482 (Dr. G. An, Washington State University,
Pullman, WN), by replacing the nopaline synthase controlled NPTII
sequence with a CaMV 35S promoter-NPTII-phaseolin terminator
selectable marker. The selectable marker was situated at the left
T-DNA border to insure that the passenger gene, inserted at the
right T-DNA border, would be transferred into the plant cell.
[0084] A BamHI fragment of pUC8-CaMVCAT was ligated with a 2.2-kb
BamHI fragment of pDOB513ro4.6K (J. L. Slightom, Pharmacia &
Upjohn), containing the NPTII coding region and octopine Ti plasmid
T-DNA ORF No. 26 transcription terminator, to produce pJTS228. The
pJTS228 construct has the 2.2-kb fragment, inserted as a
transcription fusion unit immediately downstream of the CaMV 35S
promoter of pUC8-CaMVCAT. Most of the CAT gene of pUC8-CaMVCAT was
deleted from pJTS228 by digesting with EcoRI to produce
pJTS228.DELTA.. A 4.0-kb BamHI-NcoI fragment from pJTS228 was
ligated with a 1.55 kb BamHI-NcoI fragment from pkanPhas (J. L.
SLightom, Pharmacia & Upjohn) containing the NPTII coding
sequences 5' distal to the NcoI site and the phaseolin terminator.
A resulting plasmid, in which the T-DNA transcription terminator
fused to the NPTII ORF was replaced with the phaseolin storage
protein terminator from Phaseolus vulgaris, was designated
pJTS233.
[0085] pJTS233 was digested with HindIII and flush ended. A 2.8-kb
EcoRI fragment containing the 35S promoter, NPTII coding region and
phaseolin terminator was isolated and ligated in a 3-part reaction
with SmaI-BamHI fragment of pUC9 and an 8.0-kb BamHI-EcoRI fragment
of pGA482 containing the broad host range replicon, left and right
nopaline Ti T-DNA borders and nopaline synthase promoter. The
desired construct, pJTS246, was cloned and isolated. pJTS246 was
modified to eliminate the ampicillin drug resistance contributed by
pUC9. The plasmid was digested with ScaI and HindIII, and treated
with HindIII linkers followed by HindIII digestion. The resulting
plasmid, pJTS246.DELTA., had 1730-bp of pUC sequence deleted from
pJTS246.
EXAMPLE 2.2
[0086] Synthesis of binary vector pJTL222
[0087] pJTS222 is pGA492 (Dr. G. An) in which a 2.2-kb
BamHI-HindIII fragment replaced by the 430 bp BamHI-HindIII
fragment of pUC8-CamVCAT containing the CaMV 35S promoter.
EXAMPLE 2.3
[0088] Synthesis of binary vector pJTS235
[0089] pJTS235 was a binary plasmid derived from pGA492 in which
the NPTII coding sequence and its transcription terminator were
removed and replaced with a CaMV 35S promoter-NPTII coding
sequence-phaseolin terminator selectable marker. pJTS235 was
constructed by ligating a 2.1-kb BamHI fragment of pJTS233
containing the NPTII coding sequence and phaseolin terminator into
the BamHI fragment of pJTS222. The resulting plasmid, pJTS235 had
the NPTII structural gene under the control of 35S promoter.
EXAMPLE 2.4
[0090] Synthesis of binary vector pJTS250
[0091] pJTS250 was assembled as follows. A 353-bp PstI-BamHI
fragment of pLG90 (provided by Dr. L. Gritz, Biogen, S.A., 46 Route
des Acacias, Geneva, Switzerland), which includes the entire
hygromycin phosphotransferase gene (hph) coding region from the ATG
translation start codon to 15 bp distal to the translation
terminator, was ligated with the PstI-BamHI digest of pUC9 to
produce pUC9+hph-a. Another aliquot of AvaI-digested pLG90 with
AvaI flush ended. The 670-bp PstI fragment was cloned into the
SmaI-PstI fragment of pUC9 to produce pUC9+hph-b, creating a 670-bp
fragment PstI-EcoRI fragment. A 1.18-kb NaeI-BamHI fragment
containing the phaseolin terminator (J. L. Slightom) was cloned
into the BamHI-SmaI fragment of pUC9 to create pUC9+T.sub.phas. The
above three fragments (353-, 670- and 1180-bp) were ligated with
the BamHI digest of pJTS222. The resulting binary plasmid, pJTS250,
was produced comprised of a P.sub.35S-hph-T.sub.phas plant
selectable marker, and the capability to transform plant tissue to
hygromycin resistance via Agrobacterium-mediated gene transfer.
EXAMPLE 2.5
[0092] Synthesis of binary vector pGA482.DELTA.+HYG.sup.R
[0093] pGA482.DELTA.+HYG.sup.R was produced from the following
plasmids: pGA470 (Dr. G. An); pJTS262, including the entire T-DNA
of pGA470 and a broad host range replicon; pJTS222; pJTS250, a
binary plasmid that includes HYG.sup.R constructed by ligation of
four fragments, including 353-bp PstI-BamHI fragment encoding part
of the hph coding region, 670-bp PstI fragment encoding the
remainder of the hph coding region, 1180-bp NaeI-BamHI fragment
constituting T.sub.phas and pJTS222 digested with BamHI;
pUC19B2-P.sub.nos; pUC19B2+hph-T.sub.phas; p.sub.nos-hph-T.sub.pha-
s expression cassette; and pGA482G (Dr. G. An).
[0094] The pGA482A+HYG.sup.R was constructed as follows: SalI
fragments of pGA470 were ligated into SalI-digested pBR322. The
resulting construct, pJTS262, is comprised of the entire T-DNA of
pGA470 (from right to left border) and a second fragment containing
part of the broad host range replicon. The 345-bp BclI-BamHI
fragment of the resulting plasmid, having the nopaline synthase
promoter (P.sub.nos) fused to the 5' 42-bp of nopaline synthase (14
N-terminal amino acids), was inserted into the BamHI site of
pUC19B2, having the SmaI site of pUC19 converted to a BglII site.
The resultant recombinant plasmid, pUC19B2+P.sub.nos, had the
P.sub.nos segment within the BamHI-BglII fragment.
[0095] A 2.2-kb BamHI fragment containing the hph coding region
from bacterial transposon Tn5 and the phaseolin transcription
terminator (hph-T.sub.phas) was isolated from pJTS250. The 2.2-kb
hph-T.sub.phas fragment was inserted into the BamHI site of
pUC19B2. The pUC19B2-P.sub.nos was digested with BamHI and HindIII.
pUC19B2+hph-T.sub.phas was partially digested with BamHI and
completely with HindIII to produce a 2.2-kb fragment with
BamHI-HindIII ends. The fragment was ligated with BamHI-HindIII
digested pUC19B2-P.sub.nos plasmid. The resulting construct, a
P.sub.nos-hph-T.sub.phas expression cassette, pUC19B2+HYG.sup.R,
was partially digested with BamHI; a resulting 5.3-kb fragment was
digested with BglII to produce a 2.6-kb fragment. Separately,
HindIII-EcoRI-digested pGA482 was ligated with
HindIII-EcoRI-digested pSL1180, lacking a mcs. The resulting
construct was further restricted to delete 2.5-kb of the original
T-DNA containing the mcs. This binary was digested with BglII and
ligated with the BamHI-BglII-ended 2.6-kb P.sub.nos-hph-T.sub.phas
fragment to produce pGA482.DELTA.+HYG.sup.R.
EXAMPLE 3
[0096] Geminivirus DNA Insertion into Expression Vector
Constructs
EXAMPLE 3.1
[0097] Synthesis of Wild-Type ToMoV-FL AC1 ORF
[0098] ToMoV was collected from infected tomato plants in
Bradenton, Fla. and inoculated into Nicotiana benthamiana and
tomato. DNA was isolated from infected plants and viral DNA was
isolated by preparative agarose gels. Viral DNA was digested with
BglII, inserted into BglII-digested pSP72 to produce a full-length
A-component clone (Seq ID 17). Similarly, a full-length DNA-B clone
was produced from viral DNA digested with BamHI and inserted into
BamHI-digested pBluescript II KS+ (Seq ID 18). DNA of either clone
inoculated into N. benthamiana produced symptoms similar to the
original virus.
[0099] A dimer clone in which DNA-A was inserted as a direct,
tandem duplicate into the cloning vector was made by removing the
single insert from its original vector with BglII and reinserting
it into BglII-digested pSP72. The ApaI fragment of the resulting
plasmid comprising the cloned DNA-A was inserted into the ApaI site
of pBluescript II KS+.
EXAMPLE 3.3
[0100] Synthesis of ToMoV-AC1
[0101] Wild type AC1 sense ORF and antisense (as) ORF of Table 1A
were constructed from the AC1 ORF (SEQ ID 1 and 2) and part of the
intergenic region was amplified by PCR from ToMoV-infected N.
benthamiana DNA using primers PFL-2549B (SEQ ID 9)
(5'-GGATCCGAGTAACTCATCTGGAGTACC-3') and PFL-1108B (SEQ ID 10)
(5'-GGATCCGGAAGTAGATGGAGCACCCGC-3'). The 1.1-kb PCR product was
BamHI-digested and inserted into the BamHI site pBluescript II KS+
to produce pTFAC1.
EXAMPLE 3.4
[0102] Synthesis of ToMoV-AC1dlm
[0103] For the production of the mutated ORF, the AC1 ORF and part
of the intergenic region was PCR amplified from ToMoV-infected N.
benthamiana DNA by PCR using primers PFL-2549H (SEQ ID 16)
(5'-TATCA+E,uns AAGCTTGAGTAACTCATCTGGAGTACC-3') and PFL-1108B (SEQ
ID 10) (5'-TATC+E,uns GGATCCGGAAGTAGATGGAGCACCCGC-3') to produce a
HindIII site near the translation start codon and a BamHI site near
the translation terminator codon. The HindIII-BamHI-digested
product was ligated with HindIII-BamHI-digested pBluescript II KS+
in a sense orientation relative to the f1 origin of replication.
Mutations were generated in the NTP binding motifs of AC1 of this
clone.
[0104] Trans-dominant lethal mutants (dlm) of AC1 protein (SEQ ID 3
and 4) were created by Kunkel mutagenesis. The above pBluescript
plasmid was transformed into CJ236 (Invitrogen Co.), a dut-,
ung-strain, so that the amplified plasmid DNA contains uracil.
Single-stranded DNA was produced by transfecting the above
transformed cells with helper phage M13-K07. The complementary
sense strand of the ssDNA was synthesized in vitro using
deoxynucleotides, including dTTP, and two mutagenic primers:
PFAC1-680c (SEQ ID 11) (5'-CAAGAACAGGGcAcACGATGTGGG-3') and
PFAC1-781c (SEQ ID 12) (5'-GTATAACGTCATTaAatACATCGCACCGC-3'). The
lower case letters indicate altered nucleotides. The product was
treated with T4 DNA ligase and transformed into XL1 Blue E. coli
(Stratagene) to amplify plasmids containing the mutations produced
by the mutagenic primers, which resulted in the mutations 2(a),
3(b) and 3(c), described above.
EXAMPLE 3.5
[0105] Synthesis of ToMoV-AC1dlm1
[0106] The 1.1-kb BamHI fragment of pTFAC1, containing wild type
AC1 ORF, was inserted to the BamHI site of pRT101e to produce a
sense (pRTAC1-S) construct. The AC1 triple mutant (AC1 dlm) ORF was
removed as a 1.1-kb XhoI-BamHI fragment from its vector and
inserted in the sense orientation into XhoI-BamHI-digested pRT101e
to produce pRT101e+AC1dlm. Plasmids pRTAC1-S and pRT101e+AC1dlm
were cleaved at the unique PmlI site. After an additional digestion
with ScaI, 1.6- and 3.2-kb fragments were isolated from each
digest. The 1.6-kb fragment from pRTAC1-S was ligated with the
3.2-kb fragment from pRT101e+AC1dlm to produce a construct
comprising the sequence designated as ToMoV-AC1dlm1 (SEQ ID 5 and
6) in Table 1A, mutation 2a described above.
EXAMPLE 3.6
[0107] Synthesis of ToMoV-AC1dlm23
[0108] Plasmids PRTAC1-S and pRT101e+AC1dlm were cleaved at the
unique PmlI site. After an additional digestion with ScaI, 1.6- and
3.2-kb fragments were isolated from each digest. The 3.2-kb
fragment from pRTAC1-S was ligated with the 1.6-kb fragment from
pRT101e+AC1dlm to produce a construct comprising the sequence
designated as ToMoV-AC1dlm23 (SEQ ID 7 and 8) in Table 1A, double
mutations 3(b) and 3(c) described above.
EXAMPLE 3.7
[0109] Synthesis of ToMoV-AC1-AC2-AC3
[0110] A construct containing the AC1-AC2-AC3 fragments was
produced by ligating a BamHI-HindIII fragment of a binary plasmid
comprised of a dimer of the full-length, infectious ToMoV
A-component with BamHI-HindIII-digested pJTS222. The BamHI-HindIII
fragment from this construct was inserted into
BamHI-HindIII-digested pBluescript II KS+. A 1.24 kb BglII-SphI
fragment of the resulting plasmid, containing the complete AC2 and
AC3 coding sequences and the C-terminal two-thirds of the AC1 ORF
(SEQ ID 15), was ligated into BglII-SphI-digested pSL1180. The
resulting plasmid contained the .DELTA.AC1-AC2-AC3 fragment from
ToMoV-A.
EXAMPLE 4
[0111] TYLCV-IS-EG Wild Type and Mutant Sequences.
EXAMPLE 4.1
[0112] Synthesis of TYLCV-C1
[0113] Tomato leaves with TYLCV symptoms were collected in Fayoum,
Giza and Ismailia, Egypt. They were grafted to Geneva 80 tomatoes
and N. benthamiana. The tomatoes and tobacco developed symptoms
typical of TYLCV. Infectious TYLCV (TYLCV-IS-EG1) DNA was isolated
from the infected N. benthamiana. The C1 ORF of TYLCV-IS-EG1 (SEQ
ID 19 and 20) was produced as a 1.1-kb fragment by PCR
amplification of infected plant DNA. The primers used were pTYIRc4
(SEQ ID 21) (5'-GGCCATAGAGCTTTGAGGGATCC CGATTCATTTC-3') and
PTYC2v1679 (SEQ ID 22) (5'-GGTAGTAT GAGGATCCACAGTCTAGGTCT-3').
After BamHI-digesting the PCR products, they were ligated with
BamHI-digested pBluescript II KS+ to produce pEGAL1-AS1, which
contained the C1 ORF, as TYLCV-C1.
EXAMPLE 4.2
[0114] Synthesis of TYLCV-.DELTA.C2as
[0115] A truncated C2 ORF (.DELTA.C2) was produced as a 365 bp
fragment by PCR amplification of TYLCV-IS-EG1-infected N.
benthamiana DNA. The primers PTYC2v1499 (SEQ ID 32)
(5'-ATTTGTGGATCCTGATTACCTTCCTGATGTTGTGG-3,- ) and PTYC2c1814 (SEQ
ID 35) (5'-AAACGGATCCTTGAAAAATTGGGC-3') were used. The primers were
BamHI-digested and ligated into BamHI-digested pBluescript II KS+
to produce pTYC2-25-1, which contained the .DELTA.C2 ORF in
antisense orientation.
EXAMPLE 4.3
[0116] Synthesis of TYLCV-V1
[0117] A truncated V1 ORF was produced as a 625-bp fragment by PCR
amplification of TYLCV-IS-EG1 infected N. benthamiana DNA. The
primers used were PTYAR1v466 (SEQ ID 33)
(5'-TTAGGATCCTATATCTGTTGTAAGGGC-3') and PTYAR1c1046 (SEQ ID 34)
(5'-TTAACTAATGCAGGATCCTACATTCCAGAGGGC-3').
[0118] The primers were BamHI-digested and ligated into
BamHI-digested pBluescript II KS+ to produce pTYV1-6-1, which
contains the V1 ORF.
EXAMPLE 4.4
[0119] Synthesis of TYLCV-C1-.DELTA.C2-.DELTA.C3
[0120] A 1.3-kb fragment of the TYLCV-IS-EG1 genome from nt 1471 to
nt 20 via nt 2787 (Navot et al 1991) was produced by PCR
amplification of infected N. benthamiana DNA. The primers used were
PTYIRc4 and PTYC2v1499. The primers were BamHI-digested and
inserted into BamHI-digested pBluescript II KS+ to produce
pTYEGC4.
EXAMPLE 4.5
[0121] Synthesis of TYLCV ORF Mutations
[0122] A full-length infectious clone of TYLCV-IS-EG1 (pTYEG14) was
created to serve as the basis for TYLCV ORF constructs and for
agroinoculation (see below). DNA from a tomato infected with
TYLCV-IS-EG1 was used as template in two PCR amplification
reactions. The first used primers PTYC1c2196 (SEQ ID 37)
(5'-AAATCTGCAGATGAACTAGAAGAGTGGG-3') and PTYV1v1164 (SEQ ID 36)
(5'-GTACGAGAACCATACTGAAAACGCCT-3') to amplify a fragment. The
PstI-SphI-digested fragment was ligated with PstI-SphI-digested
pGEM-5zf+ (Promega) to produce plasmid pEGI1A.
[0123] The second amplification reaction employed primers
PTYC1v2182 (SEQ ID 39) (5'-TAGGCCATGGCCGCGCAGCGGAATACACG-3') and
PTYC3c1320 (SEQ ID 38) (5'-GGTTCTGCAGCAGAGCAGTTGATCATGTATTG-3').
The PstI-NcoI-digested fragment was ligated with PstI-NcoI-digested
pGEM-5zf+to produce pEGI1-7B.
[0124] To assemble the full-length virus, the PstI-NcoI fragment of
pEGI1-7B was ligated with the PstI-NcoI fragment of pEGI1A to
produce a construct comprising full-length 2.7-kb viral DNA. The
full-length construct was tested for infectivity by biolistic
delivery into tobacco cells and found to create symptoms identical
to the original disease. This clone was called pTYEG14. Orientation
of insertion with respect to the f1 origin of replication was
confirmed by DNA sequencing. Three mutant C1 ORFs were constructed,
each having one or two base changes altering the amino acid
specificity of one codon by Kunkel mutagenesis using the plasmid
representing the full-length infectious clone of TYLCV-IS-EG1
(pTYEG14) as template. The mutagenic primers (all viral sense)
were: PC1v2467 (SEQ ID 25) (5'-GTTTCCGTCTcgCTCCACGTAGG-3');
PC1v2101 (SEQ ID 28) (5'-GGCCCACATTGTTgCGCCTGTTCTGC-3'); and
PC1v2000 (SEQ ID 31) (5'-GGGTCTACGTCTctAATGACGTTGTACC-3'). (Lower
case letters indicate altered nucleotides.) The resulting DNA was
treated with T4 DNA ligase and transformed into XLI Blue E. coli
cells to produce the following constructs: pTYK.sup.104R #1 (SEQ ID
23 and 24), mutation 1(c); pTYK.sup.225A #4 (SEQ ID 26 and 27),
mutation 2(b); and pTYD.sup.259R #5 (SEQ ID 29 and 30), mutation
3(a), described above.
[0125] The three mutant C1 ORFs were cloned into pCRII
(Invitrogen). The C1 ORF for each mutant was PCR amplified using
primers PTYIRc4 (SEQ ID 21)
(5'-GGCCATAGAGCTTTGAGGATCCCGATTCATTTC-3') and PTYCv1707 (SEQ ID 42)
(5'-GGTAGTATGAGGATCCACAGTCTAGGTCT-3'). The amplified fragments were
ligated with pCRII to produce: pC1K.sup.104R #2, mutation 1(c);
pC1K.sup.225A #4, mutation 2(b); and pC1D29R #2, mutation 3(a),
described above.
[0126] These three ORF in BamHI fragments of their respective
vectors provided the mutant C1 ORFs for expression cassettes for
Agrobacterium mediated transformation.
EXAMPLE 5
[0127] BGMV Constructions
[0128] Wild-type and mutated versions of BGMV C1 (replication
protein) ORF have been prepared. The wild-type sequence (SEQ ID 43
and 44) was mutated by Kunkel mutagenesis. Mutations in BGMV-C1
disclosed here include:
4 ORF Mutant SEQ ID Mutagenic Primer BGAC190 control 45 47 BGAC221
mutation 2(c) 48 50 BGAC228 mutation 2(a) 51 53 BGAC262 mutation
3(a) 54 56
[0129] SEQ ID NOS. 45, 48, 51, and 54, refer to mutagenized BGMV-C1
ORF DNA sequences presented in the Sequence Listing. These encode
protein sequences 46, 49, 52, and 55, respectively. The mutant
sequences were derived from wildtype DNA by Kunkel mutagenesis with
mutagenic primers 47, 50, 53, and 56, respectively.
[0130] A 1.8 Kb BamHI-XhoI fragment containing the 35S promoter
transcriptionally fused to a mutated AC1 ORF from BGMV-GA followed
by the nopaline synthase transcription terminator was removed from
WRG2398 (Dr. D. R. Russell, Agracetus Corp., Middleton, Wis.). The
AC1 coding sequence was mutated in vitro using Kunkel mutagenesis
to produce double mutations 2(c) and 2(a). This fragment was
ligated with pRT101e digested with the same enzymes and the
ligation mix used to transform E. coli DH5 cells. Some
transformants yielded desired recombinant plasmids that had the
entire expression cassette from WRG2398 inserted into PRT101e
(pJTS364). The new expression cassette was removed as a 2.9-Kb
fragment from one of the recombinant plasmids by partial digestion
with HindIII. It was ligated with pJTS246.DELTA. that has been
digested with HindIII and treated with CIAP. After transformation
of DH5 cells, one recombinant among the transformants was
identified that had the expression cassette inserted in the binary
vector. DNA of this binary vector was transformed into A.
tumefaciens LBA4404 and one transformant containing the binary was
called strain At.sup.364.
[0131] Plasmid pJTS364 was digested with EcoRV to eliminate the
duplicated 35S promoters (P.sub.355) and the cleaved DNA ligated. A
fraction of the rejoined molecules have a deletion for the fragment
between the EcoRV sites which contains the 35S enhancer (e.sub.35S)
from WRG2398 and P.sub.35S from pRT101e. The ligation mix was used
to transform DH5 cells. Among the transformants, the desired
deleted plasmid was found and called pJTS365. The 2.5-Kb expression
cassette was removed and ligated with HindIII-digested, CIAP
treated pJTS246.DELTA.. The ligation mix was used to transform DH5
cells. Recombinant binary plasmids were identified among the
transformants and one of these was used as a source of DNA which
was transformed into A. tumefaciens LBA4404. The transformed
agrobacterium having the recombinant binary was called
At.sup.365.
[0132] The listed BGMV ORF are installed into appropriate promoter
vectors and then into binary plasmids for Agrobacterium-mediated
transformation into Phaseolus plants. Additionally, expression
vectors are delivered into plants by biolistic acceleration or
other methods by which plants can be transformed. Regenerated
transformed plants are evaluated for levels of transgene RNA
accumulation by RNA blot analysis to verify activity of the
transgene. Subsequently, progeny are evaluated for ability to
resist BGMV infection.
EXAMPLE 6
[0133] Expression Cassettes and Agrobacterium strains.
[0134] The following ToMoV constructs were produced.
EXAMPLE 6.1
[0135] RTSC and RTAC
[0136] The 1.1-kb BamHI fragment of pTFAC1, containing wild type
AC1 ORF was inserted to the BamHI site of pRT101e. Antisense
(pRTAC1-A) and sense (pRTAC1-S) constructs were produced. HindIII
fragments of each plasmid were each inserted into the HindIII site
of pJTS246.DELTA. in the same transcriptional direction as the
NPTII selectable marker. The binary vectors were transformed into
LBA4404 to produce RTAC (antisense) and RTSC (sense).
EXAMPLE 6.2
[0137] DHSC and DHAC
[0138] The wild type AC1 ORF was also inserted as a. BamHI fragment
into BamHI-digested pDH51 in both orientations creating pDHAC1-S
(sense) and pDHAC1-AS (antisense). The expression cassette of each
recombinant was removed with EcoRI and inserted into EcoRI-digested
pJTS235. Recombinant binary plasmids were selected that had the
expression cassette inserted such that the directions of
transcription as the selectable marker. These binary plasmids were
introduced into LBA4404 by transformation to produce DHSC (sense)
and DHAC (antisense).
EXAMPLE 6.3
[0139] RTSFS
[0140] pRTAC1-S was digested with BglII and flush ended by filling
out. The resulting plasmid, pRTAC1-S.DELTA.BglII, lacked a BglII
site but retained a core 4-base Sau3A site. This mutation shifted
the translation reading frame by adding four nucleotides thereby
producing a translation stop codon, and truncating the polypeptide
(SEQ ID 13 and 14). A 2.1-kb HindIII fragment of
pRTAC1-S.DELTA.BglII, which contains the expression cassette, was
inserted in both orientations into the HindIII site of
pJTS246.DELTA., unidirectional or divergent respecting the sense of
selectable marker. A plasmid having an unidirectional orientation
was introduced into LBA4404 by transformation to produce RTSFS.
EXAMPLE 6.4
[0141] RT3AA
[0142] The 1.24-kb BglII-KpnI fragment of
pSL1180+.DELTA.AC1-AC2-AC3 was ligated into BglII-KpnI-digested
pRTAC1-A to produce, pRT3AA, a pRT101e-like construct with the AC1,
AC2 and AC3 ORFs inserted in antisense orientation. The 2.7 kb
HindIII fragment of the pRT3AA was inserted into the HindIII site
of pJTS246.DELTA. in unidirectional orientation. The construct was
introduced into LBA4404 by transformation to produce RT3AA.
EXAMPLE 6.5
[0143] LASD and LASU
[0144] The 600-bp EcoRI-HincII fragment of pSL1180+PLAT52 was
ligated with EcoRI-HincII-digested pRTAC1-S to replace the 800-bp
35S EcoRI-HincII promoter fragment by the 600-bp LAT52 EcoRI-HincII
promoter fragment. After linearizing the plasmid with NcoI, the ATG
start codon was destroyed by mung bean nuclease. The resulting
plasmid contained an EcoRI and HindIII, but lacked a NcoI site.
Accordingly, the sequences flanking the mutated NcoI site were the
same as in the original LAT52 promoter untranslated 5' leader. The
5' untransformed leader was lengthened to 181 bp and included 68%
A/T nucleotides. HindIII cut plasmid fragment containing the
expression cassette was inserted into the HindIII site of
pJTS246.DELTA. in both unidirectional and divergent orientations
respecting the sense of the selectable marker. One binary of each
type was transformed into LBA4404 creating strains LASU and LASD,
respectively.
EXAMPLE 6.6
[0145] MEU and MEU2
[0146] The AC1 triple mutant (dlmAC1) ORF was removed as a 1.1-kb
Xho I-BamHI fragment from its vector and inserted in the sense
orientation into Xho I-Bam HI-digested pRT101e. A 2.1-kb expression
cassette thus created was removed from pRT101e+AC1dlm by
incompletely digesting the recombinant vector with HindIII and
isolating a 2.1-kb fragment. This fragment was inserted into the
HindIII site of pJTS246.DELTA. to produce a mutated enhanced
unidirectional (MEU) vector. A second binary involving the same
expression cassette which was tandemly duplicated in the
unidirectional orientation was called MEU2. Both of the above
binary vectors were transformed into LBA4404 to produce MEU and
MEU2, respectively.
EXAMPLE 6.7
[0147] MUA and MUB
[0148] ToMoV-AC1dlm1 was partially digested with HindIII and the
2.1-kb expression cassette was isolated. ToMoV-AC1dlm3 was
completely digested with HindIII and the 2.1-kb cassette isolated.
Each cassette was inserted into the HindIII site of pJTS246.DELTA..
The recombinants were transformed into LBA4404 creating the
Agrobacterium strains MUA and MUB, respectively.
EXAMPLE 6.8
[0149] MUAIN and MUBIN
[0150] The 1.2 kb XhoI-BamHI-fragment of pRT101e+AC1 dlm1
containing the AC1dlm1 ORF was ligated with the XhoI-BamHI-fragment
of pRTIN+Geneblock in a sense orientation. This construct was
incompletely digested with HindIII followed by complete digestion
with ScaI to produce a 2.6-kb fragment comprising the expression
cassette. They were ligated with HindIII-digested pJTS246.DELTA. in
a divergent orientation respecting the selectable marker. The
resulting Agrobacterium strain was called MUAIN.
[0151] The 2.1 kb BamHI fragment of pRT101e+AC1dlm23, containing
the AC1dlm23, was ligated with BamHI-digested OpRTIN+Geneblock
plasmid in the sense orientation. This plasmid was digested with
HindIII and ScaI producing a 2.6-kb expression cassette fragment
which inserted into HindIII-digested pJTS246.DELTA. in an
unidirectional direction. Plasmid DNA from this clone was
transformed into LBA4404 to produce MUBIN.
EXAMPLE 6.9
[0152] CODLM
[0153] The 1.1 kb BamHI fragment containing the wild type AC1 ORF
was inserted into the BamHI site of p.DELTA.1CO35 in a sense
orientation to produce p.DELTA.1CO35+AC1S. The 4.5 kb ApaI-BglII
fragment of p.DELTA.1CO35+AC1S was restricted to delete a 475-bp
comprising wild-type AC1 ORF and ligated to the ApaI-BglII fragment
of pRT101e+AC1 dlm1 to replace the wild type internal fragment by
the mutated fragment. The recombinant (p.DELTA.1CO35+AC1 dlm) was
incompletely digested with HindIII, the 2.4-kb fragment containing
the expression cassette isolated and inserted into the HindIII site
of pJTS246.DELTA. in an unidirectional orientation. The plasmid was
transformed into LBA4404 cells to produce CODLM.
EXAMPLE 7
[0154] Constructs Containing TYLCV-IS-EG1
EXAMPLE 7.1
[0155] LCA
[0156] The 1.1 kb BamHI fragment of pEGAL1-AS1 containing the C1
ORF was inserted in the BamHI site of pRT101e in an antisense
orientation to produce pRTLCA1-A. A 2.1 kb HindIII fragment of
pRTLCA1-A was inserted into the HindIII of pJTS246.DELTA. in the
unidirectional (U) orientation with regard to directions of
transcription. LBA4404 cells were transformed with the resulting
plasmid to produce LCA.
EXAMPLE 7.2
[0157] LCR'
[0158] A 350-bp BamHI fragment encoding part of the C2 ORF of
TYLCV-IS-EG1 was removed from pTYC2-25-1 and ligated into the BamHI
site of pRT101e. The resulting construct contained the truncated C2
ORF inserted in an antisense orientation with respect to P.sub.35S.
The 1.3-kb expression cassette removed by HindIII digestion was
inserted into the HindIII site of pJTS246.DELTA.. Plasmid DNA of
the resulting recombinant was partially digested with HindIII and
ligated with the C1 antisense expression cassette. The desired
plasmid had one copy of each the expression cassette inserted such
that the directions of transcription of all cassettes was
unidirectional. DNA of this binary plasmid was transformed into
L3A4404 to produce a strain, LCR', comprising the two-cassette
recombinant binary plasmid.
EXAMPLE 7.3
[0159] LCR"
[0160] A 620-bp BamHI fragment of pTYV1-6-1 encoding part of the V1
ORF of TYLCV-IS-EG1 was ligated into the BamHI site of pRT101e in
an antisense orientation with respect to the 35S promoter. A
HindIII fragment of the resulting plasmid was ligated into the
HindIII site of pJTS246.DELTA. in an unidirectional direction
respecting the selectable marker. Plasmid DNA of this recombinant
was transformed into LBA4404 to produce LCR".
EXAMPLE 7.4
[0161] RT3CA
[0162] The 1.3 kb BamHI fragment of pTYEGC4 containing the
C1+AC2+AC3C structure was inserted into the BamHI site of pRT101e
in an antisense manner with respect to the direction of
transcription of the 35S promoter. The 2.3-kb HindIII fragment of
pTYEGC4 the resulting plasmid containing the expression cassette
was inserted into the HindIII site of pJTS24GA in an unidirectional
transcription directions. LBA4404 transformed with this plasmid to
produce strain RT3CA.
[0163] The 1.3 kb BamHI fragment of pTYEGC4 comprising C1+AC2+AC3
DNA was ligated into the BamHI site of p.DELTA.1Co35 in an
antisense orientation with respect to the Commelina yellow mottle
virus promoter. The 2.8 kb HindIII fragment of the resulting
plasmid containing the expression cassette was inserted into the
HindIII site of pJTS246.DELTA. in an unidirectional orientation
respecting the selectable marker. Plasmid DNA transformed into
LBA4404 produced CO3CA.
[0164] The mutated 1.2 kb fragments containing C1 ORF were removed
from their pCRII vectors and directionally ligated into the
EcoRV-HindIII fragment of eP.sub.mas-mcs-T.sub.phas.
[0165] The resulting constructs were digested with XhoI and NaeI.
HindIII fragments of pGA482.DELTA.+HYG.sup.R were flush ended by
filling out, and digested with XhoI. The XhoI-NaeI expression
cassettes were ligated into the binary vector that had an XhoI
cohesive end and a blunt end to produce three constructs,
C.sup.104, C.sup.225 and C.sup.259. DNA of each of the constructs
were transformed into LBA4404 to produce the strains LC104, LC225
and LC259 and into Agrobacterium strain EHA105 (Mogen
International, N.V.) to produce strains EC104, EC225 and EC259.
EXAMPLE 7.5
[0166] RT3CS
[0167] The 1.3 kb BamHI fragment of pTYEGC4 containing the
C1+.DELTA.C2+.DELTA.C3C structure was inserted into the BamHI site
of pRT101e in an sense manner with respect to the direction of
transcription of the 35S promoter. The 2.3-kb HindIII fragment of
pTYEGC4 the resulting plasmid containing the expression cassette
was inserted into the HindIII site of pJTS246.DELTA. in an
unidirectional transcription directions. LBA4404 transformed with
this plasmid to produce strain RT3CS.
EXAMPLE 8
[0168] Production of Transgenic Plants Containing Disclosed
Constructions and Analysis of Transgene expression
[0169] Transgenic plant were produced by
Agrobacterium-co-cultivation procedures well known to those skilled
in the art.
[0170] The media of compositions used are here defined, for 1
liter:
[0171] 1/2.times. basal: 1/2.times. MS salts (Gibco), 10 g sucrose,
7 g agar, pH 5.8;
[0172] TCM: 1.times. MS salts, 30 g sucrose, 0.2 g
KH.sub.2PO.sub.4, 1.times. N&N vitamins (Gibco), 0.1 mg 2,4-D,
0.05 mg kinetin, 20 mg acetosyringone, 7 g agar, pH 5.8
[0173] 1Z: 1.times. MS salts, 30 g sucrose, 1.times. N&N
vitamins, 1 mg zeatin, 100 mg kanamycine sulfate, 500 mg
carbenicillin, 7 g agar, pH 5.8;
[0174] TR1: 1.times. MS salts, 30 g sucrose, 1.times. N&N
vitamins, 3 mg glycine, 0.17 g NaH.sub.2PO.sub.4.H2O, 40 mg
acetosyringone, pH 5.8;
[0175] MK5: 1/2.times. MS salts, 10 g sucrose, 1.times. N&N
vitamins, 3 mg glycine, 0.17 g NaH.sub.2PO.sub.4.H2O, 50 mg
kanamycin sulfate, 500 mg carbenicillin, 7 g agar, pH 5.8;
[0176] C: 1.times. MS salts, 30 g sucrose, 1.times. N&N
vitamins, 3 mg glycine, 0.8 g NH.sub.4NO.sub.3, 2 mg BAP, 0.5 mg
IAA, 100 mg kanamycine sulfate, 250 mg carbenicillin, 7 g agar, pH
5.8.
[0177] Seeds were sterilized by briefly rinsing in 70% EtOH and
then in a solution of 20% chlorox plus Tween-20. The seeds were
dried in vacuo and then rinsed several times with sterile water.
Washed seeds were transferred into 1/2.times. basal media and
incubated in a Magenta box for about 7 days in 16 hour photoperiods
daily.
[0178] Fully expanded cotyledons were cut aseptically under water.
Two cuts were made at the end, and the tip of the cotyledon piece
and the center piece was retained and used. A culture of
Agrobacterium containing the appropriate binary plasmid was
initiated 24 hours before co-cultivation. Bacteria in 4-5 ml of the
culture were collected by centrifugation and resuspended in
TR1-liquid media. The suspension was poured onto cut cotyledon
pieces and incubated for about 25 min. The cotyledon pieces were
placed on sterile filter papers and placed compactly on TCM medium.
The plates were kept in the dark at room temperature for about 48
hours, after which they were placed on plates containing 1Z medium.
The plates were incubated in 16 hours light daily at about
24.degree. C. for about 21 days.
[0179] Calli that formed on cotyledon pieces were transferred to
fresh 1Z plates and shoots were removed as they formed to 1/2-X
MK-5 tubes for rooting. A 4-mm piece of leaf from the shoot was
also placed on C medium for callus formation. Twelve to fourteen
days after the plating on C medium, calli were scored as "-" or
"+". About 60 to 70% of shoots with +callus root in MK5 media.
Those that have +callus but did not root were trimmed off at the
end and re-rooted on fresh MK5 tubes. About 80% of these will root
on the second attempt.
[0180] Rooted shoots were removed to potting soil when a strong
root system has developed, usually about 3 weeks after rooting. The
plants were kept in a closed plastic bags for about 3 days, the
bags were opened slowly after that to acclimatize the young plant.
A 6- to 8-mm piece of leaf tissue was collected for the NPTII ELISA
assay. The NPTII positive plants were transferred to the greenhouse
for seed production. About 4 to 5 weeks in the greenhouse leaf
tissues were collected for RNA isolation and Northern blots were
done for these plants.
EXAMPLE 9
[0181] Analysis of Transgenic Plants
[0182] Transgene RNA expression in transgenic tomato lines was
accomplished by estimating steady state transcription levels using
Northern blot hybridization. The level of transgene expression was
used to select lines for agroinoculation. Total RNA was isolated
from leaves and stems of young plants and electrophoresed on
agarose gels.
[0183] The appropriate ORF DNA probe was radio-labeled and
hybridized to RNA blotted on paper. After washing the RNA was
visualized by autoradiography on X-ray film.
[0184] The following Tables 2, 3, and 4 summarize results showing
plants produced with geminivirus constructs described above. The
following symbols are used:
[0185] No+ or No-, Northern blot positive or negative;
[0186] So+ or So-, Southern blot positive or negative;
[0187] *, no data;
[0188] R.sub.0 and R.sub.1, primary and progeny lines.
[0189] Table 2 summarizes the transgenic tomato plants produced by
transfer of wildtype ToMoV ORF DNA into the plant by Agrobacterium
infection. For example, several tomato plants (TGM-1 to -17, -20,
-24, -28, -29, -33 to -41, -47 to -49, -53, -54, -59 to -67, -70 to
-131; TTGV92-1 to -5, -10, -13 to -20) were produced by
Agrobacterium containing the RTAC construct. As shown in Table 1A,
this construct is comprised of the ToMoV AC1 OFR in an antisense
configuration. The predominant characteristic of these
RTAC-containing plants is the presence of ToMoV DNA in the plant
tissue (i.e., So+), transcribed RNA (i.e., No+), and transmitted
these traits to their progeny (R.sub.1 RNA). Table 2 also described
transgenic plants with DHAC and RT3AA constructs, comprised of
ToMoV AC1 and AC1-AC2-AC3 antisense ORF, respectively (Table
1A).
[0190] Table 3 describes transgenic tomato plant containing mutant
ToMoV ORF. These include the meu, meu2, Codlm, mub, mua, mubin,
mauin, rtsfs, lasu, and lasd constructs described in Table 1A.
[0191] Table 4 describes transgenic tomato plant containing TYLCV
ORF. These include LCA, LCR, RT3CA, RT3CS and Co3CA constructs of
Table 1A, comprising TYLCV C1, C2 and C3 ORF.
[0192] These results establish that the methods described herein
produce transgenic plants using DNA constructs containing gemini
virus ORF.
5TABLE 2 TOMATO PLANTS TRANSFORMED WITH ToMoV R.sub.0 R.sub.1
R.sub.0 Product Gene RNA RNA DNA Tgm-1, 10, 12, 14, 20, 29, 39, 53,
54, 64, RTAC No+ No+ So+ 66, 70, 71, 80, 81, 82, 127 Tgm-3, 8, 13,
16, 17, 24, 28, 33, 34, 36, RTAC * * So+ 40, 41, 47, 48, 49, 65,
114 Tgm-35 RTAC * * So- Tgm-59, 79, 102, 88, 116 RTAC No- No- So+
Tgm-67 RTAC No+ No- So+ Tgm-84, 90, 93, 94, 97, 98, 99, 100, 101,
RTAC No+ * So+ 103, 106, 107, 108, 112, 113, 115, 117, 120, 121,
122, 123, 125, 129, 131 Tgm-18, 19, 26, 42, 55, 58, 68 DHAC * * So+
Tgm-23, 31, 44, 51 DHAC No+ No+ So+ Tgm-27 DHAC No- No+ So+ 3AA-3,
7, 9, 12, 13, 18, 21, 22, 23, RT3AA No+ * * 26, 27, 30 3AA-4, 11,
16 RT3AA No- * * TTGV92-1 RTAC No+ No- So+ TTGV92-2, 5, 15, 17 RTAC
No- * So+ TTGV92-3, 13, 19 RTAC No- * So- TTGV92-4, 20 RTAC No+ No+
So+ TTGV92-6, 20 DHAC No+ No+ So+ TTGV92-7 DHAC * No+ So+ TTGV92-10
RTAC No+ No+ * TTGV92-11 DHAC No+ * So+ TTGV92-14 RTAC No- * *
TTGV92-16 RTAC * * So-
[0193]
6TABLE 3 TOMATO PLANTS TRANSFORMED WITH ToMoV REP ORF DOMINANT
LETHAL MUTANT CONSTRUCTS Con- R.sub.0 R.sub.1 Product struct RNA
RNA TTGV92-26, 28, 36 meu2 * * TTGV92-27 meu2 No- * TTGV92-42 meu2
* No+ DLM2, 39, 42, 46, 47, 48, 49, 51, 52, 55, 58, meu No+ * 60,
62, 64, 66, 68, 70, 72, 74, 76, 79, 80, 81, 82, 83, 85, 88, 89, 90,
91, 93, 95, 96, 97, 98, 99, 100, 101, 102, 104, 106, 107, 108, 109,
110, 112, 116, 117, 118, 119, 120, 122, 124, 126, 127, 130, 133,
135, 136, 137, 139, 144, 148, 149, 151, 180, 155, 159, 162, 167,
170, 172, 173, 177, 192, 198, 200, 201, 204, 206, 211, 215, 217,
219, 220 DLM3, 5, 7, 9, 10, 12, 14, 15, 18, 21, 26, 30, meu No+ No+
31 DLM16 meu2 No- No- DLM17, 29 meu * * DLM22 meu No- No- DLM24 meu
No- No- DLM25 meu2 No+ No+ DLM27, 28 meu No- No- DLM32 meu2 No+ No+
DLM37, 38, 44, 57, 59, 61, 75, 115, 129, 131, meu No- * 138, 150,
166, 174, 179, 189, 208 DLM143 meu No- * CODLM2, 4, 5, 6, 8, 9, 10,
13, 14, 15, 16, 18, Cod1m No+ * 21, 24, 26, 28 CODLM3, 7, 19, 27
Cod1m No- * MU-2, 3, 4, 5, 6, 7, 14, 15, 19, 20, 26, 27, 30, mub
No+ * 31, 32, 33, 34, 36, 37 MU-8, 9, 12, 16, 18, 22, 28, 39, 41
mua No+ * MU-11 mua No- * MU-13, 47 mub No- * MUIN-3, 6, 7 mubin
No+ * MUIN-4, 5 muain No+ * MUIN-8, 10, 11, 14, 17, 18 mubin * *
MUIN-9, 15, 16, 19 muain * RTSFS-1, 3, 4, 6, 9, 10 rtsfs No+ *
RTSFS-7, 8 rtsfs No- * LAS-1 1asu No+ * LAS-6, 10 1asd No+ * LAS-11
1asd No- *
[0194]
7TABLE 4 TRANSGENIC TOMATO PLANTS TRANSFORMED WITH TYLCV GENE
CONSTRUCTS R.sub.0 R.sub.0 R.sub.1 Product Construct DNA RNA RNA
Lca-1, 2, 37, 39, 43 1ca So- * * Lca-5, 14, 21, 24, 29 1ca So+ *
No+ Lca-6, 35, 36, 46 1ca So+ * * Lca-8, 12, 18, 19, 20, 26, 28 1ca
So+ * No- Lca-25 1ca * * No- Lca-45 1ca So+ No+ * Lcr-1, 5, 6, 22
1cr So+ No+ * Lcr-3, 4, 17, 18, 20, 24 1cr So- No- * Lcr-12 1cr So-
No+ * Lcr-16, 31 1cr So+ No- * Lcr-25 1cr So+ * * Lcr-26, 27, 29,
32 1cr So- * * 3CA-2, 3, -4, -6, -12, -15, -17, RT3CA * No+ * -18,
-19, -21, -22 CO3CA-1, -2, -4, -5, -7, -8, -9, Co3CA * No+ * -11,
-12, -13, -14, -17, -18, -19 CO3CA-6, -10 Co3CA * No- * RT3CS-1
RT3CS * No- *
EXAMPLE 10
[0195] Viral Challenge of Transgenic Plants
EXAMPLE 10.1
[0196] ToMoV Agroinoculation Vector
[0197] A 5.6-kb fragment composed of a dimer of full-length
infectious DNA-A was ligated with BamHI-HindIII digested binary
plasmid pJTS222 to produce construct comprising the ToMoV-A dimer.
The resulting plasmid produced transformed LBA4404 cells, uses as
the A-component in agroinoculation experiments.
[0198] A 6.9-kb XbaI fragment that includes a full length
infectious clone of DNA-B and the complete pBluescript II KS+
plasmid was inserted into the XbaI site of pJTS222. The resulting
plasmid produced transformed LBA4404 cells used as the B-component
in agroinoculation experiments.
EXAMPLE 10.2
[0199] TYLC-IS-EG1 Agroinoculation Vector
[0200] The full length TYLCV-IS-EG1 DNA from infectious clone
pTYEG14 was removed from the plasmid by SphI digestion and inserted
at high molar excess into the SphI site of pGEM5Zf+(Promega). The
resulting plasmid, pTYEG7, contained a dimer of infectious
TYLCV-IS-EG1 DNA. The 6.7-kb fragment of ScaI-PstI fragment of
pTYEG7 comprised the dimer and part of pGEMZAf+. The 1.9-kb
PstI-ScaI fragment of pSL1180 was ligated with the 6.7-kb fragment
from pTYEG to produce a 8.7-kb construct with a single BglII
site.
[0201] The 7.0 kb ScaI-BamHI fragment of the resulting recombinant
plasmid was ligated with HpaI-BamHI-digested pJTS222. A resulting
construct was used to transform LBA4404 cells to produce AtLC1,
which was used in the TYLCV agroinoculation experiments.
10.3
[0202] Agroinoculation Procedure
[0203] R.sub.1 plants from self pollinated R.sub.0 primary
regenerants were agroinoculated 3 weeks after sowing. For bipartite
geminiviruses, agroinoculation involves delivery of
greater-than-full-length (must contain 2 common regions) ToMoV
DNA-A and DNA-B into the seedlings using Agrobacterium. A small
amount of a mixture of two Agrobacterium strains each containing a
binary vector having in its T-DNA a partial or full tandem
duplication of infectious geminivirus DNA was injected into the
plant. For monopartite geminiviruses, only one agrobacterial strain
is required if it carries a binary vector comprising a full or
partial duplication of a full length infectious DNA.
[0204] Overnight cultures of Agrobacteria were diluted, and
injected into stems of one month old tomato seedlings. About 100
hours later, a second inoculation identical to the first is
performed.
[0205] Detection of NPTII by ELISA was taken as an indicium of the
presence of the transgene. Agroinoculation experiments, summarized
in Tables 5 to 10, show an array of resistance phenotypes. The data
show several transgenic tomatoes resistant to ToMoV infection,
including DLM12, TTGV92-42, CODLM6, CODLM8, CODLM13, CODLM14, MUA9,
MUB20, MUA8, MUA18, MUA28, and MUA41.
8TABLE 5 ToMoV Agroinoculations - DLM Transgenics Fraction of
symptom-free Days Part and virus-free plants Line Inoculation NPTII
positives NPTII negatives (Generation) observation visual blot
visual blot TTGV92-36 20 2/16 2/16 0/2 0/2 (R1) TTGV92-42 20 9/11
8/11 3/7 3/7 (R1) untransformed 20 * * 0/17 0/17 DLM3 (R1) 26 *
5/16 * 0/2 DLM7 (R1) 26 * 4/15 * 1/3 DLM9 (R1) 26 * 0/14 * 1/2
DLM10 (R1) 26 * 2/16 * 0/2 DLM12 (R1) 26 * 10/17 * 0/1
untransformed 26 * * * 0/20 DLM12 (R1) 23 8/11 6/11 * *
TTGV92-42-(R2) 23 * * 3/18 2/18 TTGV92-42-(R2) 23 6/13 4/13 0/5 0/5
TTGV92-42 23 13/15 10/15 0/3 0/3 (R1) untransformed 23 * * 1/24
1/24 DLM12 (R1) 21 12/20 13/20 1/5 1/5 DLM14 (R1) 21 6/18 4/18 * *
DLM15 (R1) 21 0/14 0/14 0/4 0/4 DLM27 (R1) 21 0/15 0/15 0/3 0/3
DLM28 (R1) 21 1/16 1/16 0/1 0/1 untransformed 21 * * 0/15 0/15 DLM5
(R1) 18 0/13 1/13 0/5 0/5 DLM17 (R1) 18 1/5 1/5 3/9 3/9 DLM22 (R1)
18 0/15 0/15 0/3 0/3 DLM26 (R1) 18 2/3 2/3 1/5 1/5 DLM29 (R1) 18
0/13 0/13 1/3 1/3 DLM30 (R1) 18 3/14 3/14 1/4 1/4 DLM31 (R1) 18
4/12 4/12 0/6 0/6 TTGV92-42-17(R2) 18 7/13 6/13 1/4 1/4
TTGV92-42(R2) 18 17/18 17/18 * * untransformed 18 * * 0/20 0/20
DLM16 (R1) 18 0/13 0/13 0/5 0/5 DLM18 (R1) 18 2/16 2/16 0/2 0/2
DLM21 (R1) 18 0/18 0/18 * * DLM24 (R1) 18 1/11 1/11 0/7 0/7 DLM25
(R1) 18 7/16 6/16 0/2 0/2 DLM32 (R1) 18 1/16 1/16 0/2 0/2
untransformed 18 * * 0/13 0/13 DLM39 (R1) 30 0/15 1/15 1/4 1/4
DLM46 (R1) 18 0/15 0/15 0/5 0/5 DLM47 (R1) 18 5/17 4/17 0/3 0/3
DLM48 (R1) 18 0/15 0/15 0/5 0/5 DLM49 (R1) 18 1/16 9/16 0/4 0/4
DLM55 (R1) 18 0/14 0/14 0/6 0/6 DLM58 (R1) 18 0/14 1/14 0/6 0/6
untransformed 30 * * 0/9 0/9
[0206]
9TABLE 6 ToMoV Agroinocu1ations: 3AA Transgenics Fraction of
symptoms and virus free plants Line DPI NPTII positives NPTII
negatives (Generation) observation visual blot visual blot 3AA3
(R1) 25 1/14 1/14 0/6 0/6 3AA7 (R1) 25 3/18 3/18 0/2 0/2 3AA9 (R1)
25 1/19 1/19 0/1 0/1 3AA12 (R1) 25 0/14 0/14 0/6 0/6 3AA13 (R1) 25
1/4 0/4 0/4 0/4 3AA16 (R1) 25 1/11 1/11 0/9 0/9 3AA18 (R1) 25 0/4
0/4 2/20 2/20 untransformed 25 * * 0/15 0/15 3AA13 (R1) 22 2/19
2/19 0/1 0/1 3AA21 (R1) 25 3/13 3/13 0/7 0/7 3AA22 (R1) 22 6/16
9/16 0/4 0/4 3AA23 (R1) 25 3/9 4/9 0/1 0/1 3AA26 (R1) 25 0/16 0/16
0/4 0/4 3AA27 (R1) 25 0/10 0/10 0/4 0/4 3AA30 (R1) 25 2/18 5/18 0/2
0/2 untransformed 25 * * 0/15 0/15
[0207]
10TABLE 7 ToMoV Agroinoculations: LAS Transgenics Fraction of
symptoms and virus free plants Line DPI NPTII positives NPTII
negatives (Generation) observation visual blot visual blot LAS6
(R1) 25 8/11 8/11 0/9 0/9 LAS1 (R1) 25 0/10 0/10 0/10 0/10 LAS10
(R1) 25 1/14 1/14 1/6 1/6 LAS11 (R1) 25 * * 0/20 0/20 untransformed
25 * * 0/14 0/14
[0208]
11TABLE 8 ToMoV Agroinoculations: CODLM Transgenics Fraction of
symptom- and virus-free plants Line DPI NPTII positives NPTII
negatives (Generation) observation visual blot visual blot CODLM2
20 0/14 0/14 0/6 0/6 (R1) CODLM5 " 0/15 0/15 0/5 0/5 (R1) CODLM6 "
9/14 3/14 0/6 0/6 (R1) CODLM8 " 8/20 1/20 No NPTII- * (R1) plants
CODLM9 " 0/18 0/18 0/2 0/2 (R1) CODLM10 " 0/17 0/17 0/3 0/3 (R1)
CODLM13 " 11/20 0/20 No NPTII- * (R1) plants CODLM14 " 7/16 0/16
0/4 0/4 (R1) untransformed " * * 1/9 1/9
[0209]
12TABLE 9 ToMoV Agroinoculations: MUA and MUB Transgenics Fraction
of symptom- and virus-free plants Line DPI NPTII positives NPTII
negatives (Generation) observation visual blot visual blot MUA9 22
8/14 10/14 0/6 0/6 MU820 " 10/20 1/20 No * NPTII.sup.- MUB37 " 1/14
0/14 0/6 0/6 MUB3 20 0/14 0/14 0/6 0/6 MUB5 " 1/7 0/17 0/3 0/3 MUB7
" 1/18 1/18 0/2 0/2 MUA8 " 6/6 5/6 0/14 0/14 MUA12 " No * 0/20 0/20
NPTII.sup.+ MUB14 " 2/15 1/15 0/5 0/5 MUB15 " 5/20 3/20 No *
NPTII.sup.- MUA16 " 5/14 4/14 0/6 0/6 MUA18 22 * 6/6 * 0/14 MUB19 "
* 1/11 * No NPTII.sup.- MUB26 " * 0/15 * 0/5 MUA22 21 * 11/11 * 0/9
MUB33 " * 0/15 * 0/5 MUB30 " * 4/12 * 0/8 MUA28 " * 12/12 * 0/7
MUA41 " * 6/9 * 0/11 MUB36 " * 1/15 * 0/5 MUA39 " * 5/14 * 4/6
MUB31 " * 2/16 * 0/4 MUB34 " * No * 0/20 NPTII.sup.+ MUB32 " * 3/16
* 0/4 untransformed " * * 0/10 0/10
[0210]
13TABLE 10 ToMoV Agroinoculations: RTFS Transgenics Fraction of
symptom- and virus-free plants Line DPI NPTII positives NPTII
negatives (Generation) observation visual blot visual blot RTFS1 20
5/12 2/12 0/8 0/8 RTFS3 " 0/15 0/15 0/5 0/5 RTFS4 " 5/16 1/16 0/4
0/4 RTFS6 " 10/12 10/12 0/8 0/8 RTFS9 " 5/13 3/13 0/7 0/7 RTFS10 "
No NPTII.sup.+ * 0/20 0/20 untransformed " * * 0/10 0/10
EXAMPLE 10.4
[0211] Squash Blot Assay of Geminivirus
[0212] Approximately 3 weeks after agroinoculation, visible
symptoms were monitored and compared to untransformed tomato lines.
At the same time, two samples per plant of leaf extract were
applied to a hybridization membrane. This was done by squashing a
leaf disc about 1/8 inch diameter on the membrane such that leaf
sap thoroughly impregnated the membrane. After the membrane was
treated to denature the DNA in the extract, it was hybridized
according to the same protocol as used for Northern blots with a
radioactive probe that would detect the DNA-B component of ToMoV or
the C1 ORF of TYLCV. The presence of viral DNA in the plant sap
could be detected by autoradiography.
[0213] The presence of viral DNA was highly correlated with
appearance of symptoms, an indicia of susceptibility to infection.
The virus-free phenotype was correlated with the presence of the
marker in families of transgenic tomatoes segregating the NPTII
marker.
[0214] FIG. 1 shows that expression of the ToMoV AC1dlm transgene
is required for resistance to ToMoV infection mediated by
agroinoculation. High expression is necessary but in itself does
not ensure resistance.
EXAMPLE 10.4
[0215] Viruliferous Whitefly Inoculations
[0216] Ten whiteflies carrying ToMoV were put on each eight-day old
seedling. Twenty-five seedlings were used per family. In those
families of seedlings which were not homozygous for the transgene,
NPTII assays were correlated with squash blot results. Twenty-one
to thirty-one days after inoculation, samples of each plant were
taken for biochemical and molecular hybridization assays. The
results are-summarized in Table 11. The Visual Rating gives the
average of are plants, in which "0" is no symptoms and "4" is with
most marked symptoms. The squash blot results give the fraction of
the plants that were virus free.
14TABLE 11 Florida Greenhouse Whitefly ToMoV Inoculations Fraction
of symptom- and virus-free plants NPTII NPTII DPI Positives
Negatives Squash Line obser- Blot Blot Visual Blot (Generation)
vation Blot Blot Ratings Results TGM44 (R2) 21 7/20 0/6 2.2 13/26
TGM44 (R2) 21 6/17 0/9 1.6 10/26 untransformed 21 * 7/25 3.6 7/25
DLM12 (R2) 31 10/26 * 1.0 20/26 DLM12 (R2) 31 8/26 * 2.0 18/26
untransformed 31 * 1/19 3,7 3/19 DLM12 (R2) 31 8/20 * 0.8 8/20
DLM14 (R2) 31 3/11 * 1.7 3/11 DLM14 (R2) 31 9/23 * 3.0 12/23
untransformed 31 * 0/16 2.9 0/16 TTGV92-42 (R2) 32 7/21 1/5 2.5
23/26 TTGV92-42 (R3) 32 22/26 * 0 26/26 untransf. 32 * * 3.8 6/15
XPH5978 32 * 10/26 2.9 No Data XPH5979 32 * 7/26 2.7 No Data
EXAMPLE 11
[0217] Transdominance in plant cell lines
[0218] A mutated form of AC1 protein of BGMV inhibits replication
of DNA-A in a tobacco suspension cell system. To evaluate AC1
protein mutants for their potential to interfere with viral
replication, a transient assay was used to detect trans-dominant
interference activity of the mutant viral ORF. (Table 12 and FIG.
2.).
15TABLE 12 Effects of BGMV AC1 Mutations on Replication and
Transdominance Mutation Replication Trans-dominance WT AC1 + 0%
mutation 1(a) - 90% mutation 1(c) - 90% I.sup.190R + 0% mutation
2(c) + 0%* mutation 2(a) - 50-80% mutation 3(a) - >95% mutations
2(a) and (c) - 50-80%
[0219] NT-1 cells were inoculated with wildtype DNA-A or a lethal
mutant of DNA-A of BGMV-GA (ADM; double mutations 2(a) and (c) in
combination with carrier DNA (PBS) or AC1 transexpression vectors
containing mutated forms of AC1 ORF. Total DNA was harvested from
the NT-1 tobacco cells at 72 hours after inoculation,
electrophoresed in an agarose gel, blotted onto paper and probed
with a radiolabeled DNA probe corresponding to the coat protein of
BGMV-GA DNA-A. The results demonstrate that wildtype AC1 protein
produced in trans can replicate a lethal AC1 mutant of DNA-A. More
importantly, the results show that codon changes in the nicking
motif of the AC1 ORF abolished infectivity and replication. In the
transient assay for trans-dominance interference, double mutations
1(a) and 1(c) showed trans-dominance interference (Table 12).
[0220] Additional experimental treatments included:
[0221] A+PBS: wildtype BGMV-DNA-A was introduced into NT-1 cells
with PBS at DNA weight ratios of 1:100 and 5:95 wildtype:PBS;
[0222] A+TDM: BGMV-DNA-A was introduced into NT-1 cells with
transexpression vector coding for double mutations 2(a) and 2(c) at
ratios of 1:100 and 5:95;
[0223] A+TD.sup.262R: BGMV-DNA-A was introduced with
transexpression vector coding for mutation 3(a) at ratios of 1:100
and 5:95;
[0224] ADM+PBS: DNA-A containing double mutations 2(a) and 2(c)
with PBS at 5:95;
[0225] ADM+TAC1: DNA-A containing double mutations 2(a) and 2(c)
with transexpression vector coding for wildtype AC1 at a ratio of
5:95.
[0226] The transexpression vectors used in these experiments
express AC1 in the proper context for replication.
[0227] FIG. 1 represents the results of these experiments. The
mutations created in the 35S promoter driven AC1 ORF are listed in
the first column. These ORF are used in trans with wild-type DNA-A
of BGMV-GA to determine transdominance interference. Replication
was tested in an NT-1 cell system. Replication is presented as the
amount of reduction in replication in comparison to wild-type
replication level. Trans-dominance was determined by engineering
each mutation into a AC1 transexpression vectors which contained
the AC1 ORF under control of the CaMV 35S promoter. Mutant AC1
expression vectors were coinoculated into NT-1 cells along with WT
DNA-A and reductions in DNA-A replication were estimated from
autoradiograms. Trans-dominance data are expressed as the observed
reduction in DNA-A replication when co-inoculated with each AC1
mutant. Mutation 2(c) confers a temperature sensitive phenotype for
replication, supporting replication at 23.degree. C. but not at
28.degree. C.
[0228] Replication was observed in inoculations with wildtype
BGMV-DNA-A plus carrier DNA (A+PBS) (FIG. 1). No replication was
observed in inoculations with a mutant of DNA-A containing double
mutations 2(a) and 2(c) coinoculated with carrier DNA (ADM+PBS).
Replication of double mutations 2(a) and 2(c) was, however,
complemented by transexpression of wildtype AC1 in the transient
expression vector (ADM+TAC1). Replication of BGMV-DNA-A in the
presence of two different AC1 mutants, treatments A+TDM and
A+TD.sup.262R reduced replication of virus DNA-A compared to the
A+PBS treatments. Accordingly, transexpression of AC1 mutants can
inhibit replication of BGMV-DNA-A. Further lethal mutants of AC1
inhibit replication when expressed in trans to DNA-A.
[0229] The results show that non-lethal mutants do not exhibit
detectable transdominant activity. While levels of transdominance
varied among different AC1 mutants, only replication-lethal mutants
exhibited transdominant interference. Levels of AC1 expression
directly relate to levels of trans-dominance and replication (FIG.
1). Thus, AC1 expression, results in production of a protein that
mediates the "trans"-effective suppression. That is, this protein
likely binds to the CR region which mediates its suppressive effect
by inhibiting the binding of the wildtype AC1 protein.
Sequence CWU 0
0
* * * * *