U.S. patent application number 13/624294 was filed with the patent office on 2018-02-01 for citrus tristeza virus based vectors for foreign gene/s expression.
This patent application is currently assigned to UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC.. The applicant listed for this patent is UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC.. Invention is credited to William O. Dawson, Choaa El-Mohtar, Svetlana Y. Foliminova.
Application Number | 20180030461 13/624294 |
Document ID | / |
Family ID | 47915097 |
Filed Date | 2018-02-01 |
United States Patent
Application |
20180030461 |
Kind Code |
A9 |
Dawson; William O. ; et
al. |
February 1, 2018 |
CITRUS TRISTEZA VIRUS BASED VECTORS FOR FOREIGN GENE/S
EXPRESSION
Abstract
Disclosed herein are viral vectors based on modifications of the
Citrus Tristeza virus useful for transfecting citrus trees for
beneficial purposes. Included in the disclosure are viral vectors
including one or more gene cassettes that encode heterologous
polypeptides. The gene cassettes are positioned at desirable
locations on the viral genome so as to enable expression while
preserving functionality of the virus. Also disclosed are methods
of transfecting plants and plants transfected with viral vector
embodiments.
Inventors: |
Dawson; William O.; (Winter
Haven, FL) ; Foliminova; Svetlana Y.; (Winter Haven,
FL) ; El-Mohtar; Choaa; (Lake Alfred, FL) |
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Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC. |
Gainesville |
FL |
US |
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Assignee: |
UNIVERSITY OF FLORIDA RESEARCH
FOUNDATION, INC.
Gainesville
FL
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20130125254 A1 |
May 16, 2013 |
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Family ID: |
47915097 |
Appl. No.: |
13/624294 |
Filed: |
September 21, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12174159 |
Jul 16, 2008 |
8629334 |
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13624294 |
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61537154 |
Sep 21, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8203
20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A CTV viral vector engineered to comprise at least one gene
cassette comprising a polynucleotide encoding a heterologous
polypeptide, the CTV viral vector engineered such that the gene
cassette is positioned at CTV genome regions p13-p20, p20-p23 or
p23-3'NTR.
2. The CTV viral vector of claim 1, wherein said at least one gene
cassette is positioned at CTV genome region p13-p20.
3. The CTV viral vector of claim 1, wherein said at least one gene
cassette is positioned at CTV genome region p20-p23.
4. The CTV viral vector of claim 1, wherein said at least one gene
cassette is positioned CTV genome region p23-3'NTR.
5. The CTV viral vector of claim 1, wherein the at least one gene
cassette further comprises a subgenomic mRNA controller element
positioned upstream of said polynucleotide encoding a heterologous
polypeptide.
6. A plant comprising at least one cell transfected with the CTV
viral vector of claim 1.
7. The plant of claim 6, wherein said plant is a tree.
8. The plant of claim 6, wherein said plant is a citrus tree.
9. A method of infecting a tree to express a heterologous
polypeptide, said method comprising transfecting at least one cell
of said tree with a CTV viral vector engineered to comprise at
least one gene cassette comprising a polynucleotide encoding a
heterologous polypeptide, the CTV viral vector engineered such that
the gene cassette is positioned at CTV genome regions p13-p20,
p20-p23 or p23-3'NTR.
10. The method of claim 8, wherein said tree is a citrus tree.
11. A CTV viral vector engineered to comprise a gene cassette
comprising a polynucleotide encoding a heterologous polypeptide,
the CTV viral vector engineered such that the gene cassette is
inserted in place of the CTV p13 gene.
12. The CTV viral vector of claim 11, wherein the gene cassette
further comprises a subgenomic mRNA controller element positioned
upstream of said polynucleotide encoding a heterologous
polypeptide.
13. A plant comprising at least one cell transfected with the CTV
viral vector of claim 11.
14. The plant of claim 13, wherein said plant is a tree.
15. The plant of claim 13, wherein said plant is a citrus tree.
16-33. (canceled)
34. A CTV viral vector engineered to comprise a first gene cassette
comprising a polynucleotide sequence encoding a first heterologous
polypeptide and a first controller element upstream of said first
heterologous sequence; and a second gene cassette comprising a
polynucleotide sequence encoding a second heterologous polypeptide
and a second control element upstream of said second heterologous
sequence.
35-42. (canceled)
43. The vector of claim 1, wherein said CTV viral vector is
engineered to comprise multiple gene cassettes located at one or
multiple positions.
44. The vector of claim 43, wherein said CTV viral vector comprises
at least two gene cassettes at one position and at least one gene
cassette at a different location.
45. The method of claim 9, wherein said CTV viral vector is
engineered to comprise multiple gene cassettes located at one or
multiple positions.
46. The method of claim 43, wherein said CTV viral vector comprises
at least two gene cassettes at one position and at least one gene
cassette at a different position.
47-51. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to U.S. Provisional Application
No. 61/537,154 filed Sep. 21, 2011, to which priority is claimed
under 35 USC 119 and which is incorporated herein by reference.
BACKGROUND
[0002] The early development of viral vectors was aimed at the
inexpensive production of high levels of specialty proteins that
could be scaled up in the field. The first attempt at a plant viral
vector utilized Cauliflower mosaic virus, a dsDNA virus (Brisson et
al., 1984; Gronenborn et al., 1981). However, this vector was too
unstable to be useful (Futterer et al., 1990). The development of
reverse genetics systems amenable for manipulation of RNA viruses
made many more viruses candidates for vector development (Ahlquist
et al., 1984).
[0003] Virus vectors are key ingredients in basic research and have
great potential for commercial applications. Lack of stability of
foreign inserts has been a major drawback for potential
applications of virus vectors for commercial protein expression in
field applications.
SUMMARY
[0004] The present disclosure is based on multiple studies testing
the vector limits of using CTV to express foreign genes ranging
from 806 to 3480 nucleotides in size. In one embodiment, gene
cassettes were introduced into the CTV genome as replacement of the
p13 gene. In other embodiments, a gene was inserted at different
locations (e.g., p13-p20, p20-p23 and p23-3'NTR (non-translated
region)). In another embodiment, a fusion to p23 and protease
processing were tested. In alternative embodiments, genes were
inserted behind IRES sequences to create bi-cistronic messages.
[0005] Twenty seven expression vectors have been created and tested
in Nicotinia benthamiana protoplasts and plants. Remarkably, most
of the newly developed vector constructs disclosed herein
replicated, spread systemically in plants, and produced their
foreign gene(s). The highest expressing vectors tested include the
"add a gene" constructs having an insertion between the p13 and p20
genes or between the p23 gene and the 3'NTR. Similarly, the vectors
with the inserted gene replacing the p13 gene effectively expressed
different reporter genes. However, optimal expression of the
reporter gene depended both on the size and location of the
insertion. Optimal expression of smaller genes are from positions
nearer the 3' terminus, whereas larger genes are optimally
expressed from more internal positions.
[0006] Efficient expression of two genes simultaneously from the
same vector has been accomplished in both N. benthamiana and
citrus. The novel CTV constructs disclosed herein have genomes with
unique elasticity capable of accommodating and expressing foreign
gene/s by different strategies.
[0007] Engineering an effective vector requires a balance between
different factors. The vector needs to be designed such that
replication and systemic movement in the plant are reduced
minimally while the level of expression of the foreign protein is
maximal (Shivprasad et al., 1999). The final factor is the
stability of the vector. In general, the vector's usefulness is
directly correlated with its stability. Stability is a product of
reduced recombination and increased competitiveness of the vector
with the resulting recombinants that have lost part or all of the
inserted sequences.
BRIEF DESCRIPTION OF DRAWINGS
[0008] FIG. 1. GFP replacement of p13 to produce CTV based
expression vectors. (A) Schematic representation of CTV9R.DELTA.p33
(Boxes represent open reading frames with blue outline of boxes
represent the replication gene block whereas the red outline
represent the closterovirus conserved gene block (Karasev, 2000).
The black circle and black boxes outline represent silencing
suppressors (Lu et al., 2004). Gold box outline represent genes
dispensible for the infection of some citrus genotypes (Tatineni et
al., 2008). Filled black rectangle represents the deletion of the
p33 controller elements and ORF (nts 10858-11660 Genebank Accession
# AY170468) (Satyanarayana et al., 1999; 2000; 2003)). Arrows
indicate the processing of the leader proteases of CTV, LP1 and LP2
are two tandem leader protease, MT (methyl transferase), Hel
(Helicase), RdRp (RNA dependent RNA polymerase, 433 (deletion of
the 33 kda protein sequence), p6 (6 kda protein), Hsp70h (heat
shock protein 70 homologue), p61 (61 kda protein), CPm (minor coat
protein), CP (major coat protein, inter cellular silencing
suppressor), p18 (18 kda protein), p13 (13 kda protein), p20 (20
kda protein, inter/intra cellular silencing suppressor), p23 (23
kda protein, intracellular silencing suppressor) and modification
to produce expression vectors CTV33-.DELTA.13-BY-GFP-57 (C57),
CTV33-.DELTA.13-G-GFP-65 (C65), CTV33-.DELTA.13-B-GFP-66 (C66) with
the CP-CE of BYSV, GLRaV-2 and BYV driving GFP, respectively. (B)
Northern blot analysis of wild type CTV (WT) and CTV based
expression vector transfected to N. benthamiana protoplast (T) and
passaged to a new set of protoplasts (P). (C) Representative sample
of fluorescence in N. benthamiana infected with either of the three
constructs CTV33-.DELTA.13-BY-GFP-57, CTV33-.DELTA.13-G-GFP-65,
CTV33-.DELTA.13-B-GFP-66 magnified under a fluorescent stereoscope.
(D) Representative sample of fluorescence in the phloem of citrus
bark pieces infected with constructs CTV33-.DELTA.13-G-GFP-65 and
CTV33-.DELTA.13-B-GFP-66 with high (left) and low (right)
magnification under a fluorescent stereoscope.
[0009] FIG. 2 GUS replacement of p13 to produce CTV based
expression vectors. (A) Schematic representation of CTV9R.DELTA.p33
and its modification creating expression vector
CTV33-.DELTA.13-BY-GUS-61 in which the p13 and its controller
element is replaced by GUS under the control of CP-CE of BYSV. (B)
Northern blot hybridization analysis of wild type CTV (WT) and CTV
based expression vector CTV33-.DELTA.13-BY-GUS-61 (C61) transfected
to N. benthamiana protoplast (T) and passaged to a new set of
protoplasts (P). (C) Representative sample of GUS activity in the
bark pieces of citrus trees infected with construct
CTV33-.DELTA.13-BY-GUS-61 (right) and the GUS solution before
fixing of the bark pieces (left) (A=Healthy control, B=infect).
[0010] FIG. 3 GFP insertion between p13 and p20 to produce CTV
based expression vectors. (A) Schematic representation of
CTV9R.DELTA.p33 and modification by inserting between p13 and p20
of GFP ORF under the control of BYSV creating expression vector
CTV33-13-BY-GFP-69 (B) Northern blot hybridization analysis of
transfected protoplast with the wild type virus (WT) and expression
vector CTV33-13-BY-GFP-69 (C69) from transcripts (T) and their
passages (P). Representative sample of fluorescence in N.
benthamiana (C) and peeled bark phloem pieces of C. macrophylla (D)
infected with CTV33-13-BY-GFP-69 magnified under a fluorescent
stereoscope.
[0011] FIG. 4 GFP insertion between p20 and p23 to produce CTV
based expression vectors. (A) Schematic representation of
CTV9R.DELTA.p33 and its modification producing expression vector
CTV33-20-B-GFP-49 and CTV33-20-BY-GFP-58, respectively. (B)
Northern blot hybridization analysis of transfected protoplast with
the wild type virus (WT) and expression vectors CTV33-20-B-GFP-49
(C49) and CTV33-20-BY-GFP-58 (C58) from transcripts (T) and their
passages (P). (C) Flourescence under UV light of protoplast (right)
and the leaf (left) showing lack of efficient movement of the
vector. (D) Western blot analysis of the same gene inserted at
different locations in the CTV genome. BCN5 (Folimonov et al.,
2007) original CTV vector (contains GFP under BYV promoter between
CPm and CP), constructs CTV33-23-BY-GFP-37 (C37, insertion of BYSV
driving GFP behind p23), CTV33-20-BY-GFP-58 (C58, insertion of BYSV
driving GFP between p20 and p23), CTV33-13-BY-GFP-69 (C69,
insertion of BYSV driving GFP between p13 and p20),
CTV33-.DELTA.13-BY-GFP-57 (C57, replacement of p13 gene with BYSV
CP-CE driving GFP) and CTV33-27-BY-GFP-63 (C63, Insertion of BYSV
CP-CE driving GFP ORF between CPm and CP).
[0012] FIG. 5 GFP insertion between p23 and 3'NTR to produce CTV
based expression vectors. (A) Schematic representation of
CTV9R.DELTA.p33 and its modification by insertion of GFP behind p23
under control of CP-CE of BYSV, GLRaV-2 and BYV creating expression
CTV33-23-BY-GFP-37 (C37), CTV33-23-G-GFP-40 (C40) and
CTV33-23-B-GFP-42 (C42), respectively. (B) Northern blot
hybridization analysis of transfected protoplast with the wild type
virus (WT) and expression vectors CTV33-23-BY-GFP-37,
CTV33-23-G-GFP-40 and CTV33-23-B-GFP-42 from transcripts (T) and
their passages (P). (C) Representative sample of fluorescence in N.
benthamiana infected with either of the three constructs
CTV33-23-BY-GFP-37, CTV33-23-G-GFP-40 and CTV33-23-B-GFP-42
magnified under a fluorescent stereoscope. (D) Representative
sample of fluorescence in the phloem tissue of Citrus macropylla
infected with constructs CTV33-23-BY-GFP-37 and
CTV33-23-G-GFP-40.
[0013] FIG. 6 GUS insertion between p23 and 3'NTR insertion between
p23 and 3'NTR to produce CTV based expression vectors. (A)
Schematic representation of CTV9R.DELTA.p33 and modification by
insertion of GUS ORF under control of BYSV CP-CE between p23 and
3'NTR creating expression vector CTV33-23-BY-GUS-60 (C60). (B)
Northern blot hybridization analysis of transfected protoplast with
the wild type virus (WT) and expression vectors CTV33-23-BY-GUS-60
from transcripts (T). (C) Enzymatic activity of the GUS protein in
N. benthamiana tissue and citrus phloem bark pieces (Blue color
indicate infected plant and colorless tissue and solution indicate
healthy control and GUS solution subject to the same treatment.
[0014] FIG. 7 GFP inserted behind IRES sequences to create CTV
based expression vectors. (A) Schematic representation of
CTV9R.DELTA.p33 and CTV.DELTA.Cla 333R and their modification
behind p23 creating expression vectors CTV33-23-ITEV-GFP-41;
CTV33-23-I3XARC-GFP-43 represent the TEV 5'NTR IRES and 3xARC-1
IRES, respectively and CTVp333R-23-ITEV-GFP; CTVp333R-23-I3XARC-GFP
representing the TEV 5'NTR IRES and 3xARC-1 IRES, respectively. (B)
1--Northern blot hybridization analysis from tranfected N.
benthamiana protoplast with wild type virus (WT),
CTV33-23-ITEV-GFP-41 (C41) and CTV33-23-I3XARC-GFP-43 (C43); T=RNA
isolated from transcript transfected protoplast and P=RNA isolated
from virion transfected protoplast isolated from RNA transfected
protoplast. 2-Northern blot hybridization analysis from protoplast
transfected with CTVp333R-23-ITEV-GFP (Lane A);
CTVp333R-23-I3XARC-GFP (lane B), CTVp333R (lane C) and
CTVp333R-23-B-GFP (BYV CP-CE driving the expression of GFP behind
p23) (Lane D).
[0015] FIG. 8 GFP and a protease fused to p23 to create CTV based
expression vectors. (A) Schematic representation of CTV9R.DELTA.p33
and the modifications by fusing two TEV proteases (NIa and HC-Pro)
and their recognition sequences to create expression vectors
CTV33-23-HC-GFP-72, CTV33-23-NIa-GFP-73, CTV33-23-HCO-GFP-74 and
CTV33-23-NIaO-GFP-75.
[0016] FIG. 9 Comparison of Florescence in N. benthamiana. (A)
Comparison of fluoresce in infiltrated leaves of representative
samples of constructs CTV33-23-HC-GFP-72, CTV33-23-NIa-GFP-73,
CTV33-23-HCO-GFP-74 and CTV33-23-NIaO-GFP-75 (GFP fused) and
CTV33-23-BY-GFP-37, CTV33-23-G-GFP-40 and CTV33-23-B-GFP-42 (free
GFP) under hand held UV light (Right) and the same leaves under
white light (left). (B) Comparison on whole plant level between
representative samples of constructs CTV33-23-HC-GFP-72 and
CTV33-23-NIa-GFP-73 (fused GFP) and CTV33-23-BY-GFP-37,
CTV33-23-G-GFP-40 and CTV33-23-B-GFP-42 (GFP under its own
controller element behind p23 (Free GFP)) under hand held UV light
(Right) and same plants under white light (Left). (C) Comparison
between the abaxial (Lower) and adaxial (upper) leaf surfaces of
the same representative leaf sample of constructs
CTV33-23-HC-GFP-72 and CTV33-23-NIa-GFP-73 under hand held UV light
(Right) and white light (Left).
[0017] FIG. 10 Western blot analysis of different expression
vectors infiltrated into N. benthamiana leaves using GFP antibody.
A=CTV9R.DELTA.p33GFP (GFP inserted under the BYV CP-CE controller
element between CPm and CP (produces free GFP) (Tatineni et al.,
2008)), B=CTV33-23-BY-GFP-HC-GUS-51, C=CTV33-23-G-GFP-NIa-GUS-54,
D=Empty well; E=CTV33-.DELTA.13-BY-GFP-NIa-GUS-78,
F=CTV33-23-HC-GFP-72, G=CTV33-23-NIa-GFP-73.
[0018] FIG. 11 Hybrid gene (GFP/Protease/GUS fusion) replacement of
p13 to create expression vectors. (A) Schematic representation of
CTV9R A p33 and its modification to create expression vectors
CTV33-.DELTA.13-BYGFP-HC-GUS-77 and
CTV33-.DELTA.13-BYGFP-NIa-GUS-78 with the two fusion genes under
the control of BYSV CP-CE with TEV HC-Pro and NIa spanned by their
proteolysis recognition sequence separating GFP and GUS,
respectively. (B) Activity of the reporter genes in N. benthamiana
and Citrus macrophylla. (a.) Representative sample of N.
benthamiana plant infected with either
CTV33-.DELTA.13-BYGFP-HC-GUS-77 or CTV33-.DELTA.13-BYGFP-NIa-GUS-78
N. benthamiana under white light and (b.) the same plant under UV
light (c.) Two pictures of peeled phloem bark pieces of C.
macrophylla infected with construct
CTV33-.DELTA.13-BYGFP-NIa-GUS-78 under a flourescent stereoscope
(d.) Representative sample of GUS activity in systemic N.
benthamiana leaves, control leaf (Left) and infected leaf (right)
(e.) Peeled bark phloem pieces and GUS solution of healthy C.
macrophylla plant (f.) Peeled bark phloem pieces of C. macrophylla
plant infected with construct CTV33-.DELTA.13-BYGFP-NIa-GUS-78.
[0019] FIG. 12 Stability of Constructs in N. benthamiana. (A) Upper
leaf from Agro-inoculated N. benthamiana plants carrying the binary
vector CTV33-.DELTA.13-BYGFP-HC-GUS-77 (GFP/HC-Pro/GUS) pictured
under fluorescent microscope. (B) The same leaf was tested for GUS
activity indicating almost perfect overlap between the two reporter
genes.
[0020] FIG. 13 Hybrid gene (GFP/Protease/GUS fusion) between p23
and 3'NTR to create expression vectors. (A) Schematic
representation of CTV9R A p33 and its modification to produce
expression vectors CTV33-23-BY-GFP-HC-GUS-51 and
CTV33-23-BY-GFP-NIa-GUS-52 has the BYSV CP-CE driving the hybrid
genes that contain HC-Pro and NIa proteases respectively;
CTV33-23-G-GFP-HC-GUS-53 (C53) and CTV33-23-G-GFP-NIa-GUS-54 (C54)
are GLRaV-2 driven fusion genes that contain the HC-Pro and NIa
proteases, respectively; CTV33-23-BY-GFP-HC-GUS-55 (C55) and
CTV33-23-BY-GFP-NIa-GUS-56 (C56) are BYV driven fusion genes that
contain HC-Pro and NIa proteases, respectively. (B) Northern blot
hybridization analysis of transfected protoplast with wild type
virus (WT), C53, C54, C55 and C56 constructs.
[0021] FIG. 14 Activity of reporter genes generated by insertion of
the Hybrid gene (GFP/Protease/GUS fusion) behind p23. (A) Activity
of the reporter genes in N. benthamiana. plants (a.) Representative
sample of N. benthamiana plant infected with
CTV33-23-BY-GFP-HC-GUS-51, CTV33-23-G-GFP-HC-GUS-53,
CTV33-23-BY-GFP-NIa-GUS-52 or CTV33-23-G-GFP-NIa-GUS-54 under white
light and (b.) the same plant under hand held UV light (c.)
Representative sample of GUS activity in infected systemic N.
benthamiana leaves and control leaves (tubes 1 &2 represent the
solution before fixing and tissues in fixing solution, respectively
from healthy leaves whereas 3&4 represent the solution and
tissues from infected leaves, respectively, G tube is the GUS assay
buffer (B.) Activity of reporter genes in C. macrophylla (a.)
Picture of peeled phloem bark pieces of C. macrophylla infected
with construct CTV33-23-BY-GFP-HC-GUS-51 under a flourescent
stereoscope (b.) Peeled bark phloem pieces GUS activity in infected
and healthy C. macrophylla plants (tubes 1 &2 represent the
solution and tissues in fixing solution from healthy leaves whereas
3&4 represent the solution and tissues from infected leaves,
respectively.
[0022] FIG. 15 Bimolecular Flouresence complementation (BiFC) prove
of concept. (A) Schematic representation of CTV.DELTA. Cla 333R
(Gowda et al., 2001, Satyanarayana et al., 2003) replicon and its
modification to create expression replicons: (a.) Insertion of both
BiFC genes between p23 and 3'NTR giving rise to
CTVp333R-23-BYbJunN-GbFosC and the controls with one gene behind
p23, CTVp333R-23-BYbJunN(b.) or CTVp333R-23-GbFosC(c.). (B)
Northern blot hybridization analysis of transfected protoplast with
CTVp333R-23-BYbJunN-GbFosC (Lane a.), CTVp333R-23-BYbJunN (Lane c.)
and CTVp333R-23-GbFosC (Lane b.). (C) Flourescence of a transfected
protoplast when pictured under a stereoscope (Upper) or a laser
scanning confocal microscope (lower) indicating the flourescence
from the nucleus.
[0023] FIG. 16 BiFC gene replacement of p13 to produce CTV based
expression vectors. (A) Schematic representation of CTV9R.DELTA.p33
and modification to produce vector
CTV33-.DELTA.13-BYbJunN-GbFosC-76 and the control vectors
CTV33-23-G-bFosC-98 and CTV33-23-BY-bJunN-97 (insertion behind p23
nts 19020-19021). (B) Representative sample of N. benthamiana
fluorescence in systemically infected plants.
[0024] FIG. 17 CTV based expression vector built to simultaneously
express two genes from two controller elements. (A) Schematic
representation of CTV9R.DELTA.p33 and its modification to produce
expression vectors CTV33-23-BYbJunN-GbFosC-59 and
CTV33-.DELTA.13-BYbJunN-23-GbFosC-67. (B) Northern blot
hybridization analysis of the RNA transfected protoplast with the
wild type virus (WT,T), two clones of
CTV33-.DELTA.13-BYbJunN-23-GbFosC-67 (C67,T1 and T2) and two clones
of CTV33-23-BY-bJunN-Gb-FosC-59 (C59, T3 and T4) probed with
3'NTR+p23 (Satyanarayana et al., 1999). (C) Flourescence of N.
benthamiana plant parts under a flourescent stereo microscope
(CTV33-23-BY-bJunN-Gb-FosC-59=a., b., c. and d;
CTV33-.DELTA.13-BYbJunN-23-GbFosC-67=e.) (a.) bud (b.) Corolla,
(c.) systemic leaves, (d.) peeled bark phloem pieces and (e.)
infiltrated leaf
[0025] FIG. 18 CTV based expression vector built to simultaneously
express two genes from two controller elements. (A) Schematic
representation of CTV9R.DELTA.p33 and its modification to produce
expression vectors CTV33-.DELTA.13-BYGUS-23-GGFP-71. (B) Northern
blot hybridization analysis of the RNA transfected protoplast with
the wild type virus (WT) and the CTV33-.DELTA.13-BYGUS-23-GGFP-71
(C71) expression vector probed with 3'NTR+p23 (Satyanarayana et
al., 1999). (C) Biological activity of reporter genes in N.
benthamiana and Citrus. N. benthamiana plant under white light (a.)
and hand held UV light (b.). (c.) GUS activity from healthy (tube 1
(assay solution) &2 (tissue) and infected N. benthamiana (tube
3 (assay solution) and tube 4 (tissue). (d.) Peeled bark phloem
pieces under flourescent microscope and (e.) GUS assay activity in
citrus similar to (c.)
[0026] FIG. 19 Western blot analysis of the different constructs in
citrus to evaluate the expression of GFP and GUS. (A) GFP and CP
antibody used to determine the level of expression of GFP relative
to CP in citrus 708 plant infected with .DELTA.p33CTV9R (Tatineni
et al., 2008), 1808 plant infected with BCN5 (Folimonov et al.,
2007), 1916 plant infected with CTV33-23-G-GFP-40, 1874 plant
infected with CTV33-23-BY-GFP-37, 1934, 1935, 1937 infected with
CTV33-13-BY-GFP-69, 1931 and 1939 infected with construct
CTV33-.DELTA.13-G-GFP-65 and CTV33-.DELTA.13-B-GFP-66,
respectively. (B) GUS and CP antibody used to determine the level
of expression of GUS relative to CP in citrus 2084, 2085, 2086,
2087 plants infected with construct CTV33-.DELTA.13-BYGUS-61, 2132
plant infected with construct CTV33-23-BYGUS-60, 2096 plant
infected with expression vector CTV33-.DELTA.13-BYGFP-NIa-GUS-78,
E=empty well and buffer=-iveC.
[0027] FIG. 20 CTV based expression vector built to simultaneously
express four genes from four controller elements. (A) A schematic
representation of CTV9R. (B) Modification of CTV9R to create
expression vector CTV.DELTA.13-BRFP-GbFosC-BYbJunN-CTMVCP-118 which
expresses 4 genes from different locations within the CTV genome.
The first gene is the red flourescent protein gene (tagRFP)
expressed from between the minor and major coat proteins under the
control of the Beet yellows virus (BYV) coat protein controller
element (CP-CE), the second and third genes are the truncated
mammalian transcription factors bFos and bJun fused to the C and N
terminus of EYFP (Hu et al., 2002) under the control of Grape vine
leaf roll associated virus-2 (GLRaV-2) and Beet yellow stunt virus
(BYSV) CP-CE respectively replacing the p13 gene and the fourth
gene is the CP of TMV expressed from behind p23 under the control
of the duplicated major CP-CE of CTV.
[0028] FIG. 21 CTV based expression vector built to simultaneously
express three genes from three controller elements. (A) A schematic
representation of CTV9R. (B) Modification of CTV9R to create
expression vector CTV.DELTA.13-GbFosC-BYbJunN-CTMVCP-129 which
expresses 3 genes from different locations within the CTV genome.
The first and second genes are the truncated mammalian
transcription factors bFos and bJun fused to the C and N terminus
of EYFP (Hu et al., 2002) under the control of Grape vine leaf roll
associated virus-2 (GLRaV-2) and Beet yellow stunt virus (BYSV)
CP-CE respectively replacing the p13 gene and the fourth gene is
the CP of TMV expressed from behind p23 under the control of the
duplicated major CP-CE of CTV.
[0029] FIG. 22 CTV based expression vector built to simultaneously
express three genes from three controller elements. (A) A schematic
representation of CTV9R. (B) Modification of CTV9R to create
expression vector CTV-BRFP-BYGFP-CTMVCP-117 which expresses 3 genes
from different locations within the CTV genome. The first gene is
the red flourescent protein gene (tagRFP) expressed from between
the minor and major coat proteins under the control of the Beet
yellows virus (BYV) coat protein controller element (CP-CE), the
second gene is the Green fluorescent protein (GFPC3) under the
control of Beet yellow stunt virus (BYSV) CP-CE inserted between
p13-p20 gene and the third gene is the CP of TMV expressed from
behind p23 under the control of the duplicated major CP-CE of
CTV.
[0030] FIG. 23 CTV based expression vector built to simultaneously
express three genes from three controller elements. (A) A schematic
representation of CTV9R. (B) Modification of CTV9R to create
expression vector CTV-BASL-BYPTA-CP7-119 which expresses 3 genes
from different locations within the CTV genome. The first gene is a
lectin from Allium sativum (ASL) expressed from between the minor
and major coat proteins under the control of the Beet yellows virus
(BYV) coat protein controller element (CP-CE), the second gene is
an agglutinin from Pinellia ternata (PTA) under the control of Beet
yellow stunt virus (BYSV) CP-CE inserted between p13-p20 gene and
the third gene is an antimicrobial peptide from Tachypleus
tridentatus (P7) expressed from behind p23 under the control of the
duplicated major CP-CE of CTV.
[0031] FIG. 24 CTV based expression vector built to simultaneously
express three genes from three controller elements. (A) A schematic
representation of CTV9R. (B) Modification of CTV9R to create
expression vector CTV-BASL-BYPTA-CP10-120 which expresses 3 genes
from different locations within the CTV genome. The first gene is a
lectin from Allium sativum (ASL) expressed from between the minor
and major coat proteins under the control of the Beet yellows virus
(BYV) coat protein controller element (CP-CE), the second gene is
an agglutinin from Pinellia ternata (PTA) under the control of Beet
yellow stunt virus (BYSV) CP-CE inserted between p13-p20 gene and
the third gene is an antimicrobial peptide from Sus scorfa (P10)
expressed from behind p23 under the control of the duplicated major
CP-CE of CTV.
[0032] FIG. 25 CTV based expression vector built to simultaneously
express three genes from three controller elements. (A) A schematic
representation of CTV9R. (B) Modification of CTV9R to create
expression vector CTV-BASL-BYP10-CP7-131 which expresses 3 genes
from different locations within the CTV genome. The first gene is a
lectin from Allium sativum (ASL) expressed from between the minor
and major coat proteins under the control of the Beet yellows virus
(BYV) coat protein controller element (CP-CE), the second gene is
an antimicrobial peptide from Sus scorfa (P10) under the control of
Beet yellow stunt virus (BYSV) CP-CE inserted between p13-p20 gene
and the third gene is a second antimicrobial peptide from
Tachypleus tridentatus (P7) expressed from behind p23 under the
control of the duplicated major CP-CE of CTV.
[0033] FIG. 26 CTV based expression vector built to simultaneously
express three genes from three controller elements. (A) A schematic
representation of CTV9R.DELTA.p33. (B) Modification of CTV9R
.DELTA.p33 to create expression vector CTV33-BGFP-BYGUS-GTMVCP-79
which expresses 3 genes from different locations within the CTV
genome. The first gene is a green flourescent protein expressed
from between the minor and major coat proteins under the control of
the Beet yellows virus (BYV) coat protein controller element
(CP-CE), the second gene is a .beta.-Glucuronidase (GUS) gene from
Eisherchia coli under the control of Beet yellow stunt virus (BYSV)
CP-CE inserted between p13-p20 gene and the third gene is the CP of
TMV expressed from behind p23 under the control of Grape vine leaf
roll associated virus-2 (GLRaV-2) CP-CE.
[0034] FIG. 27 CTV based expression vector built to simultaneously
express four genes from four controller elements. (A) A schematic
representation of CTV9R.DELTA.p33. (B) Modification of
CTV9R.DELTA.p33 to create expression vector
CTV33-BGFP-GbFosC-BYbJunN-81 which expresses 3 genes from different
locations within the CTV genome. The first gene is the green
flourescent protein gene (GFPC3) expressed from between the minor
and major coat proteins under the control of the Beet yellows virus
(BYV) coat protein controller element (CP-CE), the second and third
genes are the truncated mammalian transcription factors bFos and
bJun fused to the C and N terminus of EYFP (Hu et al., 2002) under
the control of Grape vine leaf roll associated virus-2 (GLRaV-2)
and Beet yellow stunt virus (BYSV) CP-CE respectively. The bFosC
gene is inserted behind p23 gene.
[0035] FIG. 28 CTV based expression vector built to simultaneously
express four genes from four controller elements. (A) A schematic
representation of CTV9R.DELTA.p33. (B) Modification of
CTV9R.DELTA.p33 to create expression vector
CTV33-.DELTA.13-BGFP-BYbJunN-GbFosC-82 which expresses 3 genes from
different locations within the CTV genome. The first gene is the
green flourescent protein gene (GFPC3) expressed from between the
minor and major coat proteins under the control of the Beet yellows
virus (BYV) coat protein controller element (CP-CE), the second
gene is the truncated mammalian transcription factor bJun to the N
terminus of EYFP (bJunN) (Hu et al., 2002) under the control of
Beet yellow stunt virus (BYSV) CP-Cereplacing the p13 gene of CTV
and the third gene is the truncated mammalian transcription factor
bFos fused to the C-terminus of EYFP (bFosC) under the control of
Grape vine leaf roll associated virus-2 (GLRaV-2) CP-CE inserted
behind p23.
[0036] FIG. 29 Negative staining Electron microscopy pictures from
leaf dips of infiltrated N. benthamiana Leaves. (A) Leaf dips from
infiltrated N. benthamiana leaves with construct
CTV33-BGFP-BYGUS-GTMVCP-79 reveals the formation of CTV vector
virions and TMV pseudo virions indicating the expression of the TMV
coat protein gene. (B) Leaf dip from Infiltrated N. benthamiana
leaves with construct CTV33-.DELTA.13-BGFP-BYbJunN-GbFosC-82
reveals the formation of virions.
DETAILED DESCRIPTION
[0037] The early development of viral vectors was aimed at the
inexpensive production of high levels of specialty proteins that
could be scaled up in the field. The first attempt at a plant viral
vector utilized Cauliflower mosaic virus, a dsDNA virus (Brisson et
al., 1984; Gronenborn et al., 1981). However, this vector was too
unstable to be useful (Futterer et al., 1990). The development of
reverse genetics systems amenable for manipulation of RNA viruses
made many more viruses candidates for vector development (Ahlquist
et al., 1984). There was considerable controversy concerning the
value of RNA viruses for vectors (Siegel, 1983, 1985; Van
Vluten-Dotting, 1983 Van Vluten-Dotting et al., 1985). It was
argued that the lack of proof-reading of the RNA virus replicases
would result in too rapid sequence drift to maintain foreign
sequences during replication. However, subsequent development and
use of RNA virus-based vectors demonstrated that this concern was
overstated.
[0038] Ongoing efforts have been underway to create virus-based
vectors for citrus trees based on Citrus tristeza virus (CTV). CTV
has the largest reported RNA of a plant virus of approximately 20
kb (Karasev et al., 1995; Pappu et al., 1994). It has two conserved
gene blocks associated with replication and virion formation
(Karasev, 2000). The replication gene block occupies the 5' half of
the genome. Its proteins are expressed from the genomic RNA via a
poly protein strategy with a +1 ribosomal frame shift to
occasionally express the RNA dependent RNA polymerase (Karasev et
al., 1995). The filamentous virions of CTV are encapsidated by two
coat proteins, with the major coat protein (CP) encapsidating about
97% of the virion and the 5' .about.700 nts encapsidated by the
minor coat protein (CPm) (Satyanarayana et al., 2004). Virion
formation is a complex process requiring two proteins (Hsp70h and
p61) in addition to the coat proteins (Satyanarayana et al., 2000,
2004; Tatineni et al., 2010). These four genes as well as the 6
remaining genes are differentially expressed via a nested set of 3'
co-terminal sub genomic (sg) RNAs (Hilf et al., 1995). Upstream of
each ORF there is a controller element (CE) that determines the
transcription level (Gowda et al., 2001). Levels of transcription
are also associated with the +1 transcription start site (Ayllon et
al., 2003), the presence of a non-translated region upstream of the
ORF (Gowda et al., 2001), and the closeness of the ORF to the 3'
terminus (Satyanarayana et al., 1999).
[0039] The first generations of CTV vector examined three different
strategies that were fusion of the CP gene, insertion of an extra
gene, and replacement of the p13 ORF (Folimonov et al., 2007).
Replacement of the p13 ORF and fusion to the coat protein ORF did
not result in effective vectors, but the addition of an extra gene
resulted in viable vectors that produce relative large amounts of
foreign gene and were stable in citrus trees for years. However,
the first efforts in designing vectors based on CTV examined only a
few of the many possibilities for expressing foreign genes in this
large virus. In this work, the inventors attempted to examine the
limitations of CTV to be manipulated into a vector. The inventors
examined whether the virus allowed insertions in different
positions within the genome and which resulted in maximal
expression with different sizes of inserts. The inventors also
examined whether different fusion strategies with different viral
genes are viable and whether multiple foreign genes can be
expressed. The CTV constructs disclosed herein are amazingly
tolerant to manipulation at several positions within the genome
giving a multitude of different vector strategies that are
viable.
[0040] Once citrus is infected with a CTV vector containing a
foreign gene, it is easy to move the vector to other citrus trees
by grafting. However, a limitation of the CTV vector system is the
difficulty of initially getting citrus infected with new vector
constructs. Directly inoculating citrus from the cDNA clones,
either by agro-inoculation, particle bombardment, or mechanical
inoculation with RNA transcripts is extremely difficult and
unpredictable (Gowda et al., 2005; Satyanarayana et al., 2001). An
alternative has been to inoculate with virions purified from
Nicotiana benthamiana protoplasts (Folimonov et al., 2007;
Robertson et al., 2005; Satyanarayana et al., 2001; Tatineni et
al., 2008). However, infection of only approximately 0.01-0.1% of
protoplasts with in vitro transcribed RNA has been achieved
(Satyanarayana et al., 2001). Yet, since virions are much more
infectious to the protoplasts than RNA (Navas-Castillo et al.,
1997), the inventors were able to amplify the infection by
sequential passage in protoplasts (Folimonov et al., 2007;
Robertson et al., 2005; Satyanarayana et al., 2001; Tatineni et
al., 2008). Although workable, this is an extremely difficult
system. The inventors are now able to agro-inoculate N.
benthaminana plants that result in systemic infection. This result
allows analysis of the vector constructs more quickly in these
plants and provides copious amounts of recombinant virus for
inoculation of citrus. Thus, the inventors report the activity of
the different vector constructs in N. benthamina and Citrus.
[0041] According to one embodiment, the invention pertains to a CTV
viral vector engineered to comprise a gene cassette comprising a
polynucleotide encoding a heterologous polypeptide. The gene
cassette is located at a targeted position on the CTV genome. In a
more specific embodiment, the CTV viral vector is engineered such
that the gene cassette is positioned at CTV genome regions p13-p20,
p20-p23 or p23-3'NTR. In other embodiments, the CTV viral vector is
engineered to include multiple genes at one or multiple positions.
It is shown herein that CTV viral vectors can successfully be
engineered to include up to 3 or at least 4 genes that are
expressible by the vector, while maintaining the proper function
and infectivity of the vector.
[0042] In related embodiments, the invention pertains to a plant
that includes at least one cell transfected with the CTV viral
vector engineered to comprise a gene cassette comprising a
polynucleotide encoding a heterologous polypeptide, the CTV viral
vector engineered such that one or more gene cassettes are
positioned at CTV genome regions p13-p20, p20-p23 or p23-3'NTR.
Other related embodiments pertain to methods of expressing at least
one heterologous polypeptide in a plant by infecting the plant with
the specified vector.
[0043] In a further embodiment, the invention is directed to a CTV
viral vector engineered to comprise at least one gene cassette that
includes a polynucleotide encoding a heterologous polypeptide,
wherein the CTV viral vector engineered such that the gene cassette
is inserted in place of the CTV p13 gene. In related embodiments,
the invention pertains to a plant that includes at least one cell
transfected with the CTV viral vector or to methods of expressing
the heterologous polypeptide in a plant by infecting the plant with
the specified vector.
[0044] In another embodiment, the invention relates to a CTV viral
vector engineered to comprise at least one gene cassette comprising
a polynucleotide encoding heterologous polypeptide and IRES
sequence conjugated thereto. In related embodiments, the invention
pertains to a plant that includes at least one cell transfected
with the CTV viral vector or to methods of expressing the
heterologous polypeptide in a plant by infecting the plant with the
specified vector.
[0045] In further embodiments, the invention relates to a CTV viral
vector engineered to comprise a gene cassette comprising a
polynucleotide sequence with continuous amino acid codons extending
from the p23 ORF encoding a first heterologous polypeptide
(protease) with cleavage sites on each side plus a second
heterologous polypeptide. In related embodiments, the invention
pertains to a plant that includes at least one cell transfected
with the CTV viral vector or to methods of expressing the
heterologous polypeptide in a plant by infecting the plant with the
specified vector.
[0046] In further embodiments, the polynucleotide further comprises
a sequence encoding a first control element upstream of said first
heterologous polypeptide, a second sequence encoding a protease
with cleavage sites engineered on each side, and a sequence
encoding a second heterologous polypeptide.
[0047] According to another embodiment, the invention is directed
to CTV viral vector engineered to comprise a first gene cassette
comprising a polynucleotide sequence encoding a first heterologous
polypeptide and a first controller element upstream of said first
heterologous polypeptide encoding sequence; and a second gene
cassette comprising a polynucleotide sequence encoding a second
heterologous polypeptide and a second control element upstream of
said second heterologous polypeptide encoding sequence. Optionally,
the CTV viral vector further comprises a third gene cassette
comprising a polynucleotide sequence encoding a third heterologous
polypeptide and a third controller element upstream of said third
heterologous polypeptide encoding sequence; and a fourth gene
cassette comprising a polynucleotide sequence encoding a fourth
heterologous polypeptide and a fourth controller element upstream
of said fourth heterologous polypeptide encoding sequence. Those
skilled in the art will appreciate that additional gene cassettes
can be added to the vector so long as function and infectivity of
the vector is maintained. In related embodiments, the invention
pertains to a plant that includes at least one cell transfected
with the CTV viral vector or to methods of expressing the
heterologous polypeptide in a plant by infecting the plant with the
specified vector.
[0048] Examples of controller elements (CE) useful in accordance
with the teachings herein include but are not limited to controller
elements homologous to CTV or heterologous control elements.
Heterologous controller elements include, but are not limited to,
coat protein controller elements (CP-CEs) of three closteroviruses:
Beet yellows virus (BYV) (94 nts from 13547-13640 Genbank accession
# AF190581) (Peremyslov et al., 1999), Beet yellow stunt virus
(BYSV) (101 nts from 8516-8616 Genbank accession # U51931) (Karasev
et al., 1996) and Grape vine leaf roll associated virus-2 (GLRaV-2)
(198 nts from 9454-9651 Genbank accession # DQ286725). It will be
evident to those skilled in the art, in view of the teachings
herein, that other controller elements may be implemented, and in
particular control elements having strong promoter like
activity.
[0049] These and other embodiments are further described below and
encompassed within the appended claims.
[0050] Materials and Methods for Examples 1-7 Below
Plasmids Construction
[0051] pCTV9R.DELTA.p33 and pCTV.DELTA.Cla 333R (Gowda et al.,
2001; Satyanarayana et al., 1999, 2000, 2003; Tatineni et al.,
2008) were used as base plasmids for developing all expression
vectors that were used in the protoplast reverse genetics system.
The numbering of the nucleotides (nts) is based on the full length
T36 clone (Genbank Accession # AY170468) (Satyanarayana et al.,
1999, 2003). CTVp333R-23-ITEV-GFP and CTVp333R-23-I3XARC-GFP (FIG.
7A) were created by fusing 5' non translated region (NTR) of
Tobacco etch virus (TEV) (nucleotides (nts) 2-144 Genbank accession
# DQ986288) (Carrasco et al., 2007) and 3xARC-1 (Active ribosome
complementary sequence) (Akergenov et al., 2004) behind the p23
stop codon (between nts19020-19021 in full length T36 clone) using
overlap extension polymerase chain reaction (PCR) (Horton et al.,
1989). For creating expression vectors by gene addition and/or
substitution at different locations, heterologous controller
elements (CE) were selected from coat protein controller elements
(CP-CEs) of three closteroviruses: Beet yellows virus (BYV) (94 nts
from 13547-13640 Genbank accession # AF190581) (Peremyslov et al.,
1999), Beet yellow stunt virus (BYSV) (101 nts from 8516-8616
Genbank accession # U51931) (Karasev et al., 1996) and Grape vine
leaf roll associated virus-2 (GLRaV-2) (198 nts from 9454-9651
Genbank accession # DQ286725) to drive the ORFs for cycle 3 GFP
(GFP) (Chalife et al., 1994; Crameri et al., 1996),
.beta.-Glucuronidase (GUS) ORF of Eisherchia coli, bFosYC155-238
(bFosC), bJunYN1-154 (bJunN). CTVp333R-23-BYbJunN-GbFosC,
CTVp333R-23-BYbJunN, CTVp333R-23-GbFosC (FIG. 15A) were created by
overlap extension PCR from plasmids pBiFC-bFosYC155 and
pBiFC-bJunYN155 (Hu et al., 2002) and CTV9R (Satyanarayana et al.,
1999; 2003). Since two NotI sites exist within the bimolecular
fluorescence genes (BiFC), the overlap extension PCR products were
digested partially by NotI restriction endonuclease. The PCR
products were introduced into a StuI and NotI digested
pCTV.DELTA.Cla 333R (FIGS. 7A & 3-15A).
[0052] The expression vectors created in pCTV9R.DELTA.p33 were
introduced into the CTV genome by digesting the plasmid with PstI
(nts 17208-17213) and NotI or StuI (introduced behind 19,293 the
final CTV nucleotide). Overlap extension PCR (Horton et al., 1989)
was used to introduce the appropriate genes at the different
locations. Replacement of the p13 gene was done by deletion of nts
17293-17581 in the p13 ORF and (CE) by overlap extension PCR (FIG.
3-1A, 3-2A, 3-11A, 3-16A, 3-17A & 3-18A). Similarly, insertion
between p13 and p20 (nts #17685-17686) (FIG. 3A), p20-p23 (nts
#18312-18313) (FIG. 4A) and p23-3'NTR (nts #19020-19021) (FIG.
3-5A, 3-6A, 3-13A, 3-16A, 3-17A & 3-18A) were done by overlap
extension PCR. A hybrid gene created by fusing the GFP ORF (Chalife
et al., 1994; Crameri et al., 1996) and GUS ORF separated by the
HC-Pro protease motif (nts 1966-2411 Genbank accession # M11458)
(Allison et al., 1985; Carrington et al., 1989) and its recognition
sequence fused to the N terminus of GUS
(ATGAAAACTTACAATGTTGGAGGGATG (nts 2412-2438 Genbank accession #
M11458) (Allison et al., 1985; Carrington et al., 1989) (Amino acid
sequence (A.A.) MKTYNVG.dwnarw.GM) (arrow indicate processing site)
and C terminus of GFP (ATGAAGACCTATAACGTAGGTGGCATG) was created and
inserted behind p23 (FIG. 13A) or as replacement of p13 (FIG.
3-11A) under different controller elements. A similar hybrid gene
was created by using the NIa protease motif of TEV (nts 6270-6980
Genbank accession # M11458) (Allison et al., 1985) and its
recognition sequence (GAGAATCTTTATTTTCAGAGT (nts 8499-8519 Genbank
accession # M11458) (A.A. ENLYFQ.dwnarw.S) (arrow indicate
processing site) (Carrington and Dougherty, 1988) at C terminus of
GFP and GAAAACCTATACTTCCAATCG at N terminus of GUS). The redundancy
of the amino acid genetic code was used to eliminate complete
duplication of the nucleotide sequences of the recognition motifs.
A similar strategy was used to create a hybrid gene between p23 ORF
and GFP ORF in construct CTV33-23-HC-GFP-72 and CTV33-23-NIa-GFP-73
(FIG. 8). Switching the recognition motif of the proteases
generated control vectors CTV33-23-HCO-GFP-74 and
CTV33-23-NIaO-GFP-75 (FIG. 8).
[0053] The binary plasmid pCAMBIACTV9R (Gowda et al., 2005) was
modified to eliminate the p33 gene by deleting nts 10858-11660
(Satyanarayana et al., 2000; Tatineni et al., 2008) and introducing
a SwaI site behind the ribozyme engineered based on subterranean
clover mottle virusoid (Turpen et al., 1993). PCR products
amplified from the expression vectors in the pCTV9R.DELTA.p33
back-bone were introduced into the modified binary plasmid
pCAMBIACTV9R.DELTA.p33 digested with PstI (Forward primer C-749)
and SwaI (Reverse primer C-1894). When introducing the bimolecular
fluorescence complementation (BiFC) genes into constructs
CTV33-23-BYbJunN-GbFosC-59 (FIG. 17),
CTV33-.DELTA.13-BYbJunN-23-GbFosC-67 (FIG. 17),
CTV33-.DELTA.13-BYbJunN-GbFosC-76 (FIG. 16), CTV33-23-GbFosC-98
(FIG. 16) and CTV33-23-BYbJunN-97 (FIG. 16) a primer was used
switching the PstI to the compatible NsiI (primer C-2085) for ease
of cloning (the bFosC gene sequence contains one PstI site while
the bJunN gene sequence contains two PstI sites). Preliminary
screening for the right inserts in the different expression vectors
was done by restriction digestion using the appropriate enzymes.
The junctions where the foreign genes were introduced into the
expression vectors were confirmed by sequencing at the
Interdisciplinary Center for Biotechnology Research (ICBR)
(University of Florida, Gainesville, Fl). All primers are listed in
Table 1-1.
TABLE-US-00001 TABLE1-1 List of primers used in building expression
vector ##STR00001## ##STR00002## ##STR00003## ##STR00004##
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019##
##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029##
##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034##
##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039##
##STR00040##
Polymerase Chain Reaction (PCR)
[0054] PCR was performed using diluted plasmids (1:50) as templates
using Vent DNA polymerase (New England Biolabs, Ipswich, Ma.)
according to the manufacturer recommendations.
Agro-Injection/Infiltration
[0055] Agro-inoculation of Nicotiana benthamiana was performed
according to the procedure developed by Gowda et al., (2005) with
minor modifications. Agrobacterium tumefaciens EHA 105 was
transformed with the binary plasmid containing CTV, variants
(expression vectors) and silencing suppressors (p19 of Tomato bushy
stunt virus (Gowda et al., 2005); p24 of GLRaV-2 (Chiba et al.,
2007), P1/HC-Pro of Turnip mosaic virus (Kasschau et al., 2003) and
p22 of Tomato chlorosis virus (Canizares et al., 2008) by heat
shock method (37.degree. C. for 5 minutes) and subsequently were
grown at 28.degree. C. for 48 hours (hrs) on luria burtani (LB)
(Sigma-Aldrich, St Louis, Mo.) plates supplemented with antibiotics
(kanamycin (50 microgram (.mu.g)/milliliter (ml)) and Rifampicilin
((50 .mu.g/ml)). The colonies (two individual colonies per
construct) were grown overnight as seed cultures in LB medium
supplemented with antibiotics. On the next day 0.5 ml of the seed
culture was used to inoculate 35 ml of LB medium supplemented with
antibiotics for overnight growth. The bacterial culture was
centrifuged at 6,000 rotation per minute (rpm) and resuspended in
10 milli molar (mM) MgCL.sub.2 and 10 mM MES. The pellet was washed
with 10 mM MgCL.sub.2 and 10 mM MES and suspended in induction
medium; 10 mM MgCL.sub.2 and 10 mM MES containing acetosyringone at
a final concentration of 150 .mu.M. The suspension was incubated in
the induction medium for at least 5 hrs before injection into the
stem or infiltration into the abaxial (lower) surface of N.
benthamiana leaves.
Plant Growth conditions
[0056] N. benthmaiana plants maintained in a growth-room
(21.degree. C. with 16 hrs of light in a 24 hr period) were used
for agro-injection/agro-infiltration four weeks after
transplanting.
Infection of Citrus Plants
[0057] Recombinant virions of CTV for infection of citrus plants
were obtained from infiltrated and/or systemic leaves of N.
benthamiana. The virions were partially purified and enriched by
concentration over a sucrose cushion in a TL 100 or SW41 rotor
(Robertson et al., 2005). Virions of constructs expressing two
foreign proteins were concentrated two times over a step gradient
followed by a cushion gradient in SW28 and SW41 rotors,
respectively (Garnsey and Henderson, 1982). Inoculation of citrus
plants was carried out by bark flap inoculation into 1-1.5 year old
Citrus macrophylla seedlings (Robertson et al., 2005) which were
grown in a greenhouse with temperatures ranging between
approximately 25-32.degree. C.
Protoplast Preparation, Transfection, RNA Isolation and Northern
Blot Analysis
[0058] N. benthamiana leaf mesopyhll protoplasts were prepared
according to the procedure previously developed by Nava-Castillo et
al., (1997). Surface sterilized leaves from three week old N.
benthamiana plants were gently slashed on the lower side with a
sterile blade and incubated overnight in the dark (16-20 hrs) in
0.7M MMC (0.7M mannitol, 5 mM MES, 10 mM CaCl.sub.2) supplemented
with the 1% cellulose (Yakult Honsh, Tokyo, Japan) and 0.5%
macerase pectinase enzymes (Calbiochem, La Jolla, Calif.).
[0059] Capped in vitro RNA transcripts from NotI or StuI linearized
plasmid DNA were generated (Satyanarayana et al., 1999) using Sp6
RNA polymerase (Epicentre Technologies, WI) and were transfected
into the protoplasts using PEG (poly ethylene glycol) as described
by Satyanarayana et al., (1999). Four days after transfection,
protoplasts were used for preparation of total RNA for northern
blot hybridization analysis and isolation of virions. Protoplasts
were pelleted in equal amounts in two 1.5 ml eppendorf tubes. The
first tube was flash frozen in liquid nitrogen and stored at
-80.degree. C. for isolation of virions to subsequently inoculate a
new batch of protoplasts to amplify virions (Satyanarayana et al.,
2000). The second tube was used for RNA isolation by the buffard
buffer disruption of protoplasts followed by phenol: chloroform:
isoamyl alcohol (25:24:1) extraction and ethanol precipitation as
previously described by Navas-Castillo et al., (1997) and Robertson
et al., (2005). Total RNA was resuspended in 20 .mu.l DNAse/RNAase
free water and used in Northern blot hybridization analysis as
previously described by Lewandowski and Dawson (1998). In brief,
isolated RNA was heat denatured in denaturing buffer (8.6%
formaldehyde, 67% formamide in 1XMOPS (5 mM sodium acetate, 1 mM
EDTA, 0.02M MOPS pH=7.0) separated in a 0.9% agarose gel in 1XMOPS
containing 1.9% formaldehyde, and transferred onto a nylon membrane
(Boehringer Mannheim, Germany) by electroblotting.
Pre-hybridization (at least 1 hr) and hybridization (overnight)
were carried out in a hybridization oven (Sigma-Aldrich, St. Louis,
Mo.) at 68.degree. C. A 900 nts digoxigenin labeled RNA probe
corresponding to the 3' end of the CTV genome (plus strand specific
CTV RNA probe) (Satyanarayana et al., 1999) was used for
hybridization except when the insertion of the foreign genetic
material was behind p23 in which case a digoxigenin labeled RNA
probe was produced from PCR amplified DNA (reverse primer contain
3'NTR of CTV and SP6 phage promoter (C-1982) according to the
manufacturer recommendation (Boehringer Mannheim, Germany) that is
complimentary to the sequence inserted behind p23 in addition to
the 3'NTR sequence of CTV.
Western Blots
[0060] After powdering the plant tissue in liquid nitrogen via
grinding in a mortar and pestle, laemmli buffer (50 mM Tris-C1, pH
6.8, 2.5% 2-mercaptoethanol, 2% SDS, 0.1% bromophenol blue, 10%
glycerol) was added (100 .mu.l per 100 mg tissue). The sample was
transferred to a 1.5 ml centrifuge tube and boiled in a water bath
for 3 minutes followed by centrifugation at maximum speed for 2
minutes. The supernatant was transferred to a new tube and stored
at -20.degree. C. until further use. The electrophoresis was
carried out in a 12% SDS-Polyacrylamide gel (Bio-Rad, Hercules,
Calif.) followed by two hours of semi-dry blotting to transfer the
protein onto a nitrocellulose membrane (Bio-Rad, Hercules, Calif.).
The membrane was blocked for 1 hr at room temperature followed by
incubation with the primary antibody of either CP (1:5000), GFP
(1:100) (Clontech Laboratories, Palo Alto, Calif.) or GUS (1:1000)
(Molecular probes, Eugene, Or.) for an hour followed incubation for
1 hr in horseradish peroxidase conjugated donkey anti-rabbit
secondary antibody (1:10,000) (Amersham, Buckinghamshire, United
Kingdom). Finally, the chemiluminescent system for western blot
(Amersham, Buckinghamshire, United Kingdom) development on an X-ray
film (Kodak, Rochester, N.Y.) was used according to the
manufacturer recommendations.
Plant and Protoplast Photos
[0061] Plant pictures under UV or white light were taken with a
Canon Camera (Canon EOS Digital Rebel XTi 400D, Lake Success, New
York). Close up fluorescent pictures of plant parts or protoplast
were taken using a fluorescent dissecting microscope (Zeiss Stemi
SV 11 UV-fluorescence dissecting microscope, Carl Zeiss Jena,
GmbH., Jena, Germany). High resolution protoplast pictures were
taken using a confocal scanning microscope (Leica TCS SL, Leica
Microsystems, Inc., Exton, Pa.).
Enzyme Linked Immunosorbent Assay (ELISA)
[0062] Double antibody sandwiched ELISA was used according to the
procedure developed by Garnsey and Cambra (1991). A rabbit
polyclonal antibody (1 .mu.g/ml) was used for coating the ELISA
plate. The plant tissue sample was diluted at a 1:20 in PBS-T
(phosphate buffer saline-1% Tween 20) extraction buffer. The
detection antibody used was Mab ECTV 172 (1:100K dilution).
GUS Assay
[0063] Citrus bark pieces or systemic leaves from Agro-inoculated
N. benthamiana plants that were surface sterilized in alcohol (70%
ethanol) followed by Sodium hypo chloride (10% solution) and
washing three times in sterile distilled water before staining for
GUS. The samples were incubated overnight in an EDTA-phosphate
buffer (0.1M Na.sub.2HPO.sub.4, 1 mM Na.sub.2EDTA) containing 1
mg/ml X-gluc (cyclohexylammounium salt: Gold Biotechnology, St
Louis, Mo.). Fixing of the tissue was done in 95% ethanol: glacial
acetic acid solution (3:1.
Example 1
Systems Used to Examine CTV-Based Expression Vectors
[0064] CTV-based expression vectors were examined in three systems,
N. benthamiana mesophyll protoplasts as well as whole plants of N.
benthaminia and Citrus macropylla. The full-length cDNA clone of
CTV (pCTV9R) and a mutant with most of the p33 gene deleted
(pCTV9R.DELTA.p33), which has a PstI restriction site removed
making cloning easier and still retaining the ability to infect
most citrus varieties (Tatineni et al., 2008), was used for
building constructs to infect whole plants. Relatively quick assays
were done in N. benthamiana protoplasts, which require constructs
to be built in the SP6 transcription plasmid (Satyanarayana et al.,
1999). A mini-replicon pCTV.DELTA.Cla 333R (Gowda et al., 2001),
with most of the 3' genes removed, was convenient to use in
protoplasts. The ultimate goal to obtain citrus trees infected with
the different CTV expression vectors was much more difficult and
time consuming. So far, agro-inoculate citrus trees has proven
difficult. Thus, to avoid this difficulty virions are amplified and
concentrated for inoculation of citrus trees by stem-slashing or
bark-flap inoculation (Robertson et al., 2005; Satyanarayana et
al., 2001). N. benthamiana protoplasts can be inoculated with in
vitro produced transcripts of recombinant CTV constructs and the
virus amplified by successively passaging virions in crude sap
through a series of protoplasts (Folimonov et al., 2007;
Satyanarayana et al., 2001; Tatineni et al., 2008). Also,
recombinant CTV can be amplified in N. benthamiana plants after
agro-inoculation (Gowda et al., 2005). The virus can infect
mesophyll cells of agro-inoculated areas of leaves, but as the
virus moves systemically into upper non-inoculated leaves, it is
limited to vascular tissues and usually induces vein clearing and
later vein necrosis. All of the vector constructs were examined
during systemic infection of N. benthamiana plants. Since CTV
virions do not resuspend after centrifugation to a pellet, virions
have to be concentrated by centrifugation through a sucrose step
gradient (Garnsey et al., 1977; Robertson et al., 2005). After
inoculation, the tops of citrus plants were removed, and viral
systemic infections were monitored in new growth after 2-3 months.
Once trees were infected, inoculum (buds, leaf pieces, or shoots)
from the first infected plants was then used to propagate new
plants for experimentation. The whole process takes approximately
one year. For this reason, the inventors chose to examine only the
most promising vector constructs in citrus trees. Some of the later
developed constructs are not yet in citrus.
Example 2
Addition of an Extra Gene at Different Locations within the CTV
Genome
[0065] Insertions at the p13 Gene Site
[0066] The effective CTV vector developed previously (Folimonov et
al., 2007) has the additional gene inserted between the two coat
protein genes, positioning the foreign gene as the sixth gene from
the 3' terminus. Yet, the most highly expressed genes of CTV tend
to be closer to the 3' terminus. Thus, it appeared that positioning
an inserted gene closer to the 3' terminus could result in higher
levels of expression. P13, the third gene from the 3' terminus, is
a relatively highly expressed gene that is not necessary for the
infection of most of the CTV host range (Tatineni et al., 2008;
Tatineni et al., in preparation). Yet, replacement of the p13 ORF
with the GFP ORF was not successful in previous attempts (Folimonov
et al., 2007). There were possible reasons for the failure. The
previous construct was designed with the assumption that
translation initiated at the first start codon, but the p13 ORF has
a second in-frame AUG. Translation might normally start at the
second AUG. However, fusion of the GFP ORF behind the second in
frame AUG also did not express the reporter gene (Gowda et al.,
unpublished result). A second possibility is that the p13
controller element (CE) might extend into the p13 ORF or that
ribosome recruitment is directed from within the ORF. Here, the
inventors deleted the p13 CE and ORF and inserted a new ORF behind
a heterologous CE in the p13 position. The GFP ORF controlled by
the CP-CE from BYSV (101 nts from 8516-8616 accession # U51931),
GLRaV-2 (198 nts from 9454-9651 accession # DQ286725) or BYV were
engineered into pCTV9R.DELTA.p33 as a replacement for nts
17293-17581 (CTV33-.DELTA.13-BY-GFP-57, CTV33-.DELTA.13-G-GFP-65,
CTV33-.DELTA.13-B-GFP-66 respectively) (FIG. 1 A). RNA transcripts
were used to inoculate a series of protoplasts to determine whether
the constructs could replicate and whether virions formed
sufficiently for passage in crude sap to a new batch of
protoplasts. The fluorescence of infected protoplasts (data not
presented) and northern blot hybridization analysis demonstrated
the successive passage of the expression vectors through the
protoplast transfers (FIG. 1B). Furthermore, the level of the GFP
mRNA was similar to that of CP. Vectors sequences
CTV33-.DELTA.13-BY-GFP-57, CTV33-.DELTA.13-G-GFP-65 and
CTV33-.DELTA.13-B-GFP-66 then were transferred into the
Agrobacterium binary plasmid for agro-inoculation of N. benthamiana
plants. All three vectors infected and moved systemically in
vascular tissue of the N. benthamiana plants as indicated by
fluorescence in leaves, buds, flowers and corolla (FIG. 1C), vein
clearing phenotype in early stages, as well as confirmed by ELISA
(Data not presented).
[0067] CTV33-.DELTA.13-G-GFP-65 and CTV33-.DELTA.13-B-GFP-66 were
amplified and used to inoculate Citrus macrophylla plants. The
initially infected plants exhibited bright fluorescence in vascular
tissue (FIG. 1D). Fluorescence continued in these plants 2 years
after inoculation.
[0068] The GFP ORF (720 nts) was replaced with the GUS ORF (1812
nts) in the same position to examine the expression of a larger
foreign gene. The BYSV CP-CE was selected to drive the GUS ORF in
expression vector CTV33-.DELTA.13-BY-GUS-61 (FIG. 2A). RNA
transcripts of this construct were transfected into protoplast
where the virus replicated and passaged efficiently from one
protoplast batch to another as indicated by northern blot
hybridization analysis (FIG. 2B). In addition, it revealed that the
level of accumulation of GUS mRNA was identical to the CP mRNA, and
the CP and CPm mRNAs of vector were similar to that of the wild
type virus. Agro-inoculation of N. benthamiana plants revealed that
the construct infected and spread throughout the vascular tissue of
the plants based on GUS staining and confirmed by ELISA (Data not
presented) and the vein clearing phenotype.
[0069] Virions isolated from infiltrated leaves of N. benthamiana
plants of CTV33-.DELTA.13-BY-GUS-61 infected Citrus macrophylla
plants as confirmed by ELISA (Data not presented) and the
bioactivity of the GUS protein (FIG. 2C). The GUS gene was still
biologically active in citrus 1.5 year after inoculation.
[0070] Technically, the above constructs replaced a gene (p13)
rather than added an extra gene. To examine a vector with an extra
gene between p13 and p20, the CP-CE of BYSV controlling the GFP ORF
was inserted between nts 17685-17686 to yield CTV33-13-BY-GFP-69
(FIG. 3A). This vector should produce an extra subgenomic RNA
between the subgenomic RNAs of p13 and p20. Vector
CTV33-13-BY-GFP-69 was examined in N. benthamiana protoplasts and
plants. In the protoplast system, CTV33-13-BY-GFP-69 replicated
efficiently and was successfully passaged from one protoplast batch
to another demonstrating efficient replication and virion formation
as indicated by fluorescence (Data not presented) and northern blot
hybridization analysis (FIG. 3B). The foreign mRNA accumulated at a
relatively high level but the CP mRNA was reduced. Similar to the
replacement of p13 constructs, agro-inoculation of the expression
vector CTV33-13-BY-GFP-69 into N. benthamiana plants enabled the
new vector to infect and spread throughout the vascular tissue
(FIG. 3C).
[0071] Construct CTV33-13-BY-GFP-69 infected C. macrophylla plants
as indicated by strong fluorescence throughout the vascular tissue
(FIG. 3C) and confirmed by ELISA (Data not presented). The plants
were still fluorescencing 2 years after inoculation.
[0072] Insertion Between p20 and p23
[0073] To examine expression of a foreign gene closer to the 3' NTR
of CTV, an extra gene was inserted between the p20 and p23 genes
(nts 18312-18313). The BYV or BYSV CP-CE was used to drive the GFP
mRNA in two vectors based on T36 CTV9R.DELTA.p33 (CTV33-20-B-GFP-49
and CTV33-20-BY-GFP-58) (FIG. 3-4A). The new vectors produced an
extra sgRNA mRNA between the p20 and p23 sgRNAs (FIG. 4B). However,
the accumulation of the p20 sg mRNA was substantially reduced. Both
vectors replicated and were passaged in protoplasts, but the
protoplast passage was reduced as demonstrated by reduced numbers
of cells with GFP fluorescence and northern blot hybridization
(FIG. 4B &C). When both CTV33-20-B-GFP-49 or CTV33-20-BY-GFP-58
vectors were infiltrated into N. benthamiana leaves for transient
expression, the vectors replicated and produced abundant amounts of
GFP as indicated by fluorescence (Data not presented) and western
blot analysis (FIG. 4D).However, when agro-inoculated into N.
benthamiana plants, the constructs replicated but movement into
upper non-inoculated leaves was random and often unsuccessful.
Since systemic infection of N. benthamiana plants was marginal, no
attempt was made to inoculate citrus.
[0074] Insertion Between p23 and 3'NTR
[0075] The next position to be examined was to make the inserted
gene the 3'-most gene. Since CTV gene expression tends to be
highest for genes positions nearer the 3' terminus, this position
could be expected to result in the highest level of expression of a
foreign gene (Navas-Castillo et al., 1997; Hilf et al., 1995).
Although the 3' NTR has been analyzed (Satyanarayana et al.,
2002a), it was not known what effect an extra gene in this area
would have on the efficiency of replication. The insertion of an
extra gene between the CP gene and the 3'NTR in Tobacco mosaic
virus (TMV) and Alfalfa mosaic virus (AMV) failed to produce viable
vectors (Dawson et al., 1989; Sanchez-Navarro et al., 2001). The
CP-CE of BYSV, GLRaV-2 or BYV in front of the GFP ORF was inserted
between nucleotides 19020 and 19021 creating vectors
CTV33-23-BY-GFP-37, CTV33-23-G-GFP-40 and CTV33-23-B-GFP-42,
respectively (FIG. 5A). All of the constructs when transfected into
the protoplast replicated and were passaged efficiently as
indicated by northern blot hybridization analysis (FIG. 5B) and GFP
fluorescence (Data not presented). The GFP mRNA was the highest
accumulating mRNA, with only slight decreases to the other mRNAs
compared to that of the wild type virus (FIG. 5B). Furthermore, the
constructs with a GFP insertion 3' of the p23 ORF had the highest
accumulation of the foreign gene mRNA among the constructs
examined. CTV33-23-BY-GFP-37, CTV33-23-G-GFP-40 and
CTV33-23-B-GFP-42 constructs were agro-inoculated into N.
benthamiana plants. The infections spread systemically throughout
the vascular tissue as demonstrated by the fluorescence (FIG. 5C),
phenotype (vein clearing followed by necrosis), and ELISA (Data not
presented). The fluorescence in the vascular tissue of N.
benthamiana plants was extremely bright and continued for the life
of the infected plants (FIG. 5C)
[0076] Construct CTV33-23-BY-GFP-37 was amplified by passage
through 12 protoplast sets before citrus inoculation. C macrophylla
plants that were bark-flap inoculated with the concentrated virions
became infected. The infection of citrus was confirmed by
fluorescence of GFP (FIGS. 3-5D) and ELISA (Data not presented).
Inoculation of citrus with constructs CTV33-23-G-GFP-40 was done
via amplification in agro-inoculated N. benthamiana plants. The
infection rate was in 1 of 4 C. macrophylla plants as indicated by
fluorescence (FIG. 5D) and confirmed by ELISA (Data not presented).
Similar to N. benthamiana, citrus plants expressed bright
fluorescence in the vascular tissue 12 weeks after inoculation and
were still fluorescing 2.5 years later (FIG. 5D).
[0077] To examine the ability of the vector to express a larger
gene at this position, the GUS ORF behind the BYSV CP-CE was
inserted 3' of the p23 gene resulting in construct
CTV33-23-BY-GUS-60 (FIG. 6A). The construct replicated in
successfully transfected protoplasts. However, the accumulation
levels of all the CTV subgenomic RNAs were decreased profoundly
compared to the wild type virus as demonstrated by northern blot
hybridization analysis (FIG. 6B). Also, the CTV33-23-BY-GUS-60
construct passaged poorly in protoplasts (Data not presented). Yet,
after agro-inoculation of N. benthamiana plants, the vector
replicated and moved systemically as demonstrated by the systemic
symptoms (vein clearing followed by necrosis), ELISA (Data not
presented) and GUS assays. The activity of GUS in the N.
benthamiana plants was continuously produced in old and new leaves
until the death of the plant (FIG. 7C). Similar to
CTV33-.DELTA.13-BY-GUS-61, the location between p23 and 3'NTR was
able to accommodate moderately to long genes albeit with a
differential effect on sg RNA levels of upstream genes (FIG. 5B
& FIG. 6B)
[0078] Concentrated virions from Construct CTV33-23-GUS-60 were
used to inoculate C. macropyhlla plants, which became infected as
confirmed by ELISA (Data not presented) and activity of the GUS
gene (FIG. 6C). Furthermore, GUS activity and western blot analysis
revealed the presence of the GUS gene in citrus 1.3 years after
inoculation (FIG. 6C, FIG. 19).
Example 3
Production of an Extra Polypeptide without Producing an Extra
Subgenomic mRNA
Internal Ribosome Entry Site Strategy (IRES)
The Tobacco Etch Virus (TEV) IRES
[0079] The 5'NTR of TEV mediates cap independent translation of the
viral mRNA. Studies on the 5'NTR of TEV demonstrate its ability to
initiate translation at an internal ORF in a bi-cistronic mRNA
(Gallie, 2001; Niepel and Gallie, 1999). The 5'NTR of TEV (nts
2-144 Genbank accession # DQ986288) was inserted into a CTV
mini-replicon behind the p23 ORF (between nts 19020-19021) followed
by the GFP ORF (CTVp333R-23-ITEV-GFP) (FIG. 7A) to examine whether
a bicistronic subgenomic mRNA would work with this virus. Although
northern blot hybridization analysis demonstrated that the
mini-replicon replicated and produced abundant amounts of the
bicistronic mRNA in transfected N. benthamiana protoplasts (FIG.
7C), GFP fluorescence was not observed, suggesting a lack of
translation of the second ORF in the bicistronic mRNA. The
inventors also examined the 5'NTR TEV IRES construct in full length
CTV in N. benthamiana protoplasts and plants. Construct
CTV33-23-ITEV-GFP-41 was passaged efficiently from protoplast to
the next protoplast sets (FIG. 7B), indicating the good replication
and formation of virions, but no fluorescing protoplasts were
observed demonstrating that this IRES did not work well in CTV
(data not presented). This construct infected and moved
systemically in N. benthamiana plants based on the systemic
symptoms of vein clearing followed by necrosis and ELISA (Data not
presented), but no GFP fluorescence was observed under UV light
(Data not presented).
Active ribosome complementary sequence (ARC) IRES
[0080] Insertion of an IRES consensus sequence obtained from
analysis of host and viral mRNAs (the engineered 3xARC-1 (86 nts)
IRES (Akbergenov et al., 2004)) was next examined for activity in
CTV. This IRES was fused behind the p23 ORF (nts 19020-19021) in
both the CTV mini-replicon (CTVp333R-23-I3XARC-GFP) and
.DELTA.p33CTV9R (CTV33-23-I3XARC-GFP-43) as described above (FIG. 7
A). However, after infection of protoplasts and plants, no GFP
fluorescence was observed even though the virus replicated well in
both (FIGS. 7B&C).
Poly-Peptide Fusion
[0081] P23, the highest expressed gene of CTV, is a multifunctional
protein that is essential for citrus infection. P23 is a silencing
suppressor and controls plus to minus RNA ratio in infected cells
via an RNA binding domain constituted of positive charged amino
acid residues and Zn finger domain present between amino acid 50-86
(Lopez et al., 2000; Satyanarayana et al., 2002b; Lu et al., 2004).
In order to create a gene fusion the HC-Pro or NIa protease motifs
of TEV were selected to be fused at the C-terminus of p23 (between
nts 19017 and 19018) (FIG. 8). The protease recognition sequence of
the HC-Pro and NIa was duplicated between p23 and the protease and
between the protease and GFP creating vectors CTV33-23-HC-GFP-72
and CTV33-23-NIa-GFP-73, respectively (FIG. 8). The processing of
the protease motif from p23 should release the p23 with 7 extra
amino acids at its C-terminus in the case of HC-Pro and 6 amino
acids in the case of NIa. The GFP protein should have two extra and
one extra amino acid after being cleaved from HC-Pro and NIa,
respectively. The recognition sequences were switched between
HC-Pro and NIa creating vectors CTV33-23-HCO-GFP-74 and
CTV33-23-NIaO-GFP-75 as controls that are unable to be cleaved
(FIG. 8). All the polypeptide fusion vectors were created in CTV
binary vectors for infection of plants because in protoplast it was
shown that p23 fusion did not affect the ability to replicate and
pass between protoplast sets (Tatineni and Dawson, unpublished
result). In N. benthamiana infiltrated leaves, all constructs
fluoresced similarly to each other and to the free GFP constructs
behind p23 (FIG. 9A). Furthermore, western immune-blot analysis
from infiltrated leaves indicated a near-perfect processing of the
reporter gene from the polypeptide fusion (FIG. 10). The GFP
protein did not localize to the nucleus unlike the fusion to p23
without a protease processing releasing the reporter gene. Upon
agro-inoculation of plants, only constructs with the protease and
its homologous processing sites were able to move systemically into
upper non-inoculated leaves. The fluorescence in upper
non-inoculated leaves was weaker than those for the expression
vectors CTV33-23-BY-GFP-37, CTV33-23-G-GFP-40 and CTV33-23-B-GFP-42
carrying GFP under its own controller element (FIG. 9B).
Furthermore, it was easier to visualize fluorescence on the abaxial
rather than the adaxial leaf surface (FIG. 9C). Upon inoculation of
citrus with construct CTV33-23-HC-GFP-72, one plant became positive
with relatively low ELISA value compared to others (Data not
presented). The reporter gene activity was not detected.
Example 4
Production of More than One Extra Foreign Protein from CTV
Vectors
[0082] Use of Single Controller Elements to Express Multiple
Proteins
[0083] In order to exploit the polypeptide strategy to express
multiple genes driven by the same controller element in a CTV based
vector, a fusion polypeptide was created consisting of GFP/Protease
(Pro)/GUS. Two different protease motifs were used in the different
constructs, HC-Pro and NIa, with their proteolytic motifs and
recognition sequences separating GFP ORF from the GUS ORF (FIGS.
14A & 3-16) (Carrington and Dougherty, 1988; Carrington et al.,
1989). Theoretically, in case the NIa was the protease motif in the
fusion, six extra amino acids are coupled with the N-terminal
protein (GFP) at its C-terminus whereas only one extra amino acid
is added to the N-terminus of GUS. Similarly, where HC-Pro was the
protease within the fusion poly-peptide, 7 extra amino acids are
added to the C-terminus of GFP and two extra amino acids added to
the N-terminus of GUS. The fusion genes ranged in size between 3127
and 3480 nts.
[0084] Replacement of p13 Gene
[0085] The two fusions of GFP/Pro/GUS described above were
engineered into the p13 site of CTV in the agro-inoculation binary
vector under the control of the BYSV CP-CE
(CTV33-.DELTA.13-BYGFP-HC-GUS-77 with HC-Pro protease motif and
CTV33-.DELTA.13-BYGFP-NIa-GUS-78 with NIa protease motif) (FIG.
11A). The constructs were agro-inoculated to N. benthamiana for
monitoring the ability to systemically infect the plant and produce
GUS and GFP. Both genes were produced based on their assays (FIG.
11B). Western immune-blot analysis indicated the efficient
processing of the GFP protein from the polypeptide fusion (FIG.
10). The virus multiplied and spread to high titers in N.
benthamiana plants as indicated by symptom development in the upper
leaves (FIG. 11B) and ELISA. However, the level of GFP fluorescence
was less than that of vectors CTV33-.DELTA.13-BY-GFP-57,
CTV33-.DELTA.13-G-GFP-65 and CTV33-.DELTA.13-B-GFP-66 expressing
the GFP alone and spread more slowly into the upper non-inoculated
leaves than those vectors (Data not presented). In N. benthamiana
plants, overlapping fluorescence and enzymatic activity of GUS were
demonstrated 7 months after the injection of the construct
revealing their stability (FIG. 12).
[0086] Insertion Between p23 and 3'NTR
[0087] In an attempt to improve the expression level of GFP and
GUS, the fusion polypeptide was moved closer to the 3'NTR. The
fusion gene with either BYSV, GLRaV-2 or BYV CP-CE with the
protease of HC-Pro was inserted between p23 and 3'NTR referred to
as CTV33-23-BY-GFP-HC-GUS-51, CTV33-23-G-GFP-HC-GUS-53 and
CTV33-23-BY-GFP-HC-GUS-55 whereas with the NIa protease constructs
were named, CTV33-23-BY-GFP-NIa-GUS-52, CTV33-23-G-GFP-NIa-GUS-54
and CTV33-23-BY-GFP-NIa-GUS-56, respectively (FIG. 13). After N.
benthamiana plants were agro-inoculated, all the constructs
multiplied and spread into the upper non-inoculated leaves as
indicated by GFP fluorescence (FIG. 14A) and GUS activity (FIG.
14A). Similar to constructs CTV33-.DELTA.13-BYGFP-HC-GUS-77 and
CTV33-.DELTA.13-BYGFP-NIa-GUS-78, fluorescence overlapping with GUS
enzymatic activity was demonstrated 7 months after injection
indicating the stability of the fusion. However, C. macrophylla
plants infected with construct CTV33-23-BY-GFP-HC-GUS-51 revealed
only faint fluorescence and almost no GUS activity (FIG. 14B) and
high ELISA values.
Example 5
Use of Multiple Promoters to Express Foreign Genes
Simultaneously
[0088] Bimolecular Fluorescence Complementation (BiFC) in CTV.
[0089] For examination of the insertion of two CP-CE controlling
different ORFs, the BiFC system, which produces visible
fluorescence only when the two proteins accumulate in the same
cell, was used. This system was developed using the bJun fused to
N-terminus of EYFP (A.A. 1-154) (referred to as bJunN) and bFos ORF
fused to C-terminus of EYFP (A.A. 155-238) (referred to as bFosC)
(Hu et al., 2002).
[0090] Both proteins are transported to the nucleus where they
directly interact enabling the EYFP protein to regain its wild type
folding pattern and results in emission of fluorescence upon
activation by a blue light source (Excitation wave length is 525 nm
and emission wavelength is 575 nm) (Hu et al., 2002). One or both
components of BiFC were introduced into the CTV mini-replicon 3' of
the p23 ORF (between nts #19020 and 19021 Genbank Accession #
AY170468) referred to as CTVp333R-23-BYbJunN, CTVp333R-23-GbFosC
and CTVp333R-23-BYbJunN-GbFosC (FIG. 15 A). Northern blot
hybridization analysis demonstrates the successful transfection of
all three constructs into N. benthamiana protoplast (FIG. 15B). The
two transcription factors interacted in the plant cell as
demonstrated by nuclear fluorescence observed only in protoplasts
infected with CTVp333R-23-BYbJunN-GBFosC (FIG. 15C). It is worth
noting that the size of the two inserted genes is approximately
identical to that of the GUS ORF.
[0091] As a control for the BiFC experiments, the inventors also
introduced the genes individually into .DELTA.p33CTV9R behind p23
creating vectors CTV33-23-BYbJunN-97 and CTV33-23-GbFosC-98 so that
only one component would be produced (FIG. 16B). Neither construct
exhibited fluorescence in the nucleus.
[0092] Expression of Multiple Foreign Genes Simultaneously at the
Same Location
[0093] P13 Replacement.
[0094] Both genes were introduced into a .DELTA.p33CTV9R
(Satyanarayana et al., 1999, 2000, 2003; Tatineni et al., 2008) as
a replacement of the p13 gene (replacement of the nucleotides
deleted between 17292 and 17581), resulting in
CTV33-.DELTA.13-BYbJunN-GbFosC-76 (FIG. 16A). Transfection of
protoplasts with the RNA transcripts of
CTV33-.DELTA.13-BYbJunN-GbFosC-76 resulted in the nuclear
fluorescence of infected protoplasts (Data not presented).
Similarly, infiltrated leaves of N. benthamiana plants with full
length CTV33-.DELTA.13-BYbJunN-GbFosC-76 emitted nuclear
fluorescence (FIG. 16B). On the contrary, infiltrated leaves with
constructs CTV33-23-BYbJunN-97 and CTV33-23-GbFosC-98 did not show
any nuclear fluorescence (Data not presented). Monitoring stem
phloem and leaf veins of N. benthamiana plants infiltrated with
CTV33-.DELTA.13-BYbJunN-GbFosC-76 seven weeks after infiltration
revealed fluorescence of the vascular tissue indicating the ability
of this construct to systemically infect upper leaves of N.
benthamiana (FIG. 16B).
[0095] Insertion Between p23 and 3'NTR.
[0096] The next step was to examine expression of the two genes
when positioned closer to the 3' terminus. The two gene components
of the BiFC system were introduced into CTV.DELTA.p33 behind p23
(between nts #19020 and 19021), CTV33-23-BYbJunN-GbFosC-59 (FIG.
3-17A). Upon RNA transfection of construct
CTV33-23-BYbJunN-GbFosC-59, nuclear flourescence of infected
protoplast was observed under the fluorescent microscope. However,
it was difficult to pass the new construct from one protoplast
batch to another, similar to GUS and the GFP/Pro/GUS fusion genes
inserted at the same location. Upon agro-infiltration of N.
benthamiana plants with CTV33-23-BYbJun-GbFosC-59 in full length
CTV, fluorescence was observed in infiltrated areas. Systemic
symptoms similar to that expected for infection of N. benthamiana
by CTV was extremely delayed. However, monitoring upper
non-inoculated leaves and phloem tissue of the stem at seven weeks
after agro-infiltration of leaves revealed fluorescence of nuclei
of the vascular tissue, demonstrating systemic infection by the
vector (FIG. 17C). These results confirmed by ELISA, indicate that
the position between p23 and 3'NTR can accommodate two extra genes
without affecting the ability of CTV to systemically invade the
plants. Similar to both genes replacing p13 in construct
CTV33-.DELTA.13-BYbJunN-GbFosC-76 there was a delay in the time
frame of colonizing the upper vascular tissues by construct
CTV33-23-BYbJunN-GbFosC-59. Nuclear fluorescence of systemic stem
phloem tissue indicates that CTV33-A13-BYbJunN-GbFosC-76 infected
more cells than construct CTV33-23-BYbJunN-GbFosC-59 (FIG. 16B
&FIG. 17C). This difference in the number of cells infected
indicates the better ability of CTV33-.DELTA.13-BYbJunN-GbFosC-76
to move in N. benthamiana as compared to
CTV33-23-BYbJunN-GbFosC-59.
Example 6
Expression of Multiple Foreign Genes Simultaneously from Different
Locations
[0097] To express multiple foreign genes from two different
positions, the inventors elected to replace the p13 gene and insert
a second gene behind p23. CTV33-.DELTA.13-BYbJunN-23-GbFosC-67
(FIG. 17A) was created via replacement of the p13 gene with the
BYSV CP-CE driving the bJunN ORF and the GLRaV-2 CP-CE controlling
the bFosC ORF inserted between the p23 ORF and the 3'NTR.
CTV33-.DELTA.3-BYbJunN-23-GbFosC-67 was transfected into
protoplasts and Northern blot analysis revealed the replication of
the virus (FIG. 17B). However, accumulation of the p23 mRNA was
greatly reduced. CTV33-.DELTA.13-BYbJunN-23-GbFosC-67 was
agro-inoculated into N. benthamiana. The infiltration into the
leaves indicated nuclear fluorescence of infected cells (FIG. 17C)
which were much fewer in number compared to constructs
CTV33-.DELTA.13-BYbJunN-GbFosC-76 and CTV33-23-BYbJunN-GbFosC-59.
Isolation of virions from leaves and transfection of protoplast was
carried out resulting in nuclear fluorescence of infected
protoplast indicating the successful formation of biologically
active virions. However, systemic infection was not achieved in N.
benthamiana as indicated by the lack of nuclear fluorescence in the
stem and upper non-inoculated leaves of N. benthamiana and
confirmed by ELISA.
[0098] In order to further study simultaneous multiple gene
expression from the different locations as above,
CTV33-.DELTA.13-BYGUS-23-GGFP-71 was engineered such that the GUS
ORF under the control of the BYSV CP-CE replaced the p13 gene(nts
17292-17582) and the GFP ORF under the control of the GLRaV-2 CP-CE
was inserted between the p23 and 3'NTR (nts 19020 and 19021) (FIG.
18A). RNA transcripts of CTV33-.DELTA.p13-BYGUS-23-GGFP-71 were
transfected into N. benthamiana protoplasts and northern blot
analysis indicated efficient replication of the construct in
protoplasts (FIG. 18B). Leaf infiltration of N. benthamiana plants
with construct CTV33-.DELTA.p13-BYGUS-23-GGFP-71 resulted in
replication of the virus as indicated by visible fluorescence under
a UV light and by GUS activity (Data not presented). The
agro-inoculated plants began to exhibit GUS activity and
fluorescence in the upper non-inoculated leaves 6 weeks after
infiltration (FIG. 3-18C). The systemic infection of upper leaves
was slightly slower than constructs with only GFP alone. Also, the
phenotype of vein clearing followed by necrosis associated with CTV
infection of N. benthamiana vascular tissue occurred later than
that of single gene vectors. The level of fluorescence when
observed UV light appeared to be slightly less than that of the
single gene constructs. However, the GFP fluorescence was more in
plants infected with construct CTV33-.DELTA.p13BYGUS-23GGFP-71,
which was controlled by its own CE, compared to that of the fusion
in constructs (CTV33-23-BY-GFP-HC-GUS-51,
CTV33-23-BY-GFP-NIa-GUS-52, CTV33-23-G-GFP-HC-GUS-53,
CTV33-23-G-GFP-NIa-GUS-54, CTV33-.DELTA.13-BYGFP-HC-GUS-77 and
CTV33-.DELTA.13-BYGFP-NIa-GUS-78). The activity of both genes
continued until the death of the N. benthamiana plants. Similarly,
in citrus the expression of both genes were better than the same
genes in constructs CTV33-.DELTA.13-BYGFP-NIa-GUS-78 and
CTV33-23-BY-GFP-HC-GUS-51.
Example 7
Level of Foreign Gene Expression of the Different Constructs in
Citrus
[0099] It is difficult to directly compare foreign gene expression
from the different vectors in citrus due to the differences in the
times of infection, the ages of the tissue and the effects of the
inserted foreign gene cassette on the replication of the virus.
Yet, protein presence in citrus is the best measure of expression
level. Thus, western blot analysis was used to compare the relative
level of expression of the different GFP and GUS constructs in
citrus to that of CP protein, a house keeping gene to determine the
replication levels. Western blots using the GFP antibodies and the
CP antibody revealed a trend which confirms the relative higher
expression levels near the 3' end of the genome and a lower
expression level when the inserted gene is moved further away from
the 3' end with the exception for the insertion between p13 and p20
(FIG. 19A). In contrary, the GUS expression in citrus revealed a
higher relative expression level as replacement of p13 rather than
insertion behind p23 (FIG. 19B).
Example 8
Multiple Gene Vectors
Plasmid Construction:
[0100] Three and four gene vectors were developed by introducing
different combination of gene cassettes into the CTV genome at
different locations. Three of the vectors were developed in
CTV9R.DELTA.p33 in the pCAMBIA 1380 background
(CTV33-BGFP-BYGUS-GTMVCP-79, CTV33-BGFP-GbFosC-BYbJunN-81 and
CTV33-.DELTA.13-BGFP-BYbJunN-GbFosC-82). The other three gene
vectors (CTV-BASL-BYPTA-CP7-119, CTV-BASL-BYP10-CP7-131,
CTV-BASL-BYPTA-CP10-120 and CTV-BRFP-BYGFP-CTMVCP-117) and one four
gene vector (CTV.DELTA.13-BRFP-GbFosC-BYbJunN-CTMVCP-118) were
developed by modifying CTV9R in the background of pCAMBIA1380
altered by replacing the hygromycin ORF with the p22 ORF of Tomato
chlorosis virus. For the ease of cloning the PstI restriction site
in p33 ORF in full length CTV9R was eliminated by introducing a
silent mutation using overlap extension PCR using primers 1749 and
1750 in combination with primer C-1436 and C-253 followed by
digestion of both the overlap PCR product and CTV9R with XmaI and
PmeI. Most of the gene cassettes were introduced into their
locations by overlap extension PCR using the primers listed in
table 1. The only exception was the insertion of green fluorescent
protein cycle 3 in between the CPm and CP gene. Introducing the
GFPC3 gene cassette into that location was done by restriction
digestion of 9-47RGFP plasmid and point mutated CTV9R in
pCAMBIA1380 with PmeI and PstI.
Expression of Three and Four Foreign Genes Simultaneously
[0101] After successfully expressing two genes in N. benthamiana
and citrus with one and two different controller elements we are
building vectors to express three and four foreign genes from three
and four different controller elements, respectively. The reporter
genes used in different combinations were the green fluorescent
protein (cycle 3 GFP, GFPC3), red fluorescent protein (tag red
fluorescent protein, RFP), Bimolecular fluorescence complementation
using the bFos and bJun mammalian transcription factors (Hu et al.,
2002), .beta.-glucuronidase (GUS) gene from Escherichia coli and
the Tobacco mosaic virus (TMV) coat protein gene (CP). Similarly,
three gene vectors were built in different combinations to express
two antimicrobial peptides (AMPs) from Tachypleus tridentatus and
Sus scorfa, Allium sativum lectin (ASL) and Pinellia ternata
agglutinin (PTA). The three gene vectors were either expressed from
two or three locations within the CTV genome
Expression of Three Foreign Genes from Three Different Locations
Simultaneously:
[0102] Six vectors were built to express three foreign genes from
three different locations. The vectors were built to express the
genes either from CTV9R.DELTA.p33 or full length CTV9R.
[0103] Vectors Built to Express Three Genes from Three Different
Locations in CTV9R.DELTA.p33
[0104] Two vectors were built by inserting the three extra gene
cassettes into CTV9R.DELTA.p33 creating expression vectors
CTV33-BGFP-BYGUS-GTMVCP-79 (FIG. 26) and
CTV33-.DELTA.13-BGFP-BYbJunN-GbFosC-82 (FIG. 28).
CTV33-BGFP-BYGUS-GTMVCP-79 expresses the three ORFs of GFP
(insertion between CPm and CP), GUS (insertion between p13 and p20)
and the coat protein of TMV (insertion between p23 and 3'UTR) under
the CP-CE of BYV, BYSV and GLRaV-2, respectively.
CTV33-.DELTA.13-BGFP-BYbJunN-GbFosC-82 expresses the three ORFs of
GFP (insertion between CPm and CP), bJunN ORF (replacement of p13)
and bFosC (insertion between p23 and 3'UTR) under the CP-CE of BYV,
BYSV and GLRaV-2, respectively. The two vectors were infiltrated
into N. benthamiana leaves in combination with silencing
suppressors and inoculated into citrus using the procedure of Gowda
et al., 2005. As leaves were cut and grinded to isolate virions
over 70% sucrose cushion gradient just 5 days after infiltration
into the N. benthamiana leaves it was not likely that these plants
will get systemically infected, thus they were discarded. The
fluorescence of infiltrated leaves under hand held UV indicated the
expression of the GFP protein in both CTV33-BGFP-BYGUS-GTMVCP-79
and CTV33-.DELTA.13-BGFP-BYbJunN-GbFosC-82 indicating the ability
of the created vector to replicate in the N. benthamiana leaves.
Electron microscope grids prepared from leaf dips of infiltrated N.
benthamiana leaves for construct CTV33-BGFP-BYGUS-GTMVCP-79 and
CTV33-.DELTA.13-BGFP-BYbJunN-GbFosC-82 indicated the formation of
virions a prerequisite for the successful mechanical inoculation of
citrus seedlings with CTV. Furthermore, in the case of
CTV33-BGFP-BYGUS-GTMVCP-79 and not
CTV33-.DELTA.13-BGFP-BYbJunN-GbFosC-82 there was the formation of
rod-shaped structures referred to as TMV pseudo-virions a
characteristic of the expression of the TMV coat protein.
[0105] Vectors Built to Express Three Genes from Three Different
Locations in CTV9R
[0106] Four vectors were built to express three foreign genes from
the same three different locations within the CTV genome. The three
locations selected were insertion between CPm and CP, p13 and p20
and p23 and 3'UTR. For the ease of cloning into the full length CTV
infectious clone a the PstI site within the p33 ORF was eliminated
by introducing a silent point mutation by overlap extension PCR.
Three of the four vectors were created by using different
combinations of the two AMPs, ASL and PTA resulting in expression
vectors CTV-BASL-BYPTA-CP7-119, CTV-BASL-BYP10-CP7-131 and
CTV-BASL-BYPTA-CP10-120. The fourth vector named
CTV-BRFP-BYGFP-CTMVCP-117 was created by inserting the ORFs of GFP,
RFP and TMV CP under the control of BYV, BYSV and duplicated CP-CE
of CTV. All the vectors were infiltrated into N. benthamiana to
monitor the development of systemic infection.
CTV-BASL-BYPTA-CP7-119 developed efficient systemic infection in 1
N. benthamiana plant. Plants infiltrated with vector
CTV-BRFP-BYGFP-CTMVCP-117 revealed fluorescence in systemic leaves
under hand held UV. Upon development of pronounced systemic
infection, virions from CTV-BRFP-BYGFP-CTMVCP-117 will be
concentrated over a sucrose step gradient and a sucrose cushion in
order to inoculate citrus plants similar to the procedure recently
followed for vector CTV-BASL-BYPTA-CP7-119
Expression of Three Foreign Genes from Two Different Locations
Simultaneously:
[0107] Two vectors were created for the simultaneous expression of
three genes from two different locations within the CTV genome. One
vector was built in CTV9R.DELTA.p33 creating expression vector
CTV33-BGFP-GbFosC-BYbJunN-81 whereas the other vector was built in
full length CTV9R named CTV.DELTA.13-GbFosC-BYbJunN-CTMVCP-129.
[0108] Vector Built to Express Three genes from Two different
locations in CTV9R.DELTA.p33:
[0109] CTV33-BGFP-GbFosC-BYbJunN-81 (FIG. 27) was engineered
through modifying CTV9R.DELTA.p33 by inserting a single gene
cassette between CPm and CP (GFP ORF under the control of BYV
CP-CE) and a double gene cassette (bFosC ORF followed by bJunN ORF
under the control of GLRaV-2 and BYSV CP-CE, respectively) as an
insertion between p23 and 3'UTR. A 1:1 mixture of 4 different
silencing suppressors and CTV33-BGFP-GbFosC-BYbJunN-81 were
infiltrated into N. benthamiana leaves. Electron microscopy from
grids of leaf dips revealed the formation of virions similar to
constructs CTV33-BGFP-BYGUS-GTMVCP-79 and
CTV33-.DELTA.13-BGFP-BYbJunN-GbFosC-82. In addition, the
infiltrated leaves revealed strong fluorescence under hand held UV
light. Infiltrated leaves were used to concentrate virions on a 70%
sucrose cushion in an attempt to infect citrus seedlings.
[0110] Vector Built to Express Three genes from Two different
locations in CTV9R:
[0111] CTV9R was modified by inserting a double gene cassette
(bFosC ORF followed by bJunN ORF under the control of GLRaV-2 and
BYSV CP-CE, respectively) as replacement of p13 and a gene cassette
(TMV CP ORF under the control of the duplicated CP-CE) as an
insertion between p23 and 3'UTR creating expression vector
CTV.DELTA.13-GbFosC-BYbJunN-CTMVCP-129 (FIG. 21). This vector is
recently infiltrated into N. benthamiana leaves. After systemic
infection of N. benthamiana the virions will be concentrated to
enable the inoculation of citrus plants.
Expression of Four Foreign Genes from Three Different Locations
Simultaneously:
[0112] In order to build the four gene vector we used four gene
cassettes located at three different locations within the CTV
genome. The RFP ORF was introduced between CPm and CP under the
control of the BYV CP-CE, the two BiFC components bFosC and bJunN
under the control of GLRaV-2 and BYSV respectively were introduced
as a replacement of the p13 gene and the TMV ORF under the control
of the duplicated CP-CE of CTV was introduced behind p23. The four
gene vector named CTV.DELTA.13-BRFP-GbFosC-BYbJunN-CTMVCP-118 was
infiltrated into the N. benthamiana leaves for the development of
systemic infection. Upon systemic infection virion concentration
will be carried out over a sucrose step gradient and cushion for
the infection of the citrus trees.
Discussion Related to Examples 1-8
[0113] In this work, CTV constructs that are extraordinarily
permissive in allowing insertion of foreign sequences at different
places in the 3' portion of the genome are disclosed. Numerous
different potential vector constructs to express foreign genes via
additional subgenomic RNAs, di-cistronic mRNAs, or protease
processing of fusion proteins were created and examined.
Remarkably, most of these constructs functioned as vectors.
Additionally, that the CTV constructs disclosed herein are capable
of simultaneously producing large amounts of multiple foreign
proteins or peptides.
[0114] The ultimate goal was to develop high expressing and stable
vectors for the natural CTV host, citrus. Thus, virions were
concentrated from N. benthamiana plants infected with 12 different
constructs that spread and expressed moderate to high levels of the
foreign protein(s) and used to inoculate citrus. C macrophylla
plants became positive for infection between 6-60 weeks after
inoculation depending on the insert length in the virus and the
amount of virions concentrated from the N. benthamiana leaves that
were used for inoculation. Most of the constructs that infected
citrus produced moderate levels of the reporter gene/s.
[0115] Several approaches were examined for expression of foreign
genes from CTV. The first approach was the "add-a-gene" strategy
that involved the addition or duplication of a controller element
and an additional ORF, which resulted in an additional subgenomic
RNA. The "add-a-gene" approach was developed initially in TMV via
duplicating the CP subgenomic promoter controlling a foreign gene
(Dawson et al., 1989; Donson et al., 1991; Shivprasad et al.,
1999). An advantage of this strategy is that it expresses the exact
protein with no additional amino acids added to the N or/and C
terminus which could affect its biological activity, at relatively
high levels. However, there are limitations of this strategy that
should be considered. Duplication of the controller element can
lead to homologous recombination resulting in the loss of the gene
of interest (Chapman et al., 1992; Dawson et al., 1989). Although
this made the TMV insert unstable, it appeared to have little
effect on the stability in CTV (Folimonov et al., 2007). The use of
a heterologous controller element from related viruses stabilized
the TMV insertions. However, heterologous controller elements
usually are differentially recognized by the replicase complex of
the virus (Folimonov et al., 2007; Shivprasad et al., 1999). This
observation can be utilized to regulate the levels of desired gene
expression (Shivprasad et al., 1999). An important consideration is
that there can be competition between the different subgenomic RNAs
of a virus. With TMV, the extra gene competed with the coat protein
gene and the movement gene. There appeared to be a maximal capacity
for production of subgenomic RNAs that was divided among the three
RNAs. Manipulations that resulted in increases in one resulted in
decreases in the others. One solution was to reduce coat protein
production to allow optimal foreign gene and movement gene
expression (Shivprasad et al., 1999; Girdishivelli et al., 2000).
Yet, CTV subgenomic mRNAs appeared to be much less competitive
(Folimonov et al., 2007; Ayllon et al., 2003).
[0116] In previous work, a CTV vector was created that expressed an
extra gene between the CP and CPm genes that was an effective and
stable vector in citrus trees. The foreign gene was in position 6
from the 3' terminus (Folimonov et al., 2007). The position of the
extra gene was chosen arbitrarily. Here the inventors continued
vector design in an attempt to define the limits of manipulation of
the CTV genome in producing extra proteins or peptides. The virus
expresses its ten 3' genes via sg mRNAs (Hilf et al., 1995). One
rule of CTV gene expression is that genes nearer the 3' terminus
are transcribed higher than internal genes. For example,
transcription of the p33 gene, which is at position 10 from the 3'
terminus, is very low in its native position, but transcription
became very high when the p33 gene was moved near the 3' terminus
(Satyanarayana et al., 1999). Thus, expression of foreign genes
from positions nearer the 3' terminus might result in higher levels
than from the position 6 arbitrarily chosen in the first vector
(Folimonov et al., 2007). Yet, based on results from other viruses,
only certain positions within the viral genome are likely to
tolerate extra gene insertions. For example, with TMV or Alfalfa
mosaic virus the location between CP and 3'NTR did not accommodate
an insert (Dawson et al., 1989; Lehto and Dawson, 1990;
Sanchez-Navarro et al., 2001). Remarkably, almost all of the
constructs with insertions in CTV within the p13 deletion, between
p13 and p20, and between p23 and the 3' NTR were viable. In
contrast, it was found that the only position the virus did not
tolerate insertions was between the p20 and p23 genes. It is
possible that these insertions interfered with the transcription of
either of the adjacent genes.
[0117] Another strategy to express foreign genes in a viral vector
consists of in-frame fusion of an ORF of interest to a viral ORF at
either the N or C terminus. The two proteins can be released by
engineering a protease and processing sites between the two
proteins (Dolja et al., 1997; Gopinath et al., 2000). It was first
adapted in the potyviridae, tobacco etch virus (Dolja et al.,
1992). The major advantage of polyprotein fusion strategy is that
the foreign protein is expressed in 1:1 ratio with the viral
protein. A major limitation is that this process adds extra amino
acids at the N and/or C termini of both proteins, which may affect
their biological activities.
[0118] A series of constructs utilizing the HC-Pro or NIa proteases
from potyviruses to enable post translational processing of the
engineered polyprotein to release free GFP, protease, and the p23
protein were created. These vectors were able to systemically
infect N. benthamiana. The systemic movement of these constructs
was slower than the expression vector constructs containing only
the GFP ORF as an extra gene. The slower systemic movement and the
lower levels of GFP expression in the systemic leaves partially
could be attributed to the extra C-terminal amino acids of p23
reduced its activity in RNA silencing suppression or amplification
of viral RNAs or the protease processing delayed its activity.
Although these constructs did not produce the maximal levels of
foreign protein, they were viable vectors expressing substantial
amounts of GFP.
[0119] Upon identifying the locations within the CTV genome that
could accommodate foreign gene inserts, strategies were designed to
construct viral vectors that express multiple genes. The first
strategy depended on the use of a single controller element driving
the transcription of a polypeptide gene. The fusion gene that
consisted of GFP/Pro/GUS, ranged in size from 3127 nts to 3480 nts.
Other strategies utilized two extra CEs to produce two extra sg
RNAs simultaneously. This strategy gave the flexibility to insert
the two genes in tandem in the same location or in two different
locations. Both strategies worked.
[0120] Heterologous protein expression in whole plant is usually
accomplished by development of transgenic plants by insertion of
foreign DNA into the plastid or nuclear genome. Plastid
transformation has been successful for only a few annual crops.
Time and success of nuclear transformation varies among the
different crops. Certain plants are more recalcitrant to
transformation and subsequent regeneration than others. There are
other disadvantages, particularly in perennial crops. For example,
citrus has a long juvenile stage after regeneration that prolongs
the time necessary to evaluate the horticultural characteristics
and delays the time to commercial use. Another major disadvantage
is that transformation is limited to the next generation of
plants.
[0121] The inventors have now developed a series of different CTV
vectors, each with different characteristics that are more
effective under specific conditions. For example, with the
"add-a-gene" vectors, the inventors would advocate the expression
of a small gene in 3' of the p23 gene in CTV for maximal
expression. A medium gene could be more efficiently expressed from
within the p13 area. A large gene probably would be better
accommodated as an insertion between CP and CPm where it would
disrupt the viral subgenomic RNAs less and result in better
systemic invasion of the plant. For expression of smaller proteins,
peptides, or RNAs to target RNA silencing, it is possible that the
virus could accommodate 3 or 4 different genes. Different
combinations of extra sg RNAs and protease processing can be
chosen. Although two foreign proteins have been produced from other
viruses, CTV is unique in usefulness because of its stability. The
original vector has been continuously producing GFP for 8
years.
[0122] The uses of the CTV based expression vector have evolved
since its inception. It was initially developed as a laboratory
tool for citrus improvement. The vector was designed to express
potential genes for transformation of citrus. Results of the effect
of the heterologous gene in citrus, particularly if the effect was
expected in mature tissue or fruit, could be obtained by the virus
years before results would come from direct transformation.
However, conditions and needs of the citrus industry have changed
due to the invasion of a new bacterial disease referred to as
Huanglongbing (HLB). This disease has spread so rapidly and is so
damaging that the survival of the citrus industry is threatened.
Initially, the CTV vector was used to identify antimicrobial
peptides with activity against the HLB bacterium for transformation
into citrus. However, the disease is spreading so rapidly that
transgenic plants may not be available in time to save the
industry. Due to the remarkable stability, the CTV vector now is
being considered for use in the field to protect citrus trees and
to treat infected trees until resistant transgenic plants become
available. The CTV vector as a tool in the field to fight an
invading disease of citrus is only one example of what viral
vectors can do for agriculture. The possibilities are many for very
stable vectors like those of CTV and perennial crops, particularly
trees. Many trees are productive for 100 years or more. During the
lifespan of the trees technologies changes and disease and pest
pressures change. To improve trees by traditional transformation
methods requires removing all of the present trees from the field
and replanting. The use of a viral vector could add new genes to
the existing trees.
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[0230] While various disclosed embodiments have been described
above, it should be understood that they have been presented by way
of example only, and not limitation. Numerous changes to the
subject matter disclosed herein can be made in accordance with this
Disclosure without departing from the spirit or scope of this
Disclosure. In addition, while a particular feature may have been
disclosed with respect to only one of several implementations, such
feature may be combined with one or more other features of the
other implementations as may be desired and advantageous for any
given or particular application.
[0231] Thus, the breadth and scope of the subject matter provided
in this Disclosure should not be limited by any of the above
explicitly described embodiments. Rather, the scope of this
Disclosure should be defined in accordance with the following
claims and their equivalents.
[0232] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting. As
used herein, the singular forms "a," "an," and "the" are intended
to include the plural forms as well, unless the context clearly
indicates otherwise. Furthermore, to the extent that the terms
"including," "includes," "having," "has," "with," or variants
thereof are used in either the detailed description and/or the
claims, such terms are intended to be inclusive in a manner similar
to the term "comprising."
[0233] Unless otherwise defined, all terms (including technical and
scientific terms) used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which embodiments
belong. It will be further understood that terms, such as those
defined in commonly used dictionaries, should be interpreted as
having a meaning that is consistent with their meaning in the
context of the relevant art and will not be interpreted in an
idealized or overly formal sense unless expressly so defined
herein.
[0234] The teachings of any patents, patent applications, technical
or scientific articles or other references are incorporated herein
in their entirety to the extent not inconsistent with the teachings
herein.
Sequence CWU 1
1
78127DNATobacco etch virus 1atgaaaactt acaatgttgg agggatg
2729PRTTobacco etch virus 2Met Lys Thr Tyr Asn Val Gly Gly Met 1 5
327DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 3atgaagacct ataacgtagg tggcatg
27421DNATobacco etch virus 4gagaatcttt attttcagag t 2157PRTTobacco
etch virus 5Glu Asn Leu Tyr Phe Gln Ser 1 5 621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6gaaaacctat acttccaatc g 21733DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
7agtcctcgag aaccacttag ttgtttagct atc 33842DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8ttatgcggcc gcaggccttg gacctatgtt ggccccccat ag 42942DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9taatcgtact tgagttctaa tatggctagc aaaggagaag aa
4210100DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10gccgcactag tatttaaatc ccgtttcgtc ctttagggac
tcgtcagtgt actgatataa 60gtacagactg gacctatgtt ggccccccat agggacagtg
1001144DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11atggatgagc tctacaaatg attgaagtgg acggaataag ttcc
441244DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 12ggaacttatt ccgtccactt caatcatttg tagagctcat ccat
441384DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13gcacgttgtg ctatagtacg tgccataata gtgagtgcta
gcaaagtata aacgctggtg 60tttagcgcat attaaatact aacg
8414101DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 14cagcttgctt ctacctgaca cagttaagaa gcggcataaa
tcgaagccaa accctaaatt 60ttgcaactcg atcaattgta acctagagcg aagtgcaatc
a 1011545DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15tttagcgcat attaaatact aacgatggct agcaaaggag
aagaa 451645DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 16actgtgtcag gtagaagcaa gctgtcagat
gaagtggtgt tcacg 451745DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 17ttggatttag gtgacactat
agtggaccta tgttggcccc ccata 451845DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 18gtaacctaga gcgaagtgca
atcaatggct agcaaaggag aagaa 451986DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 19gcctaagctt acaaatactc
ccccacaaca gcttacaata ctcccccaca cagcttacaa 60atactccccc acaacagctt
gtcgac 862045DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 20ctccgtgaac accacttcat ctgaaaataa
caaatctcaa cacaa 452145DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 21ttgtgttgag atttgttatt
ttcagatgaa gtggtgttca cggag 452245DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 22ggagtatttg taagcttagg
ctcagatgaa gtggtgttca cggag 452345DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 23ccccacaaca gcttgtcgac
atggctagca aaggagaaga acttt 452445DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 24cgtgaacacc acttcatctg
attcgacctc ggtcgtctta gttaa 452545DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 25ttaactaaga cgaccgaggt
cgaatcagat gaagtggtgt tcacg 4526137DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26ggcgatcacg acagagccgt gtcaattgtc gcggctaaga atgctgtgga tcgcagcgct
60ttcactggag gggagagaaa aatagttagt ttgtatgcct taggaaggaa ctaagcacgt
120tgtgctatag tacgtgc 1372745DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 27tgacacggct ctgtcgtgat
cgcctcagat gaagtggtgt tcacg 452845DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 28gccacctacg ttataggtct
tcattttgta gagctcatcc atgcc 452945DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 29aagacctata acgtaggtgg
catgaaggct caatattcgg atcta 453045DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 30atgaaaactt acaatgttgg
agggatgtta cgtcctgtag aaacc 453145DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 31ggtttctaca ggacgtaaca
tccctccaac attgtaagtt ttcat 453245DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 32ccgcagcagg gaggcaaaca
atgattgaag tggacggaat aagtt 453345DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 33aacttattcc gtccacttca
atcattgttt gcctccctgc tgcgg 453445DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 34cttactctga aaataaagat
tctctttgta gagctcatcc atgcc 453545DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 35aaagagaatc tttattttca
gagtaaggga ccacgtgatt acaac 453645DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 36cgattggaag tataggtttt
cttgcgagta caccaattca ctcat 453745DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 37caagaaaacc tatacttcca
atcgatgtta cgtcctgtag aaacc 453845DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 38gtcactttgt ttagcgtgac
ttagcagctt gcttctacct gacac 453945DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 39gtgtcaggta gaagcaagct
gctaagtcac gctaaacaaa gtgac 454045DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 40ttagtctctc catcttgcgt
gtagcagctt gcttctacct gacac 454145DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 41gtgtcaggta gaagcaagct
gctacacgca agatggagag actaa 454245DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 42atggatgagc tctacaaatg
agtttcagaa attgtcgaat cgcat 454345DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 43atgcgattcg acaatttctg
aaactcattt gtagagctca tccat 454445DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 44atggatgagc tctacaaatg
agttaatacg cttctcagaa cgtgt 454545DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 45acacgttctg agaagcgtat
taactcattt gtagagctca tccat 454645DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 46tttagcgcat attaaatact
aacgatgtac ccatacgatg ttcca 454745DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 47tggaacatcg tatgggtaca
tcgttagtat ttaatatgcg ctaaa 454845DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 48actgtgtcag gtagaagcaa
gctgttactt gtacagctcg tccat 454945DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 49gtaacctaga gcgaagtgca
atcaatggac tacaaagacg atgac 455045DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 50gtcactttgt ttagcgtgac
ttagggcgat cacgacagag ccgtg 455145DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 51cacggctctg tcgtgatcgc
cctaagtcac gctaaacaaa gtgac 455245DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 52gtcactttgt ttagcgtgac
ttagttcgac ctcggtcgtc ttagt 455345DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 53actaagacga ccgaggtcga
actaagtcac gctaaacaaa gtgac 455445DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 54cacaacgtct atatcatggc
ctaggtttca gaaattgtcg aatcg 455545DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 55cgattcgaca atttctgaaa
cctaggccat gatatagacg ttgtg 455645DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 56ggcatggacg agctgtacaa
gtaattgaag tggacggaat aagtt 455745DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 57aacttattcc gtccacttca
attacttgta cagctcgtcc atgcc 455845DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 58tcgctcttac cttgcgataa
ctagcagctt gcttctacct gacac 455945DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 59gtaacctaga gcgaagtgca
atcaatgtta cgtcctgtag aaacc 456047DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 60ggtttctaca ggacgtaaca
ttgattgcac ttcgctctag gttacaa 476145DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
61ccgcagcagg gaggcaaaca atgagtttca gaaattgtcg aatcg
456245DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 62cgattcgaca atttctgaaa ctcattgttt gcctccctgc
tgcgg 456345DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 63gtgtcaggta gaagcaagct gctagttatc
gcaaggtaag agcga 456445DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 64atggatgagc tctacaaatg
aagtctactc agtagtacgt ctatt 456545DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 65aatagacgta ctactgagta
gacttcattt gtagagctca tccat 456645DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 66gcggatgcat tatttggttt
tacaacaacg gtacgtttca aaatg 456745DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 67atgaaaactt acaatgttgg
agggatggct agcaaaggag aagaa 456845DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 68ttcttctcct ttgctagcca
tccctccaac attgtaagtt ttcat 456943DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 69gagaatcttt attttcagag
taagggacca cgtgattaca acc 437044DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 70gaaaacctat acttccaatc
gatggctagc aaaggagaag aact 447144DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 71agttcttctc ctttgctagc
catcgattgg aagtataggt tttc 447245DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 72aagacctata acgtaggtgg
catgaaggga ccacgtgatt acaac 457345DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 73ccctccaaca ttgtaagttt
tcatttgcga gtacaccaat tcact 457445DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 74gagaatcttt attttcagag
taaggctcaa tattcggatc taaag 457545DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 75cgattggaag tataggtttt
cttcggattc caaacctgaa tgaac 457645DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 76gccacctacg ttataggtct
tcatgatgaa gtggtgttca cggag 457745DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 77actctgaaaa taaagattct
cgatgaagtg gtgttcacgg agaac 457839DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 78catttacgaa cgatagccat
ggctagcaaa ggagaagaa 39
* * * * *