U.S. patent application number 11/759603 was filed with the patent office on 2008-02-21 for plant virus.
This patent application is currently assigned to De Ruiter Seeds R&D B.V.. Invention is credited to Annette Maria Dullemans, Paulus Cornelis Maris, Johannes Franciscus Johanna Maria van den HEUVEL, Rene Andries Antionius van Der Vlugt, Marinus Verbeek.
Application Number | 20080044427 11/759603 |
Document ID | / |
Family ID | 36061519 |
Filed Date | 2008-02-21 |
United States Patent
Application |
20080044427 |
Kind Code |
A1 |
van den HEUVEL; Johannes Franciscus
Johanna Maria ; et al. |
February 21, 2008 |
PLANT VIRUS
Abstract
The invention relates to the field of virology. The invention
provides an isolated plant virus (ToTV) named Tomato torrado virus
(ToTV), and components thereof. The invention further relates to
methods of producing a ToTV-resistant plant comprising the steps of
identifying a ToTV-resistant donor plant, crossing said
ToTV-resistant donor plant with a recipient plant, and selecting
from an offspring plant a resistant plant.
Inventors: |
van den HEUVEL; Johannes Franciscus
Johanna Maria; (Rotterdam, NL) ; Maris; Paulus
Cornelis; (Benthuizen, NL) ; Verbeek; Marinus;
(Andel, NL) ; Dullemans; Annette Maria;
(Wageningen, NL) ; van Der Vlugt; Rene Andries
Antionius; (Rhenen, NL) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
28 STATE STREET
28th FLOOR
BOSTON
MA
02109-9601
US
|
Assignee: |
De Ruiter Seeds R&D
B.V.
Bergschenhoek
NL
|
Family ID: |
36061519 |
Appl. No.: |
11/759603 |
Filed: |
June 7, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/NL06/00009 |
Jan 9, 2006 |
|
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11759603 |
Jun 7, 2007 |
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Current U.S.
Class: |
424/159.1 ;
435/183; 435/194; 435/235.1; 435/320.1; 435/5; 530/350; 530/387.1;
536/23.72; 800/265; 800/301 |
Current CPC
Class: |
C12N 2770/00022
20130101; C12N 15/8203 20130101; G01N 33/56983 20130101; C12N
2770/00021 20130101; C12N 7/00 20130101; C07K 14/005 20130101; A61P
31/12 20180101; C12N 15/8283 20130101; A61P 37/04 20180101; A01H
1/04 20130101 |
Class at
Publication: |
424/159.1 ;
435/183; 435/194; 435/235.1; 435/320.1; 435/005; 530/350;
530/387.1; 536/023.72; 800/265; 800/301 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A01H 1/02 20060101 A01H001/02; C07H 21/00 20060101
C07H021/00; C07K 16/08 20060101 C07K016/08; C12N 7/00 20060101
C12N007/00; C12N 9/12 20060101 C12N009/12; C12Q 1/70 20060101
C12Q001/70; C12N 9/00 20060101 C12N009/00; C12N 15/63 20060101
C12N015/63; C07K 14/00 20060101 C07K014/00; A01H 5/00 20060101
A01H005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 2005 |
EP |
05075725.1 |
Jan 7, 2005 |
EP |
05075042.1 |
Claims
1. A plant virus named Tomato torrado virus (ToTV), deposited with
the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on
24 Nov. 2004 under depositors reference number ToTV-E01 (DSM
16999).
2. A plant virus named Tomato Marchitez virus (TMarV), to be
deposited with a depository under the Budapest Treaty under
depositors reference number PRI-TMarV0601.
3. A virus comprising at least one nucleic acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:15, SEQ ID NO:16 and sequences having a nucleotide sequence
homology of at least 50% thereof.
4. The virus according to claim 3, wherein said virus is associated
with one or more tomato diseases selected from the group consisting
of "Torrado," "Marchitez" and "Chocolate spot disease," or wherein
said virus, based on numerical taxonomic analysis of taxonomic
descriptors essentially as defined in Table 1, shows to be more
closely related to the virus as defined in claim 1 than to any
other virus, and wherein said virus is associated with a disease
that causes necrotic lesions in tomato.
5. An isolated or recombinant nucleic acid or ToTV-specific
fragment thereof comprising a nucleic acid sequence selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ
ID NO:16, sequences having a nucleotide sequence homology of at
least 50% to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15 or SEQ ID NO:16
or their complementary strands and ToTV-specific fragments
thereof.
6. An expression vector comprising a nucleic acid according to
claim 5.
7. A polynucleotide capable of hybridizing under stringent
conditions to a nucleic acid according to claim 5.
8. An isolated or recombinant polypeptide obtainable from a virus
according to claim 1 or a ToTV-specific fragment thereof.
9. The polypeptide according to claim 8, wherein said polypeptide
is selected from the group consisting of 23 kDa capsid protein, 26
kDa capsid protein, 35 kDa capsid protein, helicase, RNA-dependent
RNA polymerase and putative movement protein (MP) of ToTV or
ToTV-specific fragments thereof.
10. An antigen comprising a polypeptide according to claim 8.
11. An antibody specifically directed against the antigen according
to claim 10.
12. A method for producing an antibody against ToTV comprising the
steps of: a) providing a ToTV virus, or a protein or peptide
fragment thereof; b) immunizing an appropriate vertebrate host with
said virus, said protein or said peptide fragment; and c)
harvesting from blood or splenocytes of said vertebrate host
antibodies against said virus, said protein or said peptide
fragment.
13. The method according to claim 12, further comprising the steps
of selecting one antibody-producing splenocyte, fusing said
splenocyte to an immortalized hybridoma cell line to create a
hybridoma fusion and allowing said hybridoma fusion to produce
monoclonal antibodies.
14. An antibody obtainable by a method according to claim 12.
15. A method for identifying a viral isolate as a ToTV virus
comprising reacting said viral isolate or a component thereof with
an antibody according to claim 11.
16. A method for identifying a viral isolate as a ToTV virus
comprising reacting a viral isolate or a component thereof with a
polynucleotide according to claim 7.
17. A method for detecting the presence of ToTV in a sample
comprising determining in said sample the presence of a ToTV virus
or component thereof by reacting said sample with a polynucleotide
according to claim 6.
18. A method for identifying a ToTV-resistant plant, comprising the
steps of: a) exposing a plant or plant part to an infective dosage
of ToTV; and b) identifying said plant as a ToTV-resistant plant
when, after said exposure, either disease-symptoms in said plant or
plant part remain absent or are delayed in expression or are at
least reduced in severity or are localized relative to a
susceptible control plant, or ToTV virus or ToTV genomic sequences
are not present in said plant or plant part or the presence of ToTV
virus is at least quantitatively reduced relative to a susceptible
control plant.
19. The method according to claim 18, wherein in step b) the
presence of ToTV in said plant or plant part is determined by
performing a method according to claim 17.
20. A method of producing a ToTV-resistant plant comprising the
steps of: a) identifying a ToTV-resistant donor plant by performing
a method according to claim 18; b) crossing said ToTV-resistant
donor plant with a recipient plant; and c) selecting from offspring
plants a resistant plant by performing a method according to claim
18.
21. The method according to claim 20, wherein said donor plant is a
plant of the family Solanaceae or Cucurbitaceae.
22. The method according to claim 20, wherein said donor plant is a
tomato plant, an eggplant plant, a pepper plant, a melon plant, a
watermelon plant or a cucumber plant.
23. The method according to claim 20, wherein said recipient plant
is a plant of the family Solanaceae or Cucurbitaceae.
24. The method according to claim 20, wherein said recipient plant
is a tomato plant, an eggplant plant, a pepper plant, a melon
plant, a watermelon plant or a cucumber plant.
25. The method according to claim 20, wherein said recipient plant
is a tomato plant of the species Solanum lycopersicum, more
preferably an S. lycopersicum line that possess commercially
desirable characteristics.
26. A ToTV-resistant plant, or part thereof, obtainable by a method
according to claim 20.
27. A diagnostic kit for performing the method according to claim
16, comprising at least one component selected from the group
consisting of a virus according to claim 1, a virus according to
claim 2, a virus according to claim 3, a virus according to claim
4, a polynucleotide according to claim 7, a polypeptide according
to claim 8, a polypeptide according to claim 9, an antigen
according to claim 10, an antibody according to claim 11, an
antibody according to claim 12, an antibody according to claim 13
and an antibody according to claim 14.
28. Use of at least one component selected from the group
consisting of a virus according to claim 1, a virus according to
claim 2, a virus according to claim 3, a virus according to claim
4, a polynucleotide according to claim 7, a polypeptide according
to claim 8, a polypeptide according to claim 9, an antigen
according to claim 10, an antibody according to claim 11 and an
antibody according to claim 14 for the production of a diagnostic
composition.
29. A diagnostic composition comprising at least one component
selected from the group consisting of a virus according to claim 1,
a virus according to claim 2, a virus according to claim 3, a virus
according to claim 4, a polypeptide according to claim 8, a
polypeptide according to claim 9, an antigen according to claim 10,
an antibody according to claim 11 and an antibody according to
claim 14.
30. Use of a virus according to claim 1, its genome, or
resistance-conferring parts thereof as antiviral agent.
31. Use of a virus according to of claim 1, or its genome, or parts
thereof as expression vector.
32. Use of an attenuated form of a virus according to claim 1, or
its genome, or parts thereof, for premunition of a plant.
33. An isolated or recombinant polypeptide obtainable from a virus
according to claim 2 or a ToTV-specific fragment thereof.
34. An isolated or recombinant polypeptide obtainable from a virus
according to claim 3 or a ToTV-specific fragment thereof.
35. A method for identifying a viral isolate as a ToTV virus
comprising reacting said viral isolate or a component thereof with
an antibody according to claim 14.
36. A method for detecting the presence of ToTV in a sample
comprising determining in said sample the presence of a ToTV virus
or component thereof by reacting said sample with an antibody
according to claim 11.
37. A method for detecting the presence of ToTV in a sample
comprising determining in said sample the presence of a ToTV virus
or component thereof by reacting said sample with an antibody
according to claim 14.
38. Use of a virus according to claim 2, its genome, or
resistance-conferring parts thereof as antiviral agent.
39. Use of a virus according to claim 3, its genome, or
resistance-conferring parts thereof as antiviral agent.
40. Use of a virus according to claim 2, or its genome, or parts
thereof as expression vector.
41. Use of a virus according to claim 3, or its genome, or parts
thereof as expression vector.
42. Use of an attenuated form of a virus according to claim 2, or
its genome, or parts thereof, for premunition of a plant.
43. Use of an attenuated form of a virus according to claim 3, or
its genome, or parts thereof, for premunition of a plant.
44. The method according to claim 25, wherein said Solanum
lycopersicum is an S. lycopersicum line that possesses one or more
commercially desirable characteristics.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of
International application PCT/NL2006/000009 published under the PCT
and filed on 17 Aug. 2006, which claims priority from European
applications EP 05075042.1 filed 7 Mar. 2005, and EP 05075725.1
filed 29 Mar. 2005, each of which is incorporated herein by
reference in its entirety.
FIELD
[0002] The present invention is in the field of plant disease. More
in particular, the invention relates to a new plant virus isolated
from tomato, to methods for detecting said virus, to methods of
detecting resistant plants and to methods for producing resistant
plants.
BACKGROUND
[0003] The tomato Solanum lycopersicum (formerly Lycopersicon
esculentum) is susceptible to a large number of viral species. Some
of the most prominent tomato viruses include Tomato spotted wilt
virus (TSWV; genus Tospovirus); Pepino mosaic virus (PepMV; genus
Potexuirus), and Tomato yellow leaf curl virus (TYLCV; genus
Begomovirus). The damage that these diseases inflict on the plant
range from discoloration of leaves and necrotic lesions, to severe
crop loss and death of the plant.
[0004] The ability to provide resistant plants is of utmost
importance to commercial breeders, and for some of the economically
most damaging viruses, resistant plant varieties have been
produced. However, from time to time, new viruses emerge that may
inflict considerable damage on crops.
[0005] In 1996 a new tomato virus was reported which had infected
tomato plants in the USA and Italy since 1993, and was named Tomato
infectious chlorosis virus (TICV; genus Crinivirus; Duffus et al.,
1996). Another new tomato virus of the same genus was reported in
1998. This virus was shown to have infected tomato plants in the
USA since 1989 and was named Tomato chlorosis virus (ToCV; Wisler
et al., 1998). Both these new viruses proved to be spread by a
whitefly, the insect being a very effective disease-transmission
vector.
[0006] It is generally believed that the geographic distribution of
known viruses will increase and that new viruses will continue to
appear, partly as a result of recombination of different viruses to
form new strains or new viruses. The development of resistant
cultivars can play an important role in the successful management
of these diseases.
SUMMARY
[0007] Recently, a new virus was discovered on tomato plants from
Spain, which caused symptoms that could not be attributed to any
known virus. The plants exhibited necrotic lesions on leaves and
brown rings on fruits and showed reduced growth. Serological tests
(ELISA) indicated the presence of Pepino mosaic virus (PepMV).
Electron microscopic investigations indeed revealed the rod-like
particles typical for potexviruses. However, also spherically
shaped viral particles were found in infected leaf tissue. The
inventors were able to separate the new virus from the complex with
PepMV. The new virus was tentatively named Tomato torrado virus
(ToTV).
[0008] Following the discovery of the ToTV virus, a new and very
related virus was isolated from a tomato plant from the state of
Sinaloa in Mexico. This plant showed symptoms locally known as
`marchitez disease,` including severe leaf necrosis, beginning at
the base of the leaflets, and necrotic rings on the fruits. (This
disease should not be confused with another virus known to cause
what is called in Mexico "marchitez manchada del tomate," the
causative agent of which is tomato spotted wilt virus (TSWV) or
Virus de la Marchitez Manchada del Tomate (VMMT), [see e.g. De La
Torre-Almaraz et al. Agrociencia 36: 211-221. 2002]). Virus
particles isolated from the infected plants are isometric with a
diameter of approximately 28 nm. The viral genome consists of two
(+)ssRNA molecules of 7221 (RNA1) and 4906 nucleotides (RNA2). The
viral capsid contains three coat proteins of 35, 26 and 24 kDa
respectively. The above mentioned characteristics; symptoms,
morphology, number and size of coat proteins, and number of RNAs,
are similar to the previously described tomato torrado virus
(ToTV). Sequence analysis of the entire viral genome shows that
this new virus is related to, but distinct from ToTV and, together
with ToTV belongs to a new plant virus genus. For this new virus
the name tomato marchitez virus (TMarV) is proposed.
[0009] For being able to trace its origin, monitor its epidemiology
and prevent possible spreading of the disease, it is of great
importance to be able to recognise the disease in an early stage.
Only then sufficient measures can be taken to isolate plants and
initiate phytosanitairy precautions. At this moment no diagnostic
tools are available. Consequently, there is a need for developing
diagnostic tools for this disease. Furthermore, at present there
are no plants known that harbour specific resistance to this new
virus, while there is a need for developing such resistant
plants.
[0010] The invention provides in a first aspect a plant virus
tentatively named Tomato torrado virus (ToTV), deposited with the
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) in
Braunschweig, Germany, on 24 Nov. 2004 under depositors reference
number ToTV-E01 (DSM 16999).
[0011] The invention provides in another aspect a plant virus
tentatively named tomato marchitez virus (TMarV), soon to be
deposited with an international depository under the Budapest
treaty under depositors reference number "PRI-TMarV0601."
[0012] When reference is made herein to "ToTV," the broad genus of
viruses sharing the homology levels as indicated herein is
intended, unless otherwise specified. This broad genus includes
ToTV-E01 (DSM 16999) as well as PRI-TMarV0601.
[0013] The virus causes disease-symptoms in tomato plants as well
as in other plants, and may cause the symptoms by itself, or in a
complex with other viruses or diseases.
[0014] The first systemic symptoms consist of necrotic spots at the
top of the plant, starting at the base of the leaves of a leaflet.
Necrotic spots expand and are surrounded by a light-green or yellow
area (see FIG. 1). Not all systemic infected leaves show symptoms,
however, the virus can be detected in these leaves, for instance by
electron microscopy. Fruits infected with ToTV exhibit necrotic
rings. The growth of infected plants may be reduced compared to
non-infected plants.
[0015] The above description relates to plants newly infected with
the isolated virus and need not necessarily reflect the exact
symptoms encountered in the field. Factors such as plant race or
variety, development stage, additional disease pressure, and
abiotic factors (e.g. temperature and relative humidity) will
eventually determine the expression and characteristics of the
symptoms.
[0016] The viral particles are spherical (icosahedral) in shape
with a diameter of approximately 28 nm (see FIG. 2). Virus
particles consist of at least three capsid proteins of
approximately 23, 26 and 35 kDa (see FIG. 3). Upon purification,
the virus displays at least two visible bands in a cesium sulfate
gradient. The upper visible band (virus top fraction) contains an
RNA molecule of approximately 5.5 kb (more precisely 5.2 kb) and
the lower visible band (bottom fraction) contains an RNA molecule
of approximately 8 kb (more precisely 7.7 kb) (see FIG. 4). The
inoculation of both bands combined on tobacco plants results in an
infection.
[0017] ToTV is mechanically transmissible to several Nicotiana
species. A standard inoculation buffer (e.g. a 0.03 M phosphate
buffer at pH 7.7) is suitable. For propagation of ToTV, N.
glutinosa, N. tabacum and N. benthamina are preferred. The tobacco
species N. hesperis `67A` and N. occidentalis `P1` are very
sensitive to ToTV and show systemic symptoms after 3-4 days. These
tobacco species become very necrotic in short time and are
therefore more suitable for use as an indicator plant than as
propagation host. N. glutinosa reacts with chlorotic local lesions
and a systemic chlorosis and mild deformation of the leaves. N.
benthamiana does not show local symptoms and reacts with systemic
chlorosis and deformation of the leaves.
[0018] The invention further provides a virus comprising at least
one nucleic acid sequence selected from the group consisting of SEQ
ID NO:1 and SEQ ID NO:2 and sequences having a nucleotide sequence
homology of at least 30, preferably at least 40, preferably at
least 50%, preferably at least 60%, more preferably at least 70%,
more preferably at least 80%, still more preferably at least 90%,
still more preferably at least 95%, still more preferably at least
98%, and most preferably at least 99% thereto. Such viruses are
also encompassed by the term ToTV as used herein.
[0019] In a preferred embodiment of a virus of the invention having
the above referred sequence homology, said virus is associated with
tomato diseases known under the names of "Torrado," "Marchitez"
and/or "Chocolate spot disease," and/or said virus shows, based on
numerical taxonomic analysis of taxonomic descriptors essentially
as defined in Table 1, to be more closely related to the virus as
defined in claim 1 than to any other viral isolate available in
public collections, and said virus has as an essential
characteristic that it is associated with a disease that causes
necrotic lesions in tomato.
[0020] In another aspect, the invention provides an isolated or
recombinant nucleic acid comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,
sequences having a nucleotide sequence homology of at least 50%,
preferably at least 70%, more preferably at least 80%, still more
preferably at least 90%, still more preferably at least 95%, still
more preferably at least 98%, and most preferably at least 99% to
SEQ ID NO:1 or SEQ ID NO:2, and their complementary strands and
ToTV-specific fragments thereof. Such a nucleic acid may be
obtained from a virus according to the invention.
[0021] In another aspect, the invention provides a polynucleotide
capable of hybridizing under stringent conditions to the isolated
or recombinant nucleic acid of the invention as described
above.
[0022] In another aspect, the invention provides an isolated or
recombinant polypeptide obtainable from a virus according to the
invention, or a ToTV-specific fragment thereof. In a preferred
embodiment said polypeptide is selected from the group consisting
of the 23, 26 and 35 kDa capsid proteins of ToTV and ToTV-specific
fragments thereof.
[0023] In another aspect, the invention provides an antigen
comprising a polypeptide or ToTV-specific fragment thereof
according to the invention.
[0024] In another aspect, the invention provides an antibody
specifically directed against an antigen according to the
invention.
[0025] In another aspect, the invention provides a method for
producing an antibody against ToTV comprising the steps of: a)
providing a ToTV virus, or a (recombinant) protein or peptide
fragment thereof; b) immunizing an appropriate vertebrate host with
said virus, protein or peptide fragment, and c) harvesting from
blood (including serum) or splenocytes of said vertebrate host
antibodies against said virus, protein or peptide fragment. In a
preferred embodiment said method further comprises the steps of d)
selecting one antibody-producing splenocyte, e) fusing said
splenocyte to an immortalized hybridoma cell line and f) allowing
said hybridoma fusion to produce monoclonal antibodies.
[0026] In another aspect, the invention provides an antibody
obtainable by a method of producing an antibody against ToTV
according to the invention.
[0027] In another aspect, the invention provides a method for
identifying a viral isolate as a ToTV virus comprising reacting
said viral isolate or a component thereof with an antibody
according to the invention.
[0028] In another aspect, the invention provides a method for
identifying a viral isolate as a ToTV virus comprising reacting
said viral isolate or a component thereof with a polynucleotide
according to the invention.
[0029] In another aspect, the invention provides a method for
detecting the presence of ToTV in a sample comprising determining
in said sample the presence of a ToTV virus or component thereof by
reacting said sample with a polynucleotide or an antibody according
to the invention.
[0030] In another aspect, the invention provides a method for
identifying a ToTV-resistant plant comprising the steps of: a)
exposing a plant or plant part to an infective dosage of ToTV, and
b) identifying said plant as a ToTV-resistant plant when, after
said exposure, either i) disease-symptoms in said plant or plant
part remain absent or are delayed in expression or are at least
reduced in severity relative to a susceptible control plant, and/or
ii) ToTV virus or ToTV genomic sequences are not present in said
plant or plant part or the presence of ToTV virus is at least
quantitatively reduced in said plant relative to a susceptible
control plant. Step a) includes an incubation period for a duration
sufficiently long to allow for the establishment of detectable
disease-symptoms in susceptible control plants exposed to a
comparable infective dosage of virus. By performing this method all
forms of resistance, including full resistance, partial resistance,
hypersensitivity and tolerance may be identified in a plant. In
order to confirm tolerance, the (systemic) presence of the virus in
the plant (cells) must be confirmed. Step b) may involve performing
a method for detecting the presence of ToTV in a sample of said
plant or plant part according to the present invention wherein use
is made of an antibody or polynucleotide according to the invention
in standard methods for nucleotide hybridization assays or
immunoassays, well known to the skilled person. Alternatively, step
b) may comprise contacting a part of said exposed plant with a
susceptible indicator plant. In this way, one may detect the
presence of a systemic or local infection in said exposed plant
through observing the emergence of disease in the indicator plant
or even a further contacted indicator plant contacted with said
first contacted indicator plant.
[0031] In another aspect, the invention provides a method of
producing a ToTV-resistant plant comprising the steps of
identifying a ToTV-resistant donor plant by performing either of
the above methods for identifying a ToTV-resistant plant according
to the invention, crossing said ToTV-resistant donor plant with a
recipient plant (which recipient plant may be either
ToTV-susceptible or ToTV-resistant, but is suitably a
ToTV-resistant plant in case the resistant phenotype is brought
about by a recessive gene), and selecting from an offspring plant
(e.g. an F.sub.1, an F.sub.2, and a selfed plant) a resistant plant
by performing a method for identifying ToTV resistance in a plant
as described above. In the instance that the resistance trait is a
recessive trait, resistant plants may be found among offspring
plants of the selfings of the F1 or F2 or still further
generations. In preferred embodiments of this aspect, said
ToTV-resistant donor plant and the recipient plant are plants of
the family Solanaceae or family Cucurbitaceae. In other preferred
embodiments of this aspect, said recipient plant is a tomato plant,
eggplant plant, pepper plant, melon plant, watermelon plant or
cucumber plant, more preferably a plant of the species Solanum
lycopersicum, most preferably an S. lycopersicum line that
possesses commercially desirable characteristics.
[0032] In another aspect, the invention provides a ToTV-resistant
plant, preferably a tomato plant, eggplant plant, pepper plant,
melon plant, watermelon plant or cucumber plant, or part thereof,
such as a seed, obtainable by a method of producing a
ToTV-resistant plant according to the invention.
[0033] In another aspect, the invention provides a diagnostic kit
for detecting the presence of ToTV in a sample or for identifying
ToTV resistance in a plant comprising a virus, a polynucleotide, a
polypeptide, an antigen and/or an antibody according to the
invention.
[0034] In another aspect, the invention provides the use of a
virus, a polynucleotide, a polypeptide, an antigen or an antibody
according to the invention for the production of a diagnostic
composition.
[0035] In another aspect, the invention provides a diagnostic
composition comprising a virus, a polynucleotide, a polypeptide, an
antigen or an antibody according to the invention.
[0036] In another aspect, the invention provides the use of ToTV,
or parts of the ToTV viral genome, as an expression vector.
[0037] In another aspect, the invention provides the use of ToTV,
or parts of the ToTV viral genome, for producing pathogen-derived
resistance in plants.
[0038] In another aspect, the invention relates to the use of an
attenuated form of a ToTV virus, or its genome, or parts thereof,
for premunition of a plant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The foregoing and other features and advantages of the
present invention will be more fully understood from the following
detailed description of illustrative embodiments taken in
conjunction with the accompanying drawings in which:
[0040] FIG. 1 depicts a photograph of symptoms of the ToTV-disease
on leaves of a tomato plant caused by ToTV-E01.
[0041] FIG. 2 depicts an electron micrograph of purified ToTV-E01
particles. Particles are about 28 nm in diameter.
[0042] FIG. 3 depicts the result of a silver-stained PAGE gel of
purified ToTV-E01 virus top (TAgV-T) and bottom (TAgV-B) fractions
indicating the three capsid proteins of approximately 23, 26 and 35
kDa.
[0043] FIG. 4 depicts the result of denaturing agarose gel
electrophoresis. Sizes of the segments are indicated in kilobases
(kb). The gel was stained using ortho-toluidine blue. M: molecular
size standard. TAgV: 1 .mu.g of isolated ToTV-E01 RNA.
[0044] FIG. 5 depicts the complete sequence of the ToTV-E01 RNA 2
molecule (SEQ ID NO:1).
[0045] FIG. 6 depicts the complete sequence of the ToTV-E01 RNA 1
molecule (SEQ ID NO:2).
[0046] FIG. 7 schematically depicts the general structure of the
ToTV-E01 virus indicating the site of annealing of the various
primer sets as used in Example 2 to RNA 2.
[0047] FIG. 8 depicts the complete sequence of the PRI-TMarV0601
RNA 1 molecule (SEQ ID NO:15).
[0048] FIG. 9 depicts the complete sequence of the PRI-TMarV0601
RNA 2 molecule (SEQ ID NO:16).
[0049] FIGS. 10A-10B depict the typical symptoms of tomato
marchitez virus in (A) tomato leaves: necrosis surrounded by a
yellow or bright green area, beginning at the base of the leaflets,
and (B) tomato fruits: necrotic rings and patches.
[0050] FIGS. 11A-11B depict in panel A) the result of denaturing
polyacrylamide gel electrophoresis (SDS-PAGE) of TMarV capsid
proteins. Proteins were visualized by silver staining. 1) molecular
weight markers (Bio-Rad Precision Plus Protein Standards (note: the
prestained 37 kDa and 50 kDa standards do not stain in the used
silver staining method), 2) TMarV purified virions after
Cs.sub.2SO.sub.4 buoyant density gradient centrifugation; in panel
B) the result of denaturing agarose gel electrophoresis of RNA
extracted from TMarV virions and stained with ortho-toluidine blue.
1: molecular size standard (Invitrogen 0.24-9.5-kb RNA Ladder); 2:
RNA purified from TMarV virions. Arrows indicate positions of the
.about.3.5-kb and .about.7.5-kb RNA bands.
[0051] FIG. 12 schematically depicts the genome organization of
TMarV.
[0052] FIGS. 13A-13B depict a phylogenetic analysis of TMarV
(PRI-TMarV0601) and related viruses (e.g. ToTV-E01) based on the
alignment of (A) the region between the protease CG motif and GDD
RdRp motif of RNA1; and (B) the helicase region between motifs A
and C of RNA1.
DETAILED DESCRIPTION
Definitions
[0053] As used herein the term "ToTV," is to be interpreted as
referring to the new genus of viruses sharing the RNA homology
levels as indicated herein, unless expressly stated or intended
otherwise (for instance when TMarV and ToTV are compared, the term
ToTV should be interpreted as referring to the species ToTV-E01.
This broad genus includes at least the species ToTV-E01 (DSM 16999)
as well as the PRI-TMarV0601. The genus is defined by the homology
levels between RNA sequences of the viral genome (or translated
into DNA sequences); or the homology levels between amino acid
sequences of open reading frames within the viral genome.
[0054] As used herein the term "ToTV-resistant," is to be
interpreted as referring to the resistance of a plant, in
particular a tomato plant to the establishment of an infection, or
the establishment of a disease caused by a viral species of the new
genus of viruses sharing the RNA homology levels as indicated
herein, unless expressly stated or intended otherwise.
[0055] The term "ToTV-specific fragment" as used herein refers to a
nucleic acid fragment having a sequence that is specific for the
genus of viruses comprising ToTV and TMarV as described herein,
With the term "specific" is meant that the nucleic acid is capable
of hybridizing specifically under stringent hybridization
conditions to the nucleic acid of said virus.
[0056] As used herein the term "TMarV" is to be interpreted as
referring to a species within the new genus of ToTV viruses which
genus shares the RNA homology levels as indicated herein, unless
expressly stated or intended otherwise (for instance when TMarV and
ToTV are compared herein, the term TMarV should be interpreted as
referring to the species PRI-TMarV0601). The term "TMarV" is also
to be interpreted as referring to a virus belonging to this novel
species within the new genus of ToTV viruses which genus wherein
said novel species shares homology levels on nucleotide or amino
acid sequence levels generally accepted among virologists to
indicate different isolates (strains) belonging to the same
species. Such intraspecific homology levels are usually above 80 to
90%, for instance above 93% for nucleotide sequences, and possibly
even higher for amino acid sequences.
[0057] As used herein, the term "plant part" indicates a part of a
plant, including single cells and cell tissues such as plant cells
that are intact in plants, cell clumps and tissue cultures from
which plants can be regenerated. Examples of plant parts include,
but are not limited to, single cells and tissues from pollen,
ovules, leaves, embryos, roots, root tips, anthers, flowers,
fruits, stems shoots, and seeds; as well as pollen, ovules, leaves,
embryos, roots, root tips, anthers, flowers, fruits, stems, shoots,
scions, rootstocks, seeds, protoplasts, calli, and the like.
[0058] The term "sample" includes a sample from a plant, from a
plant part or from a transmission vector, or a soil, water or air
sample.
[0059] The term "transmission vector" as used herein refers to the
disease-spreading agent or substance. Transmission vectors of ToTV
in the field may comprise, but are not limited to animals such as
Arthropoda (in particular those of the classes Insecta and
Arachnida), Nematoda (in particular those of the class
Adenophorea), but also larger animals such as for instance birds,
rabbits and mice, fungi (i.e. phylum Eumycota, in particular fungi
of the class Phycomycota), (parasitic) plants (including members of
the family Cuscutaceae), pollen, seed, water, soil particles, and
even human hands, equipment and shoes.
[0060] The term "offspring" plant refers to any plant resulting as
progeny from a vegetative or sexual reproduction from one or more
parent plants or descendants thereof. For instance an offspring
plant may be obtained by cloning or selfing of a parent plant or by
crossing two parent plants and include selfings as well as the F1
or F2 or still further generations. An F.sup.1 is a
first-generation offspring produced from parents at least one of
which is used for the first time as donor of a trait, while
offspring of second generation (F2) or subsequent generations (F3,
F4, etc.) are specimens produced from selfings of F1's, F2's etc.
An F1 may thus be (and usually is) a hybrid resulting from a cross
between two true breeding parents (true-breeding is homozygous for
a trait), while an F2 may be (and usually is) an offspring
resulting from self-pollination of said F1 hybrids.
[0061] The term "resistant," as used herein, refers to a plant that
is capable of resisting the multiplication in its cells and/or the
(systemic) movement of the virus to other cells and/or the
development of disease symptoms after infection with said virus,
which virus is capable of infecting and multiplying in
corresponding non-resistant or susceptible varieties of said plant.
The term is used to include such separately identifiable forms of
resistance as "full resistance," "immunity," "partial resistance,"
"hypersensitivity" and "tolerance."
[0062] "Full resistance" is referred to as complete failure of the
virus to develop after infection, and may either be the result of
failure of the virus to enter the cell (no initial infection) or
may be the result of failure of the virus to multiply in the cell
and infect subsequent cells (no subliminal infection, no spread).
The presence of full resistance may be determined by establishing
the absence of viral particles or viral RNA in cells of the plant,
as well as the absence of any disease symptoms in said plant, upon
exposure of said plant to an infective dosage of virus (i.e. after
`infection`). Among breeders, this phenotype is often referred to
as "immune." "Immunity" as used herein thus refers to a form of
resistance characterized by absence of viral replication even when
virus is actively transferred into cells by e.g.
electroporation.
[0063] An "infective dosage" is defined as a dosage of viral
particles or virus nucleic acid capable of infecting a plant, which
dosage may vary between plants and between ToTV-isolates tested.
Theoretically, an amount of about 1 to 10 to an amount of about
500-5000 viral particles of said virus or the nucleic acids thereof
will be sufficient. Infection in this way may be achieved by
mechanical inoculation of purified virus particles or virus nucleic
acid on plants.
[0064] "Partial resistance" is referred to as reduced
multiplication of the virus in the cell, as reduced (systemic)
movement of the virus, and/or as reduced symptom development after
infection. The presence of partial resistance may be determined by
establishing the systemic presence of low titres of viral particles
or viral RNA in the plant and the presence of decreased or delayed
disease-symptoms in said plant upon exposure of said plant to an
infective dosage of virus. Virus titres may be determined by using
a quantitative detection method (e.g. an ELISA method or a
quantitative reverse transcriptase-polymerase chain reaction
[RT-PCR]). Among breeders, this phenotype is often referred to as
"intermediate resistant."
[0065] The term "hypersensitive" refers to a form of resistance
whereby the infection remains local and does not systemically
spread, for instance due to local necrosis of infected tissue or
lack of systemic movement beyond inoculated tissue. Hypersensitive
plants show local, but severe disease symptoms and the local
presence of the virus can be established in such plants.
[0066] "Tolerant" is used herein to indicate a phenotype of a plant
wherein disease-symptoms remain absent upon exposure of said plant
to an infective dosage of virus, whereby the presence of a systemic
or local viral infection, virus multiplication, at least the
presence of viral genomic sequences in cells of said plant and/or
genomic integration thereof can be established. Tolerant plants are
therefore resistant for symptom expression but symptomless carriers
of the virus. Sometimes, viral sequences may be present or even
multiply in plants without causing disease symptoms. This
phenomenon is also known as "latent infection." Some DNA and RNA
viruses, may become undetectable following a primary infection only
to reappear later and produce acute disease. In latent infections,
the virus may exist in a truly latent non-infectious occult form,
possibly as an integrated genome or an episomal agent (so that
viral particles cannot be found in the cytoplasm, while PCR
protocols may indicate the present of viral nucleic acid sequences)
or as an infectious and continuously replicating agent. A
reactivated virus may spread and initiate an epidemic among
susceptible contacts. The presence of a "latent infection" is
indistinguishable from the presence of a "tolerant" phenotype in a
plant.
[0067] The term "susceptible" is used herein to refer to a plant
having no resistance to the virus resulting in entry of the virus
into the plant's cells and multiplication and systemic spread of
virus, resulting in disease symptoms. The term "susceptible" is
therefore equivalent to "non-resistant." A susceptible plant
exhibits normal virus titres in its cells upon infection.
Susceptibility may thus be determined by establishing the presence
of normal (i.e. relative to other viral infections in plants)
titres of viral particles or of viral RNA in cells of the plant and
the presence of normal disease symptoms (i.e. relative to the
disease symptoms as herein described for the plant from which ToTV
was first isolated) in said plant upon exposure of said plant to an
infective dosage of virus.
[0068] The term "sensitive" reflects the symptomatic reaction of a
susceptible plant upon virus infection. The reaction or symptoms
can be more or less severe depending on the level of sensitivity of
the plant. If the plant is injured or even killed by the virus,
said plant is qualified as "sensitive."
[0069] Plants artificially inoculated with attenuated virus strains
are subsequently protected from closely related virulent viruses.
As protective viruses, either naturally occurring mild strains or
an attenuated strain (an artificially-induced mild mutant) may be
used. Preferably, in order to attain premunition of a plant against
ToTV, an attenuated strain of ToTV may be used that is symptomless
or that shows at least reduced symptom expression in an infected
plant, relative to a virulent strain of ToTV. Methods of producing
attenuated virus may for instance include random mutagenesis of the
ToTV genome and screening for strains with symptom attenuation.
Express reference is made to the methods for producing attenuated
plant viruses as described in the articles of Takeshita et al.,
2001; Lu et al., 2001; Hagiwara, et al., 2002; and Hirata et al.,
2003.
[0070] As used herein, the term "tomato" means any plant, line or
population of Lycopersicon or Solanum that includes, but is not
limited to, those presented in the Listing below. Recently,
nomenclature of Lycopersicon has been changed.
[0071] The new nomenclature for Lycopersicon is provided in the
following Listing (from: Peralta, Knapp & Spooner, unpublished
monograph (see: http://www.sgn.cornell.edu "Guide to revised
Solanum nomenclature")). TABLE-US-00001 Name in tomato monograph
(Peralta et al., in preparation for publication in Systematic
Botany Monographs) Lycopersicon equivalent Solanum juglandifolium
Dunal Lycopersicon juglandifolium (Dunal) J. M. H. Shaw Solanum
ochranthum Dunal Lycopersicon ochranthum (Dunal) J. M. H. Shaw
Solanum sitiens I. M. Johnst. Lycopersicon sitiens (I. M. Johnst.)
J. M. H. Shaw Solanum lycopersicoides Dunal Lycopersicon
lycopersicoides (Dunal in DC.) A. Child ex J. M. H. Shaw Solanum
pennellii Correll Lycopersicon pennellii (Correll) D'Arcy Solanum
habrochaites S. Knapp & D. M Spooner Lycopersicon hirsutum
Dunal Solanum `N peruvianum` to be described by Part of
Lycopersicon peruvianum (L.) Miller Peralta (4 geographic races:
humifusum, lomas, (incl. var. humifusum and Marathon races)
Marathon, Chotano-Yamaluc) Solanum `Callejon de Huaylas` to be
described Part of Lycopersicon peruvianum (L.) Miller by Peralta
(from Ancash, alogn Rio Santa) Solanum neorickii D. M. Spooner, G.
J. Anderson Lycopersicon parviflorum C. M. Rick, & R. K. Jansen
Kesicki, Fobes & M. Holle Solanum chmielewskii (C. M. Rick,
Kesicki, Lycopersicon chmielewskii C. M. Rick, Fobes & M.
Holle) D. M. Spooner, G. J. Anderson Kesicki, Fobes & M. Holle
& R. K. Jansen Solanum corneliomuelleri J. F. Macbr. (1 Part of
Lycopersicon peruvianum (L.) geographic race: Misti nr. Arequipa)
Miller; also known as Lycopersicon glandulosum C. F. Mull. Solanum
peruvianum L. Lycopersicon peruvianum (L.) Miller Solanum chilense
(Dunal) Reiche Lycopersicon chilense Dunal Solanum cheesmaniae (L.
Riley) Fosberg Lycopersicon cheesmaniae L. Riley (published as
cheesmanii) Solanum galapagense S. Darwin & Peralta Part of
Lycopersicon cheesmaniae L. Riley (previously known as forma or
var. minor) Solanum lycopersicum L. Lycopersicon esculentum Miller
Solanum pimpinellifolium L. Lycopersicon pimpinellifolium (L.)
Miller
[0072] An "expression vector" is defined as a nucleic acid molecule
containing a gene, usually a heterologous gene, that is expressed
in a host cell. Typically, this gene comprises a protein encoding
sequence. Gene expression is always placed under the control of a
promoter, and such a gene is said to be "operably linked to" the
promoter. The term "heterologous" refers to a DNA molecule, or a
population of DNA molecules, that does not exist naturally within a
given host cell.
[0073] The term "polynucleotide" as used herein, is interchangeable
with the term "nucleic acid," and refers to a nucleotide multimer
or polymeric form of nucleotides having any number of nucleotides,
e.g., deoxyribonucleotides or ribonucleotides, or compounds
produced synthetically (e.g. PNA as described in U.S. Pat. No.
5,948,902 and the references cited therein) and can be either
double- or single-stranded. A polynucleotide can hybridize with
naturally occurring polynucleotides in a sequence specific manner
analogous to that of two naturally occurring polynucleotides, e.g.
can participate in Watson-Crick base pairing interactions. The term
also includes modified, for example by methylation and/or by
capping, and unmodified forms of the polynucleotide.
[0074] The terms "ribonucleic acid" and "RNA" as used herein mean a
polymer composed of ribonucleotides.
[0075] The terms "deoxyribonucleic acid" and "DNA" as used herein
mean a polymer composed of deoxyribonucleotides.
[0076] The term "oligonucleotide" refers to a short sequence of
nucleotide monomers (usually 6 to 100 nucleotides) joined by
phosphorous linkages (e.g., phosphodiester, alkyl and
aryl-phosphate, phosphorothioate), or non-phosphorous linkages
(e.g., peptide, sulfamate and others). An oligonucleotide may
contain modified nucleotides having modified bases (e.g., 5-methyl
cytosine) and modified sugar groups (e.g., 2'-O-methyl ribosyl,
2'-O-methoxyethyl ribosyl, 2'-fluoro ribosyl, 2'-amino ribosyl, and
the like). Oligonucleotides may be naturally-occurring or synthetic
molecules of double- and single-stranded DNA and double- and
single-stranded RNA with circular, branched or linear shapes and
optionally including domains capable of forming secondary
structures (e.g., stem-loop, pseudo knots and kissing loop
structures).
[0077] The term "nucleotide sequence homology" as used herein
denotes the presence of homology between two polynucleotides.
Polynucleotides have "homologous" sequences if the sequence of
nucleotides in the two sequences is the same when aligned for
maximum correspondence. Sequence comparison between two or more
polynucleotides is generally performed by comparing portions of the
two sequences over a comparison window to identify and compare
local regions of sequence similarity. The comparison window is
generally from about 20 to 200 contiguous nucleotides. The
"percentage of sequence homology" for polynucleotides, such as 50,
60, 70, 80, 90, 95, 98, 99 or 100 percent sequence homology may be
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may include additions or
deletions (i.e. gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by: (a) determining
the number of positions at which the identical nucleic acid base
occurs in both sequences to yield the number of matched positions;
(b) dividing the number of matched positions by the total number of
positions in the window of comparison; and (c) multiplying the
result by 100 to yield the percentage of sequence homology. Optimal
alignment of sequences for comparison may be conducted by
computerized implementations of known algorithms, or by visual
inspection. Readily available sequence comparison and multiple
sequence alignment algorithms are, respectively, the Basic Local
Alignment Search Tool (BLAST) (Altschul et al., 1990; Altschul et
al., 1997) and ClustalW programs, both available on the internet.
Other suitable programs include, but are not limited to, GAP,
BestFit, PlotSimilarity, and FASTA in the Wisconsin Genetics
Software Package (Genetics Computer Group (GCG), Madison, Wis.,
USA) (Devereux et al., 1984).
[0078] As used herein, "substantially complementary" means that two
nucleic acid sequences have at least about 65%, preferably about
70%, more preferably about 80%, even more preferably 90%, and most
preferably about 98%, sequence complementarity to each other. This
means that primers and probes must exhibit sufficient
complementarity to their template and target nucleic acid,
respectively, to hybridise under stringent conditions. Therefore,
the primer and probe sequences need not reflect the exact
complementary sequence of the binding region on the template and
degenerate primers can be used. For example, a non-complementary
nucleotide fragment may be attached to the 5'-end of the primer,
with the remainder of the primer sequence being complementary to
the strand. Alternatively, non-complementary bases or longer
sequences can be interspersed into the primer, provided that the
primer has sufficient complementarity with the sequence of one of
the strands to be amplified to hybridize therewith, and to thereby
form a duplex structure which can be extended by the polymerizing
means. The non-complementary nucleotide sequences of the primers
may include restriction enzyme sites. Appending a restriction
enzyme site to the end(s) of the target sequence would be
particularly helpful for cloning of the target sequence. A
substantially complementary primer sequence is one that has
sufficient sequence complementarity to the amplification template
to result in primer binding and second-strand synthesis. The
skilled person is familiar with the requirements of primers to have
sufficient sequence complementarity to the amplification
template.
[0079] The term "hybrid" in the context of nucleic acids refers to
a double-stranded nucleic acid molecule, or duplex, formed by
hydrogen bonding between complementary nucleotide bases. The terms
"hybridise" or "anneal" refer to the process by which single
strands of nucleic acid sequences form double-helical segments
through hydrogen bonding between complementary bases.
[0080] The term "hybrid" in the context of plant breeding refers to
a plant that is the offspring of genetically dissimilar parents
produced by crossing plants of different lines or breeds or
species.
[0081] The term "probe" refers to a single-stranded oligonucleotide
sequence that will recognize and form a hydrogen-bonded duplex with
a complementary sequence in a target nucleic acid sequence analyte
or its cDNA derivative.
[0082] The term "primer" as used herein refers to an
oligonucleotide which is capable of annealing to the amplification
target allowing a DNA polymerase to attach, thereby serving as a
point of initiation of DNA synthesis when placed under conditions
in which synthesis of primer extension product is induced, i.e., in
the presence of nucleotides and an agent for polymerization such as
DNA polymerase and at a suitable temperature and pH. The
(amplification) primer is preferably single stranded for maximum
efficiency in amplification. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
agent for polymerization. The exact lengths of the primers will
depend on many factors, including temperature and composition (A/T
en G/C content) of primer. A pair of bi-directional primers
consists of one forward and one reverse primer as commonly used in
the art of DNA amplification such as in PCR amplification.
[0083] It will be understood that "primer," as used herein, may
refer to more than one primer, particularly in the case where there
is some ambiguity in the information regarding the terminal
sequence(s) of the target region to be amplified. Hence, a "primer"
includes a collection of primer oligonucleotides containing
sequences representing the possible variations in the sequence or
includes nucleotides which allow a typical base pairing.
[0084] The oligonucleotide primers may be prepared by any suitable
method. Methods for preparing oligonucleotides of specific sequence
are known in the art, and include, for example, cloning and
restriction of appropriate sequences, and direct chemical
synthesis. Chemical synthesis methods may include, for example, the
phospho di- or tri-ester method, the diethylphosphoramidate method
and the solid support method disclosed in e.g. U.S. Pat. No.
4,458,066. The primers may be labeled, if desired, by incorporating
means detectable by for instance spectroscopic, fluorescence,
photochemical, biochemical, immunochemical, or chemical means.
[0085] Template-dependent extension of the oligonucleotide
primer(s) is catalyzed by a polymerizing agent in the presence of
adequate amounts of the four deoxyribonucleotide triphosphates
(dATP, dGTP, dCTP and dTTP, i.e. dNTPs) or analogues, in a reaction
medium which is comprised of the appropriate salts, metal cations,
and pH buffering system. Suitable polymerizing agents are enzymes
known to catalyze primer- and template-dependent DNA synthesis.
Known DNA polymerases include, for example, E. coli DNA polymerase
I or its Klenow fragment, T4 DNA polymerase, and Taq DNA
polymerase. The reaction conditions for catalyzing DNA synthesis
with these DNA polymerases are known in the art.
[0086] The products of the synthesis are duplex molecules
consisting of the template strands and the primer extension
strands, which include the target sequence. These products, in
turn, serve as template for another round of replication. In the
second round of replication, the primer extension strand of the
first cycle is annealed with its complementary primer; synthesis
yields a "short" product which is bound on both the 5'- and the
3'-ends by primer sequences or their complements. Repeated cycles
of denaturation, primer annealing, and extension result in the
exponential accumulation of the target region defined by the
primers. Sufficient cycles are run to achieve the desired amount of
polynucleotide containing the target region of nucleic acid. The
desired amount may vary, and is determined by the function which
the product polynucleotide is to serve.
[0087] The PCR method is well described in handbooks and known to
the skilled person.
[0088] After amplification by PCR, the target polynucleotides may
be detected by hybridization with a probe polynucleotide which
forms a stable hybrid with that of the target sequence under
stringent to moderately stringent hybridization and wash
conditions. If it is expected that the probes will be essentially
completely complementary (i.e., about 99% or greater) to the target
sequence, stringent conditions will be used. If some mismatching is
expected, for example if variant strains are expected with the
result that the probe will not be completely complementary, the
stringency of hybridization may be lessened. However, conditions
are chosen which rule out non-specific/adventitious binding.
Conditions which affect hybridization, and which select against
non-specific binding are known in the art, and are described in,
for example, Sambrook et al., (2001). Generally, lower salt
concentration and higher temperature increase the stringency of
binding. For example, it is usually considered that stringent
conditions are incubations in solutions which contain approximately
0.1.times.SSC, 0.1% SDS, at about 65.degree. C. incubation/wash
temperature, and moderately stringent conditions are incubations in
solutions which contain approximately 1-2.times.SSC, 0.1% SDS and
about 50.degree.-65.degree. C. incubation/wash temperature. Low
stringency conditions are 2.times.SSC and about
30.degree.-50.degree. C.
[0089] The terms "stringency" or "stringent hybridization
conditions" refer to hybridization conditions that affect the
stability of hybrids, e.g., temperature, salt concentration, pH,
formamide concentration and the like. These conditions are
empirically optimised to maximize specific binding and minimize
non-specific binding of primer or probe to its target nucleic acid
sequence. The terms as used include reference to conditions under
which a probe or primer will hybridize to its target sequence, to a
detectably greater degree than other sequences (e.g. at least
2-fold over background). Stringent conditions are sequence
dependent and will be different in different circumstances. Longer
sequences hybridise specifically at higher temperatures. Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequence
at a defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of a
complementary target sequence hybridises to a perfectly matched
probe or primer. Typically, stringent conditions will be those in
which the salt concentration is less than about 1.0 M Na.sup.+ ion,
typically about 0.01 to 1.0 M Na.sup.+ ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes or primers (e.g. 10 to 50
nucleotides) and at least about 60.degree. C. for long probes or
primers (e.g. greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. Exemplary low stringent conditions or "conditions of
reduced stringency" include hybridization with a buffer solution of
30% formamide, 1 M NaCl, 1% SDS at 37.degree. C. and a wash in
2.times.SSC at 40.degree. C. Exemplary high stringency conditions
include hybridization in 50% formamide, 1M NaCl, 1% SDS at
37.degree. C., and a wash in 0.1.times.SSC at 60.degree. C.
Hybridization procedures are well known in the art and are
described by e.g. Ausubel et al., 1998 and Sambrook et al.,
2001.
[0090] The term "antigen" refers to a substance capable of
triggering an immune response in a vertebrate, resulting in
production of an antibody as part of the defence against said
substance. Antigens can be virus proteins that can provoke the
antibody production in for instance blood cells, cells of lymph
nodes, and spleen of vertebrates.
[0091] The term "antibody" includes reference to antigen binding
peptides and refers to antibodies, monoclonal antibodies, to an
entire immunoglobulin or antibody or any functional fragment of an
immunoglobulin molecule. Examples of such peptides include complete
antibody molecules, antibody fragments, such as Fab, F(ab')2,
complementarity determining regions (CDRs), VL (light chain
variable region), VH (heavy chain variable region), and any
combination of those or any other functional portion of an antibody
peptide. The term "antibody" refers to a polypeptide substantially
encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof which specifically bind and recognize an analyte
(antigen). However, while various antibody fragments can be defined
in terms of the digestion of an intact antibody, one of skill will
appreciate that such fragments may be synthesized de novo either
chemically or by utilizing recombinant DNA methodology. Thus, the
term antibody, as used herein, also includes antibody fragments
such as single chain Fv, chimeric antibodies (i.e., comprising
constant and variable regions from different species), humanized
antibodies (i.e., comprising a complementarity determining region
(CDR) from a non-human source) and heteroconjugate antibodies
(e.g., bispecific antibodies).
[0092] The terms "substantially pure" and "isolated," are used
interchangeably and describe a protein, peptide or nucleic acid
which is substantially separated from other (sub)cellular
components which naturally accompany it. The term embraces a
nucleic acid or protein which has been removed from its naturally
occurring environment, and includes recombinant or cloned DNA
isolates and chemically synthesized analogues or analogues
biologically synthesized by heterologous systems. Generally, the
term refers to a purified protein and nucleic acid having a purity
of at least about 75%, for example 85%, 95% or 98% by weight. Minor
variants or chemical modifications typically share the same
polypeptide or nucleotide sequence. A substantially pure protein or
nucleic acid will typically comprise about 85 to 100% (w/w) of a
protein or nucleic acid sample, more usually about 95%, and
preferably will be over about 99% pure. Protein or nucleic acid
purity or homogeneity can be indicated by a number of means well
known in the art, such as polyacrylamide gel electrophoresis of a
protein sample, followed by visualizing a single polypeptide band
on a polyacrylamide gel upon staining, or by agarose gel
electrophoresis of a nucleic acid sample, followed by visualizing a
single polynucleotide band on an agarose gel upon staining.
"Staining" may either refer to the use of a-specific peptide or
nucleic acid stains such as silver and Coomassie stains, or
ethidium bromide and SYBR.RTM. stains, or may refer to the use of
specific peptide or nucleic acid stains such as contacting the
peptide with an antibody and visualizing the antibody using a
labeled secondary antibody (e.g. conjugated to alkaline
phosphatase) in the case of proteins or peptides, or contacting the
nucleic acid with a complementary probe labelled for visualizing
the presence of hybridization between the nucleic acid and the
probe. For certain purposes higher resolution can be provided by
using high performance liquid chromatography (HPLC) or a similar
means for purification. Such methods are in the area of common
general knowledge (see e.g. Katz, et al., 1998).
Identification and Taxonomy
[0093] The invention provides an isolated virus comprising at least
one nucleic acid sequence according to SEQ ID NO:1 and/or SEQ ID
NO:2 and sequences having a nucleotide sequence homology of at
least 30%, 35%, 40%, 45%, 50%, 55%, 60% or preferably 65% thereto.
As stated above, polynucleotides have "homologous" sequences if the
sequence of nucleotides in the two sequences is the same when
aligned for maximum correspondence. BLAST searches using nucleotide
sequences obtained from the ToTV virus isolate previously revealed
no significant homologies with any of the known viral or non-viral
sequences in the GenBank and EMBL databases. Highest percentage
homology found thus far was 46.5% between the helicase motifs in
ORF1 on SEQ ID NO:2 corresponding to ToTV RNA1 with helicase motifs
of other plant viruses. (see Examples below).
[0094] TMarV shares virion characteristics and its genome
organization with ToTV-E01, but based on levels of nucleotide and
amino acid sequence identities the two viruses are related but
distinct. BLAST searches in the GenBank and EMBL databases recently
revealed a significant homology with another recently isolated
virus. Remarkably high levels of identities are observed between
TMarV RNA1 and RNA2 and partial sequences of RNA1 and RNA 2 of a
virus of which the sequence was deposited on 16 Oct. 2006 in the
NCBI database under the name of tomato apex necrosis virus (Genbank
accessions EF063641 and EF063642; hereinafter ToANV). This virus
appears also to be obtained from the Culiacan area (Sinaloa,
Mexico). No additional data on the virus from which these partial
sequences were derived are available. However, the relative low
levels of nucleotide sequence identities (less than 90%) in the
3'-NTRs of both RNA1 and RNA2 (88.5% and 85.6% respectively), as
well less then 90% aa identity (89%) in the largest putative CP
(Vp36) between TMarV and ToANV, suggests that the two viruses are
not identical and may be strains or isolates of the same virus.
Additional biological and molecular data on the virus from which
the ToANV sequence was derived will be needed to determine its
precise relationship to TMarV.
[0095] Phylogenetic analysis based the aa region between the CG
protease motif [Bazan J F, Fletterick R J (1988) Proc. Natl. Acad.
Sci. USA, 85: 7872-7876] and the GDD RdRp active site [Argos P
(1988) Nucleic Acids Res., 16:9909-9916] in the RNA1-ORF were
performed to determine the relationships between tomato marchitez
virus, ToTV and other viruses from the genera Sadwavirus,
Cheravirus and the families Sequiviridae, Comoviridae,
Dicistroviridae and Picornaviridae. This region is proposed to be a
good taxonomic predictor for classifying picorna-like viruses
[Ikegami M, et al. (2002) Taxonomy of recognized and putative
species in the family Comoviridae. XIIth IUMS Virology Meeting
Paris, France, 27 Jul.-12 Aug. 2002]. The resulting dendrogram
(FIG. 13) shows that TMarV virus clusters with ToTV and ToANV and
confirms the separate taxonomic position of these viruses from the
genus Cheravirus. A similar phylogenetic analysis on basis of the
helicase region between the motifs A and C [Gorbalenya A E, Koonin
E V, Wolf Y I (1990) FEBS Lett. 262: 145-148](aa 397-494) confirms
the taxonomic positions of TmarV (FIG. 13). For this motif these
viruses seem to be more closely related to the genera Waikavirus
and Sequivirus.
[0096] Tomato marchitez virus (TMarV) is a new picorna-like plant
virus, related to, but distinct from, tomato torrado virus (ToTV).
TMarV and ToTV clearly separate from other plant picorna-like
viruses but based on standard virological criteria, they belong to
the same genus. ToTV is the tentative new type species of a novel
genus of plant viruses: Torradovirus.
[0097] It should be understood that homologies may be large when
two sequences are compared over a small comparison window since
local regions of sequence similarity can often be found when two
long nucleotide sequences are compared. However, the skilled person
is aware that sequence homology requires the establishment of
common motifs between the sequences, among which the sequence
identity may locally be as high as 35 to 100%, but may be as low as
10-20% in other parts of the genomic sequence of the same ORF.
Thus, when reference is made herein that a sequence has a
nucleotide sequence homology of at least 50% to SEQ ID NO:1 or SEQ
ID NO:2, this may refer to the sequence homology between regions
amongst common motifs, where homology is greatest, but also between
the coding sequence of two homologous proteins. Note that sequence
homologies may differ between the various genes in the genome (see
Examples).
[0098] As an indication of the relatedness between the newly
identified ToTV virus isolate and other viruses (including viruses
to be compared therewith) a phylogenetic analysis may normally be
performed based on (part of the) the genomic sequence information
of the viruses.
[0099] Several such analyses are presented in the Examples below.
The information obtained thus far indicates that ToTV shows the
highest level of homology with viruses from the genera Sequivirus
and Waikavirus (Sequiuiridae) and the genera Cheravirus and
Sadwavirus. Viruses from these genera are distinguished on the
basis of the number of viral RNAs--Sequiuiridae have 1 RNA while
Cheraviruses and Sadwaviruses have two RNAs--and the number of
capsid proteins--Sadwaviruses have two CPs, Cheraviruses have three
CPs. These criteria suggest that Tomato torrado virus is most
likely to group within the genus Cheravirus, However, phylogenetic
analyses using several different motifs from the putative RdRp and
helicase proteins, position ToTV clearly separate from the genera
Cheravirus and Sadwavirus and in fact suggest that it is more
closely related to the Sequiviridae. Unfortunately, full sequences
from only one Cheravirus (CRLV) and two Sadwaviruses (SDV and SMoV)
are currently available. Preliminary data on vector transmission
suggest that ToTV might be transmitted by whiteflies while the
natural vectors of CRLV and Sadwaviruses are unknown. On the basis
of the presently available information, including sequence
information as well as additional taxonomical information as
presented in Table 1, below, the newly found virus is most likely a
novel genus. TABLE-US-00002 TABLE 1 Taxonomic descriptors for the
newly discovered ToTV virus (as listed by the Internationally
accepted methods from the handbook "Matthew's Plant Virology"
("Matthew's Plant Virology," Fourth Edition, Roger Hull (ed.)
Academic Press, San Diego, p 15, Table 2.1 therein) (The numbering
of the Table in Matthews' is adhered to in Table 1 below) I Virion
Properties A. Morphological properties of virions 1. 28 nm in size
2. Spherical (icosahedral) in shape 3. Envelop absent B. Physical
properties of the virions (not investigated) C. Properties of the
genome 1. RNA type nucleic acid 2. Single stranded RNA 3. Linear
RNA 4. Positive sense coding 5. At least 2 segments (RNA 2; SEQ ID
NO: 1 and RNA 1; SEQ ID NO: 2) 6. 5.5 kb (more precisely 5.2 kb or
5389 nucleotides excluding the poly(A)tail) and 8 kb (more
precisely 7.7 kb or 7793 nucleotides excluding the poly(A)tail) 7.
5' terminal cap unknown 8. 5' terminalcovalently linked polypeptide
unknown 9. Poly(A) tract present at both segments 10. Nucleotide
sequence comparisons: RNA 1 contains an ORF1 with motifs typical of
helicase (Hel), Protease and RNA-dependent RNA polymerase (RdRp).
Level of homology with helicase and RdRp motifs of other viruses is
described in detail in the Examples. RNA 2 contains two ORFs of
which one has at its N-terminus a motif typical of putative
movement protein (MP) and which contains at the C- terminus the
sequences of the three capsid (coat) proteins (CP). The level of
homology with capsid proteins of other viruses is described in
detail in the Examples. D. Properties of the proteins 1. 3 .times.
CP; Hel; RdRp; Protease; putative MP 2. CPs: 35, 26 and 23 kDa 3.
(For functional activities, see above and Examples below) 4. (For
amino acid sequence comparison see Examples below) II. Genomic
organization and replication 1. Genomic organization see FIG. 7 6.
Cytopathology: At least the epidermis of mesophyl cells or
accompanying cells of phloem III. Antigenic properties. 1. No known
serological relationships IV. Biological properties 1a. Natural
host range: Tomato; 1b. Experimental host range: Local symptoms/
Tested alternative host plants for ToTV systemic symptoms
Chenopodium quinoa --/-- Gomphrena globosa --/-- Nicotiana
benthamiana --/c Nicotiana clevelandii --/c Nicotiana glutinosa
--/c Nicotiana hesperis 67A nl/c, n, mf Nicotiana occidentalis P1
nl/c, n, mf Nicotiana rustica --/--(la) Nicotiana tabacum `White
Burley` --/--(la) Physalis floridana nl/c, n, mf, do c = chlorosis;
n = necrosis; l = lesions; mf = malformation; do = die off; la =
latent infection, -- = no symptoms. 2. Pathogenicity Symptoms on
natural host plant: Necrotic lesions finalizing in burn-like, full
necrosis of plant material and death of the plant; concentric rings
of necrotic spots on fruits. Association with disease: Torrado;
presumably also Chocolate spot; presumably also Marchitez 3. Tissue
trophism: At least the epidermis of mesophyl cells or accompanying
cells of phloem 4. Mode of transmission in nature unknown 5. Vector
relationships: Presumably white fly 6. Geographical distribution:
Mediterranean; America's (Central America, Mexico and Southern part
of North America)
[0100] It was recently found by the present inventors that genomic
sequences of the associated virus of a tomato disease in Central
America (e.g. Mexico and Guatemala) by the name of "Chocolate spot
disease," "Chocolate" or "Marchitez" were identical to those of the
herein described ToTV (see Example 2). Therefore, the causal agent
associated with this disease is an aspect of the present
invention.
[0101] As more viral sequences become available, both from non-ToTV
viruses that may or may not be closely related thereto, or from
viruses closely related to or essentially resembling the ToTV
virus, phylogenetic analysis will prove a valuable way of
determining the breadth of the taxon or lade of ToTV based on
phylogenetic relatedness between isolates.
[0102] Phylogenetic relatedness may for instance be determined
based on any one or all of the nucleotide sequences of RNA 1 and/or
RNA 2 of the viral genome or on capsid protein (gene) sequence
data. Phylogenetic analyses are well known to the skilled person
and may for instance comprise analysis by distance-based
tree-reconstruction (e.g. neighbor joining), maximum likelihood or
parsimony analysis methods by using such programs as ClustalX
(Thompson et al., 1997), PAUP (Swofford, 2000) or PHYLIP
(Felsenstein, 1989).
[0103] In order to perform an analysis of phylogenetic relatedness
between a novel isolate, the sequence of ToTV as provided herein,
and reference sequences from viral strains from, for instance,
GenBank, EMBL, or DDBJ databases, genomic RNA from said novel
isolate may be extracted directly from infected plants and genomic
sequences may be amplified therefrom. Reverse transcription with
PCR amplification methods (RT-PCR) may for instance be conducted
using degenerate oligonucleotide primers, such primers being for
instance capable of acting as amplification primer for
amplification of nucleic acid sequences from the genomes of
divergent ToTV isolates as well as from the genomes of closely
related viral species. Preferably, but not necessarily, full-length
genomic amplification products may thus be obtained from reference
strains (e.g. divergent isolates), test-strains (ToTV-suspected
isolates) and closely related species. Preferably, specific genetic
regions of interest are amplified for comparison. The amplification
products (DNA) may then be sequenced by for instance direct
double-stranded nucleotide sequencing using fluorescently labeled
dideoxynucleotide terminators (Smith et al., 1986) with the
degenerate oligonucleotide primers used for RT-PCR. Nucleotide
sequence editing, analysis, optional prediction of amino acid
sequences, and alignments may be conducted with software packages
available, such as with the LaserGene sequence analysis package
version 5 (DNASTAR, Inc., Madison, Wis.) and IntelliGenetics
GeneWorks version 2.5.1 (IntelliGenetics, Mountain View, Calif.)
software. Phylogenetic analyses may then be completed with
phylogenetic analysis using for instance parsimony (PAUP) software
with a neighbor-joining algorithm using absolute distances
following a heuristic search and 1,000 bootstrap replicates, and a
phylogenetic tree may be generated by parsimony analysis of the
aligned contiguous nucleotide or amino acid sequences, whereby in
such trees the numbers generally represent bootstrap confidence
levels. Following 1,000 replications, a confidence level of above
60%, preferably above 70%, more preferably above 80%, 90%, 95% or
98%, within a phylogenetic tree, are to be considered sufficient
proof of correct phylogenetic inference (placement of isolates in a
certain lade), provided that the tree is sufficiently branched
whereby optionally branching may be improved by rooting to a
suitably out-group species. In this way it can be determined which
isolates are most closely related to the ToTV sequences as provided
herein. Relatedness is generally expressed in terms of percentage
sequence similarity, herein termed sequence homology.
[0104] Although phylogenetic analyses provide a convenient method
of identifying a virus in case of sufficient nucleic acid homology,
with known viruses several other possibly more straightforward
albeit somewhat more coarse methods for identifying said virus or
viral proteins or nucleic acids from said virus are herein also
provided. As a rule of thumb, a ToTV virus can be identified by the
percentages of homology of the viral proteins or nucleic acids to
be identified in comparison with viral proteins or nucleic acids
identified herein by sequence. It is generally known that virus
species, especially RNA virus species, often constitute a quasi
species wherein a cluster of said viruses displays heterogeneity
among its members. Thus it is expected that each isolate may have a
somewhat different percentage homology with the sequences of the
isolate as provided herein. Therefore, other viral isolates that
exhibit sufficient sequence homology to ToTV (e.g. more than 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence homology)
are considered to belong to the same virus. The ToTV virus of the
present invention is therefore a virus having at least 50 or 60%
homology, preferably at least 70%, more preferably at least 80%,
still more preferably at least 90%, still more preferably at least
95%, still more preferably at least 98%, and most preferably at
least 99% homology to the protein or nucleic acid sequences
provided herein and causes ToTV-induced disease-symptoms in
Solanaceae, more particularly in Solanum and Nicotiana, which
disease-symptoms may or may not be similar to those described
herein. In Cucurbitaceae the virus appears not to cause visible
symptoms although the virus is capable of propagation in the
plants.
[0105] When one wishes to compare a separate virus isolate with the
protein or nucleic acid sequences described herein, the invention
provides an isolated virus (ToTV) identifiable as phylogenetically
corresponding to ToTV by determining a protein or nucleic acid
sequence of said separate virus isolate and determining that said
protein or nucleic acid sequence has a percentage sequence homology
of at least 60%, preferably at least 70%, more preferably at least
80%, still more preferably at least 90%, still more preferably at
least 95%, still more preferably at least 98%, and most preferably
at least 99% to the sequences as listed herein.
[0106] Viral isolates having individual proteins or nucleic acids
with higher homology than these mentioned maximum values are
considered as phylogenetically corresponding and thus taxonomically
corresponding to ToTV virus, and generally the proteins will be
encoded by a nucleic acid sequence structurally corresponding with
a sequence listed herein. Herewith the invention provides a virus
phylogenetically corresponding to the isolated virus of which the
sequences are listed herein.
[0107] It should be noted that, similar to other viruses, a certain
degree of variation can be expected to be found between
ToTV-viruses isolated from different sources.
[0108] Also, the nucleotide or amino acid sequence of the ToTV
virus or fragments thereof as provided herein for example show less
than 95%, preferably less than 90%, more preferably less than 80%,
more preferably less than 70% and most preferably less than 60%
nucleotide sequence homology, or less than 95%, preferably less
than 90%, more preferably less than 80%, more preferably less than
70% and most preferably less than 60% amino acid sequence homology
with the respective nucleotide or amino acid sequence of any
non-ToTV virus or closest relative.
[0109] Sequence divergence of ToTV strains around the world may be
somewhat higher, in analogy with other plant viruses.
[0110] The invention provides an isolated virus (ToTV) identifiable
as phylogenetically corresponding thereto by determining a nucleic
acid sequence of a suitable fragment of the genome of said virus
and testing it in phylogenetic analyses wherein maximum likelihood
trees are generated using for instance 1000 bootstraps as described
above and finding it to be phylogenetically more closely related to
a virus isolate comprising the sequences of SEQ ID NO:1 or SEQ ID
NO:2, as listed herein for ToTV than it is related to a virus
isolate of a non-ToTV reference strain or closest relative.
[0111] Suitable nucleic acid genome fragments each useful for such
phylogenetic analyses are for example any portion of nucleic acid
sequences of the 5.5 kb or 8 kb RNA fragments (respectively termed
herein RNA2 and RNA1) as described in the Example.
[0112] With the provision of the sequence information of this ToTV
virus, the invention provides diagnostic means and methods to be
employed in the detection of ToTV virus in a sample. Preferably,
the detection of ToTV virus is performed with reagents that are
most specific for ToTV virus. This by no means however excludes the
possibility that less specific, but sufficiently cross-reactive
reagents are used instead, for example because they are more easily
available and sufficiently address the task at hand.
[0113] The invention for example provides a method for detecting
the presence of ToTV in plants, preferably in tomato plants, more
preferably in plants of S. lycopersicum. Said method may for
instance comprise determining in said sample the presence of a ToTV
virus or component thereof by reacting said sample with a ToTV
specific nucleic acid or antibody according to the invention.
Although contact-infection of an indicator plant is also a suitable
method for detecting the presence of virus in a test plant.
[0114] The invention provides the partial nucleotide sequence of a
novel isolated virus (herein also called ToTV virus) and ToTV
virus-specific components or synthetic analogues thereof.
Additional genomic sequences of the ToTV virus to those provided
herein may be determined by sequencing methods known to the skilled
person. Genomic sequence determination is well within reach of the
skilled person now that the present invention provides the ToTV
virus, as well as partial genomic sequences thereof. These methods
comprise for instance those described in the Example below. In
general such sequencing methods include the isolation of the viral
genome nucleic acids by nucleic acid isolation procedures, and the
determination of the nucleotide sequence of the isolated nucleic
acid, for instance by dideoxy chain termination methods (Sanger et
al., 1977) optionally preceded by reverse transcription of RNA into
DNA.
[0115] The invention provides among others an isolated or
recombinant nucleic acid or virus-specific functional fragment
thereof obtainable from a virus according to the invention. The
isolated or recombinant nucleic acids comprise the sequences as
listed herein or sequences of homologues, which are able to
hybridise with those under stringent conditions. In particular, the
invention provides primers and/or probes suitable for identifying a
ToTV virus nucleic acid. Additional probes and primers capable of
hybridising to a nucleic acid sequence of the ToTV virus may be
developed by methods known to the skilled person.
Expression Vectors and Expression of Viral Encoding Genes
[0116] Furthermore, the invention relates to an expression vector
comprising a nucleic acid according to the invention. To begin
with, expression vectors such as plasmid vectors containing (parts
of) a double stranded sequence of the ToTV viral genome, viral
vectors containing (parts of) the genome of ToTV (for example, but
not limited to vaccinia virus, retroviruses, baculovirus), or ToTV
virus containing (parts of) the genome of other viruses or other
pathogens are part of the present invention.
[0117] The expression vector may comprise a ToTV genomic sequence
or part thereof that is under control of or operatively linked to a
regulatory element, such as a promoter. The segment of DNA referred
to as the promoter is responsible for the regulation of the
transcription of DNA into mRNA. The expression vector may comprise
one or more promoters suitable for the expression of the gene,
preferably for the expression of viral protein-encoding gene, in
plant cells, fungal cells, bacterial cells, yeast cells, insect
cells or other eukaryotic cells. Expression vectors of the
invention are very useful to provide antigens of the virus in gene
expression systems.
[0118] Also, the invention pertains to a host cell comprising a
nucleic acid or an expression vector according to the invention.
Plasmid or viral vectors containing the nucleic acids encoding
protein components of ToTV virus may be generated in prokaryotic
cells for the expression of the components in relevant cell types
(plant cells, fungal cells, bacteria, insect cells, plant cells or
other eukaryotic cells). Plasmid or viral vectors containing
full-length or partial copies of the ToTV virus genome may be
generated in prokaryotic cells for the expression of viral nucleic
acids in vitro or in vivo.
Methods for Isolation and Purification of ToTV
[0119] The ToTV virus may be isolated from infected plants or other
sources by any method available. Isolation may comprise purifying
or partially purifying ToTV viral particles from a suitable source.
A wide range of methods is available for virus isolation and
purification (for instance, see Dijkstra and De Jager, 1998).
Purification of ToTV may for instance be performed by using
standard procedures for e.g. nepoviruses or luteoviruses (with the
aid of organic solvents) (see e.g. Walker, 2004). Although such
protocols may result in loss of infectivity of the virus, these
procedures may still be useful to obtain virus material for other
purposes.
[0120] Preferably, in order to maintain viral integrity, a mild
purification method is used to purify ToTV. Such a mild
purification method may for instance comprise homogenizing infected
material of a plant (such as leaves) in a homogenization buffer
(comprising for instance 0.1 M TRIS-HCl buffer at a pH of about 8,
about 20 mM of sodium sulphite [Na.sub.2SO.sub.3], about 10 mM of
sodium diethyldithiocarbamate [Na-DIECA] and about 5 mM of sodium
ethylenediaminetetraacetate (Na-EDTA)), separating the debris by
centrifugation (for instance for 30 min. at 49,000 g), placing the
supernatant on a suitable sucrose cushion (e.g. 20%) and pelleting
the viral particles (for instance by centrifuging for 1.5 h at
70,000 g). Thereafter, the pellet comprising the viral particles
may be resuspended in a suitable buffer (e.g. in TRIS-HCl, pH 8),
and the virus-containing fraction may be separated from the
remainder of the suspension by using density centrifugation in a
sucrose gradient (e.g. 10-40% sucrose in homogenization buffer
centrifuged for 2 h at 110,000 g). The virus-containing fraction
may be determined by using infection experiments. Further
purification may occur by using density centrifugation in a cesium
sulfate gradient (e.g. 10-40% cesium sulfate gradient in TRIS-HCl,
pH 8, centrifuged for 16 h at 125,000 g). Enriched viral bands may
then be collected from the gradient and further concentrated by
centrifugation or dialysis (e.g. against 0.1 M TRIS-HCl, pH 8).
[0121] Infectivity of ToTV after purification may be checked by
inoculation onto a sensitive plant (e.g. N. hesperis `67A`).
Methods for Purification of ToTV Associated Proteins and Amino Acid
Sequencing
[0122] Having prepared purified or partially purified ToTV virus it
is possible to prepare a substantially pure preparation of a
viral-associated protein (e.g. virus-encoded proteins). Although
numerous methods and strategies for protein purification are known
in the art it will be most convenient to purify ToTV viral
proteins, such as viral coat proteins, by either electrophoresis
using for instance a sodium dodecylsulphate-polyacrylamide gel
(SDS-PAGE) or by affinity chromatography. Each of these methods
will be described below.
[0123] Proteins of interest of ToTV may be separated by
electrophoresis using for instance Tricine-SDS-PAGE (Schagger and
Von Jagow, 1987) or Glycine-SDS-PAGE (Laemmli, 1970). Other
electrophoresis systems that are capable of resolving the various
proteins comprised in the virus isolate, or transcribed from its
genome and expressed in a suitable expression system, may of course
also be employed, such as non-denaturing gel electrophoresis. The
area of the PAGE gel including the target protein may be excised
and the target polypeptides may be eluted therefrom, for instance
by using an Elutrap.RTM. device (Schleicher & Schuell, Dassel,
Germany). A target protein may be identified by its mobility
relative to reference polypeptides in a gel. To increase purity the
eluted protein may be run on a second SDS-PAGE gel and eluted a
second time. The protein or peptide contained in the excised gel
fragment may then be eluted again and is suitable for use in
immunization or in protein sequencing.
[0124] Proteins of interest of ToTV may also be purified by
affinity chromatography using an antibody (such as a monoclonal
antibody) that specifically binds to a ToTV protein. The antibody
may be covalently coupled to solid supports such as celluloses,
polystyrene, polyacrylamide, cross-linked dextran, beaded agarose
or controlled pore glass using bifunctional coupling agents that
react with functional groups on the support and functional groups
(i.e., reactive amino acid side chains) on the antibody molecule.
Such methods are readily available to the skilled person. The
resulting antibody-bearing solid phase is contacted with purified
or partially purified virus under reducing conditions using pH,
ionic strength, temperature and residence times that permit the
protein of interest to bind to the immobilized antibody. The virus
or protein is eluted from the column by passing an eluent that
dissociates hydrogen bonds through the bed. Buffers at specific pH
or NaCl solutions above about 2 M are commonly used eluents.
[0125] Methods for carrying out affinity chromatography using
antibodies as well as other methods for immunoaffinity purification
of proteins (such as viral capsid proteins) are well known in the
art (see e.g., Harlow and Lane, 1988).
[0126] With the teachings provided herein, the skilled person is
capable of isolating a virus-specific protein of ToTV, determining
the amino acid sequence of for instance the N-terminal part of said
protein, designing a set of degenerate probes (for the degeneracy
of the genetic code) to hybridise with the DNA coding for a region
of said protein, using these probes on an array of genes in a
genomic library produced from the virus, obtaining positive
hybridisations and locating the corresponding genes. The skilled
person is then capable of identifying the structural region of the
gene and optionally upstream and downstream sequences thereof.
Thereafter the skilled person is capable of establishing the
correct sequence of the amino acid residues that form the
protein.
Antibody Production
[0127] Antibodies, either monoclonal or polyclonal, can be
generated to a purified or partially purified protein or peptide
fragment of the ToTV virus in a variety of ways known to those
skilled in the art including injection of the protein as an antigen
in animals, by hybridoma fusion, and by recombinant methods
involving bacteria or phage systems (see Marks et al., 1992a; Marks
et al., 1992b; Lowman et al., 1991; Lerner et al., 1992, each of
which reference discloses suitable methods).
[0128] Antibodies against viral particles, proteins or peptides of
the virus may be produced by immunizing an appropriate vertebrate,
preferably mammalian host, e.g., rabbits, goats, rats, chicken and
mice with the particles, proteins or peptides alone or in
conjunction with an adjuvant. Usually two or more immunizations
will be involved, and the blood or spleen will be harvested a few
days after the last injection. For polyclonal antisera, the
immunoglobulins may be precipitated, isolated and (affinity)
purified. For monoclonal antibodies, the splenocytes will normally
be fused with an immortalized lymphocyte, e.g., a myeloid line,
under selective conditions for hybridomas. The hybridomas may then
be cloned under limiting dilution conditions and their supernatants
screened for antibodies having the desired specificity. Techniques
for producing (monoclonal) antibodies and methods for their
preparation and use in various procedures are well known in the
literature (see e.g. U.S. Pat. Nos. 4,381,292, 4,451,570, and
4,618,577; Harlow and Lane, 1988; Ausubel, et al., 1998; Rose et
al., 1997; Coligan et al., 1997). Typically, an antibody directed
against a virus-associated protein will have a binding affinity of
at least 1.times.10.sup.5-1.times.10.sup.7 M.sup.-1.
[0129] A recombinant protein derived from the ToTV virus, such as
may be obtained by expressing a protein-encoding genomic sequence
of the virus in a suitable expression system, is preferred as the
antigen. However, purified proteins may also be used. Antigens
suitable for antibody detection include any ToTV protein that
combines with any ToTV-specific antibody of a mammal exposed to or
infected with ToTV virus. Preferred antigens of the invention
include those that bring about the immune response in mammals
exposed to ToTV, which therefore, typically are recognised most
readily by antibodies of a mammal. Particularly preferred antigens
include the capsid proteins of ToTV. Structural proteins from
purified virus are the most preferred.
[0130] Methods for cloning genomic sequences, for manipulating the
genomic sequences to and from expression vectors, and for
expressing the protein encoded by the genomic sequence in a
heterologous host are well-known, and these techniques can be used
to provide the expression vectors, host cells, and the cloned
genomic sequences encoding antigens, which sequences are to be
expressed in a host to produce antibodies for use in diagnostic
assays (see for instance Sambrook et al., 2001 and Ausubel, et al.,
1998).
[0131] A variety of expression systems may be used to produce ToTV
antigens. For instance, a variety of expression vectors suitable to
produce proteins in E. coli, B. subtilis, yeast, insect cells,
plant cells and mammalian cells have been described, any of which
might be used to produce a ToTV antigen suitable to produce an
anti-ToTV antibody or fragment thereof. Of course ToTV itself may
also be used as an expression vector for this purpose.
[0132] One use of antibodies of the invention is to screen cDNA
expression libraries for identifying clones containing cDNA inserts
that encode proteins of interest or structurally-related,
immuno-cross-reactive proteins. Such screening of cDNA expression
libraries is well known in the art (see e.g. Young and Davis,
1983), to which reference is made in this context, as well as other
published sources. Another use of these antibodies is for use in
affinity chromatography for purification of ToTV proteins. These
antibodies are also useful for assaying for ToTV infection.
[0133] The present invention thus provides a ToTV-specific viral
protein or a fragment thereof hereinafter termed proteinaceous
molecule. Useful proteinaceous molecules are for example derived
from any of the genomic sequences or fragments thereof derivable
from a virus according to the invention. Such proteinaceous
molecules, or antigenic fragments thereof, as provided herein, are
for example useful in diagnostic methods or kits and in diagnostic
compositions. Particularly useful are those proteinaceous molecules
that are encoded by recombinant nucleic acid fragments that are
identified for eliciting ToTV virus specific antibodies, whether in
vivo (e.g. for providing diagnostic antibodies) or in vitro (e.g.
by phage display technology or another technique useful for
generating synthetic antibodies or parts thereof).
[0134] Also provided herein are antibodies, be it natural
polyclonal or monoclonal, or synthetic antibodies (e.g. (phage)
library-derived binding molecules) that specifically react with an
antigen comprising a proteinaceous molecule or ToTV virus-specific
functional fragment thereof, such as a capsid protein according to
the invention.
Methods for Identifying a Viral Isolate as a ToTV Virus
[0135] Apart from the detection of ToTV virus, which involves
diagnostic methods, the present invention also relates to methods
for identification, i.e. confirmation that the isolate is ToTV.
Such methods may be based on phylogenetic inference as described
above, and determining the level of nucleotide or amino acid
sequence homology between an unidentified viral isolate and one or
more reference strains of confirmed ToTV virus and non-ToTV virus.
Such methods may for instance comprise sequencing (part of) the
genome of a viral isolate or of a capsid protein and comparing the
level of homology of that sequence to the sequences as provided
herein for ToTV. An isolate having more than 50% sequence homology
with SEQ ID NO:1 or SEQ ID NO:2 as provided herein is considered
taxonomically corresponding to ToTV or belonging to the ToTV viral
taxon. Such a virus is part of the present invention.
[0136] In order to identify a virus as a Tomato torrado virus
(ToTV), one may also make use of the taxonomic descriptors as
presented in Table 1 above, and find by comparison that a new
isolate belongs to the presumptive novel genus, of which the ToTV
strain identified by the nucleic acid sequences of SEQ ID NOs: 1
and 2 provided herein, and deposited with the Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH on 24 Nov. 2004 under
depositors reference number ToTV-E01 (DSM 16999) may be assigned as
the type species. Thus, it is not essential that a sequence
comparison is performed in order to assess that a virus is ToTV.
Rather, a method of identifying a virus as a Tomato torrado virus
(ToTV) may comprise the steps of assessing the presence of the
combination of taxonomic descriptors selected from the group
consisting of
[0137] a) morphological descriptors, such as spherical,
non-enveloped virion particles of approximately 28 nm in
diameter;
[0138] b) genome properties descriptors, such as having single
stranded linear positive sense RNA virus properties based on two
RNA segments that comprise poly(A)-tails, that encode polyproteins
of 5.5 and 8 kDa, respectively, and that comprise coding regions or
motifs for 3 capsid proteins, helicase, protease, RdRP and putative
movement protein and which RNA segments and/or polyproteins and/or
motifs have homologies based on sequence comparison essentially as
described herein, and
[0139] c) biological properties descriptors, such as producing the
necrotic lesions and burn-like symptoms in tomato, having a host
range, vector relationship and/or geographical distribution
essentially as described herein, and being associated with the
diseases of tomato plants locally known under such names as
torrado, marchitez and/or chocolate spot.
[0140] The combination of taxonomic descriptors that results in a
positive identification of a virus isolate as being a Tomato
torrado virus (ToTV) is that combination which shows the isolate as
being more closely related (based on numerically taxonomic methods
well known to the skilled artisan) to ToTV as described herein,
than to other viruses, and wherein said isolate preferably produces
the disease symptoms in tomato typical of torrado as described
herein.
[0141] Thus, a virus which, based on numerical taxonomic analysis
of taxonomic descriptors essentially as defined in Table 1, shows
to be more closely related to the virus as defined in claim 1
herein than to any other virus known at the time of filing of the
present application, and which virus is associated with a disease
that causes necrotic lesions in tomato, is considered herein to be
a ToTV and falls within the scope of the present invention.
[0142] In this way the invention provides a viral isolate
identifiable with a method according to the invention as a plant
virus taxonomically corresponding to a virus identifiable as likely
belonging to the ToTV viral taxon. Depending upon the phylogenetic
relatedness, or distinctness with other viral taxa, the ToTV viral
taxon may be an isolate, a species, a genus or even a family of
virus.
[0143] Alternatively, methods for identifying a viral isolate as a
ToTV virus may be based on symptomatology, i.e. recognition of the
virus by its disease-symptoms.
[0144] However, in a preferred embodiment, antibodies of the
invention are used in a method for identifying a viral isolate as a
ToTV virus, provided cross-reactivity of such antibodies with
related non-ToTV strains has been effectively ruled out. Such
methods comprise the step of reacting said viral isolate or a
component thereof with an antibody as provided herein. Reacting is
herein referred to as allowing the occurrence of antibody-antigen
bonding. This can for example be achieved by using purified or
non-purified ToTV virus or parts thereof (proteins, peptides).
Preferably, infected cells or cell cultures are used to identify
viral antigens using any suitable immunological method.
Specifically useful in this respect are antibodies raised against
ToTV viral capsid proteins.
[0145] Other preferred methods for identifying a viral isolate as a
ToTV virus comprise reacting said viral isolate or a component
thereof with a virus specific polynucleotide according to the
invention, which polynucleotide is capable of hybridizing under
stringent conditions to a nucleic acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, sequences having a
nucleotide sequence homology of at least 60% to SEQ ID NO:1 or SEQ
ID NO:2, and their complementary strands and ToTV-specific
fragments thereof. Such a hybridization reaction may be performed
in any format available to the skilled person and will generally
involve tissue printing, dot blot methods, Southern/Northern
blotting or hybridization, in situ hybridization, PCR, RT-PCR and
the like.
Immunological Detection Methods
[0146] Methods of the invention in which antigens are detected can
in principle be performed by using any immunological method, such
as for instance classical immunofluorescence (IF),
immunohistochemical techniques or comparable antigen detection
assay formats. Preferred ToTV detection methods based on detection
of the viral coat protein may for instance comprise such methods as
precipitation and agglutination tests, radio-immunoassay (RIA),
immunogold labeling, immunosorbent electron microscopy (ISEM),
enzyme-linked immunosorbent assay (ELISA), Western blotting and
immunoblotting. Examples of types of immunoassays that can utilize
antibodies of the invention are competitive and non-competitive
immunoassays in either a direct or indirect format. Antibodies can
be utilized in immunoassays in the liquid phase or bound to a solid
phase carrier. In addition, the antibodies in these immunoassays
can be detectably labeled in various ways. Those skilled in the art
will know, or can readily discern, suitable immunoassay formats
without undue experimentation. Assay formats are well known in the
literature and are described, for example, in Harlow and Lane
(1988).
[0147] A variety of immunoassay formats may be used to select
antibodies specifically reactive with a particular polypeptide
according to the invention, such as ToTV viral coat proteins. For
example, solid-phase ELISA immunoassays are routinely used for this
purpose. See Harlow and Lane (1988), for a description of
immunoassay formats and conditions that can be used to determine
selective binding.
[0148] Antibodies can be bound to many different carriers and used
to detect the presence of the target molecules. Alternatively,
antigens may be bound to many different carriers and used to detect
the presence of the antibody. Examples of well-known carriers
include glass, polystyrene, polypropylene, polyethylene, dextran,
nylon, amylases, natural and modified celluloses, polyacrylamides,
agaroses and magnetite. The nature of the carrier can be either
soluble or insoluble for purposes of the invention. Those skilled
in the art will know of other suitable carriers for binding
antibodies, or will be able to ascertain such using routine
experimentation.
[0149] Western blot assays are described generally in Harlow and
Lane (1988). According to this method the viral proteins (and other
proteins in the virus preparation) are separated by gel
electrophoresis and transferred to a solid phase (i.e., a membrane
such as nitrocellulose). The immobilized antigen is subsequently
reacted with an antibody and detection system (e.g., an alkaline
phosphatase-conjugated second antibody). As will be apparent to
those of skill, it will be advantageous to include appropriate
negative and positive control materials (such as substantially
purified antigen or ToTV virus) in the assay.
[0150] Enzyme-Linked Immuno-Sorbent Assays (ELISA) are described
generally in Harlow and Lane (1988). The assay involves the
reaction of a viral component (e.g., a capsid protein) with an
antibody. In one embodiment the sample may comprise a plant tissue
which is ground and reacted to the antibody that has been coated
onto a solid phase such as a test plate. If the virus is present in
the sample, an enzyme-labeled specific antibody will bind to the
antibody-virus complex, and it will be detected by an enzyme
substrate reaction that produces a color reaction. Preferred
methods of ELISA analysis are direct double antibody sandwich (DAS)
ELISA (Clark and Adams, 1977), DAS indirect ELISA (Vela et al.
1986), or TAS-ELISA. It will be apparent to those skilled in the
art that in an ELISA or any other type of assay it will sometimes
be desirable to determine the presence of more than one viral
capsid protein in a single reaction, for example by mixing two or
more antibodies with different specificities and assaying for
either or all of the protective capsid-associated proteins of the
invention.
Nucleic Acid Based Detection Methods
[0151] ToTV is composed of at least two ribonucleic acids (RNAs).
There are strong indications for the presence of three protective
capsid proteins. The methods described above are focused on
immunological detection of viral proteins for the detection of the
virus. By recombinant DNA technology it is possible to produce
probes that directly or indirectly hybridize to the viral RNAs (or
their complement), or cDNA produced therefrom by reverse
transcription, and which can be used in assays for the detection of
the virus. Nucleic acid amplification techniques allow the
amplification of fragments of viral nucleic acids, which may be
present in very low amounts.
[0152] In order to develop nucleic acid-based detection methods,
virus-specific sequences must be determined for which primers or
probes may then be developed. To detect ToTV by nucleic acid
amplification and/or probe hybridization, the capsid protein of
ToTV may be sequenced or, alternatively, the viral genomic RNA may
be isolated from purified virus, reverse transcribed into cDNA and
directly cloned and/or sequenced. Using either the cloned nucleic
acid as a hybridization probe, using sequence information derived
from the clone, or by designing degenerative primers based on the
amino acid sequence of the ToTV protein, nucleic acid hybridization
probes and/or nucleic acid amplification primers may be designed an
used in a detection assay for detecting the presence of the virus
in a sample as defined herein.
[0153] Methods of the invention in which nucleic acids are detected
can in principle be performed by using any nucleic acid
amplification method, such as the Polymerase Chain Reaction (PCR;
Mullis and Faloona, 1987; U.S. Pat. Nos. 4,683,195; 4,683,202; and
4,800,159) or by using amplification reactions such as Ligase Chain
Reaction (LCR; Barany, 1991; EP 0 320 308), Self-Sustained Sequence
Replication (3SR; Guatelli et al., 1990), Strand Displacement
Amplification (SDA; Walker et al., 1992; U.S. Pat. Nos. 5,270,184
and 5,455,166), Transcriptional Amplification System (TAS; Kwoh et
al., 1989), Q-Beta Replicase (Lizardi et al., 1988), Rolling Circle
Amplification (RCA; U.S. Pat. No. 5,871,921), Nucleic Acid Sequence
Based Amplification (NASBA; Compton, 1991), Cleavase Fragment
Length Polymorphism (U.S. Pat. No. 5,719,028), Isothermal and
Chimeric Primer-initiated Amplification of Nucleic Acid (ICAN),
Ramification-extension Amplification Method (RAM; U.S. Pat. Nos.
5,719,028 and 5,942,391) or other suitable methods for
amplification of nucleic acids.
[0154] Since the virus is an RNA virus (i.e. the sequences of FIGS.
5 and 6 are the DNA equivalent of the viral RNA genome), a suitable
detection method may comprise isolating the viral nucleic acids
from a sample, for instance from an infected plant, by using
methods known per se to the skilled person (e.g. Chomczynski and
Sacchi, 1987; Boom et al., 1990) or commercially available systems
(e.g. the RNeasy total RNA isolation kit or RNeasy plant RNA
isolation kit from QIAGEN GmbH, Hilden, Germany, or the
High-Pure-RNA-Isolation-Kit.RTM. (Roche Diagnostics, a division of
F. Hoffmann-La Roche Ltd, Basel, Switzerland).
[0155] Total RNA may for instance be extracted from leaf material
or protoplasts of plant cells and the total RNA, or specifically
the viral genomic RNA, or a part thereof, may then be reverse
transcribed into cDNA by using for instance an Auian myeloblastosis
virus (AMV) reverse transcriptase or Moloney murine leukemia virus
(M-MuLV) reverse transcriptase. A suitable method may for instance
include mixing into a suitable aqueous buffering system (e.g. a
commercially available RT buffer) a suitable amount of total RNAs
(e.g. 1 to 5 .mu.g), a suitable amount (e.g. 10 pmol) of a reverse
transcription primer, a suitable amount of dNTPs and the reverse
transcriptase, denaturing the nucleic acids by boiling for 1 min,
and chilling them on ice, followed by reverse transcription at for
instance 45.degree. C. for 1 h as recommended for the specific
reverse transcriptase used, to obtain cDNA copies of the viral
sequences.
[0156] As a reverse transcription primer a polynucleotide according
to the present invention may be used, for instance an 18-25-mer
oligonucleotide comprising a nucleotide sequence complementary to
the ToTV genomic sequence or preferably at least capable of
hybridizing under stringent conditions to the nucleic acid sequence
of SEQ ID NO:1 or SEQ ID NO:2, or a ToTV-specific fragment thereof.
Alternatively, an a-specific polyT primer (oligo dT primer) may be
used in order to start reverse transcription from polyA RNA
motifs.
[0157] Following the RT-step, the cDNA obtained may be PCR
amplified by using for instance Pfu and Taq DNA polymerases and
amplification primers specific for the viral genomic cDNA
sequences. Also complete commercially available systems may be used
for RT-PCR (e.g. the Access and AccessQuick.TM. RT-PCR Systems of
Promega [Madison Wis., USA], or the Titan.TM. One Tube RT-PCR
System or two-step RT-PCR systems provided by Roche Diagnostics [a
division of F. Hoffmann-La Roche Ltd, Basel, Switzerland]).
[0158] In order to amplify a nucleic acid with a small number of
mismatches to one or more of the amplification primers, an
amplification reaction may be performed under conditions of reduced
stringency (e.g. a PCR amplification using an annealing temperature
of 38.degree. C., or the presence of 3.5 mM MgCl.sub.2). The person
skilled in the art will be able to select conditions of suitable
stringency.
[0159] The primers herein are selected to be "substantially"
complementary (i.e. at least 65%, more preferably at least 80%
perfectly complementary) to their target regions present on the
different strands of each specific sequence to be amplified. It is
possible to use primer sequences containing e.g. inositol residues
or ambiguous bases or even primers that contain one or more
mismatches when compared to the target sequence. In general,
sequences that exhibit at least 65%, more preferably at least 80%
homology with the target DNA or RNA oligonucleotide sequences, are
considered suitable for use in a method of the present invention.
Sequence mismatches are also not critical when using low stringency
hybridization conditions.
[0160] The detection of the amplification products can in principle
be accomplished by any suitable method known in the art. The
amplified fragments may be directly stained or labelled with
radioactive labels, antibodies, luminescent dyes, fluorescent dyes,
or enzyme reagents. Direct DNA stains include for example
intercalating dyes such as acridine orange, ethidium bromide,
ethidium monoazide or Hoechst dyes.
[0161] Alternatively, the DNA or RNA fragments may be detected by
incorporation of labelled dNTP bases into the synthesized
fragments. Detection labels which may be associated with nucleotide
bases include e.g. fluorescein, cyanine dye, digoxigenin (DIG) or
bromodeoxyuridine (BrdUrd).
[0162] When using a probe-based detection system, a suitable
detection procedure for use in the present invention may for
example comprise an enzyme immunoassay (EIA) format (Jacobs et al.,
1997). For performing a detection by manner of the EIA procedure,
either the forward or the reverse primer used in the amplification
reaction may comprise a capturing group, such as a biotin group for
immobilization of target DNA PCR amplicons on e.g. a streptavidin
coated microtiter plate wells or streptavidin coated Dynabeads.RTM.
(Dynal Biotech, Oslo, Norway) for subsequent EIA detection of
target DNA-amplicons. The skilled person will understand that other
groups for immobilization of target DNA PCR amplicons in an EIA
format may be employed.
[0163] Probes useful for the detection of the target nucleic acid
sequences as disclosed herein preferably bind only to at least a
part of the nucleic acid sequence region as amplified by the
nucleic acid amplification procedure. Those of skill in the art can
prepare suitable probes for detection based on the nucleotide
sequence of the target nucleic acid without undue experimentation
as set out herein. Also the complementary nucleotide sequences,
whether DNA or RNA or chemically synthesized analogues, of the
target nucleic acid may suitably be used as type-specific detection
probes in a method of the invention, provided that such a
complementary strand is amplified in the amplification reaction
employed.
[0164] Suitable detection procedures for use herein may for example
comprise immobilization of the amplicons and probing the nucleic
acid sequences thereof by e.g. Northern and Southern blotting.
Other formats may comprise an EIA format as described above. To
facilitate the detection of binding, the specific amplicon
detection probes may comprise a label moiety such as a fluorophore,
a chromophore, an enzyme or a radio-label, so as to facilitate
monitoring of binding of the probes to the reaction product of the
amplification reaction. Such labels are well known to those skilled
in the art and include, for example, fluorescein isothiocyanate
(FITC), .beta.-galactosidase, horseradish peroxidase, streptavidin,
biotin, digoxigenin, .sup.35S, .sup.14C, .sup.32P or .sup.125I.
Other examples will be apparent to those skilled in the art.
[0165] Detection may also be performed by a so called reverse line
blot (RLB) assay, such as for instance described by Van den Brule
et al. (2002). For this purpose RLB probes are preferably
synthesized with a 5' amino group for subsequent immobilization on
e.g. carboxyl-coated nylon membranes. The advantage of an RLB
format is the ease of the system and its speed, thus allowing for
high throughput sample processing.
[0166] The use of nucleic acid probes for the detection of RNA or
DNA fragments is well known in the art. Mostly these procedures
comprise the hybridization of the target nucleic acid with the
probe followed by post-hybridization washings. Specificity is
typically the function of post-hybridization washes, the critical
factors being the ionic strength and temperature of the final wash
solution. For nucleic acid hybrids, the Tm can be approximated from
the equation of Meinkoth and Wahl (1984): Tm=81.5.degree. C.+16.6
(log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of
monovalent cations, % GC is the percentage of guanosine and
cytosine nucleotides in the nucleic acid, % form is the percentage
of formamide in the hybridization solution, and L is the length of
the hybrid in base pairs. The Tm is the temperature (under defined
ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. Tm is reduced by
about 1.degree. C. for each 1% of mismatching; thus, the
hybridization and/or wash conditions can be adjusted to hybridize
to sequences of the desired identity. For example, if sequences
with >90% identity are sought, the Tm can be decreased
10.degree. C. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (Tm) for
the specific sequence and its complement at a defined ionic
strength and pH. However, severely stringent conditions can utilize
a hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than
the thermal melting point (Tm); moderately stringent conditions can
utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C.
lower than the thermal melting point (Tm); low stringency
conditions can utilize a hybridization and/or wash at 11, 12, 13,
14, 15, or 20.degree. C. lower than the thermal melting point (Tm).
Using the equation, hybridization and wash compositions, and
desired Tm, those of ordinary skill will understand that variations
in the stringency of hybridization and/or wash solutions are
inherently described. If the desired degree of mismatching results
in a Tm of less than 45.degree. C. (aqueous solution) or 32.degree.
C. (formamide solution) it is preferred to increase the SSC
concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, 1993; Ausubel et al., 1998.
[0167] In another aspect, the invention provides oligonucleotide
probes for the detection of ToTV RNA or cDNA. The detection probes
herein are selected to be "substantially" complementary to a single
stranded RNA molecule, or to one of the strands of the double
stranded nucleic acids generated by an amplification reaction of
the invention. Preferably the probes are substantially
complementary to the, optionally immobilized (e.g. biotin labelled)
antisense strands of the amplicons generated from the target RNA or
DNA.
[0168] It is allowable for detection probes of the present
invention to contain one or more mismatches to their target
sequence. In general, sequences that exhibit at least 65%, more
preferably at least 80% homology with the target oligonucleotide
sequences are considered suitable for use in a method of the
present invention.
ToTV Resistant Plants
[0169] The invention further relates to a method for identifying a
ToTV-resistant plant, or part thereof. There are various
possibilities of identifying ToTV-resistant plants. In a first set
of embodiments of such a method, active/infectious virus or
full-length infectious clones may be used, whereas in an
alternative embodiment only virus-detection means are used.
[0170] A first step of a method for identifying a ToTV-resistant
plant using active/infectious virus comprises exposing a plant or
plant part, such as a leaf or stem segment, to a infective dosage
of ToTV, the aim of which is to achieve an infection. The exposure
may in many cases involve the establishment of physical contact. An
infective dosage may vary between plants and between ToTV-isolates
tested. Theoretically, an amount of about 1 to 10 to an amount of
about 500-5000 viral particles of said virus or the nucleic acids
thereof will be sufficient. Infection in this way may be achieved
by mechanical inoculation of purified virus particles or virus
nucleic acid on healthy plants.
[0171] Alternatively, infection may be achieved by, for instance:
[0172] growing a healthy scion on a ToTV-infected rootstock, or
vice versa; [0173] exposing a healthy plant to transmission vectors
containing the virus (including infected plants, e.g. parasitic
plants like Cuscuta spp.); [0174] introducing into a healthy plant
an expression vector harbouring a coding region of the ToTV virus
genome; [0175] the use of agro-infectious clones, such as
Agrobacterium tumefaciens strains containing an expression vector
harbouring a coding region of the ToTV virus genome.
[0176] In the context of the present invention, methods for
exposing a plant or plant part to an infective dosage of ToTV are
not limited to any particular method.
[0177] As stated, infection may comprise mechanical inoculation of
the virus on healthy plants. For instance, a portion of a diseased
leaf may be rubbed directly onto a leaf of a plant that is to be
infected. In an alternative procedure, an inoculum may for instance
be prepared by grinding virus-containing plant tissue, preferably
young leaves showing symptoms, with a mortar and pestle, or any
other suitable type of homogeniser, in for instance a buffer
suitable for inoculation (e.g. a 0.03 M phosphate buffer, pH 7.7).
After grinding, the obtained homogenate (the sap) is preferably
filtered, e.g. through cheese cloth. The sap may then be
inoculation, for instance by gently contacting leaves with an
amount of the sap. The leaves are preferably pre-treated in order
to damage the lower epidermis and enhance entry of the virus. This
may for instance be achieved by pre-dusting the leaves with
carborundum powder. Excessive wounding is preferably avoided.
Preferably a carborundum powder is used having microscopically
small angular particles of silicon carbide (400-500 mesh).
Carborundum powder may also be added directly to the sap, in which
case the pre-treatment is omitted. The sap may, for instance, be
applied by the forefinger, a pad of sap-soaked foam or fabric, or
even with the pestle used for grinding, a glass spatula, a stiff
brush, or a spray gun. After inoculation, the leaves are preferably
immediately washed with water.
[0178] A second step of a method for identifying a ToTV-resistant
plant comprises identifying said plant as a ToTV-resistant plant
when, after said exposure, either i) disease-symptoms in said plant
or plant part remain absent or are delayed in expression or are at
least reduced in severity or are localized relative to a
susceptible and/or sensitive control plant, and/or ii) ToTV virus
or ToTV genomic sequences are not present in said plant or plant
part or the presence of ToTV virus is at least quantitatively
reduced in said plant relative to a susceptible control plant. As
used herein the term localized means limited to the inoculated
leaf.
[0179] Determining the development of ToTV-induced disease-symptoms
in infected plants may be performed by quantitative methods, e.g.
wherein the period required for the development of discernible
(e.g. visible) disease-symptoms is noted, or by qualitative methods
wherein, after a certain period has lapsed, the plant is inspected
for symptom expression and the presence or severity of the symptoms
is indicated.
[0180] In addition to determining the development of ToTV-induced
disease-symptoms or as an alternative thereto, depending on the
type of ToTV-resistance to be detected, the presence of the virus
is detected in the plant or plant part. In order to detect the
absence of virus in the test plants, any method may in principle be
used. For instance, a method may be employed wherein a ToTV
specific antibody, primer-set or probe according to the present
invention is used. Alternatively, a portion of the test plant may
be brought into contact with a susceptible indicator plant (e.g. N.
hesperis `67A`) to establish whether virus is present or absent in
the test plant. The skilled person will understand that for such
methods it is important to decontaminate the surface of the test
plant, in order to distinguish between a transmission vector, a
tolerant test-plant and a resistant test plant, since only the
presence of virus in the plant cells needs to be established.
[0181] In performing the second step of a method for identifying a
ToTV-resistant plant, the following results may be obtained. If,
after successful inoculation (e.g. after the establishment of a
plant-virus contact under conditions that would result in infection
in a susceptible and sensitive control plant):
[0182] i) disease-symptoms remain absent; or viral particles, or
viral RNA cannot be detected: the plant is resistant;
[0183] ii) disease-symptoms are delayed or reduced in severity; or
systemic low titres of viral particles or viral RNA can be
detected: the plant is partially resistant;
[0184] iii) disease-symptoms are severe, but remain local, limited
to the inoculated leaf and do not systemically spread beyond
inoculated tissue; or viral particles, or viral RNA can only be
detected locally: the plant is hypersensitive;
[0185] iv) if disease-symptoms remain absent; and viral particles,
or viral RNA can be detected: the plant is tolerant.
[0186] v) if the plant develops disease-symptoms and has high
systemic virus titres, then the plant is susceptible and sensitive.
Examples of such plants are the plants from which the virus of the
present invention was isolated. These plants may serve as suitable
control plants in methods of the present invention.
[0187] For the purpose of producing resistant plants, and from a
viewpoint of phytosanitation, only outcome i), ii) and iii) may be
considered of interest. For the purpose of obtaining plants
suitable for the production of symptomless crops and products,
outcome iv) may also be of particular commercial interest.
[0188] In an alternative embodiment of a method for identifying a
ToTV-resistant plant only virus-detection means are used. For
instance, a ToTV-resistant plant may be identified in the field by
observing or identifying a symptomless plant among symptomatic
plants and determining the absence of virus in said plant by
performing any of the virus detection methods according to the
present invention. In fact, this corresponds to a method for
identifying a ToTV-resistant plant wherein step a) of exposing a
plant or plant part to a infective dosage of ToTV, is performed
passively (e.g. naturally). When such a method is performed it is
preferred that a method for detecting the presence of ToTV in a
sample according to the present invention is used wherein the
presence of a ToTV virus or component thereof is performed by
reacting said sample with a polynucleotide or an antibody according
to the present invention. Preferably, a method of identifying a
ToTV-resistant plant requires the use of either the virus of the
present invention or a polynucleotide or antibody according to the
present invention.
[0189] The invention further relates to a method of producing a
ToTV-resistant plant, or part thereof. Once a ToTV-resistant plant
has been identified, this plant may serve as a donor plant of
genetic material which is to be transferred from said donor plant
to a recipient plant in order to provide said recipient plant with
the genetic material. Transfer of genetic material from a donor
plant to a recipient plant may occur by any suitable method known
in the art. The genetic material will in most cases be genomic
material. It is important however, that at least the
resistance-conferring parts of the donor plant's genome are
transferred. In the absence of methods for determining which parts
of the donor plant's genome confer the ToTV resistance, the
transfer may suitably occur by transferring complete chromosomes.
Preferably, the ToTV-resistant plant serves as a male or female
parent plant in a cross for producing resistant offspring plants,
the offspring plant thereby receiving genomic material from the
resistant donor and acting as the recipient plant. Although a
susceptible parent in crosses is sensu stricto not necessarily a
recipient plant, such a susceptible parent will herein also be
included in the term recipient plant.
[0190] In a method for producing a ToTV-resistant plant, protoplast
fusion can also be used for the transfer of resistance-conferring
genomic material from a donor plant to a recipient plant, i.e. as a
manner of crossing said plants. Protoplast fusion is an induced or
spontaneous union, such as a somatic hybridization, between two or
more protoplasts (cells of which the cell walls are removed by
enzymatic treatment) to produce a single bi- or multi-nucleate
cell. The fused cell, that may even be obtained with plant species
that cannot be interbred in nature, is tissue cultured into a
hybrid plant exhibiting the desirable combination of traits. More
specifically, a first protoplast can be obtained from a tomato
plant or other plant line that exhibits resistance to infection by
ToTV. For example, a protoplast from a ToTV-resistant (tomato,
eggplant, pepper, melon, watermelon or cucumber) line may be used.
A second protoplast can be obtained from a susceptible second plant
line, optionally from another plant species or variety, preferably
from the same plant species or variety, that comprises commercially
desirable characteristics, such as, but not limited to disease
resistance, insect resistance, valuable fruit characteristics, etc.
The protoplasts are then fused using traditional protoplast fusion
procedures, which are known in the art to produce the cross.
[0191] Alternatively, embryo rescue may be employed in the transfer
of resistance-conferring genomic material from a donor plant to a
recipient plant i.e. as a manner of crossing said plants. Embryo
rescue can be used as a procedure to isolate embryo's from crosses
wherein plants fail to produce viable seed. In this process, the
fertilized ovary or immature seed of a plant is tissue cultured to
create new plants (this method is described in detail in Pierik,
1999).
[0192] A method of producing a ToTV-resistant plant thus comprises
in one embodiment the steps of identifying a ToTV-resistant donor
plant as described herein above and crossing said ToTV-resistant
donor plant with a recipient plant, as described above, thereby
producing resistant offspring plants.
[0193] A method of producing a ToTV-resistant plant further
comprises the step of selecting from offspring plants a resistant
plant by performing a method for identifying a ToTV-resistant plant
as described earlier.
[0194] Preferably, said ToTV-resistant donor plant is a plant of
the family Solanaceae or Cucurbitaceae, even more preferably a
tomato plant, an eggplant plant, a pepper plant, melon plant,
watermelon plant or a cucumber plant.
[0195] Preferably, said recipient plant is a plant of the family
Solanaceae or Cucurbitaceae, even more preferably a tomato plant,
an eggplant plant, a pepper plant, a melon plant, a watermelon
plant or a cucumber plant. Still more preferably, said recipient
plant is a tomato plant of the species Solanum lycopersicum, more
preferably an S. lycopersicum plant that possess commercially
desirable characteristics. The recipient plant may be a
ToTV-susceptible plant, a ToTV sensitive plant or a ToTV resistant
recipient plant. As explained above, the choice of the plant will
primarily be determined by whether the resistance trait is dominant
or recessive. The skilled person is aware of the various
methodologies available to resolve such issues.
[0196] Also an aspect of the present invention is a ToTV-resistant
plant, or a part thereof, obtainable by a method of the
invention.
[0197] As stated, a preferred embodiment of a method for producing
a ToTV-resistant plant comprises the transfer by introgression of
said resistance-conferring nucleic acid sequence from a
ToTV-resistant donor plant into a recipient plant by crossing said
plants. Resistant plants developed according to this preferred
embodiment can advantageously derive a majority of their traits
from the recipient plant, and derive ToTV-resistance from the donor
plant.
[0198] In one method, which is referred to as pedigree breeding, a
donor plant that exhibits resistance to ToTV is crossed with a
recipient plant that preferably exhibits commercially desirable
characteristics, such as, but not limited to, disease resistance,
insect resistance, valuable fruit characteristics, etc. The
resulting plant population (representing the F.sub.1 hybrids) is
then self-pollinated and allowed to set seed (F.sub.2 seeds). The
F.sub.2 plants grown from the F.sub.2 seeds are then screened for
resistance to ToTV. The population can be screened in a number of
different ways, preferably by performing a method of the present
invention for visual inspection.
[0199] Because the identification of ToTV-resistant plants has only
at first been possible by the present invention, the method for
producing the resistant plant is an aspect of the invention. Also
an aspect of the present invention is a ToTV-resistant plant, or a
part thereof, obtainable by a method of the invention.
[0200] The present invention provides for methods of preventing the
spreading of ToTV infection in tomato plants by providing resistant
tomato plants as well as by eliminating plants that carry the ToTV
virus. These measures may form a part of a general strategy to
improve phytosanitation in relation to ToTV virus. Tolerant plants
may thus be identified and eliminated in order to eliminate such
sources of the ToTV virus.
[0201] In one embodiment of a method for producing a ToTV-resistant
plant, or part thereof, the present invention provides a method of
producing a ToTV-tolerant plant. A tolerant plant may provide
valuable crop, fruits and seeds, since, although the plant may
harbour the virus, it does not exhibit disease symptoms. Such a
method will involve the identification of tolerant plants, and the
use of such tolerant plants as sources or donors of the desired
genetic material. The aim is not to provide a plant capable of
withstanding entry or multiplication of the virus in its cells, but
to provide a plant which does not suffer from symptoms.
[0202] Thus, the present invention relates to a method for
identifying a ToTV-tolerant plant, comprising the steps of a)
exposing a plant or plant part to a infective dosage of ToTV, and
b) identifying said plant as a ToTV-tolerant plant when, after said
exposure, disease-symptoms in said plant or plant part remain
absent, and ToTV is present in said plant or plant part.
[0203] Determining the development of ToTV-induced disease-symptoms
in infected plants may be performed by quantitative methods, e.g.
wherein the period required for the development of discernible
(e.g. visible) disease-symptoms is noted, or by qualitative methods
wherein, after a certain period has lapsed, the plant is inspected
for the absence of symptom expression or the reduction in severity
of the symptoms is indicated.
[0204] In a preferred embodiment, the presence of ToTV in said
plant or plant part is determined in step b) by performing a method
comprising determining in said plant or plant part the presence of
a ToTV virus or component thereof by reacting said plant or plant
part with a polynucleotide according to the invention or an
antibody according to the invention.
Diagnostic Kits
[0205] Methods and means provided herein are particularly useful in
a diagnostic kit for diagnosing a ToTV virus infection by
virological diagnosis. Such kits or assays may for example comprise
a virus, a nucleic acid, a proteinaceous molecule or fragment
thereof, and/or an antibody according to the invention.
[0206] The invention also provides a diagnostic kit for diagnosing
a ToTV infection comprising a ToTV virus, a ToTV virus-specific
nucleic acid, proteinaceous molecule or fragment thereof and/or an
antibody according to the invention, and preferably a means for
detecting said ToTV virus, ToTV virus-specific nucleic acid,
proteinaceous molecule or fragment thereof and/or an antibody, said
means for example comprising an excitable group such as a
fluorophore or enzymatic detection system used in the art (examples
of suitable diagnostic kit format comprise IF, ELISA,
neutralization assay, RT-PCR assay, hybridisation assays). Suitable
detection assays include direct and indirect assays, sandwich
assays, solid phase assays such as those using plates or beads
among others, and liquid phase assays. Assays suitable include
those that use primary and secondary antibodies, and those that use
antibody binding reagents such as protein A. Moreover, a variety of
detection methods can be used in the invention, including
calorimetric, fluorescent, phosphorescent, chemiluminescent,
luminescent and radioactive methods.
[0207] To determine whether an as yet unidentified virus component
or synthetic analogue thereof such as nucleic acid, proteinaceous
molecule or fragment thereof can be identified as
ToTV-virus-specific, it suffices to analyse the nucleic acid or
amino acid sequence of said component, for example for a stretch of
said nucleic acid or amino acid, preferably of at least 10, more
preferably at least 25, more preferably at least 40 nucleotides or
amino acids (respectively), by sequence homology comparison with
the provided ToTV viral sequences and with known non-ToTV viral
sequences (preferably the closest phylogenetic relative of ToTV is
used) using for example phylogenetic analyses as provided herein.
Depending on the degree of relationship with said ToTV or non-ToTV
viral sequences, the component or synthetic analogue can be
identified.
[0208] A kit for detecting a ToTV virus may, depending on the assay
format, include one or more antibodies specific for a protein,
preferably specific for at least one capsid protein of ToTV, and
preferably also includes a substantially purified ToTV protein or
anti idiotypic antibody for use as a positive control.
Antiviral Agents
[0209] The invention also provides methods to obtain an antiviral
agent useful in the treatment of ToTV infection in plants
comprising establishing a cell culture or experimental plant
comprising a virus according to the invention, treating said
culture or plant with an candidate antiviral agent, and determining
the effect of said agent on said virus or its infection of said
culture or plant. An example of such an antiviral agent comprises a
ToTV-neutralising antibody, or functional component thereof, as
provided herein, but antiviral agents of other nature may be
obtained as well.
[0210] There are different antiviral agents used in plants, such as
chemical products, bacteria, fungus, insects and virus. Most of
them are related to systemic acquired resistance (SAR). The present
invention contemplates the use of the ToTV genome or a part thereof
as an inductor of systemic acquired resistance in plants. The
systemic acquired resistance may be directed to ToTV or to other
diseases. In this aspect, ToTV, its genome, or
resistance-conferring parts thereof can be used as antiviral
agent.
[0211] The invention also provides use of an antiviral agent
according to the invention for the preparation of a treatment
composition, in particular for the treatment of ToTV infection in
plants, and provides a pharmaceutical composition comprising an
antiviral agent according to the invention, useful in a method for
the treatment or prevention of a ToTV virus infection, said method
comprising providing such a treatment composition to an individual
plant.
[0212] The invention also relates to a plant model usable for
testing of treatment methods and/or compositions. It has appeared
that several Nicotiana species can be infected with the ToTV virus,
thereby showing disease-symptoms dissimilar to those found in
tomato plants suffering from the ToTV virus. Subjecting plants of
Nicotiana to an antiviral treatment either before or during
infection with the virus may have predictive value for application
of such an antiviral agent in tomato plants.
[0213] The invention also relates to the use of ToTV, or parts of
the ToTV viral genome, as expression vector, for instance for use
in virus-induced gene silencing (VIGS). VIGS is a technology that
exploits an RNA-mediated antiviral defence mechanism in plants. In
plants infected with unmodified viruses the mechanism is
specifically targeted against the viral genome. By using viral
expression vectors carrying inserts derived from host genes the
mechanism can also be used to target against the corresponding
plant RNAs. VIGS has been used widely in plants for analysis of
gene function and has been adapted for high-throughput functional
genomics. Until now most applications of VIGS have been in
Nicotiana benthamiana. However, the present invention contemplates
the use of ToTV as new expression vector systems that allows for
the analysis of gene function in other plants, such as tomato or
other species of the family Solanaceae, such as pepper and potato
and in species of the family Cucurbitaceae.
[0214] The invention is further explained in the Example without
limiting it thereto.
EXAMPLES
Example 1
Isolation and Characterization of ToTV from Tomato Plants
Methods
Introduction
[0215] Samples of tomato plants from Spain were received for
diagnostic research. Symptoms on tomato plants consisted of
necrotic spots and chlorosis on the leaves and brown rings on the
fruits. Serological tests (ELISA) pointed out the presence of
Pepino mosaic virus (PepMV), but, considering the symptoms, it was
likely that another, still undefined, agent was present.
[0216] Spherical virus particles were found in electron microscopic
studies of infected leaf tissue.
[0217] Subsequently an infection test was conducted and multiple
accessions of tomato were found to be susceptible for ToTV, several
of which reacted with clear symptoms (necrosis of the leaves,
beginning at the base of the individual leaflets (see FIG. 1).
Virus Transmission and Propagation
[0218] ToTV was isolated as described below from a diseased plant
obtained from Spain. ToTV was mechanically transmissible to several
Nicotiana species. A standard inoculation buffer (e.g. 0.03 M
phosphate buffer, pH 7.7) proved suitable. Throughout this Example,
ToTV was mechanically inoculated onto and propagated in N.
glutinosa or N. benthamiana. Virus purification was carried out
approximately 14 days after inoculation.
Virus Purification
[0219] Several attempts were made to purify ToTV according standard
protocols for e.g. nepoviruses or luteoviruses (with the aid of
organic solvents). These protocols resulted always in loss of
infectivity of the virus. Moreover, ToTV tended to aggregate when
low temperatures were used in centrifugation steps (below 5.degree.
C.).
[0220] Eventually a very mild purification method resulted in
reasonable clean virus preparations on which further experiments
could be carried out.
[0221] The following procedure was used to purify ToTV (all
centrifugation steps were performed at 6.degree. C.). Infected
leaves of N. glutinosa or N. benthamiana were homogenized in 5
parts of 0.1 M TRIS-HCl (pH 8) plus 20 mM Na.sub.2SO.sub.3, 10 mM
Na-DIECA en 5 mM Na-EDTA (homogenization buffer) and the homogenate
was centrifuged for 30 min. at 49,000.times.g. The supernatant was
placed on a 20% sucrose cushion and centrifuged for 1.5 h at
70,000.times.g. The pellet was resuspended in 2 ml TRIS-HCl, pH 8,
and the suspension was placed onto a sucrose gradient (10-40%
sucrose in homogenization buffer) and centrifuged for 2 h at
110,000.times.g. Since a virus band was not visible, the gradient
was aliquoted into discrete fractions and the presence of virus in
each fraction was determined by inoculation of a portion of said
fraction on leaves of N. hesperis `67A` as described herein and
observing the occurrence of infection. The virus-containing
fraction was placed onto a 10-40% cesium sulfate gradient (in
TRIS-HCl, pH 8) and centrifuged for 16 h at 125,000.times.g. The
virus bands were collected and concentrated by centrifugation or
dialyzed against 0.1 M TRIS-HCl, pH 8.
[0222] Infectivity of ToTV after each purification step was checked
by inoculation onto N. hesperis `67A` (cesium sulfate gradient
fractions were dialyzed against TRIS-HCl, pH 8, prior to
inoculation).
Electron Microscopy
[0223] Virus suspensions were "mounted" on a grid coated with
Formvar.RTM., otherwise known as polyvinyl formal, stained with 2%
uranylacetate and examined in a Philips CM12 electron
microscope.
PAGE Analysis
[0224] Viral proteins were separated by 12% denaturing
polyacrylamide gel electrophoresis (SDS-PAGE, Laemmli, 1970) and
silver-stained.
Nucleic Acid Isolation and Evaluation
[0225] Purified virus was concentrated by centrifugation (2 h at
115,000 g). Pellets were subjected to RNA extraction according to
the Qiagen RNeasy MinElute Cleanup procedure (Qiagen, Hilden,
Germany). RNA concentration was determined in a
UV-spectrophotometer (Beckman Coulter, Inc., Fullerton, USA).
[0226] RNA integrity was checked by agarose gel electrophoresis.
After electrophoresis for 2 hrs at 60V, the RNA was stained using
orthotoluidine blue.
cDNA Synthesis and Cloning
[0227] cDNA was synthesised using the Invitrogen Superscript Choice
system (Invitrogen, Breda, The Netherlands) for cDNA synthesis
according to the manufactures instructions. First strand cDNA was
primed using either Oligo-d(T) or random hexamer primers. After
second strand synthesis, EcoRI adapters were ligated to facilitate
cloning. Following phosphorylation of the EcoRI-adapted cDNA,
unligated linkers were removed by column chromatography. The
resulting cDNA was ligated in pBluescript II EcoRI pre-digested
expression vector (Stratagene, La Jolla, USA) and the ligation mix
transformed to Top10 competent cells (Invitrogen).
[0228] The 5' terminal end of the ToTV sequence was determined
using the 5'RACE System for Rapid Amplification of cDNA Ends
(LifeTechnologies) using dCTP according to the manufacturer's
instructions.
Analysis of cDNA
[0229] Upon transformation recombinant clones were analysed for the
presence of inserts by PCR using both T3 and T7 specific primers.
PCR products were analysed for size on a 1% agarose gel. Clones
containing inserts of about 1500 nucleotides were used for further
sequence analysis. Resulting sequence data were analysed using the
DNASTAR program package.
Determination of Amino Acid Sequences of the Three Capsid Proteins
of ToTV
[0230] Purified ToTV particles were loaded onto a denaturing PAGE
gel and capsid proteins were separated. Separated capsid protein
bands were isolated from the gel, treated with trypsin and the
digests were analyzed using a tandem mass spectrometer (MSMS) using
essentially the method as described by Kinter & Sherman, 2000.
This resulted in amino acid (AA) sequences of small peptides each
of which showed homology with the amino acid sequence predicted
from the RNA2 nucleotide sequence of ToTV. Comparison with other
virus sequence data was done with programs from the PHYLIP
package.
Results
Virus Transmission and Propagation
[0231] For ToTV propagation N. glutinosa and N. benthamina could be
used. The tobacco species N. hesperis `67A` and N. occidentalis
`P1` are very susceptible to ToTV and showed symptoms after 3-4
days. These tobacco species became very necrotic in short time and
were therefore considered more suitable as indicator plant than as
propagation host. N. glutinosa and N. benthamiana react with
chlorotic local lesions and a systemic chlorosis and mild
deformation of the leaves.
Virus Purification
[0232] Purification of virions from infected leaf tissue turned out
to be rather difficult. ToTV cannot resist organic solvents and
tends to aggregate when centrifuged at low temperatures. The
purification protocol used (see 1.2.) resulted in two visible
virus-containing bands in the cesium sulfate gradient. The bands
appeared in the bottom part of the gradient, indicating a rather
high buoyant density in Cs.sub.2SO.sub.4 of equal to or greater
than 1.4 g/cm.sup.2.
[0233] The infectivity of ToTV is affected by cesium sulphate, but
not completely lost when the virus concentration in the starting
material was high. Fractions of the cesium sulphate-gradient
containing both virus bands were infectious. Infectivity of
individual bands was not determined.
Electron Microscopy
[0234] The two bands were examined by electron microscopy and both
contained virus particles of about 28 nm in diameter (FIG. 2).
PAGE Analysis
[0235] Polyacryl Amide Gel Electrophoresis (PAGE) of purified virus
fractions, followed by silver-staining of the gel showed three
capsid proteins (CP) of approximately 23, 26 and 35 kDa (see FIG.
3, indicating purified virus top (TAgV-T) and bottom (TAgV-B)
fractions).
Nucleic Acid Isolation and Analysis of cDNA
[0236] RNA isolation of both virus bands together revealed two
RNAs: approximately 5.5 kb and 8 kb (see FIG. 4). RNA isolation of
the upper band resulted in a 5.5 kb RNA fragment only.
[0237] The 5.5 kb RNA from the top band was used as the template
for cDNA synthesis and cloning. Sequence reactions were performed
using forward and reverse sequence primers (T3/T7 or M13F/M13R) on
different clones. A 5' Rapid Amplification of c-DNA Ends (RACE) was
performed to determine the exact 5'-end of the RNA. Analysis of the
resulting sequence data, using the Lasergene.RTM. software package
(DNASTAR, Inc., Madison, Wis., USA), resulted in the complete
sequence of RNA2 of the virus (SEQ ID NO:1; see FIG. 5). The size
of the RNA is 5389 nucleotides excluding the poly A tail. On the
RNA two open reading frames (ORFs) are found. ORF1 is located from
nucleotide 182 to 742, is 561 nucleotides in length, and codes for
a protein of 187 amino acids. No homologies were found for this
sequence in the NCBI databases on protein and nucleotide level.
[0238] ORF2 stretches from nucleotide 702 to 4298, is 3597
nucleotides in length, and encodes a protein of 1199 amino acids.
After a BLAST search in the NCBI database, low homologies were
found with several viral polyproteins. For the found homologies,
the accession numbers from the NCBI database are provided together
with the virus name and the type of protein. Both the nucleic acid
sequences and derived amino acid sequences in all three reading
frames were used in a BLAST analysis. MAPDRAW from Lasergene.RTM.
software package was use to identify the ORFs.
ORF Maps of Both RNAs
RNA1
[0239] RNA1 contains one ORF (ORF1) with motifs typical of
helicase, RNA dependent RNA polymerase (RdRp). In addition a low
level of amino acid (aa) sequence homology was observed with a
protease co-factor (Pro-Co) of Patchouli mild mosaic virus (PatMMV)
(NP647592.1), for the amino acid positions 106-338, with an
identity of 22%.
[0240] Typical Helicase motifs A (GKS), B (D), C(N) were identified
at aa positions 398-400, 444 and 495 of the putative
polypeptide.
[0241] For the helicase region the closest identities were found
with Rice tungro spherical virus (RTSV; 42% identical in 140 aa
overlap), Maize chlorotic dwarf virus (MCDV; 43% identical in 137
aa overlap), Strawberry mottle virus (SMoV; 42% identical in 135 aa
overlap) and Parsnip yellow fleck virus (PYFV; 42% in 138aa
overlap). In the putative VpG region no sequence similarity with
other viruses was found.
[0242] The highest similarity in protease is found for aa
1000-1100, 25% identity is found with Potato virus V (PVV; NIa
protease in 86 aa overlap).
[0243] The RdRp region between motifs I (KDE) to VII (FLSR) was
found between aa 1303-1554 (Koonin 1991).
[0244] The closest identities of the polyprotein sequence were
found with: Rice tungro spherical virus (RTSV) with 29% identity in
a 751 aa overlap, Maize chlorotic dwarf virus (MCDV) with 28%
identity in a 742 aa overlap, Parsnip yellow fleck virus (PYFV)
with a 33% identity in 501 aa, Apple latent spherical virus (ALSV)
with 32% identity in 472 aa, Strawberry mottle virus (SMoV) with
30% identity in 680 aa and Cherry rasp leaf virus (CRLV) with 33%
in 465 aa TABLE-US-00003 TABLE 2 Overall levels of homology (in %)
between the RdRp motifs in ORF1 on ToTV RNA1 with RdRp motifs of
other plant viruses. NIMV PYFV RTSV SDV SMoV ToTV ALSV CRLV MCDV
NIMV 100 32.7 33.9 88.6 49.4 33.1 39.6 39.6 35.1 PYFV 100 43.7 33.2
32.0 35.2 36.4 36.0 42.9 RTSV 100 34.3 32.7 35.1 37.5 35.1 69.4 SDV
100 50.2 33.1 38.4 38.4 36.7 SMoV 100 35.7 40.6 38.5 38.5 ToTV 100
38.1 38.1 33.7 ALSV 100 79.5 35.5 CRLV 100 35.1 MCDV 100 NIMV =
Navel orange Infectious mottling virus (Sadwa); PYFV = Parsnip
yellow fleck virus (Sequivirus); RTSV = Rice tungro sperical virus
(Waikavirus); SDV = Satsumae dwarf virus (Sadwavirus); SMoV =
Strawberry mottle virus (Sadwavirus); ToTV--Tomato torrado virus
(genus proposed); ALSV = Apple latent spherical virus (Cheravirus);
CRLV = Cherry rasp leaf virus (Cheravirus); MCDV = Maize chlorotic
dwarf virus (Waikavirus)
[0245] TABLE-US-00004 TABLE 3 Overall levels of homology (in %)
between the helicase motifs in ORF1 on ToTV RNA1 with helicase
motifs of other plant viruses. SDV SMoV ToTV ALSV CRLV MCDV PYFV
RTSV SDV 100 42.5 37.2 31.9 33.6 39.8 39.8 38.1 SMoV 100 42.5 31.0
31.9 32.7 33.6 32.7 ToTV 100 36.0 34.2 46.5 40.4 44.7 ALSV 100 86.7
34.5 31.0 30.1 CRLV 100 34.5 30.1 31.0 MCDV 100 39.8 69.0 PYFV 100
42.5 RTSV 100 PYFV = Parsnip yellow fleck virus (Sequivirus); RTSV
= Rice tungro sperical virus (Waikavirus); SDV = Satsumae dwarf
virus (Sadwavirus); SMoV = Strawberry mottle virus (Sadwavirus);
ToTV--Tomato torrado virus (genus proposed); ALSV = Apple latent
spherical virus (Cheravirus); CRLV = Cherry rasp leaf virus
(Cheravirus); MCDV = Maize chlorotic dwarf virus (Waikavirus)
RNA2
[0246] RNA2 contains two potential ORFs (FIG. 7). ORF1 encodes a
predicted protein of 187 aa with a molecular weight of 20 kDa.
Sequence analysis revealed no homologies with any protein from the
EMBL databases. It is not known whether this ORF codes for an
actual protein.
[0247] The second ORF which partially overlaps with ORF1 starts
with three ATG start codons in frame. It encodes a putative protein
of 1198 aa with a predicted molecular weight of 134 kDa. Protein
identification by mass spectrometry of the isolated coat proteins
clearly maps the three coat protein cistrons at the C-terminus of
RNA2-ORF2.
[0248] The N-terminal region of the RNA2-ORF2 polyprotein most
likely codes for the putative movement protein (MP) since a motif
LRVPML highly similar to the proposed movement protein consensus
sequence LxxPxL (Mushegian, 1994) was found at aa position 262-267.
No other sequence homologies were found in the N-terminus of the
RNA2 ORF2.
[0249] ORF2 putatively encodes four proteins which are must be
cleaved from the polyprotein precursor by proteolytic cleavage.
However, no apparent homologies with known polyprotein cleavage
sites could be identified. The exact positions of these cleavage
sites remain to be determined.
[0250] For CP1 polyprotein sequence we found only any homology with
Human parechovirus (HPeV) with 21% identity in 103 aa.
[0251] The closest identities of the CP2 polyprotein sequence were
found with: Rhopalosiphum padi virus (RhPV) with 25% identity in
168aa, Avian encephalomyeliltis virus (AEV) with 33% in 74 aa,
Black queen cell virus (BQCV) with 43% identity in 37 aa and
Solenopsis invicta virus (SINV-1) with 30% identity in 51 aa. With
the CP3 polyprotein sequence no homologies were found.
Untranslated Regions (UTRs)
[0252] The 3'UTRs of RNA1 and RNA2 are 1210 nt and 1092 nt
respectively. There is 98% identity in the final 988 nucleotides of
both RNAs. To confirm that the 3'-regions of the UTRs of both RNAs
are identical, RT-PCRs were performed on total viral RNA with one
reverse primer derived from the identical 3'-UTR region and two RNA
specific forward primers. Resulting PCR products were
sequenced.
[0253] From these results, the following was concluded: The virus
isolated from tomato plants and tentatively named Tomato torrado
virus (ToTV) has spherical (icosahedral) particles of approximately
28 nm in diameter. Upon purification the virus displays at least
two bands in a cesium sulphate-gradient. Both bands combined are
infectious when inoculated on tobacco plants. Virus particles
appear to consist of at least three capsid proteins of
approximately 23, 26 and 35 kDa.
[0254] The cesium sulphate-gradient top fraction of the virus
contains an RNA molecule of approximately 5.5 kb (RNA 2; SEQ ID
NO:1) and the bottom fraction an RNA molecule of approximately 8 kb
(RNA 1; SEQ ID NO:2).
[0255] cDNA synthesis and cloning, using the 5.5 kb RNA from the
viral top fraction, and subsequent analysis of sequence
information, resulted in the compilation of several contigs into
SEQ ID NO:1. Two contigs clearly contained a poly-A tail suggesting
the viral RNA has a poly-A tail. BLAST analysis of the nucleotide
and derived amino acid sequences did not reveal any significant
homology with any known virus from the EMBL database.
[0256] The information above indicates that ToTV is a new and so
far undescribed virus. The information obtained so far does not yet
allow a grouping of the virus in a particular virus family or
genus.
[0257] For virus detection and identification purposes two
RT-PCR-primer sets have been designed (Table 4) on the basis of the
sequence of SEQ ID NO:1. TABLE-US-00005 TABLE 4 RT-PCR primers for
the detection of ToTV. length/ temp Sequence SEQ ID No. Primer set
A: forward primer 17-mer 5'-GAGAGCCGGCATTCACA-3' SEQ ID NO:3
reverse primer 17-mer 5'-GCACAGCTTGGCGACAC-3' SEQ ID NO:4 Product
length 493 bp Optimal annealing temp. 54.8.degree. C. Primer set B:
forward primer 24-mer 5'-CCCATCATCACCCTCCTCTTCGTA-3' SEQ ID NO:5
reverse primer 22-mer 5'-TTCCAGTAATGATCCAACCAAT-3' SEQ ID NO:6
Product length 585 bp Optimal annealing temp. 54.9.degree. C.
[0258] A suitable RT-PCR Protocol would comprise the following.
Total RNA is isolated and purified from about 100 .mu.g of infected
leaf material using a RNA purification kit, such as for instance
Qiagen RNA-Easy. Of this total RNA, an amount of about 1 .mu.g of
is used in a 50 .mu.l reaction mixture of the Superscript One-Step
RT-PCR reaction (Invitrogen), which reaction mixture further
comprises 25 .mu.l 2.times. reaction mix, 1 .mu.l (100 ng) of both
upper primer and down primer, 1 .mu.l of RT/Taq mix and 22 .mu.l of
MilliQ water. In order to reverse transcribe the RNA into cDNA and
amplify this cDNA the following RT-PCR programme may be used: Step
1: 30 min @ 50.degree. C. (Reverse transcription reaction); Step 2:
3 min @ 94.degree. C. (activation of Taq Polymerase); Step 3: 30
sec @ 94.degree. C.; Step 4: 30 sec @ 55.degree. C.; Step 5: 1 min
@ 72.degree. C.; Step 6: Repeat steps (3 to 5) 40.times.; Step 7:
10 min @ 72.degree. C.; Step 8: 10.degree. C. as long as needed.
PCR products may be analysed on a 1% agarose gel in TAE or TBE
buffer.
2.6. Determination of Amino Acid Sequences of the Three Capsid
Proteins of ToTV
[0259] Fragments of the largest coat protein (CP1: approximately 35
kDa) could be aligned with parts of an area in the ORF2 of RNA2 (AA
487-729). Fragments of the middle coat protein band (CP2: app. 26
kDa) could be aligned with an area of ORF2 of RNA2 between AA 730
and 983.
[0260] Fragments of the smallest coat protein (CP3: app. 23 kDa)
could be aligned with the C-terminus of ORF2 of RNA2 (AA 984-1195).
From these results it can be concluded that the coding sequences of
the three capsid proteins are located on the ORF2 of RNA2 (5.5 kb)
of ToTV, and thus that the isolated viral RNA molecules are part of
the ToTV virus particles.
Example 2
Isolation and Characterization of the Causal Agent of Marchitez
[0261] In 2003, tomato plants grown in Central America (Mexico and
Guatemala) with symptoms similar to symptoms of ToTV were found.
The causal agent was suspected to be viral. The disease is locally
known under the names "Chocolate," "Marchitez (virus)" or
"Chocolate spot disease." Susceptible plants grown in the field
become heavily infected from 2003 onwards.
[0262] The goal of the present investigation was to compare the
sequence of the so-far unknown virus causing the "Chocolate spot
disease" to the sequence of ToTV as isolated in Example 1.
Methods
[0263] RNA was isolated from about 100 .mu.g of "Chocolate spot
disease" infected leaf material using the Total RNA Isolation
System (Promega SV 96). Two .mu.l of the total RNA was used in a 50
.mu.l reaction mixture of the Superscript One-Step RT-PCR reaction
(Invitrogen), which reaction mixture further comprises 25 .mu.l
2.times. reaction mix, 1 .mu.l (100 ng) of both forward primer and
reverse primer, 1 .mu.l of RT/Taq mix and 22 .mu.l of MilliQ water.
In order to reverse transcribe the RNA into cDNA and amplify this
cDNA the following RT-PCR program was used: Step 1: 30 min
50.degree. C. (reverse transcription reaction); Step 2: 3 min @
94.degree. C. (activation of Taq Polymerase); Step 3: 30 sec @
94.degree. C.; Step 4: 30 sec @ 55.degree. C.; Step 5: 1 min @
72.degree. C.; Step 6: Repeat steps (3 to 5) 40.times.; Step 7: 10
min @ 72.degree. C.; Step 8: 10.degree. C. as long as needed. PCR
products were analysed on a 1% agarose gel in TAE or TBE
buffer.
[0264] Different primer sets were used in the RT-PCR, which were
known to anneal at different locations with the RNA1 or RNA2 of the
ToTV genome. Table 5. RT-PCR primers based on the RNA-2 sequence of
ToTV used for the characterization of the causal virus of Chocolate
spot disease as used in Example 2 and as indicated in FIG. 7.
TABLE-US-00006 Sequence SEQ ID No. Primer set P1048/1049: forward
primer 5'-CAAGCCATCACGGAACCTAC-3' SEQ ID NO:7 reverse primer
5'-AGCATCTTCTTCCTCCGCT-3' SEQ ID NO:8 Product length From base
36-544 = 508 bases Primer set P1056/1057: forward primer
5'-TGCTGAGGTGCTATCACTGG-3' SEQ ID NO:9 reverse primer
5'-CACACTTTCCACGATTTCCA-3' SEQ ID NO:10 Product length From base
2422-2955 = 523 bases Primer set P1060/1061: forward primer
5'-AAAGGAAAGAAGCAGCCACA-3' SEQ ID NO:11 reverse primer
5'-GGAAATCTTGGTCAAGCCAG-3' SEQ ID NO:12 Product length From base
3599-4170 = 571 bases Primer set P1064/1065: forward primer
5'-GCAATGCCAGTGGTTCAGAG-3' SEQ ID NO:13 reverse primer
5'-GGTCAAATGGATACTCGGGA-3' SEQ ID NO:14 Product length From base
4820-5327 = 507 bases
Results
[0265] The primer sets used promoted the amplification of part of
the ToTV sequence as expected, but the use of four different primer
sets based on the RNA-2 sequence (P1048/1049, P1056/1057,
P1060/1061 and P1064/1065) in the RT-PCR resulted also in
amplification of part of the "Chocolate spot disease" genome. The
site of annealing of these primer sets is illustrated in FIG.
1.
[0266] The RT-PCR-product of the four primer sets that amplified
fragments of "Chocolate spot disease" were sequenced, and showed
100% identity with the corresponding parts of the sequence of the
viral genome of the virus as described in Example 1.
Example 3
Isolation and Characterization of the Causal Agent of Marchitez
[0267] Following the Experiments described in Example 2, additional
diseased plant material from the same location in Mexico (Sinaloa)
was investigated. The disease referred to is locally known as
"Marchitez," meaning wilted or withered. Because of the resemblance
in leaf symptoms, it was assumed that Marchitez disease was caused
by ToTV and initial experiments described in Example 2 confirmed
this notion. However, testing of further material, ToTV could not
be detected using PCT with ToTV-specific primers. In the further
tested material, a spherical virus was found that shared
characteristics with ToTV. In this Example we describe and
characterize this new virus, related to but distinct from ToTV.
This new virus is tentatively named tomato marchitez virus
(TMarV).
Material and Methods
Virus Transmission and Propagation
[0268] TMarV was isolated in 2005 from a tomato plant from
Culiacan, Sinaloa, Mexico, that showed severe necrosis. The isolate
was designated PRI-TMarV0601. Using a 0.03 M sodium/potassium
phosphate buffer, pH 7.7, the virus was mechanically inoculated to
and maintained in Nicotiana glutinosa `PRI,` Physalis floridana or
N. benthamiana.
Virus Purification
[0269] All centrifugation steps were carried out at 6.degree. C.
Systemically infected leaves of N. benthamiana were harvested
approximately 14 days post inoculation and homogenized in 5 volumes
(W/V) extraction buffer (0.1 M Tris-HCl, pH8+20 mM
Na.sub.2SO.sub.3, 10 mM Na-DIECA en 5 mM Na-EDTA) and squeezed
through cheesecloth. Three volumes of an 1:1 mixture of
Chloroform/Butanol were added and after mixing, the suspension was
centrifuged at 16,500 g for 10 min. 1% Triton X-100 was added to
the water phase and stirred for 30 min. 5% PEG 6000 and 2.3% NaCl
was added and the suspension was stirred for 1 h. The suspension
was left to settle for 1 h and centrifuged for 15 min at 21,500 g.
The pellets were resuspended in a total of 80 ml extraction buffer,
centrifuged for 10 min at 15,000 g and the supernatant was placed
on a 30% sucrose cushion. After centrifugation for 3 h at 70,500 g,
the pellets were resuspended in a total of 2 ml Tris buffer pH 8.0
and centrifuged for 2 min at 14,000 g. The virus suspension was
loaded onto a 10-40% Cesium sulfate gradient and centrifuged for 16
h at 126,000 g. Virus bands were collected and dialyzed against 0.1
M Tris-HCl, pH 8.0.
Electron Microscopy
[0270] Virus suspensions were mounted on formvar-carbon coated
grids, stained with 2% uranyl acetate and examined in a Philips
CM12 electron microscope.
Polyacrylamide Gel Electrophoresis
[0271] Viral proteins were separated by subjecting purified virus
particles to 12% denaturing polyacrylamide gel electrophoresis
(SDS-PAGE) [Laemmli U K (1970) Nature 277: 680-685], and visualized
by silver staining.
Nucleic Acid Isolation and Evaluation
[0272] Purified virus was concentrated by centrifugation (at
115,000 g for 2 h). RNA was extracted from pelleted virus particles
using a Qiagen RNeasy kit (Qiagen) according to the manufacturer's
instructions.
[0273] RNA concentration was determined in a Beckmann
UV-spectrophotometer. Viral RNA integrity and size was checked on a
1% agarose gel using a formaldehyde/formamide/HEPES buffer system.
After electrophoresis, the RNA was stained using ortho-toluidine
blue.
[0274] Total RNA for RT-PCRs, was isolated with the RNeasy plant
mini kit (Qiagen) from TMarV infected Nicotiana occidentalis `P1`
plants.
RT-PCR and 5' RACE
[0275] PCR fragments were obtained by one-tube RT-PCR (Access
RT-PCR system, Promega). RT-PCRs were initiated using a universal
oligo dT primer [Van der Vlugt et al. (1999) Phytopathology
89:148-155] and various primers derived from the ToTV RNA 1 and RNA
2 sequences (respectively GenBank accession numbers DQ388879 and
DQ388880 The 5' regions of the TMarV RNAs were determined by
walking towards the 5' end of the viral genome through repeated use
of a 5' RACE kit (Roche) in combination with the Expand high
fidelity PCR system (Roche), essentially as described previously
[Ongus J R, et al. (2004). J. Gen. Virol., 85:3747-3755; Valles S
M, et al. (2004) Virology 328:151-157]. cDNA primers for the
5'-RACE strategy and primer sets for additional RT-PCR reactions
were based on newly obtained TMarV sequence data.
Nucleotide Sequencing and Sequence Analysis
[0276] All PCR products the 5' RACE) were purified using the
QIAquick PCR Purification Kit (Qiagen) and directly sequenced.
[0277] Sequence analysis was performed with an Applied Biosystems
3100 Genetic Analyser, using the DYEnamic ET Terminator Cycle
Sequencing Kit (Amersham) and the primers that were used for
amplification. For longer PCR fragments TMarV specific primers were
used for primer walking sequencing.
[0278] Nucleotide and amino acid sequence data were analyzed and
assembled using the DNASTAR package (Lasergene).
[0279] Sequence comparisons with other viruses were performed with
programs from the PHYLIP package. Multiple alignments and
phylogenies were performed with the CLUSTAL X program after
bootstrapping in 1000 replicates. Neighbour-joining consensus
phylogenies were viewed by the NJplot program [Thompson J D, et al.
(1997) Nucleic Acids Res 25: 4876-4882] and printed by using
TreeView [Page R D M (1996) Computer Applications in the
Biosciences 12: 357-358].
Results and Discussion
Virus Characterization
[0280] TMarV was easily transmitted to a number of indicator plants
by mechanical inoculation. In table 1 an overview is given of the
indicator plants used and their reactions to TMarV and ToTV. Most
symptoms of TMarV and ToTV in indicator plants resemble each other,
except for the reactions in P. floridana and Chenopodium quinoa.
ToTV causes severe necrosis and die off in P. floridana, where
TMarV induces only occasionally necrosis in the local infected
leaves and a systemic mottle. C. quinoa shows no reaction when
inoculated with ToTV, but TMarV induces necrotic pin point lesions
in the inoculated leaves.
[0281] Electron microscopical studies in tomato showing Marchitez
symptoms and in systemically infected leaves of Nicotiana
occidentalis `P1` revealed the presence of spherical virus
particles with a diameter of 28-30 nm. The particles of TMarV
clearly resemble the particles of ToTV in shape and size.
[0282] In initial attempts to purify TMarV the purification
protocol designed for ToTV was used (see Example 1). This protocol
did not lead to visible virus bands in the final cesium sulfate
(CsSO.sub.4) gradient. Electron microscopical analysis of gradient
fractions revealed that virus particles were present in the bottom
part of the gradient. However, this part of the gradient contained
also plant components, veiling the virus bands. Therefore another
purification protocol was designed in which the separation of the
virus from plant components was aided by the use of a
chloroform-butanol mixture. This protocol resulted in one diffuse
band in the CsSO.sub.4 gradient. This result is in contract with
that obtained for ToTV purifications, which always yielded two
distinct bands in CsSO.sub.4 gradient centrifugation. The TMarV
band was collected from the gradient and checked by electron
microscopy. Virions of the expected size of 28 nm were present in
the collected band, and were infectious when mechanically
transmitted to test plants. Purified virions were also mechanically
transmitted to tomato plants, which showed characteristic symptoms
of Marchitez disease two weeks after inoculation. Presence of TMarV
in these plants was confirmed by electron microscopy and
RT-PCR.
[0283] Purified virions were subjected to SDS-PAGE and three viral
proteins were detected with estimated sizes of 35, 26 and 24 kDa,
named respectively Vp35, Vp26 and Vp24 (FIG. 11A). The number and
estimated molecular sizes of the viral coat proteins of TMarV are
the same as previously found for ToTV (see Example 1).
[0284] Purified virus preparations of tomato marchitez virus showed
two RNA molecules on a denaturing RNA-gel:RNA1 with an estimated
size of 7.5 kb and RNA2 with an estimated size of 4 kb (FIG. 11B).
The number of RNAs found is in accordance with ToTV, but their
estimated sizes are smaller (8.5 and 5.5 kb for ToTV RNA1 and RNA2
respectively).
Viral RNA Analysis
[0285] Based on biological and structural data like indicator plant
symptoms, particle sizes and morphology, number and sizes of coat
proteins, and number of RNAs obtained for TMarV, a possible
relationship with ToTV was suspected. Therefore different upstream
primers based on the ToTV RNA1 and RNA2 sequences (DQ388879 and
DQ388880) in combination with a general oligo-dT primer, were used
to perform RT-PCR reactions. This resulted in a limited number of
PCR fragments indicating possible differences in RNA sequences
between the two viruses. Sequence analyses of these fragments
revealed low levels of homology with the ToTV RNAs 1 and 2. Based
on the obtained sequence information new cDNA primers were
generated and used to obtain additional sequence information in a
5'-RACE sequence walking strategy.
[0286] TMarV specific primers were used for RT-PCRs to confirm
sequences of both RNAs in two orientations.
RNA1
[0287] RNA1 (FIG. 8) consists of 7221 nucleotides (nts), without
poly(A) tail and contains one open reading frame (RNA1-ORF1) of
6453 nts encoding a predicted polyprotein of 2151 amino acids (aa)
with a molecular mass of 237 kDa (FIG. 12). The first in-frame AUG
is found at nt positions 141-143. The ORF has an UGA stop codon at
positions 6594-6596. The putative polyprotein sequence contains
several conserved regions with motifs typical for a protease
co-factor, helicase, protease and RNA-dependent RNA polymerase
(RdRp).
[0288] Typical helicase motifs A (GKS), B (D), C(N) were identified
at aa positions 397-399, 443 and 494 of the putative protein. The
RdRp region could be identified between aa 1305 and 1553 by the
presence of the typical motifs I (KDE) to VII (FLSR) [Koonin E V
(1991). J Gen Virol 72: 2197].
[0289] The ORF1 encoding region shows 65% overall identity with the
ToTV-RNA1 (DQ388879) at both the nucleotide level (6474 nts) and
the amino acid level (2158 aa).
[0290] The helicase region between motifs A and C [Gorbalenya A E,
et al. (1990). FEBS Lett 262: 145-148] in the C-terminal part of
RNA1-ORF1 showed 95.6% aa identity with the corresponding region
from ToTV and significantly lower levels of identity with other
viruses ranging from 46.9% for maize chlorotic dwarf virus (MCDV,
genus Waikavirus, AAV86083) to 25.4% for acute bee paralysis virus
(ABPV, unassigned species in the family Dicistrouiridae,
NP.sub.--066241).
[0291] Levels of aa identity for the RdRp motif in ORF1 of RNA1
were relatively high with ToTV (78.2%) and significantly lower with
other viruses, ranging from 15.3% for avian encephalomyelitis virus
(AEV, genus Hepatovirus, NP.sub.--653151) to 25.9% for strawberry
latent ringspot virus (SLRSV, genus Sadwavirus, NC.sub.--006764).
These levels of identity indicate a close level of relationship
between TMarV and ToTV.
[0292] A BLAST search with our available data also revealed a
remarkably high level of overall sequence homology (95%) with a
partial sequence in the NCBI data base of tomato apex necrosis
virus (ToANV, Genbank accession number EF063641) which became only
available during the course of our work. In the coding region of
RNA1 the overall level of identity between TMarV and ToANV is 96%
at the nt level and 99% at the protein level with levels of aa
identity of 99.3 and 100% for the RdRp and Helicase motifs
respectively. These percentages suggest a close level of
relationship between TMarV and the partial virus sequence submitted
under the name of apex necrosis virus (ToANV).
RNA2
[0293] RNA2 (FIG. 9) consists of 4906 nt, and in analogy with ToTV,
contains two open reading frame (RNA2-ORF1 and RNA2-ORF2) encoding
two predicted polyproteins (FIG. 12). The overall level of sequence
identity between RNA2-ORF1 of TMarV and ToTV is 62.1%.
[0294] The highest sequence identity (80%) was found with the
published partial sequence of ToANV-RNA2 (Gen bank accession no.
EF063642). In the coding region the identity is 78% at nt level and
90% at protein level.
[0295] In analogy with ToTV, the N-terminal region of the RNA2-ORF1
polyprotein most likely codes for the putative MP since a motif
LRVPTL highly similar to the proposed movement protein consensus
sequence LxxPxL [Mushegian A R (1994). Arch Virol 135: 437-441] was
found.
[0296] The sequence of the putative movement protein region of
ToANV is not available.
[0297] The relative order of the three putative coat proteins of
tomato marchitez virus on the RNA2-ORF1 was derived from direct
sequence comparisons with the ToTV CPs. No obvious homologies could
be identified with regions which were identified earlier as
putative CP cleavage sites for ToTV CPs [Verbeek M, et al. (2007).
Arch Virol 152:881-890].
[0298] For ToTV the coat proteins were sequenced to locate the coat
protein genes on the RNA2 sequence. The amino acid sequences of the
individual coat proteins of TMarV, ToTV and ToANV are compared.
TMarV-Vp35 shows 89% and 72% identity to ToANV and ToTV.
TMarV-Vp26 shows 98% and 86% identity to ToANV and ToTV.
TMarV-Vp24 shows 94% and 71% identity to ToANV and ToTV.
5'- and 3'-Untranslated Regions (UTRs)
[0299] The 5'-UTR size of RNA1 is 140 nt. That of RNA2 could not
yet be reliably determined. The 3'-UTRs of RNA1 and RNA2 are 628 nt
and 655 nt, respectively, in length and share an overall level of
identity of 91%. The 553 most 3'-terminal nucleotides of both RNAs
are almost perfectly conserved (99% identity). The near identical
regions in the 3'-part of both 3'-UTRs is a characteristic that
tomato marchitez virus shares with ToTV. However direct sequence
comparisons between the total 3'-UTRs of RNA1 and RNA2 of TMarV and
ToTV reveals only 49.0% and 48.9. % sequence identity respectively
between the two viruses.
[0300] A comparison to the 3'-UTRs of ToANV with respective lengths
of 630 nts and 650 nts for RNA1 and RNA2, shows 88.5% and 85.6%
sequence identity with RNA1 and RNA2 of TMarV. This is
significantly lower then the levels of identity observed in the
Helicase and CG-GDD regions found in the ORF encoded on RNA1.
Taxonomic Position of TMarV
[0301] Tomato marchitez virus shares virion characteristics and its
genome organization with ToTV but based on levels of nt and aa
sequence identities the two viruses are related but distinct.
Remarkably high levels of identities are observed between TMarV
RNA1 and RNA2 and a sequence deposited in the NCBI database under
the name of tomato apex necrosis virus (ToANV). No additional data
on the virus from which these partial sequences were derived are
available. However, the relative low levels of nucleotide sequence
identities (less than 90%) in the 3'-NTRs of both RNA1 and RNA2
(88.5% and 85.6% respectively), as well less then 90% aa identity
(89%) in the largest putative CP (Vp36) between the two viruses,
suggests that the two viruses are not identical and may be strains
or isolates of the same virus. Additional biological and molecular
data on the virus from which the ToANV sequence was derived will be
needed to determine its precise relationship to TMarV.
[0302] Phylogenetic analysis based the aa region between the CG
protease motif [Bazan J F, Fletterick R J (1988) Proc. Natl. Acad.
Sci. USA, 85:7872-7876] and the GDD RdRp active site [Argos P
(1988) Nucleic Acids Res., 16:9909-9916] in the RNA1-ORF were
performed to determine the relationships between tomato marchitez
virus, ToTV and other viruses from the genera Sadwavirus,
Cheravirus and the families Sequiuiridae, Comouiridae,
Dicistrouiridae and Picornauiridae. This region is proposed to be a
good taxonomic predictor for classifying picorna-like viruses
[Ikegami M et al. (2002) Taxonomy of recognized and putative
species in the family Comoviridae. XIIth IUMS Virology meeting
Paris, France, 27 Jul.-12 Aug. 2002]. The resulting dendrogram
(FIG. 13A) shows that TMarV virus clusters with ToTV and ToANV and
confirms the separate taxonomic position of these viruses from the
genus Cheravirus. A similar phylogenetic analysis on basis of the
helicase region between the motifs A and C [Gorbalenya A E, et al.
(1990). FEBS Lett 262: 145-148] (aa 397-494) confirms the taxonomic
positions of TMarV, ToTV and ToANV (FIG. 13B). For this motif these
viruses seem to be more closely related to the genera Waikavirus
and Sequivirus.
[0303] The data we present in this Example describe Tomato
marchitez virus (TMarV) as a new picorna-like plant virus, related
to but distinct from tomato torrado virus (ToTV) [Verbeek M, et al.
(2007) Arch Virol 152:881-890] TMarV and ToTV clearly separate from
other plant picorna-like viruses and are likely to belong to the
same yet unnamed new genus.
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Sequence CWU 1
1
20 1 5404 DNA Tomato torrado virus 1 tttaaaagaa taattttata
caatatttat gtgatccaag ccatcacgga acctacgtcg 60 ttcctaagat
attgtatcaa ttatctttgc tgttaccatt ttgagtgcac cgtcaactga 120
aggaacctac gccgtttcca aggacagtgt atcttttgtt atttattttg gttacatctc
180 tatgtcattt atttctcgtt tgaatacatc tcttgaagag gaggcatttc
acaagcaagt 240 tgcagactct cagtgggtgt gttcggttga tacaggctct
ggcataataa acagtgatcc 300 aactctagac ttcaagattt gtcccaagac
aggtggagca atttctgttc tatctgtttc 360 gtggcaaaac aatagccctc
aactggttcc tggtcattat ttactgcgaa gtggaacttg 420 gccaattact
ggcgttaagc tatctggctt actagttcat agatcaattc gtttggaaac 480
caccagaaaa cttctagaag ctcagaggat ttctgtatcc cagcaagcat cttcttcctc
540 cgctgctggt gctgcaggaa aacaaccaca agtaacgctt acacaattac
aagaggagct 600 tgacgaggcc aaaactcgct tagctctcaa agagaaagag
ttacttgaag ctctatccga 660 aatatccaaa ttaagattgc aattgtccaa
ccaactaagt aatgatgatg tcttcagtgg 720 ctggacagag gaagggccca
agtgataaaa gtgcagctgt atctgagcaa ttaattgctc 780 agattacagc
tgcagttgaa gcaggcaaca aaaacctgct tcgcaaactg ggcatgggtt 840
catatgggat tctctatggc gcccaagaga agaaagccat ggaactgttt gaccctgagg
900 atgtacataa tataacctca ttgtggtctt catttaagaa aactttcact
tcctcgaggg 960 atcatggaaa tctattcttc catttgtatg gagttatgtt
tttcatggtt cctcatgtac 1020 atggtgggga aggtagtgta aaaattagtt
tatgttccag taatgatcca accaatcctg 1080 ttctgcagga gaaggttttg
tatttttccg ggggggcaca ggcagtgtta atgagtccga 1140 ccattacact
accttttgtt aaaagaggcc ccatgttcta ctacacaatg gagtgccttg 1200
gcactcgggc tcaaattcct tgctcagtag tggccatttg gaaacaaaag attgatatcc
1260 gtagtgccat ttactcaaag caggaaacaa tgtcttgggc tattgaggct
cttcaccgac 1320 ctcaattttt ccaggataga caggaggcag cacagtacat
atcatcagta tattctaatg 1380 ctacctcctc agcaactgat tcagtgcttc
cgtttgtggg agcacagctt ggcgacacaa 1440 agatgaatgt gccaagcgaa
gcaagaatga tccggtcctc ctcgctccgg gtgcccatgc 1500 tcaaggtgca
gagtaagcgg ttttcctcaa tggaaatacc atctacctca actgcacacc 1560
tccttggcac aacgcgtgat gaaactgtaa tacaggagga aagtaggtac gaagaggagg
1620 gtgatgatgg ggttttgttc cccgttaaaa aggctcaagg tctgaactat
tcccatgtgt 1680 gggataatct tggtattgag tcctttgtag atgttgagct
gcccgaaaac tgggatgagc 1740 tgtcggtgag acagcaagtt gctgcagcaa
tgatagcatt tgcaaacaag ggtgtttgct 1800 tggtcccaaa acatataatc
aaccgggaca agcacaatat ccacttggag aatatcaccg 1860 agcacaacta
cttggtgata ctggaaaggt acggcattgt gaatgccggc tctctggcca 1920
gaactgaaaa ctggtacaat cttaccttgg cacagagagt ggaagagcta atttatcaaa
1980 gggacgatgc atatttcatg tttggtgata acaccaaccc atatcctccc
tttgattgtt 2040 atgatggctt aacgctcaaa gttcgcagtg agctagagcg
tgtggcaaag gagcaggcgc 2100 gccaaagatt ttacaaggag gcggccaggg
ctcaggtgaa aaacaaggtg gcccaaacta 2160 gtgtggaaga aataccatcc
acctcattcg ccacaaaggt tgccatggag agtggcagtg 2220 tggatagcat
gaaaattgct attcaagctg aagctgccaa tgaagctgta aggcccaatg 2280
aagttatgtt tgaatttggg caagaaatga ataatgaagg tgcgacagag ctggaattac
2340 aacaaccagc ctgcgtggcc agtaactcct tcttcaatgt tggagttttt
gagtttgcat 2400 ggaagaaaag cagttctgtt gctgctgagg tgctatcact
ggcgctccct gcggctctct 2460 ttggtaaatc caaggagatg tcaatgggat
cgcaaatgct aaggtattat gatgccgcat 2520 taattatgta caaggtcatc
ttgtatattt ccggcatggg tgcaatttcg ggtcagctgg 2580 ccctggtttg
ggatgagtgc aatgtgctca acagaaagaa ggagttcatc aacattgcct 2640
ctctgtatgc cagcaaacat aggctggttt cagcatctga acagagtagt ggggaatttt
2700 gttttacacc tacgggtatc ggcaaattcg ttccacttga tccagcctcg
ggggcttatg 2760 atctgggtag catacgggtg tttgtgacgc accccttggc
tagtgctact gaattggaga 2820 gcataccttg tcacatccat ctgcagtgta
aagtgttgtc gaccaacatc atgcagcctc 2880 ctcgtttgcg agcacaggca
caatttggta tgaaaccaga ccagacacac tttccacgat 2940 ttccaacaaa
tcaggtactg ttacactaca attggggagt ggctgctagc atgggtacta 3000
ccttagttag catcttctct ccgtcaggca tatatgaaag tgacggtacg ctgcaaccat
3060 ccttgcttgg aaacatagca cgcaattgca aatggtggac tggcacttgc
gtgtttgaga 3120 tttgtattga gaagactcag ttccattctg gtagtttggc
cattggactg ggtacactga 3180 acacaagcat gtccacccct catgacattt
taaatatgcc gcatgttatt tgtaaccttg 3240 agatgggacg aaagttctat
ttcaggtgta cgataaccaa ttggaatggg aaaaatcttt 3300 tgaccactgg
tcggaagagt tctctaccgc ggcccaagca tatgtctcac atgaggttgt 3360
ttgctacagt cttgaaaccc ctggtatcaa cttcaataca tctagatacg gtcggggtaa
3420 cagtgcagct taaatgcata gaagatttgg tccttggggg cactgtgtct
gttaaaccca 3480 tttacggaca ctggactaaa ggaaagaatg ctgtggactt
cctattctct gagatggact 3540 tgtctcagcg caaagaaatt gagaaattac
gcaaggaaaa cgttgagaca tttgatgaga 3600 aaggaaagaa gcagccacag
gtacaagtgc cgctcagaga caagttttca tatggggctg 3660 tacaatattt
tgtgatgaat tggaaggacg aagagcgact gttggtttta ccatgcgcac 3720
cctggtccgt aagattccct cagggggcac tggtacagga ggccatcaca tgcccattca
3780 ttgattggtg ctcttccttc tgttattggt ctggaagtct tgaatacacc
attattgtac 3840 atagagtgca gacttccaat aacataggag gagtgctgaa
catcacctta gattcatcag 3900 ggtacccttt tcctcttgga atctcaaagg
gcacctatgt tgtctctgct ggtggaggag 3960 caaaatgggc tttcacttat
ggtatgagcg acaacatctt ttcttttgtg gtgcatgatg 4020 atgagttttt
tcctagacgc cataccaaag ccagagcaat agatccaaat gcttcaagaa 4080
taatgactct gcaagatcga ctaggaaatc tcataataaa tttaccagcc aaagatgtga
4140 taagctctct ggaaatcttg gtcaagccag gacctgattt caaattgcaa
ctggctcaag 4200 ctccttcagc aaatcatgag aagcatttgg gtgatatgca
aacgcatacc tacctttata 4260 ctcctgattt ttcagaacta aggagttttg
aaaattaact atcctgtacg tgatatagga 4320 gccgtgagtc cggcatgtga
ctcatcatgt gcagttaaca tgtgtactgt gaataatgtt 4380 aactgtaagg
atagttaatt ttaacggaga gtactgactt taactagttg ggagtccggc 4440
tccatttgga gtaccaatga atctacattg gttaaagaga tttgcacgcc tctctttaaa
4500 tatggatcgt gtaccctgct tggttagaaa gtacctttac ttaggtttac
aaagtacggg 4560 gagcactccc tggttaacat agtgcaggtg ctatcccatg
atagtccttt aactcaaggg 4620 ttgagttcgg tttgcatctt ttgccgtgat
gaaagatgag gtagcttccc ccttattggg 4680 aggctgaaac tacacatatg
tagtgggttt gactgagtcc tataatcagt ccgtttgaaa 4740 ttcgataatt
ttccgtagct tgcgtcaagc tgctcacgtt aggggcgtga gtgaagatgc 4800
gccgtaccac gccttccccg gcaatgccag tggttcagag cgggccctca gaatagaggt
4860 taaaactagt gtgatggtgt atatcacgat aaaaagtgac acccgggttg
tgctgcgcct 4920 agttaacacg agcacaggtc ccaccctata gtggaagaaa
cttggttgag ttttaaagaa 4980 caacccgttt gagcgacgac aaagttccgt
agcttgcgac aagctgtttg tgttaggggc 5040 acaaatgaag ttatagcaca
ccacttcttc cctaggttcg tccagtggtt tcacagtgct 5100 atcctcagaa
aagaggttaa atctagtgtg atggtgtata tcacgataaa gaatgacacc 5160
cgggatgtgc ttctcctagt taactcgagc acggttttca caccacagtg aaactatctt
5220 actgctttaa atgttgtttg tttgttttac ttgttttatt gagtgtttaa
tatcatgcat 5280 atttgctgtt gaaggttctg tcgatgaggt caaatggata
ctcgggaact acagtaactt 5340 tgcatttgaa ttgttttcaa ttcagtgtgt
gagtttgaat atgtatttta aaaaaaaaaa 5400 aaaa 5404 2 7809 DNA Tomato
torrado virus 2 ttaaaagagt tattttgaga atataaccta cgtcgttatt
cacagaccaa gtctctgtta 60 atcaaaatct ccctttaaaa ctcattctac
ttttacattt ggcaacatgt ctttttccaa 120 gatgttcccc ggtttcaact
cagttactga aaagtgcgct accagctcct ctggttcttt 180 cttttcagag
cttactgcta gtattagtaa tttctcccgc actctgtcca atgttaccaa 240
ggtttcatct caaatttctt ctcacattga agatttgaag ccttcagtta cagatgctgc
300 ttcttccttt accagcacct gtaactctgt tactaaattg ttagataaga
taatgacttt 360 aattgaaccc ttcatcaagg cttactcttt tgtcgcatcc
atgtacaaat caatttgtga 420 tatggttgca aagattgttg caagcatcaa
agataagttc acacttggtt ttaactgggt 480 gttggacaaa tctgaggatg
tggatgtttt agttatagct tttcttattt ttgcaatttc 540 tatgttaata
attgttttca tttgtccaag tagtgtacta gatggagttg tacagatgac 600
tcacatagtt tttaatacag taggtaactt cttttcagct ttgtacaaat tagactggtt
660 accgacatgg tcccaaaagt tttcaatgat ggcacaagcc aatgttctac
caggggaatc 720 catgtcacac tcaccacttt cacaagtggt agcatccctc
atagcctttg ggatttctac 780 ccttgtgttc gtggctgtac ctggtagacc
caatggtcta tccaacccgc tatcaaaaat 840 tctgtactca gcgggaagtg
gtgctcaaca gtgcaatcaa ttgtttaccc tgttcaggaa 900 tatgaaagat
tgtacttccc aggccttttc ctgggttctg gaaataatag tagacatttt 960
tggttttaag aatccagttt tgtctgctat tagtgccaca ttgtctacgg acttattcac
1020 gtggatggaa gaggtggatg cagtgtgtga tccagcacat cgcttggaaa
attttgcaaa 1080 ccctgcattc actatcaagc tccaacatct aagggagcag
gctcttaaaa tctctgctta 1140 cattgctaca catcctgtag cagcctttat
gagccacagg gtaacggcag caatarccca 1200 tcttgataag atatatgggg
agaactgcca gcacactggc gtgggccaat acagggcaga 1260 accttttatg
gttcaatggt atggtgccag tggttgtggg aaatccacca gcatgcggct 1320
gttcatcaat gatgttttgg accgcatgga agagccaaag ttgaacaggc tctatgctgt
1380 gagtaagcga gatgcttact ggtcaaatta tgcccatcaa actgctatcc
tgatggatga 1440 catgggcgct ctgagagatg gggcagggca atgccaggac
attaaggatt tgatagatat 1500 caagtccact caaccagccc ccttgcccat
ggcagcagtg gaggataaag gccgccactt 1560 tacctcccga tatatatttg
ccacatccaa ccttatctca gctcctgctc agtgtggtct 1620 aacctacccc
gatgcttttg agagaagacg ggatgttctt gtggagtgca ggaaggtggg 1680
tgagtttaac actgatgctc ccacttctca ccttgaattt gatgtggttg agagcaaaag
1740 accccatgca ataacacaca ggggtttgag ctatgatgat ttgctggaat
atgtggttgc 1800 caagtgcaag gttcatgcag aaatctcagg aaaactgtat
ggcgccacat caggcaaggt 1860 ggcacaggtt gatgtgtcac ctgaggaaat
aatagcatcc atggatatgt tgaatatcca 1920 agataccaag caggatgcta
agcttcccgt ggttgttgta agtgaagagg atagggtggc 1980 ctactcycag
gagttgacag tggaagcttt gaaatatgct tatcagggta gtctgaatcc 2040
cgcagcatat ttccctcatg acatgcacaa acaggccata tttgatgtgc taagtgaatc
2100 cgccaaagaa acattcacca ggtgggttaa tgacatgctg tatcaaggtt
gctgtaatga 2160 gaactatcgc tggctgataa agaatatccc agctgattat
atcatgcact ttaagagctt 2220 catctacgct tccacaatca atgagcgtag
ctttgacgtt cagaagcagc tgcctgatgg 2280 aatggcgcac cgtgccatag
atgctgatgt ggacacactg atatgcgtgg agcagatgcc 2340 tgcccacgta
caattcttgt acacagcatt tgtgaggtat tggtgccgca gaaagatgga 2400
acagcctagg caatcctggg tggtagtttg ctaccacagt attgtggatt atatcaagaa
2460 tgcttggtat gatttaccgt acatcttgag agttctgatt aaggctggcc
ttattttaat 2520 tgcactcaat ggtgcatttg gggctgtcac agcattctgt
gcttgttggc aatccaacac 2580 tttcccttca gcagaaggaa gaggagggat
taccaacgag tcaaatagca tctccagccg 2640 gaagaacaag ggaaagagca
tctttgctcg atctttgctg gcacaagcca agggtgatat 2700 gctggagaaa
tgggcaagtg atgatggctt catcaatgaa ggattgaaga aaaacctagt 2760
cgtcttgaga ctaggtgaag gtgtctactt cagaggcacc tatgtctgct cgggctgggt
2820 gatgacagtg gctcatgctt tttcaagcct ccgtgatggc acaactttct
caataataca 2880 tgcccagtca atttccaagg tgcaatacaa tgccaaaact
gcacggttct tgaaggagca 2940 agatattgtc ctgctcaatg ttggaaaccc
cgatggtccc aagcctgata ttcgcaaaca 3000 ctttcctgta cgggatggtg
tttgtttttc taagggcact caaggggtat gtgtgagagc 3060 agtagcatca
aaggatgctt cgcaaggaaa tcttgagtac ttgcgtttta atgtgatgat 3120
gtccaagggt taccttgaaa aggtaacgta ccagatggac tctagttcct ttaaactgga
3180 gtctcaagca tcttatgagt atcacatgaa tggtgaaaat ggtgattgtg
gtactcttct 3240 tcttttgccc aacgtgcaag acaaacaacc atgcattgtg
ggtattcatt gtgcttctta 3300 tgatgaagaa gctgcgcaca aagggtttgt
agcatccaat gctacagcta ttttccgaga 3360 tcagttggaa gatcttccga
ctggtccggt taaagtagca atggtaaggt gccagctcct 3420 taaggatcta
cgagccaggg atgcggctct ttttgaagaa aaacaggtgg cttttgttgg 3480
cacattgcca gctgaacaag cagccacggt tccccacaaa acaacgctgc gaaggagtgg
3540 cttgtttgaa gcttttgggc ctgcagaaac tgctccatct atcatttcag
cttcagacaa 3600 acgtggggaa ggttttgatc cgtacgtggc tggcatacaa
aaatacaatg aaacagcaca 3660 aaattttgat gaggacattg cgaggctagc
ctatgaaggg ctacgtcaag caattttgcc 3720 tgtgctgcac tcccagcgag
ttccttttgg aaagcccgtc acacagaatg aagatgtggt 3780 gctcaatggt
gttgatgggt ttgactattt tgacgggatg gagttgagta cctcttgcgg 3840
gtatccgtac aacaagttgg gtatgggcac tagcaagaga gagtttgtgg agccaagtgg
3900 agatggagat cgagtccaac tcaaaaggac cactccaatt tttgatgact
gggaggcttt 3960 ggatgtggaa attcgcaaag gaaactttgt ggaactggtc
accacccaat gtgccaaaga 4020 tgagcgcttg ccgttggaaa aggtttttgg
gaagcggaaa acccgtttgt ttgaaattct 4080 tcccttccat tacaatatgt
tggttaggaa gtatttcctg gatttttccg ccagtctgat 4140 ggcatcccac
aatgctctgc catgcaaagt gggcattaat cctggaggta ttgaatggac 4200
tctgttggct aatggcttca gagcagtctc tgatacagga ttttctgctg actattccag
4260 ttttgatggg agagctccca tctttgcctt tcaatggttt tgtgatcttg
tggatgacta 4320 ctatggatca cctcctggtt ctccagactc caatgccaga
catgtgcttc ttatgatggc 4380 ttcatgccat tatactattt gtgagaacaa
ggtttttagg ttggtgggag gtatgccttc 4440 aggatttgca ctcaccgtta
tcttcaactc tcttctcaat gagttttata tgcgttatgc 4500 atttatttct
ctattgagaa gaccacatat agcagctcaa gctatagggt gcaaaccctc 4560
tgatttcaac aagctatttg tggcagtcta tggtgatgac aatctagttg cagttcccat
4620 ggaattgcat tggtatactc tgccagctat tgcccaagaa ttggagatgg
tgaatgttat 4680 tataaagaat ggcatcgaca agaacatgga tgttagcagt
tccaaaatgc tagacttgtc 4740 tgagctaaca tttctaagca gaggttttaa
gaggcaccgt ctaggatacg ttcaagctcc 4800 tctgaaatgg gtatctatca
tagaaccaat gtactggata aggccttctg ttggttgtcc 4860 cgatgctctc
gctatgttgg aaaacataga cacgggagtt agagaggcat ttcaccatgg 4920
gcctcaggtt tttgaaaagt tggtgacaga tgttcaaaac gctctcaagg agcggtgttt
4980 cccagccacc acatttccta catattttga attggagcag gactggctgg
tggaggttac 5040 aggaaatcca gccattgggc tcatcaagga acttcatatt
gcagcttcag cttttgtgcc 5100 tttgccccca ggcaatactg ttctgaattt
ttctgatgga gtgcatactt ttgctgaccg 5160 agtgagtttc tgctcctcgc
gaacagctgc tgcacagcag tgggacacca ccactgtttt 5220 ggtgaactgc
actggggcaa agagacccac atgggtaaga gggcccacca catggaggga 5280
ctttgaaggg cttatttggc cttacacaat ggctgcaatc aaggaccaca tctgcagcat
5340 tgtaaccaaa ggggtgacca aaccacatgt ggtttttgtt tgtggcaatg
ggtatgctat 5400 tggtccagtg tgcgctgcat tgtactgtct gtccactggc
caatattctt ctcaggatgt 5460 tgttgtgaga ttgagaacca tagcagatgt
tacagatctc agtcaatatc caggaggttg 5520 tgccaagtat cttctgaaat
gtgctgatac aagagaagaa gagcttgcag atacatgtaa 5580 aattgcacaa
gccaagggtg agacaccagc atacatacct caaggaggat tttcccttgg 5640
taattttaga attgtgcaag ggagaattga tctacagttg gcccagcgct tgccttttac
5700 agtgggacct tatgggggat ggggtcaaca cactactaga gagcttaagt
tgctgctcaa 5760 ggacatggag aagatatatc aaattttagt ccaaagagag
agcttcatca ctctctactt 5820 tgactatctc agttcagagc aggtgatgtt
gttggttgac tttcttaggc tccaagggtt 5880 ttttccycgc caaaatgatg
tggattactt gcttaaagcc tttaagctga gcaagcagag 5940 gcacaataag
gaaaactgtc atacggttta ctttagaaag ccttttctct caaggaaaat 6000
gaccatgggg tccaaagaaa ttctgtccgc aacagctgct gagtcattgt ttggtatgga
6060 tgtttccgct aatgtgctca agagtaggct acttcatctt cagaagccca
taaagtgttc 6120 atccatggag ttggccttta aaatttattg tgtcatccag
ggccacctga gcaaggaagt 6180 tgtaactcac ttccaacgca tgtaccaaca
agatctgaca gaagggatca tagagaaagt 6240 gatattgtgg ttaaccgcca
cactgtcgga gagctttcca gtggatcttg ttgatgtacc 6300 tttaggcttg
gataacatag agatccagga taaaggtttt tccctaaatc caaataatat 6360
aaatatgaat gcatgtgatg ccatcttgtt tcaactcact gagtgttaca accgatcaac
6420 aaagaaacat gtgttctgtc gctacacgac tgcatcctct cttgttgttg
cctatgtgct 6480 tgcacataga catcagacaa ttgatgagtt gccgtccttc
tatgcaacac acccagatgt 6540 gttgcttttg acaccaatcc taacaggcta
caaagcgcct tgagtcgggc taaatgactc 6600 agcttgtaca tgcaatatgt
gtactgtgaa taatattgca tgaggattaa cggagagtac 6660 tgacttttgc
tagtcgggag tccgacccac tatatgggta cctgtgaatc tacacgggtt 6720
aggagatttg cacgcctctc cataaatatg gttcgtgtgc cctgccttgg ttagacagcc
6780 ttccatgccg gaagtaaatg gcctataacg gagagtactg actttaacta
gttgggagtc 6840 cggctccatt tggagtacca atgaatttac attggttaaa
gagatttgca cgcctctctt 6900 taaatatgga tcgtgtaccc tgcttggtta
gaaagttcct ttacttaggt ttacaaagta 6960 cggggagcac tccctggtta
acatagtgca ggtgctatcc catgatagtc ctttaactca 7020 agggttgagt
tcggtttgca tcttttgccg tgatgaaaga tgaggtagct tcccccttat 7080
tgggaggctg aaactacaca tatgtagtgg gtttgactga gtcctataat cagtccgttt
7140 gaaattcgat aattttccgt agcttgcgtc aagctgctca cgttaggggt
gtgagtgaag 7200 atgcgccgta ccacgtcttc cccggcaatg ccagtggttc
agagcgggcc ctcagaatag 7260 aggttaaaac tagtgtgatg gtgtatatca
cgataaaagt gacacccggg ttgtgctgcg 7320 cctagttaac acgagcacag
gtcccaccct atagtggaag aaacttggtt gagttttaaa 7380 agaacaaccc
gtttgagcga cgacaaagtt ccgtagcttg cgacgagctg tttgtgttag 7440
gggcacaaat gaagttatag cacaccactt cttccctagg ttcgtccaga tggtttcaca
7500 gtgctatcct cagaaaagag gttaaatcta gtgtgatggt gtatatcacg
ataaagaatg 7560 acacccgggg tgtgcttctc ctagttaact cgagcacggt
tttcacacca cagtgaaact 7620 atcttattgc tttaaatgtt gtttgtttgt
tttacttgtt ttattgtgtg tttaatatca 7680 tgcatatttg ctgttgaagg
ttctgtcgat gaggtcaaat ggatactcgg gaactacagt 7740 aactttgcat
ttgaattgtt ttcaattcag tgtgtgagtt tgaatatgta ttttaaaaaa 7800
aaaaaaaaa 7809 3 17 DNA Artificial Sequence primer 3 gagagccggc
attcaca 17 4 17 DNA Artificial Sequence primer 4 gcacagcttg gcgacac
17 5 24 DNA Artificial Sequence primer 5 cccatcatca ccctcctctt cgta
24 6 22 DNA Artificial Sequence primer 6 ttccagtaat gatccaacca at
22 7 20 DNA Artificial Sequence primer 7 caagccatca cggaacctac 20 8
19 DNA Artificial Sequence primer 8 agcatcttct tcctccgct 19 9 20
DNA Artificial Sequence primer 9 tgctgaggtg ctatcactgg 20 10 20 DNA
Artificial Sequence primer 10 cacactttcc acgatttcca 20 11 20 DNA
Artificial Sequence primer 11 aaaggaaaga agcagccaca 20 12 20 DNA
Artificial Sequence primer 12 ggaaatcttg gtcaagccag 20 13 20 DNA
Artificial Sequence primer 13 gcaatgccag tggttcagag 20 14 20 DNA
Artificial Sequence primer 14 ggtcaaatgg atactcggga 20 15 7229 DNA
PRI-TMarV0601 RNA 1 15 aaagaattat atatcaagat tgcaacctac gtcgttgttc
acagactacg cctctgttaa 60 ttgatatttg attcttgtaa acttttccaa
catcttcttc tcttcttact cgaccattat 120 ttaaactcct caattttcca
atggcttttt caaagatgtt ctccaaggtt gctggtgttg 180 atgacgccag
ttcttcttcc accacttcat tctttggtga actttcaaat tctatttcca 240
aattttcaaa agcggttagt aatattacca ctatgtcaca gaagatttca gatcatcttg
300 aggatctgaa accatctgtg actgatgtgt ccacatcttt tgtggccact
tgtagttcac 360 ttaataaagt tttggacaaa attggtgcac ttatagatcc
atttcttaaa gcgtactctt 420 tcttgtccac aatgtacaaa tctatcaagg
atatggtttt aaaattgttt gagagtttgt 480 ccaataaagt taaattagga
tttgcttggg tgttaaataa
aagtgaagat gtagatatag 540 ttgtatatgg ttttcttgta tttgctataa
cattgttagt gcttctattt gtttgtccta 600 gtgatattgt tgagggtgtt
tctaacactg tcaaaaatat ctttttgata gtcggaaaca 660 tgttttcaag
tctatacaat ttagattggt ttccaaaatg ggccgaacga ttcaccatgg 720
tggcccaagc tagtatgctt cctggggaat cagtttcaca tacaccaacg tctcagttga
780 tgtccacaat attggctttt gggatttcga cgttggtgtt cattgcagtg
cctggtagac 840 caaatggctt gagcaaccca ttgtccaaaa ttctctattc
gacaggaagt ggtgctcagc 900 agtgcaatca actattcact ctttaccgta
atatgaaaga ttgcacctcc caggctttct 960 catgggttct tgaaataata
gtcggcacct ttggattcaa aaatcctgtg ttgtcagcta 1020 taagtgcaac
gctgtccact gatctgttcg agtggatgca ggaggttgat gcagtgtgtg 1080
atcctgcaac gcgccttgag aactttgcca ataaggcttt ccctaccaaa ttgaaccatc
1140 tgagggaaga agctctcaag atctcagctt acattgcaac ccatccagtt
gcggccttca 1200 tgagccacag ggttagtgct gccattgcgc agttagaaaa
agtttatgct gaaagttgta 1260 ggcacatggg cgtgggccag tatcgtattg
aaccttttat ggtacaatgg ttcgggtcca 1320 gtgggtgtgg taaatctaca
tccatgcgct tatttattaa tgatgtgttg gacagaatgg 1380 gtgagccaaa
actcaatcgg ctatatgcag taagtaagag ggatgcatat tggtccaact 1440
atgctcacca aactgctatc ctgatggatg acatgggagc attgcgagat ggggctgggc
1500 agtgccaaga tattaaagat ctgattgata tcaaatcaac acaaccagca
cccttaccaa 1560 tggccgcagt tgaggacaaa ggcaggcatt tcacctccaa
gtatatattt gccacatcca 1620 atctgatctc agctcctgcc caatgtggtc
ttacatatcc agatgctttt gagcggagaa 1680 gggacatcct ggtggagtgt
atgaaggagg gcgagttttc cactgaagat cctacgggac 1740 atctcagatt
taacatagtg gagagcagaa gacctcatgc tatcactcac aggaatttga 1800
cctatagtga tcttttggag tatgtggtag ccaaatgtca ggtacatgca gaagtatcaa
1860 agcaattgtt tgaagctgaa tctggcataa gtcctaagat agctcaggtt
caagtttctg 1920 cagatgatgt gatagcgtct gtggatgggg ctagattgcg
caccaagcaa gatgaaccaa 1980 tattggtgcc cactgttgtg agtgaaaatg
atagagtgat ctatgcaaga gagctcacgg 2040 tagaggcatt gaaatatgca
taccagggca gtttggaccc tgaggaactt tttcctcatg 2100 accatcataa
gcaggccatg tatgattctc ttgatgatga acataaagag attttcaaca 2160
aatggagagt caacatgttg tatagaggag cagatgctga acagtatcga tggttggtac
2220 aaaacattcc tgatgactat ataatgcact ttaagagttt catttatgca
tcaaccatct 2280 ctgagaagaa actagcagtg cagacagaga tgaggactgg
atttgcacat tcttgcattg 2340 atgcagatgt tgacaccctc atatgtatcg
agcagatgcc accctttgtt cagtttttat 2400 atacagcatt tgtgagatac
tggtgcaaca aagtatcaaa agagccaaag gaatcatgga 2460 tcaaaatttg
ctaccataaa atcgttgaat atatcaagga tacatggtgg agccttccct 2520
atgcactgag attgctcatc aaagcaggtc tgattataat ggctcttaat ggagtttttg
2580 gaggcattac agcatttttg gcgtgctggc agagtaactc tttccccaat
gcatcgggca 2640 gaggaggtgt gaccaatgaa tccaacagta tatccagtaa
gaagaacaag ggtaataagc 2700 tcagaaatct tctcgttggt caaagttctc
aatcattggc acaagattgg gctgctgaag 2760 atggatttgt aaatcagagc
ctcaagaaaa atttggtggt gttaagactt ggtgaaggag 2820 tgtactttag
aggcacctac gtgtgttctg gttggataat gaccgtagct catgcattcc 2880
acaatgctcg agatggtact ccatttacaa tcatccatgc caattctcga tctaaagttc
2940 aatacaacgc cagagaatca aggattattg agggccaaga catcattctg
ttgcgcgttg 3000 gtgatccaga tggtccaaag cctgacatcc gtaaacactt
cccaagaagg gatgaggtgt 3060 gcttcacaaa gggctcacaa ggattgtgct
gtagagctgt tgcgtctaca gatccacgtc 3120 ttggcaattt agagtttctc
aagatgccag tgatgatgtc aaagggatac acagttaaag 3180 tggaatatga
actgaactcc tccagtttta agatttgctc tcaacaatct tatgaatacc 3240
acataaatgg ggaaaatggt gactgtggca cgttgctact gttaccaagt gttcagaata
3300 agcaacctgt gatcgttggc atccactgtg catcatatga tggcgtagca
gctgaacgtg 3360 gatttatctc ttcaaatgct acagctatct acagggaaca
actagaggat ttgccaactg 3420 ggccggtcaa agcagcaatg gtacgctgtg
atattctgaa gtcaattaga agcagagaaa 3480 cacagctttt tgaggaaaac
caagtgtact accttggaac agttccacag gagttggccg 3540 ccacagttcc
ccacaagacc actctgcgga aaagccaatt gtttgaagca ttcggacctg 3600
cagagacagc accatccatt ctaacagttc atgacaaaag aggtgatggt tttgacccct
3660 atgtggctgg ggtaatgaaa tacaatgaaa cagcttgtgg atttgatgat
gacattgcca 3720 aactagcatt cgaaaatctc aagtgctcgc tgctacctat
catgcgtagc cagaagatcc 3780 ctgggggacg tccatgtgaa agggatgagg
atgtagtgct caatggaata gatggatgtg 3840 attactatga tggcatggag
ctgagcacat cttgcggata tcccttcaac aagatgggga 3900 tggggatgaa
caagagagaa tttgtgcaat ccactggcga aggagagaga gtggaactca 3960
aaagagacac tcctgtattt gaagcatggg aagagctaga tgtgcagatt aggaaaggca
4020 tccatgtgga tctggtcacc acccaatgcg ccaaagatga acgcctccca
cttgagaaaa 4080 tctatggcaa gagaaagacc aggctctttg agatacttcc
tttccattac aacatgttgg 4140 tcaggaagta ttttcttgat ttctcagcca
cattgatggc tttgcacaat gctataccat 4200 gcaaagttgg tattgatcct
acaagttctg agtggacatt gttggcaaat gggtttagag 4260 ctgtgtcaga
cgtgggattt tcagctgatt attccagctt tgatggaaga gcacctgttt 4320
ttgcttttca gtggttttgt gatttggtgg atgaatacta cggatcaaag cctggcagtc
4380 ctgattccaa tgctcgacat gcacttttaa tgatggcatc ttgtcattac
acactgtgcg 4440 aggataaagt gtttaggttg gttgggggca tgccatcagg
atttgcacta acggtcatct 4500 tcaattctct cctcaatgag ttttatatgc
gatatgcctt tatatcattg ttaagaagac 4560 cccatattgc tgccagggct
ataggagtta aaccaagtga tttcaatcag ctattcatag 4620 ctgtttatgg
agatgacaat cttgttgctg taccattaca tctccagtgg tattctctgc 4680
caaatatagc acatgagtta gaactggtca atgtaatcat taagaatggt cttgacaaat
4740 cgatggatgt taatgaggta caatttcaag atttgtctga gctaactttt
ctgagtagag 4800 gttttaaacg acatgctctt ggataccaca tggctcctct
caagtgggtt tcaatcattg 4860 agcccatgta ttggataaga cctgccccag
gttgtcctga cactcaggcc atgatggaaa 4920 atgtggaaac aggaatacgt
gaagctttcc atcacggtcg tgtggcttat gacaagcttg 4980 tcttagatgt
tcagacggcg ttggatgaaa ggggtttcag agctgtgatc tttccttcct 5040
atttggaagt ggaacaggaa tggattgcaa aggtaacagg ggattcaagt gccctgacaa
5100 tttgtgaaat ggcaaaagca gctatttcct atacgccatt ggacgcaggt
gagaagatca 5160 caaattttga gcgtgatctg aattggtttg caccaaacat
tggtttttgt tcagcacgta 5220 ctgcagccca ctacacgtgg gatgaagggt
acattattgt caactgtaca ggtgcaaaga 5280 agtccaattg ggttagaggt
ccagccaact ggaaggactt tgaagggaaa atgtggccgt 5340 acactatgtc
agctataatg gatgcccaaa agaatgtgtt ggcaggagga catgtagcaa 5400
ccaatgtcgt ttttgtgtgt ggaaatggat atgctgtcgg cccaatttgt gcagcgctga
5460 tggccttagc aacaaggcaa tattgtgtgg aggacataat agttagattg
cgcacaattg 5520 gaaatgtgct tgacctcaat acctatcctg gaggctgtgt
gcagtatttt cttcaatgtg 5580 tgcctcatgg agacaaagtg gctcaaagtg
gtgcatcgct ccacagtagt tttatgcacc 5640 aagggtttga attgggcaac
ctccgcatta tacatggtga tttagcaaaa cagacagcaa 5700 tgcggatgcc
atatgtggta ggaccacatg gaggatgggg aaatttttcc acacaggatc 5760
ttgagagttt gctacactat ttggagcagg gatatgcaga gttaattcaa aagaatacaa
5820 aactaactct gtatttcaaa gagctgagta tggaaaatgt gcaacaactg
atagattttg 5880 ttaagcttca ggggtttttc ccaaaagaaa ctaccattca
gaagctcaaa aattttgttg 5940 atgctgaatg tctaacattc aaagcaagga
gctttaggca cgtagttttc aaaaagaaac 6000 ttttgagttc cacatggaaa
atgtgtggtg aaagtattgt tgcttcaagg tctgcagaga 6060 gtctattccc
tggcaatttg tctgcttccg tattgaaaac attattggaa agacacacaa 6120
gaagcatgag ttgtcagagt atggagcttg ctctgaaaat atatctatta aacttccaag
6180 taataacaag tgagatatta aagaagtttg aagatatatt tcaggagaag
atatccacca 6240 ccctactaat caaagtattc ttgtggcttg aggagagtta
tcagactgag atctgtatag 6300 attctcaggt gctccagagg ataaactccc
agaaattcag agtgcaggaa ggtggttttt 6360 atttgcatcc tgaaggaatc
aacatgaatg ccgtagatgc aataatattt tctctgtggg 6420 agtcctatag
ccgagatagt aactcctatt ctgtgtcaac tcccatcaaa ctggggtgtt 6480
tcattttcct tgtcgtgcta gatagtcaag gcaaggaaga tcatcctgtt cggaggtttt
6540 caacggtttt ccttaaaaac tgtgaaacca ttcttacaaa ttacaaggag
ccttgagtca 6600 ggcattatga ctcaatttgt gcgatatgca tgtgtactgt
tagtaatgta tatcgttagg 6660 atggttttgt taagggagag tactgtcttt
taatagatgg gagtcccctc cactttatgg 6720 agaccaatga atctacattg
gtatagaggt ttgcacgctc ctctttaaat aagtttcgtg 6780 tgccctgctt
ggttagaaag catgtggtga ttaacactac tcgtttggag tataagcaat 6840
agacctcatg atgtctaact catgcgtgat tgctcatgta cgaaataaat gagccgtttg
6900 gaactcgata atttccctta gctgctgcac agctgtcact attaggggta
gtggcgaagt 6960 cgtgagtccc ctcttcctcc ctaggttcgt ccagagggtt
caaagattca ccttctttgt 7020 caagaagcaa tgaatgacac gtgttgcgtc
gacaaagcac cgttctacgt ggttagtaga 7080 aagttatatt atattagatt
tgttagtcaa ttgtgtgatt tctttcttag attaggaagt 7140 tttccgtggc
gataggaagg gtttgtcctt ttaccttctt tgctatgctg gacacaaaaa 7200
gattttcttt tcttttattt taaaaaaaa 7229 16 4906 DNA PRI-TMarV0601 RNA
2 16 aaagaattgt aatatatatg wgtcatgcaa cctacgtcgt tgtgcgatct
acgtcatcgc 60 tcgcagatat aaaattaaat ccttttagct tgagaactac
gtcttctcat tttcttttcc 120 tttgttttgt tgttctttat gtcattcatt
gggcgtttga atacatctgt agacgaacaa 180 gcttttcatc atcaagttgc
cacctcgaat tggatttgtt ctgttgatgt tggcaccggt 240 ttgataaata
gcaatcctac ccttgacttc aaagttgttc ctccatcagg tggtgctgtc 300
tcagttttaa ccgtttcttg ggagaattca acgccacaat tagtgcctgg acactatttg
360 ttgcgtagtg gtaattggcc cattaagaac gtaaagcttt ctggtttact
tgtccatcgt 420 tcagtgcgtc ttgaaacaac cagaaaggtc ctagaggaaa
acaaggtttc tatttcagca 480 tcatcttcat cttctccttc ttcttctgac
agtaaaggca agagtaaagt agagcaaccc 540 acacgagagg atctcattaa
agaagttgag gttctcaaac gtgagttaga gagatttcag 600 aaagagttgg
caagtcaaaa atctgaaaat cagaaactac aacttcaact ctccaatcaa 660
gttagtaata atgacatctt ctcaggttgg actgaaagtg ggccccagta atcaccagca
720 ggaggaagcc tccgagaggt tgatatcaca aataacagct gctgtggagg
ccggaaacaa 780 aaatttgctc agaaagcttg gcatgggttc ctacggggtt
ctttatggtt cccaagagaa 840 gagagcaatg gagttrtttg acccagatga
tgtatcaaag atcacatccc tttggtcaac 900 attcaaacat aagtttgttg
agtctaagga ccatgcaaac ttgttttttc atctgtatgg 960 tgttcttttc
ttcatggtcc cacatgttca tagtggggag ggaagggtta agataagtct 1020
ttgttcaagt aatgatcccc tctcaccggt aatacaagaa aagacactgt ccttggccga
1080 tggagcgcag gcagttctca tgagccccag cataacattg ccctttctca
aaagagggcc 1140 catgttttac tacaccctgg agtgccaaaa taccagggca
cagattccgt gttccgtggt 1200 tgccatatgg aaacaaaaga ttgacacaag
gagtgcagtt tattcacagc aagaaacaat 1260 gtcttgggcc atagaagcac
tcaatcgtcc gcaatttttc caggatagac aggaggctgc 1320 tcaatatata
gcttctgttt attcgagtgg gcaaagtact caaatggctc ttgaaaacaa 1380
agcctttgtt ggagaacagc ttgggggcac aagaatggac gtcatgaatg aaagcactat
1440 gatccggagt tcatctttga gagtcccaac tctcaaagtg cagagcaagc
gcttcccgtc 1500 catggaactg ccggctgtaa gtacatcatc cttactcagc
acacgtgaag aaacagttca 1560 tgatgaggat gattgtgggg gattgtttcc
agcacccaag aaaaagggcc aagccttcaa 1620 catgggtgcc atttgggata
atttgggcat ggagtcgttc gcacacattg atttcccaga 1680 tgattggaca
gaacgaacaa tagctcagca ggtacaattt atcctcttta gtgaggctaa 1740
aagaggaaac gttattgtgc caagacatgt tgcaaagcgg catctgcata atatcaatag
1800 ggagcacata accgaggaca attatgtaga aatacttgaa ggttatggtg
ttaccaatat 1860 ccaaggcttg actcgcactt tcaattggta tgctatgtct
ctcaaggaga gagttgtgga 1920 gctcgtccat caaagagatc attccttcta
tattcaaggg cagactaaca atccaatgcc 1980 gaattttgac tgctatgatg
gattaacctt gaaagagcgt cagttgattg tggaggagca 2040 agtggcccaa
agaaggtctg aaagacaagc acaggtgaca gcaaggggca tagctgaaag 2100
tcagcctgaa gacagagtga ctgattcctt tgtgtcaact accacgatgg aagatcccac
2160 caagccagat aagatagaaa ttgttgcaga gggagcagaa gaagaaactc
aacctggtga 2220 tgttattttt gactttggac cagaaatgga cacatccatg
gcagttgaac tggatatgca 2280 gcaaccggtg tgtgtagcta gcaatgattt
cttcaatgtt ggagtttttg aattcgtttg 2340 ggagaagtcc gctaatgttg
ctgagcaagt aatgagcttg gctttgcccg ctgccctatt 2400 ctcaaaaagc
aaagaaactt caatgggtgc gcaaatgctt aagtattacg atgcagctct 2460
aatcatgtac aaaataatac tttatgtttc tggagttgga gctatctctg gtcaactggc
2520 tttggtgtgg gatgaatgta atgtgcttaa tcgaaagaag gaattcatca
acatcgccac 2580 attgtatgcc agtaagcaca cattggtttc agcttcacaa
cagaacagtg aggagttttg 2640 ctttacccca acagggatag gcaagtacgt
gcctctggat gaaggtacag gagccactga 2700 tttaggtagt gtgagagtgt
ttgtgacaca ccccttatct agtgcaactg agttgaatag 2760 tgtaccatgc
catttgcact tacagtgcaa agtgttatca accaatatac ttcaacctcc 2820
acgaatgata gcacaggctc aatatggcat gaaggcgggg cagacatatt ttccaaggtt
2880 tccaactaat caggttttgt tacattataa ttgggggaca tcatccccaa
tgggaactac 2940 attggtaagc atattttcac catcaggaat atatgagagt
gatggcacct tgcagccgtc 3000 tttgcttggt aacatagcca ggaattgcaa
gtggtggact ggaacttgtg tttttgaaat 3060 ttgcattgag aaaaccttgt
ttcattcagg tagtctggca attggacttg gaactctgaa 3120 caccaaaatg
accaatgctc atgatatatt taacatgcca catgtggtat gcaatcttga 3180
aatgggtcga aaatttcggt tccggtgttc tattacaaat tggaatggaa aaaatttgct
3240 ttccacaggg cgaaagagtt ccttgccaag accacagcac ttttcccact
tgcgcttgtt 3300 tgcaacggta atgaagccac tcgtttcaac gtccatacat
ctggattccg ttggtgtcac 3360 agtgcagttg aagtgccttg aaaatcttac
tttgggtggc acagtatctg tgaaaccaat 3420 atatgggcat tggacaaaag
gcaaaagctc agttgatttc cttttctctg aaatggattt 3480 atcacagcgt
aaggaaattg aaaagttgag aaaggacaac attgaggagt accaggagaa 3540
aggcaaagat ccgcccaaga aggctcaaag tattctgtcc ataagagaga aattttccta
3600 tggtgctgta caatatttct gcatgggttg gaaggatgac gaaagattgt
tggtaattcc 3660 ttgtgcacca tggtccataa ggtttgaagg gcacagtcct
gttaaggagg caatcacttg 3720 tccatttata gattggtgta catcattttg
ttattggtca ggtagtttga attattcaat 3780 tgtgatacac agagtacaat
ccagtcctaa tgttggaggt gtactaaatg ttgcttttga 3840 tgcctcaggc
tatccttttc cagctgggct taataaagga aattatgtgg tatcagcagg 3900
tggaggcaca aaatgggatt tttcatacgg tgtggcaaca aatacgttct cattcactgt
3960 gcaagatgat gagtttttcc caaggcggca tacaaggatg agggaattct
caagcaagca 4020 atcccgcatc atgtcactac aggataggct tggaaatctg
atcataaatt tgcctccttc 4080 cgccatagtg agttccattg agatacttat
atctcctgga cttgatttca agttggagtt 4140 ggcccaacct ccttctgcca
accatgaaaa atatcttggc aatatgcaaa ctcacaccta 4200 tcagtatacc
tcagattttt ctgagctacg tgattttgcg atttgaaaaa gtaccatgct 4260
actgggtata tagtagccaa atacacttat ctacgtgtac tgttagtagt agctaagtgg
4320 tgtttgtttg caatgataag ggagagttaa gggagagtac tgtcttttaa
tagatgggag 4380 tcccctccac ttcatggaga ccaatgaatc tacattggta
tagaggtttg cacgctcctc 4440 tttaaataag tttcgtgtgc cctgcttggt
tagaaagcat gtggtgatta acactactcg 4500 tttggagtat aagcaataga
cctcatgatg tctaactcat gcgtgattgc tcatgtacga 4560 aataaatgag
ccgtttggaa ctcgataatt tcctttagct gctgtacagc tgtcactatt 4620
aggggtagtg gcgaagttgt gagtcccctc ttcctcccta ggttcgtcca gagggttcaa
4680 agattcacct tctttgtcaa gaagcgatga atgacacgtg ttgcgtcgac
aaagcaccgt 4740 tctacgtggt tagtagaaag ttatattata ttagatttgt
tagtcaattg tgtgatttct 4800 ttcctagatt aggaagtttt ccgtggcgat
aggaagggtt tgtcctttta ccttctttgc 4860 tatgctggac acaaaaagat
tttcttttct tttattttaa aaaaaa 4906 17 6 PRT Artificial Sequence
motif 17 Leu Arg Val Pro Met Leu 1 5 18 6 PRT Artificial Sequence
VARIANT 2, 3, 5 Xaa = Any Amino Acid consensus sequence VARIANT 2,
3, 5 Xaa = Any Amino Acid 18 Leu Xaa Xaa Pro Xaa Leu 1 5 19 6 PRT
Artificial Sequence motif 19 Leu Arg Val Pro Thr Leu 1 5 20 6 PRT
Artificial Sequence VARIANT 2, 3, 5 Xaa = Any Amino Acid consensus
sequence VARIANT 2, 3, 5 Xaa = Any Amino Acid 20 Leu Xaa Xaa Pro
Xaa Leu 1 5
* * * * *
References