U.S. patent application number 14/386441 was filed with the patent office on 2015-02-19 for agrobacterium for transient transfection of whole plants.
The applicant listed for this patent is Nomad Bioscience GmbH. Invention is credited to Luisa Bortesi, Anatoli Giritch, Yuri Gleba, Patrick Roemer, Doreen Tiede.
Application Number | 20150052638 14/386441 |
Document ID | / |
Family ID | 48170407 |
Filed Date | 2015-02-19 |
United States Patent
Application |
20150052638 |
Kind Code |
A1 |
Roemer; Patrick ; et
al. |
February 19, 2015 |
AGROBACTERIUM FOR TRANSIENT TRANSFECTION OF WHOLE PLANTS
Abstract
A process of transiently transfecting a plant or leaves on a
plant, comprising contacting said plant or said leaves with a
suspension comprising Agrobacterium cells of strain CryX or a
derivative strain of strain CryX, wherein said derivative strain
has the chromosomal background of strain CryX or said derivative
strain contains the vir plasmid of strain CryX or a derivative of
said vir plasmid.
Inventors: |
Roemer; Patrick; (Zoerbig,
DE) ; Bortesi; Luisa; (Aachen, DE) ; Tiede;
Doreen; (Kothen, DE) ; Giritch; Anatoli;
(Halle, DE) ; Gleba; Yuri; (Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nomad Bioscience GmbH |
Munchen |
|
DE |
|
|
Family ID: |
48170407 |
Appl. No.: |
14/386441 |
Filed: |
April 3, 2013 |
PCT Filed: |
April 3, 2013 |
PCT NO: |
PCT/EP2013/000994 |
371 Date: |
September 19, 2014 |
Current U.S.
Class: |
800/294 ;
435/252.2 |
Current CPC
Class: |
C12N 15/8205
20130101 |
Class at
Publication: |
800/294 ;
435/252.2 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 3, 2012 |
EP |
12 002 402.1 |
Claims
1. A process of transiently transfecting a plant or leaves on a
plant, comprising contacting said plant or said leaves with a
suspension comprising Agrobacterium cells of strain CryX or a
derivative strain of strain CryX, wherein said derivative strain
has the chromosomal background of strain CryX or said derivative
strain contains the vir plasmid of strain CryX or a derivative of
said vir plasmid.
2. A process of transiently expressing a DNA sequence of interest
in a plant, comprising contacting said plant or said leaves on said
plant with a suspension comprising Agrobacterium cells of strain
CryX or a derivative strain of strain CryX, wherein said derivative
strain has the chromosomal background of strain CryX or said
derivative strain contains the vir plasmid of strain CryX or a
derivative of said vir plasmid.
3. The process according to claim 1, wherein said strain CryX or
said derivative strain contains a binary vector having T-DNA
comprising a DNA sequence of interest to be transfected into cells
of said plant or leaves.
4. The process according to claim 1, wherein said strain CryX or
said derivative strain contains a disarmed vir plasmid or disarmed
Ti-plasmid.
5. The process according to claim 1, wherein said derivative strain
of strain CryX contains a vir plasmid that is a derivative of the
vir plasmid of strain CryX, and said derivative strain achieves at
least 70% of the T-DNA transfer efficiency from a T-DNA-containing
binary vector into plant cells of the T-DNA transfer efficiency
with strain CryX.
6. The process according to claim 1, wherein said derivative of the
vir plasmid of strain CryX encodes the virG protein of strain
CryX.
7. The process according to claim 6, wherein said derivative of the
vir plasmid of strain CryX contains the vir region of the vir
plasmid of strain CryX.
8. The process according to claim 3, wherein said binary vector
comprises a virG gene expressible in said strain CryX or said
derivative strain.
9. The process according to claim 8, wherein said virG gene encodes
a VirG protein from Agrobacterium tumefaciens strain LBA4404 of SEQ
ID NO: 1, or is an N54D mutant of the VirG protein encoded by the
virG gene from A. tumefaciens strain LBA4404.
10. The process according to claim 1, wherein said derivative of
the vir plasmid of strain CryX is obtained by introducing a T-DNA
of interest into the vir plasmid of strain CryX.
11. The process according to claim 1, wherein said plant or said
leaves on a plant are contacted with said suspension by spraying or
by vacuum infiltrating said plant or said leaves on a plant with
said suspension.
12. The process according to claim 1, wherein said plant is a dicot
plant such as Nicotiana benthamiana, tobacco, cotton, soybean,
rapeseed, pepper, potato, tomato, or a monocot plant.
13. The process according to claim 1, wherein said Agrobacterium
strain CryX is the strain having DSM accession No: DSM25686.
14. Agrobacterium strain CryX having DSM accession No: DSM25686 or
a derivative strain of strain CryX, wherein said derivative strain
has the chromosomal background of strain CryX, or said derivative
strain contains the vir plasmid of strain CryX or a derivative of
said vir plasmid; or an Agrobacterium cell of strain CryX or of
said derivative strain.
15. The Agrobacterium cell according to claim 14, wherein said cell
further comprises a binary vector comprising a T-DNA comprising a
DNA sequence of interest to be transfected into a plant.
16. A kit comprising an Agrobacterium cell of said strain or said
derivative strain as defined in claim 14 and a vector containing in
T-DNA a DNA sequence of interest to be transfected into cells of a
plant.
17. Vir plasmid of Agrobacterium strain CryX having DSM accession
No: DSM25686.
18. Agrobacterium cells having the chromosome of strain CryX having
DSM accession No: DSM25686.
19. Aqueous cell suspension of Agrobacterium strain CryX having DSM
accession No: DSM25686 or a derivative strain of strain CryX, said
suspension having a cell concentration of at most 1.110.sup.6
cfu/ml of the suspension, preferably at most 4.410.sup.5 cfu/ml of
the suspension, and more preferably of at most 1.110.sup.5 cfu/ml
of the suspension.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a process of transiently
transfecting a plant or leaves on a plant. The invention also
relates to a process of transiently expressing a DNA sequence of
interest in a plant or in leaves on a plant. Further, the invention
relates to an Agrobacterium strain.
BACKGROUND OF THE INVENTION
[0002] Current genetic engineering processes for agriculture are
all based on stable genetic modification of crop species,
demonstrated first in 1983 (Fraley et al 1983; Barton et al 1983)
and commercialized since 1996. Although agriculture processes based
on plant stable genetic transformation is a reality today and is a
basis of successful new practices, it has multiple limitations, the
main ones being very long time and high cost required for
development of transgenic crops. General consensus among the
companies involved in plant biotechnology is that the R&D
process requires, depending on the crop species, between 8 and 16
years, and the total average development cost is estimated to be
between $100 and $150 million. Because of these limitations, after
more than 25 years since the discovery of a plant genetic
transformation process, only a handful traits and few GM crop
species have been commercialized thus far.
[0003] It is known that plant cells and whole plants can also be
re-programmed transiently (i.e. without stable integration of new
genetic material on a plant chromosome), and the transient
processes, such as viral infections, are fast. Such transient
processes could in principle allow a very fast modification of
plant metabolism in favor of certain products that are of interest
to the user. Such processes require a DNA or RNA vector (a virus or
a bacterium), that has been engineered to effectively and safely
transfect the plant. Earlier attempts to use vectors based on plant
viruses have been partially successful in that they allow
transfection of plants for manufacturing of high-value recombinant
proteins such as certain biopharmaceuticals (Gleba et al 2007,
2008; Lico et al 2008). Use of viruses for manipulation of other
traits, such as input traits (for example, herbicide resistance,
Shiboleth et al 2001; Zhang and Ghabiral 2006) have been described
in the literature, but virus transfection introduces so many
undesired changes in the infected host that this kind of transient
process is not pursued anymore for input traits. Transient
processes can also be built around the ability of Agrobacterium
species to transfer part of their Ti plasmid to eukaryotic, in
particular, plant cells. Use of Agrobacterium-based transfection is
a basis for genetic manipulations such as genetic transformation
protocols and of laboratory transient transfection assays.
Industrial applications of Agrobacterium-based transfection have
also been limited to recombinant protein manufacturing, because the
optimal application conditions such as vacuum infiltration of
plants with bacterial suspensions cannot be used on a large scale
in the field, whereas spraying aerial parts or watering plants with
bacterial solutions results in a supposedly very small proportion
of plant cells to be transfected, and previous studies simply did
not address that specific question.
[0004] Agrobacterium tumefaciens and A. rhizogenes are broadly used
in research laboratories worldwide for transient transfection and
stable genetic transformation of plants. These applications are
based on the ability of Agrobacterium to transfer genetic
information to eukaryotic cells. Many of the transgenic plants
cultivated today, such as soybeans, canola and cotton, have been
generated through Agrobacterium-mediated genetic transformation.
The essential difference between the transient and stable
transformation is that in the process of stable transformation,
Agrobacterium-delivered DNA is eventually integrated into a plant
chromosome, and is afterwards inherited by the plant progeny. Such
integration events are rare even in laboratory experiments
specifically designed to provide massive contacts between plant
cells and bacteria; thus for the selection of stable transformants,
specific selective screening methods have to be utilized and
specific plant explants (rich in meristematic tissues) selected for
optimum transformation and regeneration into whole plants are
employed. Subsequently, the knowledge accumulated in this science
domain is of limited value to those interested in transient
processes where many cells of the plant body should be affected
without selection for transfected cells.
[0005] Transient transfection, on the other hand, takes into
account only earlier steps of Agrobacterium-driven DNA delivery
into a nucleus of a plant cell, along with the fact that such
delivered DNA molecules can be transcribed in a nucleus even in the
absence of DNA integration into a plant chromosome, such expression
resulting in a transient metabolic reprogramming of a plant cell.
Such reprogramming has been developed into a laboratory tool for
rapid evaluation of different genetic experiments. Whereas there is
considerable body of knowledge about Agrobacterium-mediated DNA
transfer to plant cells, that information is invariably limited to
laboratory scale experiments, and thus far, there were very few
attempts to develop industrial scale applications involving
Agrobacterium as a DNA vector.
[0006] One of the limitations of laboratory applications is the
fact that Agrobacterium-based DNA delivery requires certain
treatments that are difficult or impossible to apply in open field
or on a large scale. In typical transient experiments, cultured
plant cells or parts of plants (explants) are treated with an
excess of bacteria to provide for maximum delivery. In typical
research experiments, one is also interested in expression levels
that are not economically viable if done on an industrial scale. In
general, the research done in this domain has led the inventors to
the conclusion that the parameters seriously affecting transient
expression are those allowing for the best interaction access of
agrobacteria to plant cells within a plant body. Most such studies
utilize vacuum infiltration, injection into plant leaf or
surfactant treatment, wounding of plant surface e.g. with razor
blades, or combination thereof. In fact, the only group that is
developing an Agrobacterium-based transfection process for
commercial production of recombinant proteins that does not involve
further (virus-based) amplification of the original DNA, is the
group of Medicago (D'Aoust et al 2008, 2009; Vezina et al, 2009).
Their process relies entirely on vacuum infiltration as a delivery
method. However, because of being based on great excess of bacteria
to plant cell ratio, current laboratory protocols used for
transient transfection of plants do not have serious translational
value, i.e. they cannot be directly replicated on an industrial
level. Except in few cases (e.g. Vaquero et al, 1999, D'Aoust et
al, 2008, 2009) they also have not addressed quantitatively the
issue of efficiency of the transient transfection process.
(Examples of such research are multiple, we provide a citation for
just a few representative ones: Li et al, 1992; Liu et al, 1992;
Clough and Bent, 1998; De Buck et al, 1998, 2000; Chung et al,
2000; Yang et al, 2000; Zambre et al, 2003; Wroblewski et al, 2005;
Lee and Yang, 2006; Zhao et al, 2006; Shang et al, 2007; Jones et
al., 2009; Li et al, 2009; De Felippes and Weigel, 2010).
[0007] One of the industrial processes being under development
today is magnifection, a process that is based on
vacuum-infiltration of agrobacteria into leaves of plants. The
magnifection process (trademarked by Icon Genetics GmbH as
magnICON.RTM. and covered by several patents/patent applications)
is a simple and indefinitely scalable protocol for heterologous
protein expression in plants, which is devoid of stable genetic
transformation of a plant, but instead relies on transient
amplification of viral vectors delivered to multiple areas of a
plant body (systemic delivery) by Agrobacterium as DNA precursors.
Such a process is in essence an infiltration of whole mature plants
with a diluted suspension of agrobacteria carrying T-DNAs encoding
viral RNA replicons. In this process, the bacteria assume the
(formerly viral) functions of primary infection and systemic
movement, whereas the viral vector provides for cell-to-cell (short
distance) spread, amplification and high-level protein expression.
The scale-up (industrial) version is built around fully assembled
viral vectors (rather than pro-vectors requiring in planta
assembly) and requires apparatuses for high-throughput
Agrobacterium delivery to whole plants by vacuum infiltration. The
process can be scaled up but it requires submersion of aerial parts
of plants into bacterial suspension under vacuum (the process
involves inverting plants grown in pots or in trays), a procedure
that imposes limitations on the volumes of biomass that can be
treated in this way, on the throughput of the process, on the ways
the plants can be cultivated prior to treatment, and it also
carries certain costs that limit the use of the process to
high-cost products, such as recombinant biopharmaceuticals only.
The magnifection process is efficient as it allows transfection of
almost all leaf cells in treated plants, or approximately 50% of
the total aerial plant biomass (the rest being stems and leaf
petioles). The process has been optimized in many ways, see e.g.
Marillonnet et al, 2005. However, the current process has been
built entirely around bacterial delivery methods such as injection
into a plant leaf or vacuum-infiltration (e.g. Simmons et al,
2009), wounding of leaves (Andrews and Curtis, 2005), or pouring
agrobacteria into soil (`agrodrenching`, Ryu et al, 2004; Yang et
al, 2008), but these methods can not be applied for the mass
treatment of the plants in a field (reviewed in Gleba et al, 2004,
2007, 2008; Gleba & Giritch, 2010, 2011; Lico et al, 2008;
original articles of our group include Giritch et al. 2006;
Marillonnet et al., 2004, 2005; Santi et al, 2006; and
ideologically similar papers from other research groups--Voinnet et
al, 2003; Sudarshana et al, 2006; Gao et al, 2006; Mett et al,
2007; Lindbo, 2007a,b; Plesha et al, 2007, 2009; Huang et al, 2006;
Regnard et al 2009; Green et al, 2009; Shoji et al, 2009).
[0008] Attempts to use Agrobacterium treatment on whole plants (in
planta) without vacuum-infiltration have resulted in a very low
number of initially transfected cells, thus greatly limiting the
practical application of the process. Moreover, since no selection
for transfected plant cells is done in transient transfection
systems, the entire transient transfection process is of too low
efficiency for large scale applications if vacuum-infiltration is
to be avoided. Further, several plant species such as soybean or
rape seed are difficult to transfect by Agrobacterium, unless
specific plant tissue is used, whereby in planta transient
transfection has not been achieved to a significant extent.
SUMMARY OF THE INVENTION
[0009] Departing from the prior art, it is an object of the present
invention to provide an efficient process of transient in planta
transfection. It is another object of the invention to provide an
efficient process of transiently expressing a DNA sequence of
interest in planta. Further, it is an object of the invention to
provide an efficient process allowing transient plant .
transfection using Agrobacterium on a large (industrial) scale
(i.e. to many plants in parallel) without the need for the
application of pressure differences to introduce Agrobacterium into
the intercellular space of plants. It is also an object to provide
an Agrobacterium cell and strain suitable for this purpose.
[0010] These problems are solved by a process of transiently
transfecting a plant or leaves on a plant, comprising contacting
said plant or said leaves with a suspension comprising
Agrobacterium cells of strain CryX deposited under accession No:
DSM25686 or a derivative strain of strain CryX, wherein said
derivative strain has the chromosomal background of strain CryX or
said derivative strain contains the vir plasmid of strain CryX or a
derivative of said vir plasmid.
[0011] Further provided is a process of transiently expressing a
DNA sequence of interest in a plant, comprising contacting said
plant or said leaves on said plant with a suspension comprising
Agrobacterium cells of strain CryX deposited under accession No:
DSM25686 or a derivative strain of strain CryX, wherein said
derivative strain has the chromosomal background of strain CryX or
said derivative strain contains the vir plasmid of strain CryX or a
derivative of said vir plasmid.
[0012] The invention also provides an Agrobacterium strain CryX
having DSM accession No: DSM25686 or a derivative strain of strain
CryX, wherein said derivative strain has the chromosomal background
of strain CryX, or said derivative strain contains the vir plasmid
of strain CryX or a derivative of said vir plasmid; or an
Agrobacterium cell of strain CryX or of said derivative strain.
[0013] The invention also provides Agrobacterium cells of strain
CryX having DSM accession No: DSM25686 or a derivative thereof,
said cells containing a binary vector containing in T-DNA a DNA
sequence of interest to be transfected into cells of a plant,
wherein the binary vector may encode a VirG protein from strain
CryX or a closely related VirG protein as defined below.
[0014] The invention further provides a kit comprising: [0015] an
Agrobacterium cell of said strain or said derivative strain as
defined above and [0016] a binary vector containing in T-DNA a DNA
sequence of interest to be transfected into cells of a plant. The
binary vector may encode a VirG protein from strain CryX or a
closely related VirG protein as defined below.
[0017] The invention also provides the vir plasmid of strain CryX
and Agrobacterium cells having the chromosome of strain CryX.
[0018] The invention further provides an aqueous cell suspension of
Agrobacterium strain CryX having DSM accession No: DSM25686 or a
derivative strain of strain CryX (as defined herein), said
suspension having a cell concentration of at most 1.110.sup.6
cfu/ml of the suspension, preferably at most 4.410.sup.5 cfu/ml of
the suspension, and more preferably of at most 1.110.sup.5 cfu/ml
of the suspension.
[0019] The inventors of the present invention have found a way of
strongly increasing the transient transfection efficiency of plants
by Agrobacterium. The inventors have identified an Agrobacterium
strain (Agrobacterium strain CryX) that achieves particularly high
efficiency in transient transfection in planta with a wide variety
of plants. Notably, strain CryX achieves much higher transient
transfection efficiency in planta than other Agrobacterium strains
that are used as a standard for plant transformation or
transfection such as strain LBA4404 or EHA105 (see page 64 of
Slater et al., in: Plant Biotechnology, 2.sup.nd edition, Oxford
University Press, 2008). The inventors have further found that
strain CryX achieves higher transient transfection efficiency than
related Agrobacterium strains such as Chry5/KYRT1. Moreover, the
inventors have found that a particularly high transfection
efficiency can be obtained when a virG gene, notably the virG gene
from Agrobacterium strain LBA4404 or a virG gene that is closely
related to that from LBA4404 is expressed in chrysopine or
succinamopine-type Agrobacterium tumefaciens strains.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIGS. 1A and 1B show T-DNA regions with DNA sequences of
interest of vectors used in the examples. Pact2: promoter of
Arabidopsis actin2 gene; o: 5'-end from TVCV (turnip vein clearing
virus); RdRp: RNA-dependent RNA polymerase open reading frame (ORF)
from cr-TMV (crucifer-infecting tobamovirus); MP: movement protein
ORF from cr-TMV; N: 3'-non-translated region from cr-TMV; Tnos or
nos: nopaline synthase terminator; white segments interrupting grey
segments in the RdRp and MP ORFs indicate introns inserted into
these ORFs for increasing the likelihood of RNA replicon formation
in the cytoplasm of plant cells, which is described in detail in
WO2005049839; GUS: coding sequence of GUS protein; GFP: green
fluorescent protein coding sequence; fs: frame-shift deleting
cell-to-cell movement ability; P35S: 35S promoter; P19: gene
silencing suppressor of tomato bushy stunt virus (cf. Plant J. 33,
949-56); Tocs: ocs terminator; LB: left T-DNA border; RB: right
T-DNA border.
[0021] FIG. 2 shows a comparison of different Agrobacterium
tumefaciens strains for their transient transfection efficiency.
Photographs show GFP fluorescence 4 dpi (days post infection) under
uv light due to TMV-based GFP expression after syringe infiltration
of Nicotiana benthamiana leaves with diluted agrobacterial cultures
as described in Example 2. Numerals 10.sup.-2, 10.sup.-3 and
10.sup.-4 show the concentration factors of the overnight
agrobacterial cultures of OD=1.3 at 600 nm that correspond to
10.sup.2-fold, 10.sup.3-fold and 10.sup.4-fold dilutions,
respectively. The composition of the buffer for infiltration is 5
mM MES, pH5.5 and 10 mM MgSO.sub.4. Each infiltration was performed
in triplicate using three independent leaves of the same plant.
[0022] (A) TMV-based vector capable of cell-to-cell movement:
TMV(MP)-GFP (pNMD560). [0023] (B) TMV-based vector lacking
cell-to-cell movement ability: TMV(fsMP)-GFP (pNMD570). [0024]
1--Agrobacterium tumefaciens strain AGL1; [0025] 2--Agrobacterium
tumefaciens strain EHA105; [0026] 3--Agrobacterium tumefaciens
strain GV3101; [0027] 4--Agrobacterium tumefaciens strain ICF320;
[0028] 5--Agrobacterium tumefaciens strain CryX; [0029]
6--Agrobacterium tumefaciens strain LBA4404; [0030]
7--Agrobacterium tumefaciens strain LBA9402.
[0031] FIG. 3 demonstrates the influence of virG gene
overexpression on transient transfection efficiency for AGL1,
EHA105, ICF320 and GV3101 strains. virG sequences from GV3101 and
LBA4404 strains carrying the N54D mutation as well as native
sequence from LBA4404 strain were used for comparison. Photographs
show GFP fluorescence 4 (FIG. 3A) and 6 (FIG. 3B) dpi under uv
light due to TMV-based GFP expression after syringe infiltration of
Nicotiana benthamiana leaves of the same age from 3 independent
plants with diluted agrobacterial cultures as described in Example
3. Numerals 10.sup.-2, 10.sup.-3 and 10.sup.-4 indicate the factors
by which the cell concentrations of the overnight agrobacterial
cultures of OD=1.3 at 600 nm were reduced. Thus, the factors
10.sup.-2, 10.sup.-3 and 10.sup.-4 correspond to 10.sup.2-,
10.sup.3- and 10.sup.4-fold dilutions, respectively. The
composition of the buffer for infiltration: 5 mM MES, pH5.3 and 10
mM MgCl.sub.2. In all cases TMV-based vectors capable of
cell-to-cell movement were used. [0032] 1--pNMD560 (no virG) in
GV3101; [0033] 2--pNMD064 (virGN54D/GV3101) in GV3101; [0034]
3--pNMD063 (virGN54D/LBA4404) in GV3101; [0035] 4--pNMD062
(virG/LBA4404) in GV3101; [0036] 5--pNMD560 (no virG) in ICF320;
[0037] 6--pNMD064 (virGN54D/GV3101) in ICF320; [0038] 7--pNMD063
(virGN54D/LBA4404) in ICF320; [0039] 8--pNMD062 (virG/LBA4404) in
ICF320; [0040] 9--pNMD560 (no virG) in EHA105; [0041] 10--pNMD064
(virGN54D/GV3101) in EHA105; [0042] 11--pNMD063 (virGN54D/LBA4404)
in EHA105; [0043] 12--pNMD062 (virG/LBA4404) in EHA105; [0044]
13--pNMD560 (no virG) in AGL1; [0045] 14--pNMD064 (virGN54D/GV3101)
in AGL1; [0046] 15--pNMD063 (virGN54D/LBA4404) in AGL1; [0047]
16--pNMD062 (virG/LBA4404) in AGL1.
[0048] FIGS. 4A and 4B show the influence of virG gene
overexpression on the transient transfection efficiency of GV3101
and CryX strains. virG sequences from GV3101 and LBA4404 strains
carrying N54D mutation as well as native sequence from LBA4404
strain were used for comparison. Photographs show GFP fluorescence
3, 4 and 5 dpi under uv light due to TMV-based GFP expression after
the syringe infiltration of Nicotiana benthamiana leaves of the
same age from 3 independent plants with diluted agrobacterial
cultures as described in Example 3. Numerals 10.sup.-4 and
10.sup.-5 indicate the concentration factors of the overnight
agrobacterial cultures of OD=1.3 at 600 nm. The composition of the
buffer for infiltration: 5 mM MES, pH5.3 and 10 mM MgCl.sub.2.
TMV-based vectors capable of cell-to-cell movement were used.
[0049] 1--pNMD560 (no virG) in GV3101 strain; [0050] 2--pNMD064
(virGN54D/GV3101) in GV3101 strain; [0051] 3--pNMD063
(virGN54D/LBA4404) in GV3101 strain; [0052] 4--pNMD560 (no virG) in
CryX strain; [0053] 5--pNMD064 (virGN54D/GV3101) in CryX strain;
[0054] 6--pNMD063 (virGN54D/LBA4404) in CryX strain.
[0055] FIG. 5 shows a comparison of transient transfection
efficiencies for CryX and GV3101 strains in a range of dilutions of
overnight agrobacterial cultures from 10.sup.-3 to 10.sup.-7 using
syringe infiltration of Nicotiana benthamiana leaves. Numerals
10.sup.-3, 10.sup.-4, 10.sup.-5, 10.sup.-6 and 10.sup.-7 indicate
the concentration factors of the overnight agrobacterial cultures
of OD=1.3 at 600 nm. These correspond to 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6 and 10.sup.7-fold dilutions, respectively. The
composition of the buffer for infiltration: 5 mM MES, pH5.3 and 10
mM MgCl.sub.2 in water. Photographs are taken at 4 dpi. [0056]
1--pNMD560 (no virG) in GV3101 strain; [0057] 2--pNMD064
(virGN54D/GV3101) in GV3101 strain; [0058] 3--pNMD063
(virGN54D/LBA4404) in GV3101 strain; [0059] 4--pNMD560 (no virG) in
CryX strain; [0060] 5--pNMD064 (virGN54D/GV3101) in CryX strain;
[0061] 6--pNMD063 (virGN54D/LBA4404) in CryX strain.
[0062] FIG. 6 shows the results of testing different Agrobacterium
strains for transient transfection of soybean using spraying with
suspension of agrobacterial cells. pNMD2190 construct (35S:GUS;
35S:p19 and virGN54D/LBA4404 in the plasmid backbone) was used with
AGL1, EHA105, CryX and LBA4404 strains; pNMD2180 construct
(35S:GUS; 35S:p19 and virGN54D/GV3101in the plasmid backbone) was
used with GV3101 and ICF320 strains. Staining of leaves for the GUS
activity was performed at 11 dps. [0063] (A) For spraying, liquid
Agrobacterium cultures of OD.sub.600=1.3 were diluted in the ratio
1:10 with buffer containing 5 mM MES, pH5.3; 10 mM MgCl.sub.2and
0.05% (v/v) Tween.RTM.20. [0064] (B) For spraying, liquid
Agrobacterium cultures of OD.sub.600=1.3 were diluted in ratios
1:10, 1:100 and 1:1000 with buffer containing (5 mM MES, pH5.3; 10
mM MgCl.sub.2 and 0.05% (v/v) Tween.RTM.20) supplemented with
silicon carbide particles of size 800 in the concentration 0.3%
(w/v).
[0065] FIG. 7 shows test results of CryX and EHA105 strains for
transient transfection of cotton Gossipium hirsutum L. using
spraying with suspension of agrobacterial cells. For spraying,
liquid Agrobacterium cultures of OD.sub.600=1.3 were diluted in the
ratio 1:10 with buffer containing 5 mM MES pH5.3; 10 mM MgCl.sub.2
and 0.25% (v/v) Silwet L-77. For testing, constructs pNMD1971
(35S:GUS; 35S:p19), pNMD2180 (35S:GUS; 35S:p19 and virGN54D/GV3101
in the plasmid backbone) and pNMD2190 (35S:GUS; 35S:p19 and
virGN54D/LBA4404 in the plasmid backbone) were used. GUS activity
test was performed at 6 dps.
[0066] FIG. 8 shows a comparison of two accessions of Agrobacterium
tumefaciens Chry5/KYRT1 strains received from different
laboratories using a TMV-based vector capable of cell-to-cell
movement (TMV-GFP, pNMD560 vector). Photographs show GFP
fluorescence 4 dpi (days post infection) under uv light due to
TMV-based GFP expression after syringe infiltration of Nicotiana
benthamiana leaves with diluted overnight agrobacterial cultures of
OD=1.3 at 600 nm as described in Example 8. The strain obtained
from the laboratory of Dr. G. Collins in the University of Kentucky
(Lexington, USA) is infiltrated on the right-hand side of each
leaf. The accession from the Institute of Cell Biology and Genetics
Engineering (ICBGE, Kiev, Ukraine), is infiltrated on the left-hand
side of each leaf. The composition of the buffer for infiltration
is 5 mM MES, pH5.5 and 10 mM MgSO.sub.4. Each infiltration was
performed in triplicate using three independent leaves of the same
plant. [0067] 1--ICBGE accession, concentration factor 10.sup.-1
(10-fold dilution); [0068] 2--ICBGE accession, concentration factor
10.sup.-2; [0069] 3--ICBGE accession, concentration factor
10.sup.-3; [0070] 4--Kentucky University accession, concentration
factor 10.sup.-1; [0071] 5--Kentucky University accession,
concentration factor 10.sup.-2; [0072] 6--Kentucky University
accession, concentration factor 10.sup.-3.
DETAILED DESCRIPTION OF THE INVENTION
[0073] In the present invention, a particular class of
Agrobacterium tumefaciens strains is used for transient
transfection of plants such as leaves on a plant. This class of
Agrobacterium comprises A. tumefaciens strain CryX and derivative
strains thereof as defined below. Strain CryX was deposited with
DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,
Inhoffenstra.beta.e 7B, 38124 Braunschweig, Germany on Feb. 23,
2012 under the Budapest Treaty. Accession number DSM 25686 has been
assigned to it. Strain CryX has a chromosomally integrated
rifampicin resistance.
[0074] Strain CryX is related to the Chrysanthemum
morifolium-derived Agrobacterium strain Chry5 that has been
identified by Busch & Puepke in 1991. It has been shown in
their paper that the strain is a biotype I by traditional biotype
tests and that it produces tumors on at least 10 plant species. It
has been characterized as unusual because of its ability to form
efficiently large tumors on soybean (Glycine max) and for this
reason, it has been subsequently further characterized in a number
of papers by various groups. Chry5 is unable to utilize octopine or
mannopine as a carbon source; instead it is able to catabolize a
single isomer each of nopaline and succinamopine, at the same time
it is insensitive to agrocin 84 (Busch & Puepke, 1991). In
addition, Chry5-strain-induced tumors produce a family of
Amadori-type opines that includes deoxyfructosyl glutamine (Dfg)
and its lactone, chrysopine (Chy) (Palanichelvam et al., 2000). The
isolates of Chry5 have been shown to contain at least two plasmids,
one with a homology with pTiB6. Torisky et al. (1997) have
partially disarmed the strain by removing approx. 16.5-kb segment
from the 285-kb Ti plasmid of Chry5, including approx. 4 kb of the
oncogenic T-DNA, through homologous recombination. This deletion
mutant, named KYRT1, has been shown to be an efficient vector
organism, and this partially disarmed derivative of Chry5 has since
been used by some researchers. More recently, Palanichelvamet et
al. (2000) have developed a fully disarmed derivative.
[0075] In research that led to the present invention, the inventors
have initially tested two accessions of Chry5/KYRT1 received from
different laboratories. The strain obtained from the laboratory of
Dr. G. Collins (Torisky et al., 1997) did not show any superiority
over standard comparator strains EHA105 and GV3101 in our transient
studies and was excluded from further studies. An accession from
the Institute of Cell Biology and Genetics Engineering (Kiev,
Ukraine), on the other hand, has been found to be unusually active
in its transient transfection and expression efficiency and has
been used in the present invention. This latter accession was
deposited under the Budapest treaty in the official depository
DSMZ-Leibniz-Institut Deutsche Sammlung von Mikroorganismen and
Zellkulturen GmbH, Braunschweig, Germany under the name CryX, to
reflect the fact that there is no clear provenance information on
it.
[0076] The original and subsequent papers have characterized the
Chry5 strain in more detail. These studies aimed at standard
characterization of molecular biology and genetics of the strain,
as well as its comparative ability to induce tumors, to cause
genetic transformation of different plant species, as well as its
ability to cause transient expression in Agrobacterium-treated
explants. Results of these studies are briefly summarized
below.
Methods of Comparison Used to Characterize Agrobacterium
tumefaciens Chry5
1. Data on Oncogenicity
[0077] In the original paper of Bush & Puepke (1991), it has
been established that the Chry5 strain is able to cause tumors on
10 plant species representing 7 plant families. The test involved
semi-quantitative evaluation of the number of plants with tumors
caused by this strain versus the common laboratory strain B6. There
were no significant differences in tumorigenicity between the
strains in 6 out of 9 species (beets, kalanchoe, marigold,
sunflower, tobacco and tomato). On collard, Chry5 has been approx.
two times more efficient, and on soybean--approx. three times more
efficient, whereas on pea, it was somewhat less efficient than B6.
Torisky et al. (1997) provided additional data on tumor formation
on stems of tobacco and tomato; in this study, the Chry5 strain and
its partially disarmed derivative KYRT1 have been compared with two
other Agrobacterium strains often used in transformation studies,
including A281, a succinamopine-type strain containing Bo542 Ti
plasmid in the C58 chromosomal background and its disarmed
derivative EHA105 strain. It has been shown in that study that
whereas the original Chry5 and the other succinamopine strain used,
A281, are both highly tumorigenic, the partially disabled
derivative KYRT1 and EHA105 were not active.
2. Data on Transformation Efficiency Using Partially Disabled and
Fully Disabled Strains
[0078] Torisky et al. (1997) demonstrated that KYRT1 successfully
transfers the beta-glucuronidase (GUS) gene into tobacco leaf
explants, producing GUS-expressing callus which could be
regenerated into viable plants. In these experiments, the
transformation efficiency of KYRT1 strain was approximately the
same as was shown for EHA105. Grant et al. (2003) found the KYRT1
strain to be on average threefold more efficient than AGL 1 for
producing transgenic plants of pea using for evaluation
cotyledonary explants of three different plant genotypes.
[0079] In the work of Palanichelvam et al. (2000), it has been
shown that KYRT1 derivative is only partially disarmed and contains
all of oncogenic T-right and the fragment of T-left regions. A
Chry5 derivative with completely disarmed Ti plasmid, pKPSF2
(Palanichelvam et al., 2000), was, however, less efficient for the
stable transformation of soybean, as the KYRT1 strain retains some
hormonal effect on plant explants enhancing somatic embryogenesis
in soybean (Ko et al., 2004).
3. Data Characterizing Transient Activity
[0080] Again, Torisky et al. (1997) were the first to study
transient expression of .beta.-glucuronidase transgene caused by
the Chry5 derivative KYRT1 by using a quantitative assay of GUS
expression in cotyledonary node explants of soybean. These data
indicated that KYRT1 derivative was approx. 2.5 times more
efficient in causing transient expression when compared to EHA105
or GV3850. KYRT1 was on average 2.8-fold more efficient than EHA105
and C58C1 for producing transient .beta.-glucuronidase (GUS) gene
(gus) expression on cotyledonary petioles of a recalcitrant legume
plant, lentil (Lens culinaris M.) (Akcay et al., 2009). Akbulut et
al. (2008) have measured GUS activity in explants derived from
wounded seedlings after treatment with KYRT1 and two other common
vectors, C58C1 and EHA105. The quantitative evaluation of GUS
expressing spots has shown that after 16 hours of imbibition, there
are no statistically significant differences between KYRT1 and
C58C1, and after 40 hours, KYRT1 is better by ca. 50%, but only in
one of two measurement points.
[0081] Interpretation of the above mentioned data in its entirety
is difficult because different authors used different plant
species, different plant explants, different strains of
Agrobacterium for comparison, and have made their conclusions based
on three different activity methods: tumorigenic activity,
efficiency of genetic transformation and efficiency of transient
expression. The process of interaction of a plant cell and an
Agrobacterium is very complex, and it involves transfer of
T-DNA-protein complex, transfer of proteins such as VirE2 (via a
separate secretion system), transient expression of T-DNA genes in
a plant cell, hormonal effect of expressed genes, integration of
some T-DNA molecules into a plant chromosomal DNA, etc. Any of
these intermediate processes can influence the end result;
therefore, data on tumorigenicity and on transformation efficiency
gives no information with regard to the efficiency of T-DNA
transfer and transient expression. The presented data on transient
activity are, on the other hand, limited and the slight differences
observed are inconclusive or not practically relevant.
[0082] A major difference in the processes and strains described
herein and the methods used in the prior art described above is the
different biology of the plant material used for transient
expression studies. Whereas all previous authors used in vitro
cultured or excised plant explants rich in meristematic tissues
(the ultimate goal being ability to transform a plant cell and to
regenerate a whole plant from said transgenic cell), such as
excised embryo, parts of young seedlings, etc., the present
invention relates to transient transfection of intact, developed
plants which interact with Agrobacterium differently. On entire
plants, agrobacteria enter the leaf via stomata (that is absent in
other organs and in meristematic tissues) and not via wounds on
plant explants. As mentioned before, all authors without exception
were using high bacteria densities when treating plant
explants.
[0083] Strain CryX of the present invention contains a vir plasmid
that is at least partially disarmed. "Disarmed" means that the vir
plasmid and its host Agrobacterium is not oncogenic, i.e. it does
not insert oncogenes or genes for the production of opines into
plant cells, either because it does not contain such genes or it
cannot transfer such genes. A vir plasmid comprises the vir genes
(virulence genes) required for T-DNA transfer into plant cells. Vir
genes and their functions in T-DNA transfer are known in the art
and are e.g. summarized in the book of Slater et al., Plant
Biotechnology, 2.sup.nd edition; Oxford University Press, 2008; see
also Hellens et al., Trends in Plant Science 5 (2000) 446-451.
[0084] Strain CryX of the present invention is a binary strain,
i.e. the vir genes required for transfer of T-DNA into plant cells
and the T-DNA are on separate plasmids (see e.g. the book of Slater
et al. and the article of Hellens et al. regarding binary
Agrobacterium strains and vector systems). In the context of a
binary Agrobacterium strain, the plasmid containing the vir genes
is referred to herein as "vir plasmid" or "vir helper plasmid". The
plasmid containing the T-DNA to be transfected is referred to as
"vector" or "binary vector". The term "strain" or "Agrobacterium
strain" relates to components of the Agrobacterium other than the
binary vector. Thus, herein, a binary Agrobacterium strain not
containing a binary vector and after introduction of a binary
vector are referred to by the same strain name. Deposited strain
CryX contains a vir plasmid, but does not contain a binary
vector.
[0085] The invention also relates to derivative strains of strain
CryX. In an embodiment (i), a derivative strain of strain CryX has
the chromosomal background of strain CryX. It may have the same
chromosome as CryX. In another embodiment (ii), a derivative strain
of strain CryX contains the vir plasmid of strain CryX. In a
further embodiment (iii), a derivative strain of strain CryX
contains a derivative of the vir plasmid of strain CryX, and may
have the chromosome of strain CryX. In embodiment (ii), the strain
is a binary strain and is used in the processes of the invention
after introduction of a binary vector containing a T-DNA of
interest. In embodiments (i) and (iii), the strains may be binary
strains. If they are binary strains, they are used in the processes
of the invention after introduction of a binary vector containing a
T-DNA of interest. Alternatively, in embodiments (i) and (iii), the
T-DNA to be transferred into plant cells in the processes of the
invention may be inserted into the vir plasmid, such that the vir
genes and the T-DNA are present on one and the same plasmid
molecule. However, it is generally more convenient and thus
preferred to use binary strains.
[0086] The term "chromosomal background" is a standard term in the
art of Agrobacterium transformation or transfection (cf. Hellens et
al., Trends in Plant Science 5 (2000) 446-451). It relates to
genetic material of said Agrobacterium strain other than the Ti
plasmids, vir helper plasmids and binary vectors. In one
embodiment, a derivative strain of strain CryX has the same
chromosome as strain CryX. A derivative strain having the
chromosomal background of strain CryX may differ from CryX, for
example, in the vir plasmid compared to the vir plasmid of CryX.
Thus, a derivative strain of strain CryX may contain a vir plasmid
that is a derivative of the vir plasmid of strain CryX.
[0087] Whether a given Agrobacterium strain that has a chromosome
that is non-identical to that of strain CryX has a chromosomal
background of strain CryX can be tested experimentally by comparing
the T-DNA transfer efficiency (or transfection efficiency) from a
T-DNA-containing binary vector of strain CryX with the strain to be
tested that contains the vir plasmid of CryX and the same binary
vector. The strain to be tested is considered having the
chromosomal background of CryX if it achieves at least 70%,
preferably at least 80% of the T-DNA transfer efficiency of strain
CryX. Transfection efficiencies can be determined as described in
Example 2. Alternatively, transfection efficiencies can be
determined as in Example 2 but using a binary vector encoding a
TMV-viral replicon not capable of cell-to-cell movement such as
pNMD570. T-DNA transfer efficiency can also be determined by
protoplast counting as described in WO 2005049839. In one
embodiment, the chromosome of an Agrobacterium strain having the
chromosomal background of strain CryX has a chromosome that is the
same in base sequence as that of strain CryX.
[0088] A derivative of the vir plasmid of strain CryX achieves,
when present in Agrobacterium cells having the same chromosome as
strain CryX, a similar efficiency of T-DNA transfer into plant
cells from a T-DNA-containing binary vector present in these cells.
For this purpose, the derivative vir plasmid has a vir region
sufficiently similar to that of the vir plasmid of strain CryX. The
derivative vir plasmid may encode the virG protein of strain CryX
or a closely related virG protein. Preferably, the derivative vir
plasmid contains the virG gene of strain CryX. In one embodiment,
the derivative vir plasmid contains genes encoding at least two of
the following virulence proteins of CryX: VirA, VirG, VirB1-BirB11,
VirC1, VirD1, VirD2, VirD4, VirE1, VirE2, VirF and VirJ. In a
further embodiment, a derivative vir plasmid contains at least the
genes encoding the following virulence proteins of CryX: VirA,
VirG, VirD2, and VirE2. In a further embodiment, a derivative vir
plasmid contains the entire vir region, i.e. all vir genes of the
vir plasmid of strain CryX. The derivative vir plasmid may differ
in the plasmid backbone from the vir plasmid of CryX. For example,
the derivative vir plasmid may have a different or additional
selective marker gene or may have deleted further nucleic acid
portions from outside the vir region. In one embodiment, the
derivative vir plasmid is pKYRT1 (U.S. Pat. No. 5,929,306), notably
if the derivative strain has the same chromosome as CryX.
[0089] Whether a given vir plasmid is a derivative vir plasmid in
the sense of the present invention may be tested experimentally by
comparing the T-DNA transfer efficiency from a T-DNA-containing
binary vector between Agrobacterium of strain CryX and
Agrobacterium having the chromosome of strain CryX but the vir
plasmid to be tested under otherwise identical conditions. In one
embodiment, the Agrobacterium containing a derivative plasmid
according to the invention achieves at least 70%, preferably at
least 80%, more preferably at least 90% of the T-DNA transfer
efficiency of strain CryX. Transfection efficiencies can be
determined as described in Example 2 and as mentioned above.
[0090] "Closely related virG protein" to the virG protein of CryX
means a virG protein that differs from the virG protein of CryX in
at most 3 non-conservative amino acid substitutions or in at most
6, preferably at most 3 conservative amino acid substitutions. The
non-conservative amino acid substitutions may be at positions
corresponding to positions 6, 7 or 106 of the amino acid sequence
of SEQ ID NO: 1 which is the VirG protein from Agrobacterium strain
LBA4404, all other amino acid residues being as in SEQ ID NO:1. In
addition, the closely related virG protein may have an asparagine
to aspartate substitution at the position corresponding to position
54 of SEQ ID NO: 1. The conservative amino acid substitutions may
be at positions 6, 7 and/or 106 of the amino acid sequence of SEQ
ID NO: 1.
[0091] Herein, conservative substitutions are substitutions of
amino acid residues within each of the following four groups:
[0092] Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr [0093] Val, Ile,
Leu, Met [0094] Lys, Arg, His [0095] Phe, Tyr, Trp All other amino
acid residue substitutions are considered non-conservative.
[0096] Herein, a "T-DNA of interest" is a DNA containing, between
T-DNA left and right border sequences, a DNA sequence of interest.
A T-DNA of interest may be present or may have been incorporated by
sub-cloning into a vir plasmid such as the vir plasmid of strain
CryX. In the processes of the invention, it is preferred to use a
binary vector system. Therefore, a T-DNA of interest is preferably
present or will have been incorporated into into a binary
vector.
[0097] The binary vector to be used in the present invention is a
DNA molecule comprising a DNA sequence of interest to be
transfected into plant cells. The DNA sequence of interest
typically encodes a protein or an RNA to be expressed in cells of
the transfected plants. The binary vector is generally produced by
inserting or cloning a nucleic acid construct containing the DNA
sequence of interest into a cloning site within T-DNA of a
precursor binary vector, as generally done in
Agrobacterium-mediated plant transfection. After said insertion,
the nucleic acid construct is flanked by T-DNA left and right
border sequences for allowing transfection of said plant with said
T-DNA. In the T-DNA of the binary vector, the DNA sequence of
interest is present such as to be expressible in plant cells. For
this purpose, the DNA sequence of interest is, e.g. in said nucleic
acid construct, typically under the control of a promoter active in
plant cells. Examples of the DNA sequence of interest are a DNA
sequence encoding a DNA viral replicon or an RNA viral replicon or
a gene to be expressed. The gene may encode an RNA of interest or a
protein of interest to be expressed in cells of the plant(s). Also
the viral replicons typically encode an RNA or a protein of
interest to be expressed in plants. The DNA construct may comprise,
in addition to the DNA sequence of interest, other sequences such
as regulatory sequences for expression of the DNA sequence of
interest. Binary vectors usable in the invention are known to the
skilled person, e.g. from the references cited in the introduction
or from text books on plant biotechnology such as Slater, Scott and
Fowler, Plant Biotechnology, second edition, Oxford University
Press, 2008. The binary vector typically has an antibiotic
resistance gene for allowing selection in bacteria such as E.
coli.
[0098] For increasing transfection efficiency, the binary vector
may comprise, outside the T-DNA, a virG gene expressible in said
Agrobacterium strain. Alternatively, an additional plasmid may be
inserted into said Agrobacterium strain, whereby said additional
plasmid contains a virG gene expressible in said Agrobacterium
strain (Pazour et al., Proc. Natl. Acad. Sci. USA 88 (1991)
6941-6945). The virG gene preferably encodes a VirG protein from
Agrobacterium tumefaciens strain LBA4404 or a closely related VirG
protein. Further, the VirG protein may have the N54D mutation at
the position corresponding to position 54 of SEQ ID NO: 1, i.e. the
VirG protein from A. tumefaciens strain LBA4404. The N54D mutation
in a VirG protein from another Agrobacterium strain was described
by Jung et al., Current Microbiology 49 (2004) 334-340.
[0099] The closely related VirG protein may be
[0100] (i) a protein comprising at least 235, preferably at least
239 consecutive amino acids of the amino acid sequence of SEQ ID
NO: 1 or of the amino acid sequence of the N54D mutant of the amino
acid sequence of SEQ ID NO: 1; or
[0101] (ii) a protein comprising an amino acid sequence having not
more than 3 non-conservative amino acid substitutions and not more
than 10 conservative amino acid substitutions of the amino acid
sequence of SEQ ID NO: 1 or the N54D mutant thereof; or
[0102] (iii) a protein comprising an amino acid sequence having not
more than 20, preferably not more than 10, conservative amino acid
substitutions of the amino acid sequence of SEQ ID NO: 1 or the
N54D mutant thereof.
[0103] In items (ii) and (iii), said protein preferably has an
asparagine or aspartate residue at the position corresponding to
position 54 of SEQ ID NO: 1
[0104] Possible positions for the conservative amino acid
substitutions in the embodiments mentioned above are positions 6,
7, 18, 35, 38, 42, 44, 66, 69, 73, 81, 86, 89, 97, 106, 107, 122,
124, 133, 135, 143, 147, 150, 165, 188, 208, 212, 213, 232, 235,
and 238 of SEQ ID NO: 1, while amino acid residues at other
positions are those as in SEQ ID NO:1. Possible positions for
non-conservative substitutions are positions 6, 7 and 106 of the
amino acid sequence of SEQ ID NO: 1.
[0105] In embodiments wherein strong expression of a protein or RNA
is desired or wherein accumulation of viral nucleic acids to high
amounts in cells of said plant and possible negative effects on
plant health is not a concern, the nucleic acid construct or DNA
sequence of interest may encode a replicating viral vector that can
replicate in plant cells. In order to be replicating, the viral
vector contains an origin of replication that can be recognized by
a nucleic acid polymerase present in plant cells, such as by the
viral polymerase expressed from the replicon. In case of RNA viral
vectors, the viral replicons may be formed by transcription, under
the control of a plant promoter, from the DNA construct after the
latter has been introduced into plant cell nuclei. In case of DNA
viral replicons, the viral replicons may be formed by recombination
between two recombination sites flanking the sequence encoding the
viral replicon in the DNA construct, e.g. as described in
WO00/17365 and WO 99/22003. If viral replicons are encoded by the
DNA construct, RNA viral replicons are preferred. Use of DNA and
RNA viral replicons has been extensively described in the
literature at least over the last 15 years. Some examples are the
following patent publications by Icon Genetics: WO2008028661,
WO2007137788, WO 2006003018, WO2005071090, WO2005049839,
WO02097080, WO02088369, and WO02068664. An example of DNA viral
vectors are those based on geminiviruses. For the present
invention, viral vectors or replicons based on plant RNA viruses,
notably based on plus-sense single-stranded RNA viruses are
preferred. Examples of such viral vectors are tobacco mosaic virus
(TMV) and potexvirus X (PVX) used in the examples. Potexvirus-based
viral vectors and expression systems are described in EP2061890.
Many other plant viral replicons are described in the patent
publications mentioned above.
[0106] When performing the process of the invention, the binary
vector containing in T-DNA the DNA sequence of interest may be
introduced into the Agrobacterium strain containing the vir plasmid
or its derivative by conventional methods such as electroporation.
A culture of the strain containing the binary vector is then grown
in suitable media, typically in the presence of a selective agent
for selecting Agrobacterium cells containing the binary vector,
and, optionally, sub-cultured to produce the desired amount of an
aqueous suspension comprising the Agrobacterium cells. The obtained
suspension may be diluted to the desired concentration with water,
a suitable buffer or media and be used for transfecting a plant or
leaves on the plant. Alternatively, if the T-DNA is part of the vir
plasmid, the T-DNA containing vir plasmid is introduced into
Agrobacterium by conventional methods such as electroporation and
further treated as described for the binary system.
[0107] In the processes of the invention, in planta transfection is
used. In planta means that the processes are performed on whole
living plants after the seedling stage, preferably on fully
developed plants, rather than on excised or in vitro cultivated
plant tissues or organs. Preferably, the process is applied to many
plants in parallel such as plants growing on a field.
[0108] Said plants may be contacted with the suspension of
Agrobacterium cells by infiltration with or without application of
vacuum. In one embodiment, notably when applied to multiple plants
in parallel, the plants may be contacted with the suspension by
spraying. The aqueous suspension used in the processes of the
invention may have a concentration of Agrobacterium cells of at
most 1.110.sup.9 cfu/ml, which corresponds approximately to an
Agrobacterium culture in LB-medium of an optical density at 600 nm
of 1. Due to the high transfection efficiency achieved in the
invention, much lower concentrations may, however, be used, which
allows treatment of many plants such as entire farm fields without
the need for huge fermenters for Agrobacterium production. Thus,
the concentration is preferably at most 2.210.sup.7 cfu/ml, more
preferably at most 1.110.sup.7 cfu/ml, more preferably at most
4.410.sup.6 cfu/ml. In one embodiment, the concentration is at most
1.110.sup.6 cfu/ml of the suspension. In a further embodiment, the
concentration is at most 4.410.sup.5 cfu/ml of the suspension, and
in a further embodiment, the concentration is at most 1.110.sup.5
cfu/ml of the suspension
[0109] For avoiding determination of cell concentrations in terms
of cfu/ml, concentrations of agrobacterial suspensions are
frequently assessed by measuring the apparent optical density at
600 nm using a spectrophotometer. Herein, the concentration of
1.110.sup.7 cfu/ml corresponds to a calculated optical density at
600 nm of 0.01, whereby the calculated optical density is defined
by a 100-fold dilution with water or buffer of a suspension having
an optical density of 1.0 at 600 nm. Similarly, the concentrations
of 4.410.sup.6 cfu/ml, 1.110.sup.6 cfu/ml, 4.410.sup.5 cfu/ml and
1.110.sup.5 cfu/ml of the suspension correspond to a calculated
optical density at 600 nm of 0.004, 0.001, 0.0004, and 0.0001
respectively, whereby the calculated optical densities are defined
by a 250-fold, 1000-fold, 2500-fold, or 10000-fold, respectively,
dilution with water or buffer of a suspension having an optical
density of 1.0 at 600 nm.
[0110] Thus, in a particularly preferred embodiment, the invention
provides a process, and Agrobacterium cell suspension therefor, of
transiently expressing a DNA sequence of interest in a plant,
comprising contacting said plant or said leaves on said plant with
a suspension comprising Agrobacterium cells of strain CryX or a
derivative strain of strain CryX, wherein said derivative strain
has the chromosomal background of strain CryX or said derivative
strain contains the vir plasmid of strain CryX or a derivative of
said vir plasmid, wherein said suspension has any of the maximum
Agrobacterium cell concentrations mentioned in any one of the
preceding two paragraphs. In this embodiment, the Agrobacterium
strain is preferably a binary strain containing a binary vector
comprises a virG gene expressible in said strain CryX or said
derivative strain. Said virG gene may encodes a VirG protein from
Agrobacterium tumefaciens strain LBA4404 of SEQ ID NO: 1, or is an
N54D mutant of the VirG protein encoded by the virG gene from A.
tumefaciens strain LBA4404.
[0111] It is possible to include an abrasive into the suspension
for increasing the transfection efficiency. The abrasive is a
particulate material that is essentially insoluble in the aqueous
suspension of Agrobacterium cells. The abrasive is believed to
weaken, notably if used together with a wetting agent, the surface
of plant tissue such as leaves, and thereby facilitates penetration
of Agrobacterium cells into the intercellular space of plant
tissue. Regarding possible abrasive usable in the presence
invention, particle sizes thereof, concentrations and possible
commercial products, reference is made to International patent
application published as WO 2012/019669 and disclosure regarding
abrasives of this publication is incorporated herein.
[0112] The aqueous suspension of the invention may contain an
agricultural spray adjuvant. The spray adjuvant may be a surfactant
or wetting agent. The surfactant and wetting agent has multiple
advantages in the present invention. It reduces the surface tension
of the water of the aqueous suspension and makes the waxy surface
of plant leaves more permeable for agrobacteria. It further
improves the stability of the suspension and reduces settling of
the abrasive in the suspension. Surfactants usable in the processes
of the present invention are not particularly limited, and are
disclosed in International patent application published as WO
2012/019669. Preferred surfactants are nonionic surfactants of an
HLB value of 12 or greater, preferably at least 13. As noninionic
surfactants, organo-silicone surfactants such as
polyalkyleneoxide-modified heptamethyltrisiloxane are most
preferred in the present invention. A commercial product is Silwet
L77.TM. spray adjuvant from GE Advanced Materials.
[0113] Surfactants such as those disclosed in WO 2012/019669 may be
used singly or in combination of two or more surfactants. Notably,
the preferred organo-silicone surfactants may be combined with
other surfactants. The total concentration of surfactants in the
aqueous suspension of the invention may be easily tested by
conducting comparative spraying experiments, similarly as done in
the examples. However, in general, the total concentration of
surfactants may be between 0.005 and 2 volume-%, preferably between
0.01 and 0.5 volume-%, more preferably between 0.025 and 0.2
volume-% of said suspension. Since the density of surfactants is
generally close to 1.0 g/ml, the total concentration of surfactants
may be defined as being between 0.05 and 20 g per liter of said
suspension, preferably between 0.1 and 5.0 g, more preferably
between 0.25 and 2.0 g per liter of said suspension (including
abrasive). If the above organo-silicone surfactants such as
polyalkyleneoxide-modified heptamethyltrisiloxane are used, the
concentration of the organo-silicone surfactant in the
agrobacterial suspension used for spraying may be between 0.01 and
0.5 volume-%, preferably between 0.05 and 0.2 volume-%.
Alternatively, the concentration of the organo-silicone surfactant
in the agrobacterial suspension used for spraying may be defined as
being between 0.1 and 5.0 g, preferably between 0.5 and 2.0 g per
liter of said suspension.
[0114] In order to improve the physical properties of the aqueous
suspension, it is possible to add highly dispersed sub-micron size
silicic acid (silica) or porous polymers such as urea/formaldehyde
condensate (Pergopak.TM.). Notably, where the median particle size
of the abrasive is between 0.1 and 30 .mu.m, or in one of the
preferred sub-ranges of this range given above, it is possible to
add a highly dispersed sub-micron size silica to the suspension.
Herein, sub-micron size silica is silica having a median particle
size between 0.01 and 0.5 .mu.m, preferably between 0.02 and 0.5
.mu.m, more preferably between 0.02 and 0.1 .mu.m. Highly dispersed
silicic acid such as Hi-Sil.TM. 233 (PPG Industries) can contribute
to the abrasive properties of the aqueous suspension (see Jensen et
al., Bull. Org. mond. Sante, Bull. Wld Hlth Org. 41 (1969)
937-940). These agents may be incorporated in an amount of from 1
to 10 g per liter of the suspension of the invention.
[0115] Further possible additives to the agrobacterial suspension
are buffer substances to maintain the pH of the suspension used for
spraying at a desired pH, typically between 4.5 and 6.5, preferably
between 5.0 and 5.5. Further, inorganic soluble salts such as
sodium chloride may be added to adjust the ionic strength of the
suspension. Nutrient broth such as LB medium may also be contained
in the suspension.
[0116] The aqueous suspension for contacting with plants may be
produced as follows. In one method, the Agrobacterium strain or
cells containing the desired binary vector to be used in the
process of the invention is inoculated into culture medium and
grown to a high cell concentration. Larger cultures may be
inoculated with small volumes of a highly concentrated culture
medium for obtaining large amounts of the culture medium.
Agrobacteria are generally grown up to a cell concentration
corresponding to an OD at 600 nm of at least 1, typically of about
1.5. Such highly concentrated agrobacterial suspensions are then
diluted to achieve the desired cell concentration. For diluting the
highly concentrated agrobacterial suspensions, water is used. The
water may contain a buffer. The water may further contain the
surfactant of the invention. Alternatively, the concentrated
agrobacterial suspensions may be diluted with water, and any
additives such as the surfactant and the optional buffer substances
are added after or during the dilution process. The abrasive may be
added before, during or after dilution. It is however preferred to
agitate the suspension during addition of the abrasive to uniformly
disperse the abrasive in the agrobacterial suspension. The step of
diluting the concentrated agrobacterial suspension may be carried
out in the spray tank of the sprayer used for spraying the diluted
suspensions.
[0117] Said plants, notably leaves on said plant are then contacted
with the suspension of Agrobacterium cells to effect transient
transfection of cells of the plant. As explained above, contacting
may be done by spraying. The sprayer to be used in the process of
the invention mainly depends on the number of plants or the area to
be sprayed. For one or a small number of plants to be sprayed, pump
sprayers as widely used in household and gardening can be used.
These may have volumes of the spray tank of between 0.5 and 2
liters. For applications on a medium scale, manually operated
hydraulic sprayers such as lever-operated knapsack sprayers or
manually operated compression sprayers may be used. However, the
high transfection efficiency achieved in the invention has its full
potential in the transfection of many plants such as plants growing
on a farm field or in a greenhouse. For this purpose,
power-operated hydraulic sprayers such as tractor-mounted hydraulic
sprayers equipped with spray booms can be used. Aerial application
techniques using helicopters or airplanes are also possible for
large fields. All these types of sprayers are known in the art and
are described for example in the book "Pesticide Application
Methods" by G. A. Matthews, third edition, Blackwell Science, 2000.
In order to ensure a homogeneous suspension in the spray tanks of
the sprayers, small or medium size sprayers may be shaken at
regular intervals or continuously during spraying. Large sprayers
such as the tractor-mounted sprayers should be equipped with an
agitator in the spray tank.
[0118] Considering the presence of agrobacterial cells and abrasive
in the suspensions to be sprayed, sprayers used in the invention
should produce spray of a droplet size at least of fine spray.
Also, medium spray or coarse spray in the classification of sprays
used in "Pesticide Application Methods" by G. A. Matthews, third
edition, Blackwell Science, 2000, page 74, may be used. The main
purpose of the spraying in the invention is wetting of plant tissue
with the suspension. Thus, the exact droplet size is not critical.
However, the transfection efficiency may be further improved by
providing the spray to plant surfaces with increased pressure.
[0119] In the process of the invention, at least parts of plants
are sprayed. In an important embodiment, plants growing in soil on
a field are sprayed, i.e. plants not growing in movable pots or
containers. Such plants cannot be turned upside down and dipped
into agrobacterial suspension for vacuum infiltration. At least
parts of plants are sprayed such as leaves. Preferably, most leaves
are sprayed or entire plants.
[0120] The present invention is used for transient transfection of
plants or for transient with a DNA sequence of interest that may
then be transiently expressed. The term "transient" means that the
no selection methods are used for selecting cells or plants
transfected with the DNA sequence of interest in the background of
non-transfected cells or plants using, e.g. selectable agents and
selectable marker genes capable of detoxifying the selectable
agents. As a result, the transfected DNA sequence of interest is
generally not stably introduced into plant chromosomal DNA.
Instead, transient methods make use of the effect of transfection
in the very plants transfected.
[0121] The invention is generally used for transfecting
multi-cellular plants, notably, higher plants. Both monocot and
dicot plants can be transfected, whereby dicot plants are
preferred. Plants for the use in this invention include any plant
species with preference given to agronomically and horticulturally
important crop species. Common crop plants for the use in present
invention include alfalfa, barley, beans, canola, cowpeas, cotton,
corn, clover, lotus, lentils, lupine, millet, oats, peas, peanuts,
rice, rye, sweet clover, sunflower, sweetpea, soybean, sorghum
triticale, yam beans, velvet beans, vetch, wheat, wisteria, and nut
plants. The plant species preferred for practicing this invention
include, but not restricted to, representatives of Gramineae,
Compositeae, Solanaceae and Rosaceae.
[0122] Further preferred species for the use in this invention are
plants from the following genera: Arabidopsis, Agrostis, Allium,
Antirrhinum, Apium, Arachis, Asparagus, Atropa, Avena, Bambusa,
Brassica, Bromus, Browaalia, Camellia, Cannabis, Capsicum, Cicer,
Chenopodium, Chichorium, Citrus, Coffea, Coix, Cucumis, Curcubita,
Cynodon, Dactylis, Datura, Daucus, Digitalis, Dioscorea, Elaeis,
Eleusine, Festuca, Fragaria, Geranium, Glycine, Helianthus,
Heterocallis, Hevea, Hordeum, Hyoscyamus, Ipomoea, Lactuca, Lens,
Lilium, Linum, Lolium, Lotus, Lycopersicon, Majorana, Malus,
Mangifera, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis,
Oryza, Panicum, Pelargonium, Pennisetum, Petunia, Pisum, Phaseolus,
Phleum, Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus,
Saccharum, Salpiglossis, Secale, Senecio, Setaria, Sinapis,
Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella,
Triticum, Vicia, Vigna, Vitis, Zea, and the Olyreae, the Pharoideae
and others.
[0123] Preferably, the processes of the invention are applied to
dicot plant such as Nicotiana benthamiana, tobacco, cotton,
soybean, rapeseed, pepper, potato, or tomato.
[0124] In one embodiment, the process of the invention can be used
for producing a protein of interest in a plant or in many plants
growing on a field. For this purpose, the plants may be sprayed
with the suspension comprising the Agrobacterium cells containing
the desired binary vector at a desired growth state of the plants.
If the main aim is to achieve the highest possible expression
levels followed by harvesting plants for obtaining plant material
containing high amounts of the protein, viral vectors may be used,
since they generally give the highest expression levels.
[0125] In another embodiment, the process of the invention is used
for generating or altering a trait in a plant such as an input
trait. In this embodiment, excessive expression of a protein or RNA
of interest may not be desired for avoiding deleterious effects on
plant health. For such embodiments, non-replicating vectors (also
referred to herein as "transcriptional vectors"), i.e. vectors
lacking a functional origin of replication recognised by a nucleic
acid polymerase present in the plant cells are preferred. Another
application of the invention is RNA expression, e.g. for RNA
interference, wherein the interference signal can spread in the
plant from cells having expressed the signal to other cells. An
example is the targeting of undesired viral DNA in plants as
described by Pooggin in Nat. Biotech. 21 (2003) 131. An example of
oncogene silencing that can be adapted to a transient system is
described by Escobar et al. Proc. Natl. Acad. Sci. USA 98 (2001)
13437-13442. A further example is the control of coleopteran insect
pests through RNA interference similar as described by Baum et al.,
Nat. Biotech. 25 (2007) 1322-1326 that can be adapted to the
transient process of the invention by transiently transfecting
pest-infested plants with a DNA sequence of interest encoding the
dsRNA such that it can be expressed. Further methods applicable to
the transient process of the invention are those described by Huang
et al., Proc. Natl. Acad. Sci. USA 103 (2006) 14302-14306; Chuang
et al., Proc. Natl. Acad. Sci. USA 97 (2000) 4985-4990.
[0126] Further, the process of the invention allows altering at a
desired point in time traits relating to the regulation of
flowering time or fruit formation such as tuberisation in potato
(Martinez-Garcia et al., Proc. Natl. Acad. Sci. USA 99 (2002)
15211-15216) or the regulation of the flavonoid pathway using a
transcription factor (Deluc et al., Plant Physiol. 147 (2008)
2041-2053). Flowering may be induced by transiently expressing the
movable florigen protein FT (Zeevaart, Current Opinion in Plant
Biology 11 (2008) 541-547; Corbesier et al., Science 316 (2007)
1030-1033). Parthenocarpic fruits in tomatoes may by produced on a
large scale using the invention and the method described by
Pandolfini et al., BMC Biotechnology 2 (2002). Further applications
of the invention are in the context of altering cotton fiber
development by way of MYB transcription factors as described by Lee
et al., Annals of Botany 100 (2007) 1391-1401 or activation of
plant defensive genes (Bergey et al., Proc. Natl. Acad. Sci. USA 93
(1996) 12053-12058.
[0127] The invention also provides a process of protecting crop
plants on a field from a pest. In such process, infestation of at
least one of the plants from a plurality of plants growing in a lot
or farm field may be determined. Due to the rapidness of the
process of the invention expression of a protein or RNA detrimental
to the pest needs to be caused only if infestation by the pest is
determined. Thus, strong and constitutive expression of pest toxins
or dsRNA for RNAi even in the absence of a risk of infestation is
not necessary. Transient expression of Bacillus thuringiensis
endotoxins after the spraying with diluted agrobacterial cultures
harbouring corresponding PVX-based expression vectors protected
Nicotiana benthamiana plants from feeding damage by larvae of the
tobacco hornworm Manduca sexta (cf. FIG. 30 of WO 2012/019660).
EXAMPLES
Reference Example 1
[0128] Determination of Agrobacterium Cell Concentration in Liquid
Culture in Terms of Colony Forming Units (cfu)
[0129] The concentration of Agrobacterium cells in liquid
suspension in terms of colony forming units per ml (cfu/ml) of
liquid suspensions can be determined using the following protocol.
Cells of Agrobacterium tumefaciens strain ICF 320 transformed with
construct pNMD620 were grown in 7.5 ml of liquid LBS medium
containing 25 mg/L kanamycin (AppliChem, A1493) and 50 mg/L
rifampicin (Carl Roth, 4163.2). The bacterial culture was incubated
at 28.degree. C. with continuous shaking. Absorbance or optical
density of bacterial culture expressed in absorbance units (AU) was
monitored in 1-ml aliquots of the culture using a spectrophotometer
at 600 nm wavelength (OD600). The cell concentration estimated as a
number of colony-forming units per milliliter of liquid culture
(cfu/ml) can be analyzed at OD600 values 1; 1.3; 1.5; 1.7 and 1.8.
For this purpose 250-.mu.l aliquots of liquid culture were diluted
with LBS-medium to achieve a final volume of 25 ml (dilution
1:100). 2.5 ml of such 1:100 dilution were mixed with 22.5 ml of
LBS to achieve the dilution 1:1000. Liquid culture dilutions 1:100;
1:1,000; 1:10,000; 1:100,000; 1:1,000,000; 1:10,000,000 and
1:100,000,000 were prepared similarly. Aliquots of last three
dilutions were spread on agar-solidified LBS medium supplemented
with 25 mg/L kanamycin and 50 mg/L rifampicin (250 82 l of
bacterial culture per plate of 90 mm diameter). Plating of aliquots
for each dilution was performed in triplicate. After 2 days
incubation at 28.degree. C., bacterial colonies were counted for
each plate. Plating of 1:1,000,000 and 1:10,000,000 dilutions
resulted in few hundred and few dozen colonies per plate,
respectively. So far as dilution 1:100,000,000 provided just few
colonies per plate, this dilution was not used for calculation of
cell concentration. The cell concentration was estimated according
to the formula: cfu/ml=4.times.number of colonies per
plate.times.dilution factor.
[0130] For transforming cell concentrations as measured by
absorbance measurements at 600 nm (in LB medium) and in terms of
cell-forming units, the following relation is used herein: an OD600
of 1.0 corresponds to 1.1.times.10.sup.9 cfu/ml.
LBS Medium (Liquid)
[0131] 1% soya peptone (papaic hydrolysate of soybean meal;
Duchefa, S1330) [0132] 0.5% yeast extract (Duchefa, Y1333) [0133]
1% sodium chloride (Carl Roth, 9265.2) dissolved in water, and the
is adjusted to pH 7.5 with 1M NaOH (Carl Roth, 6771.2)
[0134] To prepare the solid LBS medium, liquid LBS medium was
supplemented with 1.5% agar (Carl Roth, 2266.2). Media were
autoclaved at 121.degree. C. for 20 min.
EXAMPLE 1
Vectors Used in the Following Examples
[0135] In this study we used TMV- and PVX-based viral vectors as
well as standard transcriptional vectors based on 35S CaMV
promoter.
[0136] TMV-based vectors with cell-to-cell movement ability
pNMD560, pNMD062, pNMD063 and pNMD064 (FIG. 1A) were created on the
basis of vectors described in Marillonnet et al. (2006). All these
vectors are designed for the expression of GFP as a reporter gene;
they contain the insertion of a coding sequence of sGFP that is
modified green fluorescent protein (GFP) from jelly fish Aequorea
victoria (GeneBank accession no. EF030489). The pNMD560 construct
contains, in sequential order, a fragment from the Arabidopsis
actin 2 (ACT2) promoter (GenBank accession no. AB026654); the 5'
end of TVCV (GenBank accession no. BRU03387, base pairs 1-5455) and
a fragment of cr-TMV [GenBank accession no. Z29370, base pairs
5457-5677, both together containing 16 intron insertions]; a sGFP
coding sequence; cr-TMV 3' nontranslated region (3' NTR; GenBank
accession no. Z29370), and the nopaline synthase (Nos) terminator.
The entire fragment was cloned between the T-DNA left and right
borders of binary vector.
[0137] The pNMD062 plasmid was created on the basis of pNMD560
construct. For this purpose, a DNA fragment comprising the coding
sequence and a 5'-upstream genomic region of a virG gene of
octopine-type Ti-plasmid from LBA4404 strain of Agrobacterium
tumefaciens (GenBank accession no. AF242881.1, base pairs
160603-161600) flanked by the sequence ctgtcgatc from the
5'-terminus and the sequence aagatcgacag (SEQ ID NO: 8) from the 3'
terminus was amplified by PCR and inserted into the plasmid
backbone using AfeI restriction site.
[0138] The pNMD063 construct was identical to pNMD062 except for
the N54D mutation.
[0139] To create pNMD064 construct, a DNA fragment comprising the
coding sequence containing the N54D mutation and 5'-upstream
genomic region of virG gene of nopaline-type Ti-plasmid from GV3101
strain of Agrobacterium tumefaciens (GenBank accession no.
AE007871.2, base pairs 194307-193333) was amplified by PCR and
inserted into the plasmid backbone of pNMD560 construct using AfeI
restriction site.
[0140] The pNMD570 construct (TMV-based vectors lacking cell-to
cell movement ability) was identical to pNMD560 except for a point
mutation in the MP-coding sequence leading to an open reading frame
shift that distorts MP translation (FIG. 1A).
[0141] The pNMD620 construct, a PVX-based vector without
cell-to-cell and systemic movement abilities for GFP expression,
contained, in sequential order, a 35S CaMV promoter, coding
sequences of the RNA-dependent RNA polymerase, triple gene block
modules comprising 25 kDa, 12 kDa and 8 kDa proteins, an sGFP
coding sequence and a 3'-untranslated region. The entire fragment
was cloned between the T-DNA left and right borders of binary
vector (FIG. 1B).
[0142] All transcriptional vectors were created on the basis of
pICBV10, a pBIN19-derived binary vector (Marillonnet et al., 2004,
2006). They contained two expression cassettes inserted within
right and left borders of the same T-DNA region (FIG. 1B). In the
case of the pNMD1971 construct, an expression cassette adjacent to
the right border comprised, in sequential order, the Cauliflower
mosaic virus (CAMV) 35S promoter, omega translational enhancer from
Tobacco Mosaic Virus, coding sequence of beta-glucuronidase from
Escherichia coli (GenBank accession no. S69414) containing the
intron from Petunia hybrids PSK7 gene (GenBank accession no.
AJ224165.1, base pairs 4411-4484), and the terminator from the
nopaline synthase gene of Agrobacterium tumefaciens. The second
expression cassette was inserted between the first one and the
T-DNA left border. It contained, in sequential order, the
Cauliflower mosaic virus (CAMV) 35S promoter, omega translational
enhancer from Tobacco Mosaic Virus, coding sequence of P19
suppressor of silencing from Tomato Bushy Stunt Virus (TBSV)
(GenBank accession no. CAB56483.1) and terminator from octopine
synthase gene of Agrobacterium tumefaciens.
[0143] The pNMD2180 construct was created on the basis of the
pNMD1971 vector. For this purpose, the NotI/NdeI fragment of the
pNMD1971 construct was replaced with same fragment of pNMD064
construct containing virGN54D gene of nopaline-type Ti-plasmid from
GV3101 strain of Agrobacterium tumefaciens flanked by 5'-upstream
genomic region.
[0144] The pNMD2190 was created in a similar way. The NotI/NdeI
fragment of pNMD1971 construct was replaced with same fragment of
pNMD063 vector containing virGN54D gene of octopine-type Ti-plasmid
from LBA4404 strain of Agrobacterium tumefaciens flanked by
5'-upstream genomic region.
EXAMPLE 2
Strain CryX Shows Stronger Transient Transfection of Nicotiana
benthamiana if Compared with Commonly Used Agrobacterium
tumefaciens Strains
[0145] We tested the number of Agrobacterium tumefaciens strains
including AGL1, EHA105, GV3101, ICF320, CryX, LBA4404 and LBA9402
for the transient transfection of Nicotiana benthamiana plants. For
this purpose plant leaves were infiltrated using a needleless
syringe with 10.sup.-3 and 10.sup.-4 dilutions of OD.sub.600=1.3 of
agrobacterial cultures of the seven above-mentioned strains
harboring a GFP expression TMV-based vector capable of cell-to-cell
movement (TMV-GFP, pNMD560 construct) as it is shown in FIG. 2A. In
parallel, leaves were infiltrated with 10.sup.-2 and 10.sup.-3
dilutions of the overnight agrobacterial cultures of same strains
carrying TMV-based vector lacking cell-to-cell movement ability
(TMV(fsMP)-GFP, pNMD570 construct) as it is shown in FIG. 2B.
[0146] Based on the density of fluorescing spots and the intensity
of GFP fluorescence, we proved the efficient transient transfection
for several strains (e.g., AGL1, EHA105, and LBA9402) however the
transient transfection efficiency of CryX strain was significantly
higher for both constructs with all tested dilutions of
agrobacterial cultures if compared with any other tested
strain.
[0147] To provide a quantitative evaluation of transient
transfection efficiency for the CryX strain, we estimated the ratio
between the number of cells in the bacterial suspension infiltrated
in leaves and the number of produced GFP fluorescent spots
considered as a single transfection event. For this purpose, leaves
of 6-weeks old Nicotiana benthamiana plants were infiltrated using
a syringe without needle with 200 .mu.l of agrobacterial cultures
of OD600=1.3 diluted by dilution factors of 10.sup.-5, 10.sup.-6
and 10.sup.-7 with a buffer consisting of 5 mM MES, pH 5.3 and 10
mM MgCl.sub.2. CryX, EHA105, GV3101 and ICF320 agrobacterial cells
carried constructs pNMD560 (TMV-GFP vector). For the scoring of
bacterial cells, 100 .mu.l aliquots of bacterial suspensions used
for leaf infiltration were plated in triplicate on LB-agar plates
containing 50 .mu.l/l rifampicin and 50 .mu.l/l kanamycin. Plates
were incubated for 2 days at 28.degree. C. and after that the
number of cfu (colony forming units) was counted. According to our
estimation, 100 .mu.l of bacterial cultures of CryX, EHA105, GV3101
and ICF320 contained 7.38+/-1.72, 5.00+/-1.50, 2.53+/-0.87 and
6.17+/-1.37 cfu (Table 1). In parallel at 4 dpi Nicotiana
benthamiana leaves were scored for GFP fluorescent spots. On
average, 7.38+/-1.72, 5.00+/-1.50, 2.53+/-0.87 and 6.17+/-1.37
fluorescent spots were produced per 100 .mu.l of 10.sup.-7 dilution
of infiltrated agrobacterial culture for CryX, EHA105, GV3101 and
ICF320 strains, respectively. Each agrobacterial cell produced 0.46
transfection loci for CryX strain and 0.01 transfection loci for
all other tested strains, CryX strain being 46 times more efficient
than EHA105, GV3101 and ICF320 strains.
TABLE-US-00001 TABLE 1 Transfection efficiency of CryX in
comparison with other strains at concentration factor 10.sup.-7 of
agrobacterial culture (pNMD560 construct, 4 dpi). CryX EHA105
GV3101 ICF320 GFP spots/ 2.85 +/- 0.83 0.06 +/- 0.02 0.03 +/- 0.01
0.06 +/- 0.02 100 .mu.l input culture cfu/100 .mu.l 7.38 +/- 1.72
5.00 +/- 1.50 2.53 +/- 0.87 6.17 +/- 1.37 input culture GFP spots/
0.46 0.01 0.01 0.01 cfu of input culture
EXAMPLE 3
Overexpression of virG Gene from LBA4404 Strain Increases the
Transient Transfection Efficiency of CryX Strain
[0148] We tested the influence of overexpression of virG genes on
the transient transfection efficiency of several Agrobacterium
strains. For this purpose we created TMV-based vectors carrying the
insertion of virG genes either from GV3101 or LBA4404 strains in
their plasmid backbones (FIG. 1A). First, we tested AGL1, EHA105,
ICF320 and GV3101 strains using vectors harboring virGN54D genes
from GV3101 and LBA404 strains (pNMD063 and pNMD064, respectively)
as well as a vector with native virG gene sequence from LBA4404
strain (pNMD062). Nicotiana benthamiana leaves were infiltrated
using syringe without needle with 10.sup.2, 10.sup.3 and
10.sup.4-fold dilutions of OD.sub.600=1.3 of agrobacterial
cultures. Photographs showing GFP fluorescence in the uv light were
taken at 4th (FIG. 3A) and 6th dpi (FIG. 3B). Based on the visual
evaluation, we demonstrated the strain-specific increase of the
transient transfection efficiency. As it is summarized in Table 2,
overexpression of virGN54D from GV3101 strain increased the
transient transfection efficiency for GV3101 and ICF320 strains;
virGN54D from LBA4404 strain improved the transient transfection
efficiency of AGL1, EHA105 and LBA4404 strains.
[0149] The CryX strain was tested similarly, as shown in FIG. 4.
Nicotiana benthamiana leaves were infiltrated using syringe without
needle with liquid CryX and GV3101 agrobacterial cultures of
OD600=1.3 diluted 10.sup.-4 and 10.sup.-5 with buffer for
infiltration. GFP expression TMV-based vectors containing virGN54D
genes from GV3101 and LBA404 strains in the plasmid backbone
(pNMD063 and pNMD064, respectively) were compared with pNMD560
vector containing no virG gene insertion. FIG. 4A depicts the GFP
fluorescence for the dilution 10.sup.-4 at 3 and 4 dpi; FIG. 4B
shows the GFP fluorescence for the dilution 10.sup.-5 at 4 and 5
dpi. We demonstrated a significant increase of transient
transfection efficiency for CryX strain in combination with
virGN54D gene from LBA4404; in contrast, the overexpression of
virGN54D gene from GV3101 strain had a negative impact on
CryX-mediated transient transfection.
TABLE-US-00002 TABLE 2 Effect of virG overexpression on the T-DNA
transfer efficiency Agrobacterium strain virGN54D/GV3101
virGN54D/LBA4404 AGL1 no effect increase GV3101 increase no effect
ICF320 increase no effect EHA105 no effect increase LBA4404 no
effect increase
EXAMPLE 4
CryX in Combinations with virGN54D/LBA4404 Provides Efficient
Transient Transfection of Nicotiana benthamiana in up to
10.sup.7-Fold Dilutions Using Leaf Infiltration
[0150] To find the maximal effective dilutions of CryX liquid
cultures for transient transfection of Nicotiana benthamiana
plants, we performed syringe infiltration of leaves with liquid
CryX and GV3101 agrobacterial cultures of OD600=1.3 diluted to
10.sup.-3, 10.sup.4, 10.sup.-5, 10.sup.-6 and 10.sup.-7 with buffer
for infiltration.
[0151] In our experiments, Agrobacterium tumefaciens strain CryX in
combination with virGN54D from LBA4404 strain provided the most
efficient transfection of Nicotiana benthamiana plants we ever
observed, resulting in the reasonable number of fluorescing spots
even at the 10.sup.-7 concentration factor of overnight culture
(FIG. 5). It allows increasing the dilution of agrobacterial
culture used for plant infiltration approx. 100 to 1000-fold
compared with 10.sup.3-fold dilution typically used in
Magnicon.RTM. system.
[0152] To provide a quantitative evaluation of transient
transfection efficiency for CryX strain, we estimated the ratio
between the number of cells in the bacterial suspension infiltrated
in leaves and the number of produced GFP fluorescent spots
considered as a single transfection event. For this purpose leaves
of 6-weeks old Nicotiana benthamiana plants were infiltrated using
syringe without needle with 200 .mu.l of agrobacterial cultures of
OD600=1.3 diluted 10.sup.-7 with an aqueous buffer containing 5 mM
MES, pH 5.3 and 10 mM MgCl.sub.2. CryX agrobacterial cells carried
constructs pNMD560 (TMV-GFP vector) and pNMD062 (TMV-GFP vector
containing virGN54D from LBA4404 strain in the plasmid backbone).
For scoring of bacterial cells, 100 .mu.l aliquots of bacterial
suspensions used for leaf infiltration were plated in triplicate on
LB-agar plates containing 50 .mu.l/l rifampicin and 50 .mu.l/l
kanamycin. Plates were incubated for 2 days at 28.degree. C. and
after that the number of cfu (colony forming units) was counted.
According to our estimation, 100 .mu.l of bacterial cultures
contained 14.7+/-4.4 and 13.9+/-3.4 cfu for pNMD560 and pNMD062
constructs, respectively (Table 3). In parallel at 5 dpi Nicotiana
benthamiana leaves were scored for GFP fluorescent spots. In
average, 16.3+/-1.5 and 24.0+/-0.0 fluorescent spots were produced
per 100 .mu.l of infiltrated agrobacterial culture for pNMD560 and
pNMD062 constructs, respectively. In other words, each
agrobacterial cell harboring the pNMD560 construct produced about
1.1 transfection loci; for the pNMD062 construct, this value was
higher, 1.7, showing an increase of the transfection efficiency due
to the overexpression of virG gene.
EXAMPLE 5
Agrobacterium tumefaciens Strain CryX in Combination with
virGN54D/LBA4404 Shows High Spraying Transfection Efficiency for
Soybean
[0153] We tested the number of Agrobacterium tumefaciens strains
including AGL1, EHA105, GV3101, ICF320, CryX and LBA4404 for the
transient transfection of soybean Glycine max L. using spraying of
plants with suspension of agrobacterial cells. For this purpose,
liquid cultures harboring GUS expression vectors were grown to
OD600=1.3 and diluted for spraying with buffer containing 5 mM MES
pH5.3; 10 mM MgCl.sub.2 and 0.05% (v/v) Tween.RTM.20 in the ratio
1:10. For testing of AGL1, EHA105, CryX and LBA4404 strains, we
used pNMD2190 construct (35S:GUS; 35S:p19 and virGN54D/LBA4404 in
the plasmid backbone). GV3101 and ICF320 strains were tested with
pNMD2180 vector (35S:GUS; 35S:p19 and virGN54D/GV3101 in the
plasmid backbone). Staining of leaves for the GUS activity was
performed at 11 days post spraying. Compared to other tested
strains which showed no or little transfection, CryX provided
significantly higher transient transfection rate as revealed by GUS
staining (FIG. 6A).
[0154] Combining surfactant and abrasive treatment, we achieved
efficient transient transfection of soybean with CryX strain for up
to 10.sup.-2 dilutions of agrobacterial cultures when GUS
expression transcriptional vector pNMD2190 was used (FIG. 6B).
EXAMPLE 6
Agrobacterium tumefaciens Strain CryX in Combination with
virGN54D/LBA4404 Shows Enhanced Transient Spraying Transfection for
Cotton
[0155] We tested the transient transfection of cotton with EHA105,
GV3101, ICF320, and CryX strains using spraying. For this purpose
liquid Agrobacterium cultures of OD.sub.600=1.3 were diluted in the
ratio 1:10 with buffer containing 5 mM MES, pH5.3; 10 mM MgCl.sub.2
and 0.25% (v/v) Silwet L-77. pNMD2180 construct (35S:GUS; 35S:p19
and virGN54D/GV3101in the plasmid backbone) was used with GV3101
and ICF320 strains, and pNMD2190 (35S:GUS; 35S:p19 and
virGN54D/LBA4404 in the plasmid backbone) was used with EHA105 and
CryX strains. pNMD1971 construct (35S:GUS; 35S:p19) was applied as
a control with all strains. GUS staining test was performed at 6
days post spraying. After the staining, few blue dots were found
for GV3101 and ICF320 strains (data not shown). Very low
transfection efficiency was shown also for EHA105 and CryX strains
used with pNMD1971 construct. Compared with all other tested
strains, CryX in combination with virGN54D from LBA4404 strain
(pNMD2190) demonstrated increased transient transfection efficiency
(FIG. 7).
TABLE-US-00003 TABLE 3 Transfection efficiency of CryX in
combination with virGN54D/LBA4404 (pNMD062 construct) pNMD062
pNMD560 (virGN54D/ (no virG) LBA4404) Dilution of input
agrobacterial culture 10-7 10-7 cfu/100 .mu.l input culture 14.7
+/- 4.4 13,9 +/- 3.4 GFP spots/100 .mu.l input culture, 5dpi 16.3
+/- 1.5 24.0 +/- 0.0 GFP spots/cfu of input culture 1.1 1.7
EXAMPLE 7
ICBGE Accession of Agrobacterium tumefaciens Chry5/KYRT1 Strain
Shows Stronger Transient Transfection of Nicotiana benthamiana
Compared to Kentucky University Accession of Same Strain
[0156] We tested two accessions of Agrobacterium tumefaciens
Chry5/KYRT1 strain received from different laboratories, the
laboratory of Dr. G. Collins in the University of Kentucky
(Lexington, USA) and the accession from the Institute of Cell
Biology and Genetics Engineering (ICBGE, Kiev, Ukraine) for the
transient transfection of Nicotiana benthamiana plants. For this
purpose, plant leaves were infiltrated using needleless syringe
with dilutions using concentration factors of 10.sup.-1, 10.sup.-2
and 10.sup.-3 of an OD.sub.600=1.3 of agrobacterial cultures of
both strain accessions harboring GFP expression TMV-based vector
capable of cell-to-cell movement (TMV-GFP, pNMD560 construct) as it
is shown in FIG. 8. Based on the density of fluorescing spots and
the intensity of GFP fluorescence, we showed higher transient
transfection efficiency for ICBGE accession if compared with
Kentucky University accession.
[0157] The content of European patent application No. 12 002 402.1
filed on Apr. 3, 2012 is herein incorporated by reference in its
entirety, including description, claims, figures and sequence
listing.
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ANNEX
TABLE-US-00004 [0214] SEQ ID NO: 1: Amino acid sequence from
Agrobacterium LBA4404 virG protein. N54 is shown in bold.
LKHVLLVDDDVAMRHLIIEYLTIHAFKVTAVADSTQFTRVLSSATVDVVVVDLNLGREDGLEIVRNLAAKSDIP-
IIIISGDRLEETDKVVALELGASDFI
AKPFSIREFLARIRVALRVRPNVVRSKDRRSFCFTDWTLNLRQRRLMSEAGGEVKLTAGEFNLLLAFLEKPRDV-
LSREQLLIASRVRDEEVYDRSIDVLI
LRLRRKLEADPSSPQLIKTARGAGYFFDADVQVSHGGTMAA SEQ ID NO: 2: T-DNA
region sequences of pNMD560, pNMD062, pNMD063, pNMD064
cctgtggttggcacatacaaatggacgaacggataaaccttttcacgcccttttaaatatccgattattctaat-
aaacgctcttttctcttaggtttacc
cgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacgacaatctgatctaagctagcttg-
gaattggtaccacgcgtttcgacaaa
atttagaacgaacttaattatgatctcaaatacattgatacatatctcatctagatctaggttatcattatgta-
agaaagttttgacgaatatggcacga
caaaatggctagactcgatgtaattggtatctcaactcaacattatacttataccaaacattagttagacaaaa-
tttaaacaactattttttatgtatgc
aagagtcagcatatgtataattgattcagaatcgttttgacgagttcggatgtagtagtagccattatttaatg-
tacatactaatcgtgaatagtgaata
tgatgaaacattgtatcttattgtataaatatccataaacacatcatgaaagacactttctttcacggtctgaa-
ttaattatgatacaattctaatagaa
aacgaattaaattacgttgaattgtatgaaatctaattgaacaagccaaccacgacgacgactaacgttgcctg-
gattgactcggtttaagttaaccact
aaaaaaacggagctgtcatgtaacacgcggatcgagcaggtcacagtcatgaagccatcaaagcaaaagaacta-
atccaagggctgagatgattaattag
tttaaaaattagttaacacgagggaaaaggctgtctgacagccaggtcacgttatctttacctgtggtcgaaat-
gattcgtgtctgtcgattttaattat
ttttttgaaaggccgaaaataaagttgtaagagataaacccgcctatataaattcatatattttcctctccgct-
ttgaagttttagttttattgcaacaa
caacaacaaattacaataacaacaaacaaaatacaaacaacaacaacatggcacaatttcaacaaacaattgac-
atgcaaactctccaagccgctgcggg
acgcaacagcttggtgaatgatttggcatctcgtcgcgtttacgataatgcagtcgaggagctgaatgctcgtt-
ccagacgtcccaaggtaataggaact
ttctggatctactttatttgctggatctcgatcttgttttctcaatttccttgagatctggaattcgtttaatt-
tggatctgtgaacctccactaaattt
ttggttttactagaatcgatctaagttgaccgatcagttagctcgattatagctaccagaatttggcttgacct-
tgatggagagatccatgttcatgtta
cctgggaaatgatttgtatatgtgaattgaaatctgaactgttgaagttagattgaatctgaacactgtcaatg-
ttagattgaatctgaacactgtttaa
ggttagatgaagtttgtgtatagattcttcgaaactttaggatttgtagtgtcgtacgttgaacagaaagctat-
ttctgattcaatcagggtttatttga
ctgtattgaactctttttgtgtgtttgcaggtccacttctccaaggcagtgtctacggaacagaccctgattgc-
aacaaacgcatatccggagttcgaga
tttcctttactcatacgcaatccgctgtgcactccttggccggaggccttcggtcacttgagttggagtatctc-
atgatgcaagttccgttcggttctct
gacgtacgacatcggcggtaacttttccgcgcaccttttcaaagggcgcgattacgttcactgctgcatgccta-
atctggatgtacgtgacattgctcgc
catgaaggacacaaggaagctatttacagttatgtgaatcgtttgaaaaggcagcagcgtcctgtgcctgaata-
ccagagggcagctttcaacaactacg
ctgagaacccgcacttcgtccattgcgacaaacctttccaacagtgtgaattgacgacagcgtatggcactgac-
acctacgctgtagctctccatagcat
ttatgatatccctgttgaggagttcggttctgcgctactcaggaagaatgtgaaaacttgtttcgcggcctttc-
atttccatgagaatatgcttctagat
tgtgatacagtcacactcgatgagattggagctacgttccagaaatcaggtaacattccttagttacctttctt-
ttctttttccatcataagtttataga
ttgtacatgctttgagatttttctttgcaaacaatctcaggtgataacctgagcttcttcttccataatgagag-
cactctcaattacacccacagcttca
gcaacatcatcaagtacgtgtgcaagacgttcttccctgctagtcaacgcttcgtgtaccacaaggagttcctg-
gtcactagagtcaacacttggtactg
caagttcacgagagtggatacgttcactctgttccgtggtgtgtaccacaacaatgtggattgcgaagagtttt-
acaaggctatggacgatgcgtggcac
tacaaaaagacgttagcaatgcttaatgccgagaggaccatcttcaaggataacgctgcgttaaacttctggtt-
cccgaaggtgctcttgaaattggaag
tcttcttttgttgtctaaacctatcaatttctttgcggaaatttatttgaagctgtagagttaaaattgagtct-
tttaaacttttgtaggtgagagacat
ggttatcgtccctctctttgacgcttctatcacaactggtaggatgtctaggagagaggttatggtgaacaagg-
acttcgtctacacggtcctaaatcac
atcaagacctatcaagctaaggcactgacgtacgcaaacgtgctgagcttcgtggagtctattaggtctagagt-
gataattaacggtgtcactgccaggt
aagttgttacttatgattgttttcctctctgctacatgtattttgttgttcatttctgtaagatataagaattg-
agttttcctctgatgatattattagg
tctgaatgggacacagacaaggcaattctaggtccattagcaatgacattcttcctgatcacgaagctgggtca-
tgtgcaagatgaaataatcctgaaaa
agttccagaagttcgacagaaccaccaatgagctgatttggacaagtctctgcgatgccctgatgggggttatt-
ccctcggtcaaggagacgcttgtgcg
cggtggttttgtgaaagtagcagaacaagccttagagatcaaggttagtatcatatgaagaaatacctagtttc-
agttgatgaatgctattttctgacct
cagttgttctctttgagaattatttcttttctaatttgcctgatttttctattaattcattaggttcccgagct-
atactgtaccttcgccgaccgattgg
tactacagtacaagaaggcggaggagttccaatcgtgtgatctttccaaacctctagaagagtcagagaagtac-
tacaacgcattatccgagctatcagt
gcttgagaatctcgactcttttgacttagaggcgtttaagactttatgtcagcagaagaatgtggacccggata-
tggcagcaaaggtaaatcctggtcca
cacttttacgataaaaacacaagattttaaactatgaactgatcaataatcattcctaaaagaccacacttttg-
ttttgtttctaaagtaatttttactg
ttataacaggtggtcgtagcaatcatgaagtcagaattgacgttgcctttcaagaaacctacagaagaggaaat-
ctcggagtcgctaaaaccaggagagg
ggtcgtgtgcagagcataaggaagtgttgagcttacaaaatgatgctccgttcccgtgtgtgaaaaatctagtt-
gaaggttccgtgccggcgtatggaat
gtgtcctaagggtggtggtttcgacaaattggatgtggacattgctgatttccatctcaagagtgtagatgcag-
ttaaaaagggaactatgatgtctgcg
gtgtacacagggtctatcaaagttcaacaaatgaagaactacatagattacttaagtgcgtcgctggcagctac-
agtctcaaacctctgcaaggtaagag
gtcaaaaggtttccgcaatgatccctctttttttgtttctctagtttcaagaatttgggtatatgactaacttc-
tgagtgttccttgatgcatatttgtg
atgagacaaatgtttgttctatgttttaggtgcttagagatgttcacggcgttgacccagagtcacaggagaaa-
tctggagtgtgggatgttaggagagg
acgttggttacttaaacctaatgcgaaaagtcacgcgtggggtgtggcagaagacgccaaccacaagttggtta-
ttgtgttactcaactgggatgacgga
aagccggtttgtgatgagacatggttcagggtggcggtgtcaagcgattccttgatatattcggatatgggaaa-
acttaagacgctcacgtcttgcagtc
caaatggtgagccaccggagcctaacgccaaagtaattttggtcgatggtgttcccggttgtggaaaaacgaag-
gagattatcgaaaggtaagttctgca
tttggttatgctccttgcattttaggtgttcgtcgctcttccatttccatgaatagctaagattttttttctct-
gcattcattcttcttgcctcagttct
aactgtttgtggtatttttgttttaattattgctacaggtaaacttctctgaagacttgattttagtccctggg-
aaggaagcttctaagatgatcatccg
gagggccaaccaagctggtgtgataagagcggataaggacaatgttagaacggtggattccttcttgatgcatc-
cttctagaagggtgtttaagaggttg
tttatcgatgaaggactaatgctgcatacaggttgtgtaaatttcctactgctgctatctcaatgtgacgtcgc-
atatgtgtatggggacacaaagcaaa
ttccgttcatttgcagagtcgcgaactttccgtatccagcgcattttgcaaaactcgtcgctgatgagaaggaa-
gtcagaagagttacgctcaggtaaag
caactgtgttttaatcaatttcttgtcaggatatatggattataacttaatttttgagaaatctgtagtatttg-
gcgtgaaatgagtttgctttttggtt
tctcccgtgttataggtgcccggctgatgttacgtatttccttaacaagaagtatgacggggcggtgatgtgta-
ccagcgcggtagagagatccgtgaag
gcagaagtggtgagaggaaagggtgcattgaacccaataaccttaccgttggagggtaaaatttgaccttcaca-
caagctgacaagttcgagttactgga
gaagggttacaaggtaaagtttccaactttcctttaccatatcaaactaaagttcgaaactttttatttgatca-
acttcaaggccacccgatctttctat
tcctgattaatttgtgatgaatccatattgacttttgatggttacgcaggatgtgaacactgtgcacgaggtgc-
aaggggagacgtacgagaagactgct
attgtgcgcttgacatcaactccgttagagatcatatcgagtgcgtcacctcatgttttggtggcgctgacaag-
acacacaacgtgttgtaaatattaca
ccgttgtgttggacccgatggtgaatgtgatttcagaaatggagaagttgtccaatttccttcttgacatgtat-
agagttgaagcaggtctgtctttcct
atttcatatgtttaatcctaggaatttgatcaattgattgtatgtatgtcgatcccaagactttcttgttcact-
tatatcttaactctctctttgctgtt
tcttgcaggtgtccaatagcaattacaaatcgatgcagtattcaggggacagaacttgtttgttcagacgccca-
agtcaggagattggcgagatatgcaa
ttttactatgacgctcttcttcccggaaacagtactattctcaatgaatttgatgctgttacgatgaatttgag-
ggatatttccttaaacgtcaaagatt
gcagaatcgacttctccaaatccgtgcaacttcctaaagaacaacctattttcctcaagcctaaaataagaact-
gcggcagaaatgccgagaactgcagg
taaaatattggatgccagacgatattctttcttttgatttgtaactttttcctgtcaaggtcgataaattttat-
ttttttggtaaaaggtcgataatttt
tttttggagccattatgtaattttcctaattaactgaaccaaaattatacaaaccaggtttgctggaaaatttg-
gttgcaatgatcaaaagaaacatgaa
tgcgccggatttgacagggacaattgacattgaggatactgcatctctggtggttgaaaagttttgggattcgt-
atgttgacaaggaatttagtggaacg
aacgaaatgaccatgacaagggagagcttccaggtaaggacttctcatgaatattagtggcagattagtgttgt-
taaagtctttggttagataatcgatg
cctcctaattgtccatgttttactggttttctacaattaaaggtggctttcgaaacaagagtcatctacagttg-
gtcagttagcggactttaactttgtg
gatttgccggcagtagatgagtacaagcatatgatcaagagtcaaccaaagcaaaagttagacttgagtattca-
agacgaatatcctgcattgcagacga
tagtctaccattcgaaaaagatcaatgcgattttcggtccaatgttttcagaacttacgaggatgttactcgaa-
aggattgactcttcgaagtttctgtt
ctacaccagaaagacacctgcacaaatagaggacttcttttctgacctagactcaacccaggcgatggaaattc-
tggaactcgacatttcgaagtacgat
aagtcacaaaacgagttccattgtgctgtagagtacaagatctgggaaaagttaggaattgatgagtggctagc-
tgaggtctggaaacaaggtgagttcc
taagttccatttttttgtaatccttcaatgttattttaacttttcagatcaacatcaaaattaggttcaatttt-
catcaaccaaataatatttttcatgt
atatataggtcacagaaaaacgaccttgaaagattatacggccggaatcaaaacatgtctttggtatcaaagga-
aaagtggtgatgtgacaacctttatt
ggtaataccatcatcattgccgcatgtttgagctcaatgatccccatggacaaagtgataaaggcagctttttg-
tggagacgatagcctgatttacattc
ctaaaggtttagacttgcctgatattcaggcgggcgcgaacctcatgtggaacttcgaggccaaactcttcagg-
aagaagtatggttacttctgtggtcg
ttatgttattcaccatgatagaggagccattgtgtaatacgatccgcttaaactaatatctaagttaggttgta-
aacatattagagatgttgttcactta
gaagagttacgcgagtctttgtgtgatgtagctagtaacttaaataattgtgcgtatttttcacagttagatga-
ggccgttgccgaggttcataagaccg
cggtaggcggttcgtttgctttttgtagtataattaagtatttgtcagataagagattgtttagagatttgttc-
tttgtttgataatgtcgatagtctcg
tacgaacctaaggtgagtgatttcctcaatctttcgaagaaggaagagatcttgccgaaggctctaacgaggtt-
aaaaaccgtgtctattagtactaaag
atattatatctgtcaaggagtcggagactttgtgtgatatagatttgttaatcaatgtgccattagataagtat-
agatatgtgggtatcctaggagccgt
ttttaccggagagtggctagtgccagacttcgttaaaggtggagtgacgataagtgtgatagataagcgtctgg-
tgaactcaaaggagtgcgtgattggt
acgtacagagccgcagccaagagtaagaggttccagttcaaattggttccaaattactttgtgtccaccgtgga-
cgcaaagaggaagccgtggcaggtaa
ggatttttatgatatagtatgcttatgtattttgtactgaaaagcatatcctgcttcattgggatattactgaa-
agcatttaactacatgtaaactcact
tgatgatcaataaacttgattttgcaggttcatgttcgtatacaagacttgaagattgaggcgggttggcagcc-
gttagctctggaagtagtttcagttg
ctatggtcaccaataacgttgtcatgaagggtttgagggaaaaggtcgtcgcaataaatgatccggacgtcgaa-
ggtttcgaaggtaagccatcttcctg
cttatttttataatgaacatagaaataggaagttgtgcagagaaactaattaacctgactcaaaatctaccctc-
ataattgttgtttgatattggtcttg
tattttgcaggtgtggttgacgaattcgtcgattcggttgcagcatttaaagcggttgacaactttaaaagaag-
gaaaaagaaggttgaagaaaagggtg
tagtaagtaagtataagtacagaccggagaagtacgccggtcctgattcgtttaatttgaaagaagaaaacgtc-
ttacaacattacaaacccgaataatc
gataactcgagtatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattacatttacaatt-
atcatggtgagcaagggcgaggagct
gttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcg-
agggcgagggcgatgccacctacggc
aagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcag-
ctacggcgtgcagtgcttcagccgct
accccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatc-
ttcttcaaggacgacggcaactacaa
gacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaagg-
aggacggcaacatcctggggcacaag
ctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaactt-
caagatccgccacaacatcgaggacg
gcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaac-
cactacctgagcacccagtccgccct
gagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacg-
gcatggacgagctgtacaagtaaagc
ggcccctagagcgtggtgcgcacgatagcgcatagtgtttttctctccacttgaatcgaagagatagacttacg-
gtgtaaatccgtaggggtggcgtaaa
ccaaattacgcaatgttttgggttccatttaaatcgaaaccccttattcctggatcacctgttaacgcacgttt-
gacgtgtattacagtgggaataagta
aaagtgagaggttcgaatcctccctaaccccgggtaggggcccagcggccgctctagctagagtcaagcagatc-
gttcaaacatttggcaataaagtttc
ttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaat-
aattaacatgtaatgcatgacgttat
ttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgc-
gcaaactaggataaattatcgcgcgc
ggtgtcatctatgttactagatcgacctgcatccaccccagtacattaaaaacgtccgcaatgtgttattaagt-
tgtctaagcgtcaatttgtttacacc
acaatatatcctgccaccagccagccaacagctccccgaccggcagctcggcacaaaatcaccactcgatacag-
gcagcccatcag SEQ ID NO: 3: Sequence of T-DNA region of pNMD570
cctgtggttggcacatacaaatggacgaacggataaaccttttcacgcccttttaaatatccgattattctaat-
aaacgctcttttctcttaggtttacc
cgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacgacaatctgatctaagctagcttg-
gaattggtaccacgcgtttcgacaaa
atttagaacgaacttaattatgatctcaaatacattgatacatatctcatctagatctaggttatcattatgta-
agaaagttttgacgaatatggcacga
caaaatggctagactcgatgtaattggtatctcaactcaacattatacttataccaaacattagttagacaaaa-
tttaaacaactattttttatgtatgc
aagagtcagcatatgtataattgattcagaatcgttttgacgagttcggatgtagtagtagccattatttaatg-
tacatactaatcgtgaatagtgaata
tgatgaaacattgtatcttattgtataaatatccataaacacatcatgaaagacactttctttcacggtctgaa-
ttaattatgatacaattctaatagaa
aacgaattaaattacgttgaattgtatgaaatctaattgaacaagccaaccacgacgacgactaacgttgcctg-
gattgactcggtttaagttaaccact
aaaaaaacggagctgtcatgtaacacgcggatcgagcaggtcacagtcatgaagccatcaaagcaaaagaacta-
atccaagggctgagatgattaattag
tttaaaaattagttaacacgagggaaaaggctgtctgacagccaggtcacgttatctttacctgtggtcgaaat-
gattcgtgtctgtcgattttaattat
ttttttgaaaggccgaaaataaagttgtaagagataaacccgcctatataaattcatatattttcctctccgct-
ttgaagttttagttttattgcaacaa
caacaacaaattacaataacaacaaacaaaatacaaacaacaacaacatggcacaatttcaacaaacaattgac-
atgcaaactctccaagccgctgcggg
acgcaacagcttggtgaatgatttggcatctcgtcgcgtttacgataatgcagtcgaggagctgaatgctcgtt-
ccagacgtcccaaggtaataggaact
ttctggatctactttatttgctggatctcgatcttgttttctcaatttccttgagatctggaattcgtttaatt-
tggatctgtgaacctccactaaatct
tttggttttactagaatcgatctaagttgaccgatcagttagctcgattatagctaccagaatttggcttgacc-
ttgatggagagatccatgttcatgtt
acctgggaaatgatttgtatatgtgaattgaaatctgaactgttgaagttagattgaatctgaacactgtcaat-
gttagattgaatctgaacactgttta
aggttagatgaagtttgtgtatagattcttcgaaactttaggatttgtagtgtcgtacgttgaacagaaagcta-
tttctgattcaatcagggtttatttg
actgtattgaactctttttgtgtgtttgcaggtccacttctccaaggcagtgtctacggaacagaccctgattg-
caacaaacgcatatccggagttcgag
atttcctttactcatacgcaatccgctgtgcactccttggccggaggccttcggtcacttgagttggagtatct-
catgatgcaagttccgttcggttctc
tgacgtacgacatcggcggtaacttttccgcgcaccttttcaaagggcgcgattacgttcactgctgcatgcct-
aatctggatgtacgtgacattgctcg
ccatgaaggacacaaggaagctatttacagttatgtgaatcgtttgaaaaggcagcagcgtcctgtgcctgaat-
accagagggcagctttcaacaactac
gctgagaacccgcacttcgtccattgcgacaaacctttccaacagtgtgaattgacgacagcgtatggcactga-
cacctacgctgtagctctccatagca
tttatgatatccctgttgaggagttcggttctgcgctactcaggaagaatgtgaaaacttgtttcgcggccttt-
catttccatgagaatatgcttctaga
ttgtgatacagtcacactcgatgagattggagctacgttccagaaatcaggtaacattccttagttacctttct-
tttctttttccatcataagtttatag
attgtacatgctttgagatttttctttgcaaacaatctcaggtgataacctgagcttcttcttccataatgaga-
gcactctcaattacacccacagcttc
agcaacatcatcaagtacgtgtgcaagacgttcttccctgctagtcaacgcttcgtgtaccacaaggagttcct-
ggtcactagagtcaacacttggtact
gcaagttcacgagagtggatacgttcactctgttccgtggtgtgtaccacaacaatgtggattgcgaagagttt-
tacaaggctatggacgatgcgtggca
ctacaaaaagacgttagcaatgettaatgccgagaggaccatcttcaaggataacgctgcgttaaacttctggt-
tcccgaaggtgctcttgaaattggaa
gtcttcttttgttgtctaaacctatcaatttctctgcggaaatttatttgaagctgtagagttaaaattgagtc-
ttttaaacttttgtaggtgagagaca
tggttatcgtccctctctttgacgcttctatcacaactggtaggatgtctaggagagaggttatggtgaacaag-
gacttcgtctacacggtcctaaatca
catcaagacctatcaagctaaggcactgacgtacgcaaacgtgctgagcttcgtggagtctattaggtctagag-
tgataattaacggtgtcactgccagg
taagttgttacttatgattgttttcctctctgctacatgtattttgttgttcatttctgtaagatataagaatt-
gagttttcctctgatgatattattag
gtctgaatgggacacagacaaggcaattctaggtccattagcaatgacattcttcctgatcacgaagctgggtc-
atgtgcaagatgaaataatcctgaaa
aagttccagaagttcgacagaaccaccaatgagctgatttggacaagtctctgcgatgccctgatgggggttat-
tccctcggtcaaggagacgcttgtgc
gcggtggttttgtgaaagtagcagaacaagccttagagatcaaggttagtatcatatgaagaaatacctagttt-
cagttgatgaatgctattttctgacc
tcagttgttctcttttgagaattatttcttttctaatttgcctgatttttctattaattcattaggttcccgag-
ctatactgtaccttcgccgaccgatt
ggtactacagtacaagaaggcggaggagttccaatcgtgtgatctttccaaacctctagaagagtcagagaagt-
actacaacgcattatccgagctatca
gtgcttgagaatctcgactcttttgacttagaggcgtttaagactttatgtcagcagaagaatgtggacccgga-
tatggcagcaaaggtaaatcctggtc
cacacttttacgataaaaacacaagattttaaactatgaactgatcaataatcattcctaaaagaccacacttt-
tgtthgtttctaaagtaatttttact
gttataacaggtggtcgtagcaatcatgaagtcagaattgacgttgcctttcaagaaacctacagaagaggaaa-
tctcggagtcgctaaaaccaggagag
gggtcgtgtgcagagcataaggaagtgttgagcttacaaaatgatgctccgttcccgtgtgtgaaaaatctagt-
tgaaggttccgtgccggcgtatggaa
tgtgtcctaagggtggtggtttcgacaaattggatgtggacattgctgatttccatctcaagagtgtagatgca-
gttaaaaagggaactatgatgtctgc
ggtgtacacagggtctatcaaagttcaacaaatgaagaactacatagattacttaagtgcgtcgctggcagcta-
cagtctcaaacctctgcaaggtaaga
ggtcaaaaggtttccgcaatgatccctctttttttgtttctctagthcaagaatttgggtatatgactaacttc-
tgagtgttccttgatgcatatttgtg
atgagacaaatgtttgttctatgttttaggtgcttagagatgttcacggcgttgacccagagtcacaggagaaa-
tctggagtgtgggatgttaggagagg
acgttggttacttaaacctaatgcgaaaagtcacgcgtggggtgtggcagaagacgccaaccacaagttggtta-
ttgtgttactcaactgggatgacgga
aagccggtttgtgatgagacatggttcagggtggcggtgtcaagcgattccttgatatattcggatatgggaaa-
acttaagacgctcacgtcttgcagtc
caaatggtgagccaccggagcctaacgccaaagtaattttggtcgatggtgttcccggttgtggaaaaacgaag-
gagattatcgaaaaggtaagttctgc
atttggttatgctccttgcattttaggtgttcgtcgctcttccatttccatgaatagctaagattttttttctc-
tgcattcattcttcttgcctcagttc
taactgtttgtggtatttttgttttaattattgctacaggtaaacttctctgaagacttgattttagtccctgg-
gaaggaagcttctaagatgatcatcc
ggagggccaaccaagctggtgtgataagagcggataaggacaatgttagaacggtggattccttcttgatgcat-
ccttctagaagggtgtttaagaggtt
gtttatcgatgaaggactaatgctgcatacaggttgtgtaaatttcctactgctgctatctcaatgtgacgtcg-
catatgtgtatggggacacaaagcaa
attccgttcatttgcagagtcgcgaactttccgtatccagcgcattttgcaaaactcgtcgctgatgagaagga-
agtcagaagagttacgctcaggtaaa
gcaactgtgttttaatcaatttcttgtcaggatatatggattataacttaatttttgagaaatctgtagtattt-
ggcgtgaaatgagtttgctttttggt
ttctcccgtgttataggtgcccggctgatgttacgtatttccttaacaagaagtatgacggggcggtgatgtgt-
accagcgcggtagagagatccgtgaa
ggcagaagtggtgagaggaaagggtgcattgaacccaataaccttaccgttggagggtaaaattttgaccttca-
cacaagctgacaagttcgagttactg
gagaagggttacaaggtaaagtttccaactttcctttaccatatcaaactaaagttcgaaactttttatttgat-
caacttcaaggccacccgatctttct
attcctgattaatttgtgatgaatccatattgacttttgatggttacgcaggatgtgaaractgtgcacgaggt-
gcaaggggagacgtacgagaagactg
ctattgtgcgcttgacatcaactccgttagagatcatatcgagtgcgtcacctcatgttttggtggcgctgaca-
agacacacaacgtgttgtaaatatta
caccgttgtgttggacccgatggtgaatgtgatttcagaaatggagaagttgtccaatttccttcttgacatgt-
atagagttgaagcaggtctgtctttc
ctatttcatatgtttaatcctaggaatttgatcaattgattgtatgtatgtcgatcccaagactttcttgttca-
cttatatcttaactctctctttgctg
tttcttgcaggtgtccaatagcaattacaaatcgatgcagtattcaggggacagaacttgtttgttcagacgcc-
caagtcaggagattggcgagatatgc
aattttactatgacgctcttcttcccggaaacagtactattctcaatgaatttgatgctgttacgatgaatttg-
agggatatttccttaaacgtcaaaga
ttgcagaatcgacttctccaaatccgtgcaacttcctaaagaacaacctattttcctcaagcctaaaataagaa-
ctgcggcagaaatgccgagaactgca
ggtaaatattggatgccagacgatattctttcttttgatttgtaactttttcctgtcaaggtcgataaatttta-
ttttttttggtaaaaggtcgataatt
tttttttggagccattatgtaattttcctaattaactgaaccaaaattatacaaaccaggtttgctggaaaatt-
tggttgcaatgatcaaaagaaacatg
aatgcgccggatttgacagggacaattgacattgaggatactgcatctctggtggttgaaaagttttgggattc-
gtatgttgacaaggaatttagtggaa
cgaacgaaatgaccatgacaagggagagcttctccaggtaaggacttctcatgaatattagtggcagattagtg-
ttgttaaagtctttggttagataatc
gatgcctcctaattgtccatgttttactggttttctacaattaaaggtggctttcgaaacaagagtcatctaca-
gttggtcagttagcggactttaactt
tgtggatttgccggcagtagatgagtacaagcatatgatcaagagtcaaccaaagcaaaagttagacttgagta-
ttcaagacgaatatcctgcattgcag
acgatagtctaccattcgaaaaagatcaatgcgattttcggtccaatgttttcagaacttacgaggatgttact-
cgaaaggattgactcttcgaagtttc
tgttctacaccagaaagacacctgcacaaatagaggacttcttttctgacctagactcaacccaggcgatggaa-
attctggaactcgacatttcgaagta
cgataagtcacaaaacgagttccattgtgctgtagagtacaagatctgggaaaagttaggaattgatgagtggc-
tagctgaggtctggaaacaaggtgag
ttcctaagttccatttttttgtaatccttcaatgttattttaacttttcagatcaacatcaaaattaggttcaa-
ttttcatcaaccaaataatatttttc
atgtatatataggtcacagaaaaacgaccttgaaagattatacggccggaatcaaaacatgtctttggtatcaa-
aggaaaagtggtgatgtgacaacctt
tattggtaataccatcatcattgccgcatgtttgagctcaatgatccccatggacaaagtgataaaggcagctt-
tttgtggagacgatagcctgatttac
attcctaaaggtttagacttgcctgatattcaggcgggcgcgaacctcatgtggaacttcgaggccaaactctt-
caggaagaagtatggttacttctgtg
gtcgttatgttattcaccatgatagaggagccattgtgtattacgatccgcttaaactaatatctaagttaggt-
tgtaaacatattagagatgttgttca
cttagaagagttacgcgagtctttgtgtgatgtagctagtaacttaaataattgtgcgtatttttcacagttag-
atgaggccgttgccgaggttcataag
accgcggtaggcggttcgtttgctttttgtagtataattaagtatttgtcagataagagattgtttagagattt-
gttctttgtttgataatgtcgatagt
ctcgtacgaacctaaggtgagtgatttcctcaatctttcgaagaaggaagagatcttgccgaaggctctaacga-
ggttaaaaaccgtgtctattagtact
aaagatattatatctgtcaaggagtcggagactttgtgttgatatagatttgttaatcaatgtgccattagata-
agtatagatatgtgggtatcctagct
aggagccgtttttaccggagagtggctagtgccagacttcgttaaaggtggagtgacgataagtgtgatagata-
agcgtctggtgaactcaaaggagtgc
gtgattggtacgtacagagccgcagccaagagtaagaggttccagttcaaattggttccaaattactttgtgtc-
caccgtggacgcaaagaggaagccgt
ggcaggtaaggatttttatgatatagtatgcttatgtattttgtactgaagcatatcctgcttcattgggatat-
tactgaaagcatttaactacatgtaa
actcacttgatgatcaataaacttgattttgcaggttcatgttcgtatacaagacttgaagattgaggcgggtt-
ggcagccgttagctctggaagtagtt
tcagttgctatggtcaccaataacgttgtcatgaagggtttgagggaaaaggtcgtcgcaataaatgatccgga-
cgtcgaaggtttcgaaggtaagccat
cttcctgcttatttttataatgaacatagaaataggaagttgtgcagagaaactaattaacctgactcaaaatc-
taccctcataattgttgtttgatatt
ggtcttgtattttgcaggtgtggttgacgaattcgtcgattcggttgcagcatttaaagcggttgacaacttta-
aaagaaggaaaaagaaggttgaagaa
aagggtgtagtaagtaagtataagtacagaccggagaagtacgccggtcctgattcgtttaatttgaaagaaga-
aaacgtcttacaacattacaaacccg
aataatcgataactcgagtatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattacatt-
tacaattatcatggtgagcaagggcg
aggagctgttcaccggggtggtgcccatcctggacggcgacgtaaacggccacaagttcagcgtgtccggcgag-
ggcgagggcgatgccacctacggcaa
gctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcagct-
acggcgtgcagtgcttcagccgctac
cccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatctt-
cttcaaggacgacggcaactacaaga
cccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggag-
gacggcaacatcctggggcacaagct
ggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttca-
agatccgccacaacatcgaggacggc
agcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaacca-
ctacctgagcacccagtccgccctga
gcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacggc-
atggacgagctgtacaagtaaagcgg
cccctagagcgtggtgcgcacgatagcgcatagtgtttttctctccacttgaatcgaagagatagacttacggt-
gtaaatccgtaggggtggcgtaaacc
aaattacgcaatgttttgggttccatttaaatcgaaaccccttatttcctggatcacctgttaacgcacgtttg-
acgtgtattacagtgggaataagtaa
aagtgagaggttcgaatcctccctaaccccgggtaggggcccagcggccgctctagctagagtcaagcagatcg-
ttcaaacatttggcaataaagtttct
taagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaata-
attaacatgtaatgcatgacgttatt
tatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcg-
caaactaggataaattatcgcgcgcg
gtgtcatctatgttactagatcgacctgcatccaccccagtacattaaaaacgtccgcaatgtgttattaagtt-
gtctaagcgtcaatttgtttacacca
caatatatcctgccaccagccagccaacagctccccgaccggcagctcggcacaaaatcaccactcgatacagg-
cagcccatcag SEQ ID NO: 4: Sequence of T-DNA region of pNMD620
cctgtggttggcacatacaaatggacgaacggataaaccttttcacgcccttttaaatatccgattattctaat-
aaacgctcttttctcttaggtttacc
cgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacgacaaatctgatctaagctaggca-
tgcctgcaggtcaacatggtggagca
cgacacgcttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaac-
aaagggtaatatccggaaacctcctc
ggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgcca-
tcattgcgataaaggaaaggccatcg
ttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagac-
gttccaaccacgtcttcaaagcaagt
ggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctcta-
tataaggaagttcatttcatttggag
aggagaaaactaaaccatacaccaccaacacaaccaaacccaccacgcccaattgttacacacccgcttgaaaa-
agaaagtttaacaaatggccaaggtg
cgcgaggtttaccaatcttttacagactccaccacaaaaactctcatccaagatgaggcttatagaaacattcg-
ccccatcatggaaaaacacaaactag
ctaacccttacgctcaaacggttgaagcggctaagatctagaggggttcggcatagccaccaatccctatagca-
ttgaattgcatacacatgcagccgct
aagaccatagagaataaacttctagaggtgcttggttccatcctaccacaagaacctgttacatttatgtttct-
taaacccagaaagctaaactacatga
gaagaaacccgcggatcaaggacattttccaaaatgttgccattgaaccaagagacgtagccaggtaccccaag-
gaaacaataattgacaaactcacaga
gatcacaacggaaacagcatacattagtgacactctgcacttcttggatccgagctacatagtggagacattcc-
aaaactgcccaaaattgcaaacattg
tatgcgaccttagttctccccgttgaggcagcctttaaaatggaaagcactcacccgaacatatacagcctcaa-
atacttcggagatggtttccagtata
taccaggcaaccatggtggcggggcataccatcatgaattcgctcatctacaatggctcaaagtgggaaagatc-
aagtggagggaccccaaggatagctt
tctcggacatctcaattacacgactgagcaggttgagatgcacacagtgacagtacagttgcaggaatcgttcg-
cggcaaaccacttgtactgcatcagg
agaggagacttgctcacaccggaggtgcgcactttcggccaacctgacaggtacgtgattccaccacagatctt-
cctcccaaaagttcacaactgcaaga
agccgattctcaagaaaactatgatgcagctcttcttgtatgttaggacagtcaaggtcgcaaaaaattgtgac-
atttttgccaaagtcagacaattaat
taaatcatctgacttggacaaatactctgctgtggaactggtttacttagtaagctacatggagttccttgccg-
atttacaagctaccacctgcttctca
gacacactttctggtggcttgctaacaaagacccttgcaccggtgagggcttggatacaagagaaaaagatgca-
gctgtttggtcttgaggactacgcga
agttagtcaaagcagttgatttccacccggtggatttttctttcaaagtggaaacttgggacttcagattccac-
cccttgcaagcgtggaaagccttccg
accaagggaagtgtcggatgtagaggaaatggaaagtttgttctcagatggggacctgcttgattgcttcacaa-
gaatgccagcttatgcggtaaacgca
agatttagctgcaatcaggaaaagaggacgcccgagatggatgtcggtcaagaagttaaagagcctgcaggaga-
cagaaatcaatactcaaaccctgcag
aaactttcctcaacaagctccacaggaaacacagtagggaggtgaaacaccaggccgcaaagaaagctaaacgc-
ctagctgaaatccaggagtcaatgag
agctgaaggtgatgccgaaccaaatgaaataagcgggacgatgggggcaatacccagcaacgccgaacttcctg-
gcacgaatgatgccagacaagaactc
acactcccaaccactaaacctgtccctgcaaggtgggaagatgcttcattcacagattctagtgtggaagagga-
gcaggttaaactccttggaaaagaaa
ccgttgaaacagcgacgcaacaagtcatcgaaggacttccttggaaacactggattcctcaattaaatgctgtt-
ggattcaaggcgctggaaattcagag
ggataggagtggaacaatgatcatgcccatcacagaaatggtctccgggctggaaaaagaggacttccctgaag-
gaactccaaaagagttggcacgagaa
ttgttcgctatgaacagaagccctgccaccatccctttggacctgcttagagccagagactacggcagtgatgt-
aaagaacaagagaattggtgccatca
caaagacacaggcaacgagttggggcgaatacttgacaggaaagatagaaagcttaactgagaggaaagttgcg-
acttgtgtcattcatggagctggagg
ttctggaaaaagtcatgccatccagaaggcattgagagaaattggcaagggctcggacatcactgtagtcctgc-
cgaccaatgaactgcggctagattgg
agtaagaaagtgcctaacactgagccctatatgttcaagacctctgaaaaggcgttaattgggggaacaggcag-
catagtcatctttgacgattactcaa
aacttcctcccggttacatagaagccttagtctgtttctactctaaaatcaagctaatcattctaacaggagat-
agcagacaaagcgtctaccatgaaac
tgctgaggacgcctccatcaggcatttgggaccagcaacagagtacttctcaaaatactgccgatactatctca-
atgccacacaccgcaacaagaaagat
cttgcgaacatgcttggtgtctacagtgagagaacgggagtcaccgaaatcagcatgagcgccgagttcttaga-
aggaatcccaactttggtaccctcgg
atgagaagagaaagctgtacatgggcaccgggaggaatgacacgttcacatacgctggatgccaggggctaact-
aagccgaaggtacaaatagtgttgga
ccacaacacccaagtgtgtagcgcgaatgtgatgtacacggcactttctagagccaccgataggattcacttcg-
tgaacacaagtgcaaattcctctgcc
ttctgggaaaagttggacagcaccccttacctcaagactttcctatcagtggtgagagaacaagcactcaggga-
gtacgagccggcagaggcagagccaa
ttcaagagcctgagccccagacacacatgtgtgtcgagaatgaggagtccgtgctagaagagtacaaagaggaa-
ctcttggaaaagtttgacagagagat
ccactctgaatcccatggtcattcaaactgtgtccaaactgaagacacaaccattcagttgttttcgcatcaac-
aagcaaaagatgagaccctcctctgg
gcgactatagatgcgcggctcaagaccagcaatcaagaaacaaacttccgagaattcctgagcaagaaggacat-
tggggacgttctgtttttaaactacc
aaaaagctatgggtttacccaaagagcgtattcctttttcccaagaggtctgggaagcttgtgcccacgaagta-
caaagcaagtacctcagcaagtcaaa
gtgcaacttgatcaatgggactgtgagacagagcccagacttcgatgaaaataagattatggtattcctcaagt-
cgcagtgggtcacaaaggtggaaaaa
ctaggtctacccaagattaagccaggtcaaaccatagcagccttttaccagcagactgtgatgctttttggaac-
tatggctaggtacatgcgatggttca
gacaggctttccagccaaaagaagtcttcataaactgtgagaccacgccagatgacatgtctgcatgggccttg-
aacaactggaatttcagcagacctag
cttggctaatgactacacagctttcgaccagtctcaggatggagcctgttgcaatttgaggtgctcaaagccaa-
agaccactgcataccagaggaaatca
ttcaggcatacatagatattaagactaatgcacagattttcctaggcacgttatcaattatgcgcctgactggt-
gaaggtcccacttttgatgcaaacac
tgagtgcaacatagcttacacccatacaaagtttgacatcccagccggaactgctcaagtttatgcaggagacg-
actccgcactggactgtgttccagaa
gtgaagcatagtttccacaggcttgaggacaaattactcctaaagtcaaagcctgtaatcacgcagcaaaagaa-
gggcagttggcctgagttttgtggtt
ggctgatcacaccaaaaggggtgatgaaagacccaattaagctccatgttagcttaaaattggctgaagctaag-
ggtgaactcaagaaatgtcaagattc
ctatgaaattgatctgagttatgcctatgaccacaaggactctctgcatgacttgttcgatgagaaacagtgtc-
aggcacacacactcacttgcagaaca
ctaatcaagtcagggagaggcactgtctcactttcccgcctcagaaactttctttaaccgttaagttaccttag-
agatttgaataagatggatattctca
tcagtagtttgaaaagtttaggttattctaggacttccaaatctttagattcaggacctttggtagtacatgca-
gtagccggagccggtaagtccacagc
cctaaggaagttgatcctcagacacccaacattcaccgtgcatacactcggtgtccctgacaaggtgagtatca-
gaactagaggcatacagaagccagga
cctattcctgagggcaacttcgcaatcctcgatgagtatactttggacaacaccacaaggaactcataccaggc-
actttttgctgacccttatcaggcac
cggagtttagcctagagccccacttctacttggaaacatcatttcgagttccgaggaaagtggcagatttgata-
gctggctgtggcttcgatttcgagac
gaactcaccggaagaagggcacttagagatcactggcatattcaaagggcccctactcggaaaggtgatagcca-
ttgatgaggagtctgagacaacactg
tccaggcatggtgttgagtttgttaagccctgccaagtgacgggacttgagttcaaagtagtcactattgtgtc-
tgccgcaccaatagaggaaattggcc
agtccacagctttctacaacgctatcaccaggtcaaagggattgacatatgtccgcgcaggccataggctgacc-
gctccggtcaattctgaaaaagtgta
catagtattaggtctatcatttgctttagtttcaattacctttctgctttctagaaatagcttaccccacgtcg-
gtgacaacattcacagcttgccacac
ggaggagcttacagagacggcaccaaagcaatcttgtacaactccccaaatctagggtcacgagtgagtctaca-
caacggaaagaacgcagcatttgctg
ccgttttgctactgactttgctgatctatggaagtaaatacatatctcaacgcaatcatacttgtgcttgtggt-
aacaatcatagcagtcattagcactt
ccttagtgaggactgaaccttgtgtcatcaagattactggggaatcaatcacagtgttggcttgcaaactagat-
gcagaaaccataagggccttgccgat
ctcaagcctctccgttgaacggttaagtttccattgatactcgaaagaggtcagcaccagctagcaacaaacaa-
gaaaggtatggtgagcaagggcgagg
agctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtcc-
ggcgagggcgagggcgatgccaccta
cggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccacct-
tcagctacggcgtgcagtgcttcagc
cgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcac-
catcttcttcaaggacgacggcaact
acaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttc-
aaggacgacggcaactacaagacccg
cgccgaggtgaagttcgaggggccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaac-
ttcaagatccgccacaacatcgagga
cggcagcgtgcagctcgccgaccactaccgcagaacacccccatcggcgacggccccgtgctgctgcccgacaa-
ccactacctgagcacccagtccgccc
tgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcac-
ggcatggacgagctgtacaagtaagc
ttggtcgtatcactggaacaacaaccgctgaggctgttgtcactctaccaccaccataactacgtctacataac-
cgacgcctaccccagtttcatagtat
tttctggtttgattgtatgaataatataaataaaaaaaaaaaaaaaaaaaaaaaactagtgagctcttctgtca-
gcgggcccactgcatccaccccagta
cattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgccac-
cagccagccaacagctccccgaccgg
cagctcggcacaaaatcaccactcgatacaggcagcccatcag SEQ ID NO: 5: Plasmid
backbone insertion containing virG gene of pNMD062
ctgtcgatcagatctggctcgcggcggacgcacgacgccggggcgagaccataggcgatctcctaaatcaatag-
tagctgtaacctcgaagcgtttcact
tgtaacaacgattgagaatttttgtcataaaattgaaatacttggttcgcatttttgtcatccgcggtcagccg-
caattctgacgaactgcccatttagc
tggagatgattgtacatccttcacgtgaaaatttctcaagcgctgtgaacaagggttcagattttagattgaaa-
ggtgagccgttgaaacacgttcttct
tgtcgatgacgacgtcgctatgcggcatcttattattgaataccttacgatccacgccttcaaagtgaccgcgg-
tagccgacagcacccagttcacaaga
gtactctcttccgcgacggtcgatgtcgtggttgttgatctaaatttaggtcgtgaagatgggctcgagatcgt-
tcgtaatctggcggcaaagtctgata
ttccaatcataattatcagtggcgaccgccttgaggagacggataaagttgttgcactcgagctaggagcaagt-
gattttatcgctaagccgttcagtat
cagagagtttctagcacgcattcgggttgccttgcgcgtgcgccccaacgttgtccgctccaaagaccgacggt-
ctttttgttttactgactggacactt
aatctcaggcaacgtcgcttgatgtccgaagctggcggtgaggtgaaacttacggcaggtgagttcaatcttct-
cctcgcgtttttagagaaaccccgcg
acgttctatcgcgcgagcaacttctcattgccagtcgagtacgcgacgaggaggtttatgacaggagtatagat-
gttctcattttgaggctgcgccgcaa
acttgaggcggatccgtcaagccctcaactgataaaaacagcaagaggtgccggttatttctttgacgcggacg-
tgcaggtttcgcacggggggacgatg gcagcctaagatcgacag SEQ ID NO: 6: Plasmid
backbone insertion containing virG gene of pNMD063, pNMD2190
ctgtcgatcagatctggctcgcggcggacgcacgacgccggggcgagaccataggcgatctcctaaatcaatag-
tagctgtaacctcgaagcgtttcact
tgtaacaacgattgagaatttttgtcataaaattgaaatacttggttcgcatttttgtcatccgcggtcagccg-
caattctgacgaactgcccatttagc
tggagatgattgtacatccttcacgtgaaaatttctcaagtgctgtgaacaagggttcagattttagattgaaa-
ggtgagccgttgaaacacgttcttct
tgtcgatgacgacgtcgctatgcggcatcttattattgaataccttacgatccacgccttcaaagtgaccgcgg-
tagccgacagcacccagttcacaaga
gtactctcttccgcgacggtcgatgtcgtggttgttgatctagatttaggtcgtgaagatgggctcgagatcgt-
tcgtaatctggcggcaaagtctgata
ttccaatcataattatcagtggcgaccgccttgaggagacggataaagttgttgcactcgagctaggagcaagt-
gattttatcgctaagccgttcagtat
cagagagtttctagcacgcattcgggttgccttgcgcgtgcgccccaacgttgtccgctccaaagaccgacggt-
ctttttgttttactgactggacactt
aatctcaggcaacgtcgcttgatgtccgaagctggcggtgaggtgaaacttacggcaggtgagttcaatcttct-
cctcgcgtttttagagaaaccccgcg
acgttctatcgcgcgagcaacttctcattgccagtcgagtacgcgacgaggaggtttatgacaggagtatagat-
gttctcattttgaggctgcgccgcaa
acttgaggcggatccgtcaagccctcaactgataaaaacagcaagaggtgccggttatttctttgacgcggacg-
tgcaggtttcgcacggggggacgatg gcagcctaagatcgacag SEQ ID NO: 7:
full-length nucleotide sequence of pNMD1971
ttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaat-
aattaacatgtaatgcatgacgttat
ttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgc-
gcaaactaggataaattatcgcgcgc
ggtgtcatctatgttactagatcgacctgcaggcatgccaattccaatcccacaaaaatctgagcttaacagca-
cagttgctcctctcagagcagaatcg
ggtattcaacaccctcatatcaactactacgttgtgtataacggtccacatgccggtatatacgatgactgggg-
ttgtacaaaggcggcaacaaacggcg
ttcccggagttgcacacaagaaatttgccactattacagaggcaagagcagcagctgacgcgtacacaacaagt-
cagcaaacagacaggttgaacttcat
ccccaaaggagaagctcaactcaagcccaagagctttgctaaggccctaacaagcccaccaaagcaaaaagccc-
actggctcacgctaggaaccaaaagg
cccagcagtgatccagccccaaaagagatctcctttgccccggagattacaatggacgatttcctctatcttta-
cgatctaggaaggaagttcgaaggtg
aaggtgacgacactatgttcaccactgataatgagaaggttagcctcttcaatttcagaaagaatgctgaccca-
cagatggttagagaggcctacgcagc
aggtctcatcaagacgatctacccgagtaacaatctccaggagatcaaataccttcccaagaaggttaaagatg-
cagtcaaaagattcaggactaattgc
atcaagaacacagagaaagacatatttctcaagatcagaagtactattccagtatggacgattcaaggcttgct-
tcataaaccaaggcaagtaatagaga
ttggagtctctaaaaaggtagttcctactgaatctaaggccatgcatggagtctaagattcaaatcgaggatct-
aacagaactcgccgtgaagactggcg
aacagttcatacagagtcttttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacact-
ctggtctactccaaaaatgtcaaaga
tacagtctcagaagaccaaagggctattgagacttttcaacaaaggataatttcgggaaacctcctcggattcc-
attgcccagctatctgtcacttcatc
gaaaggacagtagaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggctatcattcaaga-
tctctctgccgacagtggtcccaaag
atggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattga-
tgtgacatctccactgacgtaaggga
tgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggacac-
gctcgagtataagagctctattttta
caacaattaccaacaacaacaaacaacaaacaacattacaattacatttacaattaccatggaacgagctatac-
aaggaaacgatgctagggaacaagct
tatggtgaacgttggaatggaggatcaggaagttccacttctcccttcaaacttcctgacgaaagtccgagttg-
gactgagtggcggctacataacgatg
agacgatttcgaatcaagataatccccttggtttcaaggaaagctggggtttcgggaaagttgtatttaagaga-
tatctcagatacgacgggacggaaac
ttcactgcacagagtccttggatcttggacgggagattcggttaactatgcagcatctcgatttctcggtttcg-
accagatcggatgtacctatagtatt
cggtttcgaggagttagtgtcaccatttctggagggtcgcgaactcttcagcatctcagtgaaatggcaattcg-
gtctaagcaagaactgctacagctta
ccccagtcaaagtggaaagtgatgtatcaagaggatgccctgaaggtgttgaaaccttcgaagaagaaagcgag-
taaggatcctctagagtcctgcttta
atgagatatgcgagacgcctatgatcgcatgatatttgctttcaattctgttgtgcacgttgtaaaaaacctga-
gcatgtgtagctcagatccttaccgc
cggtttcggttcattctaatgaatatatcacccgttactatcgtatttttatgaataatattctccgttcaatt-
tactgattgtaccctactacttatat
gtacaatattaaaatgaaaacaatatattgtgctgaataggtttatagcgacatctatgatagagcgccacaat-
aacaaacaattgcgttttattattac
aaatccaattttaaaaaaagcggcagaaccggtcaaacctaaaagactgattacataaatcttattcaaatttc-
aaaagtgccccaggggctagtatcta
cgacacaccgagcggcgaactaataacgctcactgaagggaactccggttccccgccggcgcgcatgggtgaga-
ttccttgaagttgagtattggccgtc
cgctctaccgaaagttacgggcaccattcaacccggtccagcacggcggccgggtaaccgacttgctgccccga-
gaattatgcagcatttttttggtgta
tgtgggccccaaatgaagtgcaggtcaaaccttgacagtgacgacaaatcgttgggcgggtccagggcgaattt-
tgcgacaacatgtcgaggctcagcag
gacctgcataagctcttctgtcagcgggcccactgcatccaccccagtacattaaaaacgtccgcaatgtgtta-
ttaagttgtctaagcgtcaatttgtt
tacaccacaatatatcctgccaccagccagccaacagctccccgaccggcagctcggcacaaaatcaccactcg-
atacaggcagcccatcagtcagatct
cctttgcgacgctcaccgggctggttgccctcgccgctgggctggcggccgtctatggccctgcaaacgcgcca-
gaaacgccgtcgaagccgtgtgcgag
acaccgcggccgccggcgttgtggatacctcgcggaaaacttggccctcactgacagatgaggggcggacgttg-
acacttgaggggccgactcacccggc
gcggcgttgacagatgaggggcaggctcgatttcggccggcgacgtggagctggccagcctcgcaaatcggcga-
aaacgcctgattttacgcgagtttcc
cacagatgatgtggacaagcctggggataagtgccctgcggtattgacacttgaggggcgcgactactgacaga-
tgaggggcgcgatccttgacacttga
ggggcagagtgctgacagatgaggggcgcacctattgacatttgaggggctgtccacaggcagaaatccagcat-
ttgcaagggtttccgcccgtttttcg
gccaccgctaacctgtcttttaacctgcttttaaaccaatatttataaaccttgtttttaaccagggctgcgcc-
ctgtgcgcgtgaccgcgcacgccgaa
ggggggtgcccccccttctcgaaccctcccggcccgctaacgcgggcctcccatcccccccaggggctgcgccc-
ctcggccgcgaacggcctcaccccaa
aaatggcagcgctggccaattcgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatc-
cgctcatgagacaataaccctgataa
atgcttcaataatattgaaaaaggaagagtatggctaaaatgagaatatcaccggaattgaaaaaactgatcga-
aaaataccgctgcgtaaaagatacgg
aaggaatgtctcctgctaaggtatataagctggtgggagaaaatgaaaacctatatttaaaaatgacggacagc-
cggtataaagggaccacctatgatgt
ggaacgggaaaaggacatgatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggc-
atgatggctggagcaatctgctcatg
agtgaggccgatggcgtcctttgctcggaagagtatgaagatgaacaaagccctgaaaagattatcgagctgta-
tgcggagtgcatcaggctctttcact
ccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagccgaattggattacttactgaat-
aacgatctggccgatgtggattgcga
aaactgggaagaagacactccatttaaagatccgcgcgagctgtatgattttttaaagacggaaaagcccgaag-
aggaacttgtcttttcccacggcgac
ctgggagacagcaacatctttgtgaaagatggcaaagtaagtggctttattgatcttgggagaagcggcgggcg-
gacaagtggtatgacattgccttctg
cgtccggtcgatcagggaggatatcggggaagaacagtatgtcgagctattttttgacttactggggatcaagc-
ctgattgggagaaaataaaatattat
attttactggatgaattgttttagctgtcagaccaagtttactcatatatactttagattgatttaaaacttca-
tttttaatttaaaaggatctaggtga
agatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgta-
gaaaagatcaaaggatcttcttgaga
tcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg-
atcaagagctaccaactctttttccg
aaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccactt-
caagaactctgtagcaccgcctacat
acctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactca-
agacgatagttaccggataaggcgca
gcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacc-
tacagcgtgagctatgagaaagcgcc
acgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgaggga-
gcttccagggggaaacgcctggtatc
tttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagc-
ctatggaaaaacgccagcaacgcggc
ctttttacggttcctggcagatcctagatgtggcgcaacgatgccggcgacaagcaggagcgcaccgacttctt-
ccgcatcaagtgttttggctctcagg
ccgaggcccacggcaagtatttgggcaaggggtcgctggtattcgtgcagggcaagattcggaataccaagtac-
gagaaggacggccagacggtctacgg
gaccgacttcattgccgataaggtggattatctggacaccaaggcaccaggcgggtcaaatcaggaataagggc-
acattgccccggcgtgagtcggggca
atcccgcaaggagggtgaatgaatcggacgtttgaccggaaggcatacaggcaagaactgatcgacgcggggtt-
ttccgccgaggatgccgaaaccatcg
caagccgcaccgtcatgcgtgcgccccgcgaaaccttccagtccgtcggctcgatggtccagcaagctacggcc-
aagatcgagcgcgacagcgtgcaact
ggctccccctgccctgcccgcgccatcggccgccgtggagcgttcgcgtcgtctcgaacaggaggcggcaggtt-
tggcgaagtcgatgaccatcgacacg
cgaggaactatgacgaccaagaagcgaaaaaccgccggcgaggacctggcaaaacaggtcagcgaggccaagca-
ggccgcgttgctgaaacacacgaagc
agcagatcaaggaaatgcagctttccttgttcgatattgcgccgtggccggacacgatgcgagcgatgccaaac-
gacacggcccgctctgccctgttcac
cacgcgcaacaagaaaatcccgcgcgaggcgctgcaaaacaaggtcattttccacgtcaacaaggacgtgaaga-
tcacctacaccggcgtcgagctgcgg
gccgacgatgacgaactggtgtggcagcaggtgttggagtacgcgaagcgcacccctatcggcgagccgatcac-
cttcacgttctacgagctttgccagg
acctgggctggtcgatcaatggccggtattacacgaaggccgaggaatgcctgtcgcgcctacaggcgacggcg-
atgggcttcacgtccgaccgcgttgg
gcacctggaatcggtgtcgctgctgctgcaccgcttccgcgtcctggaccgtggcaagaaaacgtcccgttgcc-
aggtcctgatcgacgaggaaatcgtc
gtgctgtttgctggcgaccactacacgaaattcatatgggagaagtaccgcaagctgtcgccgacggcccgacg-
gatgttcgactatttcagctcgcacc
gggagccgtacccgctcaagctcaagctggaaaccttccgcctcatgtgcggatcggattccacccgcgtgaag-
aagtggcgcgagcaggtcggcgaagc
ctgcgaagagttgcgaggcagcggcctggtggaacacgcctgggtcaatgatgacctggtgcattgcaaacgct-
agggccttgtggggtcagttccggct
gggggttcagcagccagcgcctgatctggggaaccctgtggttggcacatacaaatggacgaacggataaacct-
tttcacgcccttttaaatatccgatt
attctaataaacgctcttttctcttaggtttacccgccaatatatcctgtcaaacactgatagtttaaactgaa-
ggcgggaaacgacaatctgatctaag
ctaggcatggaattccaatcccacaaaaatctgagcttaacagcacagttgctcctctcagagcagaatcgggt-
attcaacaccctcatatcaactacta
cgttgtgtataacggtccacatgccggtatatacgatgactggggttgtacaaggcggcaacaaacggcgttcc-
cggagttgcacacaagaaatttgcca
ctattacagaggcaagagcagcagctgacgcgtacacaacaagtcagcaaacagacaggttgaacttcatcccc-
aaaggagaagctcaactcaagcccaa
gagctttgctaaggccctaacaagcccaccaaagcaaaaagcccactggctcacgctaggaaccaaaaggccca-
gcagtgatccagccccaaaagagatc
tcctttgccccggagattacaatggacgatttcctctatctttacgatctaggaaggaagttcgaaggtgaagg-
tgacgacactatgttcaccactgata
atgagaaggttagcctcttcaatttcagaaagaatgctgacccacagatggttagagaggcctacgcagcaggt-
ctcatcaagacgatctacccgagtaa
caatctccaggagatcaaataccttcccaagaaggttaaagatgcagtcaaaagattcaggactaattgcatca-
agaacacagagaaagacatatttctc
aagatcagaagtactattccagtatggacgattcaaggcttgcttcataaaccaaggcaagtaatagagattgg-
agtctctaaaaaggtagttcctactg
aatctaaggccatgcatggagtctaagattcaaatcgaggatctaacagaactcgccgtgaagactggcgaaca-
gttcatacagagtcttttacgactca
atgacaagaagaaaatcttcgtcaacatggtggagcacgacactctggtctactccaaaaatgtcaaagataca-
gtctcagaagaccaaagggctattga
gacttttcaacaaaggataatttcgggaaacctcctcggattccattgcccagctatctgtcacttcatcgaaa-
ggacagtagaaaaggaaggtggctcc
tacaaatgccatcattgcgataaaggaaaggctatcattcaagatctctctgccgacagtggtcccaaagatgg-
acccccacccacgaggagcatcgtgg
aaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgacatctccactgacgtaagggatgac-
gcacaatcccactatccttcgcaaga
cccttcctctatataaggaagttcatttcatttggagaggacacgctcgagtataagagctcatttttacaaca-
attaccaacaacaacaaacaacaaac
aacattacaattatcgatgggtcagtccctatgttacgtcctgtagaaaccccaacccgtgaaatcaaaaaact-
cgacggcctgtgggcattcagtctgg
atcgcgaaaactgtggaattgatcagcgttggtgggaaagcgcgttacaagaaagccgggcaattgctgtgcca-
ggcagttttaacgatcagttcgccga
tgcagatattcgtaattatgcgggcaacgtctggtatcagcgcgaagtctttataccgaaaggtaagtagtgtt-
tttggataactgagtttgcctatgat
tttgtatttactgagatgtttgtcctctttgtgcaggttgggcaggccagcgtatcgtgctgcgtttcgatgcg-
gtcactcattacggcaaagtgtgggt
caataatcaggaagtgatggagcatcagggcggctatacgccatttgaagccgatgtcacgccgtatgttattg-
ccgggaaaagtgtacgtatcaccgtt
tgtgtgaacaacgaactgaactggcagactatcccgccgggaatggtgattaccgacgaaaacggcaagaaaaa-
gcagtcttacttccatgatttcttta
actatgccggaatccatcgcagcgtaatgctctacaccacgccgaacacctgggtggacgatatcaccgtggtg-
acgcatgtcgcgcaagactgtaacca
cgcgtctgttgactggcaggtggtggccaatggtgatgtcagcgttgaactgcgtgatgcggatcaacaggtgg-
ttgcaactggacaaggcactagcggg
actttgcaagtggtgaatccgcacctctggcaaccgggtgaaggttatctctatgaactgtgcgtcacagccaa-
aagccagacagagtgtgatatctacc
cgcttcgcgtcggcatccggtcagtggcagtgaagggccaacagttcctgattaaccacaaaccgttctacttt-
actggctttggtcgtcatgaagatgc
ggacttacgtggcaaaggattcgataacgtgctgatggtgcacgaccacgcattaatggactggattggggcca-
actcctaccgtacctcgcattaccct
tacgctgaagagatgctcgactgggcagatgaacatggcatcgtggtgattgatgaaactgctgctgtcggctt-
taacctctctttaggcattggtttcg
aagcgggcaacaagccgaaagaactgtacagcgaagaggcagtcaacggggaaactcagcaagcgcacttacag-
gcgattaaagagctgatagcgcgtga
caaaaaccacccaagcgtggtgatgtggagtattgccaacgaaccggatacccgtccgcaaggtgcacgggaat-
atttcgcgccactggcggaagcaacg
cgtaaactcgacccgacgcgtccgatcacctgcgtcaatgtaatgttctgcgacgctcacaccgataccatcag-
cgatctctttgatgtgctgtgcctga
accgttattacggatggtatgtccaaagcggcgatttggaaacggcagagaaggtactggaaaaagaacttctg-
gcctggcaggagaaactgcatcagcc
gattatcatcaccgaatacggcgtggatacgttagccgggctgcactcaatgtacaccgacatgtggagtgaag-
agtatcagtgtgcatggctggatatg
tatcaccgcgtctttgatcgcgtcagcgccgtcgtcggtgaacaggtatggaatttcgccgattttgcgacctc-
gcaaggcatattgcgcgttggcggta
acaagaaagggatcttcactcgcgaccgcaaaccgaagtcggcggcttttctgctgcaaaaacgctggactggc-
atgaacttcggtgaaaaaccgcagca
gggaggcaaacaatgaatcaacaactctcctggcgcaccatcgtcggctacagcctcgggattgggatcctcta-
gagtcaagcagatcgttcaaacattt ggcaataaagtttc
Sequence CWU 1
1
81241PRTAgrobacterium tumefaciens 1Leu Lys His Val Leu Leu Val Asp
Asp Asp Val Ala Met Arg His Leu 1 5 10 15 Ile Ile Glu Tyr Leu Thr
Ile His Ala Phe Lys Val Thr Ala Val Ala 20 25 30 Asp Ser Thr Gln
Phe Thr Arg Val Leu Ser Ser Ala Thr Val Asp Val 35 40 45 Val Val
Val Asp Leu Asn Leu Gly Arg Glu Asp Gly Leu Glu Ile Val 50 55 60
Arg Asn Leu Ala Ala Lys Ser Asp Ile Pro Ile Ile Ile Ile Ser Gly 65
70 75 80 Asp Arg Leu Glu Glu Thr Asp Lys Val Val Ala Leu Glu Leu
Gly Ala 85 90 95 Ser Asp Phe Ile Ala Lys Pro Phe Ser Ile Arg Glu
Phe Leu Ala Arg 100 105 110 Ile Arg Val Ala Leu Arg Val Arg Pro Asn
Val Val Arg Ser Lys Asp 115 120 125 Arg Arg Ser Phe Cys Phe Thr Asp
Trp Thr Leu Asn Leu Arg Gln Arg 130 135 140 Arg Leu Met Ser Glu Ala
Gly Gly Glu Val Lys Leu Thr Ala Gly Glu 145 150 155 160 Phe Asn Leu
Leu Leu Ala Phe Leu Glu Lys Pro Arg Asp Val Leu Ser 165 170 175 Arg
Glu Gln Leu Leu Ile Ala Ser Arg Val Arg Asp Glu Glu Val Tyr 180 185
190 Asp Arg Ser Ile Asp Val Leu Ile Leu Arg Leu Arg Arg Lys Leu Glu
195 200 205 Ala Asp Pro Ser Ser Pro Gln Leu Ile Lys Thr Ala Arg Gly
Ala Gly 210 215 220 Tyr Phe Phe Asp Ala Asp Val Gln Val Ser His Gly
Gly Thr Met Ala 225 230 235 240 Ala 210393DNAArtificial
SequenceT-DNA region sequences of pNMD560 2cctgtggttg gcacatacaa
atggacgaac ggataaacct tttcacgccc ttttaaatat 60ccgattattc taataaacgc
tcttttctct taggtttacc cgccaatata tcctgtcaaa 120cactgatagt
ttaaactgaa ggcgggaaac gacaatctga tctaagctag cttggaattg
180gtaccacgcg tttcgacaaa atttagaacg aacttaatta tgatctcaaa
tacattgata 240catatctcat ctagatctag gttatcatta tgtaagaaag
ttttgacgaa tatggcacga 300caaaatggct agactcgatg taattggtat
ctcaactcaa cattatactt ataccaaaca 360ttagttagac aaaatttaaa
caactatttt ttatgtatgc aagagtcagc atatgtataa 420ttgattcaga
atcgttttga cgagttcgga tgtagtagta gccattattt aatgtacata
480ctaatcgtga atagtgaata tgatgaaaca ttgtatctta ttgtataaat
atccataaac 540acatcatgaa agacactttc tttcacggtc tgaattaatt
atgatacaat tctaatagaa 600aacgaattaa attacgttga attgtatgaa
atctaattga acaagccaac cacgacgacg 660actaacgttg cctggattga
ctcggtttaa gttaaccact aaaaaaacgg agctgtcatg 720taacacgcgg
atcgagcagg tcacagtcat gaagccatca aagcaaaaga actaatccaa
780gggctgagat gattaattag tttaaaaatt agttaacacg agggaaaagg
ctgtctgaca 840gccaggtcac gttatcttta cctgtggtcg aaatgattcg
tgtctgtcga ttttaattat 900ttttttgaaa ggccgaaaat aaagttgtaa
gagataaacc cgcctatata aattcatata 960ttttcctctc cgctttgaag
ttttagtttt attgcaacaa caacaacaaa ttacaataac 1020aacaaacaaa
atacaaacaa caacaacatg gcacaatttc aacaaacaat tgacatgcaa
1080actctccaag ccgctgcggg acgcaacagc ttggtgaatg atttggcatc
tcgtcgcgtt 1140tacgataatg cagtcgagga gctgaatgct cgttccagac
gtcccaaggt aataggaact 1200ttctggatct actttatttg ctggatctcg
atcttgtttt ctcaatttcc ttgagatctg 1260gaattcgttt aatttggatc
tgtgaacctc cactaaatct tttggtttta ctagaatcga 1320tctaagttga
ccgatcagtt agctcgatta tagctaccag aatttggctt gaccttgatg
1380gagagatcca tgttcatgtt acctgggaaa tgatttgtat atgtgaattg
aaatctgaac 1440tgttgaagtt agattgaatc tgaacactgt caatgttaga
ttgaatctga acactgttta 1500aggttagatg aagtttgtgt atagattctt
cgaaacttta ggatttgtag tgtcgtacgt 1560tgaacagaaa gctatttctg
attcaatcag ggtttatttg actgtattga actctttttg 1620tgtgtttgca
ggtccacttc tccaaggcag tgtctacgga acagaccctg attgcaacaa
1680acgcatatcc ggagttcgag atttccttta ctcatacgca atccgctgtg
cactccttgg 1740ccggaggcct tcggtcactt gagttggagt atctcatgat
gcaagttccg ttcggttctc 1800tgacgtacga catcggcggt aacttttccg
cgcacctttt caaagggcgc gattacgttc 1860actgctgcat gcctaatctg
gatgtacgtg acattgctcg ccatgaagga cacaaggaag 1920ctatttacag
ttatgtgaat cgtttgaaaa ggcagcagcg tcctgtgcct gaataccaga
1980gggcagcttt caacaactac gctgagaacc cgcacttcgt ccattgcgac
aaacctttcc 2040aacagtgtga attgacgaca gcgtatggca ctgacaccta
cgctgtagct ctccatagca 2100tttatgatat ccctgttgag gagttcggtt
ctgcgctact caggaagaat gtgaaaactt 2160gtttcgcggc ctttcatttc
catgagaata tgcttctaga ttgtgataca gtcacactcg 2220atgagattgg
agctacgttc cagaaatcag gtaacattcc ttagttacct ttcttttctt
2280tttccatcat aagtttatag attgtacatg ctttgagatt tttctttgca
aacaatctca 2340ggtgataacc tgagcttctt cttccataat gagagcactc
tcaattacac ccacagcttc 2400agcaacatca tcaagtacgt gtgcaagacg
ttcttccctg ctagtcaacg cttcgtgtac 2460cacaaggagt tcctggtcac
tagagtcaac acttggtact gcaagttcac gagagtggat 2520acgttcactc
tgttccgtgg tgtgtaccac aacaatgtgg attgcgaaga gttttacaag
2580gctatggacg atgcgtggca ctacaaaaag acgttagcaa tgcttaatgc
cgagaggacc 2640atcttcaagg ataacgctgc gttaaacttc tggttcccga
aggtgctctt gaaattggaa 2700gtcttctttt gttgtctaaa cctatcaatt
tctttgcgga aatttatttg aagctgtaga 2760gttaaaattg agtcttttaa
acttttgtag gtgagagaca tggttatcgt ccctctcttt 2820gacgcttcta
tcacaactgg taggatgtct aggagagagg ttatggtgaa caaggacttc
2880gtctacacgg tcctaaatca catcaagacc tatcaagcta aggcactgac
gtacgcaaac 2940gtgctgagct tcgtggagtc tattaggtct agagtgataa
ttaacggtgt cactgccagg 3000taagttgtta cttatgattg ttttcctctc
tgctacatgt attttgttgt tcatttctgt 3060aagatataag aattgagttt
tcctctgatg atattattag gtctgaatgg gacacagaca 3120aggcaattct
aggtccatta gcaatgacat tcttcctgat cacgaagctg ggtcatgtgc
3180aagatgaaat aatcctgaaa aagttccaga agttcgacag aaccaccaat
gagctgattt 3240ggacaagtct ctgcgatgcc ctgatggggg ttattccctc
ggtcaaggag acgcttgtgc 3300gcggtggttt tgtgaaagta gcagaacaag
ccttagagat caaggttagt atcatatgaa 3360gaaataccta gtttcagttg
atgaatgcta ttttctgacc tcagttgttc tcttttgaga 3420attatttctt
ttctaatttg cctgattttt ctattaattc attaggttcc cgagctatac
3480tgtaccttcg ccgaccgatt ggtactacag tacaagaagg cggaggagtt
ccaatcgtgt 3540gatctttcca aacctctaga agagtcagag aagtactaca
acgcattatc cgagctatca 3600gtgcttgaga atctcgactc ttttgactta
gaggcgttta agactttatg tcagcagaag 3660aatgtggacc cggatatggc
agcaaaggta aatcctggtc cacactttta cgataaaaac 3720acaagatttt
aaactatgaa ctgatcaata atcattccta aaagaccaca cttttgtttt
3780gtttctaaag taatttttac tgttataaca ggtggtcgta gcaatcatga
agtcagaatt 3840gacgttgcct ttcaagaaac ctacagaaga ggaaatctcg
gagtcgctaa aaccaggaga 3900ggggtcgtgt gcagagcata aggaagtgtt
gagcttacaa aatgatgctc cgttcccgtg 3960tgtgaaaaat ctagttgaag
gttccgtgcc ggcgtatgga atgtgtccta agggtggtgg 4020tttcgacaaa
ttggatgtgg acattgctga tttccatctc aagagtgtag atgcagttaa
4080aaagggaact atgatgtctg cggtgtacac agggtctatc aaagttcaac
aaatgaagaa 4140ctacatagat tacttaagtg cgtcgctggc agctacagtc
tcaaacctct gcaaggtaag 4200aggtcaaaag gtttccgcaa tgatccctct
ttttttgttt ctctagtttc aagaatttgg 4260gtatatgact aacttctgag
tgttccttga tgcatatttg tgatgagaca aatgtttgtt 4320ctatgtttta
ggtgcttaga gatgttcacg gcgttgaccc agagtcacag gagaaatctg
4380gagtgtggga tgttaggaga ggacgttggt tacttaaacc taatgcgaaa
agtcacgcgt 4440ggggtgtggc agaagacgcc aaccacaagt tggttattgt
gttactcaac tgggatgacg 4500gaaagccggt ttgtgatgag acatggttca
gggtggcggt gtcaagcgat tccttgatat 4560attcggatat gggaaaactt
aagacgctca cgtcttgcag tccaaatggt gagccaccgg 4620agcctaacgc
caaagtaatt ttggtcgatg gtgttcccgg ttgtggaaaa acgaaggaga
4680ttatcgaaaa ggtaagttct gcatttggtt atgctccttg cattttaggt
gttcgtcgct 4740cttccatttc catgaatagc taagattttt tttctctgca
ttcattcttc ttgcctcagt 4800tctaactgtt tgtggtattt ttgttttaat
tattgctaca ggtaaacttc tctgaagact 4860tgattttagt ccctgggaag
gaagcttcta agatgatcat ccggagggcc aaccaagctg 4920gtgtgataag
agcggataag gacaatgtta gaacggtgga ttccttcttg atgcatcctt
4980ctagaagggt gtttaagagg ttgtttatcg atgaaggact aatgctgcat
acaggttgtg 5040taaatttcct actgctgcta tctcaatgtg acgtcgcata
tgtgtatggg gacacaaagc 5100aaattccgtt catttgcaga gtcgcgaact
ttccgtatcc agcgcatttt gcaaaactcg 5160tcgctgatga gaaggaagtc
agaagagtta cgctcaggta aagcaactgt gttttaatca 5220atttcttgtc
aggatatatg gattataact taatttttga gaaatctgta gtatttggcg
5280tgaaatgagt ttgctttttg gtttctcccg tgttataggt gcccggctga
tgttacgtat 5340ttccttaaca agaagtatga cggggcggtg atgtgtacca
gcgcggtaga gagatccgtg 5400aaggcagaag tggtgagagg aaagggtgca
ttgaacccaa taaccttacc gttggagggt 5460aaaattttga ccttcacaca
agctgacaag ttcgagttac tggagaaggg ttacaaggta 5520aagtttccaa
ctttccttta ccatatcaaa ctaaagttcg aaacttttta tttgatcaac
5580ttcaaggcca cccgatcttt ctattcctga ttaatttgtg atgaatccat
attgactttt 5640gatggttacg caggatgtga acactgtgca cgaggtgcaa
ggggagacgt acgagaagac 5700tgctattgtg cgcttgacat caactccgtt
agagatcata tcgagtgcgt cacctcatgt 5760tttggtggcg ctgacaagac
acacaacgtg ttgtaaatat tacaccgttg tgttggaccc 5820gatggtgaat
gtgatttcag aaatggagaa gttgtccaat ttccttcttg acatgtatag
5880agttgaagca ggtctgtctt tcctatttca tatgtttaat cctaggaatt
tgatcaattg 5940attgtatgta tgtcgatccc aagactttct tgttcactta
tatcttaact ctctctttgc 6000tgtttcttgc aggtgtccaa tagcaattac
aaatcgatgc agtattcagg ggacagaact 6060tgtttgttca gacgcccaag
tcaggagatt ggcgagatat gcaattttac tatgacgctc 6120ttcttcccgg
aaacagtact attctcaatg aatttgatgc tgttacgatg aatttgaggg
6180atatttcctt aaacgtcaaa gattgcagaa tcgacttctc caaatccgtg
caacttccta 6240aagaacaacc tattttcctc aagcctaaaa taagaactgc
ggcagaaatg ccgagaactg 6300caggtaaaat attggatgcc agacgatatt
ctttcttttg atttgtaact ttttcctgtc 6360aaggtcgata aattttattt
tttttggtaa aaggtcgata attttttttt ggagccatta 6420tgtaattttc
ctaattaact gaaccaaaat tatacaaacc aggtttgctg gaaaatttgg
6480ttgcaatgat caaaagaaac atgaatgcgc cggatttgac agggacaatt
gacattgagg 6540atactgcatc tctggtggtt gaaaagtttt gggattcgta
tgttgacaag gaatttagtg 6600gaacgaacga aatgaccatg acaagggaga
gcttctccag gtaaggactt ctcatgaata 6660ttagtggcag attagtgttg
ttaaagtctt tggttagata atcgatgcct cctaattgtc 6720catgttttac
tggttttcta caattaaagg tggctttcga aacaagagtc atctacagtt
6780ggtcagttag cggactttaa ctttgtggat ttgccggcag tagatgagta
caagcatatg 6840atcaagagtc aaccaaagca aaagttagac ttgagtattc
aagacgaata tcctgcattg 6900cagacgatag tctaccattc gaaaaagatc
aatgcgattt tcggtccaat gttttcagaa 6960cttacgagga tgttactcga
aaggattgac tcttcgaagt ttctgttcta caccagaaag 7020acacctgcac
aaatagagga cttcttttct gacctagact caacccaggc gatggaaatt
7080ctggaactcg acatttcgaa gtacgataag tcacaaaacg agttccattg
tgctgtagag 7140tacaagatct gggaaaagtt aggaattgat gagtggctag
ctgaggtctg gaaacaaggt 7200gagttcctaa gttccatttt tttgtaatcc
ttcaatgtta ttttaacttt tcagatcaac 7260atcaaaatta ggttcaattt
tcatcaacca aataatattt ttcatgtata tataggtcac 7320agaaaaacga
ccttgaaaga ttatacggcc ggaatcaaaa catgtctttg gtatcaaagg
7380aaaagtggtg atgtgacaac ctttattggt aataccatca tcattgccgc
atgtttgagc 7440tcaatgatcc ccatggacaa agtgataaag gcagcttttt
gtggagacga tagcctgatt 7500tacattccta aaggtttaga cttgcctgat
attcaggcgg gcgcgaacct catgtggaac 7560ttcgaggcca aactcttcag
gaagaagtat ggttacttct gtggtcgtta tgttattcac 7620catgatagag
gagccattgt gtattacgat ccgcttaaac taatatctaa gttaggttgt
7680aaacatatta gagatgttgt tcacttagaa gagttacgcg agtctttgtg
tgatgtagct 7740agtaacttaa ataattgtgc gtatttttca cagttagatg
aggccgttgc cgaggttcat 7800aagaccgcgg taggcggttc gtttgctttt
tgtagtataa ttaagtattt gtcagataag 7860agattgttta gagatttgtt
ctttgtttga taatgtcgat agtctcgtac gaacctaagg 7920tgagtgattt
cctcaatctt tcgaagaagg aagagatctt gccgaaggct ctaacgaggt
7980taaaaaccgt gtctattagt actaaagata ttatatctgt caaggagtcg
gagactttgt 8040gtgatataga tttgttaatc aatgtgccat tagataagta
tagatatgtg ggtatcctag 8100gagccgtttt taccggagag tggctagtgc
cagacttcgt taaaggtgga gtgacgataa 8160gtgtgataga taagcgtctg
gtgaactcaa aggagtgcgt gattggtacg tacagagccg 8220cagccaagag
taagaggttc cagttcaaat tggttccaaa ttactttgtg tccaccgtgg
8280acgcaaagag gaagccgtgg caggtaagga tttttatgat atagtatgct
tatgtatttt 8340gtactgaaag catatcctgc ttcattggga tattactgaa
agcatttaac tacatgtaaa 8400ctcacttgat gatcaataaa cttgattttg
caggttcatg ttcgtataca agacttgaag 8460attgaggcgg gttggcagcc
gttagctctg gaagtagttt cagttgctat ggtcaccaat 8520aacgttgtca
tgaagggttt gagggaaaag gtcgtcgcaa taaatgatcc ggacgtcgaa
8580ggtttcgaag gtaagccatc ttcctgctta tttttataat gaacatagaa
ataggaagtt 8640gtgcagagaa actaattaac ctgactcaaa atctaccctc
ataattgttg tttgatattg 8700gtcttgtatt ttgcaggtgt ggttgacgaa
ttcgtcgatt cggttgcagc atttaaagcg 8760gttgacaact ttaaaagaag
gaaaaagaag gttgaagaaa agggtgtagt aagtaagtat 8820aagtacagac
cggagaagta cgccggtcct gattcgttta atttgaaaga agaaaacgtc
8880ttacaacatt acaaacccga ataatcgata actcgagtat ttttacaaca
attaccaaca 8940acaacaaaca acaaacaaca ttacaattac atttacaatt
atcatggtga gcaagggcga 9000ggagctgttc accggggtgg tgcccatcct
ggtcgagctg gacggcgacg taaacggcca 9060caagttcagc gtgtccggcg
agggcgaggg cgatgccacc tacggcaagc tgaccctgaa 9120gttcatctgc
accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcag
9180ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
acttcttcaa 9240gtccgccatg cccgaaggct acgtccagga gcgcaccatc
ttcttcaagg acgacggcaa 9300ctacaagacc cgcgccgagg tgaagttcga
gggcgacacc ctggtgaacc gcatcgagct 9360gaagggcatc gacttcaagg
aggacggcaa catcctgggg cacaagctgg agtacaacta 9420caacagccac
aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
9480caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
accagcagaa 9540cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
cactacctga gcacccagtc 9600cgccctgagc aaagacccca acgagaagcg
cgatcacatg gtcctgctgg agttcgtgac 9660cgccgccggg atcactcacg
gcatggacga gctgtacaag taaagcggcc cctagagcgt 9720ggtgcgcacg
atagcgcata gtgtttttct ctccacttga atcgaagaga tagacttacg
9780gtgtaaatcc gtaggggtgg cgtaaaccaa attacgcaat gttttgggtt
ccatttaaat 9840cgaaacccct tatttcctgg atcacctgtt aacgcacgtt
tgacgtgtat tacagtggga 9900ataagtaaaa gtgagaggtt cgaatcctcc
ctaaccccgg gtaggggccc agcggccgct 9960ctagctagag tcaagcagat
cgttcaaaca tttggcaata aagtttctta agattgaatc 10020ctgttgccgg
tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa
10080taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt
agagtcccgc 10140aattatacat ttaatacgcg atagaaaaca aaatatagcg
cgcaaactag gataaattat 10200cgcgcgcggt gtcatctatg ttactagatc
gacctgcatc caccccagta cattaaaaac 10260gtccgcaatg tgttattaag
ttgtctaagc gtcaatttgt ttacaccaca atatatcctg 10320ccaccagcca
gccaacagct ccccgaccgg cagctcggca caaaatcacc actcgataca
10380ggcagcccat cag 10393310397DNAArtificial SequenceT-DNA region
of pNMD570 3cctgtggttg gcacatacaa atggacgaac ggataaacct tttcacgccc
ttttaaatat 60ccgattattc taataaacgc tcttttctct taggtttacc cgccaatata
tcctgtcaaa 120cactgatagt ttaaactgaa ggcgggaaac gacaatctga
tctaagctag cttggaattg 180gtaccacgcg tttcgacaaa atttagaacg
aacttaatta tgatctcaaa tacattgata 240catatctcat ctagatctag
gttatcatta tgtaagaaag ttttgacgaa tatggcacga 300caaaatggct
agactcgatg taattggtat ctcaactcaa cattatactt ataccaaaca
360ttagttagac aaaatttaaa caactatttt ttatgtatgc aagagtcagc
atatgtataa 420ttgattcaga atcgttttga cgagttcgga tgtagtagta
gccattattt aatgtacata 480ctaatcgtga atagtgaata tgatgaaaca
ttgtatctta ttgtataaat atccataaac 540acatcatgaa agacactttc
tttcacggtc tgaattaatt atgatacaat tctaatagaa 600aacgaattaa
attacgttga attgtatgaa atctaattga acaagccaac cacgacgacg
660actaacgttg cctggattga ctcggtttaa gttaaccact aaaaaaacgg
agctgtcatg 720taacacgcgg atcgagcagg tcacagtcat gaagccatca
aagcaaaaga actaatccaa 780gggctgagat gattaattag tttaaaaatt
agttaacacg agggaaaagg ctgtctgaca 840gccaggtcac gttatcttta
cctgtggtcg aaatgattcg tgtctgtcga ttttaattat 900ttttttgaaa
ggccgaaaat aaagttgtaa gagataaacc cgcctatata aattcatata
960ttttcctctc cgctttgaag ttttagtttt attgcaacaa caacaacaaa
ttacaataac 1020aacaaacaaa atacaaacaa caacaacatg gcacaatttc
aacaaacaat tgacatgcaa 1080actctccaag ccgctgcggg acgcaacagc
ttggtgaatg atttggcatc tcgtcgcgtt 1140tacgataatg cagtcgagga
gctgaatgct cgttccagac gtcccaaggt aataggaact 1200ttctggatct
actttatttg ctggatctcg atcttgtttt ctcaatttcc ttgagatctg
1260gaattcgttt aatttggatc tgtgaacctc cactaaatct tttggtttta
ctagaatcga 1320tctaagttga ccgatcagtt agctcgatta tagctaccag
aatttggctt gaccttgatg 1380gagagatcca tgttcatgtt acctgggaaa
tgatttgtat atgtgaattg aaatctgaac 1440tgttgaagtt agattgaatc
tgaacactgt caatgttaga ttgaatctga acactgttta 1500aggttagatg
aagtttgtgt atagattctt cgaaacttta ggatttgtag tgtcgtacgt
1560tgaacagaaa gctatttctg attcaatcag ggtttatttg actgtattga
actctttttg 1620tgtgtttgca ggtccacttc tccaaggcag tgtctacgga
acagaccctg attgcaacaa 1680acgcatatcc ggagttcgag atttccttta
ctcatacgca atccgctgtg cactccttgg 1740ccggaggcct tcggtcactt
gagttggagt atctcatgat gcaagttccg ttcggttctc 1800tgacgtacga
catcggcggt aacttttccg cgcacctttt caaagggcgc gattacgttc
1860actgctgcat gcctaatctg gatgtacgtg acattgctcg ccatgaagga
cacaaggaag 1920ctatttacag ttatgtgaat cgtttgaaaa ggcagcagcg
tcctgtgcct gaataccaga 1980gggcagcttt caacaactac gctgagaacc
cgcacttcgt ccattgcgac aaacctttcc 2040aacagtgtga attgacgaca
gcgtatggca ctgacaccta cgctgtagct ctccatagca 2100tttatgatat
ccctgttgag gagttcggtt ctgcgctact caggaagaat gtgaaaactt
2160gtttcgcggc ctttcatttc catgagaata tgcttctaga ttgtgataca
gtcacactcg 2220atgagattgg agctacgttc cagaaatcag gtaacattcc
ttagttacct ttcttttctt 2280tttccatcat aagtttatag attgtacatg
ctttgagatt tttctttgca aacaatctca 2340ggtgataacc tgagcttctt
cttccataat gagagcactc tcaattacac ccacagcttc 2400agcaacatca
tcaagtacgt gtgcaagacg ttcttccctg ctagtcaacg cttcgtgtac
2460cacaaggagt tcctggtcac tagagtcaac acttggtact gcaagttcac
gagagtggat 2520acgttcactc tgttccgtgg tgtgtaccac aacaatgtgg
attgcgaaga gttttacaag 2580gctatggacg atgcgtggca ctacaaaaag
acgttagcaa tgcttaatgc cgagaggacc 2640atcttcaagg ataacgctgc
gttaaacttc tggttcccga aggtgctctt gaaattggaa 2700gtcttctttt
gttgtctaaa cctatcaatt tctttgcgga aatttatttg aagctgtaga
2760gttaaaattg agtcttttaa acttttgtag gtgagagaca tggttatcgt
ccctctcttt 2820gacgcttcta tcacaactgg taggatgtct aggagagagg
ttatggtgaa caaggacttc 2880gtctacacgg tcctaaatca catcaagacc
tatcaagcta aggcactgac gtacgcaaac 2940gtgctgagct tcgtggagtc
tattaggtct agagtgataa ttaacggtgt cactgccagg 3000taagttgtta
cttatgattg ttttcctctc tgctacatgt attttgttgt tcatttctgt
3060aagatataag aattgagttt tcctctgatg atattattag gtctgaatgg
gacacagaca 3120aggcaattct aggtccatta gcaatgacat tcttcctgat
cacgaagctg ggtcatgtgc 3180aagatgaaat aatcctgaaa aagttccaga
agttcgacag aaccaccaat gagctgattt 3240ggacaagtct ctgcgatgcc
ctgatggggg ttattccctc ggtcaaggag acgcttgtgc 3300gcggtggttt
tgtgaaagta gcagaacaag ccttagagat caaggttagt atcatatgaa
3360gaaataccta gtttcagttg atgaatgcta ttttctgacc tcagttgttc
tcttttgaga 3420attatttctt ttctaatttg cctgattttt ctattaattc
attaggttcc cgagctatac 3480tgtaccttcg ccgaccgatt ggtactacag
tacaagaagg cggaggagtt ccaatcgtgt 3540gatctttcca aacctctaga
agagtcagag aagtactaca acgcattatc cgagctatca 3600gtgcttgaga
atctcgactc ttttgactta gaggcgttta agactttatg tcagcagaag
3660aatgtggacc cggatatggc agcaaaggta aatcctggtc cacactttta
cgataaaaac 3720acaagatttt aaactatgaa ctgatcaata atcattccta
aaagaccaca cttttgtttt 3780gtttctaaag taatttttac tgttataaca
ggtggtcgta gcaatcatga agtcagaatt 3840gacgttgcct ttcaagaaac
ctacagaaga ggaaatctcg gagtcgctaa aaccaggaga 3900ggggtcgtgt
gcagagcata aggaagtgtt gagcttacaa aatgatgctc cgttcccgtg
3960tgtgaaaaat ctagttgaag gttccgtgcc ggcgtatgga atgtgtccta
agggtggtgg 4020tttcgacaaa ttggatgtgg acattgctga tttccatctc
aagagtgtag atgcagttaa 4080aaagggaact atgatgtctg cggtgtacac
agggtctatc aaagttcaac aaatgaagaa 4140ctacatagat tacttaagtg
cgtcgctggc agctacagtc tcaaacctct gcaaggtaag 4200aggtcaaaag
gtttccgcaa tgatccctct ttttttgttt ctctagtttc aagaatttgg
4260gtatatgact aacttctgag tgttccttga tgcatatttg tgatgagaca
aatgtttgtt 4320ctatgtttta ggtgcttaga gatgttcacg gcgttgaccc
agagtcacag gagaaatctg 4380gagtgtggga tgttaggaga ggacgttggt
tacttaaacc taatgcgaaa agtcacgcgt 4440ggggtgtggc agaagacgcc
aaccacaagt tggttattgt gttactcaac tgggatgacg 4500gaaagccggt
ttgtgatgag acatggttca gggtggcggt gtcaagcgat tccttgatat
4560attcggatat gggaaaactt aagacgctca cgtcttgcag tccaaatggt
gagccaccgg 4620agcctaacgc caaagtaatt ttggtcgatg gtgttcccgg
ttgtggaaaa acgaaggaga 4680ttatcgaaaa ggtaagttct gcatttggtt
atgctccttg cattttaggt gttcgtcgct 4740cttccatttc catgaatagc
taagattttt tttctctgca ttcattcttc ttgcctcagt 4800tctaactgtt
tgtggtattt ttgttttaat tattgctaca ggtaaacttc tctgaagact
4860tgattttagt ccctgggaag gaagcttcta agatgatcat ccggagggcc
aaccaagctg 4920gtgtgataag agcggataag gacaatgtta gaacggtgga
ttccttcttg atgcatcctt 4980ctagaagggt gtttaagagg ttgtttatcg
atgaaggact aatgctgcat acaggttgtg 5040taaatttcct actgctgcta
tctcaatgtg acgtcgcata tgtgtatggg gacacaaagc 5100aaattccgtt
catttgcaga gtcgcgaact ttccgtatcc agcgcatttt gcaaaactcg
5160tcgctgatga gaaggaagtc agaagagtta cgctcaggta aagcaactgt
gttttaatca 5220atttcttgtc aggatatatg gattataact taatttttga
gaaatctgta gtatttggcg 5280tgaaatgagt ttgctttttg gtttctcccg
tgttataggt gcccggctga tgttacgtat 5340ttccttaaca agaagtatga
cggggcggtg atgtgtacca gcgcggtaga gagatccgtg 5400aaggcagaag
tggtgagagg aaagggtgca ttgaacccaa taaccttacc gttggagggt
5460aaaattttga ccttcacaca agctgacaag ttcgagttac tggagaaggg
ttacaaggta 5520aagtttccaa ctttccttta ccatatcaaa ctaaagttcg
aaacttttta tttgatcaac 5580ttcaaggcca cccgatcttt ctattcctga
ttaatttgtg atgaatccat attgactttt 5640gatggttacg caggatgtga
acactgtgca cgaggtgcaa ggggagacgt acgagaagac 5700tgctattgtg
cgcttgacat caactccgtt agagatcata tcgagtgcgt cacctcatgt
5760tttggtggcg ctgacaagac acacaacgtg ttgtaaatat tacaccgttg
tgttggaccc 5820gatggtgaat gtgatttcag aaatggagaa gttgtccaat
ttccttcttg acatgtatag 5880agttgaagca ggtctgtctt tcctatttca
tatgtttaat cctaggaatt tgatcaattg 5940attgtatgta tgtcgatccc
aagactttct tgttcactta tatcttaact ctctctttgc 6000tgtttcttgc
aggtgtccaa tagcaattac aaatcgatgc agtattcagg ggacagaact
6060tgtttgttca gacgcccaag tcaggagatt ggcgagatat gcaattttac
tatgacgctc 6120ttcttcccgg aaacagtact attctcaatg aatttgatgc
tgttacgatg aatttgaggg 6180atatttcctt aaacgtcaaa gattgcagaa
tcgacttctc caaatccgtg caacttccta 6240aagaacaacc tattttcctc
aagcctaaaa taagaactgc ggcagaaatg ccgagaactg 6300caggtaaaat
attggatgcc agacgatatt ctttcttttg atttgtaact ttttcctgtc
6360aaggtcgata aattttattt tttttggtaa aaggtcgata attttttttt
ggagccatta 6420tgtaattttc ctaattaact gaaccaaaat tatacaaacc
aggtttgctg gaaaatttgg 6480ttgcaatgat caaaagaaac atgaatgcgc
cggatttgac agggacaatt gacattgagg 6540atactgcatc tctggtggtt
gaaaagtttt gggattcgta tgttgacaag gaatttagtg 6600gaacgaacga
aatgaccatg acaagggaga gcttctccag gtaaggactt ctcatgaata
6660ttagtggcag attagtgttg ttaaagtctt tggttagata atcgatgcct
cctaattgtc 6720catgttttac tggttttcta caattaaagg tggctttcga
aacaagagtc atctacagtt 6780ggtcagttag cggactttaa ctttgtggat
ttgccggcag tagatgagta caagcatatg 6840atcaagagtc aaccaaagca
aaagttagac ttgagtattc aagacgaata tcctgcattg 6900cagacgatag
tctaccattc gaaaaagatc aatgcgattt tcggtccaat gttttcagaa
6960cttacgagga tgttactcga aaggattgac tcttcgaagt ttctgttcta
caccagaaag 7020acacctgcac aaatagagga cttcttttct gacctagact
caacccaggc gatggaaatt 7080ctggaactcg acatttcgaa gtacgataag
tcacaaaacg agttccattg tgctgtagag 7140tacaagatct gggaaaagtt
aggaattgat gagtggctag ctgaggtctg gaaacaaggt 7200gagttcctaa
gttccatttt tttgtaatcc ttcaatgtta ttttaacttt tcagatcaac
7260atcaaaatta ggttcaattt tcatcaacca aataatattt ttcatgtata
tataggtcac 7320agaaaaacga ccttgaaaga ttatacggcc ggaatcaaaa
catgtctttg gtatcaaagg 7380aaaagtggtg atgtgacaac ctttattggt
aataccatca tcattgccgc atgtttgagc 7440tcaatgatcc ccatggacaa
agtgataaag gcagcttttt gtggagacga tagcctgatt 7500tacattccta
aaggtttaga cttgcctgat attcaggcgg gcgcgaacct catgtggaac
7560ttcgaggcca aactcttcag gaagaagtat ggttacttct gtggtcgtta
tgttattcac 7620catgatagag gagccattgt gtattacgat ccgcttaaac
taatatctaa gttaggttgt 7680aaacatatta gagatgttgt tcacttagaa
gagttacgcg agtctttgtg tgatgtagct 7740agtaacttaa ataattgtgc
gtatttttca cagttagatg aggccgttgc cgaggttcat 7800aagaccgcgg
taggcggttc gtttgctttt tgtagtataa ttaagtattt gtcagataag
7860agattgttta gagatttgtt ctttgtttga taatgtcgat agtctcgtac
gaacctaagg 7920tgagtgattt cctcaatctt tcgaagaagg aagagatctt
gccgaaggct ctaacgaggt 7980taaaaaccgt gtctattagt actaaagata
ttatatctgt caaggagtcg gagactttgt 8040gtgatataga tttgttaatc
aatgtgccat tagataagta tagatatgtg ggtatcctag 8100ctaggagccg
tttttaccgg agagtggcta gtgccagact tcgttaaagg tggagtgacg
8160ataagtgtga tagataagcg tctggtgaac tcaaaggagt gcgtgattgg
tacgtacaga 8220gccgcagcca agagtaagag gttccagttc aaattggttc
caaattactt tgtgtccacc 8280gtggacgcaa agaggaagcc gtggcaggta
aggattttta tgatatagta tgcttatgta 8340ttttgtactg aaagcatatc
ctgcttcatt gggatattac tgaaagcatt taactacatg 8400taaactcact
tgatgatcaa taaacttgat tttgcaggtt catgttcgta tacaagactt
8460gaagattgag gcgggttggc agccgttagc tctggaagta gtttcagttg
ctatggtcac 8520caataacgtt gtcatgaagg gtttgaggga aaaggtcgtc
gcaataaatg atccggacgt 8580cgaaggtttc gaaggtaagc catcttcctg
cttattttta taatgaacat agaaatagga 8640agttgtgcag agaaactaat
taacctgact caaaatctac cctcataatt gttgtttgat 8700attggtcttg
tattttgcag gtgtggttga cgaattcgtc gattcggttg cagcatttaa
8760agcggttgac aactttaaaa gaaggaaaaa gaaggttgaa gaaaagggtg
tagtaagtaa 8820gtataagtac agaccggaga agtacgccgg tcctgattcg
tttaatttga aagaagaaaa 8880cgtcttacaa cattacaaac ccgaataatc
gataactcga gtatttttac aacaattacc 8940aacaacaaca aacaacaaac
aacattacaa ttacatttac aattatcatg gtgagcaagg 9000gcgaggagct
gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
9060gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
aagctgaccc 9120tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
gcccaccctc gtgaccacct 9180tcagctacgg cgtgcagtgc ttcagccgct
accccgacca catgaagcag cacgacttct 9240tcaagtccgc catgcccgaa
ggctacgtcc aggagcgcac catcttcttc aaggacgacg 9300gcaactacaa
gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
9360agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
ctggagtaca 9420actacaacag ccacaacgtc tatatcatgg ccgacaagca
gaagaacggc atcaaggtga 9480acttcaagat ccgccacaac atcgaggacg
gcagcgtgca gctcgccgac cactaccagc 9540agaacacccc catcggcgac
ggccccgtgc tgctgcccga caaccactac ctgagcaccc 9600agtccgccct
gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
9660tgaccgccgc cgggatcact cacggcatgg acgagctgta caagtaaagc
ggcccctaga 9720gcgtggtgcg cacgatagcg catagtgttt ttctctccac
ttgaatcgaa gagatagact 9780tacggtgtaa atccgtaggg gtggcgtaaa
ccaaattacg caatgttttg ggttccattt 9840aaatcgaaac cccttatttc
ctggatcacc tgttaacgca cgtttgacgt gtattacagt 9900gggaataagt
aaaagtgaga ggttcgaatc ctccctaacc ccgggtaggg gcccagcggc
9960cgctctagct agagtcaagc agatcgttca aacatttggc aataaagttt
cttaagattg 10020aatcctgttg ccggtcttgc gatgattatc atataatttc
tgttgaatta cgttaagcat 10080gtaataatta acatgtaatg catgacgtta
tttatgagat gggtttttat gattagagtc 10140ccgcaattat acatttaata
cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa 10200ttatcgcgcg
cggtgtcatc tatgttacta gatcgacctg catccacccc agtacattaa
10260aaacgtccgc aatgtgttat taagttgtct aagcgtcaat ttgtttacac
cacaatatat 10320cctgccacca gccagccaac agctccccga ccggcagctc
ggcacaaaat caccactcga 10380tacaggcagc ccatcag
1039747351DNAArtificial SequenceT-DNA region of pNMD620 4cctgtggttg
gcacatacaa atggacgaac ggataaacct tttcacgccc ttttaaatat 60ccgattattc
taataaacgc tcttttctct taggtttacc cgccaatata tcctgtcaaa
120cactgatagt ttaaactgaa ggcgggaaac gacaatctga tctaagctag
gcatgcctgc 180aggtcaacat ggtggagcac gacacgcttg tctactccaa
aaatatcaaa gatacagtct 240cagaagacca aagggcaatt gagacttttc
aacaaagggt aatatccgga aacctcctcg 300gattccattg cccagctatc
tgtcacttta ttgtgaagat agtggaaaag gaaggtggct 360cctacaaatg
ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca
420gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa
gacgttccaa 480ccacgtcttc aaagcaagtg gattgatgtg atatctccac
tgacgtaagg gatgacgcac 540aatcccacta tccttcgcaa gacccttcct
ctatataagg aagttcattt catttggaga 600ggagaaaact aaaccataca
ccaccaacac aaccaaaccc accacgccca attgttacac 660acccgcttga
aaaagaaagt ttaacaaatg gccaaggtgc gcgaggttta ccaatctttt
720acagactcca ccacaaaaac tctcatccaa gatgaggctt atagaaacat
tcgccccatc 780atggaaaaac acaaactagc taacccttac gctcaaacgg
ttgaagcggc taatgatcta 840gaggggttcg gcatagccac caatccctat
agcattgaat tgcatacaca tgcagccgct 900aagaccatag agaataaact
tctagaggtg cttggttcca tcctaccaca agaacctgtt 960acatttatgt
ttcttaaacc cagaaagcta aactacatga gaagaaaccc gcggatcaag
1020gacattttcc aaaatgttgc cattgaacca agagacgtag ccaggtaccc
caaggaaaca 1080ataattgaca aactcacaga gatcacaacg gaaacagcat
acattagtga cactctgcac 1140ttcttggatc cgagctacat agtggagaca
ttccaaaact gcccaaaatt gcaaacattg 1200tatgcgacct tagttctccc
cgttgaggca gcctttaaaa tggaaagcac tcacccgaac 1260atatacagcc
tcaaatactt cggagatggt ttccagtata taccaggcaa ccatggtggc
1320ggggcatacc atcatgaatt cgctcatcta caatggctca aagtgggaaa
gatcaagtgg 1380agggacccca aggatagctt tctcggacat ctcaattaca
cgactgagca ggttgagatg 1440cacacagtga cagtacagtt gcaggaatcg
ttcgcggcaa accacttgta ctgcatcagg 1500agaggagact tgctcacacc
ggaggtgcgc actttcggcc aacctgacag gtacgtgatt 1560ccaccacaga
tcttcctccc aaaagttcac aactgcaaga agccgattct caagaaaact
1620atgatgcagc tcttcttgta tgttaggaca gtcaaggtcg caaaaaattg
tgacattttt 1680gccaaagtca gacaattaat taaatcatct gacttggaca
aatactctgc tgtggaactg 1740gtttacttag taagctacat ggagttcctt
gccgatttac aagctaccac ctgcttctca 1800gacacacttt ctggtggctt
gctaacaaag acccttgcac cggtgagggc ttggatacaa 1860gagaaaaaga
tgcagctgtt tggtcttgag gactacgcga agttagtcaa agcagttgat
1920ttccacccgg tggatttttc tttcaaagtg gaaacttggg acttcagatt
ccaccccttg 1980caagcgtgga aagccttccg accaagggaa gtgtcggatg
tagaggaaat ggaaagtttg 2040ttctcagatg gggacctgct tgattgcttc
acaagaatgc cagcttatgc ggtaaacgca 2100gaggaagatt tagctgcaat
caggaaaacg cccgagatgg atgtcggtca agaagttaaa 2160gagcctgcag
gagacagaaa tcaatactca aaccctgcag aaactttcct caacaagctc
2220cacaggaaac acagtaggga ggtgaaacac caggccgcaa agaaagctaa
acgcctagct 2280gaaatccagg agtcaatgag agctgaaggt gatgccgaac
caaatgaaat aagcgggacg 2340atgggggcaa tacccagcaa cgccgaactt
cctggcacga atgatgccag acaagaactc 2400acactcccaa ccactaaacc
tgtccctgca aggtgggaag atgcttcatt cacagattct 2460agtgtggaag
aggagcaggt taaactcctt ggaaaagaaa ccgttgaaac agcgacgcaa
2520caagtcatcg aaggacttcc ttggaaacac tggattcctc aattaaatgc
tgttggattc 2580aaggcgctgg aaattcagag ggataggagt ggaacaatga
tcatgcccat cacagaaatg 2640gtctccgggc tggaaaaaga ggacttccct
gaaggaactc caaaagagtt ggcacgagaa 2700ttgttcgcta tgaacagaag
ccctgccacc atccctttgg acctgcttag agccagagac 2760tacggcagtg
atgtaaagaa caagagaatt ggtgccatca caaagacaca ggcaacgagt
2820tggggcgaat acttgacagg aaagatagaa agcttaactg agaggaaagt
tgcgacttgt 2880gtcattcatg gagctggagg ttctggaaaa agtcatgcca
tccagaaggc attgagagaa 2940attggcaagg gctcggacat cactgtagtc
ctgccgacca atgaactgcg gctagattgg 3000agtaagaaag tgcctaacac
tgagccctat atgttcaaga cctctgaaaa ggcgttaatt 3060gggggaacag
gcagcatagt catctttgac gattactcaa aacttcctcc cggttacata
3120gaagccttag tctgtttcta ctctaaaatc aagctaatca ttctaacagg
agatagcaga 3180caaagcgtct accatgaaac tgctgaggac gcctccatca
ggcatttggg accagcaaca 3240gagtacttct caaaatactg ccgatactat
ctcaatgcca cacaccgcaa caagaaagat 3300cttgcgaaca tgcttggtgt
ctacagtgag agaacgggag tcaccgaaat cagcatgagc 3360gccgagttct
tagaaggaat cccaactttg gtaccctcgg atgagaagag aaagctgtac
3420atgggcaccg ggaggaatga cacgttcaca tacgctggat gccaggggct
aactaagccg 3480aaggtacaaa tagtgttgga ccacaacacc caagtgtgta
gcgcgaatgt gatgtacacg 3540gcactttcta gagccaccga taggattcac
ttcgtgaaca caagtgcaaa ttcctctgcc 3600ttctgggaaa agttggacag
caccccttac ctcaagactt tcctatcagt ggtgagagaa 3660caagcactca
gggagtacga gccggcagag gcagagccaa ttcaagagcc tgagccccag
3720acacacatgt gtgtcgagaa tgaggagtcc gtgctagaag agtacaaaga
ggaactcttg 3780gaaaagtttg acagagagat ccactctgaa tcccatggtc
attcaaactg tgtccaaact 3840gaagacacaa ccattcagtt gttttcgcat
caacaagcaa aagatgagac cctcctctgg 3900gcgactatag atgcgcggct
caagaccagc aatcaagaaa caaacttccg agaattcctg 3960agcaagaagg
acattgggga cgttctgttt ttaaactacc aaaaagctat gggtttaccc
4020aaagagcgta ttcctttttc ccaagaggtc tgggaagctt gtgcccacga
agtacaaagc 4080aagtacctca gcaagtcaaa gtgcaacttg atcaatggga
ctgtgagaca gagcccagac 4140ttcgatgaaa ataagattat ggtattcctc
aagtcgcagt gggtcacaaa ggtggaaaaa 4200ctaggtctac ccaagattaa
gccaggtcaa accatagcag ccttttacca gcagactgtg 4260atgctttttg
gaactatggc taggtacatg cgatggttca gacaggcttt ccagccaaaa
4320gaagtcttca taaactgtga gaccacgcca gatgacatgt ctgcatgggc
cttgaacaac 4380tggaatttca gcagacctag cttggctaat gactacacag
ctttcgacca gtctcaggat 4440ggagccatgt tgcaatttga ggtgctcaaa
gccaaacacc actgcatacc agaggaaatc 4500attcaggcat acatagatat
taagactaat gcacagattt tcctaggcac gttatcaatt 4560atgcgcctga
ctggtgaagg tcccactttt gatgcaaaca ctgagtgcaa catagcttac
4620acccatacaa agtttgacat cccagccgga actgctcaag tttatgcagg
agacgactcc 4680gcactggact gtgttccaga agtgaagcat agtttccaca
ggcttgagga caaattactc 4740ctaaagtcaa agcctgtaat cacgcagcaa
aagaagggca gttggcctga gttttgtggt 4800tggctgatca caccaaaagg
ggtgatgaaa gacccaatta agctccatgt tagcttaaaa 4860ttggctgaag
ctaagggtga actcaagaaa tgtcaagatt cctatgaaat tgatctgagt
4920tatgcctatg accacaagga ctctctgcat gacttgttcg atgagaaaca
gtgtcaggca 4980cacacactca cttgcagaac actaatcaag tcagggagag
gcactgtctc actttcccgc 5040ctcagaaact ttctttaacc gttaagttac
cttagagatt tgaataagat ggatattctc 5100atcagtagtt tgaaaagttt
aggttattct aggacttcca aatctttaga ttcaggacct 5160ttggtagtac
atgcagtagc cggagccggt aagtccacag ccctaaggaa gttgatcctc
5220agacacccaa cattcaccgt gcatacactc ggtgtccctg acaaggtgag
tatcagaact 5280agaggcatac agaagccagg acctattcct gagggcaact
tcgcaatcct cgatgagtat 5340actttggaca acaccacaag gaactcatac
caggcacttt ttgctgaccc ttatcaggca 5400ccggagttta gcctagagcc
ccacttctac ttggaaacat catttcgagt tccgaggaaa 5460gtggcagatt
tgatagctgg ctgtggcttc gatttcgaga cgaactcacc ggaagaaggg
5520cacttagaga tcactggcat attcaaaggg cccctactcg gaaaggtgat
agccattgat 5580gaggagtctg agacaacact gtccaggcat ggtgttgagt
ttgttaagcc ctgccaagtg 5640acgggacttg agttcaaagt agtcactatt
gtgtctgccg caccaataga ggaaattggc 5700cagtccacag ctttctacaa
cgctatcacc aggtcaaagg gattgacata tgtccgcgca 5760gggccatagg
ctgaccgctc cggtcaattc tgaaaaagtg tacatagtat taggtctatc
5820atttgcttta gtttcaatta cctttctgct ttctagaaat agcttacccc
acgtcggtga 5880caacattcac agcttgccac acggaggagc ttacagagac
ggcaccaaag caatcttgta 5940caactcccca aatctagggt cacgagtgag
tctacacaac ggaaagaacg cagcatttgc 6000tgccgttttg ctactgactt
tgctgatcta tggaagtaaa tacatatctc aacgcaatca 6060tacttgtgct
tgtggtaaca atcatagcag tcattagcac ttccttagtg aggactgaac
6120cttgtgtcat caagattact ggggaatcaa tcacagtgtt ggcttgcaaa
ctagatgcag 6180aaaccataag ggccattgcc gatctcaagc cactctccgt
tgaacggtta agtttccatt 6240gatactcgaa agaggtcagc accagctagc
aacaaacaag aaaggtatgg tgagcaaggg 6300cgaggagctg ttcaccgggg
tggtgcccat cctggtcgag ctggacggcg acgtaaacgg 6360ccacaagttc
agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
6420gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
tgaccacctt 6480cagctacggc gtgcagtgct tcagccgcta ccccgaccac
atgaagcagc acgacttctt 6540caagtccgcc atgcccgaag gctacgtcca
ggagcgcacc atcttcttca aggacgacgg 6600caactacaag acccgcgccg
aggtgaagtt cgagggcgac accctggtga accgcatcga 6660gctgaagggc
atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
6720ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
tcaaggtgaa 6780cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
ctcgccgacc actaccagca 6840gaacaccccc atcggcgacg gccccgtgct
gctgcccgac aaccactacc tgagcaccca 6900gtccgccctg agcaaagacc
ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt 6960gaccgccgcc
gggatcactc acggcatgga cgagctgtac aagtaagctt ggtcgtatca
7020ctggaacaac aaccgctgag gctgttgtca ctctaccacc accataacta
cgtctacata 7080accgacgcct accccagttt catagtattt tctggtttga
ttgtatgaat aatataaata 7140aaaaaaaaaa aaaaaaaaaa aaactagtga
gctcttctgt cagcgggccc actgcatcca 7200ccccagtaca ttaaaaacgt
ccgcaatgtg ttattaagtt gtctaagcgt caatttgttt 7260acaccacaat
atatcctgcc accagccagc caacagctcc ccgaccggca gctcggcaca
7320aaatcaccac tcgatacagg cagcccatca g 735151018DNAArtificial
SequencePlasmid backbone insertion containing virG gene of pNMD062
5ctgtcgatca gatctggctc gcggcggacg
cacgacgccg gggcgagacc ataggcgatc 60tcctaaatca atagtagctg taacctcgaa
gcgtttcact tgtaacaacg attgagaatt 120tttgtcataa aattgaaata
cttggttcgc atttttgtca tccgcggtca gccgcaattc 180tgacgaactg
cccatttagc tggagatgat tgtacatcct tcacgtgaaa atttctcaag
240cgctgtgaac aagggttcag attttagatt gaaaggtgag ccgttgaaac
acgttcttct 300tgtcgatgac gacgtcgcta tgcggcatct tattattgaa
taccttacga tccacgcctt 360caaagtgacc gcggtagccg acagcaccca
gttcacaaga gtactctctt ccgcgacggt 420cgatgtcgtg gttgttgatc
taaatttagg tcgtgaagat gggctcgaga tcgttcgtaa 480tctggcggca
aagtctgata ttccaatcat aattatcagt ggcgaccgcc ttgaggagac
540ggataaagtt gttgcactcg agctaggagc aagtgatttt atcgctaagc
cgttcagtat 600cagagagttt ctagcacgca ttcgggttgc cttgcgcgtg
cgccccaacg ttgtccgctc 660caaagaccga cggtcttttt gttttactga
ctggacactt aatctcaggc aacgtcgctt 720gatgtccgaa gctggcggtg
aggtgaaact tacggcaggt gagttcaatc ttctcctcgc 780gtttttagag
aaaccccgcg acgttctatc gcgcgagcaa cttctcattg ccagtcgagt
840acgcgacgag gaggtttatg acaggagtat agatgttctc attttgaggc
tgcgccgcaa 900acttgaggcg gatccgtcaa gccctcaact gataaaaaca
gcaagaggtg ccggttattt 960ctttgacgcg gacgtgcagg tttcgcacgg
ggggacgatg gcagcctaag atcgacag 101861018DNAArtificial
SequencePlasmid backbone insertion containing virG gene of pNMD063
6ctgtcgatca gatctggctc gcggcggacg cacgacgccg gggcgagacc ataggcgatc
60tcctaaatca atagtagctg taacctcgaa gcgtttcact tgtaacaacg attgagaatt
120tttgtcataa aattgaaata cttggttcgc atttttgtca tccgcggtca
gccgcaattc 180tgacgaactg cccatttagc tggagatgat tgtacatcct
tcacgtgaaa atttctcaag 240tgctgtgaac aagggttcag attttagatt
gaaaggtgag ccgttgaaac acgttcttct 300tgtcgatgac gacgtcgcta
tgcggcatct tattattgaa taccttacga tccacgcctt 360caaagtgacc
gcggtagccg acagcaccca gttcacaaga gtactctctt ccgcgacggt
420cgatgtcgtg gttgttgatc tagatttagg tcgtgaagat gggctcgaga
tcgttcgtaa 480tctggcggca aagtctgata ttccaatcat aattatcagt
ggcgaccgcc ttgaggagac 540ggataaagtt gttgcactcg agctaggagc
aagtgatttt atcgctaagc cgttcagtat 600cagagagttt ctagcacgca
ttcgggttgc cttgcgcgtg cgccccaacg ttgtccgctc 660caaagaccga
cggtcttttt gttttactga ctggacactt aatctcaggc aacgtcgctt
720gatgtccgaa gctggcggtg aggtgaaact tacggcaggt gagttcaatc
ttctcctcgc 780gtttttagag aaaccccgcg acgttctatc gcgcgagcaa
cttctcattg ccagtcgagt 840acgcgacgag gaggtttatg acaggagtat
agatgttctc attttgaggc tgcgccgcaa 900acttgaggcg gatccgtcaa
gccctcaact gataaaaaca gcaagaggtg ccggttattt 960ctttgacgcg
gacgtgcagg tttcgcacgg ggggacgatg gcagcctaag atcgacag
1018710627DNAArtificial Sequencefull-length nucleotide sequence of
pNMD1971 7ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct
gttgaattac 60gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg
ggtttttatg 120attagagtcc cgcaattata catttaatac gcgatagaaa
acaaaatata gcgcgcaaac 180taggataaat tatcgcgcgc ggtgtcatct
atgttactag atcgacctgc aggcatgcca 240attccaatcc cacaaaaatc
tgagcttaac agcacagttg ctcctctcag agcagaatcg 300ggtattcaac
accctcatat caactactac gttgtgtata acggtccaca tgccggtata
360tacgatgact ggggttgtac aaaggcggca acaaacggcg ttcccggagt
tgcacacaag 420aaatttgcca ctattacaga ggcaagagca gcagctgacg
cgtacacaac aagtcagcaa 480acagacaggt tgaacttcat ccccaaagga
gaagctcaac tcaagcccaa gagctttgct 540aaggccctaa caagcccacc
aaagcaaaaa gcccactggc tcacgctagg aaccaaaagg 600cccagcagtg
atccagcccc aaaagagatc tcctttgccc cggagattac aatggacgat
660ttcctctatc tttacgatct aggaaggaag ttcgaaggtg aaggtgacga
cactatgttc 720accactgata atgagaaggt tagcctcttc aatttcagaa
agaatgctga cccacagatg 780gttagagagg cctacgcagc aggtctcatc
aagacgatct acccgagtaa caatctccag 840gagatcaaat accttcccaa
gaaggttaaa gatgcagtca aaagattcag gactaattgc 900atcaagaaca
cagagaaaga catatttctc aagatcagaa gtactattcc agtatggacg
960attcaaggct tgcttcataa accaaggcaa gtaatagaga ttggagtctc
taaaaaggta 1020gttcctactg aatctaaggc catgcatgga gtctaagatt
caaatcgagg atctaacaga 1080actcgccgtg aagactggcg aacagttcat
acagagtctt ttacgactca atgacaagaa 1140gaaaatcttc gtcaacatgg
tggagcacga cactctggtc tactccaaaa atgtcaaaga 1200tacagtctca
gaagaccaaa gggctattga gacttttcaa caaaggataa tttcgggaaa
1260cctcctcgga ttccattgcc cagctatctg tcacttcatc gaaaggacag
tagaaaagga 1320aggtggctcc tacaaatgcc atcattgcga taaaggaaag
gctatcattc aagatctctc 1380tgccgacagt ggtcccaaag atggaccccc
acccacgagg agcatcgtgg aaaaagaaga 1440cgttccaacc acgtcttcaa
agcaagtgga ttgatgtgac atctccactg acgtaaggga 1500tgacgcacaa
tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca
1560tttggagagg acacgctcga gtataagagc tctattttta caacaattac
caacaacaac 1620aaacaacaaa caacattaca attacattta caattaccat
ggaacgagct atacaaggaa 1680acgatgctag ggaacaagct tatggtgaac
gttggaatgg aggatcagga agttccactt 1740ctcccttcaa acttcctgac
gaaagtccga gttggactga gtggcggcta cataacgatg 1800agacgatttc
gaatcaagat aatccccttg gtttcaagga aagctggggt ttcgggaaag
1860ttgtatttaa gagatatctc agatacgacg ggacggaaac ttcactgcac
agagtccttg 1920gatcttggac gggagattcg gttaactatg cagcatctcg
atttctcggt ttcgaccaga 1980tcggatgtac ctatagtatt cggtttcgag
gagttagtgt caccatttct ggagggtcgc 2040gaactcttca gcatctcagt
gaaatggcaa ttcggtctaa gcaagaactg ctacagctta 2100ccccagtcaa
agtggaaagt gatgtatcaa gaggatgccc tgaaggtgtt gaaaccttcg
2160aagaagaaag cgagtaagga tcctctagag tcctgcttta atgagatatg
cgagacgcct 2220atgatcgcat gatatttgct ttcaattctg ttgtgcacgt
tgtaaaaaac ctgagcatgt 2280gtagctcaga tccttaccgc cggtttcggt
tcattctaat gaatatatca cccgttacta 2340tcgtattttt atgaataata
ttctccgttc aatttactga ttgtacccta ctacttatat 2400gtacaatatt
aaaatgaaaa caatatattg tgctgaatag gtttatagcg acatctatga
2460tagagcgcca caataacaaa caattgcgtt ttattattac aaatccaatt
ttaaaaaaag 2520cggcagaacc ggtcaaacct aaaagactga ttacataaat
cttattcaaa tttcaaaagt 2580gccccagggg ctagtatcta cgacacaccg
agcggcgaac taataacgct cactgaaggg 2640aactccggtt ccccgccggc
gcgcatgggt gagattcctt gaagttgagt attggccgtc 2700cgctctaccg
aaagttacgg gcaccattca acccggtcca gcacggcggc cgggtaaccg
2760acttgctgcc ccgagaatta tgcagcattt ttttggtgta tgtgggcccc
aaatgaagtg 2820caggtcaaac cttgacagtg acgacaaatc gttgggcggg
tccagggcga attttgcgac 2880aacatgtcga ggctcagcag gacctgcata
agctcttctg tcagcgggcc cactgcatcc 2940accccagtac attaaaaacg
tccgcaatgt gttattaagt tgtctaagcg tcaatttgtt 3000tacaccacaa
tatatcctgc caccagccag ccaacagctc cccgaccggc agctcggcac
3060aaaatcacca ctcgatacag gcagcccatc agtcagatca ggatctcctt
tgcgacgctc 3120accgggctgg ttgccctcgc cgctgggctg gcggccgtct
atggccctgc aaacgcgcca 3180gaaacgccgt cgaagccgtg tgcgagacac
cgcggccgcc ggcgttgtgg atacctcgcg 3240gaaaacttgg ccctcactga
cagatgaggg gcggacgttg acacttgagg ggccgactca 3300cccggcgcgg
cgttgacaga tgaggggcag gctcgatttc ggccggcgac gtggagctgg
3360ccagcctcgc aaatcggcga aaacgcctga ttttacgcga gtttcccaca
gatgatgtgg 3420acaagcctgg ggataagtgc cctgcggtat tgacacttga
ggggcgcgac tactgacaga 3480tgaggggcgc gatccttgac acttgagggg
cagagtgctg acagatgagg ggcgcaccta 3540ttgacatttg aggggctgtc
cacaggcaga aaatccagca tttgcaaggg tttccgcccg 3600tttttcggcc
accgctaacc tgtcttttaa cctgctttta aaccaatatt tataaacctt
3660gtttttaacc agggctgcgc cctgtgcgcg tgaccgcgca cgccgaaggg
gggtgccccc 3720ccttctcgaa ccctcccggc ccgctaacgc gggcctccca
tccccccagg ggctgcgccc 3780ctcggccgcg aacggcctca ccccaaaaat
ggcagcgctg gccaattcgt gcgcggaacc 3840cctatttgtt tatttttcta
aatacattca aatatgtatc cgctcatgag acaataaccc 3900tgataaatgc
ttcaataata ttgaaaaagg aagagtatgg ctaaaatgag aatatcaccg
3960gaattgaaaa aactgatcga aaaataccgc tgcgtaaaag atacggaagg
aatgtctcct 4020gctaaggtat ataagctggt gggagaaaat gaaaacctat
atttaaaaat gacggacagc 4080cggtataaag ggaccaccta tgatgtggaa
cgggaaaagg acatgatgct atggctggaa 4140ggaaagctgc ctgttccaaa
ggtcctgcac tttgaacggc atgatggctg gagcaatctg 4200ctcatgagtg
aggccgatgg cgtcctttgc tcggaagagt atgaagatga acaaagccct
4260gaaaagatta tcgagctgta tgcggagtgc atcaggctct ttcactccat
cgacatatcg 4320gattgtccct atacgaatag cttagacagc cgcttagccg
aattggatta cttactgaat 4380aacgatctgg ccgatgtgga ttgcgaaaac
tgggaagaag acactccatt taaagatccg 4440cgcgagctgt atgatttttt
aaagacggaa aagcccgaag aggaacttgt cttttcccac 4500ggcgacctgg
gagacagcaa catctttgtg aaagatggca aagtaagtgg ctttattgat
4560cttgggagaa gcggcagggc ggacaagtgg tatgacattg ccttctgcgt
ccggtcgatc 4620agggaggata tcggggaaga acagtatgtc gagctatttt
ttgacttact ggggatcaag 4680cctgattggg agaaaataaa atattatatt
ttactggatg aattgtttta gctgtcagac 4740caagtttact catatatact
ttagattgat ttaaaacttc atttttaatt taaaaggatc 4800taggtgaaga
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
4860cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
tttttttctg 4920cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
cagcggtggt ttgtttgccg 4980gatcaagagc taccaactct ttttccgaag
gtaactggct tcagcagagc gcagatacca 5040aatactgtcc ttctagtgta
gccgtagtta ggccaccact tcaagaactc tgtagcaccg 5100cctacatacc
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
5160tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
gtcgggctga 5220acggggggtt cgtgcacaca gcccagcttg gagcgaacga
cctacaccga actgagatac 5280ctacagcgtg agctatgaga aagcgccacg
cttcccgaag ggagaaaggc ggacaggtat 5340ccggtaagcg gcagggtcgg
aacaggagag cgcacgaggg agcttccagg gggaaacgcc 5400tggtatcttt
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
5460tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
tttacggttc 5520ctggcagatc ctagatgtgg cgcaacgatg ccggcgacaa
gcaggagcgc accgacttct 5580tccgcatcaa gtgttttggc tctcaggccg
aggcccacgg caagtatttg ggcaaggggt 5640cgctggtatt cgtgcagggc
aagattcgga ataccaagta cgagaaggac ggccagacgg 5700tctacgggac
cgacttcatt gccgataagg tggattatct ggacaccaag gcaccaggcg
5760ggtcaaatca ggaataaggg cacattgccc cggcgtgagt cggggcaatc
ccgcaaggag 5820ggtgaatgaa tcggacgttt gaccggaagg catacaggca
agaactgatc gacgcggggt 5880tttccgccga ggatgccgaa accatcgcaa
gccgcaccgt catgcgtgcg ccccgcgaaa 5940ccttccagtc cgtcggctcg
atggtccagc aagctacggc caagatcgag cgcgacagcg 6000tgcaactggc
tccccctgcc ctgcccgcgc catcggccgc cgtggagcgt tcgcgtcgtc
6060tcgaacagga ggcggcaggt ttggcgaagt cgatgaccat cgacacgcga
ggaactatga 6120cgaccaagaa gcgaaaaacc gccggcgagg acctggcaaa
acaggtcagc gaggccaagc 6180aggccgcgtt gctgaaacac acgaagcagc
agatcaagga aatgcagctt tccttgttcg 6240atattgcgcc gtggccggac
acgatgcgag cgatgccaaa cgacacggcc cgctctgccc 6300tgttcaccac
gcgcaacaag aaaatcccgc gcgaggcgct gcaaaacaag gtcattttcc
6360acgtcaacaa ggacgtgaag atcacctaca ccggcgtcga gctgcgggcc
gacgatgacg 6420aactggtgtg gcagcaggtg ttggagtacg cgaagcgcac
ccctatcggc gagccgatca 6480ccttcacgtt ctacgagctt tgccaggacc
tgggctggtc gatcaatggc cggtattaca 6540cgaaggccga ggaatgcctg
tcgcgcctac aggcgacggc gatgggcttc acgtccgacc 6600gcgttgggca
cctggaatcg gtgtcgctgc tgcaccgctt ccgcgtcctg gaccgtggca
6660agaaaacgtc ccgttgccag gtcctgatcg acgaggaaat cgtcgtgctg
tttgctggcg 6720accactacac gaaattcata tgggagaagt accgcaagct
gtcgccgacg gcccgacgga 6780tgttcgacta tttcagctcg caccgggagc
cgtacccgct caagctggaa accttccgcc 6840tcatgtgcgg atcggattcc
acccgcgtga agaagtggcg cgagcaggtc ggcgaagcct 6900gcgaagagtt
gcgaggcagc ggcctggtgg aacacgcctg ggtcaatgat gacctggtgc
6960attgcaaacg ctagggcctt gtggggtcag ttccggctgg gggttcagca
gccagcgcct 7020gatctgggga accctgtggt tggcacatac aaatggacga
acggataaac cttttcacgc 7080ccttttaaat atccgattat tctaataaac
gctcttttct cttaggttta cccgccaata 7140tatcctgtca aacactgata
gtttaaactg aaggcgggaa acgacaatct gatctaagct 7200aggcatggaa
ttccaatccc acaaaaatct gagcttaaca gcacagttgc tcctctcaga
7260gcagaatcgg gtattcaaca ccctcatatc aactactacg ttgtgtataa
cggtccacat 7320gccggtatat acgatgactg gggttgtaca aaggcggcaa
caaacggcgt tcccggagtt 7380gcacacaaga aatttgccac tattacagag
gcaagagcag cagctgacgc gtacacaaca 7440agtcagcaaa cagacaggtt
gaacttcatc cccaaaggag aagctcaact caagcccaag 7500agctttgcta
aggccctaac aagcccacca aagcaaaaag cccactggct cacgctagga
7560accaaaaggc ccagcagtga tccagcccca aaagagatct cctttgcccc
ggagattaca 7620atggacgatt tcctctatct ttacgatcta ggaaggaagt
tcgaaggtga aggtgacgac 7680actatgttca ccactgataa tgagaaggtt
agcctcttca atttcagaaa gaatgctgac 7740ccacagatgg ttagagaggc
ctacgcagca ggtctcatca agacgatcta cccgagtaac 7800aatctccagg
agatcaaata ccttcccaag aaggttaaag atgcagtcaa aagattcagg
7860actaattgca tcaagaacac agagaaagac atatttctca agatcagaag
tactattcca 7920gtatggacga ttcaaggctt gcttcataaa ccaaggcaag
taatagagat tggagtctct 7980aaaaaggtag ttcctactga atctaaggcc
atgcatggag tctaagattc aaatcgagga 8040tctaacagaa ctcgccgtga
agactggcga acagttcata cagagtcttt tacgactcaa 8100tgacaagaag
aaaatcttcg tcaacatggt ggagcacgac actctggtct actccaaaaa
8160tgtcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac
aaaggataat 8220ttcgggaaac ctcctcggat tccattgccc agctatctgt
cacttcatcg aaaggacagt 8280agaaaaggaa ggtggctcct acaaatgcca
tcattgcgat aaaggaaagg ctatcattca 8340agatctctct gccgacagtg
gtcccaaaga tggaccccca cccacgagga gcatcgtgga 8400aaaagaagac
gttccaacca cgtcttcaaa gcaagtggat tgatgtgaca tctccactga
8460cgtaagggat gacgcacaat cccactatcc ttcgcaagac ccttcctcta
tataaggaag 8520ttcatttcat ttggagagga cacgctcgag tataagagct
catttttaca acaattacca 8580acaacaacaa acaacaaaca acattacaat
tacatttaca attatcgatg ggtcagtccc 8640ttatgttacg tcctgtagaa
accccaaccc gtgaaatcaa aaaactcgac ggcctgtggg 8700cattcagtct
ggatcgcgaa aactgtggaa ttgatcagcg ttggtgggaa agcgcgttac
8760aagaaagccg ggcaattgct gtgccaggca gttttaacga tcagttcgcc
gatgcagata 8820ttcgtaatta tgcgggcaac gtctggtatc agcgcgaagt
ctttataccg aaaggtaagt 8880agtgtttttg gataactgag tttgcctatg
attttgtatt tactgagatg tttgtcctct 8940ttgtgcaggt tgggcaggcc
agcgtatcgt gctgcgtttc gatgcggtca ctcattacgg 9000caaagtgtgg
gtcaataatc aggaagtgat ggagcatcag ggcggctata cgccatttga
9060agccgatgtc acgccgtatg ttattgccgg gaaaagtgta cgtatcaccg
tttgtgtgaa 9120caacgaactg aactggcaga ctatcccgcc gggaatggtg
attaccgacg aaaacggcaa 9180gaaaaagcag tcttacttcc atgatttctt
taactatgcc ggaatccatc gcagcgtaat 9240gctctacacc acgccgaaca
cctgggtgga cgatatcacc gtggtgacgc atgtcgcgca 9300agactgtaac
cacgcgtctg ttgactggca ggtggtggcc aatggtgatg tcagcgttga
9360actgcgtgat gcggatcaac aggtggttgc aactggacaa ggcactagcg
ggactttgca 9420agtggtgaat ccgcacctct ggcaaccggg tgaaggttat
ctctatgaac tgtgcgtcac 9480agccaaaagc cagacagagt gtgatatcta
cccgcttcgc gtcggcatcc ggtcagtggc 9540agtgaagggc caacagttcc
tgattaacca caaaccgttc tactttactg gctttggtcg 9600tcatgaagat
gcggacttac gtggcaaagg attcgataac gtgctgatgg tgcacgacca
9660cgcattaatg gactggattg gggccaactc ctaccgtacc tcgcattacc
cttacgctga 9720agagatgctc gactgggcag atgaacatgg catcgtggtg
attgatgaaa ctgctgctgt 9780cggctttaac ctctctttag gcattggttt
cgaagcgggc aacaagccga aagaactgta 9840cagcgaagag gcagtcaacg
gggaaactca gcaagcgcac ttacaggcga ttaaagagct 9900gatagcgcgt
gacaaaaacc acccaagcgt ggtgatgtgg agtattgcca acgaaccgga
9960tacccgtccg caaggtgcac gggaatattt cgcgccactg gcggaagcaa
cgcgtaaact 10020cgacccgacg cgtccgatca cctgcgtcaa tgtaatgttc
tgcgacgctc acaccgatac 10080catcagcgat ctctttgatg tgctgtgcct
gaaccgttat tacggatggt atgtccaaag 10140cggcgatttg gaaacggcag
agaaggtact ggaaaaagaa cttctggcct ggcaggagaa 10200actgcatcag
ccgattatca tcaccgaata cggcgtggat acgttagccg ggctgcactc
10260aatgtacacc gacatgtgga gtgaagagta tcagtgtgca tggctggata
tgtatcaccg 10320cgtctttgat cgcgtcagcg ccgtcgtcgg tgaacaggta
tggaatttcg ccgattttgc 10380gacctcgcaa ggcatattgc gcgttggcgg
taacaagaaa gggatcttca ctcgcgaccg 10440caaaccgaag tcggcggctt
ttctgctgca aaaacgctgg actggcatga acttcggtga 10500aaaaccgcag
cagggaggca aacaatgaat caacaactct cctggcgcac catcgtcggc
10560tacagcctcg ggaattggga tcctctagag tcaagcagat cgttcaaaca
tttggcaata 10620aagtttc 10627811DNAArtificial Sequence3'-terminus
8aagatcgaca g 11
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