Figure 1. (facing page) Schematic representation of the A. tumefaciens genome. Chromosomes are drawn to scale with plasmids represented at 5 - PowerPoint PPT Presentation

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Figure 1. (facing page) Schematic representation of the A. tumefaciens genome. Chromosomes are drawn to scale with plasmids represented at 5

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Chromosomes are drawn to scale with plasmids represented at 5 or 10 ... Colors indicate orthology to proteins in the S. meliloti replicons: blue, ... – PowerPoint PPT presentation

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Title: Figure 1. (facing page) Schematic representation of the A. tumefaciens genome. Chromosomes are drawn to scale with plasmids represented at 5


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Figure 1. (facing page) Schematic representation
of the A. tumefaciens genome. Chromosomes are
drawn to scale with plasmids represented at 5 or
10 magnification, as indicated. The outer two
bands indicate opposing transcriptional
orientations of predicted genes. Colors indicate
orthology to proteins in the S. meliloti
replicons blue, chromosome green, pSymA gold,
pSymB red, nonorthologous. The inner circle
depicts GC content for each coding region, with
lower GC content indicated by darker shading. The
vir and T-DNA regions of pTiC58 and the AT island
of pAtC58 are indicated. Orthologs were
identified by comparison of predicted proteins
for each A. tumefaciens replicon with the genome
of S. meliloti. Two proteins were considered
orthologous if their BLASTP alignment covered at
least 60 of each protein at an expect value of
less than or equal to 105. Proteins that did not
match these criteria were considered
nonorthologous (14).
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Science 1999 2841328-1333
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Science 1999 2841328-1333
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(a) Beaded amino acid sequence diagram of the
processing of the VirB2 pro-pilin into the mature
cyclic T-pilin step 1, cleavage at the signal
sequence and step 2, head-to-tail peptide-bond
formation. The putative membrane association of
the T-pilin, with its conserved membrane helical
spanning domains (TMH), is shown in (b).
Abbreviations IM, inner membrane OM, outer
membrane.
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A model pathway for T-strand nuclear import.
Plant wound signals induce expression of a
Ti-plasmid regulon that contains eight virulence
loci, virAH, necessary for plant cell
transformation. (a) The T-strand is generated and
a single molecule of VirD2 is covalently attached
to the 5' end. Although the T-strand is depicted
free in the bacterial cytoplasm, it is likely
that the T-strand is generated coincident with
export. (b) The T-complex and VirE2 are exported
to the plant cell cytoplasm by a
Ti-plasmid-encoded type IV secretion system. (c)
In the plant cytoplasm, VirE2 coats the T-strand
while VirD2 imparts polarity to the T-strand.
VirD2 is recognized by importin alpha, complexed
with importin beta, and targeted to the nuclear
pore complex (NPC). (d) VIP1 associates with
VirE2 and functions as an 'adaptor', facilitating
VirE2 nuclear localization signal (NLS)
recognition by importin alpha. Alternatively,
importin alpha recognizes VIP1, and VirE2 is
targeted to the NPC by association with VIP1. (e)
The T-strand and associated proteins pass through
the NPC. VirE2 confers an import-compatible
conformation to the T-strand. (f) The GTP-binding
protein, Ran, effects release of imported
substrates from the importin complex. (g) VirE2
binding proteins, VIP1 and VIP2, target the
T-strand to regions of chromatin, promoting
integration.
Trends in Plant Science 71-3.
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Cell 68109-118
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Cell 68109-118
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Figure 1. VirD2-AtKAP interaction in the
two-hybrid system. The indicated combinations
bait/prey were achieved by introducing into the
yeast strain Y153 the combinations of the
following plasmids VirD2/AtKAP, pGBTD2 and
pGADAtKAP VirD2NLS/AtKAP, pGBTD2NLS and
pGADAtKAP lamin C/AtKAP, pLAM5 and pGADAtKAP. No
prey combinations included the corresponding bait
and pGAD424 plasmid without insert.
Protein-protein interaction was determined by the
-galactosidase assay on a nitrocellulose filter
as described (21). All other experimental
conditions were as described for the two-hybrid
screen of the cDNA library.
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Fig. 5. AtKAP promotes nuclear import of VirD2 in
permeabilized yeast cells. Nuclear import assays
were performed as described in Materials and
Methods. The following combinations of cytosolic
fractions and the fluorescently labeled protein
were added to the permeabilized srp1-31 cells A
and B, VirD2cytosol from cells expressing pGADG
C and D, VirD2cytosol from cells expressing
pAtKAP E and F, VirD2NLScytosol from cells
expressing pAtKAP G and H, VirD2cytosol from
cells expressing pAtKAP 2 µg of a synthetic
VirD2 NLS peptide I and J, VirD2cytosol from
cells expressing pSRP1. A, C, E, G, and I,
fluorescein-labeled protein B, D, F, H, and J,
4,6-diamidino-2-phenylindole staining. The
one-letter code amino acid sequence of the VirD2
NLS is KRpredddgepseRKReR with basic residues of
the first and second domains of the bipartite
signal indicated in uppercase (11). (Bar 50
µm.)
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PNAS 8911837-11841
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A model pathway for T-strand nuclear import.
Plant wound signals induce expression of a
Ti-plasmid regulon that contains eight virulence
loci, virAH, necessary for plant cell
transformation. (a) The T-strand is generated and
a single molecule of VirD2 is covalently attached
to the 5' end. Although the T-strand is depicted
free in the bacterial cytoplasm, it is likely
that the T-strand is generated coincident with
export. (b) The T-complex and VirE2 are exported
to the plant cell cytoplasm by a
Ti-plasmid-encoded type IV secretion system. (c)
In the plant cytoplasm, VirE2 coats the T-strand
while VirD2 imparts polarity to the T-strand.
VirD2 is recognized by importin alpha, complexed
with importin beta, and targeted to the nuclear
pore complex (NPC). (d) VIP1 associates with
VirE2 and functions as an 'adaptor', facilitating
VirE2 nuclear localization signal (NLS)
recognition by importin alpha . Alternatively,
importin alpha recognizes VIP1, and VirE2 is
targeted to the NPC by association with VIP1. (e)
The T-strand and associated proteins pass through
the NPC. VirE2 confers an import-compatible
conformation to the T-strand. (f) The GTP-binding
protein, Ran, effects release of imported
substrates from the importin complex. (g) VirE2
binding proteins, VIP1 and VIP2, target the
T-strand to regions of chromatin, promoting
integration.
Trends in Plant Science 71-3.
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PNAS 861193-1197
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Science 2561802-1805
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Fig. 4. VIP1-mediated nuclear import of VirE2 in
mammalian cells. (A) COS-1 cells expressing
GFPVirE2. Dispersed fluorescence surrounding the
signal-free, black cell nucleus represents the
cytoplasmic localization of GFPVirE2. (B) COS-1
cells expressing GFPVIP1. The fluorescent signal
is concentrated exclusively in the cell nucleus.
(C) COS-1 cells co-expressing GFPVirE2 and
unlabeled VIP1. In most cells, part of the
fluorescent signal enters the nucleus and part
remains dispersed in the surrounding areas of the
cytoplasm. Bar 15 µm.
EMBO J. 203596-3607
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