Tomato golden mosaic virus (TGMV) is a geminivirus that replicates its single-stranded DNA genome through double-stranded DNA intermediates in nuclei of differentiated plant cells using host replication machinery. We analyzed the distribution of viral and plant DNA in nuclei of infected leaves using fluorescence in situ hybridization (FISH). TGMV-infected nuclei showed up to a sixfold increase in total volume and displayed a variety of viral DNA accumulation patterns. The most striking viral DNA patterns were bright, discrete intranuclear compartments, but diffuse nuclear localization was also observed. Quantitative and spatial measurements of high resolution 3-dimensional image data revealed that these compartments accounted for 1–18% of the total nuclear volume or 2–45% of the total nuclear FISH signals. In contrast, plant DNA was concentrated around the nuclear periphery. In a significant number of nuclei, the peripheral chromatin was organized as condensed prophase-like fibers. A combination of FISH analysis and indirect immunofluorescence with viral coat protein antibodies revealed that TGMV virions are associated with the viral DNA compartments. However, the coat protein antibodies failed to cross react with some large viral DNA inclusions, suggesting that encapsidation may occur after significant viral DNA accumulation. Infection by a TGMV mutant with a defective coat protein open reading frame resulted in fewer and smaller viral DNA-containing compartments. Nevertheless, nuclei infected with the mutant virus increased in size and in some cases showed chromosome condensation. Together, these results established that geminivirus infection alters nuclear architecture and can induce plant chromatin condensation characteristic of cells arrested in early mitosis.
REFERENCES
Ach
R. A.
, Durfee
T.
, Miller
A. B.
, Taranto
P.
, Hanley-Bowdoin
L.
, Zambryski
P. C.
, Gruissem
W.
(1997
). RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein.
Mol. Cell Biol
17
, 5077
–5086
Bass
H. W.
, Marshall
W. F.
, Sedat
J. W.
, Agard
D. A.
, Cande
W. Z.
(1997
). Telomeres cluster de novo before the initiation of synapsis: a three-dimensional spatial analysis of telomere positions before and during meiotic prophase.
J. Cell Biol
137
, 5
–18
Belmont
A. S.
, Braunfeld
M. B.
, Sedat
J. W.
, Agard
D. A.
(1989
). Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro.
Chromosoma
98
, 129
–143
Benedetto
J. P.
, Echchaoui
R.
, Plissonneau
J.
, Laquel
P.
, Litvak
S.
, Castroviejo
M.
(1996
). Changes of enzymes and factors involved in DNA synthesis during wheat embryo germination.
Plant Mol. Biol
31
, 1217
–1225
Bisaro
D. M.
, Hamilton
W. D. O.
, Coutts
R. H. A.
, Buck
K. W.
(1982
). Molecular cloning and characterization of the two DNA components of tomato golden mosaic virus.
Nucl. Acids Res
10
, 4913
–4922
Bravo
R.
, Frank
R.
, Blundell
P. A.
, MacDonald-Bravo
H.
(1987
). Cyclin/PCNA is the auxilliary protein of DNA polymerase-.
Nature
326
, 515
–517
Brough
C. L.
, Hayes
R. J.
, Morgan
A. J.
, Coutts
R. H. A.
, Buck
K. W.
(1988
). Effects of mutagenesis in vitro on the ability of cloned tomato golden mosaic virus DNA to infect Nicotiana benthamiana plants.
J. Gen. Virol
69
, 503
–514
Brough
C. L.
, Sunter
G.
, Gardiner
W.
, Bisaro
D.
(1992
). Kinetics of tomato golden mosaic virus replication and coat protein promoter activity in Nicotiana tabacum protoplasts.
Virology
187
, 1
–9
Cardoso
M. C.
, Leonhardt
H.
, Nadal-Ginard
B.
(1993
). Reversal of terminal differentiation and control of DNA replication: cyclin A and cdk2 specifically localize at subnuclear sites of DNA replication.
Cell
74
, 979
–992
Chen
H.
, Hughes
D. D.
, Chan
T.-A.
, Sedat
J. W.
, Agard
D. A.
(1996
). IVE (Image Visualization Environment): a software platform for all three-dimensional microscopy applications.
J. Struct. Biol
116
, 56
–60
Chow
K. N. B.
, Starostik
P.
, Dean
D. C.
(1996
). The rb family contains a conserved cyclin-dependent kinase-regulated transcriptional repressor motif.
Mol. Cell Biol
16
, 7173
–7181
Collin
S.
, Fernandezlobato
M.
, Gooding
P. S.
, Mullineaux
P. M.
, Fenoll
C.
(1996
). The two nonstructural proteins from wheat dwarf virus involved in viral gene expression and replication are retinoblastoma-binding proteins.
Virology
219
, 324
–329
de Bruyn Kops
A.
, Knipe
D. M.
(1994
). Preexisting nuclear architecture defines the intranuclear location of herpesvirus DNA replication structures.
J. Virol
68
, 3512
–3526
Elmer
J. S.
, Sunter
G.
, Gardiner
W. E.
, Brand
L.
, Browning
C. K.
, Bisaro
D. M.
, Rogers
S. G.
(1988
). Agrobacterium -mediated inoculation of plants with tomato golden mosaic virus DNAs.
Plant Mol. Biol
10
, 225
–234
Fontes
E. P. B.
, Gladfelter
H. J.
, Schaffer
R. L.
, Petty
I. T. D.
, Hanley-Bowdoin
L.
(1994
). Geminivirus replication origins have a modular organization.
Plant Cell
6
, 405
–416
Gardiner
W. E.
, Sunter
G.
, Brand
L.
, Elmer
J. S.
, Rogers
S. G.
, Bisaro
D. M.
(1988
). Genetic analysis of tomato golden mosaic virus: The coat protein is not required for systemic spread or symptom development.
EMBO J
7
, 899
–904
Gendreau
E.
, Hofte
H.
, Grandjean
O.
, Brown
S.
, Traas
J.
(1998
). Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl.
Plant J
13
, 221
–230
Grafi
G.
, Burnett
R. J.
, Helentjaris
T.
, Larkins
B. A.
, DeCaprio
J. A.
, Sellers
W. R.
, Kaelin
W. G.
Jr. (1996
). A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication.
Proc. Nat. Acad. Sci. USA
93
, 8962
–8967
Hamilton
W. D. O.
, Bisaro
D. M.
, Coutts
R. H. A.
, Buck
K. W.
(1983
). Demonstration of the bipartite nature of the genome of a single-stranded DNA plant virus by infection with the cloned DNA components.
Nucl. Acids Res
11
, 7387
–7396
Hamilton
W. D. O.
, Stein
V. E.
, Coutts
R. H. A.
, Buck
K. W.
(1984
). Complete nucleotide sequence of the infectious cloned DNA components of tomato golden mosaic virus: Potential coding regions and regulatory sequences.
EMBO J
3
, 2197
–2205
Hanley-Bowdoin
L.
, Settlage
S. B.
, Orozco
B. M.
, Nagar
S.
, Robertson
D.
(1999
). Geminiviruses—models for plant DNA replication, transcription and cell cycle regulation.
Curr. Biol
18
, 71
–106
Hendzel
M. J.
, Nichioka
W. K.
, Raymond
Y.
, Allis
C. D.
, Bazett-Jones
D. P.
, Th'ng
J. P. H.
(1998
). Chromatin condensation is not associated with apoptosis.
J. Biol. Chem
38
, 24470
–24478
Hiraoka
Y.
, Swedlow
J. R.
, Paddy
M. R.
, Agard
D. A.
, Sedat
J. W.
(1991
). Three-dimensional multiple wavelength fluorescence microscopy for the structural analysis of biological phenomena.
Semin. Cell Biol
2
, 153
–165
Houban
A.
, Wako
T.
, Furushima-Shimogawara
R.
, Presting
G.
, Kunzel
G.
, Schubert
I.
, Fukui
K.
(1999
). The cell cycle dependent phosphorylation of histone H3 is correlated with the condesation of plant mitotic chromosomes.
Plant J
18
, 675
–679
Inze
D.
, Gutierrez
C.
, Chua
N. H.
(1999
). Trends in plant cell cycle research.
Plant Cell
11
, 991
–994
Ishov
A. M.
, Maul
G. G.
(1996
). The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition.
J. Cell Biol
134
, 815
–826
Ishov
A. M.
, Stenberg
R. M.
, Maul
G. G.
(1997
). Human cytomegalovirus immediate early interaction with host nuclear structures: definition of an immediate transcript environment.
J. Cell Biol
138
, 5
–16
Kelman
Z.
(1997
). PCNA: structure, functions and interactions.
Oncogene
14
, 629
–640
Kim
K.
, Shcok
T.
, Goodman
R.
(1978
). Infection of Phaseolus vulgaris by bean golden mosaic virus: ultrastructural aspects.
Virology
89
, 22
–33
Kodama
H.
, Ito
M.
, Ohnishi
N.
, Suzuka
I.
, Komamine
A.
(1991
). Molecular cloning of the gene for plant proliferating-cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells.
Eur. J. Biochem
197
, 495
–503
Koshland
D.
, Strunnikov
A.
(1996
). Mitotic chromsome condensation.
Annu. Rev. Cell Dev. Biol
12
, 305
–333
Latham
J. R.
, Saunders
K.
, Pinner
M. S.
, Stanley
J.
(1997
). Induction of plant cell division by beet curly top virus gene C4.
Plant J
11
, 1273
–1283
Laufs
J.
, Jupin
I.
, David
C.
, Schumacher
S.
, Heyraudnitschke
F.
, Gronenborn
B.
(1995
). Geminivirus replication: genetic and biochemical characterization of Rep protein function, a review.
Biochimie
77
, 765
–773
Liu
L.
, vanTonder
T.
, Pietersen
G.
, Davies
J. W.
, Stanley
J.
(1997
). Molecular characterization of a subgroup I geminivirus from a legume in South Africa.
J. Gen. Virol
78
, 2113
–2117
Liu
L.
, Saunders
K.
, Thomas
C. L.
, Davies
J. W.
, Stanley
J.
(1999
). Bean yellow dwarf virus RepA, but not Rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif.
Virology
256
, 270
–279
Maul
G. G.
(1998
). Nuclear domain 10, the site of DNA virus transcription and replication.
BioEssays
20
, 660
–667
Nagar
S.
, Pedersen
T. J.
, Carrick
K. M.
, Hanley-Bowdoin
L.
, Robertson
D.
(1995
). A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.
Plant Cell
7
, 705
–719
Nevins
J.
(1992
). E2F- a link between the Rb tumor suppressor protein and viral oncoproteins.
Science
258
, 424
–429
Oberhammer
F. A.
, Hochegger
K.
, Froschl
G.
(1994
). Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase.
J. Cell Biol
126
, 827
–837
Pooma
W.
, Gillette
W. K.
, Jeffrey
J. L.
, Petty
I. T. D.
(1996
). Host and viral factors determine the dispensability of coat protein for bipartite geminivirus systemic movement.
Virology
218
, 264
–268
Rogers
S. G.
, Bisaro
D. M.
, Horsch
R. B.
, Fraley
R. T.
, Hoffman
N. L.
, Brand
L.
, Elmer
J. S.
, Lloyd
A. M.
(1986
). Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants.
Cell
45
, 593
–600
Rushing
A. E.
, Sunter
G.
, Gardiner
W. E.
, Dute
R. R.
, Bisaro
D. M.
(1987
). Ultrastructural aspects of tomato golden mosaic virus infection in tobacco.
Phytopathology
77
, 1231
–1236
Saunders
K.
, Lucy
A.
, Stanley
J.
(1991
). DNA forms of the geminivirus African cassava mosiac virus consistent with a rolling circle mechanism of replication.
Nucl. Acids Res
19
, 2325
–2330
Skiadopoulos
M. H.
, McBride
A. A.
(1998
). Bovine papillomavirus type1 genomes and the E2 transactivator protein are closely associated with mitotic chromatin.
J. Virol
72
, 2079
–2088
Sunter
G.
, Gardiner
W. E.
, Rushing
A. E.
, Rogers
S. G.
, Bisaro
D. M.
(1987
). Independent encapsidation of tomato golden mosaic virus A component DNA in transgenic plants.
Plant Mol. Biol
8
, 477
–484
Uprichard
S. L.
, Knipe
D. M.
(1997
). Assembly of herpes simplex virus replication proteins at two distinct intranuclear sites.
Virology
229
, 113
–125
Xie
Q.
, Suarezlopez
P.
, Gutierrez
C.
(1995
). Identification and analysis of a retinoblastoma binding motif in the replication protein of a plant DNA virus: requirement for efficient viral DNA replication.
EMBO J
14
, 4073
–4082
This content is only available via PDF.
© 2000 by Company of Biologists
2000
-PolarityCFP.png?versionId=4696)
