Barley stripe mosaic virus-encoded proteins triple-gene block 2 and γb localize to chloroplasts in virus-infected monocot and dicot plants, revealing hitherto-unknown roles in virus replication
Replication of Barley stripe mosaic virus (BSMV), genus Hordeivirus, is thought to be associated with vesicles in proplastids and chloroplasts, but the molecular details of the process and identity of virus proteins involved in establishing the virus replication complexes are unknown. In addition, BSMV encodes a triple-gene block of movement proteins (TGBs) that putatively share functional roles with their counterparts in other hordei-, pomo- and pecluviruses, but detailed information on the intracellular locations of the individual TGBs is lacking. Here, the subcellular localizations of BSMV-encoded proteins TGB2 and γb fused to green or red fluorescent proteins were examined in epidermal cells of Nicotiana benthamiana and barley (Hordeum vulgare ‘Black Hulless’). The fusion proteins were expressed from a BSMV vector or under the control of the cauliflower mosaic virus 35S promoter. The subcellular localizations were studied by confocal laser-scanning microscopy (CLSM). CLSM studies showed that both proteins were recruited to chloroplasts in the presence of viral RNA and that virus RNA, coat protein and γb protein were detected in plastid preparations from infected leaves. Electron microscope images of thin sections of virus-infected leaves revealed abnormal chloroplasts with cytoplasmic inclusions containing virus-like particles. In addition, cellular localizations of BSMV TGB2 suggest subtle differences in function between the hordei-like TGB2 proteins. The results indicate that TGB2 and γb proteins play a previously unknown functional role at the site of virus replication.
BraggJ. N.,
JacksonA. O.2004; The C-terminal region of the Barley stripe mosaic virus γ b protein participates in homologous interactions and is required for suppression of RNA silencing. Mol Plant Pathol 5:465–481[CrossRef]
BraggJ. N.,
LawrenceD. M.,
JacksonA. O.2004; The N-terminal 85 amino acids of the Barley stripe mosaic virus γ b pathogenesis protein contain three zinc-binding motifs. J Virol 78:7379–7391[CrossRef]
DonaldR. G. K.,
JacksonA. O.1994; The barley stripe mosaic virus γ b gene encodes a multifunctional cysteine-rich protein that affects pathogenesis. Plant Cell 6:1593–1606
DonaldR. G. K.,
ZhouH.,
JacksonA. O.1993; Serological analysis of barley stripe mosaic virus-encoded proteins in infected barley. Virology 195:659–668[CrossRef]
HawesC.,
Satiat-JeunemaitreB.2001; Electron microscopy. In Plant Cell Biology: a Practical Approach , 2nd edn. pp 235–266 Edited by
HawesC.,
Satiat-JeunemaitreB.
Oxford: Oxford University Press;
LacommeC.,
HrubikovaK.,
HeinI.2003; Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats. Plant J 34:543–553[CrossRef]
LawrenceD. M.,
JacksonA. O.2001a; Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus
. J Virol 75:8712–8723[CrossRef]
LawrenceD. M.,
JacksonA. O.2001b; Requirements for cell-to-cell movement of Barley stripe mosaic virus in monocot and dicot hosts. Mol Plant Pathol 2:65–75[CrossRef]
LinN.-S.,
LangenbergW. G.1984; Chronology of appearance of barley stripe mosaic virus protein in infected wheat cells. J Ultrastruct Res 89:309–323[CrossRef]
OparkaK. J.,
RobertsA. G.,
BoevinkP.& 7 other authors1999; Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves. Cell 97:743–754[CrossRef]
PettyI. T. D.,
HunterB. G.,
JacksonA. O.1988; A novel strategy for one-step cloning of full-length cDNA and its application to the genome of barley stripe mosaic virus. Gene 74:423–432[CrossRef]
PettyI. T. D.,
FrenchR.,
JonesR. W.,
JacksonA. O.1990; Identification of barley stripe mosaic virus genes involved in viral RNA replication and systemic movement. EMBO J 9:3453–3457
Prod'hommeD.,
Le PanseS.,
DrugeonG.,
JupinI.2001; Detection and subcellular localization of the turnip yellow mosaic virus 66K replication protein in infected cells. Virology 281:88–101[CrossRef]
RobinsonC.1994; Isolation of intact chloroplasts. In Plant Cell Biology: a Practical Approach Edited by
HarrisN.,
OparkaK. J.
Oxford: Oxford University Press;
RyanM. D.,
KingA. M. Q.,
ThomasG. P.1991; Cleavage of foot-and-mouth-disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J Gen Virol 72:2727–2732[CrossRef]
TorranceL.1992; Serological methods to detect plant viruses: production and use of monoclonal antibodies. In Techniques for the Rapid Detection of Plant Pathogens pp 7–33 Edited by
DuncanJ. M.,
TorranceL.
Oxford: Blackwell Scientific;
TorranceL.,
CowanG. H.,
HauptS.,
GillespieT.,
ZieglerA.2006; Role of potato mop-top virus triple gene block proteins in intracellular movement and replication. In Proceedings of 6th Symposium International Working Group on Plant Viruses with Fungal Vectors Edited by
RushC. M.
Denver, CO: American Society of Sugar Beet; Technologists (in press
VoinnetO.,
LedererC.,
BaulcombeD. C.2000; A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana
. Cell 103:157–167[CrossRef]
YelinaN. E.,
SavenkovE. I.,
SolovyevA. G.,
MorozovS. Y.,
ValkonenJ. P. T.2002; Long-distance movement, virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families. J Virol 76:12981–12991[CrossRef]
YelinaN. E.,
ErokhinaT. N.,
LukhovitskayaN. I.,
MininaE. A.,
SchepetilnikovM. V.,
LesemanD.-E.,
SchiemannJ.,
SolovyevA. G.,
MorozovS. Yu.2005; Localization of Poa semilatent virus cysteine-rich protein in peroxisomes is dispensable for its ability to suppress RNA silencing. J Gen Virol 86:479–489[CrossRef]
Barley stripe mosaic virus-encoded proteins triple-gene block 2 and γb localize to chloroplasts in virus-infected monocot and dicot plants, revealing hitherto-unknown roles in virus replication