Abstract
Tomato leaf curl Java virus-A (ToLCJV-A[ID]) from Southeast Asia is a new member of the emerging group of monopartite begomoviruses that require a betasatellite component for symptom induction. Previously, we have elucidated the role of V1 ORF encoded by ToLCJV-A[ID] in cell-to-cell movement. In this study, the role of V2 (PreCP) in localization was determined. Subcellular localization of ToLCJV-A[ID] V2 in plant tissues showed that this protein is co-localized to the cell cytoplasm, perinuclear and associated with the endoplasmic reticulum network. The results obtained from deletion analysis indicate that fusion of N-terminal part of the V2, containing the nuclear export signals (NES), directed the accumulation of fluorescence towards the cell cytoplasm. Furthermore, functionality of the NES (20LAVKYLQLV29) in the N-terminal part of the V2 protein was confirmed by one-hybrid yeast system. Taken together, these results suggest that V2 enhances the coat protein-mediated nuclear export of ToLCJV-A[ID] and is consistent with the model in which V2 mediates viral DNA export from the nucleus to the plasmodesmata.
Similar content being viewed by others
References
Adachi M, Fuikuda M, Nishida E (1999) Two co-existing mechanisms of nuclear import of MAP kinase: passive diffusion of a monomer and active transport of a dimer. EMBO J 18:5347–5358
Briddon RW, Mansoor S, Bedford ID, Pinner MS, Markham PG (2000) Clones of cotton leaf curl geminivirus induce symptoms atypical of cotton leaf curl disease. Virus Genes 20:17–24
Bull SE, Briddon RW, Sserubombwe WS, Ngugi K, Markham PG (2007) Infectivity, pseudorecombination and mutagenesis of Kenyan cassava mosaic begomoviruses. J Gen Virol 88:1624–1633
Carrington JC, Kasschau KD, Mahajan SK, Schaad MC (1996) Cell-to-cell and long-distance transport of viruses in plants. Plant Cell 8:1669–1681
Chowda-Reddy RV, Achenjang F, Felton C, Etarock MT, Anangfac M, Nugent P, Fondong VN (2008) Role of geminivirus AV2 protein putative protein kinase C motif on subcellular localization and pathogenicity. Virus Res 135:115–124
Fauquet CM, Briddon RW, Brown JK, Moriones E, Stanley J, Zerbini M, Zhou X (2008) Geminivirus strain demarcation and nomenclature. Arch Virol 153:783–821
Fischer U, Huber J, Boelens WC, Mattaj IW, Luhrmann R (1995) The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82:475–483
Fridell RA, Fischer U, Luhrmann R, Meyer BE, Meinkoth JL, Malim MH, Cullen BR (1996) Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency type 1 Rev. Proc Natl Acad Sci USA 93:2936–2940
Gafni Y, Epel BL (2002) The role of host and viral proteins in intra- and intercellular trafficking of geminiviruses. Mol Plant Pathol 60:231–241
Glick E, Zrachya A, Levy Y, Mett A, Gidoni D, Belausov E, Citovsky V, Gafni Y (2008) Interaction with host SGS3 is required for suppression of RNA silencing by tomato leaf curl virus V2 protein. Proc Natl Acad Sci USA 105:157–161
Gorlich D, Mattaj IW (1999) Nucleocytoplasmic transport. Science 271:1513–1518
Hamilton WD, Bisaro DM, Coutts RH, Buck KW (1983) Demonstration of the bipartite nature of the genome of a single-stranded DNA plant virus by infection with the cloned DNA components. Nucleic Acids Res 11:7387–7396
Ia Cour T, Kiemer KD, Molgaard A, Gupta R, Skiver K, Brunak S (2004) Analysis of prediction of leucine-rich nuclear export signals. Protein Eng Des Sel 17:527–536
Kaiser C, Michaelis S, Mitchell A (1994) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Kalderon D, Richardson WD, Markham AF, Smith AE (1984) Sequence requirements for nuclear localization of SV40 large T antigen. Nature 311:33–38
Kon T (2006) Molecular characterization of Tomato infecting begomoviruses associated with DNAβ and a recombinant DNA from Indonesia. Ph.D. Thesis. 150p
Kon T, Sharma P, Ikegami M (2007) Suppressor of RNA silencing encoded by the monopartite tomato leaf curl Java begomovirus. Arch Virol 152:1273–1282
Kong LJ, Hanley-Bowdoin L (2002) A Geminivirus replication protein interacts with a protein kinase and a motor protein that display different expression patterns during plant development and infection. Plant Cell 14:1817–1832
Kotlizky G, Boulton MI, Pitaksutheepong C, Davies JW, Epel BL (2000) Intracellular and intercellular movement of maize streak geminivirus V1 and V2 proteins transiently expressed as green fluorescent protein fusions. Virology 274:32–38
Kumar PP, Usha R, Zrachya A, Levy Y, Spanov H, Gafni Y (2006) Protein–protein interactions and nuclear trafficking of coat protein and βC1 protein associated with bhendi yellow vein mosaic disease. Virus Res 122:127–136
Kunik T, Palanichelvam K, Czosnek H, Citovsky V, Gafni Y (1998) Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells. Plant J 13:393–399
Laporte C, Vetter G, Loudes AM, Robinson DG, Hillmer S, Stussi-Garaud CS, Ritzenthaler C (2003) Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fan leaf virus movement protein in tobacco BY-2 cells. Plant Cell 15:2058–2075
Lazarowitz SG (1999) Probing plant cell structure and function with viral movement proteins. Curr Opin Plant Biol 2:332–338
Lazarowitz SG, Beachy RN (1999) Viral movement proteins as probes for intracellular and intercellular trafficking in plants. Plant Cell 11:535–548
Liu H, Boulton MI, Thomas CL, Prior DA, Oparka KJ, Davies JW (1999) Maize streak virus coat protein is karyophyllic and facilitates nuclear transport of viral DNA. Mol Plant Micob Interact 12:894–900
Lucas WJ (2006) Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes. Virology 344:169–184
Maraca IG (2001) Transport into and out of the nucleus. Microbiol Mol Biol Rev 65:570–594
Mattaj IW, Englmeier L (1998) Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem 67:265–306
Nakielny S, Dreyfuss G (1999) Transport of proteins and RNAs in and out of the nucleus. Cell 12:677–690
Niwa Y, Hirano T, Yoshimoto K, Shimizu M, Kobayashi H (1999) Non-invasive quantitative detection and applications of nontoxic-, S65T-type green fluorescent protein in living plants. Plant J 18:455–463
Noueiry AO, Lucas WJ, Gilbertson RL (1994) Two proteins of a plant DNA virus coordinate nuclear and plasmodesmatal transport. Cell 76:925–932
Padidam M, Beachy RN, Facquet CM (1999) The role of AV2 “precoat” and coat protein in viral replication and movement in tomato leaf curl geminivirus. Virolology 224:390–404
Pollard VW, Malim MH (1998) The HIV-1 Rev protein. Ann Rev Microb 52:491–532
Rhee Y, Gurel F, Gafni Y, Dingwall C, Citovsky V (2000) A genetic system for detection of protein nuclear import and export. Nat Biotechnol 18:433–437
Rigden JE, Krake LR, Rezaian MA, Dry IB (1994) ORF C4 of tomato leaf curl geminivirus is a determinant of symptom severity. Virology 204:847–850
Robbins J, Dilworth SM, Laskey RA, Dingwell C (1991) Two independent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64:615–623
Rojas MR, Jiang H, Salati R, Xoconostle-Cazares B, Sudarshana MR, Lucas WJ, Gilbertson RL (2001) Functional analysis of proteins involved in movement of the monopartite begomovirus. Tomato yellow leaf curl virus. Virology 291:110–125
Rothenstein D, Krenz B, Selchow O, Jeske H (2007) Tissue and cell tropism of Indian cassava mosaic virus (ICMV) and its AV2 (precoat) gene product. Virology 359:137–145
Saunders K, Bedford ID, Briddon RW, Markham PG, Wong SM, Stanley J (2000) A unique virus complex causes Ageratum yellow vein disease. Proc Natl Acad Sci USA 97:6890–6895
Saunders K, Salim N, Mali VR, Malathi VG, Briddon RW, Markham PG, Stanley J (2002) Characterization of Sri Lankan cassava mosaic virus and cassava mosaic virus: evidence for acquisition of a DNA B component by a monopartite begomovirus. Virology 293:63–74
Sekimoto J, Katahira J, Yoneda Y (2005) Nuclear import and export signals. In: Tzfira T, Citovsky V (eds) Nuclear import and export in plants and animals. Kluwer Academic/Plenum, New York, Chapter 4
Sharma P, Ikegami M (2009) Characterization of signals that dictate nuclear, nucleolar and cytoplasmic trafficking of capsid protein of tomato leaf curl Java virus. Virus Res 144:145–153
Sharma P, Ikegami M (2010) Tomato leaf curl Java virus V2 protein determines the virulence, hypersensitive response and post transcriptional gene silencing. Virology 396:85–93
Sharma P, Rishi N, Malathi VG (2005) Molecular cloning of coat protein gene of an Indian cotton leaf curl virus (CLCuV-HS2) isolate and its phylogenetic relationship with other members of Geminiviridae. Virus Genes 30:85–91
Stanley J (1983) Infectivity of the cloned geminivirus genome requires sequences from both DNAs. Nature 305:643–645
Waigmann E, Ueki S, Trutnyeva K, Citovsky V (2004) The ins and outs of nondestructive cell-to-cell and systemic movement of plant viruses. Crit Rev Plant Sci 23:195–250
Ward BM, Lazarowitz SG (1999) Nuclear export in plants. Use of geminivirus movement proteins for a cell-based export assay. Plant Cell 11:1267–1276
Wartig L, Kheyr-Pour A, Noris E, Dekouchkovsky F, Jouanneau F, Gronenborn B, Jupin I (1997) Genetic analysis of the monopartite tomato yellow leaf curl geminivirus: roles of V1, V2 and C2 ORFs in viral pathogenesis. Virology 228:132–140
Wen W, Meinkoth JL, Tsien RY, Taylor SS (1995) Identification of a signal for rapid export of proteins from the nucleus. Cell 82:463–473
Whittaker GR, Helenius A (1998) Nuclear import and export of viruses and virus genomes. Virology 246:1–23
Zrachya A, Glick E, Levy Y, Arazi T, Citovsky V, Gafni Y (2007) Suppressor of RNA silencing encoded by Tomato yellow curl virus-Israel. Virology 358:159–165
Acknowledgments
We thank Drs V. Citovsky for the yeast one-hybrid vectors and Y. Niwa for the pGFP (S65T) plasmid and grateful to the Japan Society for the Promotion of Science for awarding the JSPS Postdoctoral Fellowship to conduct this research. We are grateful to Prof. HL Sharma and N. Sharma (HPKV, Palampur) for critical reading of the manuscript and Project Director, DWR, Karnal for encouragement while preparing this manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
Handling Editor: Manfred Heinlein
Rights and permissions
About this article
Cite this article
Sharma, P., Gaur, R.K. & Ikegami, M. Subcellular localization of V2 protein of Tomato leaf curl Java virus by using green fluorescent protein and yeast hybrid system. Protoplasma 248, 281–288 (2011). https://doi.org/10.1007/s00709-010-0166-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00709-010-0166-0