The N-Terminal Domain of PMTV TGB1 Movement Protein Is Required for Nucleolar Localization, Microtubule Association, and Long-Distance Movement
Kathryn M. WrightGraham H. CowanNina I. LukhovitskayaJens TilsnerAlison G. RobertsEugene I. SavenkovL. Torrance
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Abstract:
The triple-gene-block (TGB)1 protein of Potato mop-top virus (PMTV) was fused to fluorescent proteins and expressed in epidermal cells of Nicotiana benthamiana under the control of the 35S promoter. TGB1 fluorescence was observed in the cytoplasm, nucleus, and nucleolus and occasionally associated with microtubules. When expressed from a modified virus (PMTV.YFP-TGB1) which formed local lesions but was not competent for systemic movement, yellow fluorescent protein (YFP)-TGB1 labeled plasmodesmata in cells at the leading edge of the lesion and plasmodesmata, microtubules, nuclei, and nucleoli in cells immediately behind the leading edge. Deletion of 84 amino acids from the N-terminus of unlabeled TGB1 within the PMTV genome abolished movement of viral RNA to noninoculated leaves. When the same deletion was introduced into PMTV.YFP-TGB1, labeling of microtubules and nucleoli was abolished. The N-terminal 84 amino acids of TGB1 were fused to green fluorescent protein (GFP) and expressed in epidermal cells where GFP localized strongly to the nucleolus (not seen with unfused GFP), indicating that these amino acids contain a nucleolar localization signal; the fusion protein did not label microtubules. This is the first report of nucleolar and microtubule association of a TGB movement protein. The results suggest that PMTV TGB1 requires interaction with nuclear components and, possibly, microtubules for long-distance movement of viral RNA.Keywords:
Movement protein
Plasmodesma
Bimolecular fluorescence complementation
The subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functional for movement in all host species tested localized to plasmodesmata of infected epidermal cells and to tubules extending from the surface of infected protoplasts, as for wild-type CMV 3a MP. Various mutant MPs that were either conditionally functional for movement or dysfunctional for movement did not localize to plasmodesmata and did not form tubules on the surface of infected protoplasts. Rather, they showed distribution to different extents throughout the infected cells, including the cytoplasm, nucleus or the plasma membrane. The CMV 3a MP also did not associate with microtubules.
Plasmodesma
Movement protein
Cucumovirus
Protoplast
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Several plant viruses encode movement proteins (MPs) classified in the 30K superfamily. Despite a great functional diversity, alignment analysis of MP sequences belonging to the 30K superfamily revealed the presence of a central core region, including amino acids potentially critical for MP structure and functionality. We performed alanine-scanning mutagenesis of the Ourmia melon virus (OuMV) MP, and studied the effects of amino acid substitutions on MP properties and virus infection. We identified five OuMV mutants that were impaired in systemic infection in Nicotiana benthamiana and Arabidopsis thaliana, and two mutants showing necrosis and pronounced mosaic symptoms, respectively, in N. benthamiana. Green fluorescent protein fusion constructs (GFP:MP) of movement-defective MP alleles failed to localize in distinct foci at the cell wall, whereas a GFP fusion with wild-type MP (GFP:MPwt) mainly co-localized with plasmodesmata and accumulated at the periphery of epidermal cells. The movement-defective mutants also failed to produce tubular protrusions in protoplasts isolated from infected leaves, suggesting a link between tubule formation and the ability of OuMV to move. In addition to providing data to support the importance of specific amino acids for OuMV MP functionality, we predict that these conserved residues might be critical for the correct folding and/or function of the MP of other viral species in the 30K superfamily.
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The triple-gene-block (TGB)1 protein of Potato mop-top virus (PMTV) was fused to fluorescent proteins and expressed in epidermal cells of Nicotiana benthamiana under the control of the 35S promoter. TGB1 fluorescence was observed in the cytoplasm, nucleus, and nucleolus and occasionally associated with microtubules. When expressed from a modified virus (PMTV.YFP-TGB1) which formed local lesions but was not competent for systemic movement, yellow fluorescent protein (YFP)-TGB1 labeled plasmodesmata in cells at the leading edge of the lesion and plasmodesmata, microtubules, nuclei, and nucleoli in cells immediately behind the leading edge. Deletion of 84 amino acids from the N-terminus of unlabeled TGB1 within the PMTV genome abolished movement of viral RNA to noninoculated leaves. When the same deletion was introduced into PMTV.YFP-TGB1, labeling of microtubules and nucleoli was abolished. The N-terminal 84 amino acids of TGB1 were fused to green fluorescent protein (GFP) and expressed in epidermal cells where GFP localized strongly to the nucleolus (not seen with unfused GFP), indicating that these amino acids contain a nucleolar localization signal; the fusion protein did not label microtubules. This is the first report of nucleolar and microtubule association of a TGB movement protein. The results suggest that PMTV TGB1 requires interaction with nuclear components and, possibly, microtubules for long-distance movement of viral RNA.
Movement protein
Plasmodesma
Bimolecular fluorescence complementation
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Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.
Movement protein
Bimolecular fluorescence complementation
Importin
Nucleoplasm
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SUMMARY The subcellular localization of the 11‐kDa protein (p11) encoded by ORF3 of Garlic virus X (GarVX; genus Allexivirus , family Alphaflexiviridae ) was examined by confocal microscopy. Granules with intense fluorescence were visible on the endoplasmic reticulum when p11 fused with green or red fluorescent protein (GFP or RFP) was expressed in epidermal cells of Nicotiana benthamiana . Moreover, the p11‐RFP granules moved in the cytoplasm, along the cell periphery and through the cell membranes to adjacent cells. A 17‐kDa protein (p17) of garlic interacting with p11 was identified by yeast two‐hybridization and bimolecular fluorescence complementation assay. When p17 fused to GFP was expressed in epidermal cells of N. benthamiana , it localized to the nucleolus. However, in the presence of GarVX p11, the distribution of p17 changed to that of p11, but did not appear to affect the pattern of movement of p11.
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Recent studies of the tobacco mosaic virus (TMV) P30 movement protein (MP) fused with green fluorescent protein (GFP) during TMV infection described the involvement of elements of the cytoskeleton and components of the endoplasmic reticulum (ER) in the intracellular trafficking of MP:GFP from the sites of synthesis in the cytoplasm to plasmodesmata. To examine in real-time the pattern of synthesis, accumulation and degradation of MP:GFP, we developed a method to immobilize protoplasts in agarose such that they are maintained alive for extended periods of time. The pattern of MP:GFP accumulation in single living protoplasts visualized by confocal laser scanning microscopy (CLSM) was parallel to that previously described in a population of protoplasts harvested at different times post-infection. Additionally, a network of weakly fluorescent filaments, which are apparently different from microtubules, was observed to surround the nucleus and these filaments were associated with fluorescent bodies (previously identified as ER-derived structures). Later in infection, the fluorescent bodies increased in size and coalesced to form larger structures that accumulated near the periphery of the cells while highly fluorescent non-cortical filaments were observed distributed in the cytoplasm. The putative involvement of these filaments in targeting the fluorescent bodies to the periphery of the cell is discussed. Studies of single, embedded protoplasts make it possible to observe changes in amount and subcellular localization of viral and other proteins.
Protoplast
Agarose
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Plasmodesma
Movement protein
Bimolecular fluorescence complementation
Plant cell
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ABSTRACT We have recently used a green fluorescent protein (GFP) fusion to the γb protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The γb protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAβ but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, βb (TGB1), βc (TGB3), and βd (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65–75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.
Movement protein
Plasmodesma
Brome mosaic virus
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Virus movement proteins facilitate virus entry into the vascular system to initiate systemic infection. The potato mop-top virus (PMTV) movement protein, TGB1, is involved in long-distance movement of both viral ribonucleoprotein complexes and virions. Here, our analysis of TGB1 interactions with host Nicotiana benthamiana proteins revealed an interaction with a member of the heavy metal-associated isoprenylated plant protein family, HIPP26, which acts as a plasma membrane-to-nucleus signal during abiotic stress. We found that knockdown of NbHIPP26 expression inhibited virus long-distance movement but did not affect cell-to-cell movement. Drought and PMTV infection up-regulated NbHIPP26 gene expression, and PMTV infection protected plants from drought. In addition, NbHIPP26 promoter-reporter fusions revealed vascular tissue-specific expression. Mutational and biochemical analyses indicated that NbHIPP26 subcellular localization at the plasma membrane and plasmodesmata was mediated by lipidation (S-acylation and prenylation), as nonlipidated NbHIPP26 was predominantly in the nucleus. Notably, coexpression of NbHIPP26 with TGB1 resulted in a similar nuclear accumulation of NbHIPP26. TGB1 interacted with the carboxyl-terminal CVVM (prenyl) domain of NbHIPP26, and bimolecular fluorescence complementation revealed that the TGB1-HIPP26 complex localized to microtubules and accumulated in the nucleolus, with little signal at the plasma membrane or plasmodesmata. These data support a mechanism where interaction with TGB1 negates or reverses NbHIPP26 lipidation, thus releasing membrane-associated NbHIPP26 and redirecting it via microtubules to the nucleus, thereby activating the drought stress response and facilitating virus long-distance movement.
Plasmodesma
Bimolecular fluorescence complementation
Movement protein
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