Triple gene block protein interactions involved in movement of Barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.     

Long-distance movement,virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families

RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the gammab gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the gammab protein may be a long-distance movement factor and have antisilencing activity. This was shown for gammab proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, gammab and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the gammab cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus gammab proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.     

Hijacking of the nucleolar protein fibrillarin by TGB1 is required for cell‐to‐cell movement of Barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA‐binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear‐cytoplasmic trafficking is required for BSMV cell‐to‐cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95–104) and a nuclear localization signal (NLS) (amino acids 227–238). NoLS mutations reduced BSMV cell‐to‐cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine–arginine‐rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV‐infected cells, Fib2 accumulation increased by about 60%–70% and co‐localized with TGB1 in the plasmodesmata. In addition, BSMV cell‐to‐cell movement in fib2 knockdown transgenic plants was reduced to less than one‐third of that of non‐transgenic plants. Fib2 also co‐localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1–Fib2 interactions play a direct role in cell‐to‐cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell‐to‐cell movement.     

P42 movement protein of Beet necrotic yellow vein virus is targeted by the movement proteins P13 and P15 to punctate bodies associated with plasmodesmata

Cell-to-cell movement of Beet necrotic yellow vein virus (BNYVV) is driven by a set of three movement proteins--P42, P13, and P15--organized into a triple gene block (TGB) on viral RNA 2. The first TGB protein, P42, has been fused to the green fluorescent protein (GFP) and fusion proteins between P42 and GFP were expressed from a BNYVV RNA 3-based replicon during virus infection. GFP-P42, in which the GFP was fused to the P42 N terminus, could drive viral cell-to-cell movement when the copy of the P42 gene on RNA 2 was disabled but the C-terminal fusion P42-GFP could not. Confocal microscopy of epidermal cells of Chenopodium quinoa near the leading edge of the infection revealed that GFP-P42 localized to punctate bodies apposed to the cell wall whereas free GFP, expressed from the replicon, was distributed uniformly throughout the cytoplasm. The punctate bodies sometimes appeared to traverse the cell wall or to form pairs of disconnected bodies on each side. The punctate bodies co-localized with callose, indicating that they are associated with plasmodesmata-rich regions such as pit fields. Point mutations in P42 that inhibited its ability to drive cell-to-cell movement also inhibited GFP-P42 punctate body formation. GFP-P42 punctate body formation was dependent on expression of P13 and P15 during the infection, indicating that these proteins act together or sequentially to localize P42 to the plasmodesmata.     

The N-terminal domain of PMTV TGB1 movement protein is required for nucleolar localization, microtubule association, and long-distance movement

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.     

TIP,a novel host factor linking callose degradation with the cell-to-cell movement of Potato virus X

The cell-to-cell movement of Potato virus X (PVX) requires four virus-encoded proteins, the triple gene block (TGB) proteins (TGB25K, TGB12K, and TGB8K) and the coat protein. TGB12K increases the plasmodesmal size exclusion limit (SEL) and may, therefore, interact directly with components of the cell wall or with plant proteins associated with bringing about this change. A yeast two-hybrid screen using TGB12K as bait identified three TGB12K-interacting proteins (TIP1, TIP2, and TIP3). All three TIPs interacted specifically with TGB12K but not with TGB25K or TGB8K. Similarly, all three TIPs interacted with beta-1,3-glucanase, the enzyme that may regulate plasmodesmal SEL through callose degradation. Sequence analyses revealed that the TIPs encode very similar proteins and that TIP1 corresponds to the tobacco ankyrin repeat-containing protein HBP1. A TIP1::GFP fusion protein localized to the cytoplasm. Coexpression of this fusion protein with TGB12K induced cellular changes manifested as deposits of additional cytoplasm at the cell periphery. This work reports a direct link between a viral movement protein required to increase plasmodesmal SEL and a host factor that has been implicated as a key regulator of plasmodesmal SEL. We propose that the TIPs are susceptibility factors that modulate the plasmodesmal SEL.     

Two plant-viral movement proteins traffic in the endocytic recycling pathway

Many plant viruses exploit a conserved group of proteins known as the triple gene block (TGB) for cell-to-cell movement. Here, we investigated the interaction of two TGB proteins (TGB2 and TGB3) of Potato mop-top virus (PMTV), with components of the secretory and endocytic pathways when expressed as N-terminal fusions to green fluorescent protein or monomeric red fluorescent protein (mRFP). Our studies revealed that fluorophore-labeled TGB2 and TGB3 showed an early association with the endoplasmic reticulum (ER) and colocalized in motile granules that used the ER-actin network for intracellular movement. Both proteins increased the size exclusion limit of plasmodesmata, and TGB3 accumulated at plasmodesmata in the absence of TGB2. TGB3 contains a putative Tyr-based sorting motif, mutations in which abolished ER localization and plasmodesmatal targeting. Later in the expression cycle, both fusion proteins were incorporated into vesicular structures. TGB2 associated with these structures on its own, but TGB3 could not be incorporated into the vesicles in the absence of TGB2. Moreover, in addition to localization to the ER and motile granules, mRFP-TGB3 was incorporated into vesicles when expressed in PMTV-infected epidermal cells, indicating recruitment by virus-expressed TGB2. The TGB fusion protein-containing vesicles were labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway. TGB2 also colocalized in vesicles with Ara7, a Rab5 ortholog that marks the early endosome. Protein interaction analysis revealed that recombinant TGB2 interacted with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, shown to be essential for endocytic trafficking in Caenorhabditis elegans and Drosophila melanogaster. Collectively, the data indicate the involvement of the endocytic pathway in viral intracellular movement, the implications of which are discussed.     

Subcellular Localization of the Barley Stripe Mosaic Virus Triple Gene Block Proteins

Barley stripe mosaic virus (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.Plants use macromolecular trafficking pathways through plasmodesmata (PD) as a means to regulate developmental processes and physiological functions, and they also rely on these channels as avenues to communicate and mount defense responses to pathogen challenge (2, 37, 55). Local and systemic plant virus invasion depends on the abilities of viruses to use these pathways to spread from initially infected cells to the vascular tissue and distal regions of the plant. To this end, viruses infecting plants have evolved movement proteins (MPs) that coopt host trafficking pathways to target virus genomes to the PD and to facilitate the cell-to-cell transit of infectious entities (4, 13, 36, 48, 55). Virus MPs vary in size, number, and genome organization, but they share a number of functional characteristics including localization to PD, an ability to increase the size exclusion limits of PD, and RNA binding activities (3, 7, 8, 24, 27, 58).Viruses containing triple gene block (TGB) MPs have been the subjects of a number of investigations (4, 6, 39, 53, 54). Interestingly, viruses with a range of diverse genome structures encode MPs in a TGB, but these proteins fall into two major TGB classes that have substantial differences in protein structure and variations in their physical, functional, and cellular interactions (19, 30, 39, 45, 48). For example, the hordeivirus-like TGB1 proteins contain substantial N-terminal extensions that are lacking in the potexvirus-like TGB1 proteins, but the two classes of proteins share a conserved helicase domain at their C termini (39). The available evidence also indicates that hordeivirus-like and potexvirus-like TGB1 proteins share common biochemical features, including RNA binding abilities (3, 13, 23, 35, 44, 56), RNA helicase activities (22), associated NTPase activities (3, 13, 23, 33, 35, 44), and the capacity to form homologous interactions (29, 30, 45). However, the potexvirus-like TGB1 proteins localize at the CW when expressed autonomously and also facilitate increases in PD size exclusion limits, whereas the hordeivirus-like TGB1 proteins lack both these activities (39, 53). Major differences are also evident in the organizations of the potexvirus-like and hordeivirus-like TGB3 proteins, which share no discernible relatedness, differ in the numbers of their transmembrane domains, and indeed appear to have a polyphyletic origin (39).In both TGB classes, the movement strategy employs the coordinated actions of all three proteins. However, the coat protein is dispensable for one or more phases of movement of benyvirus, hordeivirus, pecluvirus, and pomovirus, encoding hordeivirus-like (class I) MPs, but is absolutely required for cell-to-cell movement of potexvirus-like (class II) MPs encoded by allexivirus, carlavirus, foveavirus, and potexvirus (6, 19, 39, 54). These variations clearly demonstrate that the two classes of TGB proteins have profound differences in their functional properties and in their associations with other virus and host proteins. Hence, comparative analyses of the functional and biological properties of the two classes of proteins in their common hosts may reveal important activities relevant to viral pathogenesis. To provide more information about the hordeivirus-like movement mechanisms, we are investigating the TGB interactions of Barley stripe mosaic virus (BSMV).BSMV is the type member of the genus Hordeivirus, which includes Poa semilatent virus (PSLV), Lychnis ringspot virus, and Anthoxanthum latent blanching virus (6, 19). Hordeiviruses have positive-sense, single-stranded RNA genomes consisting of three segments, designated α, β, and γ. The RNAβ segment encodes the coat protein, which is translated directly from genomic RNAβ (gRNAβ), and the TGB proteins, which are expressed from two subgenomic RNAs (sgRNAs), designated sgRNAβ1 and sgRNAβ2 (60). The coat protein is dispensable for the systemic movement of BSMV (41), and mutational analyses indicate that the TGB1, TGB2, and TGB3 proteins are each essential for cell-to-cell movement in monocot and dicot hosts (28). The BSMV TGB1 (58-kDa) protein is expressed from sgRNAβ1 at higher levels than the smaller hydrophobic TGB2 (14-kDa) and TGB3 (17-kDa) proteins, which are coexpressed from the bicistronic sgRNAβ2 during replication (14, 60). BSMV TGB1 has binding activity for both single-stranded and double-stranded RNAs (13) and forms nucleoprotein complexes with each of the BSMV gRNAs and sgRNAs (30). The hordeivirus-like TGB1 proteins differ from the potexvirus-like TGB1 proteins in having longer N-terminal domains with positively charged amino acids, but both classes of proteins have conserved C-terminal NTPase/helicase domains (13, 39, 49). In BSMV, mutations of conserved amino acids within the TGB1 helicase motif abrogate cell-to-cell movement and alter subcellular localization in infected protoplasts (27). Plants infected with a BSMV β-green fluorescent protein-TGB1 (β-GFP-TGB1) reporter virus also exhibited paired foci on both sides of the CW, and the plasma membranes of infected protoplasts developed punctate foci (27). TGB1 and TGB2 are also essential for plasma membrane targeting because β-GFP-TGB1 reporter derivatives that were unable to express TGB2 or TGB3 fluoresce at perinuclear membranes of protoplasts (27). Particle bombardment studies with the related hordeivirus PSLV also suggested that the expression of TGB3 is required to shift the localization of TGB2 from the endoplasmic reticulum (ER) to the peripheral membranes (50), and transgenically expressed PSLV TGB3 appears to be associated with PD due to its colocalization with callose markers (17).We have recently shown that TGB2 and TGB3 interact physically and have identified single amino acids in each protein that are required for these interactions (19, 30). TGB3 also interacts with TGB1, and we have proposed that these interactions facilitate the transport of ribonucleoprotein (RNP) complexes to the PD (30). However, the effects of TGB protein interactions on subcellular localization have not been defined. Moreover, because of possible convergent evolution of the hordeivirus-like and potexvirus-like TGB-containing viruses (39), the mechanisms of action resulting in transport may differ among different genera or even among different virus species within a genus. To obtain more refined information about these processes, we have now expressed fluorescent TGB fusion proteins transiently in Nicotiana benthamiana leaf cells by Agrobacterium tumefaciens infiltration and have assessed the subcellular localization patterns of BSMV wild-type (wt) and mutant TGB derivatives that differ in their interactions. We also have carried out reverse genetic experiments with selected BSMV TGB mutants to provide a biological context for the localization patterns appearing during ectopic Agrobacterium expression. These findings are elaborated in a model for TGB interactions required for the cell-to-cell movement of BSMV.     

Requirements for cell-to-cell movement of Barley stripe mosaic virus in monocot and dicot hosts

The Barley stripe mosaic virus (BSMV) RNAß genome contains a series of overlapping open reading frames termed the triple gene block. The three most abundant proteins, βb, βc and βd, have been shown to have essential roles in infectivity, but their function in cell-to-cell movement has not previously been unambiguously defined, nor has the role of a minor translational read-through protein, βd' been characterized. We have now examined the direct involvement of each of these proteins in cell-to-cell movement in planta by engineering fusions of the green fluorescent protein (GFP) to a cysteine-rich regulatory protein designated γb. Microscopic examination of inoculated and systemically infected barley and oat leaves revealed high levels of fluorescence that moved rapidly through the compact striate vascular tissue without infecting epidermal cells. In contrast, a radial pattern of fluorescence spread through a large number of epidermal and mesophyll cells before entry into the reticulate vascular tissue of the dicot hosts Nicotiania benthamiana and Chenopodium amaranticolor . Mutational analyses indicated that the βb, βc and βd proteins are each essential for cell-to-cell movement in local lesion and systemic hosts, whereas the βd' protein is dispensable. Collectively, these results demonstrate conclusively that the three major triple gene block-encoded proteins act in concert to mediate cell-to-cell movement of BSMV.     

Transient coexpression of individual genes encoded by the triple gene block of potato mop-top virus reveals requirements for TGBp1 trafficking

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the "triple gene block" (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBpl to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.     

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

A dysfunctional movement protein of tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules.

The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.     

A novel procedure for the localization of viral RNAs in protoplasts and whole plants

Analysis of virus spread using co-expressed reporter proteins has provided important details on cell-to-cell and long-distance movement of viruses in plants. However, most viruses cannot tolerate insertion of large non-viral segments or loss of any open-reading frames, procedures required to detect viruses non-evasively. A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants. The technique makes use of the binding of the coat protein of MS2 bacteriophage (CPMS2) to a 19 base hairpin (hp). A fusion protein, consisting of the CPMS2, green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear-localized upon transient expression in protoplasts. However, addition of the hp to the 3' untranslated region of Turnip crinkle virus (TCV-hp) and co-transfection of the virus and fusion protein construct into protoplasts resulted in the re-location of GFP to the cytoplasm. Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions. Transgenic plants expressing the GFP-NLS-CPMS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells. Foci of GFP cytoplasmic fluorescence were detected in TCV-hp-inoculated leaves at 2 days post-inoculation. Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins. In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves. This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.     

Plant virus movement protein dynamics probed with a GFP-protein fusion

A genetic fusion between the gene encoding green fluorescent protein (GFP) from the jellyfish Aequorea victoria, with that of the Ob-tobamovirus movement protein (MP) resulted in the expression of a fluorescent fusion protein (MP: :GFP) that was fully biologically active in mediating the cell-to-cell spread of the Ob-virus. The MP::GFP fusion was used to follow in planta the subcellular trafficking of MP. GFP-tagged MP was transiently expressed and found to be associated with several subcellular compartments and structures including trans-wall structures, presumably plasmodesmata, and filament structures. The MP::GFP fusion can be used to monitor MP association with host proteins and structures, and for the isolation of interacting host components.     

The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.     

Barley stripe mosaic virus-induced gene silencing in a monocot plant

RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.     

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.     

Approaches to defining dual-targeted proteins in Arabidopsis

A variety of approaches were used to predict dual-targeted proteins in Arabidopsis thaliana . These predictions were experimentally tested using GFP fusions. Twelve new dual-targeted proteins were identified: five that were dual-targeted to mitochondria and plastids, six that were dual-targeted to mitochondria and peroxisomes, and one that was dual-targeted to mitochondria and the nucleus. Two methods to predict dual-targeted proteins had a high success rate: (1) combining the AraPerox database with a variety of subcellular prediction programs to identify mitochondrial- and peroxisomal-targeted proteins, and (2) using a variety of prediction programs on a biochemical pathway or process known to contain at least one dual-targeted protein. Several technical parameters need to be taken into account before assigning subcellular localization using GFP fusion proteins. The position of GFP with respect to the tagged polypeptide, the tissue or cells used to detect subcellular localization, and the portion of a candidate protein fused to GFP are all relevant to the expression and targeting of a fusion protein. Testing all gene models for a chromosomal locus is required if more than one model exists.     

Investigation into the use of C- and N-terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays

Reverse transfection microarrays were described recently as a high throughput method for studying gene function. We have investigated the use of this technology for determining the subcellular localization of proteins. Genes encoding 16 proteins with a variety of functions were placed in Gateway expression constructs with 3' or 5' green fluorescent protein (GFP) tags. These were then packaged in transfection reagent and spotted robotically onto a glass slide to form a reverse transfection array. HEK293T cells were grown over the surface of the array until confluent and GFP fluorescence visualized by confocal microscopy. All C-terminal fusion proteins localized to cellular compartments in accordance with previous studies and/or bioinformatic predictions. However, less than half of the N-terminal fusion proteins localized correctly. Of those that were not in concordance with the C-terminal tagged proteins, half did not exhibit expression and the remainder had differing subcellular localizations to the C-terminal fusion protein. This data indicates that N-terminal tagging with GFP adversely affects the protein localization in reverse transfection assays, whereas tagging with GFP at the C-terminal is generally better in preserving the localization of the native protein. We discuss these results in the context of developing high-throughput subcellular localization assays based on the reverse transfection array technology.