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.     

A novel block of plant virus movement genes

Hibiscus green spot virus (HGSV) is a recently discovered and so far poorly characterized bacilliform plant virus with a positive‐stranded RNA genome consisting of three RNA species. Here, we demonstrate that the proteins encoded by the ORF2 and ORF3 in HGSV RNA2 are necessary and sufficient to mediate cell‐to‐cell movement of transport‐deficient Potato virus X in Nicotiana benthamiana. These two genes represent a specialized transport module called a ‘binary movement block’ (BMB), and ORF2 and ORF3 are termed BMB1 and BMB2 genes. In agroinfiltrated epidermal cells of N. benthamiana, green fluorescent protein (GFP)‐BMB1 fusion protein was distributed diffusely in the cytoplasm and the nucleus. However, in the presence of BMB2, GFP‐BMB1 was directed to cell wall‐adjacent elongated bodies at the cell periphery, to cell wall‐embedded punctate structures co‐localizing with callose deposits at plasmodesmata, and to cells adjacent to the initially transformed cell. Thus, BMB2 can mediate the transport of BMB1 to and through plasmodesmata. In general, our observations support the idea that cell‐to‐cell trafficking of movement proteins involves an initial delivery to membrane compartments adjacent to plasmodesmata, subsequent entry of the plasmodesmata cavity and, finally, transport to adjacent cells. This process, as an alternative to tubule‐based transport, has most likely evolved independently in triple gene block (TGB), double gene block (DGB), BMB and the single gene‐coded transport system.     

Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus

We have recently used a green fluorescent protein (GFP) fusion to the gammab protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The gammab protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAbeta 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, betab (TGB1), betac (TGB3), and betad (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.     

Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

ABSTRACT: BACKGROUND: Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. RESULTS: Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. CONCLUSION: NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin.     

Plasmodesmata‐located protein overexpression negatively impacts the manifestation of systemic acquired resistance and the long‐distance movement of Defective in Induced Resistance1 in Arabidopsis

Systemic acquired resistance (SAR) is a plant defence response that provides immunity to distant uninfected leaves after an initial localised infection. The lipid transfer protein (LTP) Defective in Induced Resistance1 (DIR1) is an essential component of SAR that moves from induced to distant leaves following a SAR‐inducing local infection. To understand how DIR1 is transported to distant leaves during SAR, we analysed DIR1 movement in transgenic Arabidopsis lines with reduced cell‐to‐cell movement caused by the overexpression of Plasmodesmata‐Located Proteins PDLP1 and PDLP5. These PDLP‐overexpressing lines were defective for SAR, and DIR1 antibody signals were not observed in phloem sap‐enriched petiole exudates collected from distant leaves. Our data support the idea that cell‐to‐cell movement of DIR1 through plasmodesmata is important during long‐distance SAR signalling in Arabidopsis.     

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.     

Identification of nuclear and nucleolar localization signals of pseudorabies virus (PRV) early protein UL54 reveals that its nuclear targeting is required for efficient production of PRV

The pseudorabies virus (PRV) early protein UL54 is a homologue of herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein that is essential for HSV-1 infection. In this study, the subcellular localization and nuclear import signals of PRV UL54 were characterized. UL54 was shown to predominantly localize to the nucleolus in transfected cells. By constructing a series of mutants, a functional nuclear localization signal (NLS) and a genuine nucleolar localization signal (NoLS) of UL54 were for the first time identified and mapped to amino acids (61)RQRRR(65) and (45)RRRRGGRGGRAAR(57), respectively. Additionally, three recombinant viruses with mutations of the NLS and/or the NoLS in UL54 were constructed based on PRV bacterial artificial chromosome (BAC) pBecker2 to test the effect of UL54 nuclear targeting on viral replication. In comparison with the wild-type virus, a recombinant virus harboring an NLS or NoLS mutation of UL54 reduced viral production to different extents. However, mutations of both the NLS and NoLS targeted UL54 to the cytoplasm in recombinant virus-infected cells and significantly impaired viral replication, comparable to the UL54-null virus. In addition, a virus lacking the NLS or the NoLS displayed modest defects in viral gene expression and DNA synthesis. However, deletion of both the NLS and the NoLS resulted in severe defects in viral gene expression and DNA synthesis, as well as production of infectious progeny. Thus, we have identified a classical NLS and a genuine NoLS in UL54 and demonstrate that the nuclear targeting of UL54 is required for efficient production of PRV.     

Nucleolar localization of parafibromin is mediated by three nucleolar localization signals

Parafibromin is a putative tumor suppressor encoded by HRPT2 and implicated in parathyroid tumorigenesis. We previously reported a functional bipartite nuclear localization signal (NLS) at residues 125-139. We now demonstrate that parafibromin exhibits nucleolar localization, mediated by three nucleolar localization signals (NoLS) at resides 76-92, 192-194 and 393-409. These NoLS represent clusters of basic amino acids arginine and lysine, similar to those found in other nucleolar proteins, as well as being characteristic of NLSs. While parafibromin's bipartite NLS is the primary determinant of nuclear localization, it does not mediate nucleolar localization. In contrast, the three identified NoLSs play only a minor role in nuclear localization, but are critical for the nucleolar localization of parafibromin.     

Inhibition of in vivo Slicer activity of Argonaute protein 1 by the viral 2b protein independent of its dsRNA‐binding function

The 2b protein of Cucumber mosaic virus (CMV) has several unique properties, such as targeting to the nucleolus and interaction with both Argonautes (AGOs) and short and long double‐stranded RNA (dsRNA). We have recently uncoupled the domain requirements for dsRNA binding and nucleolar targeting from the physical interactions with AGO proteins, and have found that the direct 2b–AGO interaction is sufficient to inhibit the in vitro AGO1 Slicer function independent of the other biochemical properties of 2b. Because the AGO binding activity of 2b is not required for its suppressor function in vivo, this raises the question of whether in vivo 2b–AGO interaction is possible to inhibit the in vivo AGO Slicer function. In this study, by taking advantage of a technology for the production of artificial trans‐acting small interfering RNA (tasiRNA), a process uniquely associated with AGO1‐mediated in vivo Slicer activity, we demonstrated that the expression of the 2b protein in planta interfered with the production of tasiRNA. Through further detailed analysis with deletion mutants of 2b proteins, we found that the inhibition of in vivo AGO1 Slicer function required the nucleolar localization signal (NoLS), in addition to the AGO‐binding domain, of the 2b protein. Our finding demonstrates that in vivo 2b–AGO1 interaction is sufficient to inhibit AGO1 Slicer function independent of the dsRNA‐binding activity of the 2b protein.     

The chloroplastic DEVH‐box RNA helicase INCREASED SIZE EXCLUSION LIMIT 2 involved in plasmodesmata regulation is required for group II intron splicing

INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) encodes a putative DEVH‐box RNA helicase originally identified through a genetic screening for Arabidopsis mutants altered in plasmodesmata (PD) aperture. Depletion of ISE2 also affects chloroplasts activity, decreases accumulation of photosynthetic pigments and alters expression of photosynthetic genes. In this work, we show the chloroplast localization of ISE2 and decipher its role in plastidic RNA processing and, consequently, PD function. Group II intron‐containing RNAs from chloroplasts exhibit defective splicing in ise2 mutants and ISE2‐silenced plants, compromising plastid viability. Furthermore, RNA immunoprecipitation suggests that ISE2 binds in vivo to several splicing‐regulated RNAs. Finally, we show that the chloroplast clpr2 mutant (defective in a subunit of a plastidic Clp protease) also exhibits abnormal PD function during embryogenesis, supporting the idea that chloroplast RNA processing is required to regulate cell–cell communication in plants.     

A Proline‐Tyrosine Nuclear Localization Signal (PY‐NLS) Is Required for the Nuclear Import of Fission Yeast PAB2, but Not of Human PABPN1

Nuclear poly(A)‐binding proteins (PABPs) are evolutionarily conserved proteins that play key roles in eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, the major nuclear PABP, Pab2, functions in the maturation of small nucleolar RNAs as well as in nuclear RNA decay. Despite knowledge about its nuclear functions, nothing is known about how Pab2 is imported into the nucleus. Here, we show that Pab2 contains a proline‐tyrosine nuclear localization signal (PY‐NLS) that is necessary and sufficient for its nuclear localization and function. Consistent with the role of karyopherin β2 (Kapβ2)‐type receptors in the import of PY‐NLS cargoes, we show that the fission yeast ortholog of human Kapβ2, Kap104, binds to recombinant Pab2 and is required for Pab2 nuclear localization. The absence of arginine methylation in a basic region N‐terminal to the PY‐core motif of Pab2 did not affect its nuclear localization. However, in the context of a sub‐optimal PY‐NLS, we found that Pab2 was more efficiently targeted to the nucleus in the absence of arginine methylation, suggesting that this modification can affect the import kinetics of a PY‐NLS cargo. Although a sequence resembling a PY‐NLS motif can be found in the human Pab2 ortholog, PABPN1, our results indicate that neither a functional PY‐NLS nor Kapβ2 activity are required to promote entry of PABPN1 into the nucleus of human cells. Our findings describe the mechanism by which Pab2 is imported into the nucleus, providing the first example of a PY‐NLS import system in fission yeast. In addition, this study suggests the existence of alternative or redundant nuclear import pathways for human PABPN1.     

Proteomic identification and characterization of secreted N‐glycosylated NPC2 following cross‐linking of the high‐affinity receptor for IgE on mast cells

Allergen‐mediated cross‐linking of the high‐affinity receptor for IgE on mast cells triggers the release of diverse preformed and de novo synthesized immunoregulatory mediators that further the allergic response. A proteomic screen applied to the detection of proteins secreted by the model rat mast cell line, RBL‐2H3 (rat basophilic leukaemia, subline 2H3.1), led to the identification of the cholesterol‐binding glycoprotein, NPC2/RE1 (Niemann–Pick Type C2/epididymal secretory protein 1). Glycosylated NPC2 is secreted early in response to an IgE‐mediated stimulus and co‐localizes with the lysosomal membrane marker, CD63. NPC2 belongs to the ML (MD‐2‐related lipid‐recognition) protein family (155 members), which includes the Toll‐like receptor co‐factors, MD‐1 and MD‐2, and perhaps most interestingly, seven major house dust mite allergens of unknown function (including Der p 2 and Der f 2). Possible role(s) for the protein in the allergic response and future applications of this approach are discussed.     

Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum–derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5′ end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.     

Nuclear and nucleolar targeting of influenza A virus NS1 protein: striking differences between different virus subtypes

Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin alpha isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin alpha/beta pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine- or lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-stranded RNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues 231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.     

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.     

The Tomato spotted wilt virus cell‐to‐cell movement protein (NSM) triggers a hypersensitive response in Sw‐5‐containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw‐5b resistance gene copy

Although the Sw‐5 gene cluster has been cloned, and Sw‐5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum ‘SR1‘ plants transformed with a copy of the Sw‐5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N. benthamiana transformed with Sw‐5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non‐structural TSWV proteins tested, the TSWV cell‐to‐cell movement protein (NS_M) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non‐replicative pEAQ‐HT expression vector system. HR was induced in Sw‐5b‐transgenic N. benthamiana as well as in resistant near‐isogenic tomato lines after agroinfiltration with a functional cell‐to‐cell movement protein (NS_M) from a resistance‐inducing (RI) TSWV strain (BR‐01), but not with NS_M from a Sw‐5 resistance‐breaking (RB) strain (GRAU). This is the first biological demonstration that Sw‐5‐mediated resistance is triggered by the TSWV NS_M cell‐to‐cell movement protein.