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PubMed2006

Tobacco mosaic virus movement protein functions as a structural microtubule-associated protein.

The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

Cells, Cultured↗
PubMed2006

Mutations in the central domain of potato virus X TGBp2 eliminate granular vesicles and virus cell-to-cell trafficking.

Most RNA viruses remodel the endomembrane network to promote virus replication, maturation, or egress. Rearrangement of cellular membranes is a crucial component of viral pathogenesis. The PVX TGBp2 protein induces vesicles of the granular type to bud from the endoplasmic reticulum network. Green fluorescent protein (GFP) was fused to the PVX TGBp2 coding sequence and inserted into the viral genome and into pRTL2 plasmids to study protein subcellular targeting in the presence and absence of virus infection. Mutations were introduced into the central domain of TGBp2, which contains a stretch of conserved amino acids. Deletion of a 10-amino-acid segment (m2 mutation) overlapping the segment of conserved residues eliminated the granular vesicle and inhibited virus movement. GFP-TGBp2m2 proteins accumulated in enlarged vesicles. Substitution of individual conserved residues in the same region similarly inhibited virus movement and caused the mutant GFP-TGBp2 fusion proteins to accumulate in enlarged vesicles. These results identify a novel element in the PVX TGBp2 protein which determines vesicle morphology. In addition, the data indicate that vesicles of the granular type induced by TGBp2 are necessary for PVX plasmodesmata transport.

Amino Acid Sequence↗
PubMed1993

The cowpea mosaic virus M RNA-encoded 48-kilodalton protein is responsible for induction of tubular structures in protoplasts.

Tubular structures extending from plasmodesmata in cowpea mosaic virus (CPMV)-infected tissue have been implicated to play an important role in cell-to-cell movement of this virus. Using a cauliflower mosaic virus 35S promoter-based transient expression vector, we show that expression of only the CPMV M RNA-encoded 48-kDa protein (48K protein) in cowpea protoplasts is sufficient to induce these structures. Strikingly, expression of the 48K protein in protoplasts from a number of nonhost plant species, such as barley, Arabidopsis thaliana, and carrot, also resulted in tubular structure formation. Thus, it is not likely that the viral 48K protein, though playing a key role in cell-to-cell movement of CPMV, has a role in determining the host range of CPMV.

Fabaceae↗
PubMed1995

Role of cucumovirus capsid protein in long-distance movement within the infected plant.

Direct evidence is presented for a host-specific role of the cucumovirus capsid protein in long-distance movement within infected plants. Cucumber (Cucumis sativus L.) is a systemic host for cucumber mosaic cucumovirus (CMV). Tomato aspermy cucumovirus, strain 1 (1-TAV), multiplied to the levels of CMV (i.e., replicated, moved from cell to cell, and formed infectious particles) in the inoculated leaves of cucumbers but was completely unable to spread systemically. The defective long-distance systemic movement of 1-TAV was complemented by CMV in mixed infections. Coinfection of cucumbers with 1-TAV RNA with various combinations of transcripts from full-length cDNA clones of CMV genomic RNA 1, RNA2, and RNA3 showed that CMV RNA3 alone complemented 1-TAV long-distance movement. We obtained mutants containing mutations in the two open reading frames in CMV RNA3 encoding the 3a protein and the capsid protein (CP), both of which are necessary for cell-to-cell movement of CMV. Complementation experiments with mutant CMV RNA3 showed that only 3a protein mutants, i.e., those with an intact CP, complemented the long-distance movement of 1-TAV in cucumbers. Since CMV and TAV have common systemic host plants, the results presented here are strong evidence for an active, host-specific function of the CPs of these two cucumoviruses for long-distance spread in the phloem. The results also suggest that the plasmodesmata in the vascular system and/or at the boundary between the mesophyll and the vascular system, involved in long-distance movement through the phloem, and those in the mesophyll, involved in cell-to-cell movement, differ functionally.

Capsid↗
PubMed1996

Distinct functional domains in the cowpea mosaic virus movement protein.

Cell-to-cell movement of cowpea mosaic virus particles in plants takes place with the help of tubules that penetrate presumably modified plasmodesmata. These tubules, which are built up by the virus-encoded 48-kDa movement protein (MP), are also formed on single protoplast cells. To determine whether the MP contains different functional domains, the effect of mutations in its coding region was studied. Mutations between amino acids 1 and 313 led to complete abolishment of the tubule-forming capacity, while a deletion in the C-terminal region resulted in tubules that could not take up virus particles. From these observations, it is concluded that the MP contains at least two distinct domains, one that is involved in tubule formation and that spans amino acids 1 and 313 and a second that is probably involved in the incorporation of virus particles in the tubule and that is located in the C terminus between amino acids 314 and 331.

Comovirus↗
PubMed1999

Identification of inhibitory mutants of Cauliflower mosaic virus movement protein function after expression in insect cells.

Cauliflower mosaic virus (CaMV) encodes a movement protein (MP) which forms tubules in vivo and mediates the translocation of virus particles through plasmodesmata. The relationship between CaMV MP structure and function, in isolation from the complete virus infection, was studied by using MP expression in insect cells. The study allowed the MP domains necessary for tubule formation to be identified and potential MP-MP interactions to be investigated by using double infections with recombinant baculoviruses. Two MP domains which interfered with the ability of the wild-type MP to form tubules were identified. These mutant domains appeared to act as competitive, rather than dominant negative, inhibitors.

Animals↗
PubMed2000

Cellular targets of functional and dysfunctional mutants of tobacco mosaic virus movement protein fused to green fluorescent protein.

Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

Biological Transport↗
PubMed2001

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.

Cell Membrane↗
PubMed2002

Intramolecular complementing mutations in tobacco mosaic virus movement protein confirm a role for microtubule association in viral RNA transport.

The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green fluorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MP(P81S)-GFP and MP(P81S;T104I;R167K)-GFP differ in subcellular distribution. MP(P81S)-GFP lacks specific sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MP(P81S;T104I;R167K)-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations confirm a role for microtubules in TMV RNA cell-to-cell transport.

Biological Transport↗
PubMed2004

Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function.

Rice dwarf virus (RDV) is a member of the genus Phytoreovirus, which is composed of viruses with segmented double-stranded RNA genomes. Proteins that support the intercellular movement of these viruses in the host have not been identified. Microprojectile bombardment was used to determine which open reading frames (ORFs) support intercellular movement of a heterologous virus. A plasmid containing an infectious clone of Potato virus X (PVX) defective in cell-to-cell movement and expressing either beta-glucuronidase or green fluorescent protein (GFP) was used for cobombardment with plasmids containing ORFs from RDV gene segments S1 through S12 onto leaves of Nicotiana benthamiana. Cell-to-cell movement of the movement-defective PVX was restored by cobombardment with a plasmid containing S6. In the absence of S6, no other gene segment supported movement. Identical results were obtained with Nicotiana tabacum, a host that allows fewer viruses to infect and spread within its tissue. S6 supported the cell-to-cell movement of the movement-defective PVX in sink and source leaves of N. benthamiana. A mutant S6 lacking the translation start codon did not complement the cell-to-cell movement of the movement-defective PVX. An S6 protein product (Pns6)-enhanced GFP fusion was observed near or within cell walls of epidermal cells from N. tabacum. By immunocytochemistry, unfused Pns6 was localized to plasmodesmata in rice leaves infected with RDV. S6 thus encodes a protein with characteristics identical to those of other viral proteins required for the cell-to-cell movement of their genome and therefore is likely required for the cell-to-cell movement of RDV.

Open Reading Frames↗
PubMed2004

The movement protein of cowpea mosaic virus binds GTP and single-stranded nucleic acid in vitro.

The movement protein (MP) of Cowpea mosaic virus forms tubules in plasmodesmata to enable the transport of mature virions. Here it is shown that the MP is capable of specifically binding riboguanosine triphosphate and that mutational analysis suggests that GTP binding plays a role in the targeted transport of the MP. Furthermore, the MP is capable of binding both single-stranded RNA and single-stranded DNA in a non-sequence-specific manner, and the GTP- and RNA-binding sites do not overlap.

Animals↗
PubMed2005

Actin cytoskeleton is involved in targeting of a viral Hsp70 homolog to the cell periphery.

The cell-to-cell movement of plant viruses involves translocation of virus particles or nucleoproteins to and through the plasmodesmata (PDs). As we have shown previously, the movement of the Beet yellows virus requires the concerted action of five viral proteins including a homolog of cellular approximately 70-kDa heat shock proteins (Hsp70h). Hsp70h is an integral component of the virus particles and is also found in PDs of the infected cells. Here we investigate subcellular distribution of Hsp70h using transient expression of Hsp70h fused to three spectrally distinct fluorescent proteins. We found that fluorophore-tagged Hsp70h forms motile granules that are associated with actin microfilaments, but not with microtubules. In addition, immobile granules were observed at the cell periphery. A pairwise appearance of these granules at the opposite sides of cell walls and their colocalization with the movement protein of Tobacco mosaic virus indicated an association of Hsp70h with PDs. Treatment with various cytoskeleton-specific drugs revealed that the intact actomyosin motility system is required for trafficking of Hsp70h in cytosol and its targeting to PDs. In contrast, none of the drugs interfered with the PD localization of Tobacco mosaic virus movement protein. Collectively, these findings suggest that Hsp70h is translocated and anchored to PDs in association with the actin cytoskeleton.

Actins↗
PubMed2005

Viroids and viroid-host interactions.

Although they induce symptoms in plants similar to those accompanying virus infections, viroids have unique structural, functional, and evolutionary characteristics. They are composed of a small, nonprotein-coding, single-stranded, circular RNA, with autonomous replication. Viroid species are clustered into the families Pospiviroidae and Avsunviroidae, whose members replicate (and accumulate) in the nucleus and chloroplast, respectively. Viroids replicate in three steps through an RNA-based rolling-circle mechanism: synthesis of longer-than-unit strands catalyzed by host RNA polymerases; processing to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes; and circularization. Within the initially infected cells, viroid RNA must move to its replication organelle, with the resulting progeny then invading adjacent cells through plasmodesmata and reaching distal parts via the vasculature. To carry out these movements, viroids must interact with host factors. The mature viroid RNA could be the primary pathogenic effector or, alternatively, viroids could exert their pathogenic effects via RNA silencing.

Host-Parasite Interactions↗
PubMed1976

Cytological evidence supporting the inclusion of poplar mosaic virus in the carlavirus group of plant viruses.

The filamentous particles of poplar mosaic virus (PopMV) were detected in palisade, mesophyll and parenchyma cells of systemically infected leaves of Nicotiana clevelandii and N. megalosiphon. The particles were sometimes found scattered throughout the cytoplasm, but more often they occurred loosely associated within ovoid aggregates (up to 3.5 x 1.5 mum) adjacent to chloroplasts and/or mitochondria. PopMV did not induce the formation of 'pinwheel' inclusions, nor other conspicuous ultrastructural or cytopathic effects in infected cells. These observations provide additional evidence that PopMV is a Carlavirus and not a Potyvirus nor a mite-borne virus. PopMV particles were also detected occasionally within plasmodesmata.

Chloroplasts↗
PubMed1980

ATPase in mature and differentiating phloem and xylem.

A cytochemical study using a lead precipitation technique has been made of the distribution of adenosine triphosphatase (ATPase) in mature and differentiating phloem and xylem cells of Nicotiana tabacum and Pisum sativum. The sites of ATPase localization in tobacco phloem were the plasma membrane, endoplasmic reticulum, mitochondria, dictyosomes, plasmodesmata, and the dispersed P proteins of mature sieve elements. In pea phloem sieve elements ATPase was localized in the endoplasmic reticulum, but was not associated with the P proteins or plasma membranes at any stage of their differentiation. In pea transfer cells ATPase activity was associated with the endoplasmic reticulum at all stages of their differentiation and with the plasma membrane of transfer cells that had formed wall ingrowths. In xylem cells of both tobacco and pea the patterns of ATPase activity was similar. At early stages of differentiation ATPase activity was associated with the plasma membrane and the endoplasmic reticulum. At intermediate stages of differentiation ATPase activity continued to be associated with the endoplasmic reticulum, but was no longer associated with the plasma membrane. At later stages of xylem element differentiation ATPase activity was associated with disintegrating organelles and with the hydrolyzing cell walls.

Adenosine Triphosphatases↗
PubMed2003

Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection.

BACKGROUND: Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS), is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. RESULTS: In the ihprolC-PP197 gene (intron hair pin rolC PPV 197), a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free and symptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23-25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in transgenic plants 6 weeks after viral inoculation. CONCLUSIONS: The ihprolC-PP197 transgene confers systemic resistance to PPV disease in N. benthamiana. Local infection is unaffected. This transgene and/or similar constructs could be used to confer PPV resistance to fruit trees where systemic disease causes economic damage.

Gene Expression Regulation, Plant↗
PubMed2004

Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.

BACKGROUND: Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH. RESULTS: The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in beta-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide. CONCLUSION: A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future.

Amino Acid Sequence↗
PubMed2003

Developmental regulation and significance of KNOX protein trafficking in Arabidopsis.

Intercellular communication delivers critical information for position-dependent specification of cell fate. In plants, a novel mechanism for cell-to-cell communication involves the intercellular trafficking of regulatory proteins and mRNAs. The maize KNOTTED1 (KN1) gene acts non cell-autonomously in the maize leaf, and KN1 was the first plant protein shown to traffic cell-to-cell, presumably through plasmodesmata. We have compared the intercellular trafficking of green fluorescent protein (GFP) fusions of KN1 and Arabidopsis KN1-related homeobox proteins to that of the viral movement protein from turnip vein clearing tobamovirus. We show that there is specific developmental regulation of GFP approximately KN1 trafficking. GFP -- KN1 was able to traffic from the inner layers of the leaf to the epidermis, but not in the opposite direction, from epidermis to mesophyll. However, GFP or the GFP -- movement protein fusion moved readily out of the epidermis. GFP -- KN1 was however able to traffic out of the epidermal (L1) layer in the shoot apical meristem, indicating that KN1 movement out of the L1 was developmentally regulated. GFP -- KNAT1/BREVIPEDICELLUS and GFP -- SHOOTMERISTEMLESS fusions could also traffic from the L1 to the L2/L3 layers of the meristem. In a test for the functional significance of trafficking, we showed that L1-specific expression of KN1 or of KNAT1 was able to partially complement the strong shootmeristemless-11 (stm-11) mutant. However, a cell-autonomous GUS fusion to KN1 showed neither trafficking ability nor complementation of stm-11 when expressed in the L1. These results suggest that the activity of KN1 and related homeobox proteins is maintained following intercellular trafficking, and that trafficking may be required for their normal developmental function.

Arabidopsis↗