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1.  Study of the Plant COPII Vesicle Coat Subunits by Functional Complementation of Yeast Saccharomyces cerevisiae Mutants 
PLoS ONE  2014;9(2):e90072.
The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23–Sec24 heterodimer following the subsequent recruitment of the Sec13–Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.
doi:10.1371/journal.pone.0090072
PMCID: PMC3934973  PMID: 24587212
2.  Tubule-Guided Cell-to-Cell Movement of a Plant Virus Requires Class XI Myosin Motors 
PLoS Pathogens  2011;7(10):e1002327.
Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD), organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP) modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and provide a conduit for virion translocation. The MP of tubule-forming viruses including Grapevine fanleaf virus (GFLV) recruit the plant PD receptors called Plasmodesmata Located Proteins (PDLP) to mediate tubule assembly and virus movement. Here we show that PDLP1 is transported to PD through a specific route within the secretory pathway in a myosin-dependent manner. This transport relies primarily on the class XI myosins XI-K and XI-2. Inactivation of these myosins using dominant negative inhibition results in mislocalization of PDLP and MP and suppression of GFLV movement. We also found that the proper targeting of specific markers of the Golgi apparatus, the plasma membrane, PD, lipid raft subdomains within the plasma membrane, and the tonoplast was not affected by myosin XI-K inhibition. However, the normal tonoplast dynamics required myosin XI-K activity. These results reveal a new pathway of the myosin-dependent protein trafficking to PD that is hijacked by GFLV to promote tubule-guided transport of this virus between plant cells.
Author Summary
To establish infection, plant viruses spread cell-to-cell via narrow channels in the cell wall, the plasmodesmata (PD). Movement proteins (MP) are virus-encoded proteins essential for virus intercellular transport through PD. Plasmodesmata located plant proteins (PDLPs), are specifically recognised by the MPs of tubule-forming viruses. Here we show that PDLP targeting to PD depends on the molecular motors myosin XI-K and XI-2. Consistently, and in support of a function of PDLP as PD receptor for MP, overexpression of dominant negative myosin mutants inhibits tubule formation by Grapevine fanleaf virus (GFLV) MP and dramatically reduces virus movement.
doi:10.1371/journal.ppat.1002327
PMCID: PMC3203191  PMID: 22046131
3.  Structural Insights into Viral Determinants of Nematode Mediated Grapevine fanleaf virus Transmission 
PLoS Pathogens  2011;7(5):e1002034.
Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.
Author Summary
Numerous pathogenic viruses from animals and plants rely on vectors such as insects, worms or other organisms for their transmission from host to host. The reasons why certain vectors transmit some viruses but not others remain poorly understood. In plants, Grapevine fanleaf virus (GFLV), a major pathogen of grapes worldwide and its specific vector, the dagger nematode Xiphinema index, provides a well-established model illustrating this specificity. Here, we determined the high-resolution structures of two GFLV isolates that differ in their transmissibility. We show that this difference is due to a single mutation in a region exposed at the outer surface of the viral particles. This mutation does not alter the conformation of the particles but modifies the distribution of charges within a positively-charged pocket at the outer surface of virions which likely affects particle retention by X. index and, thereby also transmission efficiency. Therefore, we propose that this pocket is involved in the specific recognition of GFLV by its nematode vector. This work paves the way towards the characterization of the specific compound(s) within the nematodes that trigger vector specificity and provides novel perspectives to interfere with virus transmission.
doi:10.1371/journal.ppat.1002034
PMCID: PMC3098200  PMID: 21625570
4.  A Stretch of 11 Amino Acids in the βB-βC Loop of the Coat Protein of Grapevine Fanleaf Virus Is Essential for Transmission by the Nematode Xiphinema index▿ †  
Journal of Virology  2010;84(16):7924-7933.
Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) from the genus Nepovirus, family Secoviridae, cause a severe degeneration of grapevines. GFLV and ArMV have a bipartite RNA genome and are transmitted specifically by the ectoparasitic nematodes Xiphinema index and Xiphinema diversicaudatum, respectively. The transmission specificity of both viruses maps to their respective RNA2-encoded coat protein (CP). To further delineate the GFLV CP determinants of transmission specificity, three-dimensional (3D) homology structure models of virions and CP subunits were constructed based on the crystal structure of Tobacco ringspot virus, the type member of the genus Nepovirus. The 3D models were examined to predict amino acids that are exposed at the external virion surface, highly conserved among GFLV isolates but divergent between GFLV and ArMV. Five short amino acid stretches that matched these topographical and sequence conservation criteria were selected and substituted in single and multiple combinations by their ArMV counterparts in a GFLV RNA2 cDNA clone. Among the 21 chimeric RNA2 molecules engineered, transcripts of only three of them induced systemic plant infection in the presence of GFLV RNA1. Nematode transmission assays of the three viable recombinant viruses showed that swapping a stretch of (i) 11 residues in the βB-βC loop near the icosahedral 3-fold axis abolished transmission by X. index but was insufficient to restore transmission by X. diversicaudatum and (ii) 7 residues in the βE-αB loop did not interfere with transmission by the two Xiphinema species. This study provides new insights into GFLV CP determinants of nematode transmission.
doi:10.1128/JVI.00757-10
PMCID: PMC2916547  PMID: 20519403
5.  A Family of Plasmodesmal Proteins with Receptor-Like Properties for Plant Viral Movement Proteins 
PLoS Pathogens  2010;6(9):e1001119.
Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.
Author Summary
In plants, spreading virus infection occurs via small pores in the cell wall named plasmodesmata that connect adjacent cells. Two decades have passed since the first discovery of specific viral proteins (movement proteins; MP) that assist this process. However, the manner by which these proteins adapt plasmodesmata to allow the movement of relatively large viral structures remains largely unknown. Here, we show that a family of plasmodesmata-located proteins, called PDLPs, which are conserved amongst higher plants, specifically mediate this process. PDLPs bind classes of MP that assemble into tubules within plasmodesmata to promote the movement of entire virions. This class of MP occurs for a diverse range of plant virus genera and we show that representatives of these viruses have MPs that bind PDLPs. The importance of PDLPs in this process was shown when reduction in accumulation led to reduced tubule formation, delayed infection and attenuated symptoms. Altogether, our study supports a scenario whereby the PDLPs work together to support virus infection of plants and as such provide important mechanistic insights into the movement mechanism of plant viruses within their hosts.
doi:10.1371/journal.ppat.1001119
PMCID: PMC2944810  PMID: 20886105

Results 1-5 (5)