A Novel Phloem-Specific HVA22-Like Protein Facilitates Protein Movement via Plasmodesmata in Potato.

SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the "mobilitome," covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel.

The cytoplasm of most plant cells is connected by membrane-lined cell wall channels, the plasmodesmata (PD). Dynamic regulation of sugar, hormone, and protein diffusion through PD is essential for plant development and stress responses. Understanding this regulation requires knowledge of factors and mechanisms that control PD permeability through the modulation of callose levels in the cell wall around PD openings. We investigated PD regulation in leaf epidermal cells in relation to drought stress in Arabidopsis. PD-mediated cell wall permeability was decreased by drought stress and the hormone abscisic acid (ABA), and we tested how this related to several PD-associated genes with drought-responsive expression. Mutants of NON-RACE SPECIFIC DISEASE RESISTANCE/HIN1 HAIRPIN-INDUCED-LIKE 12 (NHL12) showed relatively low PD permeability that was unaffected by drought or ABA treatment. Overexpression of NHL12 in Nicotiana benthamiana epidermal cells increased PD permeability. Moreover, we showed that NHL12 can potentially interact with the callose synthase regulator NHL3 and we explored the effect of NHL12 abundance and/or lower interface permeability on ABA signaling genes. Our results indicate that NHL12 is a drought-responsive negative regulator of PD callose levels and, thereby, interface permeability. Results are discussed in relation to PD function during drought stress and the regulation of intercellular transport.

Post-translational regulation of sucrose transporters represents one possibility to adapt transporter activity in a very short time frame. This can occur either via phosphorylation/dephosphorylation, oligomerization, protein–protein interactions, endocytosis/exocytosis, or degradation. It is also known that StSUT1 can change its compartmentalization at the plasma membrane and concentrate in membrane microdomains in response to changing redox conditions. A systematic screen for protein–protein-interactions of plant sucrose transporters revealed that the interactome of all three known sucrose transporters from the Solanaceous species Solanum tuberosum and Solanum lycopersicum represents a specific subset of interaction partners, suggesting different functions for the three different sucrose transporters. Here, we focus on factors that affect the subcellular distribution of the transporters. It was already known that sucrose transporters are able to form homo- as well as heterodimers. Here, we reveal the consequences of homo- and heterodimer formation and the fact that the responses of individual sucrose transporters will respond differently. Sucrose transporter SlSUT2 is mainly found in intracellular vesicles and several of its interaction partners are involved in vesicle traffic and subcellular targeting. The impact of interaction partners such as SNARE/VAMP proteins on the localization of SlSUT2 protein will be investigated, as well as the impact of inhibitors, excess of substrate, or divalent cations which are known to inhibit SUT1-mediated sucrose transport in yeast cells. Thereby we are able to identify factors regulating sucrose transporter activity via a change of their subcellular distribution.

Plasmodesmata (PD) allow direct communication across the cellulosic plant cell wall, facilitating the intercellular movement of metabolites and signaling molecules within the symplast. In Arabidopsis thaliana embryos with reduced levels of the chloroplast RNA helicase ISE2, intercellular trafficking and the number of branched PD were increased. We therefore investigated the relationship between altered ISE2 expression and intercellular trafficking. Gene expression analyses in Arabidopsis tissues where ISE2 expression was increased or decreased identified genes associated with the metabolism of glucosinolates (GLSs) as highly affected. Concomitant with changes in the expression of GLS-related genes, plants with abnormal ISE2 expression contained altered GLS metabolic profiles compared with wild-type (WT) counterparts. Indeed, changes in the expression of GLS-associated genes led to altered intercellular trafficking in Arabidopsis leaves. Exogenous application of GLSs but not their breakdown products also resulted in altered intercellular trafficking. These changes in trafficking may be mediated by callose levels at PD as exogenous GLS treatment was sufficient to modulate plasmodesmal callose in WT plants. Furthermore, auxin metabolism was perturbed in plants with increased indole-type GLS levels. These findings suggest that GLSs, which are themselves transported between cells via PD, can act on PD to regulate plasmodesmal trafficking capacity.

Sucrose transporter regulation at the transcriptional, post-transcriptional and post-translational level

A callus is a semi-disorganized tissue that can be induced to develop from diverse tissues by the addition of exogenous hormones. The fast growth and ease of propagation have made callus cultures useful for creating a wide variety of different experimental systems.Here, we describe a detailed and simple procedure by which different, non-clonal calli from transgenic and wild-type A. thaliana plants can be co-cultured such that they form symplasmic connections via plasmodesmata (PD). We show that callus cultures can be used to study both PD formation and transport of macromolecules between non-clonal cells via PD in a tissue lacking a vasculature. Further, we include a simple protocol for a method by which calli can be sectioned to image cells and PD by confocal laser scanning microscopy.

Intracellular vesicular traffic is controlled in part by v- and t-SNAREs, integral membrane proteins which allow specific interaction and fusion between vesicles (v-SNAREs) and their target membranes (t-SNAREs). In yeast, retrograde transport from the Golgi complex to the ER is mediated by the ER t-SNARE Ufe1p, and also requires two other ER proteins, Sec20p and Tip20p, which bind each other. Although Sec20p is not a typical SNARE, we show that both it and Tip20p can be co-precipitated with Ufe1p, and that a growth-inhibiting mutation in Ufe1p can be compensated by a mutation in Sec20p. Furthermore, Sec22p, a v-SNARE implicated in forward transport from ER to Golgi, co-precipitates with Ufe1p and Sec20p, and SEC22 acts as an allele-specific multicopy suppressor of a temperature-sensitive ufe1 mutation. These results define a new functional SNARE complex, with features distinct from the plasma membrane and cis-Golgi complexes previously identified. They also show that a single v-SNARE can be involved in both anterograde and retrograde transport, which suggests that the mere presence of a particular v-SNARE may not be sufficient to determine the preferred target for a transport vesicle.

β-1,3-Glucanases (BG) have been implicated in enhancing virus spread by degrading callose at plasmodesmata (Pd). Here, we investigate the role of Arabidopsis BG in tobamovirus spread. During Turnip vein clearing virus infection, the transcription of two pathogenesis-related (PR)-BG AtBG2 and AtBG3 increased but that of Pd-associated BG AtBG_pap did not change. In transgenic plants, AtBG2 was retained in the endoplasmic reticulum (ER) network and was not secreted. As a stress response mediated by salicylic acid, AtBG2 was secreted and appeared as a free extracellular protein localized in the entire apoplast but did not accumulate at Pd sites. At the leading edge of Tobacco mosaic virus spread, AtBG2 co-localized with the viral movement protein in the ER-derived bodies, similarly to other ER proteins, but was not secreted to the cell wall. In atbg2 mutants, callose levels at Pd and virus spread were unaffected. Likewise, AtBG2 overexpression had no effect on virus spread. However, in atbg_pap mutants, callose at Pd was increased and virus spread was reduced. Our results demonstrate that the constitutive Pd-associated BG but not the stress-regulated extracellular PR-BG are directly involved in regulation of callose at Pd and cell-to-cell transport in Arabidopsis, including the spread of viruses.

A transmembrane domain determines the localization of rice stripe virus pc4 to plasmodesmata and is essential for its function as a movement protein

Here, we demonstrate that Arabidopsis thaliana Formin 2 (AtFH2) localizes to plasmodesmata (PD) through its transmembrane domain and is required for normal intercellular trafficking. Although loss-of-function atfh2 mutants have no overt developmental defect, PD’s permeability and sensitivity to virus infection are increased in atfh2 plants. Interestingly, AtFH2 functions in a partially redundant manner with its closest homolog AtFH1, which also contains a PD localization signal. Strikingly, targeting of Class I formins to PD was also confirmed in rice, suggesting that the involvement of Class I formins in regulating actin dynamics at PD may be evolutionarily conserved in plants. In vitro biochemical analysis showed that AtFH2 fails to nucleate actin assembly but caps and stabilizes actin filaments. We also demonstrate that the interaction between AtFH2 and actin filaments is crucial for its function in vivo. These data allow us to propose that AtFH2 regulates PD's permeability by anchoring actin filaments to PD.

Here, we demonstrate that Arabidopsis thaliana Formin 2 (AtFH2) localizes to plasmodesmata (PD) through its transmembrane domain and is required for normal intercellular trafficking. Although loss-of-function atfh2 mutants have no overt developmental defect, PD's permeability and sensitivity to virus infection are increased in atfh2 plants. Interestingly, AtFH2 functions in a partially redundant manner with its closest homolog AtFH1, which also contains a PD localization signal. Strikingly, targeting of Class I formins to PD was also confirmed in rice, suggesting that the involvement of Class I formins in regulating actin dynamics at PD may be evolutionarily conserved in plants. In vitro biochemical analysis showed that AtFH2 fails to nucleate actin assembly but caps and stabilizes actin filaments. We also demonstrate that the interaction between AtFH2 and actin filaments is crucial for its function in vivo. These data allow us to propose that AtFH2 regulates PD's permeability by anchoring actin filaments to PD.

BackgroundPlant sucrose transporter activities were shown to respond to changes in the extracellular pH and redox status, and oxidizing compounds like glutathione (GSSG) or H2O2 were reported to effect the subcellular targeting of these proteins. We hypothesized that changes in both parameters might be used to modulate the activities of competing sucrose transporters at a plant/pathogen interface. We, therefore, compared the effects of redox-active compounds and of extracellular pH on the sucrose transporters UmSRT1 and ZmSUT1 known to compete for extracellular sucrose in the Ustilago maydis (corn smut)/Zea mays (maize) pathosystem.Methodology/Principal FindingsWe present functional analyses of the U. maydis sucrose transporter UmSRT1 and of the plant sucrose transporters ZmSUT1 and StSUT1 in Saccharomyces cerevisiae or in Xenopus laevis oocytes in the presence of different extracellular pH-values and redox systems, and study the possible effects of these treatments on the subcellular targeting. We observed an inverse regulation of host and pathogen sucrose transporters by changes in the apoplastic pH. Under none of the conditions analyzed, we could confirm the reported effects of redox-active compounds.Conclusions/SignificanceOur data suggest that changes in the extracellular pH but not of the extracellular redox status might be used to oppositely adjust the transport activities of plant and fungal sucrose transporters at the host/pathogen interface.

Plasmodesmata (PD) provide interconnectivity between plant cells to enable the intercellular transport and communication that is requisite to multicellularity. Being at the interface of the apoplast, plasma membrane (PM), endoplasmic reticulum (ER), and symplast, PD are uniquely positioned to integrate exogenously and endogenously derived signals with plant developmental and physiological responses. The distinct membrane curvature and composition of PD allow them to function as microdomains to facilitate dynamic protein-protein interactions. Förster resonance energy transfer (FRET) combined with fluorescence lifetime imaging microscopy (FLIM) and fluorescence anisotropic decay measurements provides valuable tools to analyze these interactions in vivo and in planta. Here we describe a detailed methodology to perform FRET-FLIM and fluorescence anisotropy measurements to analyze protein-protein interactions at PD in a transient expression system using Nicotiana benthamiana; however this can be adapted to other plant species and subcellular compartments.

Information concerning endoplasmic reticulum (ER) proteins is widely dispersed and cannot be easily and rapidly processed by the biological community. We present a comprehensive database of human ER proteins, called Human ER Aperçu (Hera). The Hera database was constructed by exhaustively searching through public databases and the scientific literature for ER proteins. Hera was used for the analysis of characteristics common to all human ER proteins. Our results show that a high proportion of ER proteins (59%) have at least one transmembrane domain and display physical characteristics consistent with this observation. In addition, one-third of ER proteins contain known ER retrieval or retention signals and 70% of ER proteins contain a signal peptide or anchor. Finally, 85% of ER proteins contain at least one InterPro motif. The most abundant InterPro motifs in ER proteins represent many of the most well-characterized functions of the ER.

Plant cell-to-cell communication is mediated by nanopores called plasmodesmata (PDs) which are complex structures comprising plasma membrane (PM), highly packed endoplasmic reticulum and numerous membrane proteins. Although recent advances on proteomics have led to insights into mechanisms of transport, there is still an inadequate characterization of the lipidic composition of the PM where membrane proteins are inserted. It has been postulated that PDs could be formed by lipid rafts, however no structural evidence has shown to visualize and analyse their lipid components. In this perspective article, we discuss proposed experiments to characterize lipid rafts and proteins in the PDs. By using atomic force microscopy (AFM) and mass spectrometry (MS) of purified PD vesicles it is possible to determine the presence of lipid rafts, specific bound proteins and the lipidomic profile of the PD under physiological conditions and after changing transport permeability. In addition, MS can determine the stoichiometry of intact membrane proteins inserted in lipid rafts. This will give novel insights into the role of membrane proteins and lipid rafts on the PD structure.