Yansong Miao

Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells

Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11–24 h from protoplast production to protein detection.

EXPO, an Exocyst-Positive Organelle Distinct from Multivesicular Endosomes and Autophagosomes, Mediates Cytosol to Cell Wall Exocytosis in Arabidopsis and Tobacco Cells

Protein secretion in plant cells is generally thought to be achieved by vesicle-mediated traffic between the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and the plasma membrane. This study describes an unconventional secretory pathway involving a double-membrane exocyst-positive organelle that mediates exocytosis directly from the cytosol to cell wall. The exocyst protein complex mediates vesicle fusion with the plasma membrane. By expressing an (X)FP-tagged Arabidopsis thaliana homolog of the exocyst protein Exo70 in suspension-cultured Arabidopsis and tobacco (Nicotiana tabacum) BY-2 cells, and using antibodies specific for Exo70, we detected a compartment, which we term EXPO (for exocyst positive organelles). Standard markers for the Golgi apparatus, the trans-Golgi network/early endosome, and the multivesicular body/late endosome in plants do not colocalize with EXPO. Inhibitors of the secretory and endocytic pathways also do not affect EXPO. Exo70E2-(X)FP also locates to the plasma membrane (PM) as discrete punctae and is secreted outside of the cells. Immunogold labeling of sections cut from high-pressure frozen samples reveal EXPO to be spherical double membrane structures resembling autophagosomes. However, unlike autophagosomes, EXPOs are not induced by starvation and do not fuse with the lytic compartment or with endosomes. Instead, they fuse with the PM, releasing a single membrane vesicle into the cell wall. EXPOs are also found in other cell types, including root tips, root hair cells, and pollen grains. EXPOs therefore represent a form of unconventional secretion unique to plants.

Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis

The endomembrane system is a complex and dynamic intracellular trafficking network. It is very challenging to track individual vesicles and their cargos in real time; however, affinity purification allows vesicles to be isolated in their natural state so that their constituent proteins can be identified. Pioneering this approach in plants, we isolated the SYP61 trans-Golgi network compartment and carried out a comprehensive proteomic analysis of its contents with only minimal interference from other organelles. The proteome of SYP61 revealed the association of proteins of unknown function that have previously not been ascribed to this compartment. We identified a complete SYP61 SNARE complex, including regulatory proteins and validated the proteome data by showing that several of these proteins associated with SYP61 in planta. We further identified the SYP121-complex and cellulose synthases, suggesting that SYP61 plays a role in the exocytic trafficking and the transport of cell wall components to the plasma membrane. The presence of proteins of unknown function in the SYP61 proteome including ECHIDNA offers the opportunity to identify novel trafficking components and cargos. The affinity purification of plant vesicles in their natural state provides a basis for further analysis and dissection of complex endomembrane networks. The approach is widely applicable and can afford the study of several vesicle populations in plants, which can be compared with the SYP61 vesicle proteome.

Wortmannin induces homotypic fusion of plant prevacuolar compartments

Wortmannin, a specific inhibitor of phosphatidyl-inositol 3-kinase, is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. It has recently been demonstrated that wortmannin at 16.5 μM or 33 μM caused the prevacuolar compartments (PVCs), identified as multivesicular bodies (MVBs) by their enrichment in vacuolar sorting receptor (VSRs) proteins and the BP-80 reporter, to form small vacuoles rapidly. However, the source(s) of the membrane needed for the rapid enlargement of PVCs/MVBs has been unclear. Using both confocal immunofluorescence and immunogold EM with high pressure freeze substitution of plant samples, it has been demonstrated here that wortmannin induces homotypic fusions of PVCs/MVBs thus providing an explanation for the demand for extra membrane. In addition, possible wortmannin-induced fusions between the trans-Golgi network (TGN) and PVC, as well as between the small internal vesicles and PVC membrane, were also observed and they may also contribute to the membranes needed for PVC enlargement. In contrast to mammalian cells and yeast, wortmannin-induced fusion of PVCs appears to be unique to plants.

Localization of Green Fluorescent Protein Fusions with the Seven Arabidopsis Vacuolar Sorting Receptors to Prevacuolar Compartments in Tobacco BY-2 Cells

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1–7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.

Reaction-Based Semiconducting Polymer Nanoprobes for Photoacoustic Imaging of Protein Sulfenic Acids

Protein sulfenic acids play a key role in oxidative signal transduction of many biological and pathological processes; however, current chemical tools rely on visible fluorescence signals, limiting their utility to in vitro assays. We herein report reaction-based semiconducting polymer nanoprobes (rSPNs) with near-infrared absorption for in vivo photoacoustic (PA) imaging of protein sulfenic acids. rSPNs comprise an optically active semiconducting polymer as the core shielded with inert silica and poly(ethylene glycol) corona. The sulfenic acid reactive group (1,3-cyclohexanedione) is efficiently conjugated to the surface of nanoparticles via click chemistry. Such a nanostructure enables the specific recognition reaction between rSPNs and protein sulfenic acids without compromising the fluorescence and PA properties. In addition to in vitro tracking of the production of protein sulfenic acids in cancer cells under oxidative stress, rSPNs permit real-time PA and fluorescence imaging of protein sulfenic acids in tumors of living mice. This study thus not only demonstrates the first reaction-based PA probes with submolecular level recognition ability but also highlights the opportunities provided by hybrid nanoparticles for advanced molecular imaging.

The vacuolar transport of aleurain‐GFP and 2S albumin‐GFP fusions is mediated by the same pre‐vacuolar compartments in tobacco BY‐2 and Arabidopsis suspension cultured cells

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.

Plasma Membrane Localization and Potential Endocytosis of Constitutively Expressed XA21 Proteins in Transgenic Rice

The rice pattern recognition receptor (PRR) XA21 confers race-specific resistance in leaf infection by bacterial blight Xathomonas oryzae pv. oryzae (Xoo), and was shown to be primarily localized to the endoplasmic reticulum (ER) when expressed with its native promoter or overexpressed in the protoplast. However, whether the protein is still ER-localization in the intact cell when overexpressed remains to be identified. Here, we showed that XA21, its kinase-dead mutant XA21P(K736EP), and the triple autophosphorylation mutant XA21P(S686A/T688A/S699A) GFP fusions were primarily localized to the plasma membrane (PM) when overexpressed in the intact transgenic rice cell, and also localized to the ER in the transgenic protoplast. The transgenic plants constitutively expressing the wild-type XA21 or its GFP fusion displayed race-specific resistance to Xoo at the adult and seedling stages. XA21 and XA21P(K736EP) could be internalized probably via the SCAMP-positive early endosomal compartment in the protoplast, suggesting that XA21 might be endocytosed to initiate resistance responses during pathogen infection. We also established a root infection system and demonstrated that XA21 also mediated race-specific resistance responses to Xoo in the root. Our current study provides an insight into the nature of the XA21-mediated resistance and a practical approach using the root cell system to further dissect the cellular signaling of the PRR during the rice-Xoo interaction.

QUASIMODO 3 (QUA3) is a putative homogalacturonan methyltransferase regulating cell wall biosynthesis in Arabidopsis suspension-cultured cells

Pectins are complex polysaccharides that are essential components of the plant cell wall. In this study, a novel putative Arabidopsis S-adenosyl-L-methionine (SAM)-dependent methyltransferase, termed QUASIMODO 3 (QUA3, At4g00740), has been characterized and it was demonstrated that it is a Golgi-localized, type II integral membrane protein that functions in methylesterification of the pectin homogalacturonan (HG). Although transgenic Arabidopsis seedlings with overexpression, or knock-down, of QUA3 do not show altered phenotypes or changes in pectin methylation, this enzyme is highly expressed and abundant in Arabidopsis suspension-cultured cells. In contrast, in cells subjected to QUA3 RNA interference (RNAi) knock-down there is less pectin methylation as well as altered composition and assembly of cell wall polysaccharides. Taken together, these observations point to a Golgi-localized QUA3 playing an essential role in controlling pectin methylation and cell wall biosynthesis in Arabidopsis suspension cell cultures.

Homomeric Interaction of AtVSR1 Is Essential for Its Function as a Vacuolar Sorting Receptor

Vacuolar sorting receptors, BP80/VSRs, play a critical role in vacuolar trafficking of soluble proteins in plant cells. However, the mechanism of action of BP80 is not well understood. Here, we investigate the action mechanism of AtVSR1, a member of BP80 proteins in Arabidopsis (Arabidopsis thaliana), in vacuolar trafficking. AtVSR1 exists as multiple forms, including a high molecular mass homomeric complex in vivo. Both the transmembrane and carboxyl-terminal cytoplasmic domains of AtVSR1 are necessary for the homomeric interaction. The carboxyl-terminal cytoplasmic domain contains specific sequence information, whereas the transmembrane domain has a structural role in the homomeric interaction. In protoplasts, an AtVSR1 mutant, C2A, that contained alanine substitution of the region involved in the homomeric interaction, was defective in trafficking to the prevacuolar compartment and localized primarily to the trans-Golgi network. In addition, overexpression of C2A, but not wild-type AtVSR1, inhibited trafficking of soluble proteins to the vacuole and caused their secretion into the medium. Furthermore, C2A:hemagglutinin in transgenic plants interfered with the homomeric interaction of endogenous AtVSR1 and inhibited vacuolar trafficking of sporamin:green fluorescent protein. These data suggest that homomeric interaction of AtVSR1 is critical for its function as a vacuolar sorting receptor.