Characterization of in vivo functions of Nicotiana benthamiana RabE1

We characterized the gene expression, subcellular localization, and in vivo functions of a Nicotiana benthamiana small GTPase belonging to the RabE family, designated NbRabE1. The NbRabE1 promoter drove strong β-glucuronidase reporter expression in young tissues containing actively dividing cells and in stomata guard cells. GFP fusion proteins of NbRabE1 and its dominant-negative and constitutively active mutants were all localized to the Golgi apparatus and the plasma membrane but showed different affinities for membrane attachment. Virus-induced gene silencing of NbRabE1 resulted in pleiotropic phenotypes, including growth arrest, premature senescence, and abnormal leaf development. At the cellular level, the leaves in which NbRabE1 was silenced contained abnormal stomata that lacked pores or contained incomplete ventral walls, suggesting that NbRabE1 deficiency leads to defective guard cell cytokinesis. Ectopic expression of the dominant-negative mutant of NbRabE1 in Arabidopsis thaliana resulted in retardation of shoot and root growth accompanied by defective root hair formation. These developmental defects are discussed in conjunction with proposed functions of RabE GTPases in polarized secretory vesicle trafficking.     

PROTEINS INVOLVED IN MEMBRANE TRANSPORT BETWEEN THE ER AND THE GOLGI APPARATUS: 21 PUTATIVE PLANT HOMOLOGUES REVEALED BY DBEST SEARCHING

Numerous proteins have been identified in yeast and mammalian cells which are involved in trafficking between the endoplasmic reticulum and the Golgi apparatus. A great number of partial cDNA sequences now available from the two major plant model species, Arabidopsis thaliana and Oryza sativa, makes it possible to identify putative plant homologues of known genes/proteins from non-plant species. The authors used this approach to screen the database of Expressed Sequence Tags (dbEST) in order to detect plant homologues of proteins involved in membrane transport between ER and Golgi. Availability of these partial sequences will facilitate the screening of cDNA and genomic libraries otherwise performed using heterologous probes derived from animal and yeast genes. As the plant Golgi complex differs in many respects from its mammalian and yeast counterparts, the dbEST clones found can be directly used for various functional assays (immunoprecipitation, two-hybrid analysis, transgenic plants etc.) to test the exact roles of the encoded proteins and identify their functional partners, some of which may be specific for plants.     

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.     

AtRabD2b, a Functional Ortholog of the Yeast Ypt1, Controls Various Growth and Developmental Processes in Arabidopsis

Rab1-related guanosine-5′-triphosphatases (GTPases) regulate the intracellular endoplasmic reticulum (ER)-to-Golgi vesicle transport in yeast, mammalian and plant systems. Arabidopsis contains four Rab1 GTPases which are further divided into two functionally distinct subclasses, RabD1 and RabD2. In this study, we investigated the function of an Arabidopsis RabD2b gene. Yeast complementary assay demonstrated that AtRabD2b is a functional counterpart of the yeast Ypt1. The active GTP-bound mutant (AtRabD2b [Q67L]) maintained the complementary ability of ypt1 mutant as wild-type AtRabD2b. Both AtRabD2b and AtRabD2b [Q67L] localize on the Golgi stacks and a second subpopulation of punctate structures in tobacco leaf cells. RT-PCR assay showed that AtRabD2b is ubiquitously expressed in different plant tissues. Transgenic plants overexpressing yellow fluorescent protein (YFP)-AtRabD2b [Q67L] fusion gene exhibited dwarfism, altered morphology of rosette leaves, and upward-pointing siliques in the stems. The downregulation of AtRabD1, AtRabD2b, and AtRabD2c, due to cosuppression of the AtRabD2b gene in the YFP-AtRabD2b [Q67L] transgenic plants, resulted in stunted bushy growth phenotype, very low fertility, and the necrosis at the apical region of the stem. Together, these data indicated that AtRabD2b gene regulates the ER-to-Golgi membrane trafficking as Ypt1 and plays a role in several aspects of plant growth and development.     

Plant TGNs: dynamics and physiological functions

In all eukaryotic cells, a membrane trafficking system connects the post-Golgi organelles, including the trans-Golgi network (TGN), endosomes, and vacuoles. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. The TGN has been considered to be a compartment belonging to the Golgi apparatus, located on the trans side of the Golgi apparatus. However, in plant cells, recent studies have suggested that the TGN is an independent, dynamic organelle that possesses features different than those of TGNs in animal and yeast cells. In this review, we summarize recent progress regarding the dynamics and physiological functions of the plant TGN.     

Localization and trafficking of an isoform of the AtPRA1 family to the Golgi apparatus depend on both N- and C-terminal sequence motifs

Prenylated Rab acceptors (PRAs) bind to prenylated Rab proteins and possibly aid in targeting Rabs to their respective compartments. In Arabidopsis, 19 isoforms of PRA1 have been identified and, depending upon the isoforms, they localize to the endoplasmic reticulum (ER), Golgi apparatus and endosomes. Here, we investigated the localization and trafficking of AtPRA1.B6, an isoform of the Arabidopsis PRA1 family. In colocalization experiments with various organellar markers, AtPRA1.B6 tagged with hemagglutinin (HA) at the N-terminus localized to the Golgi apparatus in protoplasts and transgenic plants. The valine residue at the C-terminal end and an EEE motif in the C-terminal cytoplasmic domain were critical for anterograde trafficking from the ER to the Golgi apparatus. The N-terminal region contained a sequence motif for retention of AtPRA1.B6 at the Golgi apparatus. In addition, anterograde trafficking of AtPRA1.B6 from the ER to the Golgi apparatus was highly sensitive to the HA:AtPRA1.B6 level. The region that contains the sequence motif for Golgi retention also conferred the abundance-dependent trafficking inhibition. On the basis of these results, we propose that AtPRA1.B6 localizes to the Golgi apparatus and its ER-to-Golgi trafficking and localization to the Golgi apparatus are regulated by multiple sequence motifs in both the C- and N-terminal cytoplasmic domains.     

Arf1 GTPase plays roles in the protein traffic between the endoplasmic reticulum and the Golgi apparatus in tobacco and Arabidopsis cultured cells

Arf GTPases are known to be key regulators of vesicle budding in various steps of membrane traffic in yeast and animal cells. We cloned the Arabidopsis Arf1 homologue, AtArf1, and examined its function. AtArf1 complements yeast arf1 arf2 mutants and its GFP-fusion is localized to the Golgi apparatus in plant cells like its animal counterpart. The expression of dominant negative mutants of AtArf1 in tobacco and Arabidopsis cultured cells affected the localization of co-expressed GFP-tagged proteins in a variety of ways. AtArf1 Q71L and AtArf1 T31N, GTP- and GDP-fixed mutants, respectively, changed the localization of a cis-Golgi marker, AtErd2-GFP, from the Golgi apparatus to the endoplasmic reticulum but not that of GFP-AtRer1B or GFP-AtSed5. GFP-AtRer1B and GFP-AtSed5 were accumulated in aberrant structures of the Golgi by AtArf1 Q71L. A soluble vacuolar protein, sporamin-GFP, was also located to the ER by AtArf1 Q71L. These results indicate that AtArf1 play roles in the vesicular transport between the ER and the Golgi and in the maintenance of the normal Golgi organization in plant cells.     

Infection assays in Arabidopsis reveal candidate effectors from the poplar rust fungus that promote susceptibility to bacteria and oomycete pathogens

Fungi of the Pucciniales order cause rust diseases which, altogether, affect thousands of plant species worldwide and pose a major threat to several crops. How rust effectors—virulence proteins delivered into infected tissues to modulate host functions—contribute to pathogen virulence remains poorly understood. Melampsora larici‐populina is a devastating and widespread rust pathogen of poplar, and its genome encodes 1184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria, we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial (Pseudomonas syringae) and oomycete (Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of green fluorescent protein‐tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth.     

HopW1 from Pseudomonas syringae Disrupts the Actin Cytoskeleton to Promote Virulence in Arabidopsis

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.     

Null Mutants of Individual RABA Genes Impact the Proportion of Different Cell Wall Components in Stem Tissue of Arabidopsis thaliana

In Arabidopsis, and other plants, the RABA GTPases (orthologous to the Rab11a of mammals) have expanded in number and diversity and have been shown to belong to eight sub clades, some of which have been implicated in controlling vesicles that traffic cell wall polymers and enzymes that synthesise or modify them to the cell wall. In order to investigate this, we have investigated whether T-DNA insertion knockouts of individual RABA genes belonging to different sub clades, impact on the composition of the plant cell wall. Single gene knockouts of the RABA1, RABA2 and RABA4 sub clades primarily affected the percentage composition of pectin, cellulose and hemicellulose within the cell wall, respectively, despite having no obvious phenotype in the whole plant. We hypothesise that vesicles carrying specific types of cargoes from the Golgi to the cell surface may be regulated by particular sub types of RABA proteins, a finding that could have wider implications for how trafficking systems work and could be a useful tool in cell wall research and other fields of plant biology.     

Overexpression of Arabidopsis AGD7 causes relocation of Golgi-localized proteins to the endoplasmic reticulum and inhibits protein trafficking in plant cells

ADP ribosylation factor (Arf) GTPase-activating proteins (GAPs) promote the hydrolysis of GTP bound to Arfs to GDP, which plays a pivotal role in regulating Arfs by converting the active GTP-bound forms of these proteins into their inactive GDP-bound forms. Here, we investigated the biological role of AGD7, an Arf GAP homolog, in Arabidopsis (Arabidopsis thaliana). We show that AGD7 bears a highly conserved N-terminal region and a unique C-terminal region, interacts with Arf1 both in vitro and in vivo, and stimulates Arf1 GTPase activity in a phosphatidic acid-dependent manner in vitro. In plant cells, AGD7 localized to the Golgi complex, where its overexpression was found to inhibit the Golgi localization of gamma-subunit of coat proteins and promote the relocation of Golgi proteins into the endoplasmic reticulum in both protoplasts and transgenic plants. Furthermore, overexpression of AGD7 inhibited anterograde trafficking of proteins from the endoplasmic reticulum. We propose that AGD7 functions as a GAP for Arf1 in the Golgi complex and plays a critical role in protein trafficking by controlling Arf1 activity.     

Membrane protein transport between the endoplasmic reticulum and the Golgi in tobacco leaves is energy dependent but cytoskeleton independent: evidence from selective photobleaching

The mechanisms that control protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus are poorly characterized in plants. Here, we examine in tobacco leaves the structural relationship between Golgi and ER membranes using electron microscopy and demonstrate that Golgi membranes contain elements that are in close association and/or in direct contact with the ER. We further visualized protein trafficking between the ER and the Golgi using Golgi marker proteins tagged with green fluorescent protein. Using photobleaching techniques, we showed that Golgi membrane markers constitutively cycle to and from the Golgi in an energy-dependent and N-ethylmaleimide-sensitive manner. We found that membrane protein transport toward the Golgi occurs independently of the cytoskeleton and does not require the Golgi to be motile along the surface of the ER. Brefeldin A treatment blocked forward trafficking of Golgi proteins before their redistribution into the ER. Our results indicate that in plant cells, the Golgi apparatus is a dynamic membrane system whose components continuously traffic via membrane trafficking pathways regulated by brefeldin A- and N-ethylmaleimide-sensitive machinery.     

Post-Golgi protein traffic in the plant secretory pathway

The Golgi apparatus in plants is organized as a multitude of individual stacks that are motile in the cytoplasm and in close association with the endoplasmic reticulum (ER) (Boevink et al. in Plant J 15:441–447, 1998). These stacks operate as a sorting centre for cargo molecules, providing modification and redirection to other organelles as appropriate. In the post-Golgi direction, these include vacuole and plasma membrane, and specialized transport routes to each are required to prevent mislocalization. Recent evidence in plant cells points to the existence of post-Golgi organelles that function as intermediate stations for efficient protein traffic, as well as to the influence of small GTPases such as Rabs and ARFs on post-Golgi trafficking. This review focuses on the latest findings on post-Golgi trafficking routes and on the involvement of GTPases and their effectors on the trafficking of proteins in the plant secretory pathway. Sally L. Hanton and Loren A. Matheson have contributed equally to this work.     

Overexpression of Constitutively Active OsRab11 in Plants Enhances Tolerance to High Salinity Levels

Rab proteins are key regulators of intracellular trafficking between specific compartments in a cell. Among them, Rab11, a widely conserved sub-group, mainly regulates plasma membrane (PM) trafficking. Previously, we reported that Oryza sativa Rab11 (OsRab11) plays an important role in the intracellular trafficking from the trans-Golginetwork (TGN) to the plasma membrane (PM) and prevacuolar compartments (PVCs), and in the plant’s response to high salt stress. In this study, when the constitutively active mutant of OsRab11, (CA OsRab11(Q73L)) was co-transformed with Arabidopsis Ca²⁺-ATPase8-GFP (ACA8-GFP) or sporamin-GFP (Spo-GFP) into Arabidopsis protoplasts, the PM or vacuolar trafficking proportion of the reporter proteins was highly increased. Transgenic Arabidopsis plants overexpressing (OE) CA OsRab11(Q73L) exhibited enhanced tolerance to high salt stress and exogenous abscisic acid (ABA) compared to Col plants. Moreover, certain stress-responsive genes were expressed under high salt stress and ABA treatment in OEOsRab11(Q73L) plants. Thus, these results suggest that the active conformation of OsRab11 may be required to modulate plant responses to salt and ABA via the regulation of the expression of stress-responsive genes.     

Targeting of active sialyltransferase to the plant Golgi apparatus.

Glycosyltransferases in the Golgi apparatus synthesize cell wall polysaccharides and elaborate the complex glycans of glycoproteins. To investigate the targeting of this type of enzyme to plant Golgi compartments, we generated transgenic Arabidopsis plants expressing alpha-2,6-sialyltransferase, a glycosyltransferase of the mammalian trans-Golgi cisternae and the trans-Golgi network. Biochemical analysis as well as immunolight and immunoelectron microscopy of these plants indicate that the protein is targeted specifically to the Golgi apparatus. Moreover, the protein is predominantly localized to the cisternae and membranes of the trans side of the organelle. When supplied with the appropriate substrates, the enzyme has significant alpha-2,6-sialyltransferase activity. These results indicate a conservation of glycosyltransferase targeting mechanisms between plant and mammalian cells and also demonstrate that glycosyltransferases can be subcompartmentalized to specific cisternae of the plant Golgi apparatus.     

Organization of transport from endoplasmic reticulum to Golgi in higher plants

In plant cells, the organization of the Golgi apparatus and its interrelationships with the endoplasmic reticulum differ from those in mammalian and yeast cells. Endoplasmic reticulum and Golgi apparatus can now be visualized in plant cells in vivo with green fluorescent protein (GFP) specifically directed to these compartments. This makes it possible to study the dynamics of the membrane transport between these two organelles in the living cells. The GFP approach, in conjunction with a considerable volume of data about proteins participating in the transport between endoplasmic reticulum and Golgi in yeast and mammalian cells and the identification of their putative plant homologues, should allow the establishment of an experimental model in which to test the involvement of the candidate proteins in plants. As a first step towards the development of such a system, we are using Sar1, a small G-protein necessary for vesicle budding from the endoplasmic reticulum. This work has demonstrated that the introduction of Sar1 mutants blocks the transport from endoplasmic reticulum to Golgi in vivo in tobacco leaf epidermal cells and has therefore confirmed the feasibility of this approach to test the function of other proteins that are presumably involved in this step of endomembrane trafficking in plant cells.