Immunomodulation by the Pseudomonas syringae HopZ Type III Effector Family in Arabidopsis

Pseudomonas syringae employs a type III secretion system to inject 20–30 different type III effector (T3SE) proteins into plant host cells. A major role of T3SEs is to suppress plant immune responses and promote bacterial infection. The YopJ/HopZ acetyltransferases are a superfamily of T3SEs found in both plant and animal pathogenic bacteria. In P. syringae, this superfamily includes the evolutionarily diverse HopZ1, HopZ2 and HopZ3 alleles. To investigate the roles of the HopZ family in immunomodulation, we generated dexamethasone-inducible T3SE transgenic lines of Arabidopsis for HopZ family members and characterized them for immune suppression phenotypes. We show that all of the HopZ family members can actively suppress various facets of Arabidopsis immunity in a catalytic residue-dependent manner. HopZ family members can differentially suppress the activation of mitogen-activated protein (MAP) kinase cascades or the production of reactive oxygen species, whereas all members can promote the growth of non-virulent P. syringae. Localization studies show that four of the HopZ family members containing predicted myristoylation sites are localized to the vicinity of the plasma membrane while HopZ3 which lacks the myristoylation site is at least partially nuclear localized, suggesting diversification of immunosuppressive mechanisms. Overall, we demonstrate that despite significant evolutionary diversification, all HopZ family members can suppress immunity in Arabidopsis.     

Evolutionary Relationship of Disease Resistance Genes in Soybean and Arabidopsis Specific for the Pseudomonas syringae Effectors AvrB and AvrRpm1

In Arabidopsis (Arabidopsis thaliana), the Pseudomonas syringae effector proteins AvrB and AvrRpm1 are both detected by the RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) disease resistance (R) protein. By contrast, soybean (Glycine max) can distinguish between these effectors, with AvrB and AvrRpm1 being detected by the Resistance to Pseudomonas glycinea 1b (Rpg1b) and Rpg1r R proteins, respectively. We have been using these genes to investigate the evolution of R gene specificity and have previously identified RPM1 and Rpg1b. Here, we report the cloning of Rpg1r, which, like RPM1 and Rpg1b, encodes a coiled-coil (CC)-nucleotide-binding (NB)-leucine-rich repeat (LRR) protein. As previously found for Rpg1b, we determined that Rpg1r is not orthologous with RPM1, indicating that the ability to detect both AvrB and AvrRpm1 evolved independently in soybean and Arabidopsis. The tightly linked soybean Rpg1b and Rpg1r genes share a close evolutionary relationship, with Rpg1b containing a recombination event that combined a NB domain closely related to Rpg1r with CC and LRR domains from a more distantly related CC-NB-LRR gene. Using structural modeling, we mapped polymorphisms between Rpg1b and Rpg1r onto the predicted tertiary structure of Rpg1b, which revealed highly polymorphic surfaces within both the CC and LRR domains. Assessment of chimeras between Rpg1b and Rpg1r using a transient expression system revealed that AvrB versus AvrRpm1 specificity is determined by the C-terminal portion of the LRR domain. The P. syringae effector AvrRpt2, which targets RPM1 INTERACTOR4 (RIN4) proteins in both Arabidopsis and soybean, partially blocked recognition of both AvrB and AvrRpm1 in soybean, suggesting that both Rpg1b and Rpg1r may detect these effectors via modification of a RIN4 homolog.Effector triggered immunity in plants involves highly specific recognition events in which plant resistance (R) proteins detect pathogen effector proteins directly or, alternatively, the modifications that they induce on host proteins (Bonardi et al., 2012). The largest group of R proteins belongs to the nucleotide-binding (NB)-leucine-rich repeat (LRR) family (McHale et al., 2006). The NB-LRR family can be further subdivided based on N-terminal domains into the Toll-Interleukin and R protein (TIR) class and non-TIR-NB-LRR class (McHale et al., 2006). The latter most often contain a coiled-coil (CC) domain at the N terminus. The contributions of the TIR, CC, and LRR domains to R protein specificity, and how new specificities evolve, remain important questions.There are relatively few NB-LRR R proteins characterized to date that are thought to detect pathogen effectors directly; these include Pi-ta from rice (Oryza sativa), L and M variants from flax (Linum usitatissimum), and RESISTANCE TO RALSTONIA SOLANACEARUM1 and RESISTANCE TO PERONOSPORA PARASITICA1 (RPP1) from Arabidopsis (Arabidopsis thaliana; Jia et al., 2000; Deslandes et al., 2003; Dodds et al., 2006; Ueda et al., 2006; Catanzariti et al., 2010; Krasileva et al., 2010). In at least some of these examples, the R genes are found in clusters of NB-LRR paralogs in which multiple recognition specificities are represented (Ellis et al., 1995; Botella et al., 1998) or belong to allelic series (Ellis et al., 1995), arrangements that may promote evolution of recognition specificity via recombination between alleles and paralogs. Interestingly, sequence comparisons and domain swaps involving alleles at the L locus implicate both the LRR and TIR regions as determinants of recognition specificity (Ellis et al., 1999; Luck et al., 2000). Subsequently, domain swaps involving paralogs clustered at the barley (Hordeum vulgare) MILDEW A (MLA) and potato (Solanum tuberosum) Resistance to Potato Virus X (Rx)/Globodera pallida (Gpa) loci have provided additional support for the LRR domain playing a key role in conferring recognition specificity (Ellis et al., 1999; Luck et al., 2000; Shen et al., 2003; Rairdan and Moffett, 2006).Several R proteins are known to detect the presence of pathogen effectors indirectly by monitoring the activity of pathogen effectors within the plant cell. For example, the Arabidopsis RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) and RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2) R proteins detect modification of the effector target RPM1 INTERACTOR4 (RIN4), while the Arabidopsis RPS5 protein detects modification of the effector target AvrPphB SUSCEPTIBLE1 (Mackey et al., 2002, 2003; Axtell and Staskawicz, 2003; Shao et al., 2003). At least for the well-studied examples in Arabidopsis, R proteins that employ indirect recognition mechanisms are encoded by NB-LRR genes that are not members of large clusters, or allelic series, with variants encoding distinct recognition specificities. Correlated with this genomic structure, such loci are typically relatively stable, with RPM1 and RPS5 existing as presence/absence polymorphisms that have been maintained over long evolutionary periods (Stahl et al., 1999; Tian et al., 2002). Both functional and nonfunctional alleles of RPS2 have been isolated, but only a single recognition specificity has been detected at this locus, despite sequence polymorphisms between alleles (Caicedo et al., 1999).Most likely, specificity for this class of R proteins is determined by a combination of the ability to associate with the host protein targeted by the effector and the ability to detect effector-induced modification of this target. Consistent with this hypothesis, it has been shown that the CC domains from at least some R proteins interact with the host proteins they are monitoring, even in the absence of pathogen effectors, in a prerecognition complex (Mackey et al., 2002; Ade et al., 2007). Hence, evolution of recognition specificity in R proteins that employ indirect recognition mechanisms may involve evolution of both the N-terminal CC and LRR domains.To better understand the evolution and function of R proteins that detect pathogen effectors indirectly, we have been studying two soybean (Glycine max) R genes, with known recognition specificities, that are members of a complex NB-LRR cluster. The R genes involved, Resistance to Pseudomonas glycinea 1b (Rpg1b) and Rpg1r, mediate detection of the Pseudomonas syringae effector proteins AvrB and AvrRpm1, respectively (Staskawicz et al., 1984; Ashfield et al., 1995). We have previously cloned Rpg1b, which is a CC-NB-LRR (CNL) gene that maps to a cluster of R genes effective against a diverse range of pathogens (Ashfield et al., 1998, 2004). Rpg1r is present in the same cluster and maps 0.56 centiMorgans from Rpg1b (Ashfield et al., 1995); however, the evolutionary relationship shared by the two R genes is not known. The cluster is associated with numerous NB-LRR genes, of both the CC and TIR subgroups, spread over more than a megabase of soybean chromosome 13 (Peñuela et al., 2002; Hayes et al., 2004; Innes et al., 2008; Ashfield et al., 2012; Wen et al., 2013). The NB-LRR family in this region is evolving rapidly, with duplications/deletions of paralogs, recombination, and positive selection all playing a role (Ashfield et al., 2012).While soybean can distinguish between AvrB and AvrRpm1, both effectors are detected by a single R protein, RPM1, in Arabidopsis (Bisgrove et al., 1994; Grant et al., 1995). It is known that RPM1 recognizes the effector proteins indirectly by detecting effector-dependent phosphorylation of a second Arabidopsis protein, RIN4 (Mackey et al., 2002; Chung et al., 2011; Liu et al., 2011). The available evidence suggests that a related strategy is employed by soybean, at least for the Rpg1b protein, despite the AvrB recognition specificity having evolved independently in these plant species (Ashfield et al., 2004; Selote and Kachroo, 2010; Selote et al., 2013). Soybean contains four RIN4 homologs (Chen et al., 2010), three of which interact physically with Rpg1b, with two required for full resistance conferred by this R gene (Selote and Kachroo, 2010; Selote et al., 2013). It is not known whether RIN4 homologs are required for Rpg1r function.Here, we report the map-based cloning of the soybean Rpg1r gene. Comparison of the Rpg1r protein to Rpg1b, combined with structural modeling, revealed highly polymorphic surfaces in the CC and LRR domains. Transient expression of chimeric Rpg1 proteins demonstrated that specificity for AvrB versus AvrRpm1 is determined by the C-terminal LRR region. Finally, we provide evidence that Rpg1r, like Rpg1b, detects its corresponding pathogen effector indirectly, most likely by monitoring a RIN4 homolog, indicating convergent evolution of recognition mechanisms in separate plant families.     

Arabidopsis Actin-Depolymerizing Factor AtADF4 Mediates Defense Signal Transduction Triggered by the Pseudomonas syringae Effector AvrPphB

The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes with limited, indirect evidence. To date, there are no reports linking actin with resistance against phytopathogenic bacteria. The dynamic behavior of actin filaments is regulated by a diverse array of actin-binding proteins, among which is the Actin-Depolymerizing Factor (ADF) family of proteins. Here, we demonstrate that actin dynamics play a role in the activation of gene-for-gene resistance in Arabidopsis (Arabidopsis thaliana) following inoculation with the phytopathogenic bacterium Pseudomonas syringae pv tomato. Using a reverse genetics approach, we explored the roles of Arabidopsis ADFs in plant defenses. AtADF4 was identified as being specifically required for resistance triggered by the effector AvrPphB but not AvrRpt2 or AvrB. Recombinant AtADF4 bound to monomeric actin (G-actin) with a marked preference for the ADP-loaded form and inhibited the rate of nucleotide exchange on G-actin, indicating that AtADF4 is a bona fide actin-depolymerizing factor. Exogenous application of the actin-disrupting agent cytochalasin D partially rescued the Atadf4 mutant in the AvrPphB-mediated hypersensitive response, demonstrating that AtADF4 mediates defense signaling through modification of the actin cytoskeleton. Unlike the mechanism by which the actin cytoskeleton confers resistance against fungi and oomycetes, AtADF4 is not involved in resistance against pathogen entry. Collectively, this study identifies AtADF4 as a novel component of the plant defense signaling pathway and provides strong evidence for actin dynamics as a primary component that orchestrates plant defenses against P. syringae.The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes (Hardham et al., 2007). Evidence largely comes from studies using actin cytoskeleton-disrupting agents, such as cytochalasins. Treatments with a variety of cytochalasins were shown to increase the penetration rate of both adapted and nonadapted pathogens in multiple plant-pathogen systems, thereby implicating the actin cytoskeleton as having a role in basal defenses and nonhost resistance (Kobayashi et al., 1997; Yun et al., 2003; Shimada et al., 2006; Miklis et al., 2007). The actin cytoskeleton may also play a role in race-specific resistance (Skalamera and Heath, 1998). To date, no reports linking actin dynamics with resistance against phytopathogenic bacteria have been published.While the actin cytoskeleton as a virulence target of plant pathogens has not been documented, it was well characterized in mammalian pathosystems, particularly in studies investigating macrophage interactions with the pathogenic bacterium Yersinia pestis (Mattoo et al., 2007). Yersinia species deliver a suite of effectors into the target host cell, and at least four of them (YopE, YpkA/YopO, YopT, and YopH) are involved in rearrangement of the actin cytoskeleton (Aepfelbacher and Heesemann, 2001). YopT, a Cys protease, targets a plasma membrane-localized Rho GTPase in affected phagocytes (Aepfelbacher and Heesemann, 2001). Cleavage of the GTPase by YopT releases the prenylated protein from the plasma membrane and disrupts the actin cytoskeleton, effectively shutting down phagocytosis, preventing elimination of the pathogen (Iriarte and Cornelis, 1998; Shao et al., 2002). Similarly, microbial pathogens also usurp host processes for the benefit of infection, disease, and death. Listeria species hijack the host''s cytoskeleton to move around inside the infected cell through the induction of directed polymerization of actin (Pistor et al., 1994). Salmonella injects into host cells two actin-binding proteins (SipA and SipC) as well as other regulators of actin dynamics to enhance phagocytic uptake and intracellular propagation (Galan and Zhou, 2000). In short, either by preventing polymerization or by promoting it, pathogens have evolved strategies to modify the host actin cytoskeleton for purposes of evading detection or eliciting disease and death.Dynamic actin cytoskeleton rearrangements are regulated by a pool of actin-binding proteins, which sense environmental changes and modulate the cytoskeleton through various biochemical activities (Hussey et al., 2006; Staiger and Blanchoin, 2006). Among the proteins that regulate these dynamic processes are the Actin-Depolymerizing Factor (ADF) family of proteins (Maciver and Hussey, 2002). In general, ADFs bind both monomeric (G-) and filamentous (F-) actin to increase actin dynamics. They function by severing F-actin to generate more ends for polymerization and by increasing the dissociation rate of actin monomers from the pointed ends (Maciver, 1998; Maciver and Hussey, 2002). Plant ADFs play roles in pollen tube growth (Chen et al., 2003), root formation (Thomas and Schiefelbein, 2002), and cold acclimation (Ouellet et al., 2001). There is also one report linking ADFs with plant defenses (Miklis et al., 2007). In that study, ectopic expression of barley (Hordeum vulgare) HvADF3 and several isovariants of Arabidopsis (Arabidopsis thaliana) ADFs in barley epidermal cells was shown to compromise penetration resistance to powdery mildew fungi (Miklis et al., 2007).The Arabidopsis-Pseudomonas syringae interaction provides an ideal model plant-pathogen system to study plant defense signaling. Like Yersinia species, P. syringae delivers effector proteins into the host cells via the type III secretion system and relies on these proteins for pathogenesis (Alfano and Collmer, 2004). However, once these proteins (Avr) are recognized either directly or indirectly by plant resistance (R) proteins, plant immune responses are activated (Jones and Dangl, 2006). Exciting progress has been made toward understanding the indirect recognition of several pairs of Avr-R proteins; the best examples include AvrB/AvrRPM1-RPM1, AvrRpt2-RPS2, and AvrPphB-RPS5. During activation of defense mediated by AvrB/AvrRPM1-RPM1 and AvrRpt2-RPS2, the phosphorylation or elimination of a third protein, RIN4, is essential (Mackey et al., 2002; Axtell and Staskawicz, 2003). In the case of AvrPphB-RPS5 recognition, the AvrPphB Cys protease of the same family as YopT (Shao et al., 2002) cleaves the plant protein kinase PBS1, inducing a conformational change in RPS5, which in turn leads to the activation of resistance (Ade et al., 2007). Although these studies have greatly enhanced our understanding of how pathogen effectors initiate plant defense responses, the ultimate signaling processes associated with the activation of resistance remain largely unknown, due to the limited number of genetic loci identified in these pathways. In this work, we hypothesize that actin-binding proteins play a role during plant-bacteria interactions based on the functional and structural similarity between AvrPphB and YopT.There are 11 ADFs in the Arabidopsis genome (Ruzicka et al., 2007). We utilized a reverse genetics approach to identify the putative roles these proteins play in plant resistance against the bacterial pathogen P. syringae pv tomato (Pst). AtADF4 was identified as a novel signaling component in the AvrPphB-RPS5-mediated defense signal transduction pathway. Loss of AtADF4 confers on Arabidopsis enhanced susceptibility to P. syringae expressing AvrPphB. Further subcellular localization and biochemical analyses, as well as pharmacological studies, suggest that AtADF4 functions as a bona fide actin-depolymerizing factor through modifying the actin cytoskeleton. Unlike the documented mechanism by which the actin cytoskeleton plays roles in resistance against fungi and oomycetes, the resistance against P. syringae mediated by AtADF4 is not involved in hindering pathogen entry.     

Allele-Specific Virulence Attenuation of the Pseudomonas syringae HopZ1a Type III Effector via the Arabidopsis ZAR1 Resistance Protein

Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the ∼170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T–DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS–LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1–mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a.     

大豆下胚轴质膜H+-ATPase质子转运的测定

以大豆下胚轴为材料,采用改进的匀浆介质,通过两相法制得具有质子转运活力的高纯度质膜微囊.并且发现冻融处理可以促进质膜微囊的翻转而提高荧光猝灭效率.质子载体和质子转运特性分析表明,由Mg^2＋-ATP引发的荧光猝灭可以被质子载体CCCP恢复,并被质子通道抑制剂DCCD抑制;并且发现质膜H^＋-ATPase专一抑制剂钒酸钠可以完全抑制荧光猝灭,同时发现荧光猝灭依赖于Mg^2＋,并受K^＋刺激,最适pH为6.5.以上证明所测荧光猝灭是由质膜H^＋-ATPase所进行的质子转运引起的.结果同时表明,维持H^＋-ATPase合适构象和提高质膜微囊封闭性是制备具有H^＋转运活力质膜微囊的两个关键因素.     

Phospholipase A2 Is Required for PIN-FORMED Protein Trafficking to the Plasma Membrane in the Arabidopsis Root

Phospholipase A₂ (PLA₂), which hydrolyzes a fatty acyl chain of membrane phospholipids, has been implicated in several biological processes in plants. However, its role in intracellular trafficking in plants has yet to be studied. Here, using pharmacological and genetic approaches, the root hair bioassay system, and PIN-FORMED (PIN) auxin efflux transporters as molecular markers, we demonstrate that plant PLA₂s are required for PIN protein trafficking to the plasma membrane (PM) in the Arabidopsis thaliana root. PLA₂α, a PLA₂ isoform, colocalized with the Golgi marker. Impairments of PLA₂ function by PLA₂α mutation, PLA₂-RNA interference (RNAi), or PLA₂ inhibitor treatments significantly disrupted the PM localization of PINs, causing internal PIN compartments to form. Conversely, supplementation with lysophosphatidylethanolamine (the PLA₂ hydrolytic product) restored the PM localization of PINs in the pla₂α mutant and the ONO-RS-082–treated seedling. Suppression of PLA₂ activity by the inhibitor promoted accumulation of trans-Golgi network vesicles. Root hair–specific PIN overexpression (PINox) lines grew very short root hairs, most likely due to reduced auxin levels in root hair cells, but PLA₂ inhibitor treatments, PLA₂α mutation, or PLA₂-RNAi restored the root hair growth of PINox lines by disrupting the PM localization of PINs, thus reducing auxin efflux. These results suggest that PLA₂, likely acting in Golgi-related compartments, modulates the trafficking of PIN proteins.     

Membrane release and destabilization of Arabidopsis RIN4 following cleavage by Pseudomonas syringae AvrRpt2

The Arabidopsis RIN4 protein mediates interaction between the Pseudomonas syringae type III effector proteins AvrB, AvrRpm1, and AvrRpt2 and the Arabidopsis disease-resistance proteins RPM1 and RPS2. Confocal laser-scanning fluorescence microscopy following particle bombardment of tobacco leaf epidermal cells was used to examine the subcellular localization of fusions between GFP and RIN4 or several of its homologs and to examine the effects of cobombardment with AvrRpt2 or AvrRpml. This study showed that RIN4 was attached to the plasma membrane at its carboxyl terminus and that a carboxyl-terminal CCCFxFxxx prenylation or acylation (typically palmitoylation) motif, or both, was essential for this attachment. RIN4 was cleaved by AvrRpt2 at two PxFGxW motifs, one releasing a large portion of RIN4 from the plasma membrane and both exposing amino-terminal residues that destabilized the carboxyl-terminal cleavage products by targeting them for N-end ubiquitylation and proteasomal degradation. Plasma-membrane localization of RIN4 was not affected by AvrRpml. RIN4 was found to be part of a protein family comprising two full-length homologs and at least 11 short carboxyl-terminal homologs. Representatives of this family, comprising a full-length RIN4 homolog and two short carboxyl-terminal RIN4 homologs, were also attached to the plasma membrane and cleaved near their amino termini by AvrRpt2, but in contrast to RIN4, the major portions of these proteins remained on the plasma membrane. N-end degradation may play a minor role in RIN4 degradation but probably plays a major role in the degradation of RIN4 homologs and is, therefore, a major pathogenic consequence of AvrRpt2 cleavage.     

Manipulation of Mitogen-Activated Protein Kinase Kinase Signaling in the Arabidopsis Stomatal Lineage Reveals Motifs That Contribute to Protein Localization and Signaling Specificity

When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four “stomatal” MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant.     

Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca²⁺-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca²⁺-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.     

KDEL-Containing Auxin-Binding Protein Is Secreted to the Plasma Membrane and Cell Wall

The auxin-binding protein ABP1 has been postulated to mediate auxin-induced cellular changes associated with cell expansion. This protein contains the endoplasmic reticulum (ER) retention signal, the tetrapeptide lysine-aspartic acid-glutamic acid-leucine (KDEL), at its carboxy terminus, consistent with previous subcellular fractionation data that indicated an ER location for ABP1. We used electron microscopic immunocytochemistry to identify the subcellular localization of ABP1. Using maize (Zea mays) coleoptile tissue and a black Mexican sweet (BMS) maize cell line, we found that ABP1 is located in the ER as expected, but is also on or closely associated with the plasma membrane and within the cell wall. Labeling of the Golgi apparatus suggests that the transport of ABP1 to the cell wall occurs via the secretory system. Inhibition of secretion of an ABP homolog into the medium of BMS cell cultures by brefeldin A, a drug that specifically blocks secretion, is consistent with this secretion pathway. The secreted protein was recognized by an anti-KDEL peptide antibody, strongly supporting the interpretation that movement of this protein out of the ER does not involve loss of the carboxy-terminal signal. Cells starved for 2,4-dichlorophenoxyacetic acid for 72 h retained less ABP in the cell and secreted more of it into the medium. The significance of our observations is 2-fold. We have identified a KDEL-containing protein that specifically escapes the ER retention system, and we provide an explanation for the apparent discrepancy that most of the ABP is located in the ER, whereas ABP and auxin act at the plasma membrane.     

Degradation of Connexins from the Plasma Membrane Is Regulated by Inhibitors of Protein Synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Partial Purification and Characterization of An ATPase in Mung Bean Hypocotyl Plasma Membrane: Suggestion for A New Type of Higher Plant Plasma Membrane ATPase

A vanadate-sensitive and nitrate-resistant ATPase was solubilizedwith Zwittergent 3–14 from a highly purified plasma membranefraction of mung bean hypocotyls and partially purified by glyceroldensity gradient centrifugation and phenyl-Sepharose columnchromatography. Either phosphatidylcholine or phosphatidylserinein addition to Mg^{2 +} was required for the enzyme activity, whereasK⁺, phosphatidylethanolamine and lysophosphatidylcholine hadno effect on the activity. The purified enzyme preparation containedtwo major polypeptides with molecular masses of 67 and 55 kDaas analyzed by SDS-polyacrylamide gel electrophoresis. Whenthe plasma membrane fraction was incubated with [-³²P]ATP, a45-70-kDa polypeptide(s) was labeled, and the label could berapidly chased with cold ATP. When the fraction was incubatedwith [¹⁴C]N,N'-dicyclohexylcarbodiimide, an inhibitor for theATPase, a 15-20-kDa polypeptide was labeled. We propose thatthe enzyme is a new type of higher plant plasma membrane ATP-aseand is composed of 67- and 55-kDa subunits and probably alsoa 15-20-kDa subunit. ¹Present address: Takarazuka Institute, Sumitomo Chemical IndustriesLtd., Takatsukasa, Takarazuka, Hyogo 665, Japan (Received September 2, 1987; Accepted May 20, 1988)