Salicylic Acid Regulates Plasmodesmata Closure during Innate Immune Responses in Arabidopsis

In plants, mounting an effective innate immune strategy against microbial pathogens involves triggering local cell death within infected cells as well as boosting the immunity of the uninfected neighboring and systemically located cells. Although not much is known about this, it is evident that well-coordinated cell–cell signaling is critical in this process to confine infection to local tissue while allowing for the spread of systemic immune signals throughout the whole plant. In support of this notion, direct cell-to-cell communication was recently found to play a crucial role in plant defense. Here, we provide experimental evidence that salicylic acid (SA) is a critical hormonal signal that regulates cell-to-cell permeability during innate immune responses elicited by virulent bacterial infection in Arabidopsis thaliana. We show that direct exogenous application of SA or bacterial infection suppresses cell–cell coupling and that SA pathway mutants are impaired in this response. The SA- or infection-induced suppression of cell–cell coupling requires an ENHANCED DESEASE RESISTANCE1– and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1–dependent SA pathway in conjunction with the regulator of plasmodesmal gating PLASMODESMATA-LOCATED PROTEIN5. We discuss a model wherein the SA signaling pathway and plasmodesmata-mediated cell-to-cell communication converge under an intricate regulatory loop.     

Autophagy Negatively Regulates Cell Death by Controlling NPR1-Dependent Salicylic Acid Signaling during Senescence and the Innate Immune Response in Arabidopsis

Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. In higher plants, autophagy defects result in early senescence and excessive immunity-related programmed cell death (PCD) irrespective of nutrient conditions; however, the mechanisms by which cells die in the absence of autophagy have been unclear. Here, we demonstrate a conserved requirement for salicylic acid (SA) signaling for these phenomena in autophagy-defective mutants (atg mutants). The atg mutant phenotypes of accelerated PCD in senescence and immunity are SA signaling dependent but do not require intact jasmonic acid or ethylene signaling pathways. Application of an SA agonist induces the senescence/cell death phenotype in SA-deficient atg mutants but not in atg npr1 plants, suggesting that the cell death phenotypes in the atg mutants are dependent on the SA signal transducer NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. We also show that autophagy is induced by the SA agonist. These findings imply that plant autophagy operates a novel negative feedback loop modulating SA signaling to negatively regulate senescence and immunity-related PCD.     

Arabidopsis thaliana subclass I ACTIN DEPOLYMERIZING FACTORs and vegetative ACTIN2/8 are novel regulators of endoreplication

Journal of Plant Research - Endoreplication is a type of cell cycle where genome replication occurs without mitosis. An increase of ploidy level by endoreplication is often associated with cell...     

Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth

Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes.     

Signaling, Actin Dynamics and Endocytosis

Endocytosis is an essential process for normal function of all living cells. Cells get nutrients, and control the surface-expressional level of proteins as well as membrane hemostats through the endocytosis. Endocytosis process is regulated in response to functional status of a particular cell. Signaling events and the endocytosis process go hand in hand to fulfill cellular functions. Although our understanding of the endocytosis process has grown rapidly during the last decade, little is known about how it is interconnected functionally with the signaling status of cells. During endocytosis, vesicles are formed from the plasma membrane through complex molecular machinery. The location where the vesicles are formed is rich in cortical actin cytoskeleton that supports the plasma membrane. To enter cells, vesicles have to diffuse through the cortical actin cytoskeleton. The actin cytoskeleton has a very dynamic structure and actively participates a wide variety of cellular functions. In addition to its central role in cytokinesis, cell shape, cell motility, and cell polarity, a connection between the endocytosis process and the actin cytoskeleton has been implicated in both yeast and mammalian system. In recent years the knowledge on how the actin cytoskeleton participates in the generation of coordinated cellular responses to external stimuli is grown rapidly. In this review, we focus on the potential roles of the actin cytoskeleton in regulating the endocytosis process in response to signaling events.     

Chloroplast-to-Nucleus Signaling Regulates MicroRNA Biogenesis in Arabidopsis

1. Download high-res image (103KB)
2. Download full-size image

     

Sphingolipid Pathway Regulates Innate Immune Responses at the Fetomaternal Interface during Pregnancy

For a successful pregnancy, the mother''s immune system has to tolerate the semiallogeneic fetus. A deleterious immune attack is avoided by orchestration of cellular, hormonal, and enzymatic factors. However, the precise mechanisms underlying fetomaternal tolerance are not yet completely understood. In this study, we demonstrate that sphingolipid metabolism constitutes a novel signaling pathway that is indispensable for fetomaternal tolerance by regulating innate immune responses at the fetomaternal interface. Perturbation of the sphingolipid pathway by disruption of the sphingosine kinase gene (Sphk) during pregnancy caused unusually high expression of neutrophil chemoattractants, CXCL1 and CXCL2, in the decidua, leading to a massive infiltration of neutrophils into the fetomaternal interface with enhanced oxidative damage, resulting in early fetal death. Sphk-deficient mice also exhibited neutrophilia in the peripheral blood, enhanced generation of granulocytes in the bone marrow, and a decrease in the number of decidual natural killer cells. The blockage of neutrophil influx protected Sphk-deficient mice against pregnancy loss. Notably, a similar result was obtained in human decidual cells, in which Sphk deficiency dramatically increased the secretion of CXCL1 and IL-8. In conclusion, our findings suggest that the sphingolipid metabolic pathway plays a critical role in fetomaternal tolerance by regulating innate immunity at the fetomaternal interface both in mice and humans, and it could provide novel insight into the development of therapeutic strategies to treat idiopathic pregnancy loss in humans.     

Loss of Arabidopsis thaliana Dynamin-Related Protein 2B Reveals Separation of Innate Immune Signaling Pathways

Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC ⁻, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca²⁺-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC⁻. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.     

Dying Neurons Utilize Innate Immune Signaling to Prime Glia for Phagocytosis during Development

1. Download high-res image (192KB)
2. Download full-size image

     

Calpain Regulates Actin Remodeling during Cell Spreading

Previous studies suggest that the Ca²⁺-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3– derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.     

Collapsin Response Mediator Protein 4 Regulates Growth Cone Dynamics through the Actin and Microtubule Cytoskeleton

Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4−/− mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4−/− mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton.     

Innate Immune Detection of Flagellin Positively and Negatively Regulates Salmonella Infection

Salmonella enterica serovar Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is required for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by at least two independent pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems for host defense against wild type Salmonella infection is complex, and innate immune detection of flagellin contributes to both protection and susceptibility. We hypothesized that efficient modulation of flagellin expression in vivo permits Salmonella to evade innate immune detection and limit the functional role of flagellin-specific host innate defenses. To test this hypothesis, we used Salmonella deficient in the anti-sigma factor flgM, which overproduce flagella and are attenuated in vivo. In this study we demonstrate that flagellin recognition by the innate immune system is responsible for the attenuation of flgM⁻ S. Typhimurium, and dissect the contribution of each flagellin recognition pathway to bacterial clearance and inflammation. We demonstrate that caspase-1 controls mucosal and systemic infection of flgM⁻ S. Typhimurium, and also limits intestinal inflammation and injury. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum and systemic infection, but attenuates intestinal inflammation. Our results indicate that Salmonella evasion of caspase-1 dependent flagellin recognition is critical for establishing infection and that evasion of TLR5 and caspase-1 dependent flagellin recognition helps Salmonella induce intestinal inflammation and establish a niche in the inflamed gut.     

Interdependence of Endomembrane Trafficking and Actin Dynamics during Polarized Growth of Arabidopsis Pollen Tubes

During polarized growth of pollen tubes, endomembrane trafficking and actin polymerization are two critical processes that establish membrane/wall homeostasis and maintain growth polarity. Fine-tuned interactions between these two processes are therefore necessary but poorly understood. To better understand such cross talk in the model plant Arabidopsis (Arabidopsis thaliana), we first established optimized concentrations of drugs that interfere with either endomembrane trafficking or the actin cytoskeleton, then examined pollen tube growth using fluorescent protein markers that label transport vesicles, endosomes, or the actin cytoskeleton. Both brefeldin A (BFA) and wortmannin disturbed the motility and structural integrity of ARA7- but not ARA6-labeled endosomes, suggesting heterogeneity of the endosomal populations. Disrupting endomembrane trafficking by BFA or wortmannin perturbed actin polymerization at the apical region but not in the longitudinal actin cables in the shank. The interference of BFA/wortmannin with actin polymerization was progressive rather than rapid, suggesting an indirect effect, possibly due to perturbed endomembrane trafficking of certain membrane-localized signaling proteins. Both the actin depolymerization drug latrunculin B and the actin stabilization drug jasplakinolide rapidly disrupted transport of secretory vesicles, but each drug caused distinct responses on different endosomal populations labeled by ARA6 or ARA7, indicating that a dynamic actin cytoskeleton was critical for some steps in endomembrane trafficking. Our results provide evidence of cross talk between endomembrane trafficking and the actin cytoskeleton in pollen tubes.Pollen tubes of flowering plants are specialized cells that deliver immotile sperm to the proximity of female gametes for successful reproduction (Johnson and Preuss, 2002). The growth of pollen tubes is both polar and directional (Hepler et al., 2001); many cellular activities contribute to such growth, the most important being the dynamics of the actin cytoskeleton system, targeted exocytosis, and endocytosis (Hepler et al., 2001).Pollen tubes contain longitudinal actin cables along the shank, which are important for providing structural support and acting as tracks for the movement of large organelles (Staiger et al., 1994). The apical area of pollen tubes instead contains dynamic filamentous actin (F-actin), as shown by fluorescently labeled actin-binding proteins (Kost et al., 1999; Fu et al., 2001; Chen et al., 2002; Wilsen et al., 2006). The dynamics of F-actin are critical for the polarized growth of pollen tubes. Genetically manipulating the activities of the small GTPases ROP (Kost et al., 1999; Fu et al., 2001; Cheung et al., 2008) and Rab (de Graaf et al., 2005), or of actin-binding proteins such as profilin and formin (Staiger et al., 1994; Chen et al., 2002; Cheung and Wu, 2004), disrupted F-actin dynamics and inhibited tube growth and caused apical bulges. Application of drugs such as latrunculin B (LatB) and jasplakinolide (Jas) showed similar effects (Gibbon et al., 1999; Vidali et al., 2001; Cardenas et al., 2005; Hörmanseder et al., 2005; Chen et al., 2007).Targeted exocytosis delivers building materials for cell membranes and cell walls and therefore is critical for maintaining growth polarity and directionality of growing pollen tubes (Hepler et al., 2001). Because targeted exocytosis brings more membrane and wall materials than needed to the apex of a pollen tube, an active endocytic system exists to retrieve excess secreted materials. In addition to this nonselective bulk membrane retrieval, pollen tubes may have selective and regulated endocytic trafficking pathways. For example, experiments using charged gold particles indicated the existence of two distinct endocytic pathways in tobacco (Nicotiana tabacum) pollen tubes (Moscatelli et al., 2007), and other studies showed that pollen tubes are able to take in materials from the extracellular matrix (Lind et al., 1996; Goldraij et al., 2006). The axis of targeted exocytosis correlated with the direction of tube growth and it asymmetrically changed toward the new apex during tube reorientation (Camacho and Malho, 2003; de Graaf et al., 2005). Disruption of membrane trafficking altered growth trajectories (de Graaf et al., 2005). Both suggest that membrane trafficking is a critical part of polarity maintenance and reorientation.As two important cellular processes in pollen tube growth, membrane trafficking and actin polymerization are conceivably dependent on each other. For example, several studies demonstrated that dynamic actin polymerization was essential for membrane trafficking (Hörmanseder et al., 2005; Wang et al., 2005; Chen et al., 2007; Lee et al., 2008), while others explored whether membrane trafficking affected actin polymerization (de Graaf et al., 2005; Hörmanseder et al., 2005). These studies, however, were mostly done with rapidly growing pollen tubes from tobacco or lily (Lilium longiflorum). For the model plant Arabidopsis (Arabidopsis thaliana), whose pollen tubes grow slower, little is known in this regard. Given a robust protocol for Arabidopsis pollen germination (Boavida and McCormick, 2007), it is now possible to investigate the interactions between these two cellular activities.In this study, we analyzed the effects of drug treatments on Arabidopsis pollen tubes expressing fluorescent protein probes for transport vesicles, endosomes, or the actin cytoskeleton. We show that perturbing actin dynamics by LatB or Jas treatments disrupted the V-shaped distribution of transport vesicles, caused aggregation, and finally dissipation of a subpopulation of endosomes, indicating that actin dynamics are critical at some steps of endomembrane trafficking. On the other hand, disturbing endomembrane trafficking with brefeldin A (BFA) or wortmannin abolished the F-actin structure at the apical region without affecting the longitudinal actin cables at the shank. These results provide evidence that endomembrane trafficking and actin dynamics interact at certain steps during polarized growth of Arabidopsis pollen tubes.     

斑马鱼TAK1正向调控IRF7介导的天然免疫反应

在鱼类中关于转化生长因子β-激活激酶1(TAK1)在天然免疫反应中的功能研究较少。为了探究TAK1在斑马鱼天然免疫中的功能,本文克隆并获得了一种斑马鱼tak1剪接异构体(Drtak1),其开放阅读框含有1737个核苷酸,编码578个氨基酸,其中包括N端的丝氨酸/苏氨酸蛋白激酶结构域和C端的卷曲螺旋结构部。通过免疫荧光试验,证实DrTAK1是一种胞质蛋白。双荧光素酶报告试验显现在EPC细胞中单转DrTAK1不能诱导IFN的产生,但与IRF7共转时能显著提高其诱导干扰素启动子表达的能力。本文研究结果首次在斑马鱼中发现TAK1能正向调控IRF7介导的天然免疫反应,为后续DrTAK1功能研究奠定了基础。     

Modeling and Dynamical Analysis of Virus-Triggered Innate Immune Signaling Pathways

The investigation of the dynamics and regulation of virus-triggered innate immune signaling pathways at a system level will enable comprehensive analysis of the complex interactions that maintain the delicate balance between resistance to infection and viral disease. In this study, we developed a delayed mathematical model to describe the virus-induced interferon (IFN) signaling process by considering several key players in the innate immune response. Using dynamic analysis and numerical simulation, we evaluated the following predictions regarding the antiviral responses: (1) When the replication ratio of virus is less than 1, the infectious virus will be eliminated by the immune system’s defenses regardless of how the time delays are changed. (2) The IFN positive feedback regulation enhances the stability of the innate immune response and causes the immune system to present the bistability phenomenon. (3) The appropriate duration of viral replication and IFN feedback processes stabilizes the innate immune response. The predictions from the model were confirmed by monitoring the virus titer and IFN expression in infected cells. The results suggest that the balance between viral replication and IFN-induced feedback regulation coordinates the dynamical behavior of virus-triggered signaling and antiviral responses. This work will help clarify the mechanisms of the virus-induced innate immune response at a system level and provide instruction for further biological experiments.