Requirement of vesicle-associated membrane protein 721 and 722 for sustained growth during immune responses in Arabidopsis

Extracellular immune responses to ascomycete and oomycete pathogens in Arabidopsis are dependent on vesicle-associated secretion mediated by the SNARE proteins PEN1 syntaxin, SNAP33 and endomembrane-resident VAMP721/722. Continuous movement of functional GFP-VAMP722 to and from the plasma membrane in non-stimulated cells reflects the second proposed function of VAMP721/722 in constitutive secretion during plant growth and development. Application of the bacterium-derived elicitor flg22 stabilizes VAMP721/722 that are otherwise constitutively degraded via the 26S proteasome pathway. Depletion of VAMP721/722 levels by reducing VAMP721/722 gene dosage enhances flg22-induced seedling growth inhibition in spite of elevated VAMP721/722 abundance. We therefore propose that plants prioritize the deployment of the corresponding secretory pathway for defense over plant growth. Interstingly, VAMP721/722 specifically interact in vitro and in vivo with the plasma membrane syntaxin SYP132 that is required for plant growth and resistance to bacteria. This suggests that the plant growth/immunity-involved VAMP721/722 form SNARE complexes with multiple plasma membrane syntaxins to discharge cue-dependent cargo molecules.     

Arabidopsis R-SNARE proteins VAMP721 and VAMP722 are required for cell plate formation

Background

Cell plate formation during plant cytokinesis is facilitated by SNARE complex-mediated vesicle fusion at the cell-division plane. However, our knowledge regarding R-SNARE components of membrane fusion machinery for cell plate formation remains quite limited.

Methodology/Principal Findings

We report the in vivo function of Arabidopsis VAMP721 and VAMP722, two closely sequence-related R-SNAREs, in cell plate formation. Double homozygous vamp721vamp722 mutant seedlings showed lethal dwarf phenotypes and were characterized by rudimentary roots, cotyledons and hypocotyls. Furthermore, cell wall stubs and incomplete cytokinesis were frequently observed in vamp721vamp722 seedlings. Confocal images revealed that green fluorescent protein-tagged VAMP721 and VAMP722 were preferentially localized to the expanding cell plates in dividing cells. Drug treatments and co-localization analyses demonstrated that punctuate organelles labeled with VAMP721 and VAMP722 represented early endosomes overlapped with VHA-a1-labeled TGN, which were distinct from Golgi stacks and prevacuolar compartments. In addition, protein traffic to the plasma membrane, but not to the vacuole, was severely disrupted in vamp721vamp722 seedlings by subcellular localization of marker proteins.

Conclusion/Significance

These observations suggest that VAMP721 and VAMP722 are involved in secretory trafficking to the plasma membrane via TGN/early endosomal compartment, which contributes substantially to cell plate formation during plant cytokinesis.     

Luminescence detection of SNARE–SNARE interaction in Arabidopsis protoplasts

Membrane associated proteins SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) provide the minimal fusion machinery necessary for cellular vesicles to fuse to target organelle membranes in eukaryotic cells. Despite the conserved nature of the fusion machinery in all eukaryotes, it still remains challenging to identify functional SNARE pairs in higher plants. We developed a method based on a split-luciferase complementation assay for detecting changes in SNARE–SNARE interaction by luminescence within Arabidopsis protoplasts that express recombinant proteins at physiological levels in 96-well plates. The reliability of the assay was confirmed by three experiments. First, reduction of the SNARE–SNARE interaction caused by a single amino acid substitution adjacent to the SNARE motif in endosome-localized AtVAM3/SYP22 (syntaxin of plant 22) was detected by a reduction of luminescence. Second, reduction of the interaction between plasma-membrane localized SYP121 and VAMP722 in response to sodium azide was detected in real-time. Third, the results of 21 SNARE pairs investigated by this method largely agreed with the results from previously reported co-immunoprecipitation assays. Using the method, we newly identified the interaction between SYP121 and VAMP722 was significantly increased when the protoplasts were incubated in the light. Microscopic observation of transgenic Arabidopsis expressing GFP–SYP121 (green fluorescent protein tagged SYP121) from its own promoter suggested that the plasma-membrane localization of GFP–SYP121 is maintained by light. These suggested that the vesicle trafficking pathway mediated by SYP121 might be regulated by light in Arabidopsis. In general, this article demonstrated the method that can generate new biological insight of the SNARE protein interactions in plant cells.     

Arabidopsis Sec1/Munc18 Protein SEC11 Is a Competitive and Dynamic Modulator of SNARE Binding and SYP121-Dependent Vesicle Traffic

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.     

Endoplasmic reticulum stress-induced accumulation of VAMP721/722 requires CALRETICULIN 1 and CALRETICULIN 2 in Arabidopsis

Excessive demand for translation and protein folding in the endoplasmic reticulum(ER) can cause ER stress in plants. Here, we show that CALRETICULIN 1(CRT1) and CRT2 are critical components in the accumulation of VESICLE-ASSOCIATED MEMBRANE PROTEIN 721(VAMP721) and VAMP722 during ER stress responses. We show that CRT2 interacts with VAMP722 and that CRT1/2 post-translationally maintain elevated VAMP721/722 levels under ER stress.The greater growth inhibition in VAMP721/722-deficient plants, induced by tunicamycin, suggests that plants under ER stress maintain physiological homeostasis, at least in part, by regulating VAMP721/722 levels, as VAMP721/722 are known to participate in various biological processes.     

Modulation of ROS production and hormone levels by AHK5 during abiotic and biotic stress signaling

Histidine kinases have been shown to mediate responses to endogenous and exogenous stimuli in organisms such as yeast, bacteria and plants. In the model plant Arabidopsis, histidine kinases have been shown to function in hormone signaling, and abiotic and biotic stress responses. More recently, the least characterized of the Arabidopsis histidine kinases, AHK5, was demonstrated to function in resistance toward the virulent bacterium Pseudomonas syringae pv tomato DC3000 (PstDC3000) and the necrotrophic fungus Botrytis cinerea, and as a negative regulator of tolerance toward salinity. Here, we present data which indicate that AHK5 also impacts on drought stress resistance and on the outcome of an incompatible interaction with avrRpm1-expressing PstDC3000 (PstDC3000 (avrRpm1)). We present a model which proposes a role for reactive oxygen species (ROS) and hormones in integrating abiotic and biotic stress responses via AHK5.     

The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes

Plants employ multiple cell‐autonomous defense mechanisms to impede pathogenesis of microbial intruders. Previously we identified an exocytosis defense mechanism in Arabidopsis against pathogenic powdery mildew fungi. This pre‐invasive defense mechanism depends on the formation of ternary protein complexes consisting of the plasma membrane‐localized PEN1 syntaxin, the adaptor protein SNAP33 and closely sequence‐related vesicle‐resident VAMP721 or VAMP722 proteins. The Arabidopsis thaliana resistance to powdery mildew 8.2 protein (RPW8.2) confers disease resistance against powdery mildews upon fungal entry into host cells and is specifically targeted to the extrahaustorial membrane (EHM), which envelops the haustorial complex of the fungus. However, the secretory machinery involved in trafficking RPW8.2 to the EHM is unknown. Here we report that RPW8.2 is transiently located on VAMP721/722 vesicles, and later incorporated into the EHM of mature haustoria. Resistance activity of RPW8.2 against the powdery mildew Golovinomyces orontii is greatly diminished in the absence of VAMP721 but only slightly so in the absence of VAMP722. Consistent with this result, trafficking of RPW8.2 to the EHM is delayed in the absence of VAMP721. These findings implicate VAMP721/722 vesicles as key components of the secretory machinery for carrying RPW8.2 to the plant–fungal interface. Quantitative fluorescence recovery after photobleaching suggests that vesicle‐mediated trafficking of RPW8.2–yellow fluorescent protein (YFP) to the EHM occurs transiently during early haustorial development and that lateral diffusion of RPW8.2–YFP within the EHM exceeds vesicle‐mediated replenishment of RPW8.2–YFP in mature haustoria. Our findings imply the engagement of VAMP721/722 in a bifurcated trafficking pathway for pre‐invasive defense at the cell periphery and post‐invasive defense at the EHM.     

The analysis of mutant phenotypes and tissue expression reveals a role of SNAREs VAMP721 and VAMP722 in seedling growth

Membrane traffic mediated by a soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex contributes to plant growth and development. However, the functional significance of SNAREs involved in cell wall deposition and seedling development has not been sufficiently explored. In this study, we explored the roles of R-SNAREs VAMP721 (At1g04750) and VAMP722 (At2g33120) in seedling growth of Arabidopsis thaliana by histochemical staining, fluorescence labeling, and analyzing mutant phenotypes. Our results show a massive intracellular accumulation of cellulose and callose, and an abnormal deposition of callose at the expanding cell plate in vamp721vamp722 root cells compared with the wild type. Particularly, ectopic lignin accumulation was also observed in vamp721vamp722 root cells. The alteration of cell wall components was confirmed using Fourier transform infrared analysis. Plasma membrane integrity and cell viability were disturbed in the vamp721vamp722 seedling. Morphological observation shows that vamp721vamp722 mutations impaired development of roots, hypocotyl, cotyledon, and true leaf, and inhibited lateral root formation. Confocal images reveal that green fluorescent protein-tagged VAMP721 and VAMP722 showed a similar expression pattern and were expressed throughout all cells and tissues examined, including root and shoot apical meristems and cells of hypocotyls, cotyledons, and true leaves. Taken together, our results suggest that membrane traffic mediated by VAMP721 and VAMP722 is involved in seedling growth in A. thaliana.     

The impact of cytokinin on jasmonate-salicylate antagonism in Arabidopsis immunity against infection with Pst DC3000

Cytokinin has long been shown to be an essential modulator of growth and development in plants. However, its implications in plant immunity have only recently been realized. The interaction between jasmonate and salicylate pathways is regarded as a central backbone of plant immune defense. However, the effect of cytokinin on the jasmonate and salicylate mediated balance in plant immunity is still not known. Here, we analyze the impact of cytokinin on the jasmonate-salicylate antagonism in Arabidopsis immunity regarding infection with hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Systems biology analysis of a refined hormone immune pathway model provides insights into the impact of cytokinin on the balance between jasmonate and salicylic acid pathways in Arabidopsis. Targeted experiments validate model simulations monitoring bacterial growth in wild type plants as well as in jasmonate pathway mutants. An integrated analysis shows that CK promotes the SA pathway of plant immunity and does not promote JA-mediated Arabidopsis susceptibility against infection with Pst DC3000. Finally, we discuss these results in the context of an emerging model of auxin-cytokinin antagonism in plant immunity.     

Tomato SlSAP3, a member of the stress-associated protein family,is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000

Tomato stress-associated proteins (SAPs) belong to A20/AN1 zinc finger protein family, some of which have been shown to play important roles in plant stress responses. However, little is known about the functions and underlying molecular mechanisms of SAPs in plant immune responses. In the present study, we reported the function of tomato SlSAP3 in immunity to Pseudomonas syringae pv. tomato (Pst) DC3000. Silencing of SlSAP3 attenuated while overexpression of SlSAP3 in transgenic tomato increased immunity to Pst DC3000, accompanied with reduced and increased Pst DC3000-induced expression of SA signalling and defence genes, respectively. Flg22-induced reactive oxygen species (ROS) burst and expression of PAMP-triggered immunity (PTI) marker genes SlPTI5 and SlLRR22 were strengthened in SlSAP3-OE plants but were weakened in SlSAP3-silenced plants. SlSAP3 interacted with two SlBOBs and the A20 domain in SlSAP3 is critical for the SlSAP3-SlBOB1 interaction. Silencing of SlBOB1 and co-silencing of all three SlBOB genes conferred increased resistance to Pst DC3000, accompanied with increased Pst DC3000-induced expression of SA signalling and defence genes. These data demonstrate that SlSAP3 acts as a positive regulator of immunity against Pst DC3000 in tomato through the SA signalling and that SlSAP3 may exert its function in immunity by interacting with other proteins such as SlBOBs, which act as negative regulators of immunity against Pst DC3000 in tomato.     

A SNARE complex unique to seed plants is required for protein storage vacuole biogenesis and seed development of Arabidopsis thaliana

The SNARE complex is a key regulator of vesicular traffic, executing membrane fusion between transport vesicles or organelles and target membranes. A functional SNARE complex consists of four coiled-coil helical bundles, three of which are supplied by Q-SNAREs and another from an R-SNARE. Arabidopsis thaliana VAMP727 is an R-SNARE, with homologs only in seed plants. We have found that VAMP727 colocalizes with SYP22/ VAM3, a Q-SNARE, on a subpopulation of prevacuolar compartments/endosomes closely associated with the vacuolar membrane. Genetic and biochemical analyses, including examination of a synergistic interaction of vamp727 and syp22 mutations, histological examination of protein localization, and coimmunoprecipitation from Arabidopsis lysates indicate that VAMP727 forms a complex with SYP22, VTI11, and SYP51 and that this complex plays a crucial role in vacuolar transport, seed maturation, and vacuole biogenesis. We suggest that the VAMP727 complex mediates the membrane fusion between the prevacuolar compartment and the vacuole and that this process has evolved as an essential step for seed development.     

Differential metabolomic responses of PAMP-triggered immunity and effector-triggered immunity in Arabidopsis suspension cells

Introduction

The rhizobacterial tomato pathogen Pseudomonas syringae pv. tomato str. DC3000 (PstDC3000), like many plant pathogenic bacteria, can elicit hypersensitive response in non-host plant cells. PstDC3000 uses a type III protein secretion system (T3SS) to deliver effector proteins.

Objectives

We compared metabolomic responses of Arabidopsis suspension cells to a wild-type PstDC3000, a T3SS deletion mutant PstDC3000D28E, and a pathogen associated molecular pattern (PAMP) flagellin’s N-terminal domain’s 22-aa peptide (flg22) to obtain metabolomics insights into the plant cell PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI).

Methods

Using targeted HPLC-MRM-MS and untargeted GC-MS approaches, we monitored qualitative and quantitative changes of 312 metabolites in central and specialized metabolic pathways in a time-course study.

Results

The overall metabolomic changes induced by the three treatments included phenylpropanoid, flavonoid, and phytohormone biosynthetic pathways, as well as primary metabolism in amino acid and sugar biosynthesis. In addition to shared metabolites, flg22, PstDC3000D28E and PstDC3000 each caused unique metabolite changes in the course of the development of PTI and ETI.

Conclusion

PstDC3000D28E triggered PTI responses were different from those of flg22. This study has not only revealed the discernible metabolomics features associated with the flg22, PstDC3000D28E and PstDC3000 treatments, but also laid a foundation toward further understanding of metabolic regulation and responses underlying plant PTI and ETI.

     

SNARE proteins VAMP721 and VAMP722 mediate the post-Golgi trafficking required for auxin-mediated development in Arabidopsis

The plant hormone auxin controls many aspects of plant development. Membrane trafficking processes, such as secretion, endocytosis and recycling, regulate the polar localization of auxin transporters in order to establish an auxin concentration gradient. Here, we investigate the function of the Arabidopsis thaliana R-SNAREs VESICLE-ASSOCIATED MEMBRANE PROTEIN 721 (VAMP721) and VAMP722 in the post-Golgi trafficking required for proper auxin distribution and seedling growth. We show that multiple growth phenotypes, such as cotyledon development, vein patterning and lateral root growth, were defective in the double homozygous vamp721 vamp722 mutant. Abnormal auxin distribution and root patterning were also observed in the mutant seedlings. Fluorescence imaging revealed that three auxin transporters, PIN-FORMED 1 (PIN1), PIN2 and AUXIN RESISTANT 1 (AUX1), aberrantly accumulate within the cytoplasm of the double mutant, impairing the polar localization at the plasma membrane (PM). Analysis of intracellular trafficking demonstrated the involvement of VAMP721 and VAMP722 in the endocytosis of FM4-64 and the secretion and recycling of the PIN2 transporter protein to the PM, but not its trafficking to the vacuole. Furthermore, vamp721 vamp722 mutant roots display enlarged trans-Golgi network (TGN) structures, as indicated by the subcellular localization of a variety of marker proteins and the ultrastructure observed using transmission electron microscopy. Thus, our results suggest that the R-SNAREs VAMP721 and VAMP722 mediate the post-Golgi trafficking of auxin transporters to the PM from the TGN subdomains, substantially contributing to plant growth.     

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.     

The Arabidopsis R-SNARE VAMP721 Interacts with KAT1 and KC1 K+ Channels to Moderate K+ Current at the Plasma Membrane

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis, and pathogen defense. A subset of SNAREs, including the Arabidopsis thaliana SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K⁺ channels and to moderate their gating at the plasma membrane. Here, we identify a second subset of SNAREs that interact to control these K⁺ channels, but with opposing actions on gating. We show that VAMPs (vesicle-associated membrane proteins), which target vesicles to the plasma membrane, also interact with and suppress the activities of the inward-rectifying K⁺ channels KAT1 and KC1. Interactions were evident in yeast split-ubiquitin assays, they were recovered in vivo by ratiometric bimolecular fluorescence complementation, and they were sensitive to mutation of a single residue, Tyr-57, within the longin domain of VAMP721. Interaction was also recovered on exchange of the residue at this site in the homolog VAMP723, which normally localizes to the endoplasmic reticulum and otherwise did not interact. Functional analysis showed reduced channel activity and alterations in voltage sensitivity that are best explained by a physical interaction with the channel gates. These actions complement those of SYP121, a cognate SNARE partner of VAMP721, and lead us to propose that the channel interactions reflect a “hand-off” in channel control between the two SNARE proteins that is woven together with vesicle fusion.