STARCH-EXCESS4 Is a Laforin-Like Phosphoglucan Phosphatase Required for Starch Degradation in Arabidopsis thaliana

Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear α-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases α-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism.     

Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

Background

Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.

Methodology/Principal Findings

We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.

Conclusions/Significance

Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.     

PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis

The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.     

拟南芥温度诱导脂质运载蛋白TIL1参与雌配子体发育

雌配子体的正常发育是种子形成的前提条件之一,拟南芥温度诱导的脂质运载蛋白编码基因TIL1突变使胚珠败育,结实率下降明显。基因表达分析表明T-DNA插入使得TIL1基因敲除,突变体TIL1基因功能缺失;互交实验、Alexander染色、花粉离体培养和胚珠透明实验结果表明till-1突变体雄配子体发育正常、雌配子体胚囊发育有缺陷;通过遗传互补实验证明外源克隆的TIL1基因能恢复突变体的败育表型,并确定了TIL1基因主要在胚珠的胚囊中表达。实验结果表明TIL1基因参与了植物雌配子体发育这一重要的生理过程。     

Arabidopsis Cell Division Cycle 20.1 Is Required for Normal Meiotic Spindle Assembly and Chromosome Segregation

Cell division requires proper spindle assembly; a surveillance pathway, the spindle assembly checkpoint (SAC), monitors whether the spindle is normal and correctly attached to kinetochores. The SAC proteins regulate mitotic chromosome segregation by affecting CDC20 (Cell Division Cycle 20) function. However, it is unclear whether CDC20 regulates meiotic spindle assembly and proper homolog segregation. Here, we show that the Arabidopsis thaliana CDC20.1 gene is indispensable for meiosis and male fertility. We demonstrate that cdc20.1 meiotic chromosomes align asynchronously and segregate unequally and the metaphase I spindle has aberrant morphology. Comparison of the distribution of meiotic stages at different time points between the wild type and cdc20.1 reveals a delay of meiotic progression from diakinesis to anaphase I. Furthermore, cdc20.1 meiocytes exhibit an abnormal distribution of a histone H3 phosphorylation mark mediated by the Aurora kinase, providing evidence that CDC20.1 regulates Aurora localization for meiotic chromosome segregation. Further evidence that CDC20.1 and Aurora are functionally related was provided by meiosis-specific knockdown of At-Aurora1 expression, resulting in meiotic chromosome segregation defects similar to those of cdc20.1. Taken together, these results suggest a critical role for CDC20.1 in SAC-dependent meiotic chromosome segregation.     

Maternal Control of PIN1 Is Required for Female Gametophyte Development in Arabidopsis

Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of PIN1 and PIN3 during ovule development we have used mutant alleles of both genes and also perturbed PIN1 and PIN3 expression using micro-RNAs controlled by the ovule specific DEFH9 (DEFIFICENS Homologue 9) promoter. PIN1 down-regulation and pin1-5 mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or PIN1-silenced plants. We show that sporophytically expressed PIN1 is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.     

Metabolic Regulation by the Mitochondrial Phosphatase PTPMT1 Is Required for Hematopoietic Stem Cell Differentiation

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Highlights? PTPMT1 depletion causes cell cycle delay and differentiation block in HSCs ? The HSC pool in PTPMT1 knockout mice is drastically (～40-fold) expanded ? Mitochondrial metabolism is altered and AMPK is highly activated in knockout HSCs ? PTPMT1 PIP substrates directly enhance fatty-acid-induced activation of UCP2     

Calyculin A Sensitive Protein Phosphatase Is Required for Bacillus anthracis Lethal Toxin Induced Cytotoxicity

Previous studies have shown that the Bacillus anthracis lethal toxin can induce both necrosis and apoptosis in mouse macrophage-like J774A.1 cells depending on both the toxin concentration and the phosphatase activity. In this study several protein kinase or phosphatase inhibitors were employed to evaluate the hypothesis that the lethal toxin induces cell death via protein phosphorylation processes. Pretreatment with a serine/threonine phosphatase inhibitor Calyculin A (300 nM) could inhibit about 78% of cell death induced by the lethal toxin, whereas inhibitors of kinases, such as H7, HA, Sphingosine, and Genestein, but other inhibitors of phosphatases, such as Okadaic acid, Tautomycin, and Cyclosporin A, did not. In addition, recent reports have demonstrated that the MEK1 protein may serve as a proteolytic target within its N-terminus for lethal factor cleavage. In this study, Calyculin A is shown to enhance the phosphorylation of the MEK1 protein. This prevents the cleavage of the MEK1 by lethal factor. These results suggest that a putative Calyculin A-sensitive protein phosphatase is involved in anthrax toxin induced cytotoxicity and that the blocking effect of Calyculin A on lethal factor cytotoxicity may be mediated through the MEK signaling pathway. Received: 27 December 2000 / Accepted: 1 June 2001     

Arabidopsis thaliana Glyoxalase 2-1 Is Required during Abiotic Stress but Is Not Essential under Normal Plant Growth

The glyoxalase pathway, which consists of the two enzymes, GLYOXALASE 1 (GLX 1) (E.C.: 4.4.1.5) and 2 (E.C.3.1.2.6), has a vital role in chemical detoxification. In Arabidopsis thaliana there are at least four different isoforms of glyoxalase 2, two of which, GLX2-1 and GLX2-4 have not been characterized in detail. Here, the functional role of Arabidopsis thaliana GLX2-1 is investigated. Glx2-1 loss-of-function mutants and plants that constitutively over-express GLX2-1 resemble wild-type plants under normal growth conditions. Insilico analysis of publicly available microarray datasets with ATTEDII, Mapman and Genevestigator indicate potential role(s) in stress response and acclimation. Results presented here demonstrate that GLX2-1 gene expression is up-regulated in wild type Arabidopsis thaliana by salt and anoxia stress, and by excess L-Threonine. Additionally, a mutation in GLX2-1 inhibits growth and survival during abiotic stresses. Metabolic profiling studies show alterations in the levels of sugars and amino acids during threonine stress in the plants. Elevated levels of polyamines, which are known stress markers, are also observed. Overall our results suggest that Arabidopsis thaliana GLX2-1 is not essential during normal plant life, but is required during specific stress conditions.     

DEX1, a Novel Plant Protein, Is Required for Exine Pattern Formation during Pollen Development in Arabidopsis

To identify factors that are required for proper pollen wall formation, we have characterized the T-DNA-tagged, dex1 mutation of Arabidopsis, which results in defective pollen wall pattern formation. This study reports the isolation and molecular characterization of DEX1 and morphological and ultrastructural analyses of dex1 plants. DEX1 encodes a novel plant protein that is predicted to be membrane associated and contains several potential calcium-binding domains. Pollen wall development in dex1 plants parallels that of wild-type plants until the early tetrad stage. In dex1 plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. Sporopollenin is produced and randomly deposited on the plasma membrane in dex1 plants. However, it does not appear to be anchored to the microspore and forms large aggregates on the developing microspore and the locule walls. Based on the structure of DEX1 and the phenotype of dex1 plants, several potential roles for the protein are proposed.     

Gibberellin Is Required for Flowering in Arabidopsis thaliana under Short Days

Mutants of Arabidopsis thaliana deficient in gibberellin synthesis (ga1-3 and ga1-6), and a gibberellin-insensitive mutant (gai) were compared to the wild-type (WT) Landsberg erecta line for flowering time and leaf number when grown in either short days (SD) or continuous light (CL). The ga1-3 mutant, which is severely defective in ent-kaurene synthesis because it lacks most of the GA1 gene, never flowered in SD unless treated with exogenous gibberellin. After a prolonged period of vegetative growth, this mutant eventually underwent senescence without having produced flower buds. The gai mutant and the “leaky” ga1-6 mutant did flower in SD, but took somewhat longer than WT. All the mutants flowered readily in CL, although the ga1-3 mutant showed some delay. Unlike WT and ga1-3, the gai mutant failed to respond to gibberellin treatment by accelerating flowering in SD. A cold treatment promoted flowering in the WT and gai, but failed to induce flowering in ga1-3. From these results, it appears that gibberellin normally plays a role in initiating flowering of Arabidopsis.     

The Arabidopsis ZED1-Related Kinase Genomic Cluster Is Specifically Required for Effector-Triggered Immunity

CRISPR/Cas9-mediated deletion of an Arabidopsis gene cluster encoding eight kinases supports their immunity-specific roles in sensing pathogenic effectors.

Dear Editor,ZED1-related kinases (ZRKs) associate with the nucleotide binding, Leu-rich repeat (NLR) protein HOPZ-ACTIVATED RESISTANCE1 (ZAR1) to mediate effector-triggered immunity (ETI) against at least three distinct families of pathogenic effector proteins. However, it is unknown whether ZRKs specifically function in ETI or whether they also have additional roles in immunity and/or development. Eight ZRKs are clustered in the Arabidopsis (Arabidopsis thaliana) genome, including the three members with known roles in ETI. Here, we show that an ∼14-kb CRISPR-mediated deletion of the Arabidopsis ZRK genomic cluster specifically affects ETI, with no apparent defects in pattern-recognition-receptor–triggered immunity (PTI) or development.Phytopathogens deliver effector proteins into plant cells that suppress PTI and promote the infection process (Jones and Dangl, 2006). In turn, plants have evolved NLRs that recognize effectors, leading to an ETI response. This recognition often occurs indirectly, whereby NLRs monitor host “sensor” proteins for effector-induced perturbations (Khan et al., 2016). In the absence of their respective NLRs, some of these sensors are effector virulence targets that modulate immunity and development, while others appear to be decoys that mimic virulence targets, with ETI-specific roles (van der Hoorn and Kamoun, 2008; Khan et al., 2018).The ZAR1 NLR recognizes at least six type-III secreted effector (T3SE) families from bacterial phytopathogens. This remarkable immunodiversity appears to be conveyed through associations with members of the receptor-like cytoplasmic kinase XII-2 (RLCK XII-2) family, which all display characteristics of atypical kinases (Lewis et al., 2013; Roux et al., 2014). The ZAR1-mediated ETI responses against the Pseudomonas syringae T3SEs HopZ1a and HopF1r (formerly HopF2a) require ZED1 and ZRK3, whereas recognition of the Xanthomonas campestris T3SE AvrAC requires ZRK1/RKS1 (Lewis et al., 2013; Wang et al., 2015; Seto et al., 2017). ZRKs currently have no ascribed functions outside of ZAR1-associated ETI responses and are therefore considered decoy sensors or adaptors (Lewis et al., 2013; Wang et al., 2015; Khan et al., 2018). However, functional redundancy may exist among members of the ZRK family, masking phenotypes of individual mutants beyond gene-for-gene–type ETI responses (Lewis et al., 2013). We therefore utilized the CRISPR/Cas9 system to knock out the Arabidopsis genomic region containing eight of the 13 members of the RLCK XII-2, including all ZRK genes known to contribute to ETI, to investigate any non-ETI roles of ZRKs. The 14-kb ZRK gene cluster in Arabidopsis Col-0 plants includes ZRK1, ZRK2, ZRK3, ZRK4, ZED1, ZRK6, ZRK7, and ZRK10. A CRISPR/Cas9-mediated deletion of 13.3 kb was accomplished by designing guide RNAs flanking the ends of the ZRK gene cluster, which would result in a double-stranded break on both sides of the ZRK cluster, leaving only the 5′ end 63 nucleotides (21 amino acids) of ZRK10 and the 3′ end 118 nucleotides (39 amino acids) of ZRK7 (Fig. 1A). We obtained a T1 individual (zrk_1.11) homozygous for the deletion, as well as a T1 individual heterozygous for the mutation (zrk_1.10; Fig. 1B), from which we obtained homozygous T2 (zrk_2.11) and T3 (zrk_3.10) plants, respectively. Sequencing results from zrk_2.11 confirmed that the expected region had been deleted (Supplemental Fig. S1). Plants homozygous for the ZRK gene cluster deletion were morphologically indistinguishable from wild-type Col-0 plants, as well as zar1-1 plants (Fig. 1C). In addition, zrk plant fresh weight did not significantly differ from Col-0 plants (Supplemental Fig. S2), indicating that the ZRK cluster does not play a major role in vegetative plant development.Open in a separate windowFigure 1.Deletion of the ZRK gene cluster results in loss of ZRK-mediated ETI and does not significantly alter vegetative growth. A, Representation of ZRK gene cluster before (top) and after (bottom) CRISPR/Cas9-mediated deletion depicting guide RNAs and primers used for genotyping (see Supplemental Methods S1). ZRK KO primers (magenta) were used to confirm the deletion of the ZRK cluster, while ZRK3 primers (green) were used to check if the ZRK cluster was still present in T1 individuals. B, PCR genotyping for deletion of ZRK gene cluster. Amplification of product by ZRK3 F + R primers indicates lack of deletion; amplification by ZRK KO F + R indicates deletion has occurred. Examples for wild type (WT), heterozygous (HT; zrk_1.10), and homozygous for the deletion (HM KO; zrk_1.11) are shown. T1 lines (zrk_1.10 and zrk_1.11) are compared to wild-type Col-0. C, Uninfected morphology of homozygous zrk KO plants (zrk_3.10 or zrk_2.11) compared to Col-0 and zar1-1 plants. Bar = 1 cm. D, Phenotypes of zrk_2.11 plants 7 d after being sprayed with PtoDC3000(hopZ1a; left) or PtoDC3000(hopF1r; right) relative to wild-type Col-0 and zar1-1 plants. Plant immunity and disease image-based quantification of disease symptoms is presented in Supplemental Figure S3A (Laflamme et al., 2016).Next, we wanted to confirm that the deletion of the ZRK gene cluster compromised ZRK-mediated ETI responses. We sprayed the zrk_2.11 line with PtoDC3000(hopZ1a) or PtoDC3000(hopF1r), as both T3SEs require a ZRK as well as the NLR ZAR1 for their recognition in Arabidopsis (Lewis et al., 2013; Seto et al., 2017). We observed that the zrk_2.11 line was susceptible to both PtoDC3000(hopZ1a) and PtoDC3000(hopF1r), and this susceptibility was to the same level as zar1-1 plants as quantified by plant immunity and disease image-based quantification (Fig. 1D, Supplemental Fig. S3, A and C; Laflamme et al., 2016). We observed a similar phenotype for the zrk_3.10 line, confirming that the ZRK cluster deletion compromised ZRK-mediated ETI responses (Supplemental Fig. S3, B and C). Furthermore, the ZAR1-mediated ETI responses against the P. syringae T3SEs HopBA1a, HopX1i, and HopO1c were also lost in zrk_2.11, demonstrating the ZRK-dependence of these ETI responses (Supplemental Fig. S4; Laflamme et al., 2020). To ensure that the ZRK gene cluster deletion specifically impacted ZRK-related ETI responses, the zrk_3.10 and zrk_2.11 lines were also sprayed with PtoDC3000(avrRpt2), an ETI elicitor that does not require a ZRK or ZAR1 for its recognition (Mackey et al., 2003). zrk_3.10 and zrk_2.11 plants remained resistant to PtoDC3000(avrRpt2), indicating that the ZRK gene cluster deletion specifically impacts ZRK-mediated ETI responses (Supplemental Fig. S3C). In addition, growth of virulent PtoDC3000 on the zrk_3.10 and zrk_2.11 lines was unchanged compared to wild-type Col-0 plants, indicating that the ZRKs within this cluster likely do not represent virulence targets (Supplemental Fig. S5).We then examined whether knocking out the ZRK gene cluster impacted PTI. We first measured induction of peroxidase (POX) enzyme activity, as POX enzymes are produced in response to PTI (Mott et al., 2018). After treatment with the PTI elicitor flg22, addition of the POX substrate 5-aminosalicylic acid produces a brown end-product in the presence of active POX enzymes, which is quantified by reading at an optical density of 550 nm (OD₅₅₀; Mott et al., 2018). Twenty h after leaf discs were treated with flg22, zrk_3.10 and zrk_2.11 plants showed the same level of PTI-associated POX activity as wild-type Col-0 plants (Fig. 2A). To further examine the role of the ZRK gene cluster in PTI, we quantified the growth of PtoDC3000ΔhrcC, which is defective in T3SE secretion and is sensitive to altered host PTI responses under high humidity conditions such as those used in our growth assays (Guo et al., 2009; Xin et al., 2016). Growth of PtoDC3000ΔhrcC on zrk_3.10 and zrk_2.11 plants was not significantly different compared to wild-type Col-0 plants (Fig. 2B). In addition, we monitored reactive oxygen species (ROS) production and found that zrk_3.10 and zrk_2.11 plants did not show a significant difference in the ROS burst observed in wild-type Col-0 plants (Fig. 2, C and D). Finally, we treated seedlings with flg22, and found that growth of zrk_3.10 and zrk_2.11 seedlings was inhibited by the same amount as in wild-type Col-0 seedlings, indicative of a similar induction of PTI responses (Fig. 2, E and F; Gómez-Gómez et al., 1999). Together, these results indicate that the ZRK gene cluster does not play a significant role in Arabidopsis PTI responses.Open in a separate windowFigure 2.Deletion of the ZRK gene cluster does not alter pattern-recognition-receptor–triggered immune responses. A, Response to the PTI elicitor flg22 measured by POX activity. Activity from leaf discs was quantified 20 h after treatment with 1 μm of flg22 at a measurement of OD₅₅₀ (n = 6; Mott et al., 2018). B, Bacterial growth of the T3SS-compromised PtoDC3000ΔhrcC on zrk KO plants (zrk_3.10 and zrk_2.11) relative to wild-type Columbia-0 (wild-type Col-0) and zar1-1 plants 3-d postinoculation. Plants were domed for the duration of the experiment (n = 8). C, Response of Col-0, zrk KO plants (zrk_3.10 and zrk_2.11), and fls2 to the PTI elicitor flg22 measured using luminol-based detection of ROS over a time course of 60 min, with relative light units measured every 2 min (n = 12). D, Boxplots of total relative light units over a period of 30 min from treatments in C (n = 12). E, Growth inhibition of seedlings 7 d after treatment with 1 μm of flg22. F, Seedling growth inhibition was quantified by measuring fresh weight of flg22-treated seedlings as a percentage of water-treated controls (n = 4). Error bars in A, B, C, D, and F, represent se. Lowercase letters represent significantly different statistical groups by Tukey’s honest significant difference test (P < 0.05). Experiments were replicated three times with similar results.Overall, our results support an ETI-specific role for ZRKs in Arabidopsis, acting as sensors of the ZAR1 NLR. Structural insights have revealed important residues required for ZAR1-ZRK1 complex formation, and these are conserved across the RLCK XII-2 family, which includes ZRKs outside the genomic cluster (Supplemental Fig. S6; Lewis et al., 2013; Wang et al., 2019). This suggests that the ZRKs outside this genomic cluster may also play a similar role as ZAR1 sensors. As such, the ZRK family would have evolved to mimic and/or interact with the numerous kinase virulence targets of pathogenic effectors, thereby expanding the surveillance potential of ZAR1.Supplemental DataThe following supplemental materials are available.

• Supplemental Figure S1. Sequencing confirmation of the ZRK gene cluster deletion.
• Supplemental Figure S2. Fresh weight of zrk knockout (KO) plants (zrk_3.10 and zrk_2.11) relative to wild-type Columbia-0 (wild-type Col-0) and zar1-1 plants.
• Supplemental Figure S3. ZRK gene cluster deletion specifically compromises ZRK-dependent ETI responses.
• Supplemental Figure S4. ZRK gene cluster compromises the ZAR1-dependent ETI responses against HopBA1a, HopO1c, and HopX1i.
• Supplemental Figure S5. Bacterial growth of the virulent PtoDC3000 strain on zrk KO plants (zrk_3.10 and zrk_2.11) relative to wild-type Col-0 (wild-type Col-0) plants 0- and 3-d post-inoculation via syringe infiltration.
• Supplemental Figure S6. Multiple sequence alignment of RLCK XII-2 family shows high conservation of putative ZAR1-interacting residues.
• Supplemental Methods S1. Generation and characterization of ZRK cluster deletion lines.

     

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.Plant vacuoles are vital organelles for maintaining cell volume and cell turgor, regulating ion homeostasis and pH, disposing toxic materials, and storing and degrading unwanted proteins (Marty, 1999). To perform these diverse functions, vacuoles require an array of different and complex proteins. These proteins are synthesized at the endoplasmic reticulum (ER) and are transported to the vacuole through the vacuolar trafficking pathway. Perturbation of the vacuolar trafficking machinery affects many cellular processes, including tropisms, responses to pathogens, cytokinesis, hormone transport, and signal transduction (Surpin and Raikhel, 2004). The vacuolar trafficking system is comprised of several compartments: the ER, the Golgi apparatus, the trans-Golgi network (TGN), the prevacuolar compartment (PVC), and the vacuole. Vacuolar proteins synthesized at the ER are transported to the cis-Golgi via coat protein complex II (COPII) vesicles and are then transported to the TGN through the Golgi apparatus. In the TGN, proteins are sorted for delivery to their respective locations according to their targeting signal. Vacuolar proteins carrying a vacuolar sorting signal are thought to be recognized by vacuolar sorting receptors (VSRs), which are mainly located in the PVC, although sorting of vacuolar proteins may also occur at the ER and VSRs can be recycled from the TGN to the ER (Castelli and Vitale, 2005; Niemes et al., 2010). Multiple studies suggest that plant VSRs serve as sorting receptors both for lytic vacuole proteins (daSilva et al., 2005; Foresti et al., 2006; Kim et al., 2010) and for storage vacuole proteins (Shimada et al., 2003; Fuji et al., 2007; Zouhar et al., 2010).Osmotic stress is commonly associated with many environmental stresses, including drought, cold, and high soil salinity, that have a severe impact on the productivity of agricultural plants worldwide. Therefore, understanding how plants perceive and respond to osmotic stress is critical for improving plant resistance to abiotic stresses (Zhu, 2002; Fujita et al., 2013). It has long been recognized that osmotic stress can activate several signaling pathways that lead to changes in gene expression and metabolism. One important regulator of these signaling pathways is the phytohormone abscisic acid (ABA), which accumulates in response to osmotic stress. ABA regulates many critical processes, such as seed dormancy, stomatal movement, and adaptation to environmental stress (Finkelstein and Gibson, 2002; Xiong and Zhu, 2003; Cutler et al., 2010). De novo synthesis of ABA is of primary importance for increasing ABA levels in response to abiotic stress. ABA is synthesized through the cleavage of a C40 carotenoid originating from the 2-C-methyl-d-erythritol-4-phosphate pathway, followed by a conversion from zeaxanthin to violaxanthin catalyzed by the zeaxanthin epoxidase ABA1 and then to neoxanthin catalyzed by the neoxanthin synthase ABA4. Subsequently, a 9-cis-epoxycarotenoid dioxygenase (NCED) cleaves the violaxanthin and neoxanthin to xanthoxin. Xanthoxin, in turn, is oxidized by a short-chain alcohol dehydrogenase (ABA2) to abscisic aldehyde, which is converted to ABA by abscisic acid aldehyde oxidase3 (AAO3) using a molybdenum cofactor activated by the molybdenum cofactor sulfurase (ABA3; Nambara and Marion-Poll, 2005). In this pathway, it is generally thought that the cleavage step catalyzed by NCED is the rate-limiting step (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). In Arabidopsis (Arabidopsis thaliana), five members of the NCED family (NCED2, NCED3, NCED5, NCED6, and NCED9) have been characterized (Tan et al., 2003). Of those, NCED3 has been suggested to play a crucial role in ABA biosynthesis, and its expression is induced by dehydration and osmotic stress (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). Thus, understanding how the NCED3 gene is activated in response to osmotic stress is important for the elucidation of the mechanisms that govern plant acclimation to abiotic stress.We have used the firefly luciferase reporter gene driven by the stress-responsive NCED3 promoter to enable the genetic dissection of plant responses to osmotic stress (Wang et al., 2011). Here, we report the characterization of a unique regulator of ABA biosynthesis, 9-cis Epoxycarotenoid Dioxygenase Defective2 (CED2). The ced2 mutants are impaired in osmotic stress tolerance and are defective in the expression of genes required for ABA synthesis and consequently osmotic stress-induced ABA accumulation. The CED2 gene encodes VSR1, previously known to be involved in vacuolar trafficking but not known to be critical for osmotic stress induction of ABA biosynthesis and osmotic stress tolerance. Our study further suggests that intracellular pH changes might act as an early stress response signal triggering osmotic stress-activated ABA biosynthesis.     

AvrBsT Acetylates Arabidopsis ACIP1, a Protein that Associates with Microtubules and Is Required for Immunity

Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1''s defense function, which is linked to the activation of ETI.