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.     

Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2

Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associated molecular pattern-triggered immunity). However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against the potato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated cell death by binding the plant E3 ligase CMPG1. In contrast, AVR3a^KI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does not suppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamiana using the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants of AVR3a suppress defense responses triggered by flg22 and reduce internalization of activated FLS2. Moreover, we discovered that AVR3a associates with the Dynamin-Related Protein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affect internalization of the growth receptor BRI1. Our results suggest that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector that can suppress BAK1/SERK3-mediated immunity through at least two different pathways.     

ESCRT-I Mediates FLS2 Endosomal Sorting and Plant Immunity

The plant immune receptor FLAGELLIN SENSING 2 (FLS2) is present at the plasma membrane and is internalized following activation of its ligand flagellin (flg22). We show that ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT (ESCRT)-I subunits play roles in FLS2 endocytosis in Arabidopsis. VPS37-1 co-localizes with FLS2 at endosomes and immunoprecipitates with the receptor upon flg22 elicitation. Vps37-1 mutants are reduced in flg22-induced FLS2 endosomes but not in endosomes labeled by Rab5 GTPases suggesting a defect in FLS2 trafficking rather than formation of endosomes. FLS2 localizes to the lumen of multivesicular bodies, but this is altered in vps37-1 mutants indicating compromised endosomal sorting of FLS2 by ESCRT-I loss-of-function. VPS37-1 and VPS28-2 are critical for immunity against bacterial infection through a role in stomatal closure. Our findings identify that VPS37-1, and likewise VPS28-2, regulate late FLS2 endosomal sorting and reveals that ESCRT-I is critical for flg22-activated stomatal defenses involved in plant immunity.     

Differential Roles of Arabidopsis Dynamin-Related Proteins DRP3A,DRP3B,and DRP5B in Organelle Division

Dynamin-related proteins (DRPs) are key components of the organelle division machineries, functioning as molecular scissors during the fission process. In Arabidopsis, DRP3A and DRP3B are shared by peroxisomal and mitochondrial division, whereas the structurally-distinct DRP5B (ARC5) protein is involved in the division of chloroplasts and peroxisomes. Here, we further investigated the roles of DRP3A, DRP3B, and DRP5B in organelle division and plant development. Despite DRP5B's lack of stable association with mitochondria, drp5B mutants show defects in mitochondrial division. The drp3A-2 drp3B-2 drp5B-2 triple mutant exhibits enhanced mitochondrial division phenotypes over drp3A-2 drp3B-2, but its peroxisomal morphology and plant growth phenotypes resemble those of the double mutant. We further demonstrated that DRP3A and DRP3B form a supercomplex in vivo, in which DRP3A is the major component, yet DRP5B is not a constituent of this complex. We thus conclude that DRP5B participates in the division of three types of organelles in Arabidopsis, acting independently of the DRP3 complex. Our findings will help elucidate the precise composition of the DRP3 complex at organelle division sites, and will be instrumental to studies aimed at understanding how the same protein mediates the morphogenesis of distinct organelles that are linked by metabolism.     

Mutations in FLS2 Ser-938 Dissect Signaling Activation in FLS2-Mediated Arabidopsis Immunity

FLAGELLIN-SENSING 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2_S938A, while the acidic phosphomimic mutants FLS2_S938D and FLS2_S938E conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2_S938A, demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2_S938A and FLS2_S938D mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2_S938A or FLS2_D997A (a kinase catalytic site mutant), but was normally induced in FLS2_S938D plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.     

Arabidopsis dynamin-related proteins DRP3A and DRP3B are functionally redundant in mitochondrial fission, but have distinct roles in peroxisomal fission

Two similar Arabidopsis dynamin-related proteins, DRP3A and DRP3B, are thought to be key factors in both mitochondrial and peroxisomal fission. However, the functional and genetic relationships between DRP3A and DRP3B have not been fully investigated. In a yeast two-hybrid assay, DRP3A and DRP3B interacted with themselves and with each other. DRP3A and DRP3B localized to mitochondria and peroxisomes, and co-localized with each other in leaf epidermal cells. In two T-DNA insertion mutants, drp3a and drp3b , the mitochondria are a little longer and fewer in number than those in the wild-type cells. In the double mutant, drp3a/drp3b , mitochondria are connected to each other, resulting in massive elongation. Overexpression of either DRP3A or DRP3B in drp3a/drp3b restored the particle shape of mitochondria, suggesting that DRP3A and DRP3B are functionally redundant in mitochondrial fission. In the case of peroxisomal fission, DRP3A and DRP3B appear to have different functions: peroxisomes in drp3a were larger and fewer in number than those in the wild type, whereas peroxisomes in drp3b were as large and as numerous as those in the wild type, and peroxisomes in drp3a/drp3b were as large and as numerous as those in drp3a . Although overexpression of DRP3A in drp3a/drp3b restored the shape and number of peroxisomes, overexpression of DRP3B did not restore the phenotypes, and often caused elongation instead. These results suggest that DRP3B and DRP3A have redundant molecular functions in mitochondrial fission, whereas DRP3B has a minor role in peroxisomal fission that is distinct from that of DRP3A.     

A role for Arabidopsis dynamin related proteins DRP2A/B in endocytosis; DRP2 function is essential for plant growth

Endocytosis is an essential cellular process that allows cells to internalise proteins and lipid from the plasma membrane to change its composition and sense and respond to alterations in their extracellular environment. In animal cells, the protein dynamin is involved in membrane scission during endocytosis, allowing invaginating vesicles to become internalised. Arabidopsis encodes two proteins that have all the domains essential for function in the animal dynamins, Dynamin Related Proteins 2A and 2B (DRP2A and 2B). These proteins show very high sequence identity and are both expressed throughout the plant. Single mutants exhibited no obvious phenotypes but double mutants could be recovered as gametophytes carrying mutant copies of both DRP2A and DRP2B were not transmitted to the next generation. Immunolabelling localised DRP2A/B to the tips of root hairs, a site where rapid endocytosis takes place. Constitutive expression of a GTPase defective Dominant Negative form of DRP2A/B did not allow the recovery of plants expressing this protein at a detectable level, demonstrating an interference with endogenous dynamin. Using an inducible expression system Dominant Negative protein was transiently expressed at levels several fold that of the endogenous proteins. Inducible expression of the Dominant Negative protein resulted in reduced endocytosis at the tips of root hairs, as measured by internalisation of an endocytic tracer dye, and resulted in root hairs bulging and bursting. Together these data support a role for DRP2A/B in endocytosis in Arabidopsis, and demonstrates that the function of at least one of these closely related proteins is essential for plant growth.     

How does SA signaling link the Flg22 responses?

Salicylic acid (SA) has a central role in activating plant resistance to pathogens. SA levels increase in plant tissue following pathogen infection and exogenous SA enhances resistance to a broad range of pathogens. To study the relevance of the SA signaling in the flg22 response, we investigated the responses of SA-related mutants to flg22, a 22-amino acid peptide of the flagellin bacterial protein. We identified SA as an important component of the flg22-triggered oxidative burst, a very early event after flg22 detection, and gene induction, an early event. SA acted partially by enhancing accumulation of FLS2 mRNA. We also provide new evidence that NPR1 play a role in SA-induced priming event that enhances the flg22-triggered oxidative burst, which is correlated with enhancement of the flg22-induced callose deposition. Based on these observations, we conclude that SA signaling is required for early as well as late flg22 responses.     

The Arabidopsis dynamin-related protein2 family is essential for gametophyte development

Clathrin-mediated membrane trafficking is critical for multiple stages of plant growth and development. One key component of clathrin-mediated trafficking in animals is dynamin, a polymerizing GTPase that plays both regulatory and mechanical roles. Other eukaryotes use various dynamin-related proteins (DRP) in clathrin-mediated trafficking. Plants are unique in the apparent involvement of both a family of classical dynamins (DRP2) and a family of dynamin-related proteins (DRP1) in clathrin-mediated membrane trafficking. Our analysis of drp2 insertional mutants demonstrates that, similar to the DRP1 family, the DRP2 family is essential for Arabidopsis thaliana development. Gametophytes lacking both DRP2A and DRP2B were inviable, arresting prior to the first mitotic division in both male and female gametogenesis. Mutant pollen displayed a variety of defects, including branched or irregular cell plates, altered Golgi morphology and ectopic callose deposition. Ectopic callose deposition was also visible in the pollen-lethal drp1c-1 mutant and appears to be a specific feature of pollen-defective mutants with impaired membrane trafficking. However, drp2ab pollen arrested at earlier stages in development than drp1c-1 pollen and did not accumulate excess plasma membrane or display other gross defects in plasma membrane morphology. Therefore, the DRP2 family, but not DRP1C, is necessary for cell cycle progression during early gametophyte development. This suggests a possible role for DRP2-dependent clathrin-mediated trafficking in the transduction of developmental signals in the gametophyte.     

DRP1A is responsible for vascular continuity synergistically working with VAN3 in Arabidopsis

In most dicotyledonous plants, vascular tissues in the leaf have a reticulate venation pattern. We have isolated and characterized an Arabidopsis (Arabidopsis thaliana) mutant defective in the vascular network defective mutant, van3. van3 mutants show a discontinuous vascular pattern, and VAN3 is known to encode an ADP-ribosylation-factor-GTPase-activating protein that regulates membrane trafficking in the trans-Golgi network. To elucidate the molecular nature controlling the vein patterning process through membrane trafficking, we searched VAN3-interacting proteins using a yeast (Saccharomyces cerevisiae) two hybrid system. As a result, we isolated the plant Dynamin-Related Protein 1A (DRP1A) as a VAN3 interacting protein. The spatial and temporal expression patterns of DRP1AGUS and VAN3GUS were very similar. The subcellular localization of VAN3 completely overlapped to that of DRP1A. drp1a showed a disconnected vascular network, and the drp1a mutation enhanced the phenotype of vascular discontinuity of the van3 mutant in the drp1a van3 double mutant. Furthermore, the drp1 mutation enhanced the discontinuous vascular pattern of the gnom mutant, which had the same effect as that of the van3 mutation. These results indicate that DRP1 modulates the VAN3 function in vesicle budding from the trans-Golgi network, which regulates vascular formation in Arabidopsis.     

Spatio-Temporal Cellular Dynamics of the Arabidopsis Flagellin Receptor Reveal Activation Status-Dependent Endosomal Sorting

The activity of surface receptors is location specific, dependent upon the dynamic membrane trafficking network and receptor-mediated endocytosis (RME). Therefore, the spatio-temporal dynamics of RME are critical to receptor function. The plasma membrane receptor FLAGELLIN SENSING2 (FLS2) confers immunity against bacterial infection through perception of flagellin (flg22). Following elicitation, FLS2 is internalized into vesicles. To resolve FLS2 trafficking, we exploited quantitative confocal imaging for colocalization studies and chemical interference. FLS2 localizes to bona fide endosomes via two distinct endocytic trafficking routes depending on its activation status. FLS2 receptors constitutively recycle in a Brefeldin A (BFA)–sensitive manner, while flg22-activated receptors traffic via ARA7/Rab F2b– and ARA6/Rab F1–positive endosomes insensitive to BFA. FLS2 endocytosis required a functional Rab5 GTPase pathway as revealed by dominant-negative ARA7/Rab F2b. Flg22-induced FLS2 endosomal numbers were increased by Concanamycin A treatment but reduced by Wortmannin, indicating that activated FLS2 receptors are targeted to late endosomes. RME inhibitors Tyrphostin A23 and Endosidin 1 altered but did not block induced FLS2 endocytosis. Additional inhibitor studies imply the involvement of the actin-myosin system in FLS2 internalization and trafficking. Altogether, we report a dynamic pattern of subcellular trafficking for FLS2 and reveal a defined framework for ligand-dependent endocytosis of this receptor.     

Both the Extracellular Leucine-Rich Repeat Domain and the Kinase Activity of FLS2 Are Required for Flagellin Binding and Signaling in Arabidopsis

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.     

Induced Endocytosis of the Receptor Kinase FLS2

Receptor-like kinases (RLKs) that function as pattern-recognition receptors (PRRs) play a key role in plant immune responses. The receptor recognizing flagellin in Arabidopsis, FLS2, is encoded by a membrane resident RLK. FLS2 is involved in preinvasive immunity against bacterial infection. Recent observations revealed that upon flagellin perception FLS2 accumulates in intracellular mobile vesicles and is then degraded. Reminiscent of ligand-induced receptor endocytosis in animals, FLS2 internalization is Wortmannin-sensitive. Mutation of the potentially phosphorylated residue threonine-867 impaired FLS2 endocytosis and flagellin-triggered responses. Furthermore, mutation of a PEST-motif abolished FLS2 endocytosis and downstream flagellin-elicited responses were affected. Thus, FLS2 endocytosis likely involves phosphorylation and ubiquitination events and appears to be interconnected with flagellin signaling. Similarly, TLR4, the mammalian PRR recognizing bacterial lipopolysaccharides (LPS) is internalized in a ligand specific manner. In this addendum, we discuss endocytic processes of plant RLKs focussing on FLS2 and provide a brief comparison with TLR4 endocytosis.Key words: Endocytosis, RLK, FLS2, flagellin, TLR4, LPS     

Contamination Risks in Work with Synthetic Peptides: flg22 as an Example of a Pirate in Commercial Peptide Preparations

The pattern recognition receptor FLAGELLIN SENSING2 (FLS2) renders plant cells responsive to subnanomolar concentrations of flg22, the active epitope of bacterial flagellin. We recently observed that a preparation of the peptide IDL1, a signal known to regulate abscission processes via the receptor kinases HAESA and HAESA-like2, apparently triggered Arabidopsis thaliana cells in an FLS2-dependent manner. However, closer investigation revealed that this activity was due to contamination by a flg22-type peptide, and newly synthesized IDL1 peptide was completely inactive in FLS2 signaling. This raised alert over contamination events occurring in the process of synthesis or handling of peptides. Two recent reports have suggested that FLS2 has further specificities for structurally unrelated peptides derived from CLV3 and from Ax21. We thus scrutinized these peptides for activity in Arabidopsis cells as well. While responding to <1 nM flg22, Arabidopsis cells proved blind even to 100 μM concentrations of CLV3p and axY^s22. Our results confirm the exquisite sensitivity and selectivity of FLS2 for flg22. They also show that inadvertent contaminations with flg22-type peptides do occur and can be detected even in trace amounts by FLS2.During the last years, the pattern recognition receptor FLAGELLIN SENSING2 (FLS2) and its cognate microbe-associated molecular pattern (MAMP), the peptide flg22 (Felix et al., 1999), have widely been used to study plant innate immunity (Boller and Felix, 2009). Typically, in FLS2-expressing Arabidopsis thaliana cells, flg22 stimulates rapid changes of ion fluxes, including extracellular alkalinization and an induction of defense-related genes, such as FRK1, at threshold concentrations of 10 to 100 pM, while fls2 mutants lacking the receptor kinase FLS2 are completely unresponsive to flg22 (Boller and Felix, 2009). These findings demonstrate that FLS2 has an exquisite sensitivity as a flagellin receptor and that FLS2 is the only receptor for the flg22 ligand in Arabidopsis. Our previous results also indicated an exquisite selectivity of FLS2 with regard to its ligand. For example, the flg22 peptide of Agrobacterium tumefaciens (flg22^A.tum.) is completely inactive as a ligand of FLS2 or as a stimulus for FLS2-dependent responses and therefore has often been used as a negative control in assays for flg22-induced responses (Felix et al., 1999; Asai et al., 2002).Recently, we observed that a synthetic preparation of IDL1, an endogenous peptide signal involved in abscission processes (Stenvik et al., 2008), showed considerable activity as an inducer of MAMP responses and stimulated extracellular alkalinization in Arabidopsis cells at a threshold level of <5 nM (Figure 1A). More surprisingly, when examined in a cell culture of the fls2 efr double mutant, no significant medium alkalinization was detectable after treatment with the IDL1 preparation (Figure 1B). To check if FLS2 was involved in the response to the IDL1 preparation, we made use of the inhibitor flg22-Δ2, which acts as a specific antagonist of flg22 in Arabidopsis (Bauer et al., 2001). Indeed, presence of 30 μM flg22-Δ2 completely abolished the response to 50 nM IDL1 (Figure 1C).Open in a separate windowFigure 1.Effects of IDL1 Peptides on Extracellular pH in Suspension-Cultured Arabidopsis Cells.(A) Alkalinization in response to different doses of two independent preparations of the IDL1 peptide (preparations I and II). wt, wild-type.(B) Alkalinization response in cells from the fls2 efr double mutant.(C) Alkalinization response in wild-type cells to preparation I of IDL1 alone, to preparation I in combination with the flg22 antagonist flg22-Δ2 (30 μM), or to preparation I after digestion (overnight, 37°C) with endoproteinase AspN, as indicated.Based on strong genetic evidence, IDL1 is thought to act as a regulator of abscission processes via the receptor kinases HAESA and HAESA-like 2 (Stenvik et al., 2008), but why should FLS2 be involved? The IDL1 preparation was ∼100-fold less effective than authentic flg22 preparations, as indicated by the EC₅₀ values of 0.1 nM for flg22 (Bauer et al., 2001) and 10 nM for IDL1 (Figure 1A), respectively. We hypothesized that the IDL1 preparation might be contaminated by a peptide of the flg22 type. In contrast with IDL1, which has no acidic amino acid residues, flg22 contains two Asp residues that are important for its biological activity on FLS2 (Felix et al., 1999). We used this difference for selective digestion by the endoproteinase AspN, which cuts peptides N-terminal of Asp residues. Indeed, no activity was left in IDL1 after digestion (Figure 1C). This strongly indicated that the activity was not associated with the IDL1 peptide itself but rather with a flg22-type of contamination. Repurification of IDL1 by C18 reverse-phase chromatography could not separate the IDL1 peptide from the flg22 type of activity. Apart from a dominating signal for IDL1, mass spectrometry analysis of this fraction also revealed faint mass signatures characteristic for flg22 and its spontaneous derivative containing pyroglutamate at its N terminus (see Supplemental Figure 1 online). Together, these results clearly pinpointed a contamination as the source of the flg22-like activity in IDL1.Where did this contamination occur? We observed the same activity with a second, unopened tube from the same batch of the synthetic IDL1 peptide, indicating that a putative flg22 contamination had occurred prior to arrival in our lab, most likely in the company providing the peptide. We therefore resynthesized a new batch of IDL1 and found that it did not cause alkalinization in Arabidopsis wild-type cells even at a concentration of 10 μM (preparation II, Figure 1A). A further, independent, batch of an IDL1-derived peptide with a C-terminal extension by two amino acid residues similarly failed to induce alkalinization in the Arabidopsis wild-type cells (data not shown). These results made us acutely aware of a potential contamination problem when working with peptides unrelated to flg22. Indeed, two recent reports have suggested that FLS2 perceives two additional, unrelated, peptidic signals derived from either CLV3 (Lee et al., 2011) or Ax21 (Danna et al., 2011), respectively. What if these unexpected results were due to inadvertent contamination by flg22 as well?In a recent study (Mueller et al., 2012), we compared the FLS2 orthologs from Arabidopsis and tomato (Solanum lycopersicum) and their chimeras, making use of protoplasts from fls2 mutant plants transformed simultaneously with constructs encoding one of the FLS2 orthologs and a pFRK1:luciferase reporter, an assay system originally introduced by Asai et al. (2002). Protoplasts with both versions of FLS2 exhibited exquisite sensitivity to picomolar concentrations of flg22. However, they failed to respond to the hydroxylated CLV3 peptides CLV3-ΔAra₃-ΔH (12 amino acids) and CLV3-ΔAra₃ (13 amino acids) (described in Ohyama et al., 2009), termed CLV3p and CLV3p-H in our article (see Figure 1 in Mueller et al., 2012). Indeed, even when applied at a concentration of 100 μM, the 12–amino acid CLV3p caused no significant response in protoplasts expressing FLS2 from Arabidopsis (Figure 2A). A marginal transient increase in luminescence occurred in the first 2 h of the experiment, but this effect was also seen in the absence of FLS2 (Figure 3B), demonstrating that it had nothing to do with FLS2-dependent activation of the reporter gene. Our preparation of the CLV3p peptide exhibited the expected strong inhibitory effect in root growth assays with wild-type Arabidopsis and with fls2 mutants but not with the mutant clv2-1 (see Supplemental Figure 2 online).Open in a separate windowFigure 2.The CLV3p Peptide (Arg-Thr-Val-Hyp-Ser-Gly-Hyp-Asp-Pro-Leu-His-His, CLV3-ΔAra₃-ΔH in Ohyama et al., 2009) Does Not Induce Expression of the Reporter pFRK1:luciferase via the Receptor FLS2.Mesophyll protoplasts from efr×fls2 mutants were transformed with pFRK1:luciferase (pFRK1, promoter of the flagellin responsive receptor kinase 1) together with p35S:FLS2-GFP (A) or p35S:GFP (B) and tested for responsiveness to CLV3p and flg22 as indicated. GFP, green fluorescent protein; RLU, relative light units.Open in a separate windowFigure 3.Ax21-Derived Peptides axY^s22 and axY22A Show No Activity as Inducers of Oxidative Burst and Medium Alkalinization in Arabidopsis.(A) Oxidative burst in leaf pieces of Arabidopsis treated with axY^s22, axY22A, or flg22 as indicated. Reactive oxygen species (ROS) were determined by light emission (relative light units [RLU] of the luminometer) in a luminol-based assay. Values and error bars represent mean ± se of n = 6 replicates. (Error bars in all samples not treated with flg22 were smaller than 100 relative light units.)(B) Extracellular alkalinization in cell cultures of Arabidopsis treated with axY^s22, axY22A, or a control peptide (SASRSRIQDADFAAETANLSRSQILQQAGTA) in combination with flg22, as indicated.In previous work by Lee et al. (2009), the sulfated peptide axY^s22, derived from the protein Ax21 of the pathogenic bacterium Xanthomonas oryzae, but not its variant form axY22A, in which the sulfotyrosine was replaced by an Ala, have been reported to cause a resistance response in rice (Oryza sativa) expressing the receptor kinase XA21. Recently, preparations of both of these two peptides have been reported to stimulate immune responses in Arabidopsis when applied at concentrations of 1 to 100 μM (Danna et al., 2011; Figures 1 to 3). Surprisingly, this activation was dependent on the presence of a functional FLS2 receptor, suggesting that these peptides are acting as ligands for FLS2 as well. We obtained fresh preparations of both axY^s22 and axY22A. In our hands, both peptides were completely inactive at concentrations up to 100 μM in oxidative burst and alkalinization assays (Figure 3). The cells used for the alkalinization assays strongly responded to 100 pM of authentic flg22, indicating that the Ax21-related peptides were at least a million times less efficient to induce alkalinization via FLS2 (Figure 3B).The peptide flg22-Δ2 functions as a competitive antagonist that specifically inhibits flg22-induced responses in Arabidopsis (Bauer et al., 2001). Since the FLS2-dependent responses to CLV3p and the Ax21 peptides were reported to be inhibited by excess flg22-Δ2 (Danna et al., 2011; Lee et al., 2011), we also checked whether CLV3p or the Ax21 peptides might interfere with the binding of flg22 to FLS2 (Figure 4). The receptor FLS2 binds carrier-free ¹²⁵I-Tyr-flg22 with a high affinity, and this binding can be specifically competed by 10 μM unlabeled flg22 but neither by 30 μM CLV3p nor by 30 μM axY^s22 (Figure 4). Thus, we cannot confirm that CLV3p or axY^s22 can directly interact and activate FLS2. While inadvertent contamination is a possible explanation, we cannot finally explain the obvious discrepancies to the results in the Lee et al. (2011) and Danna et al. (2011) reports. Also, since our analysis focused on direct and immediate effects on FLS2, we cannot comment on effects that high concentrations of peptides like CLV3p or axY^s22 might exert on prolonged treatment. For example, induction of plant resistance is a highly complex process that develops over days and involves two living systems. Rather than on the host cells, peptides applied might act on the pathogenic bacteria and influence their synthesis of flagellin or their assembly/disassembly of flagellin subunits into flagellar structures. At least for the ax21 peptides, described as a quorum sensing type of signals for bacteria, this is an option to be considered.Open in a separate windowFigure 4.The Peptides axY^s22 and CLV3p Do Not Compete for Binding of flg22 to FLS2.Binding of ¹²⁵I-Tyr-flg22 to wild-type Arabidopsis seedlings in the absence of competitor, in the presence of 10 μM unlabeled flg22, or in the presence of 30 μM unlabeled axY^s22 or CLV3p. Bars and error bars represent radioactivity (counts per min [cpm]) bound to plant material as means and sd of n = 3 replicates.Our results confirm the exquisite sensitivity and selectivity of FLS2 for its cognate ligand, flg22. They also show that extreme care must be taken when attempting to assess the effect of peptides on responses that can also be elicited by flg22. Based on our experience, peptide preparations ordered from different commercial suppliers may occasionally be contaminated by flg22-related activity. We observed a contamination corresponding to ∼1% of flg22 equivalents in one of the IDL1 preparations (Figure 1). However, we would like to emphasize that in a peptide preparation applied at 100 μM a contamination by flg22 of only ∼0.0001% (∼1 ppm) can activate FLS2-dependent responses. Using HPLC and mass spectrometry analysis as common checks for purity, suppliers guarantee that a certain percentage, maximally 99%, of the preparation corresponds to the peptide ordered. However, as exemplified for the contaminated IDL1 preparation (see Supplemental Figure 1 online), contaminations at or below 1% can easily go unnoticed. Also, normally, the molecular characteristics of a potential contaminant are not known, so a flg22-type of activity could be present as a partial degradation product or in the form of an unknown flg22 derivative.We cannot estimate a frequency for cross-contaminations in peptide preparations, but it seems to occur surprisingly often. Over the years, we ordered >100 peptides from various commercial suppliers and had at least two further incidents with flg22-type contaminations. In one of these cases, we ordered, and obtained, flg22 and three structurally unrelated peptides. We found residual flg22-type activity in two out of the three preparations of these unrelated peptides, indicating contamination in the course of commercial peptide synthesis (in this case, by a supplier different from the provider of IDL1) or during handling of these peptides in our lab.There are reasons why contaminations by flg22 might pose a particular risk. First, flg22 has a tendency to stick to surfaces and we recommended the use of 0.1 M NaCl and 1 mg/mL BSA to prevent loss of the peptide during serial dilutions (Felix et al., 1999). In turn, flg22 adhering to tubings, columns, or glassware might provide a source of contamination for peptides getting handled subsequently. Second, we noticed that lyophilized flg22 can easily pick up electrostatic charge and is prone to float around with the slightest streams of air. This could be a particular problem also for preparations handled by robots of peptide manufacturers. Third, due to a considerable demand by an increasing number of labs, flg22-related peptides have been ordered from various peptide suppliers numerous times and, picking up this peptide as inadvertent contamination has become a considerable problem.In conclusion, our study complements and extends the commentary by Segonzac et al. (2012) by demonstrating that the receptor FLS2 has an extraordinarily high affinity and selectivity to its ligand, flg22, and that it is completely blind to the peptides IDL1, CLV3p, axY^s22, and axY22A even in our most sensitive bioassays. Our results and arguments do not apodictically exclude that a receptor like FLS2 could have a second, physiologically relevant, ligand. Also, there may be chemical structures that inadvertently act as surrogates or mimetics of the true ligand flg22. However, in view of the high selectivity of the FLS2 for its genuine ligand flg22, we think the probability of alien interactors is rather low. By contrast, contaminations with flg22-related molecules can and do occur.How can contamination of bioactive peptides be recognized and avoided? The first, probably most important, point is a sharpened awareness about in-lab and supplier-dependent sources of contamination. These risks are often ignored, in particular when working with synthetic, purified peptides. A purity of >95 or >99%, as guaranteed by suppliers, is of limited value with respect to highly active contaminants detectable even at the ppm level. Purification offered by suppliers certainly helps to remove chemicals used in the synthesis process and to get rid of many incomplete variants of the peptide ordered. However, at least theoretically, contaminated equipment used during the purification might contribute to the problem rather than to its solution. Dose–response relationships for the peptides under scrutiny are important to consider physiological relevance in general and to compare activities with published data in particular. Thereby, the higher the dose of a peptide applied, the higher the risk to pick up even spurious contaminants. Furthermore, analysis of bioactive peptides should not depend on a single peptide preparation alone. Peptide variants are crucial to elucidate the specificity of an interaction process. The use of several, independently synthesized and handled peptide preparations should help to reliably detect sporadic contamination events and to distinguish contaminants from true ligands. Finally, at least for the particular problem of contamination by flg22, we can offer testing peptide preparations using the sensitive bioassays established in our labs. As long as we have the hands and capacity to handle such requests, we certainly would like to contribute with such a service to detect pirate peptides.     

Two small protein families, DYNAMIN-RELATED PROTEIN3 and FISSION1, are required for peroxisome fission in Arabidopsis

Peroxisomes are multi-functional organelles that differ in size and abundance depending on the species, cell type, developmental stage, and metabolic and environmental conditions. The PEROXIN11 protein family and the DYNAMIN-RELATED PROTEIN3A (DRP3A) protein have been shown previously to play key roles in peroxisome division in Arabidopsis. To establish a mechanistic model of peroxisome division in plants, we employed forward and reverse genetic approaches to identify more proteins involved in this process. In this study, we identified three new components of the Arabidopsis peroxisome division apparatus: DRP3B, a homolog of DRP3A, and FISSION1A and 1B (FIS1A and 1B), two homologs of the yeast and mammalian FIS1 proteins that mediate the fission of peroxisomes and mitochondria by tethering the DRP proteins to the membrane. DRP3B is partially targeted to peroxisomes and causes defects in peroxisome fission when the gene function is disrupted. drp3A drp3B double mutants display stronger deficiencies than each single mutant parent with respect to peroxisome abundance, seedling establishment and plant growth, suggesting partial functional redundancy between DRP3A and DRP3B. In addition, FIS1A and FIS1B are each dual-targeted to peroxisomes and mitochondria; their mutants show growth inhibition and contain peroxisomes and mitochondria with incomplete fission, enlarged size and reduced number. Our results demonstrate that both DRP3 and FIS1 protein families contribute to peroxisome fission in Arabidopsis, and support the view that DRP and FIS1 orthologs are common components of the peroxisomal and mitochondrial division machineries in diverse eukaryotic species.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> dynamin-related protein DRP2B is co-localized with DRP1A on the leading edge of the forming cell plate

The Arabidopsis genome has six families of dynamin-related proteins. One of these families includes DRP2A and DRP2B. The domain structures of proteins of this family are most similar to those of the animal endocytosis protein, dynamin. In this study, the signals of GFP-tagged DRP2B were strongly detected in the cell plate of Arabidopsis root tip cells and tobacco cultured cells. Time-lapse observations of these signals during cytokinesis in tobacco cultured cells suggested that DRP2B mainly localized to the newly formed part of the cell plate, and that the localization dynamics of DRP2B was quite similar to that of DRP1A, which is an Arabidopsis dynamin-related protein that is closely related to soybean phragmoplastin. These results indicate that Arabidopsis dynamin-related proteins, DRP1A and DRP2B, from two different families, participate in membrane remodeling at a similar place in the cell plate.     

The Arabidopsis peroxisome division mutant pdd2 is defective in the DYNAMIN-RELATED PROTEIN3A (DRP3A) gene

In plants, the division of peroxisomes is mediated by several classes of proteins, including PEROXIN11 (PEX11), FISSION1 (FIS1) and DYNAMIN-RELATED PROTEIN3 (DRP3). DRP3A and DRP3B are two homologous dynamin-related proteins playing overlapping roles in the division of both peroxisomes and mitochondria, with DRP3A performing a stronger function than DRP3B in peroxisomal fission. Here, we report the identification and characterization of the peroxisome division defective 2 (pdd2) mutant, which was later proven to be another drp3A allele. The pdd2 mutant generates a truncated DRP3A protein and exhibits pale green and retarded growth phenotypes. Intriguingly, this mutant displays much stronger peroxisome division deficiency in root cells than in leaf mesophyll cells. Our data suggest that the partial GTPase effector domain retained in pdd2 may have contributed to the distinct mutant phenotype of this mutant.Key words: peroxisome division, dynamin-related protein, arabidopsisIn eukaryotic cells, peroxisomes are surrounded by single membranes and house a variety of oxidative metabolic pathways such as lipid metabolism, detoxification and plant photorespiration.^1,2 To accomplish multiple tasks, the morphology, abundance and positioning of peroxisomes need to be highly regulated. Three families of proteins, whose homologs are present across different kingdoms, have been shown to be involved in peroxisome division in Arabidopsis. The PEX11 protein family is composed of five integral membrane proteins with primary roles in peroxisome elongation/tubulation, the initial step in peroxisome division.^3–5 Although the exact function of PEX11s has not been demonstrated, these proteins are believed to participate in peroxisome membrane modification.^6,7 The FIS1 family consists of two isoforms, which are C-terminal tail-anchored membrane proteins with rate limiting functions at the fission step.^8,9 DRP3A and DRP3B belong to a superfamily of dynamin-related proteins, which are large and self-assembling GTPases involved in the fission and fusion of membranes by acting as mechanochemical enzymes or signaling GTPases.¹⁰ The function of PEX11 seems to be exclusive to peroxisomes, whereas DRP3 and FIS1 are shared by the division machineries of both peroxisomes and mitochondria in Arabidopsis.^8,9,11–16 FIS1 proteins are believed to tether DRP proteins to the peroxisomal membrane,^17,18 but direct evidence has not been obtained from plants. DRP3A and DRP3B share 77% sequence identity at the protein level and are functionally redundant in regulating mitochondrial division; however, DRP3A''s role on the peroxisome seems stronger and cannot be substituted by DRP3B in peroxisome division.^8,13,15In a continuous effort to identify components of the plant peroxisome division apparatus from Arabidopsis, we performed genetic screens in a peroxisomal marker background expressing the YFP (yellow fluorescent protein)-PTS1 (peroxisome targeting signal 1, containing Ser-Lys-Leu) fusion protein. Mutants with defects in the morphology and abundance of fluorescently labeled peroxisomes are characterized. Following our analysis of the pdd1 mutant, which turned out to be a strong allele of DRP3A,⁸ we characterized the pdd2 mutant.In root cells of the pdd2 mutant, extremely elongated peroxisomes and a beads-on-a-string peroxisomal phenotype are frequently observed (Fig. 1A and B). These peroxisome phenotypes resemble those of pdd1 and other strong drp3A alleles previously reported.^8,15 However, the peroxisome phenotype seems to be less dramatic in leaf mesophyll cells. For instance, in addition to the decreased number of total peroxisomes, peroxisomes in leaf cells are only slightly elongated or exhibit a beads-on-a-string phenotype (Fig. 1C and D). Previously, we reported the phenotypes of three strong drp3A alleles, all of which contain a large number of peroxules, long and thin membrane extensions from the peroxisome,⁸ yet such peroxisomal structures are not observed in pdd2. On the other hand, pdd2 has a more severe growth phenotype than most drp3A alleles, as it is slow in growth and has pale green leaves (Fig. 1E). Genetic analysis showed that pdd2 segregates as a single recessive mutation (data not shown).Open in a separate windowFigure 1Phenotypic analyses of pdd2 and identification of the PDD2 gene. (A–D) Confocal micrographs of root and mesophyll cells in 3-week-old wild type and pdd2 mutant plants. Green signals show peroxisomes; red signals show chloroplasts. Scale bars = 20 µm. (E) Growth phenotype of 3-week-old mutants. (F) Map-based cloning of the PDD2 gene. Genetic distance from PDD2 is shown under each molecular marker. Positions for mutations in previously analyzed drp3A alleles and pdd2 are indicated in the gene schematic. drp3A-1 and drp3A-2 are T-DNA insertion mutants, whereas pdd1 is an EMS mutant containing a premature stop codon in exon 6. (G) A schematic of the DRP3A (PDD2) protein with functional domains indicated. The pdd2 allele encodes a truncated protein lacking part of the GED domain.The unique combination of peroxisomal and growth phenotypes of pdd2 prompted us to use map-based cloning to identify the PDD2 gene, with the hope to discover novel proteins in the peroxisome division machinery. A population of approximately 6,000 F₂ plants (pdd2 × Ler) was generated. After screening 755 F₂ mutants, the pdd2 mutation was mapped to the region between markers T10C21 and F4B14 on the long arm of chromosome 4 (Fig. 1F). Since this region contains DRP3A, we sequenced the entire DRP3A gene in pdd2 and identified a G→A transition at the junction of the 18^th exon and intron (Fig. 1F). Further analysis revealed that the point mutation at this junction caused mis-splicing of intron 18, introducing a stop codon in the GTPase effector domain GED near the C terminus (Fig. 1G).DRPs share with the classic dynamins an N-terminal GTPase domain, a middle domain (MD), and a regulatory motif named the GTPase effector domain (GED) (Fig. 1G). To date, a total of 26 drp3A mutant alleles carrying missense or nonsense mutations along the length of the DRP3A gene have been isolated.^8,15 The combined peroxisomal and growth phenotype of pdd2 and the nature of the mutation in this allele are unique among all the drp3A alleles, indicating that the partial GED domain retained in pdd2 may have created some novel function for this protein. Further analysis of the truncated protein may be necessary to test this prediction.     

Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A’s in vivo function.     

Arabidopsis dynamin‐related protein 1E in sphingolipid‐enriched plasma membrane domains is associated with the development of freezing tolerance

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin‐related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol‐enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye‐labelled plasma membrane, providing evidence that DRP1E localizes non‐uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol‐enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation.