Plant genomes encode hundreds of secreted peptides; however, relatively few have been characterised. We report here an uncharacterised, stress-induced family of plant signalling peptides, which we call CTNIPs. Based on the role of the common co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) in CTNIP-induced responses, we identified in Arabidopsis thaliana the orphan receptor kinase HAESA-LIKE 3 (HSL3) as the CTNIP receptor via a proteomics approach. CTNIP-binding, ligand-triggered complex formation with BAK1, and induced downstream responses all involve HSL3. Notably, the HSL3-CTNIP signalling module is evolutionarily conserved amongst most extant angiosperms. The identification of this novel signalling module will further shed light on the diverse functions played by plant signalling peptides and will provide insights into receptor-ligand co-evolution.
Summary Plant immunity is tightly controlled by a complex and dynamic regulatory network, which ensures optimal activation upon detection of potential pathogens. Accordingly, each component of this network is a potential target for manipulation by pathogens. Here, we report that RipAC, a type III-secreted effector from the bacterial pathogen Ralstonia solanacearum , targets the plant E3 ubiquitin ligase PUB4 to inhibit pattern-triggered immunity (PTI). PUB4 plays a positive role in PTI by regulating the homeostasis of the central immune kinase BIK1. Before PAMP perception, PUB4 promotes the degradation of non-activated BIK1, while, after PAMP perception, PUB4 contributes to the accumulation of activated BIK1. RipAC leads to BIK1 degradation, which correlates with its PTI-inhibitory activity. RipAC causes a reduction in pathogen-associated molecular pattern (PAMP)-induced PUB4 accumulation and phosphorylation. Our results shed light on the role played by PUB4 in immune regulation, and illustrate an indirect targeting of the immune signalling hub BIK1 by a bacterial effector.
Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant-derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TOPGAP, to remodel the host-pathogen interface. The effector, PiE354, hijacks TOPGAP as a susceptibility factor to usurp its GAP activity on Rab8a, a key Rab GTPase crucial for defense-related secretion. By hijacking TOPGAP, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co-opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens.
The salicylic acid-induced protein kinase (SIPK) of tobacco, which is a mitogen-activated protein kinase (MAPK), is activated by various biotic and abiotic treatments. Overexpression of SIPK has been shown to trigger cell death. In this study, a targeted yeast two-hybrid approach identified the tobacco transcription factor WRKY1 as a potential substrate. SIPK phosphorylated WRKY1, which resulted in enhanced DNA-binding activity of WRKY1 to its cognate binding site, a W box sequence from the tobacco chitinase gene CHN50. SIPK-mediated enhancement of WRKY1 DNA-binding activity was inhibited by staurosporine, a general kinase inhibitor. Co-expression of SIPK and WRKY1 in Nicotiana benthamiana led to more rapid cell death than expression of SIPK alone, suggesting that WRKY1 is involved in the formation of hypersensitive response-like cell death and may be a component of the signaling cascade downstream of SIPK.
Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor alpha-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously. Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.
Abstract The oomycete pathogen Hyaloperonospora arabidopsidis ( Hpa ) causes downy mildew disease on Arabidopsis . During infection, Hpa like other biotrophic pathogens, suppresses activation of plant innate immunity by translocating effector proteins into host cells. Some of these effectors localize to the host cell nucleus where they may manipulate transcriptional reprogramming of plant defense genes. Here we report that the nuclear-localized Hpa effector HaRxL106, when expressed in Arabidopsis , induces shade avoidance and attenuates the transcriptional response to the defense signaling molecule salicylic acid. HaRxL106 interacts with RADICAL-INDUCED CELL DEATH1 (RCD1) and loss of RCD1 function renders Arabidopsis resilient against HaRxL106-mediated suppression of immunity. To further characterize the molecular functions of RCD1 we solved a crystal structure of RCD1’s Poly-(ADP-ribose)-Polymerase (PARP) domain and, based on non-conservation of amino acids constituting the active site of canonical PARPs, conclude that RCD1 has no PARP activity. We report that RCD1-type proteins are phosphorylated and identified histone-modifying Mut9-like kinases (MLKs) as RCD1-interacting proteins. A mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared to wild-type plants. Our data suggest that HaRxL106 suppresses Arabidopsis innate immunity by manipulating the function(s) of RCD1 in the host cell nucleus and point towards a role of RCD1 as a transcriptional co-regulator that integrates signals from light and pathogen sensors.
Full text Figures and data Side by side Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses. https://doi.org/10.7554/eLife.10856.001 eLife digest Plants and other living organisms can survive stress and starvation by digesting and recycling parts of their own cells. This process is known as autophagy and it involves engulfing cellular material inside spherical structures called autophagosomes, before delivering it to sites in the cell where digestive enzymes can break the material down. A form of autophagy, known as selective autophagy, can specifically degrade toxic substances such as disease-causing microbes. Selective autophagy works through proteins called autophagy cargo receptors that define which molecules are targeted for degradation. However, it was not clear whether autophagy protects plants from infections, or how much disease-causing microbes interfere with this process for their own benefit. The microbe that causes late blight of potatoes (called Phytophthora infestans) is infamous for triggering widespread famines in Ireland in the 19th century. This disease-causing microbe continues to pose a serious threat to food security today, and parasitizes plant tissues by releasing proteins called effectors that enter the plant’s cells to subvert the plant’s physiology and counteract its defenses. Dagdas, Belhaj et al. now report that an effector from P. infestans, called PexRD54, can bind to autophagy-related protein from potato, called ATG8CL, and stimulate the formation of autophagosomes. Further experiments revealed that the PexRD54 effector could outcompete a plant autophagy cargo receptor that would otherwise bind to ATG8CL. This plant cargo receptor contributes to the plant’s defences, and by preventing it from interacting with ATG8CL, PexRD54 makes the plant more susceptible to infection by P. infestans. These findings show that the PexRD54 effector has evolved to interact with an autophagy-related protein to counteract the plant’s defences. Dagdas, Belhaj et al. suggest that PexRD54 might do this by activating autophagy to selectively eliminate some of the molecules that the plant use to defend itself. Furthermore, P. infestans might also benefit from the nutrients that are released when cellular material is broken down via autophagy. Future work could test these two hypotheses and explore whether other effectors from disease-causing microbes work in a similar way. https://doi.org/10.7554/eLife.10856.002 Introduction Autophagy is conserved catabolic pathway that sequesters unwanted cytosolic components into newly formed double membrane vesicles, autophagosomes, to direct them to the cell’s lytic compartment (He and Klionsky, 2009). The process plays a vital role in survival of the organism by improving cellular adaptation to environmental and stress conditions (Shintani and Klionsky, 2004). Autophagy provides building blocks and energy for elementary cellular processes by degrading dysfunctional or unnecessary cellular components during nutrient deprivation (Shintani and Klionsky, 2004). However, even though autophagy was initially thought to be a bulk degradation process activated during starvation, recent studies showed that it can act selectively, capturing specific substrates through specialized cargo receptors to respond to a variety of environmental and stress conditions (Stolz et al., 2014). Autophagy is executed through coordinated action of more than 30 core proteins known as the ATG (autophagy-related) proteins (Lamb et al., 2013). Selective autophagy is regulated through specific interactions of autophagy cargo receptors and ATG8 proteins (Stolz et al., 2014). Autophagy cargo receptors carry a short sequence motif called ATG8-interaction motif (AIM) that binds lipidated ATG8 proteins anchored on autophagosomal membranes. Cargo receptors mediate recognition of a diverse set of cargo (Stolz et al., 2014). For instance, mammalian autophagy cargo receptors NDP52 and optineurin can recognize intracellular pathogenic bacteria and mediate their autophagic removal by sorting the captured bacteria inside the ATG8-coated autophagosomes (Boyle and Randow, 2013). Nevertheless, the precise molecular mechanisms of selective autophagy and the components that regulate it remain unknown (Huang and Brumell, 2014; Mostowy, 2013; Randow, 2011). In plants, autophagy plays important roles in stress tolerance, senescence, development, and defense against invading pathogens (Patel and Dinesh-Kumar, 2008; Lenz et al., 2011; Vanhee and Batoko, 2011; Li and Vierstra, 2012; Lv et al., 2014; Teh and Hofius, 2014). Specifically, autophagy is implicated in the accumulation of defense hormones and the hypersensitive response, a form of plant cell death that prevents spread of microbial infection (Yoshimoto et al., 2009). However, the molecular mechanisms that mediate defense-related autophagy and the selective nature of this process are poorly understood. Furthermore, how adapted plant pathogens manipulate defense-related autophagy and/or subvert autophagy for nutrient uptake is unknown. In this study, we investigated how a pathogen interferes with and coopts a plant autophagy pathway. The potato blight pathogen, Phytophthora infestans, is a serious threat to food security, causing crop losses that, if alleviated, could feed hundreds of millions of people (Fisher et al., 2012). This pathogen delivers RXLR-type effector proteins inside plant cells to enable parasitism (Morgan and Kamoun, 2007). RXLR effectors form a diverse family of modular proteins that alter a variety of host processes and therefore serve as useful probes to dissect key pathways for pathogen invasion (Morgan and Kamoun, 2007; Bozkurt et al., 2012). Here, we show that the RXLR effector PexRD54 has evolved to bind host autophagy protein ATG8CL to stimulate autophagosome formation. In addition, PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes to counteract host defenses against P. infestans. Results and discussion As part of an in plantascreen for host interactors of RXLR effectors, we discovered that the P. infestans effector PexRD54 associates with ATG8CL, a member of the ATG8 family (Materials and methods, Supplementary files 1,2). The association between PexRD54 and ATG8CL was retained under stringent binding conditions in contrast to other candidate interactors (Supplementary file 1). We validated the association with reverse coimmunoprecipitation after co-expressing the potato ATG8CL protein with the C-terminal effector domain of PexRD54 in planta (Figure 1A,B). In addition, PexRD54 expressed and purified from Escherichia coli directly bound ATG8CL in vitro with high affinity and in a one to one ratio (KD = 383 nM based on isothermal titration calorimetry) (Figure 1C). PexRD54 has two predicted ATG8 Interacting Motifs (AIMs) that match the consensus amino acid sequence W/F/Y-x-x-L/I/V (AIM1 and AIM2, Figure 1A). In planta coimmunoprecipitations of single and double AIM mutants of PexRD54 revealed that AIM2, which spans the last four amino acids of the protein (positions 378–381), is required for association with ATG8CL (Figure 1D). PexRD54AIM2 mutant also failed to bind ATG8CL in vitro (Figure 1E). In addition, ATG8CL bound with high affinity to a synthetic peptide (KPLDFDWEIV) that matches the last 10 C-terminal amino acids of PexRD54 (KD = 220 nM) (Figure 1—figure supplement 1). We conclude that the C-terminal AIM of PexRD54 is necessary and sufficient to bind ATG8CL. Figure 1 with 1 supplement see all Download asset Open asset PexRD54 binds to ATG8CL via a C-terminal ATG8 interacting motif (AIM). (A) Domain organization of PexRD54. PexRD54 is a canonical RXLR effector with five WY folds (Win et al., 2012). The amino acid sequences of candidate AIMs are highlighted in yellow color and indicated in brackets. (B) Validation of PexRD54-ATG8CL association in planta. RFP:PexRD54 or RFP:EV (Empty vector) were transiently co-expressed with GFP:ATG8CL or GFP:EV in N. benthamiana leaves. Immunoprecipitates (IPs) obtained with anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera. Stars indicate the expected band sizes. (C) PexRD54 binds ATG8CL in vitro. The binding affinity of PexRD54 to ATG8CL was determined using isothermal titration calorimetry (ITC). Upper panel shows heat differences upon injection of ATG8CL into buffer or PexRD54 and the bottom panel show integrated heats of injection (■) and the best fit (solid line) to a single site binding model using MicroCal Origin. KD=383 nM, N=0.998, ΔH= −8.966 kJ.mol-1 and ΔS = 0.092 J.mol-1.K-1. The values of KD, N, ΔH and ΔS are representative of two independent ITC experiments. (D) ATG8 Interacting Motif 2 (AIM2) mediates ATG8CL binding in planta. RFP:PexRD54, RFP:PexRD54AIM1, RFP:PexRD54AIM2 or RFP:PexRD54AIM1&AIM2 were transiently co-expressed with GFP:ATG8CL or GFP:EV in N. benthamiana leaves. IPs obtained with anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera. Stars indicate the expected band sizes. (E) AIM2 mediates ATG8CL binding in vitro. The binding affinity of PexRD54AIM2 to ATG8CL was determined using ITC. Upper panel shows heat differences upon injection of PexRD54AIM2 and the bottom panel show integrated heats of injection (■). No binding was detected between PexRD54AIM2 and ATG8CL. https://doi.org/10.7554/eLife.10856.003 ATG8 occurs as a family of nine proteins in potato (Figure 2—figure supplements 1–2). PexRD54 bound ATG8CL with ~10 times higher affinity than another ATG8 family member, ATG8IL, in both in planta and in vitro assays (Figure 2). These findings prompted us to use ATG8IL as a negative control in the subsequent experiments. Figure 2 with 2 supplements see all Download asset Open asset PexRD54 has higher binding affinity to ATG8CL than ATG8IL. (A) RFP:PexRD54, RFP:AVRblb2 or RFP:EV were transiently co-expressed with GFP:ATG8CL, GFP:ATG8IL or GFP:EV in N. benthamiana leaves and proteins were extracted two days after infiltration and used in immunoprecipitation experiments (IPs). IPs obtained with anti-GFP or anti-RFP antisera and total protein extracts were immunoblotted with appropriate antisera. RFP:AVRblb2 (Bozkurt et al., 2011), an RFP fusion to a different P. infestans RXLR effector, did not associate with ATG8CL or ATG8IL. Both the GFP and RFP IPs indicate higher binding affinity of PexRD54 to ATG8CL than ATG8IL. Stars indicate the expected band sizes. (B) PexRD54 has lower binding affinity to ATG8IL in vitro. The binding affinity of PexRD54 to ATG8IL was determined using isothermal titration calorimetry (ITC). Upper panel shows heat generated upon injection of ATG8IL into buffer or PexRD54 and lower panel shows integrated heats of injection (■) and the best fit (solid line) to a single site binding model using MicroCal Origin. The values of KD = 4100 nM, N = 0.938, ΔH = −11.305 kJ.mol-1 and ΔS= 0.064 J.mol-1K-1 are representative values of two independent ITC experiments. The data show that PexRD54 binds ATG8CL with ~10 times higher affinity than ATGIL. https://doi.org/10.7554/eLife.10856.005 We then investigated the subcellular localization of PexRD54 within plant cells. N-terminal fusions of PexRD54 to the green fluorescent protein (GFP) or red fluorescent protein (RFP) labelled the nucleo-cytoplasm and mobile punctate structures (Figure 3—figure supplement 1 and Video 1). Immunogold labelling in transmission electron micrographs of cells expressing GFP:PexRD54 revealed a strong signal in electron dense structures that are not peroxisomes (Figure 3—figure supplements 2–3, Dagdas et al., 2016). To determine whether these structures are ATG8CL autophagosomes, we transiently co-expressed RFP:PexRD54 with GFP:ATG8CL in plant cells and observed an overlap between the two fluorescent signals in sharp contrast to RFP:PexRD54AIM2 and RFP:EV negative controls (Figure 3 and Video 2). This indicates that PexRD54 localizes to ATG8CL-marked autophagosomes and its C-terminal AIM is necessary for autophagosome localization. In contrast, RFP:PexRD54 signal overlapped with GFP:ATG8IL-labelled autophagosomes in only 15–20% of observations consistent with its weaker binding affinity to ATG8IL (Figure 3—figure supplement 4). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg PexRD54 localizes to mobile endomembrane compartments. GFP:PexRD54 was transiently expressed in N. benthamiana leaves and examined by confocal laser scanning microscopy, 3 days post infiltration. (Related to Figure 3) https://doi.org/10.7554/eLife.10856.008 Figure 3 with 7 supplements see all Download asset Open asset PexRD54 localizes to ATG8CL labelled autophagosomes. Transient co-expression of GFP:ATG8CL with (A) RFP:PexRD54, (B) RFP:PexRD54AIM2 and (C) RFP control (RFP:EV) in N. benthamiana leaves. Confocal micrographs show single optical sections of RFP:PexRD54, RFP:PexRD54AIM2 and RFP:EV in red, GFP:ATG8CL in green and the overlay indicating colocalization in yellow. White arrowheads point to punctate structures and yellow arrowheads point to puncta where GFP and RFP signals overlap. Far right panels highlight the dotted square region focusing on GFP:ATG8CL labelled puncta in overlaid GFP/RFP channels. Scale bar = 10 μm; scale bar in inset = 1 μm. (D) The intensity plots represent relative GFP and RFP fluorescence signals along the dotted line connecting points a-b, c-d and e-f that span GFP:ATG8CL marked puncta at far right panels. GFP:ATG8CL fluorescence intensity peak overlapped with fluorescence intensity peak of RFP:PexRD54 (left panel) but not with RFP:PexRD54AIM2 (mid panel) or RFP:EV (right panel) validating the localization of RFP:PexRD54 at GFP:ATG8CL labelled autophagosomes. (E) Bar charts showing colocalization of GFP:ATG8CL puncta with RFP:PexRD54, RFP:PexRD54AIM2 or RFP:EV punctate structures. Data are representative of 500 individual images from two biological replicates. Each replicate consists of five independent Z stacks with 50 images each, acquired from five independent leaf areas. (***p<0.001). Error bars represent ± SD. https://doi.org/10.7554/eLife.10856.009 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg PexRD54 and ATG8CL colocalize at mobile endomembrane compartments. RFP:PexRD54 was transiently co-expressed with GFP:ATG8CL in N. benthamiana leaves and examined by confocal laser scanning microscopy, 3 days post infiltration. (Related to Figure 3) https://doi.org/10.7554/eLife.10856.017 To further confirm that PexRD54-labelled endomembrane compartments are indeed autophagosomes, we investigated the effect of the autophagy inhibitor 3-methyl adenine (3-MA) (Hanamata et al., 2013) on PexRD54 localization. Compared to water, 3-MA treatment reduced the number of PexRD54 and ATG8CL puncta but did not reduce the number of puncta of the trans-Golgi network (TGN) marker VTI12 (Geldner et al., 2009) (Figure 3—figure supplement 5). Phospholipid modification of a conserved glycine residue at the C-terminus of ATG8 proteins is required for autophagosome formation, and deletion of this terminal glycine yields a dominant negative ATG8 (Hanamata et al., 2013). We deployed a terminal glycine deletion mutant of ATG8CL (ATG8CLΔ) to determine its effect on subcellular distribution of PexRD54 (Figure 3—figure supplement 6). As expected, deletion of the terminal glycine did not affect binding of ATG8CL to PexRD54 (Figure 3—figure supplement 7A). However, GFP:ATG8CLΔ led to the depletion of RFP:PexRD54 labelled puncta presumably because the dominant negative effect of ATG8CLΔ prevented accumulation of RFP:PexRD54 in ATG8CL-labelled autophagosomes (Figure 3—figure supplement 7B–D). In contrast, GFP:ATG8ILΔ, a terminal glycine deletion mutant of ATG8IL, had no effect on the punctate localization of RFP:PexRD54 (Figure 3—figure supplement 7C–D). These experiments independently support the finding that PexRD54 accumulates in ATG8CL autophagosomes. Increase in ATG8 labelled puncta is widely used as a functional readout of autophagic activity (Hanamata et al., 2013; Bassham, 2015). In samples expressing PexRD54, we noticed a ~fivefold increase in the number of ATG8CL marked autophagosomes compared to control samples expressing PexRD54AIM2 or empty vector control (Figure 4, Video 3). In contrast, PexRD54 did not alter the number of ATG8IL autophagosomes consistent with the weak binding noted between these two proteins (Figure 4A). This indicates that PexRD54 stimulates the formation of ATG8CL autophagosomes. Figure 4 Download asset Open asset PexRD54 increases the number of ATG8CL autophagosomes. (A) Co-expression of RFP:PexRD54, but not RFP:PexRD54AIM2 or RFP:EV significantly enhanced the number of GFP:ATG8CL-labelled autophagosomes in N. benthamiana. Bar charts display the number of GFP:ATG8CL or GFP:ATG8IL-labelled autophagosomes in the presence of RFP:PexRD54, RFP:PexRD54AIM2 or RFP:EV. GFP:ATG8CL autophagosomes were significantly enhanced by the expression of RFP:PexRD54 (***p<0.001). GFP:ATG8IL autophagosomes were not significantly enhanced by expression of RFP:PexRD54 (ns=statistically not significant). The data are representative of 500 individual images from two biological replicates. Each replicate consists of five independent Z stacks with 50 images each, acquired from five independent leaf areas. (B) GFP:ATG8CL was transiently co-expressed with RFP:PexRD54, RFP:PexRD54AIM2 or RFP:EV in N. benthamiana leaves and examined by confocal laser scanning microscopy 3 days post infiltration. Maximum projections of images show that RFP:PexRD54 increases the number of GFP:ATG8CL- labelled autophagosomes. RFP:PexRD54AIM2 and RFP:EV were used as controls. Arrowheads point to punctate structures. Regions where GFP or RFP-labelled puncta do not overlap are indicated with dotted squares. Scale bar=50 μm. Zoomed single plane images shown in the second panel indicate that larger puncta co-labelled by RFP:PexRD54 and GFP:ATG8CL are ring-shaped autophagosome clusters. Scale bar=10 μm. https://doi.org/10.7554/eLife.10856.018 Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg RFP:PexRD54 and GFP:ATG8CL colocalize at mobile ring shaped clusters. RFP:PexRD54 was transiently co-expressed with GFP:ATG8CL in N. benthamiana leaves and examined by confocal laser scanning microscopy, 3 days post infiltration. (Related to Figure 4) https://doi.org/10.7554/eLife.10856.019 Next, we set out to determine the effect of PexRD54 on autophagic flux. Treatment of RFP:ATG8CL expressing leaves with the specific vacuolar ATPase inhibitor concanamycin-A (Bassham, 2015) increased the number of ATG8CL-labelled puncta both in the presence of PexRD54 or controls (PexRD54AIM2 or vector control) indicating that PexRD54 does not block autophagic flux (Figure 5A–B). We also confirmed these observations using western blot analyses. PexRD54, but neither PexRD54AIM2 or vector control, increased the levels of GFP:ATG8CL protein 3 days after co-expression in planta (Figure 5C). Treatment of three-day samples with E64d, an inhibitor of vacuolar cysteine proteases (Bassham, 2015), further increased protein levels of GFP:ATG8CL. This further confirms that PexRD54 stimulates autophagy rather than blocking autophagic flux (Figure 5C). PexRD54 did not alter the accumulation of GFP:ATG8IL or control GFP protein, confirming that PexRD54 increases ATG8CL protein accumulation specifically (Figure 5C). Consistent with these observations, we noted an increase in GFP:ATG8CL levels, but not in control GFP, during P. infestans infection relative to the mock infection (Figure 5—figure supplement 1). Figure 5 with 1 supplement see all Download asset Open asset PexRD54 does not block autophagic flux. (A–B) ConcanamycinA treatment increases the number of autophagosomes in PexRD54 expressing samples.RFP:ATG8CL was transienly coexpressed with PexRD54, PexRD54AIM2 and empty vector controls in N. benthamiana. Two days after infiltration, leaves were treated with concanamycinA (conA) or infiltration buffer and number of autophagosomes was counted 24 hr after treatment. ConA treatment significantly increased the number of autophagosomes in PexRD54 expressing cells (p<0.05), confirming PexRD54 does not block autophagic flux. Scale bar=10 μm. (C) E64D treatment increases ATG8CL protein levels in PexRD54 expressing samples. GFP:ATG8CL, GFP:ATG8IL and GFP:EV were transiently coexpressed with RFP:GUS, RFP:PexRD54 or RFP:PexRD54AIM2 in N. benthamiana leaves and protein levels in total extracts were determined two and 3 days post infiltration (dpi). RFP:PexRD54 increased protein levels of GFP:ATG8CL but not GFP:ATG8IL consistent with stronger binding affinity of PexRD54 to ATG8CL. RFP:PexRD54 did not increase protein levels of GFP:EV, suggesting that protein level increase depends on ATG8CL binding and that PexRD54 does not increase protein levels in general. The samples were also treated with E64d to measure autophagic flux. In RFP:PexRD54 coexpressed 3 dpi samples, E64d treatment increased ATG8CL protein levels even more suggesting PexRD54 does not block autophagic flux. Hence, protein level increase is a result of stimulation of autophagy. The blots were stained with Ponceau stain (PS) to show equal loading. https://doi.org/10.7554/eLife.10856.020 The presence of a functional AIM in PexRD54 prompted us to hypothesize that this effector perturbs the autophagy cargo receptors of its host plants. Recently, Joka2 was reported as a selective autophagy cargo receptor of Solanaceous plants that also binds ATG8 via an AIM (Svenning et al., 2011; Zientara-Rytter et al., 2011) (Figure 6A). Indeed, in planta coimmunoprecipitation assays confirmed that potato Joka2, but not the AIM mutant, Joka2AIM, associated with ATG8CL (Figure 6—figure supplement 1). Joka2 association with ATG8CL was somewhat specific given that this cargo receptor failed to coimmunoprecipitate with ATG8IL (Figure 6—figure supplement 2). Joka2, but not Joka2AIM, also markedly increased the number of GFP:ATG8CL autophagosomes (Figure 6—figure supplement 3, Video 4), and enhanced ATG8CL protein levels (Figure 6—figure supplement 4). This indicates that Joka2 also activates ATG8CL-mediated selective autophagy. Figure 6 with 6 supplements see all Download asset Open asset PexRD54 is a competitive antagonist of the plant selective autophagy cargo receptor Joka2. (A) Domain organization of Joka2. (B) PexRD54 reduces binding of Joka2 to ATG8CL in a dose-dependent manner. GFP:ATG8CL (OD=0.2) and Joka2:RFP (OD=0.2) were transiently co-expressed with varying Agrobacterium concentrations (from OD=0 to OD=0.4) carrying HA:PexRD54 construct in N. benthamiana (OD=optical density of Agrobacterium cells). Joka2:RFP is depleted in GFP:ATG8CL pulldowns as the expression of HA:PexRD54 increased. Immunoprecipitates (IPs) obtained with anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera. Stars indicate expected band sizes. (C) Schematic illustrations of PexRD54 variants (PexRD54J2AIM1 and PexRD54J2AIM2) with Joka2 AIM peptides. (D) Replacement of PexRD54 AIM with Joka2 AIM fragments decreases ATG8CL binding affinity. Immunoblots showing binding affinity of PexRD54, PexRD54AIM2, PexRD54J2AIM1 or PexRD54J2AIM2 to GFP:ATG8CL. IPs obtained with anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera. Stars indicate expected band sizes. https://doi.org/10.7554/eLife.10856.022 Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg RFP:ATG8CL and Joka2:GFP colocalize at mobile endomembrane compartments. RFP:ATG8CL was transiently coexpressed with Joka2:GFP in N. benthamiana leaves and examined by confocal laser scanning microscopy, 3 days post infiltration. (Related to Figure 6) https://doi.org/10.7554/eLife.10856.029 Given that both PexRD54 and Joka2 bind ATG8CL via their respective AIMs, we hypothesized that PexRD54 interferes with the Joka2-ATG8CL complex. We tested our hypothesis by performing coimmunoprecipitation experiments between Joka2:RFP and GFP:ATG8CL in the presence or absence of PexRD54. Remarkably, ATG8CL complexes were depleted in Joka2 in the presence of PexRD54 relative to the PexRD54AIM2 and vector control (Figure 6—figure supplement 5). Consistently, Joka2 binding to ATG8CL decreased with increasing PexRD54 concentrations (Figure 6B). The distinct AIMs of PexRD54 and Joka2 presumably determine the effect observed in these competition experiments. To further test this, we replaced the functional PexRD54 AIM with two sequences that cover the Joka2 AIM:GVAEWDPI (PexRD54J2AIM1) and GVAEWDPILEELKEMG (PexRD54J2AIM2) (Figure 6C). Both PexRD54J2AIM1 and PexRD54J2AIM2 associated with ATG8CL to a lesser extent than wild-type PexRD54 (Figure 6D), and were less effective than PexRD54 in depleting Joka2 out of ATG8CL complexes (Figure 6—figure supplement 6). These findings reveal that PexRD54 antagonizes Joka2 for ATG8CL binding. Finally, we investigated the degree to which activation of Joka2-ATG8CL-mediated autophagy contributes to pathogen defense. Overexpression of Joka2, but not Joka2AIM, significantly restricted the size of the disease lesions caused by P. infestans (Figure 7A–B). Conversely, virus-induced gene silencing of Joka2 resulted in increased disease lesions (Figure 7—figure supplement 1). This indicates that Joka2-mediated selective autophagy contributes to defense against this pathogen. Remarkably, PexRD54 counteracted the enhanced resistance conferred by Joka2 whereas PexRD54AIM2 failed to reverse this effect (Figure 7C–D). We conclude that PexRD54 counteracts the positive role of Joka2-mediated selective autophagy in pathogen defense. Figure 7 with 1 supplement see all Download asset Open asset PexRD54 counteracts the enhanced resistance conferred by Joka2. (A) Overexpression of Joka2:RFP limits P. infestans colonization. Halves of N. benthamiana leaves expressing RFP:EV, Joka2:RFP and Joka2AIM:RFP were infected with P. infestans and pathogen growth was determined by lesion sizes measured 6 days post-inoculation. (B) Categorical scatter plots show lesion diameters of 11 infections sites from three independent biological replicates pointed out by three different colors. Similar p values (p<0.001) were obtained in three independent biological repeats. (C) PexRD54 counteracts the effect of Joka2 on P. infestans colonization. Joka2:RFP was co-expressed with HA:EV, HA:PexRD54 or HA:PexRD54AIM2 in N. benthamiana leaves which are then inoculated with P. infestans. Joka2:RFP failed to limit pathogen growth in the presence of PexRD54, whereas it could still restrict pathogen growth in the presence of PexRD54AIM2 or vector control (EV). (D) Categorical scatter plots show lesion diameters of 11 infections sites from three independent biological replicates pointed out by three different colors. Similar p values (p<0.001) were obtained in three independent biological repeats. https://doi.org/10.7554/eLife.10856.030 As demonstrated in mammalian systems, eukaryotic cells employ autophagy to defend against invading pathogens (Boyle and Randow, 2013; Randow and Youle, 2014). In turn, pathogens can deploy effectors to avoid autophagy and enable parasitic infection (Baxt et al., 2013). For instance, to counteract antimicrobial autophagy, intracellular bacterial pathogen Legionella pneumophila secretes a type IV effector protein RavZ that impedes autophagy by uncoupling ATG8-lipid linkage (Choy et al., 2012). In this study, we show that a plant pathogen effector has evolved an ATG8 interacting motif to bind with high affinity to the autophagy protein ATG8CL and stimulate the formation of ATG8CL-marked autophagosomes. Unlike the Legionella effector RavZ, PexRD54 activates selective autophagy possibly to eliminat
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