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
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
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.
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.
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.
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).
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.
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).
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.
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.
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 eliminate defense-related compounds or to reattribute cellular resources by promoting nutrient recycling. Our results show that, in addition to disrupting, pathogens can also activate autophagy for their own benefit (Figure 8).
Additionally, we show that the effector competes with the host cargo receptor Joka2 and depletes it out of ATG8CL autophagosomes to promote disease susceptibility. Thus P. infestans coopts the host cell’s endomembrane compartment to promote its own growth at the cost of the cell’s physiology (Bozkurt et al., 2015). Joka2 could contribute to immunity by inducing autophagic removal of plant or pathogen molecules that negatively affect host defenses. It will be interesting in the future to determine the identity of potential defense-related cargo carried by Joka2 (Figure 8).
The physiological roles of selective autophagy and the molecular mechanisms involved remain to be determined both in plants and animals. Characterization of additional host cargo receptors and interactome analysis of particular ATG8 proteins should improve our understanding of how selective autophagy operates in response to a variety of stress conditions including immunity. In the potato genome, we identified nine ATG8 genes. Both Joka2 and the effector showed higher affinity to ATG8CL compared to ATG8IL (Figure 2 and Figure 6—figure supplement 2). This indicates that variation between plant ATG8 proteins may contribute to the selective nature of autophagy.
This work further highlights the intricate changes in endomembrane compartment formation that take place during plant-microbe interactions (Bozkurt et al., 2015; Lipka and Panstruga, 2005; Kwon et al., 2008; Ivanov et al., 2010; Wang et al., 2010). Future studies will need to consider pathogen-directed modulation of the spatio-temporal dynamics of autophagy and subcellular trafficking during host infection. It will be interesting to determine whether other plant pathogens also secrete effectors that evolved an ATG8 interacting motif or target autophagy in other ways. These effectors would serve as valuable tools to dissect the mechanisms of defense-related selective autophagy.
ATG8 interacting motif (AIM), also known as LC3 interacting region (LIR), mediates interaction of cargo receptors or adaptors with ATG8 proteins anchored in autophagosome membranes (Birgisdottir et al., 2013). It follows the W/F/Y-xx-L/I/V amino acid consensus. Initially, we identified two AIM candidates in PexRD54, AIM1 and AIM2 based on the manual search of the consensus sequence mentioned earlier. Then, we confirmed these AIM candidates using the recently published iLIR software (Kalvari et al., 2014). This software assigned AIM1 with an iLIR score of 12 (1.1e-1) and AIM2 with an iLIR score of 23 (3.2e-3).
To determine the ATG8 variant(s) specifically targeted by PexRD54 among various host ATG8 family members, we performed a BLASTP search (Altschul et al., 1990) against solanaceous plant proteomes including Solanum tuberosum (potato), Solanum lycopersicum (tomato) and Nicotiana benthamiana using Arabidopsis thaliana ATG8C as the query protein sequence. We found nine ATG8 members in S. tuberosum, seven in S. lycopersicum, and eight in N. benthamiana. To verify the gene calls and open reading frame predictions of the putative orthologs in these three species, we performed a sequence alignment of the family members in each species using the Clustal X program (v2) (Larkin et al., 2007) and compared it to the published A. thaliana ATG8 sequences. We found evidence of misannotation for two S. lycopersicum sequences (Solyc10g006270 and Solyc08g078820) and three others in N. benthamiana (NbS00015425g0005, NbS00003316g0005 and NbS00003005g0010). The two S. lycopersium sequences and the NbS00003005g0010 sequence from N. benthamiana carried extra nucleotides before the likely start codon, which were corrected accordingly. A TBLASTN search against the N. benthamiana scaffolds followed by a BLASTP search of the different exons allowed curation of the two other sequences. Clustal X program was used for multiple sequence alignment of ATG8 variants. Boxshade server (http://embnet.vital-it.ch/software/BOX_form.html) was used to visualize the sequence alignment.
The phylogenetic tree of ATG8 homologs in plants was constructed with the neighbor-joining method using ATG8-like curated proteins from S. tuberosum, S. lycopersium, N. benthamina, and A. thaliana. The phylogenetic tree was constructed using MEGA5 (Kumar et al., 2001) with bootstrap values based on 1000 iterations. ATG8CL variants in N. benthamiana and potato have identical amino acid sequences.
All primers used in this study are listed in Supplementary file 3. PexRD54 and Joka2 were amplified using polymerase chain reaction (PCR) from genomic DNA of Phytophthora infestans isolate T30-4 and Solanum tuberosum cv. Désirée cDNA, respectively. To amplify both amplicons, we used Phusion proof reading polymerase (New England Biolabs, UK) and primer pairs listed in Supplementary file 3. All amplicons were subsequently cloned into the pENTR/D-Topo Gateway entry vector (Invitrogen, UK). The ATG8CL, ATG8CLΔ, ATG8IL, and ATG8ILΔ entry clones were custom synthesized into pUC57-AmpR using the sequences matching S. tuberosum ATG8CL or ATG8IL genes flanked by attL1 and attL2 gateway sites (Genewiz, UK). The destination constructs GFP:PexRD54, RFP:PexRD54, Joka2:RFP, Joka2:GFP, RFP:ATG8CL, GFP:ATG8CL, GFP:ATG8CLΔ, GFP:ATG8ILΔ, HA:PexRD54 were generated by Gateway LR recombination reaction (Invitrogen) of the corresponding entry clone and Gateway destination vectors pH7WGR2 (N-terminal RFP fusion), pK7WGF2 (N-terminal GFP fusion), pB7FWR2 (C-terminal GFP fusion), pB7RWG2 (C-terminal RFP fusion) and pK7WGF2 (N-terminal HA fusion, generated in house by replacement of GFP with an HA tag), respectively (Karimi et al., 2002). The GFP:PexRD54AIM2, RFP:PexRD54AIM1, RFP:PexRD54AIM2, RFP:PexRD54AIM1+2 RFP:PexRD54KPLDFDWEIV, RFP:PexRD54J2AIM1, RFP:PexRD54J2AIM2, HA:PexRD54AIM2, HA:PexRD54AIM2, HA:PexRD54J2AIM1, HA:PexRD54J2AIM2, Joka2AIM:RFP constructs were generated as follows:PexRD54 and Joka2 mutant constructs (PexRD54AIM1, PexRD54AIM2, PexRD54AIM1+2, PexRD54KPLDFDWEIV, RFP:PexRD54J2AIM1, RFP:PexRD54J2AIM2, and Joka2AIM:RFP) were cloned into the pENTR/D-Topo Gateway entry vector (Invitrogen) by PCR amplification with the primers carrying desired mutations (Supplementary file 3) using PexRD54 and Joka2 entry clones as templates followed by TOPO cloning procedure (Invitrogen). Templates were then eliminated by one-hour Dpn-I (New England Biolabs) restriction digestion at 37°C. Next, the entry clones of PexRD54 and Joka2 mutants were recombined into destination vectors pH7WGR2 or pB7RWG2 by Gateway LR reaction (Invitrogen). pTRBO FLAG:PexRD54 construct used for coimmunoprecipitations was custom synthesized (Genscript, , New Jersey, USA).
To generate ATG8 interacting motif (AIM) mutants in PexRD54 and Joka2, the conserved tryptophan and leucine residues of the canonical AIMs were both mutated to Alanine as previously done (Zientara-Rytter et al., 2011). In PexRD54AIM1, PexRD54AIM2 and Joka2AIM, the AIM motifs e.g. 'WLRL', 'WEIV' and 'WDPI' were mutated to 'ALRA', 'AEIA' and 'ADPA', respectively.
Nicotiana benthamiana plants were grown and maintained throughout the experiments in a greenhouse at 22–25°C with high light intensity. Phytophthora infestans cultures were grown in plates with rye sucrose agar (RSA) media for 12–14 days as described elsewhere (Song et al., 2009). Sporangia were harvested from plates using cold water and zoospores were collected 1–3 hr after incubation at 4°C. Infection assays were performed by droplet inoculations of zoospore solutions of P. infestans on 3–4-week-old detached N. benthamiana leaves as described previously (Song et al., 2009; Saunders et al., 2012). For all infection assays, P. infestans isolate 88,069 was used.
Transient gene-expression in planta was performed by delivering T-DNA constructs with Agrobacterium tumefaciens GV3101 strain into 3–4-week-old N. benthamiana plants as described previously (Bozkurt et al., 2011). For transient co-expression assays, A. tumefaciens strains carrying the plant expression constructs were mixed in a 1:1 ratio in agroinfiltration medium [10 mM MgCl2, 5 mM 2-(N-morpholine)-ethanesulfonic acid (MES), pH 5.6] to a final OD600 of 0.2, unless otherwise stated.
Proteins were transiently expressed by A. tumefaciens-mediated transient expression (agroinfiltration) in N. benthamiana leaves and harvested 2 or 3 days post infiltration. Co-IP experiments and preparation of peptides for liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed as described previously (Bozkurt et al., 2011). Except the stringent PexRD54 immunoprecipitation assay (Stringent IP), all IPs were done using 150 mM NaCl buffer and 0.15% detergent concentration. For the stringent IP, the concentrations of salt and the detergent were increased to 250 mM and to 0.5%, respectively. LC-MS/MS analysis was performed with a LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, UK) and a nanoflow-HPLC system (nanoACQUITY; Waters Corp., UK) as described previously (Petre et al., 2015). LC-MS/MS data processing and protein identification were done as described previously (Petre et al., 2015).
In planta association of PexRD54 and Joka2 with either ATG8CL, ATG8CLΔ or ATG8IL constructs was tested by co-IP assays as follows: constructs were transiently co-expressed in N. benthamiana leaves by agroinfiltration followed by protein extraction 2–3 days post infiltrations. Protein extraction, purification and western blot analysis steps were performed as described previously (Saunders et al., 2012; Bozkurt et al., 2011; Oh et al., 2009). Monoclonal FLAG M2 antibody (Sigma-Aldrich, UK), polyclonal GFP/RFP antibodies (Invitrogen, UK), and polyclonal HA (Sigma-Aldrich, UK) antibody were used as primary antibodies, and anti-mouse antibody (Sigma-Aldrich, UK) and anti-rat (Sigma-Aldrich, UK) antibodies were used as secondary antibodies.
To test if PexRD54 increased protein levels of ATG8CL, GFP:ATG8CL was co-expressed with RFP:GUS, RFP:PexRD54 and RFP:PexRD54AIM2 in N. benthamiana leaves. Leaf samples were collected 2 and 3 days after infiltration. Total proteins were extracted as described previously (Saunders et al., 2012; Oh et al., 2009) and immunoblots were performed using the appropriate antisera. The same experimental setup was also used to test whether Joka2 increases protein levels of ATG8CL. Joka2:RFP, Joka2AIM:RFP and RFP:GUS constructs were co-expressed with GFP:ATG8CL construct in N. benthamina leaves and total proteins were extracted 2 and 3 days after infiltration. Immunoblots were developed using appropriate antisera. To assay ATG8CL protein levels during infection with P. infestans, N. benthamiana leaves were first infiltrated with GFP:ATG8CL construct and infected one day later with droplets from a zoospore solution of P. infestans as described earlier (Song et al., 2009). Water droplets were used as mock treatment. Protein extracts were prepared 2 and 3 days after infection and protein levels of GFP:ATG8CL were detected by immunoblots using a polyclonal GFP-HRP antibody (Santa Cruz, Texas, USA).
Joka2:RFP was transiently co-expressed with GFP:ATG8CL in the presence of HA:PexRD54, HA:PexRD54AIM, or HA:EV constructs in N. benthamiana leaves. Total proteins extracts prepared 2 days after infiltration were then used in anti-GFP Co-IPs. Purified protein complexes were separated by SDS/PAGE and immunoblotted to detect RFP, GFP and HA signals. Similar competition experiment was conveyed with increased HA:PexRD54 A. tumefaciens concentrations (OD600=0, 0.05, 0.1, 0.2 or 0.4) while Joka2:RFP and GFP:ATG8CL A. tumefaciens concentrations were kept fixed at OD600= 0.2. Protein extracts prepared two days after infiltration were used in GFP-IPs as described above. Joka2-ATG8CL binding assays were carried out by co-expressing Joka2:RFP and GFP:ATG8CL in the presence of HA:PexRD54, HA:PexRD54AIM2, HA:PexRD54J2AIM1 (PexRD54 AIM (LDFDWEIV) replaced by Joka2 AIM (GVAEWDPI)), HA:PexRD54J2AIM2 (PexRD54 AIM replaced by Joka2 AIM plus 8 additional amino acids at the C-terminus (GVAEWDPILEELKEMG)) or HA:EV. GFP-IPs were performed as described above.
Infection assays assessing the effect of Joka2 overexpression on P. infestans colonization were performed as follow: Joka2:RFP, Joka2AIM:RFP or RFP EV were transiently overexpressed side by side on either halves of independent N. benthamiana leaves. Twenty-four hours after expression, the infiltrated leaves were detached and inoculated with P. infestans 88,069 on two or three spots on each half leaf. P. infestans growth was monitored by UV photography 6 days after infection. Colonization was quantified by measuring the diameter of the lesion on each inoculated spot and values from three independent biological replicates were used to generate the scatter plots.
To demonstrate whether PexRD54 could alleviate the effect of Joka2 on pathogen growth, Joka2:RFP was co-expressed with HA:PexRD54, HA:PexRD54AIM2 or HA:EV in N. benthamiana leaves, which were infected and monitored as described above. For all infection assays, experiments were repeated three times with minimum 10 infection spots. Lesion diameters were measured 6 days after infection.
Virus induced gene silencing of Joka2 was performed in N. benthamiana as described previously (Liu et al., 2002). Two different TRV2/pYL279 constructs were designed to target both 5’-and 3’ ends of Joka2. TRV:Joka2-1 and TRV2:Joka2-2 targeted the region between 340 and 639 and between 1942 and 2241, respectively. The primers were used for generating TRV2:Joka2-1 and TRV2:Joka2-2 are listed in Supplementary file 3. TRV2:GFP was used as a negative control as described previously (Chaparro-Garcia et al., 2015).
Suspensions of Agrobacterium tumefaciens strain GV3101 harboring TRV1/pYL155 and TRV2:Joka2-1 or TRV2:Joka2-2 were mixed in a 2:1 ratio in infiltration buffer (10 mM MES (2-[N-morpholino]ethanesulfonic acid), 10 mM magnesium chloride (MgCl2), pH 5.6) to a final OD600 of 0.3. As a control, we used TRV2:GFP. Two-week-old N. benthamiana plants were infiltrated with A. tumefaciens for VIGS assays and upper leaves were used 2–3 weeks later for P. infestans infections. UV photographs were taken 5 days post infection. Each experiment was repeated at least three times with minimum 10 infection spots and disease lesion areas were measured using ImageJ. Each lesion size value was normalized following the formula 'Nr = Or * Ar /A', where N is the normalized lesion size value, O is the original lesion size value, r is the biological repeat, Xr is the average of all O values of the biological repeat r, and A is the average of all O values. Silencing levels were confirmed using RT-PCR. Total RNA was extracted using RNAeasy Plant Mini Kit (Qiagen, UK) and treated with Ambion TURBO DNA-free according to manufacturer’s protocol. 1.0 μg of DNase treated RNA was used for cDNA synthesis using SuperScript III Reverse Transcriptase (Invitrogen). RT-PCR was performed with the following program: 1 cycle with 3 min at 95°C, followed by 35 cycles with 95°C at 30 s, 56°C at 30 s and 72°C at 30 s. Primers pairs used for cDNA amplification were Joka2-TRV1-F and Joka2-TRV1-R, and Joka2-TRV2-R and Joka2-TRV2-F (described in Supplementary file 3). NbEF1α was used to normalize transcript abundance (Segonzac et al., 2011).
All microscopy analyses were performed on live leaf tissue 3 days post agroinfiltration. N. benthamiana leaf patches were cut and mounted in water and analyzed on a Leica TCS SP5 confocal microscope (Leica Microsystems, Germany) using 63x water immersion objective. The GFP and RFP probes were excited using 488 and 561 nm laser diodes and their fluorescent emissions were collected at 495–550 nm and 570–620 nm, respectively. To avoid bleed-through from different fluorophores, co-localization images were taken using sequential scanning between lines and acquired using multi-channels.
Confocal microscopy images were processed with the Leica LAS AF software, ImageJ (2.0) and Adobe PHOTOSHOP CS5 (12.0) programs. Images for quantification of autophagosome numbers were obtained from 50 Z stacks consisting of 1 μm depth field multi-layered images with similar settings for all samples. To detect and quantify punctate structures in one channel (green channel or red channel or overlay channel (green channel images superimposed with red channel ones)), the Z stacks were separated into individual images with the ImageJ (2.0) program and analyzed. The counting procedure was based on a naked-eye detection of punctate structures to avoid cytoplasm noise and dual counting of autophagosomes within the same stack. Histograms were generated with mean of punctate numbers generated from stacks obtained in two independent biological experiments. Statistical differences were assessed by means of a two-tailed t-test assuming unequal variance as implemented in StatPlus LE package (AnalystSoft, Washington, USA). Measurements were significant when p<0.05 and highly significant when p<0.001.
Leaf samples were embedded in LR White as described (Liu et al., 2002) except sections were picked up on gold grids before immunogold labeling. All labeling procedures were carried out at room temperature. Grids were floated, section-side down, on drops of 50 mM glycine/PBS (150 mM NaCl, 10 mM phosphate, pH 7.4) for 15 min then on Aurion blocking buffer (5% BSA/0.1% cold water fish gelatin/5–10% normal goat serum/15 mM NaN3/PBS, pH7.4) (Aurion, the Netherlands) for 30 min then briefly equilibrated in incubation buffer (0.1% (v/v) BSA-C (actetylated BSA; Aurion) /PBS, pH 7.3) before a 90 min’ incubation with the primary antibody. Serial sections on separate grids were labelled with either a rabbit polyclonal anti-GFP Abcam ab6556 (Abcam, UK) at 1/250, or a rabbit polyclonal anti-catalase AS09 501 (Newmarket Scientific, UK) at 1/1000. Grids were washed 6x5 min in drops of incubation buffer then placed on the drops of goat anti-rabbit secondary antibody conjugated to 10 nm gold (BioCell, Agar Scientific Ltd., Essex, UK), diluted to 1/50 in incubation buffer, for 90 min. After 6x5 min washes in incubation buffer and 2x5 min washes in PBS, the grids were briefly washed in water and contrast stained with 2% (w/v) uranyl acetate. Grids were viewed in a FEI Tecnai 20 transmission electron microscope (FEI, the Netherlands) at 200 kV and digital TIFF images were taken using an AMT XR60B digital camera (Deben, UK) to record TIFF files. Bar charts were generated by counting the clustered (>4 gold particles close to each other) gold particles on each image.
3-Methyl adenine (3-MA), a phosphatidylinositol 3-kinase (PI3K) inhibitor is widely used to inhibit autophagosome formation (Hanamata et al., 2013). N. benthamiana leaves transiently expressing GFP:ATG8CL-RFP:PexRD54, GFP:PexRD54-RFP:ATG8CL or YFP:VTI12 were infiltrated with 5 mM 3-MA. Both GFP:PexRD54 and RFP:ATG8CL constructs were expressed side by side to monitor the effect of 3-MA treatment on stimulation of autophagosome formation by PexRD54. Punctate structures were visualized using confocal microscopy 6–10 hr after 3-MA treatment. Bar charts were generated with the number of punctate structures obtained from maximum projections of Z-stack images of two independent biological experiments.
The cysteine protease inhibitor E64d is widely used for measuring autophagic flux (Bassham, 2015). To determine whether PexRD54 blocked autophagic flux, at 2 dpi we infiltrated leaves with 100 μM E64D and kept them overnight in the dark. At 3 dpi, we collected E64d treated and untreated samples and analyzed total protein levels using appropriate antisera. Concanamycin A (2 μM in agro infiltration medium) was infiltrated into leaves of N. benthamiana transiently expressing ATG8CL and PexRD54, PexRD54AIM2 or empty vector control constructs. The leaves were than incubated in dark at 20°C for 24 hr. ATG8CL-labelled puncta were visualized using confocal microscopy 24 hr after concanamycin A treatment.
DNA encoding PexRD54 residues Val92 to Val381 (lacking secretion and translocation signals) was amplified from RFP:PexRD54 (using primers shown in Supplementary file 3) and cloned into the vector pOPINS3C, resulting in an N-terminal 6xHis-SUMO tag with PexRD54, linked by a 3C cleavage site (Berrow et al., 2007). Recombinant protein was produced using E. coli BL21-Arabinose Inducible (AI) cells. Cell cultures were grown in Power Broth at 37°C to an A650 0.4–0.6 followed by induction with 0.2% (w/v) L-arabinose and overnight incubation at 18°C. Pelleted cells were resuspended in buffer A (50 mM Tris-HCl pH 8, 500 mM NaCl, 50 mM glycine, 5% (v/v) glycerol and 20 mM imidazole supplemented with EDTA free protease inhibitor tablets (one tablet per 40 ml buffer)) and lysed by sonication. The clarified cell lysate was applied to a Ni2+-NTA column connected to an AKTA Xpress system. 6xHis-SUMO-PexRD54 was step-eluted with elution buffer (buffer A containing 500 mM imidazole) and directly injected onto a Superdex 75 26/600 gel filtration column pre-equilibrated in buffer C (20 mM HEPES pH 7.5, 150 mM NaCl). The fractions containing 6xHis-SUMO-PexRD54 were pooled and concentrated to 2–3 mg/mL. The 6xHis-SUMO tag was cleaved by addition of 3C protease (10 µg/mg fusion protein) and incubated at 4°C overnight. Cleaved PexRD54 was further purified using Ni2+-NTA column (collecting eluate) followed by gel filtration. The purified protein was concentrated as appropriate, and the final concentration was judged by absorbance at 280 nm (using a calculated molar extinction coefficient of PexRD54, 57,040 M-1cm-1).
The PexRD54AIM2 variant was amplified from RFP:RD54AIM2 (using primers shown in Supplementary file 3) and cloned into the vector pOPINS3C as above. Recombinant protein was expressed and purified as for wild-type PexRD54; with protein concentration measured using a calculated molar extinction coefficient of 51,350 M-1cm-1.
DNA encoding Met1 to Ser119 of ATG8CL and Gly2 to Ser119 of ATG8IL were amplified from GFP:ATG8CL and GFP:ATG8IL (using primers shown in Supplementary file 3) and cloned into the vector pOPINF, generating a cleavable N-terminal 6xHis-tag with ATG8CL and ATG8IL. Recombinant proteins were produced using E. coli strain BL21 (DE3) grown in lysogeny broth at 37°C to an A600 of 0.4–0.6 followed by induction with 1 mM IPTG and overnight incubation at 18°C. Pelleted cells were resuspended in buffer A and pure, concentrated protein prepared as described for PexRD54 above (concentration determined using a calculated molar extinction coefficient of 7680 M-1cm-1 and 9080 M-1cm-1 for ATG8CL and ATG8IL, respectively).
Calorimetry experiments were carried out at 15°C in 20 mM HEPES pH 7.5, 500 mM NaCl, using an iTC200 instrument (MicroCal Inc.). For protein:protein interactions, the calorimetric cell was filled with 100 μM PexRD54 and titrated with 1.1 mM ATG8CL or ATG8IL from the syringe. A single injection of 0.5 μl of ATG8CL or ATG8IL was followed by 19 injections of 2 μl each. Injections were made at 150 s intervals with a stirring speed of 750 rpm. For the heats of dilution control experiments, equivalent volumes of ATG8CL or ATG8IL were injected into buffer using the parameters above. For protein:peptide interactions, the calorimetric cell was filled with 90 μM ATG8CL or ATG8IL and titrated with 1 mM peptide from the syringe. The titrations were performed at 25°C, but otherwise as above. The raw titration data were integrated and fitted to a one-site binding model using the MicroCal Origin software.
PexRD54 (PITG_09316); StATG8CL (PGSC0003DMP400038670), StATG8IL (PGSC0003DMP400009229), SlATG8CL (Solyc10g006270 and Solyc07g064680), SlATG8IL (Solyc01g068060), NbATG8CL (Nb_S00003316g0005, KR021366), NbATG8IL (Nb_S00005942g0011, KR021365), and Joka2 (XM_006344410).
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Jean T GreenbergReviewing Editor; University of Chicago, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your work entitled "An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Jean Greenberg (Reviewing Editor) and Detlef Weigel (Senior Editor).
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you focus on the additional work we consider essential for a favorable decision on this submission.
This manuscript presents exciting data suggesting that the P. infestans effector PexRD54 targets the autophagy system and may affect autophagy through a competition mechanism. We appreciate the rigor of the work presented and its clarity of exposition. However, we think there are important issues that require additional work, which is explained below.
1) The quality of the TEM experiment:
The TEM data in the manuscript show somewhat different localization compared to the confocal images. For example, the confocal image shows GFP-tagged PexRD54 mainly localized in some foci and the cytosol (Figure 3—figure supplement 1), but the TEM image (Figure 3—figure supplement 2, please correct the figure legend, which explains A and B but not panel C) does not show any particles in the cytosol. Moreover the vesicles in which particles accumulate do not have the appearance of autophagosomes (they seem more like an aggresome due to overexpression) and this cannot explain the ring-like structure of PexRD54 (Figure 4). There should be a good control photo showing an autophagosome, probably marked with ATG8CL. We suggest observing the sample expressing both constructs of PexRD54 and ATG8CL for TEM to see both at the autophagosome. Since the autophagosomes induced by PexRD54 have altered morphology (compare Figure 4 to Figure 6—figure supplement 3), please address the morphology difference in the manuscript. Differences in morphology could also affect quantification. Are the differences the same when overlap is quantified in a 50x50 micron area compared to all puncta found in the area? The TEM data is very important and should be in the main figure. Instead, Figure 2 can be in the supplement.
2) Biological relevance:
A weakness of the work is a lack of information about the relevance of autophagy for this pathosystem and the relevance of PexRD54 for virulence. We think these issues can be addressed by: (1) silencing PexRD54 to address its role in virulence and its effect on autophagy during infection; and/or (2) performing an experiment to test the importance of autophagy for this pathosystem.
You should address the following comments:
3) Figure 3—figure supplement 4 shows expression of GFP-ATG8CL, with only about 50 puncta/10,000 µm^2, while Figure 4A counts about 10 puncta/10,000 µm^2 (with EV control). Please explain the discrepancy. It is important to address this as you claim that PexRD54 enhances autophagy activity based on the ATG8CL counts (about 40 puncta/10,000 µm^2 in Figure 4).
4) Can you assay for autophagy flux biochemically to bolster your conclusion thatPexRD54 is affecting autophagy flux?
5) Figure 2 – it seems that AVRBlb2 interacts weakly with ATG8IL. Does it have an AIM domain or is ATG8IL a bit sticky? Can you please quantify binding in the protein blot?
6) All experiments are performed using transient overexpression in Nicotiana. Please comment on the caveats with this approach and consider doing some validation using native promoters to validate findings in a more biologically relevant context.
7) Since PexRD54 can stimulate the formation of ATG8CL-marked autophagosomes, you suggest that PexRD54 can activate autophagy for the pathogen's own benefit (Pro-death?). You conclude at the same time that PexRD54 counteracts the positive role of Joka2-mediated selective autophagy in pathogen defense (Pro-survival?). Is it either or both? Please clarify.
[Editors' note: further revisions were requested prior to acceptance, as described below.]
Thank you for resubmitting your work entitled "An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor" for further consideration at eLife. Your revised article has been favorably evaluated by Detlef Weigel (Senior editor), a Reviewing editor, and four reviewers. The manuscript has been improved, but there are some minor remaining issues that need to be addressed before acceptance, as outlined below:
The work is substantially improved. An EM expert has suggested a number of improvements that we would like you to implement (none of which require additional experimentation):
1) About Figure 3—figure supplement 3:
The upper and lower photos seem to have different magnification. Please add size bars in the upper images.
Legends: "Vesicles labelled with GFP antibody were different than the vesicles labelled with catalase antibody, confirming that PexRD54 labelled vesicles are not peroxisomes." Please do not use the term "vesicles" here, since it is not sure that they are not vesicles. Instead, the description can be considered as: high electron dense regions (structures)
Also the statement: "Stars indicate vesicular structures that are labelled by gold particles in the other image" is improper. The stars in two images obviously indicate different structures- the upper image indicates electron-dense structure and the lower image indicates peroxisomes (organelle). It is better to use different labels.
For anti-GFP, the upper image indicates that gold particles were labeled in the electron-dense portion, which was neither associated with vesicle-like structures nor peroxisomes.
2) About Figure 3—figure supplement 2:
Based on the total set of supplemental EM images which authors provided, Figure 3—figure supplement 2A seems not to be a typical image. Since most of images do not have vesicle like structures, the photo of number 11 seems to represent the total set of images.
3) Methods – Electron Microscopy and Immunogold labelling:
Since authors used high pressure freezing to get immunogold labelling, the authors need to describe the process in the Methods. I think authors may have cited the wrong reference (Liu, Schiff and Dinesh-Kumar et al., 2002), please check it.
In your revision, please include your archive of EM images as supplemental material so that readers can see the whole range of data.
Major reviewer comments were:
I think the authors improved the manuscript. The effector biology has a limited way of doing experiments but this manuscript dealt with it with a lot of controls, thus convincing.
The revised manuscript of Dagdas and colleagues report the novel finding that an oomycete effector (PexRD54) binds to a plant host autophagy protein (ATG8CL) to trigger autophagosome production. As a result plant defense are circumvented. The original manuscript was extensively reviewed and in general, this exciting paper was well received. However, there were two major points of concern:
1) TEM-image quality
2) Biological Relevance
My main concern was point #2; biological relevance. In my view, while this is an excellent study with numerous nuggets that will be of interest to the eLife community. This work would be significantly compromised without some discussion, experimentation to indicate that autophagy is relevant to this pathosystem.
Briefly, to demonstrate the importance of autophagy in this system the cargo receptor Joka2 was silenced and oomycete colonization, measured using two distinct constructs; thus strengthening the argument outlined in Figure 7 and Figure 7—figure supplement 1; conclusions indicating the importance of selective autophagy in this pathosystem.
The authors also discuss the very real technical difficulties in this system and the relatively high levels of variation that complicate interpretation of these studies/this system has a high degree of difficulty in and of itself.
In summary, by performing silencing experiments and demonstrating, the importance of autophagy regulation in this system, it is my view that the authors have adequately met the reviewers’ concerns regarding biological relevance.
In this revision, the authors have addressed some of the previous concerns about the manuscript. My primary concern previously was about biological relevance due to experiments using overexpression in Nicotiana. To address biological relevance, Joka2 was silenced using VIGS in Nicotiana and demonstrated enhanced susceptibility to P. infestans. These results are consistent with the observation that overexpression of Joka2 enhances resistance. The authors provide a detailed rebuttal to why they have not performed silencing in P. infestans. While I would like to see the field move away from primarily relying on overexpression to deduce phenotypes, this manuscript does present significant insight into the role of effector-modification of autophagy and is worthy of publication.
In this revised version, in response to the reviewers’ suggestions, the authors have done TEM on serial sections and confirmed that anti-catalase conjugated gold particles did not co-localize with GFP coated gold particles, indicating that cells expressing GFP:PexRD54 revealed a strong signal in certain portions that are not peroxisomes.
The movies are a good evidence to confirm that the mobile structures are vesicles.https://doi.org/10.7554/eLife.10856.056
- Abbas Maqbool
- Benjamin Petre
- Joe Win
- Sophien Kamoun
- Yasin F Dagdas
- Khaoula Belhaj
- Neftaly Cruz-Mireles
- Jan Sklenar
- Joe Win
- Frank Menke
- Sophien Kamoun
- Pooja Pandey
- Nadra Tabassum
- Tolga O Bozkurt
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank Elaine Barclay, Liliana M. Cano, Frédéric Fragnière, Marina Franceschetti, Artemis Giannakopoulou, Ricardo Oliva, Egem Ozbudak, Stephen Whisson for technical support and/or providing materials. We are grateful to Zlay Taftacs and all members of the Kamoun Lab for helpful suggestions. This project was funded by the Gatsby Charitable Foundation, European Research Council (ERC), Biotechnology Biological Sciences Research Council (BBSRC) and the John Innes Foundation.
- Jean T Greenberg, Reviewing Editor, University of Chicago, United States
© 2016, Dagdas et al
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.