An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor
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 . 10 . 7554/eLife . 10856 . 003Figure 1 . 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 00310 . 7554/eLife . 10856 . 004Figure 1—figure supplement 1 . A synthetic peptide composed of the last ten amino acids of PexRD54 is sufficient for binding to ATG8CL . ( A ) A synthetic peptide ( KPLDFDWEIV ) containing the AIM of PexRD54 binds ATG8CL as documented by ITC . ( B ) A mutant AIM peptide ( KPLDFDAEIA ) was used as negative control and did not bind ATG8CL . Heat differences upon injection of peptides and integrated heats of injection are shown for both peptides . The values of KD , ΔH and ΔS are representative of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 004 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 . 10 . 7554/eLife . 10856 . 005Figure 2 . 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 00510 . 7554/eLife . 10856 . 006Figure 2—figure supplement 1 . Amino acid sequence alignment of ATG8 proteins from Arabidopsis thaliana ( At ) , Solanum tuberosum ( St ) , Solanum lycopersicum ( Sl ) and Nicotiana benthamiana ( Nb ) . Sequences were aligned using ClustalW and visualized using Boxshade v3 . 21 . Identical amino acids are highlighted in black and amino acids with similar biochemical properties are shown in grey . Red arrowhead points to ATG8CL and blue arrowhead points to ATG8IL . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 00610 . 7554/eLife . 10856 . 007Figure 2—figure supplement 2 . Phylogenetic analysis of ATG8 proteins . A neighbor-joining tree was generated with ATG8 sequences obtained from genomes of A . thaliana , S . tuberosum , S . lycopersium , and N . benthamiana , using MEGA5 with a 1000 bootstrap value . ATG8CL and ATG8IL are highlighted in pink and the ATG8CL variant that was identified in N . benthamiana is highlighted in green . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 007 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 ) . 10 . 7554/eLife . 10856 . 008Video 1 . 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 ) DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 00810 . 7554/eLife . 10856 . 009Figure 3 . 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 00910 . 7554/eLife . 10856 . 010Figure 3—figure supplement 1 . PexRD54 localizes to vesicle like structures . GFP:PexRD54 or RFP:PexRD54 were transiently expressed in N . benthamiana and examined by confocal laser scanning microscopy 3 days post infiltration . Confocal micrographs show single optical sections of GFP:PexRD54 ( left panel ) and RFP:PexRD54 ( right panel ) . With both tags , PexRD54 localized to vesicular structures pointed with arrowheads . Scale bar=10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 01010 . 7554/eLife . 10856 . 011Figure 3—figure supplement 2 . PexRD54 localizes to high electron dense structures . ( A ) Transmission electron microscopy ( TEM ) imaging of PexRD54 . N . benthamiana cells transiently expressing GFP:PexRD54 or GFP:EV were collected 3 days post infiltration and probed with anti-GFP antibodies conjugated to gold particles . GFP:PexRD54 labelling were mainly at high electron dense structures , whereas GFP:EV showed cytosolic distribution . Scale bar=200 nm ( B ) Bar charts showing electron dense labelling in GFP:EV and GFP:PexRD54 expressing samples ( *p<0 . 05 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 01110 . 7554/eLife . 10856 . 012Figure 3—figure supplement 3 . PexRD54 labelled high electron dense structures that are not peroxisomes . Serial sections of N . benthamiana leaves transiently expressing GFP:PexRD54 were collected 3 days post infiltration and probed with anti-GFP and anti-catalase antibodies conjugated to gold particles . High electron dense structures labelled with GFP antibody were different than the regions labelled with catalase antibody , confirming that PexRD54 labelled structures are not peroxisomes . Stars indicate regions that are labelled by gold particles in the other image . Scale bar=500 nmDOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 01210 . 7554/eLife . 10856 . 013Figure 3—figure supplement 4 . PexRD54 labelled puncta rarely include GFP:ATGIL signal . GFP:ATG8IL was transiently co-expressed with RFP:PexRD54 or RFP:EV in N . benthamiana leaves and examined by confocal laser scanning microscopy 3 days post infiltration . Maximum intensity projection of confocal micrographs shows that RFP:PexRD54 only partially colocalizes with GFP:ATG8IL . White arrowheads point to vesicles labelled with only GFP:ATG8IL or RFP:PexRD54 , whereas the red-filled arrowheads point to puncta where colocalization occurs . Regions where GFP or RFP labelled puncta do not overlap are indicated with dotted squares . Scale bar=50 μm ( B ) Bar charts showing the number of RFP-labelled puncta that colocalized with GFP:ATG8IL puncta . The data are representative of 500 individual images from two biological replicates . Each replicate consists of five independent Z stack images with 50 images each , acquired from five independent leaf areas . ( ns=statistically not significant ) . Error bars represent ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 01310 . 7554/eLife . 10856 . 014Figure 3—figure supplement 5 . Inhibition of autophagy hinders PexRD54 vesicular distribution . N . benthamiana leaves transiently expressing GFP:ATG8CL/RFP:PexRD54 , GFP:PexRD54/RFP:ATG8CL or trans-golgi network marker YFP:VTI12 were infiltrated with autophagy inhibitor 3-methyl adenine ( 3-MA ) at a concentration of 5 mM or water and imaged 6 hr post infiltration . 3-MA prevented formation of punctate structures of PexRD54 and ATG8CL but not of VTI12 . Scale bar= 50 μm ( B ) Bar charts showing the number of punctate structures in 3-MA or water-treated samples . 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 . ( ***p<0 . 001 , ns=statistically not significant ) . Error bars represent ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 01410 . 7554/eLife . 10856 . 015Figure 3—figure supplement 6 . Conservation of terminal glycine residue in ATG8 proteins from various species . Amino acid sequence alignment of ATG8 variants from different species that belong to different kingdoms shows conservation of terminal glycine residue ( highlighted with an orange arrow ) . Residues that are responsible for interaction with core ATG8 interacting motif ( AIM ) residues are highlighted in purple ( W pocket ) and green ( L pocket ) . Red and blue arrowheads point to ATG8CL and ATG8IL variants , respectively . Abbreviations for species: Hs: Homo sapiens; Tb: Trypanosoma brucei; Sc:Saccharomyces cerevisiae; Ce: Caenorhabditis elegans; At: Arabidopsis thaliana; Sl: Solanum lycopersium , Nb: Nicotiana benthamiana . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 01510 . 7554/eLife . 10856 . 016Figure 3—figure supplement 7 . ATG8CL terminal glycine deletion mutant prevents formation of PexRD54 labelled puncta in a dominant negative manner . ( A ) A glycine to alanine mutation in the conserved terminal glycine residue of GFP:ATG8CL does not prevent binding to RFP:PexRD54 . GFP:ATG8CL or GFP:ATG8CLΔ were transiently co-expressed with RFP:PexRD54 or RFP:PexRD54AIM2 in N . benthamiana leaves and total proteins were extracted two days after infiltration and used in immunoprecipitation experiments ( IPs ) . IPs obtained with anti-GFP and total protein extracts were immunoblotted with appropriate antisera . Stars indicate the expected band sizes . ( B ) GFP:ATG8CL or GFP:ATG8CLΔ were transiently co-expressed with RFP:EV or RFP:PexRD54 in N . benthamiana leaves and examined by confocal laser scanning microscopy 3 days post infiltration . GFP:ATG8CL showed distinct punctate structures , whereas GFP:ATG8CLΔ was mainly cytosolic with few visible puncta . When RFP:PexRD54 was co-expressed with GFP:ATG8CLΔ , the punctate localization was lost consistent with the recruitment of PexRD54 to ATG8CL labelled autophagosomes . ( C ) Co-expression of GFP:ATG8ILΔ and RFP:PexRD54 , did not alter punctate localization of RFP:PexRD54 . Arrowheads point to puncta labelled with GFP or RFP fusion constructs . Regions where GFP or RFP labelled puncta do not colocalize are indicated with dotted squares . Scale bar=50 μm ( D ) Bar charts confirming punctate distribution of PexRD54 depends on localization at ATG8CL labelled autophagosomes . 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 . ( ***p<0 . 001 , ns=statistically not significant ) . Error bars represent ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 01610 . 7554/eLife . 10856 . 017Video 2 . 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 ) DOI: http://dx . 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 . 10 . 7554/eLife . 10856 . 018Figure 4 . 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 01810 . 7554/eLife . 10856 . 019Video 3 . 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 ) DOI: http://dx . 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 ) . 10 . 7554/eLife . 10856 . 020Figure 5 . 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 02010 . 7554/eLife . 10856 . 021Figure 5—figure supplement 1 . GFP:ATG8CL protein levels increase in N . benthamiana during P . infestans infection . N . benthamiana leaves transiently expressing GFP:ATG8CL or GFP:EV were sprayed with P . infestans zoospore solution or water control ( mock treatment ) . Protein levels of GFP:ATG8CL or GFP were determined 2 and 3 days after infection . The samples were run on the same gel and blots were developed with the same conditions . Gels were stained with coomassie brilliant blue ( CBB ) to show equal loading . Stars indicate expected band sizes . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 021 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 . 10 . 7554/eLife . 10856 . 022Figure 6 . 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 02210 . 7554/eLife . 10856 . 023Figure 6—figure supplement 1 . Joka2 has a functional ATG8 interacting motif ( AIM ) . Mutation of the predicted AIM in Joka2 ( 819-WDPI-822 ) to ADPA resulted in loss of ATG8CL binding . GFP:EV and GFP:ATG8CL were co-expressed with Joka2:RFP and Joka2AIM:RFP in N . benthamiana and proteins were extracted two days after infiltration and used in immunoprecipitation experiments ( IPs ) . IPs were obtained with anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera . Stars indicate expected band sizes . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 02310 . 7554/eLife . 10856 . 024Figure 6—figure supplement 2 . Joka2 has higher binding affinity to ATG8CL than ATG8IL . Joka2:RFP was transiently co-expressed with GFP:ATG8CL , GFP:ATG8IL or GFP:EV in N . benthamiana and proteins were extracted two days after infiltration and used in immunoprecipitation experiments ( IPs ) . IPs obtained with anti-GFP and total protein extracts were immunoblotted with appropriate antisera . IP experiments confirmed higher binding affinity of Joka2 to ATG8CL than ATG8IL . Stars indicate expected band sizes . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 02410 . 7554/eLife . 10856 . 025Figure 6—figure supplement 3 . Joka2 increases the number of ATG8CL labelled autophagasomes . ( A ) Joka2:RFP partially colocalizes with GFP:ATG8CL . Joka2:RFP and GFP:ATG8CL were transiently co-expressed in N . benthamiana leaves and examined by confocal laser scanning microscopy 3 days post infiltration . Maximum projections of stacks of images indicate that Joka2:RFP colocalizes with GFP:ATG8CL-labelled autophagosomes . Scale bar= 50 μm ( B ) Zoomed single plane images of the puncta co-labelled by Joka2:RFP and GFP:ATG8CL . Scale bar= 10 μm ( C ) Joka2AIM:RFP and GFP:ATG8CL were transiently co-expressed in N . benthamiana leaves and examined by confocal laser scanning microscopy 3 days post infiltration . Arrowheads point to puncta co-labelled by Joka2AIM:RFP and GFP:ATG8CL . Scale bar= 50 μm ( D ) EV:RFP and GFP:ATG8CL were transiently co-expressed in N . benthamiana leaves and examined by confocal laser scanning microscopy 3 days post infiltration . Arrowheads point to puncta labelled by GFP:ATG8CL . Dotted squares highlight the absence of control RFP signal at the GFP:ATG8CL puncta . Scale bar= 50 μm ( E ) Bar charts showing colocalization of GFP:ATG8CL with Joka2:RFP , Joka2AIM:RFP or EV:RFP . Joka2AIM:RFP or EV:RFP display significantly low co-localization with GFP:ATG8CL-labelled puncta in contrast to Joka2:RFP . 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 . ( *p<0 . 05 ) . Error bars represent ± SD . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 02510 . 7554/eLife . 10856 . 026Figure 6—figure supplement 4 . Joka2 increases ATG8CL protein levels . GFP:ATG8CL was transiently co-expressed with RFP:GUS , Joka2:RFP or Joka2AIM:RFP in N . benthamiana leaves and protein levels in total extracts were determined 2 and 3 days after infiltration , using appropriate antisera . At 3 dpi GFP:ATG8CL protein levels were higher in Joka2:RFP expressing samples compared to Joka2AIM:RFP or RFP:GUS expressing samples . Blots were stained with Ponceau stain ( PS ) to show equal loading . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 02610 . 7554/eLife . 10856 . 027Figure 6—figure supplement 5 . PexRD54 outcompetes Joka2 for ATG8CL binding . Joka2:RFP and GFP:ATG8CL were transiently co-expressed with HA:EV , HA:PexRD54 and HA:PexRD54AIM2 in N . benthamiana leaves and proteins were extracted two days after infiltration and used in immunoprecipitation experiments ( IPs ) . IPs obtained with anti-GFP and total protein extracts were immunoblotted with appropriate antisera . In the presence of HA:PexRD54 , Joka2:RFP was depleted from GFP:ATG8CL complexes . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 02710 . 7554/eLife . 10856 . 028Figure 6—figure supplement 6 . PexRD54 AIM has a higher affinity to ATG8CL than Joka2 AIM . GFP:ATG8CL and Joka2:RFP were transiently co-expressed with HA:PexRD54 , HA:PexRD54AIM2 , HA:PexRD54J2AIM1 , HA:PexRD54 J2AIM2 in N . benthamiana and proteins were extracted two days after infiltration and used in immunoprecipitation experiments ( IPs ) . IPs obtained with anti-GFP and total protein extracts were immunoblotted with appropriate antisera . Joka2:RFP levels in GFP:ATG8CL complexes were higher in the presence of HA:PexRD54AIM2 , HA:PexRD54J2AIM1 and HA:PexRD54J2AIM2 compared to HA:PexRD54 expressing samples suggesting PexRD54 variants carrying Joka2 AIM peptides ( HA:PexRD54J2AIM1 , HA:PexRD54J2AIM2 ) were less effective in depleting Joka2:RFP from GFP:ATG8CL complexes . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 02810 . 7554/eLife . 10856 . 029Video 4 . 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 ) DOI: http://dx . 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 . 10 . 7554/eLife . 10856 . 030Figure 7 . 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 03010 . 7554/eLife . 10856 . 031Figure 7—figure supplement 1 . Silencing of Joka2 enhances susceptibility to P . infestans . ( A ) Cartoon showing Joka2 sites targeted for VIGs . ( B ) TRV:Joka2-1 , TRV:Joka2-2 and TRV:GFP control plants were inoculated with P . infestans 88 , 069 and pathogen growth was determined by lesion areas measured 5 days post-inoculation . ( C ) Categorical scatter plots show lesion areas of 10 infections sites from four independent biological replicates pointed out by four different colors . Similar p values ( p<0 . 001 ) were obtained in four independent biological repeats . ( D ) RT-PCR confirmed Joka2 silencing . cDNA generated from silenced and control plants were used for the RT-PCR experiments . Transcript abundance was normalized using NbEF1α . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 031 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 ) . 10 . 7554/eLife . 10856 . 032Figure 8 . PexRD54 outcompetes the autophagy cargo receptor Joka2 and enhances virulence . DOI: http://dx . doi . org/10 . 7554/eLife . 10856 . 032 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 ) .
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 .
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 .