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Structural Basis of Host Autophagy-related Protein 8 (ATG8) Binding by the Irish Potato Famine Pathogen Effector Protein PexRD54*

Open AccessPublished:July 25, 2016DOI:https://doi.org/10.1074/jbc.M116.744995
      Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8 family-interacting motif (AIM) and perturbs host-selective autophagy. However, the structural basis of this interaction remains unknown. Here, we define the crystal structure of PexRD54, which includes a modular architecture, including five tandem repeat domains, with the AIM sequence presented at the disordered C terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effector's AIM sequence, and we established a model of the full-length PexRD54-ATG8CL complex using small angle x-ray scattering. Structure-informed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta. This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease.

      Introduction

      During selective autophagy, specific cellular constituents can be targeted to autophagic pathways for subcellular trafficking or degradation (
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      ,
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      ). The autophagy toolkit includes around 40 ATG (autophagy-related) proteins. Together, they help initiate, regulate, and form the constituents of autophagic pathways. The role of selective autophagy in the response to pathogen challenge in animal cells is increasingly being appreciated and includes direct elimination of microorganisms and control of immunity-related signaling (
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      ). In turn, microorganisms have developed mechanisms to perturb host-selective autophagy to either shut it down and promote infection (
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      ) or activate it and re-direct nutrients to the parasite (
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      Autophagosomes induced by a bacterial Beclin 1 binding protein facilitate obligatory intracellular infection.
      ). There is also evidence that membrane formation and trafficking, as controlled by ATG proteins, are exploited by numerous viruses (
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      ). To date, the role of host-selective autophagy in host-microbe interactions has mostly been studied in mammals. The role of host-selective autophagy in plant-microbe interactions, and how it is manipulated by plant pathogens, remains poorly understood.
      ATG8 is a ubiquitin-like protein that performs multiple functions in autophagy. It is cycled, via conjugation and deconjugation reactions, to the membrane lipid phosphatidylethanolamine, and this localization is important for autophagosome biogenesis (
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      Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion.
      ). The intracellular animal pathogen Legionella pneumophila targets this process by delivering type IV secreted effector protein RavZ, which irreversibly deconjugates ATG8 from membranes and restricts autophagy (
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      ). ATG8 also functions as an adaptor to interact with proteins containing an ATG8-interacting motif (AIM).
      The abbreviations used are: AIM, ATG8-family interacting motif; SPR, surface plasmon resonance; NTA, nitrilotriacetic acid; RFP, red fluorescent protein; ITC, isothermal titration calorimetry; SAXS, small angle x-ray scattering; NSD, normalized spatial discrepancy.
      AIM-containing proteins can serve as receptors for cargo destined for autophagosomes. The core AIM sequence is defined as ΩXXΨ, where Ω is an aromatic amino acid (Trp, Tyr, or Phe); X is any residue, and Ψ is an aliphatic amino acid (Leu, Ile, and Val) (
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      ). Frequently, residues just to the N terminus of the ΩXXΨ motif are acidic in nature. Structural studies have elucidated how the AIM sequence binds ATG8, with key features including the Ω and Ψ residues binding within hydrophobic pockets, and the motif adopting a β-strand structure that extends the β-sheet of ATG8 (
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      ). It is generally thought that AIMs adopt a disordered or flexible conformation in the absence of a binding partner (
      • Noda N.N.
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      Atg8-family interacting motif crucial for selective autophagy.
      ,
      • Noda N.N.
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      ). Mechanisms for pathogens to perturb host-selective autophagy include delivery of factors that interfere with recruitment of endogenous AIM-containing proteins to ATG8 or that re-direct additional cellular components to autophagosomes.
      Filamentous plant pathogens cause devastating diseases of crops that are of both historical significance (
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      The rise and fall of the Phytophthora infestans lineage that triggered the Irish potato famine.
      ) and relevant to global agriculture today (
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      Emerging fungal threats to animal, plant and ecosystem health.
      ). Phytophthora infestans, the Irish potato famine pathogen, facilitates disease on its hosts by delivering effector proteins that modulate host cell processes to the benefit of the parasite (
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      • et al.
      Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.
      ), a strategy used by many biotrophic plant pathogens (
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      ). Many putative P. infestans effectors contain a conserved N-terminal RXLR (Arg-Xaa-Leu-Arg) motif for host translocation (
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      A translocation signal for delivery of oomycete effector proteins into host plant cells.
      ). Furthermore, about half of these effectors are predicted to adopt the conserved WY domain fold in their C-terminal regions, which encodes their biochemical activity (
      • Boutemy L.S.
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      • Win J.
      • Hughes R.K.
      • Clarke T.A.
      • Blumenschein T.M.
      • Kamoun S.
      • Banfield M.J.
      Structures of Phytophthora RXLR effector proteins: a conserved but adaptable fold underpins functional diversity.
      ,
      • Win J.
      • Krasileva K.V.
      • Kamoun S.
      • Shirasu K.
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      Sequence divergent RXLR effectors share a structural fold conserved across plant pathogenic oomycete species.
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      • Kamoun S.
      Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes.
      ). Although recent studies have begun to elucidate the virulence-associated targets and functions of P. infestans RXLR effectors (
      • Bos J.I.
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      Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1.
      • McLellan H.
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      • Morales J.
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      • Beynon J.L.
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      An RxLR effector from Phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus.
      ,
      • Saunders D.G.
      • Breen S.
      • Win J.
      • Schornack S.
      • Hein I.
      • Bozkurt T.O.
      • Champouret N.
      • Vleeshouwers V.G.
      • Birch P.R.
      • Gilroy E.M.
      • Kamoun S.
      Host protein BSL1 associates with Phytophthora infestans RXLR effector AVR2 and the Solanum demissum immune receptor R2 to mediate disease resistance.
      ,
      • King S.R.
      • McLellan H.
      • Boevink P.C.
      • Armstrong M.R.
      • Bukharova T.
      • Sukarta O.
      • Win J.
      • Kamoun S.
      • Birch P.R.
      • Banfield M.J.
      Phytophthora infestans RXLR effector PexRD2 interacts with host MAPKKKϵ to suppress plant immune signaling.
      ,
      • Bozkurt T.O.
      • Schornack S.
      • Win J.
      • Shindo T.
      • Ilyas M.
      • Oliva R.
      • Cano L.M.
      • Jones A.M.
      • Huitema E.
      • van der Hoorn R.A.
      • Kamoun S.
      Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface.
      ,
      • Gilroy E.M.
      • Taylor R.M.
      • Hein I.
      • Boevink P.
      • Sadanandom A.
      • Birch P.R.
      CMPG1-dependent cell death follows perception of diverse pathogen elicitors at the host plasma membrane and is suppressed by Phytophthora infestans RXLR effector AVR3a.
      ,
      • Wang X.
      • Boevink P.
      • McLellan H.
      • Armstrong M.
      • Bukharova T.
      • Qin Z.
      • Birch P.R.
      A host KH RNA-binding protein is a susceptibility factor targeted by an RXLR effector to promote late blight disease.
      • Boevink P.C.
      • Wang X.
      • McLellan H.
      • He Q.
      • Naqvi S.
      • Armstrong M.R.
      • Zhang W.
      • Hein I.
      • Gilroy E.M.
      • Tian Z.
      • Birch P.R.
      A Phytophthora infestans RXLR effector targets plant PP1c isoforms that promote late blight disease.
      ), these have yet to be identified for the vast majority of these proteins.
      Recently, a P. infestans RXLR effector, PexRD54, which contains an AIM sequence Trp-Glu-Ile-Val “WEIV” positioned at the C terminus (residues 378–381), was identified (
      • Dagdas Y.F.
      • Belhaj K.
      • Maqbool A.
      • Chaparro-Garcia A.
      • Pandey P.
      • Petre B.
      • Tabassum N.
      • Cruz-Mireles N.
      • Hughes R.K.
      • Sklenar J.
      • Win J.
      • Menke F.
      • Findlay K.
      • Banfield M.J.
      • Kamoun S.
      • Bozkurt T.O.
      An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor.
      ). It was shown that PexRD54 specifically interacts with a member of the ATG8 family of proteins from potato, ATG8CL, in vitro and in planta. In plant cells, PexRD54 activates selective autophagy by increasing the number of ATG8CL-containing autophagosomes and stabilizing ATG8CL. Furthermore, PexRD54 was shown to antagonize the function of the host autophagy cargo receptor Joka2 by competing for binding with ATG8CL. As Joka2 contributed toward immunity against P. infestans, which was counteracted by PexRD54, it was concluded that this effector acts as an inhibitor of Joka2 function.
      To better understand how PexRD54 interacts with potato ATG8CL to perturb host-selective autophagy, we have investigated the structural basis of effector-host target interaction. We determined the crystal structures of PexRD54 and ATG8CL in complex with the C-terminal AIM peptide of this effector. We also obtained a structure of the PexRD54-ATG8CL complex by docking the crystal structures into an envelope derived from solution scattering data. Site-directed mutagenesis of the PexRD54 C-terminal AIM region, and ATG8CL binding to a PexRD54 AIM-based peptide array, mapped the key residues that define the PexRD54-ATG8CL interface. Finally, we used structure-informed deletions to show that the WY domains of PexRD54 are dispensable for ATG8CL binding suggesting an alternative function for these domains. Together, these data provide a mechanistic understanding of how translocated effectors engage with their host targets and offer new methods for engineering control of plant diseases.

      Discussion

      Understanding the mechanistic basis of translocated effector protein function in support of pathogen infection and colonization is a major focus of research in plant-microbe interactions. Such studies reveal how manipulation of host cell processes by pathogen-derived molecules can promote virulence and also identify plant systems, such as autophagy, whose importance in disease or general host cell physiology may be underappreciated. In a few cases, the structural basis for bacterial plant pathogen effector interaction with a host protein or peptide has been described (
      • Cheng W.
      • Munkvold K.R.
      • Gao H.
      • Mathieu J.
      • Schwizer S.
      • Wang S.
      • Yan Y.B.
      • Wang J.
      • Martin G.B.
      • Chai J.
      Structural analysis of Pseudomonas syringae AvrPtoB bound to host BAK1 reveals two similar kinase-interacting domains in a type III effector.
      • Dong J.
      • Xiao F.
      • Fan F.
      • Gu L.
      • Cang H.
      • Martin G.B.
      • Chai J.
      Crystal structure of the complex between Pseudomonas effector AvrPtoB and the tomato Pto kinase reveals both a shared and a unique interface compared with AvrPto-Pto.
      ,
      • Xing W.
      • Zou Y.
      • Liu Q.
      • Liu J.
      • Luo X.
      • Huang Q.
      • Chen S.
      • Zhu L.
      • Bi R.
      • Hao Q.
      • Wu J.W.
      • Zhou J.M.
      • Chai J.
      The structural basis for activation of plant immunity by bacterial effector protein AvrPto.
      • Desveaux D.
      • Singer A.U.
      • Wu A.J.
      • McNulty B.C.
      • Musselwhite L.
      • Nimchuk Z.
      • Sondek J.
      • Dangl J.L.
      Type III effector activation via nucleotide binding, phosphorylation, and host target interaction.
      ). However, such studies of filamentous plant pathogen effectors are lacking. The P. infestans RXLR-type effector PexRD54 (PITG_09316) perturbs host-selective autophagy for the benefit of the pathogen via interaction with ATG8CL (
      • Dagdas Y.F.
      • Belhaj K.
      • Maqbool A.
      • Chaparro-Garcia A.
      • Pandey P.
      • Petre B.
      • Tabassum N.
      • Cruz-Mireles N.
      • Hughes R.K.
      • Sklenar J.
      • Win J.
      • Menke F.
      • Findlay K.
      • Banfield M.J.
      • Kamoun S.
      • Bozkurt T.O.
      An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor.
      ). Here, we focused on the biochemical and structural basis of PexRD54's interaction with ATG8CL to understand how the pathogen co-opts autophagic pathways.
      Structural conservation in RXLR-type effectors from the oomycetes, in the absence of confidently assignable sequence similarity, has previously been established (
      • Boutemy L.S.
      • King S.R.
      • Win J.
      • Hughes R.K.
      • Clarke T.A.
      • Blumenschein T.M.
      • Kamoun S.
      • Banfield M.J.
      Structures of Phytophthora RXLR effector proteins: a conserved but adaptable fold underpins functional diversity.
      ,
      • Win J.
      • Krasileva K.V.
      • Kamoun S.
      • Shirasu K.
      • Staskawicz B.J.
      • Banfield M.J.
      Sequence divergent RXLR effectors share a structural fold conserved across plant pathogenic oomycete species.
      ). Although each of the five structurally conserved three-helical bundle (WY domain) repeats in PexRD54 adopts the same overall fold, they pack together to form a unique structure different from that of the two WY domain repeat effector ATR1 from Hyaloperonospora arabidopsidis (
      • Chou S.
      • Krasileva K.V.
      • Holton J.M.
      • Steinbrenner A.D.
      • Alber T.
      • Staskawicz B.J.
      Hyaloperonospora arabidopsidis ATR1 effector is a repeat protein with distributed recognition surfaces.
      ). Detailed analysis of the PexRD54 structure suggests trajectories for the evolution of WY domain proteins through gain or loss of functional units presented on the N or C terminus of the core three-helical bundle. First, the minimal three helix WY domain fold seen in PexRD54 is found in P. infestans effector PexRD2 (
      • Boutemy L.S.
      • King S.R.
      • Win J.
      • Hughes R.K.
      • Clarke T.A.
      • Blumenschein T.M.
      • Kamoun S.
      • Banfield M.J.
      Structures of Phytophthora RXLR effector proteins: a conserved but adaptable fold underpins functional diversity.
      ), but in other RXLR-type effectors of known structure an N-terminal helix is present resulting in a four-helical bundle. Interestingly, in PexRD54, the C-terminal helices of WY-1, WY-3, and WY-4 are positioned such that they also serve as N-terminal helical extensions to WY-2, WY-4, and WY-5 to build four-helical bundles as observed in AVR3a4 (
      • Yaeno T.
      • Li H.
      • Chaparro-Garcia A.
      • Schornack S.
      • Koshiba S.
      • Watanabe S.
      • Kigawa T.
      • Kamoun S.
      • Shirasu K.
      Phosphatidylinositol monophosphate-binding interface in the oomycete RXLR effector AVR3a is required for its stability in host cells to modulate plant immunity.
      ), AVR3a11, and ATR1. Second, in ATR1 the tandem repeats of the four helix bundle are separated by a fifth “linker” helix. When the first WY domain of ATR1 is overlaid on WY-5 of PexRD54, the fifth linker helix is positioned on the final helix of PexRD54 (brown in Fig. 3A). In both protein structures, this helix then serves to present the proximal regions, either a second WY domain as seen in ATR1 or the AIM region as seen in PexRD54. Finally, PexRD54:WY-3 does not have an N-terminal helix and does not form a four helical bundle. This correlates with a significant kink in the PexRD54 structure between WY-2 and WY-3. Each of these observations serves to highlight the plasticity of the WY-fold and how it can be utilized to deliver new template structures with the potential for functional diversification. It is interesting to note that conserved structure in the absence of confidently assignable sequence similarity is emerging as a recurring theme for filamentous plant pathogen effectors (
      • de Guillen K.
      • Ortiz-Vallejo D.
      • Gracy J.
      • Fournier E.
      • Kroj T.
      • Padilla A.
      Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi.
      ,
      • Pedersen C.
      • Ver Loren van Themaat E.
      • McGuffin L.J.
      • Abbott J.C.
      • Burgis T.A.
      • Barton G.
      • Bindschedler L.V.
      • Lu X.
      • Maekawa T.
      • Wessling R.
      • Cramer R.
      • Thordal-Christensen H.
      • Panstruga R.
      • Spanu P.D.
      Structure and evolution of barley powdery mildew effector candidates.
      ).
      Little is known about how plant autophagic pathways are controlled and manipulated by pathogens. The structure of ATG8CL bound to the PexRD54 AIM peptide revealed the fundamental mechanisms of AIM recognition by plant ATG8s are similar to those seen in other organisms. The two critical hydrophobic residues of the ΩXXΨ motif, Trp and Val in PexRD54, are bound in two hydrophobic pockets on the surface of ATG8CL (Fig. 4A). Furthermore, our mutagenesis and peptide-binding studies confirm the important roles for these residues in the interaction. The identity of the residues to the N terminus of the AIM, which in other systems comprise acidic residues (
      • Noda N.N.
      • Ohsumi Y.
      • Inagaki F.
      Atg8-family interacting motif crucial for selective autophagy.
      ), do not seem to be important in this case. Previously, it was shown that the binding of PexRD54 to another ATG8 family member, ATG8IL, was weaker in planta and in vitro. These two proteins share 50% sequence identity. Interestingly, three amino acids are changed between ATG8CL and ATG8IL at the ATG8CL/PexRD54 AIM peptide interface: I33V, L56M, and Vl64I. ATG8CL Ile-33 is located at the base of the pocket that binds PexRD54 Trp-378, whereas ATG8CL Leu-56 and ATG8CL Val-64 are both located in the second hydrophobic pocket that faces PexRD54 Val-381. The interactions between ATG8s and AIM peptides are dominated by hydrophobic interactions, and the subtle changes delivered by these mutations may be responsible for the weaker binding affinity of ATG8IL over ATG8CL, although this remains to be tested in vitro and will be the subject of future work.
      The previous study (
      • Dagdas Y.F.
      • Belhaj K.
      • Maqbool A.
      • Chaparro-Garcia A.
      • Pandey P.
      • Petre B.
      • Tabassum N.
      • Cruz-Mireles N.
      • Hughes R.K.
      • Sklenar J.
      • Win J.
      • Menke F.
      • Findlay K.
      • Banfield M.J.
      • Kamoun S.
      • Bozkurt T.O.
      An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor.
      ) and the work described here reveal the importance of the interaction between PexRD54 and ATG8CL, as mediated by the effector's C-terminal AIM region. This region includes only ∼3% of the amino acids downstream of the RXLR-dEER motif, but deletion of WY domains 1–4 does not significantly affect ATG8CL binding in vitro or in planta. This raises the following question. How do the five WY domains contribute to PexRD54 function? This effector has been shown to stimulate host autophagosome formation, and it was hypothesized that the pathogen exploits this for its own benefit in either promoting nutrient recycling or counteracting defense. Future work will address how the PexRD54 WY domains may contribute to autophagosome formation and/or act as a receptor to localize specific cellular cargo to autophagic pathways.

      Experimental Procedures

      Gene Cloning

      All constructs were verified by DNA sequencing.

      PexRD54

      For protein expression in E. coli, DNA encoding PexRD54 residues Val-92 to Val-381 was amplified from RFP-PexRD54 (
      • Dagdas Y.F.
      • Belhaj K.
      • Maqbool A.
      • Chaparro-Garcia A.
      • Pandey P.
      • Petre B.
      • Tabassum N.
      • Cruz-Mireles N.
      • Hughes R.K.
      • Sklenar J.
      • Win J.
      • Menke F.
      • Findlay K.
      • Banfield M.J.
      • Kamoun S.
      • Bozkurt T.O.
      An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor.
      ) and cloned into pOPINA or pOPINS3C (
      • Berrow N.S.
      • Alderton D.
      • Sainsbury S.
      • Nettleship J.
      • Assenberg R.
      • Rahman N.
      • Stuart D.I.
      • Owens R.J.
      A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.
      ) by In-Fusion cloning (Clontech). The resultant vectors expressed PexRD54 protein without a fusion tag (pOPINA) or with the N-terminal His6-SUMO tag (pOPINS3C), respectively. DNA encoding PexRD54 residues Arg-219 to Val-381 was amplified from pOPINA-PexRD54 and cloned into pOPINS3C. DNA encoding PexRD54 residues Ser-299 to Val-381 was amplified from pOPINA-PexRD54 (and cloned into pOPINS3C) or from pOPINS3C-PexRD54 (and cloned into pOPINA). Single point mutants within the AIM region of PexRD54 were encoded within primers that were then used to amplify the full-length construct from pOPINS3C-PexRD54 followed by ligation into pOPINS3C. For protein expression in planta, DNA encoding PexRD54 residues Arg-219 to Val-381 or Ser-299 to Val-381 were amplified from RFP-PexRD54 and cloned into pENTR (ThermoFisher, UK). The expression constructs RFP-PexRD54Δ218 and RFP-PexRD54Δ298 were generated by Gateway LR reaction (Invitrogen) using the destination vector pH7WGR2 (N-terminal RFP fusion).

      ATG8CL

      For protein expression in E. coli, DNA encoding Met-1 to Phe-119 of ATG8CL was amplified from pOPINF-ATG8CL (
      • Dagdas Y.F.
      • Belhaj K.
      • Maqbool A.
      • Chaparro-Garcia A.
      • Pandey P.
      • Petre B.
      • Tabassum N.
      • Cruz-Mireles N.
      • Hughes R.K.
      • Sklenar J.
      • Win J.
      • Menke F.
      • Findlay K.
      • Banfield M.J.
      • Kamoun S.
      • Bozkurt T.O.
      An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor.
      ) and cloned into pOPINE (
      • Berrow N.S.
      • Alderton D.
      • Sainsbury S.
      • Nettleship J.
      • Assenberg R.
      • Rahman N.
      • Stuart D.I.
      • Owens R.J.
      A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.
      ), producing ATG8CL with a non-cleavable C-terminal His6 tag. DNA encoding Ser-5 to Asn-114 of ATG8CL was amplified from pOPINF-ATG8CL and cloned into pOPINF, expressing ATG8CL with a cleavable N-terminal His6 tag (called ATG8CL* hereafter). For probing the peptide array, DNA encoding ATG8CL residues Met-1 to Phe-119 was amplified from pOPINE-ATG8CL and cloned into pOG3182 (Oxford Genetics). DNA encoding the ATG8CL-GST fusion was amplified from ATG8CL-pOG3182 and cloned into pOPINE. The resultant pOPINE-ATG8CL-GST vector expressed ATG8CL protein with a non-cleavable C-terminal GST-His6 tag. For protein expression in planta, GFP-EV and GFP-ATG8CL constructs were described previously (
      • Dagdas Y.F.
      • Belhaj K.
      • Maqbool A.
      • Chaparro-Garcia A.
      • Pandey P.
      • Petre B.
      • Tabassum N.
      • Cruz-Mireles N.
      • Hughes R.K.
      • Sklenar J.
      • Win J.
      • Menke F.
      • Findlay K.
      • Banfield M.J.
      • Kamoun S.
      • Bozkurt T.O.
      An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor.
      ).

      Heterologous Protein Production and Purification

      Purified proteins were concentrated and stored in 20 mm HEPES buffer, pH 7.5, containing 150 mm NaCl, except where stated.

      PexRD54 and Its Variants

      For analytical gel filtration and ITC, all PexRD54 proteins were produced using E. coli BL21-arabinose-inducible cells and purified as described previously (
      • Dagdas Y.F.
      • Belhaj K.
      • Maqbool A.
      • Chaparro-Garcia A.
      • Pandey P.
      • Petre B.
      • Tabassum N.
      • Cruz-Mireles N.
      • Hughes R.K.
      • Sklenar J.
      • Win J.
      • Menke F.
      • Findlay K.
      • Banfield M.J.
      • Kamoun S.
      • Bozkurt T.O.
      An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor.
      ). For SPR, the same purification protocol was followed, with the exception of the final gel filtration step, which used 20 mm HEPES, pH 7.5, 500 mm NaCl.

      ATG8CL

      ATG8CL, expressed from pOPINF, was produced in E. coli BL21(DE3) and purified as described previously (
      • Dagdas Y.F.
      • Belhaj K.
      • Maqbool A.
      • Chaparro-Garcia A.
      • Pandey P.
      • Petre B.
      • Tabassum N.
      • Cruz-Mireles N.
      • Hughes R.K.
      • Sklenar J.
      • Win J.
      • Menke F.
      • Findlay K.
      • Banfield M.J.
      • Kamoun S.
      • Bozkurt T.O.
      An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor.
      ). When produced from pOPINE, a single Ni2+-NTA capture step followed by gel filtration produced soluble protein. The same strategy was used for purifying pOPINE-ATG8CL-GST-His. For SPR, ATG8CL was purified using 20 mm HEPES, pH 7.5, 500 mm NaCl in the gel filtration step. For crystallization, pOPINF-ATG8CL* was expressed and purified as for pOPINF-ATG8CL, except auto-induction media were used to culture the E. coli.

      PexRD54-ATG8CL Complex

      For crystallization and SAXS analysis of the complex, pOPINA-PexRD54 and pOPINE-ATG8CL were co-transformed and expressed in BL21(DE3). Purification used the same protocol as for ATG8CL produced from pOPINE.

      Protein-Protein Interaction Studies

      Analytical Gel Filtration

      Analytical gel filtration chromatography was performed at 4 °C using a Superdex 75 10/300 column (GE Healthcare) pre-equilibrated in 20 mm HEPES, pH 7.5, 150 mm NaCl. 100 μl of sample was injected at a flow rate of 0.8 ml/min, and 0.5-ml fractions were collected for analysis. To study complex formation, proteins were mixed and incubated on ice for at least 1 h prior to loading.

      Surface Plasmon Resonance

      SPR experiments were performed at 18 °C using a BIAcore T200 system (GE Healthcare) and an NTA sensor chip (GE Healthcare). Protein samples were prepared in 20 mm HEPES, pH 7.5, 500 mm NaCl, and all the measurements were recorded in the same buffer at a flow rate of 30 μl/min. A single cycle kinetics approach was used to study the interaction between PexRD54 and ATG8CL. The NTA chip was activated by injecting 10 μl of 0.5 mm NiCl2 over flow cell 2, which was also used to immobilize His-tagged ATG8CL to a response level of 85 ± 2. Increasing concentrations of PexRD54 (20, 200, 600, 1000, and 2000 nm) were injected over flow cell 1 and 2 for 90 s. After the final injection, the dissociation was recorded for 300 s. Two startup cycles were run where the chip was activated and ATG8CL immobilized in the same manner, but buffer only was injected instead of PexRD54. This was subtracted to account for any dissociation of ATG8CL from the sensor chip. The sensor chip was regenerated by injecting 10 μl of 350 mm EDTA. The data were analyzed using BIAcore T200 BIAevaluation software (GE Healthcare) and then plotted with Microsoft Excel.

      Isothermal Titration Calorimetry

      Calorimetry experiments were recorded at 15 °C in 20 mm HEPES, pH 7.5, 150 mm NaCl, using an iTC200 instrument (MicroCal Inc.). The calorimetric cell was filled with 80 μm PexRD54 truncation (PexRD54Δ218 or PexRD54Δ298) and titrated with 0.8 mm ATG8CL from the syringe. A single injection of 0.5 μl of ATG8CL was followed by 19 injections of 2 μl each. Injections were made at 120-s intervals with a stirring speed of 750 rpm. The raw titration data were integrated and fitted to a one-site binding model using the MicroCal Origin software.

      In Planta Co-immunoprecipitation

      3–4-week-old N. benthamiana plants were used for transient expression experiments. T-DNA expression vectors encoding PexRD54 constructs, ATG8CL constructs, or empty vector were transformed into the A. tumefaciens GV3101 strain. Transformed agrobacteria were diluted in 5 mm MES, 10 mm MgCl2, pH 5.6, and mixed in 1:1 ratio to a final A600 of 0.2 prior to leaf infiltration.
      N. benthamiana leaves transiently expressing proteins were harvested 2 days post-infiltration. Protein extraction, immunoprecipitation, and Western blotting analyses were performed as described previously (
      • Dagdas Y.F.
      • Belhaj K.
      • Maqbool A.
      • Chaparro-Garcia A.
      • Pandey P.
      • Petre B.
      • Tabassum N.
      • Cruz-Mireles N.
      • Hughes R.K.
      • Sklenar J.
      • Win J.
      • Menke F.
      • Findlay K.
      • Banfield M.J.
      • Kamoun S.
      • Bozkurt T.O.
      An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor.
      ). For blots shown in Fig. 8, mouse monoclonal single step GFP-HRP antibody (Santa Cruz Biotechnology) was used for GFP immunoblot experiments. For RFP blots, polyclonal RFP antibody (Invitrogen) was used as primary antibody and anti-rat HRP antibody (Sigma, UK) was used as secondary antibody.

      Crystallization, Data Collection, and Structure Solution

      PexRD54 (in the Presence of ATG8CL)

      For crystallization, the PexRD54-ATG8CL complex produced by co-expression was concentrated to 10 mg/ml in 20 mm HEPES, 150 mm NaCl, pH 7.5. Crystallization experiments used 4-μl hanging drops with a 2:1 protein/precipitant ratio. For data collection, crystals were grown in 18% PEG 10K, 0.1 m sodium acetate, pH 5.0, 0.18 m tri-ammonium citrate and transferred to a cryoprotectant solution consisting of 22% PEG 10K, 0.1 m sodium acetate, pH 5.0, 0.18 m tri-ammonium citrate and 10% ethylene glycol. To enable structure solution, crystals were soaked for ∼45 s in well solution supplemented with 500 mm potassium iodide and then cryoprotected as above.
      Native and single wavelength anomalous diffraction x-ray data sets were collected at the Diamond Light Source, United Kingdom, beamline I02. The datasets were processed using the Xia2 pipeline (
      • Winter G.
      xia2: an expert system for macromolecular crystallography data reduction.
      ), see Table 1. The structure was solved using the single wavelength anomalous diffraction approach with the data collected from the crystal soaked in potassium iodide solution. Iodide sites were identified with Phenix (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). These positions were used to estimate initial phases using PHASER EP from the CCP4 suite (
      • Winn M.D.
      • Ballard C.C.
      • Cowtan K.D.
      • Dodson E.J.
      • Emsley P.
      • Evans P.R.
      • Keegan R.M.
      • Krissinel E.B.
      • Leslie A.G.
      • McCoy A.
      • McNicholas S.J.
      • Murshudov G.N.
      • Pannu N.S.
      • Potterton E.A.
      • Powell H.R.
      • et al.
      Overview of the CCP4 suite and current developments.
      ), followed by density improvement with PARROT (
      • Cowtan K.
      Recent developments in classical density modification.
      ). An initial model was built using BUCCANEER (
      • Cowtan K.
      The Buccaneer software for automated model building. 1. Tracing protein chains.
      ) followed by manual rebuilding and refinement using COOT (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of Coot.
      ) and REFMAC5 (
      • Murshudov G.N.
      • Vagin A.A.
      • Dodson E.J.
      Refinement of macromolecular structures by the maximum-likelihood method.
      ). Next, molecular replacement with Phaser, followed by the Phenix AutoBuild wizard, was used to produce an initial model of PexRD54 using the native x-ray data. The final model was produced through iterative rounds of refinement using REFMAC5 and manual rebuilding with COOT. Structure validation used the tools provided in COOT and MOLPROBITY (
      • Chen V.B.
      • Arendall 3rd, W.B.
      • Headd J.J.
      • Keedy D.A.
      • Immormino R.M.
      • Kapral G.J.
      • Murray L.W.
      • Richardson J.S.
      • Richardson D.C.
      MolProbity: all-atom structure validation for macromolecular crystallography.
      ).

      ATG8CL

      ATG8CL* mixed with a 3-fold molar excess of pentapeptide (Asp-Trp-Glu-Ile-Val) was incubated at 4 °C for 24 h and concentrated to 80 mg/ml in 20 mm HEPES, 150 mm NaCl, pH 7.5. Crystallization experiments used 2-μl sitting drops with a 1:1 protein/precipitant ratio. Crystals were produced in 0.2 m ammonium sulfate, 0.1 m Tris buffer, pH 8.0, and 36% PEG3350 and transferred to the precipitant solution with the addition of 10% ethylene glycol as a cryoprotectant. X-ray diffraction data were collected at the Diamond Light Source, UK, beamline I04, and the data were processed as above (Table 1). The structure was solved by molecular replacement using PHASER, as implemented in Phenix. The molecular replacement search model was generated by submitting the complete sequence of ATG8CL to the Phyre web server (
      • Kelley L.A.
      • Mezulis S.
      • Yates C.M.
      • Wass M.N.
      • Sternberg M.J.
      The Phyre2 web portal for protein modeling, prediction and analysis.
      ). Based on the solution, an initial model was produced using the AutoBuild wizard in Phenix. At this stage, clear electron density was apparent for the Asp-Trp-Glu-Ile-Val pentapeptide in both molecules of ATG8CL*. The final model was completed and validated as described for PexRD54. Data collection and refinement statistics for PexRD54 and ATG8CL are given in Table 1.

      SAXS Measurements, Data Processing, and Analysis

      SAXS data were collected at the ESRF beamline BM29 (Grenoble, France (
      • Pernot P.
      • Round A.
      • Barrett R.
      • De Maria Antolinos A.
      • Gobbo A.
      • Gordon E.
      • Huet J.
      • Kieffer J.
      • Lentini M.
      • Mattenet M.
      • Morawe C.
      • Mueller-Dieckmann C.
      • Ohlsson S.
      • Schmid W.
      • Surr J.
      • Theveneau P.
      • et al.
      Upgraded ESRF BM29 beamline for SAXS on macromolecules in solution.
      ,
      • Round A.
      • Felisaz F.
      • Fodinger L.
      • Gobbo A.
      • Huet J.
      • Villard C.
      • Blanchet C.E.
      • Pernot P.
      • McSweeney S.
      • Roessle M.
      • Svergun D.I.
      • Cipriani F.
      BioSAXS sample changer: a robotic sample changer for rapid and reliable high-throughput x-ray solution scattering experiments.
      )) and at the Diamond Light Source, UK, beamline B21. For BM29, measurements were made at an energy of 12.5 keV, camera length of 2.81 m, and q range 0.003–5 nm−1. For B21, measurements were made at an energy of 12.4 keV, camera length of 4.018 m, and q range 0.004–3.8 nm−1. Measurements of 40 μl of protein solution at three different concentrations (0.5, 1.0, and 2.0 mg/ml European Synchrotron Radiation Facility (ESRF); 2.5, 5.0, and 10.0 mg/ml Diamond Light Source) were made for each sample (and buffer). Matched buffer measurements taken before and after every sample were averaged and used for background subtraction. Merging of separate concentrations and further analysis steps were performed manually using the ATSAS package (
      • Konarev P.V.
      • Petoukhov M.V.
      • Volkov V.V.
      • Svergun D.I.
      ATSAS 2.1, a program package for small-angle scattering data analysis.
      ,
      • Putnam C.D.
      • Hammel M.
      • Hura G.L.
      • Tainer J.A.
      X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution.
      ). DATCMP was used to exclude any individual frames showing signs of radiation damage using standard thresholds for the beamlines. For uncomplexed PexRD54, data collected at the ESRF were used for further analysis. Inspection of the SAXS data for the PexRD54-ATG8CL complex suggested the optimum dataset incorporated both the ESRF (low angles and wide angles) and DLS (mid-range angles) data, and these were merged manually. The forward scattering I(0) and radius of gyration (Rg) for each particle were calculated from the Guinier approximation. The molecular mass of the samples was estimated using the Porod invariant (
      • Ciccariello S.
      • Goodisman J.
      • Brumberger H.
      On the Porod Law.
      ) and the maximum particle sizes (Dmax) were determined from the pair distribution function computed by GNOM (
      • Svergun D.I.
      Determination of the regularization parameter in indirect-transform methods using perceptual criteria.
      ) using PRIMUS (
      • Konarev P.V.
      • Volkov V.V.
      • Sokolova A.V.
      • Koch M.H.
      • Svergun D.I.
      PRIMUS: a Windows PC-based system for small-angle scattering data analysis.
      ). For both PexRD54 and the PexRD54-ATG8CL complex, 40 ab initio models were calculated using DAMMIN (
      • Svergun D.I.
      Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing.
      ). DAMSEL compared these models and calculated a mean normalized spatial discrepancy (NSD) of 0.545 ± 0.02 for PexRD54 (discarding only one model with NSD > mean ± 2× S.D.), and a mean NSD of 0.635 ± 0.03 for PexRD54-ATG8CL complex (no models discarded). DAMSEL also identified the most probable (lowest NSD) model. All non-discarded models were aligned, averaged, and compared using DAMSUP, DAMAVER, and DAMFILT in ATSAS for analysis. Rigid body modeling of the PexRD54-ATG8CL complex was achieved with CORAL (
      • Petoukhov M.V.
      • Franke D.
      • Shkumatov A.V.
      • Tria G.
      • Kikhney A.G.
      • Gajda M.
      • Gorba C.
      • Mertens H.D.
      • Konarev P.V.
      • Svergun D.I.
      New developments in the program package for small-angle scattering data analysis.
      ), with the inclusion of the missing residues and linker region that were not visible in the electron density maps of PexRD54 or ATG8CL. The fits of the most probable ab initio models to the experimental data were calculated by DAMMIN, the theoretical scattering of PexRD54 was calculated with CRYSOL (
      • Svergun D.
      • Barberato C.
      • Koch M.H.J.
      CRYSOL–A program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates.
      ), and the fit of the PexRD54-ATG8CL complex was as calculated by CORAL. Rigid body models of PexRD54 and the PexRD54-ATG8CL complex were overlaid with the ab initio models using SUPCOMB (
      • Kozin M.B.
      • Svergun D.I.
      Automated matching of high- and low-resolution structural models.
      ) and viewed in PyMOL.

      Peptide Library

      The PexRD54-AIM peptide library was synthesized by Kinexus (Vancouver, Canada) and included 200 peptides where each amino acid in the last 10 amino acids of PexRD54 was changed to every other amino acid. The peptides were spotted on cellulose membrane (Invatis, Germany) with free C termini. Peptide interactions with the ATG8CL-GST-His fusion protein were determined as described previously. The membrane was blocked with 5% (w/v) nonfat dried milk in TBS-T, washed with TBS-T, and overlaid with 1 μg/ml purified ATG8CL-GST-His fusion protein for 2 h at room temperature. The membrane was washed in TBS-T, and bound proteins were detected with HRP-conjugated anti-GST antibody (1:5000) (RPN1236; GE Healthcare, UK).

      Circular Dichroism Spectroscopy

      CD spectroscopy experiments were performed using a Chirascan-Plus CD spectrophotometer (Applied Photophysics). Purified proteins in 20 mm HEPES, pH 7.5, 150 mm NaCl at a concentration of at least 10 mg/ml were diluted to 0.2 mg/ml in 20 mm di-potassium phosphate, pH 7.2. CD measurements were carried out in a quartz glass cell with a 0.5-mm path length. To obtain overall CD spectra, wavelength scans between 190 and 260 nm were collected at 15 °C using a 2.0-nm bandwidth, 0.5-nm step size, and time per point of 1 s. The data were collected over four accumulations and averaged. The raw data in millidegree units were corrected for background and converted to mean residue molar ellipticity.

      Author Contributions

      A. M., R. K. H., Y. F. D., T. O. B., S. K., and M. J. B. designed the research; A. M., R. K. H., Y. F. D., N. T., and E. Z. performed the experiments; K. B. provided reagents and analytic tools; A. M., R. K. H., Y. F. D., A. R., T. O. B., and M. J. B. analyzed the data; A. M., R. K. H., and M. J. B. wrote the paper with editorial input from all authors.

      Acknowledgments

      We thank the Diamond Light Source (beamlines I02, I04, and B21 under proposals MX7641 and MX9475) and the ESRF (beamline BM29) for access to x-ray data collection facilities and Clare Stevenson (JIC Surface Plasmon Resonance facility) for help with SPR.

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