Fig 1.
The PexRD54 C-terminal AIM was acquired in the common ancestor of Phytophthora clade 1b and 1c species.
(a) Analysis of Phytophthora clade 1 PexRD54 and PexRD54-like protein sequences. Unrooted maximum-likelihood phylogenetic tree of 20 PexRD54 and PexRD54-like protein sequences (S1 Table) from an 285 amino acid alignment (MUSCLE [25]) spanning the PiPexRD54 first WY domain through the C-terminus, constructed using MEGA7 [26] and visualized using iTOL [27]. Protein sequences were gathered from strains of P. mirabilis (pink; strains P3008, P99114), P. ipomoeae (purple), P. infestans (gray; strains T30-4, KR2A1, KR2A2), P. parasitica (green; strains race 0, P10297, P1569, INRA-310), and P. cactorum (blue; strain 10300), indicated to the right. Approximate species relationships are denoted by a phylogeny adapted from Yang et al. 2017 [14] and shown in full in S1 Fig. The PexRD54 and PexRD54-like clades are denoted with shading, the bootstrap supports of the major nodes are indicated, and the scale bar indicates the evolutionary distance based on substitution rate. Protein representations correspond to an amino acid alignment of the full-length PexRD54 and PexRD54-like protein sequences (S2 Fig). Representations include predicted motifs (RxLR-dEER) and domains (WY) based on the PiPexRD54 sequence [18] and identification of key residues [16]. Predicted AIM sequences are marked in purple and were determined using the iLIR software [24]. (b) Table showing the PexRD54 C-terminal AIM amino acid (aa) sequences for each species and the AIM prediction score from iLIR, with more information in S1 Table, S1 and S2 Figs.
Fig 2.
Mirabilis jalapa ATG8s have unique evolutionary history compared to previously characterized ATG8s.
(a) Caryophylalles ATG8 isoforms are orthologous. Unrooted maximum-likelihood phylogenetic tree of 22 ATG8 isoforms with gray shading highlighting clades, and colors indicated plant species. The tree was calculated in MEGA7 [26] from a 375 nucleotide alignment (MUSCLE [25], codon-based) and visualized using iTOL [27]. The bootstrap supports of the major nodes are indicated. The scale bar indicates the evolutionary distance based on substitution rate. (b) Caryophylalles, Solanales, and Brassicales taxa have unique ATG8 subclades. Unrooted maximum-likelihood tree of 186 ATG8 isoforms, with clades collapsed based on bootstrap support and colors indicating plant order; the full tree is in the appendix, S2 Fig. The tree was calculated in MEGA7 [26] from a 445 nucleotide alignment (MUSCLE [25], codon-based) and visualized using iTOL [27]. The Solanales and Brassicales ATG8 clades are named following the conventions in Kellner et al. 2016 [29]. The major ATG8 clades are labelled along the top of the phylogeny. The bootstrap values of the major nodes are indicated by gray circles, with the scale as shown. The scale bar indicates the evolutionary distance based on nucleotide substitution rate. (c) M. jalapa ATG8 isoforms are sequence-diverse. Alignment of all M. jalapa ATG8s (MUSCLE [25]), visualized with Jalview [31], with the protein model above corresponding to the StATG8-2.2 structure, and the residues that form electrostatic contacts with AIMs are marked below (•).
Fig 3.
The P. mirabilis PexRD54 AIM polymorphism reduces binding to M. jalapa ATG8s.
Co-immunoprecipitation experiment between PexRD54 variants (PiPexRD54AIM2, PiPexRD54, PmPexRD54, PiPexRD54PmAIM) and M. jalapa ATG8s (MjATG8s). RFP:PexRD54 variants were transiently co-expressed with GFP:EV, GFP:StATG8-2.2, and all GFP:MjATG8s. Immunoprecipitates (IPs) were obtained with anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera (listed on the right). Stars indicate expected band sizes.
Fig 4.
PmPexRD54 peptide binds weakly to ATG8s in isothermal titration calorimetry experiments.
The binding affinities between the PiPexRD54 and PmPexRD54 peptides and the ATG8 isoforms ATG8-2.2, MjATG8-I, and MjATG8-III, were determined using isothermal titration calorimetry (ITC) and downstream analyses. (a) Global fit analysis of ITC data. The isotherms for each of the experimental replicates were simultaneously fit to the same single site binding model, producing a single robust equilibrium dissociation constant (KD) estimate for each PexRD54 peptide-ATG8 interaction, using AFFINImeter analysis software [32]. KD estimates are listed in nanomolar (nM) for the PiPexRD54 interactions and millimolar (mM) for the PmPexRD54 interactions. The graphs overlay the lines of best fit for the replicate isotherms (pink, grey, purple), with the integration values (ΔQ) plotted against the ratio of ligand to protein (At/Mt). (b) Individual fit analysis of ITC data. The isotherms for each of the experimental replicates were individually fit to a single site binding model, producing KD estimates for each PexRD54 peptide-ATG8 interaction replicate, using AFFINImeter analysis software [32]. The graphs show the KD estimates for the PiPexRD54 and PmPexRD54 interaction replicates visualized using ggplot2 [33], with values in nanomolar (nM) and millimolar (mM), respectively. The graphs showing the heat differences and integrated heats of injection for each replicate are shown in S5 and S6 Figs, and a table summarizing the thermodynamic information is included in S2 Table.
Fig 5.
The PexRD54 AIM central glutamate (E) residue is important for ATG8 binding.
(a) Co-immunoprecipitation experiment between PexRD54 variants (PmPexRD54, PiPexRD54, PmPexRD54PiAIM) and ATG8s. RFP:PexRD54 variants were transiently co-expressed with GFP:EV, GFP:StATG8-2.2, and GFP:MjATG8-I. Immunoprecipitates (IPs) were obtained with anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera (listed on the right). Stars indicate expected band sizes. (b) Homology model of MjATG8-I and PiPexRD54 AIM peptide complex viewed using CCP4 [34]. MjATG8-I and PiPexRD54 AIM are illustrated in cartoon and stick representation. Amino acids making electrostatic interactions (dashed lines) are labelled. (c) PiPexRD54 AIM (PiAIM) and PmPexRD54 AIM (PmAIM) amino acid sequences, including cartoon and stick representation of the differential central residue.
Fig 6.
Model of PexRD54 evolution following a host jump.
Schematic of the Phytophthora host jump from Solanum species onto Mirabilis jalapa, leading to the differentially specialized pathogens P. infestans and P. mirabilis. In this model, the ancestral state of the PexRD54 effector includes a predicted AIM at the c-terminus, which was maintained in the P. infestans lineage and lost in the P. mirabilis lineage. The PiPexRD54 AIM has been shown to mediate binding to the potato host ATG8s, whereas the single amino acid polymorphism in the PmPexRD54 AIM precludes effector binding to the M. jalapa host ATG8s.