Structures of Phytophthora RXLR effector proteins: a conserved but adaptable fold underpins functional diversity

Abstract

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The molecular mechanisms underlying how effectors evolve to gain new virulence functions and/or evade the plant immune system are poorly understood. Here, we report the crystal structures of the effector domains from two oomycete RXLR proteins, Phytophthora capsici AVR3a11 and Phytophthora infestans PexRD2. Despite sharing <20% sequence identity in their effector domains, they display a conserved core α-helical fold. Bioinformatic analyses suggest that the core fold occurs in ∼44% of annotated Phytophthora RXLR effectors, both as a single domain and in tandem repeats of up to 11 units. Functionally important and polymorphic residues map to the surface of the structures, and PexRD2, but not AVR3a11, oligomerizes in planta. We conclude that the core α-helical fold enables functional adaptation of these fast evolving effectors through (i) insertion/deletions in loop regions between α-helices, (ii) extensions to the N and C termini, (iii) amino acid replacements in surface residues, (iv) tandem domain duplications, and (v) oligomerization. We hypothesize that the molecular stability provided by this core fold, combined with considerable potential for plasticity, underlies the evolution of effectors that maintain their virulence activities while evading recognition by the plant immune system.

FIGURE 1.

RXLR effectors AVR3a11 and PexRD2 adopt a structurally conserved but adaptable fold. a, oomycete RXLR effectors are modular proteins comprising a secretion signal (cyan), RXLR translocation motif (purple), and an effector domain (green). b, structural alignment and secondary structure elements of the effector domains of AVR3a11 and PexRD2. W-motifs (cyan) and Y-motifs (lilac) are colored, with key residues (as discussed under “Results and Discussion”) boxed. c, the structure of AVR3a11 is a monomeric four-helix bundle with a hydrophobic core. Carbon atoms of key residues in the W- and Y-motifs are colored as boxed in b; loop-3 is shown in purple. d, PexRD2 is a dimer with a hydrophobic interface, including residues Val⁷³, Asp⁷⁴, Ala⁷⁷, Thr⁸³, Ile⁸⁶, Ala⁹⁰, Met⁹⁶, Gly¹⁰⁰, Met¹⁰⁵, Leu¹⁰⁸, Leu¹⁰⁹, and Leu¹¹² (shown for one monomer only). α-Helices are labeled to correspond to equivalent positions in AVR3a11. e, the AVR3a11/PexRD2-monomer overlay generated using SSM, showing the conserved fold. Protein structures are colored as in c and d with key residues of the W- and Y-motifs colored as in b.

FIGURE 2.

RXLR effectors can adopt different oligomeric states in vitro and in vivo. Fits of the analytical ultracentrifugation data (including residuals) for (a) AVR3a11 (0.3 mg/ml (circles) and 0.45 mg/ml (triangles)) (b) PexRD2 (3 mg/ml (triangles) and 6 mg/ml (circles)) to the models described confirmed that monomeric and dimeric species are the prevalent forms of these proteins in solution. c, co-immunoprecipitation (co-IP) shows that PexRD2 self-associates in planta; no oligomerization is apparent for AVR3a11, AVR3a^KI, or AVR3a^EM. RuBisCO is included as a loading control (Coomassie-stained SDS-polyacrylamide gel).

FIGURE 3.

Functionally important and polymorphic residues are presented on the surface of AVR3a11 and PexRD2. a, in AVR3a11, Glu⁷¹ and Gln⁹⁴ (equivalent to the Lys/Glu⁸⁰ and Ile/Met¹⁰³ positions of AVR3a), Ser¹¹² (equivalent to Ser¹³² of AVR3a), and a Tyr residue near the C terminus are located on the protein surface. b, in PexRD2, the polymorphic residues between the P. infestans and P. mirabilis homologues are also surface-displayed. The positions of these residues in the primary sequences of the proteins are shown as brown boxes in Fig. 1b.

FIGURE 4.

The WY-domain is a conserved unit in Phytophthora RXLR effectors. a, the MEME motif spanning the WY-domain identified by searching the RXLR repertoire of P. infestans, P. ramorum, and P. sojae. Boxed residues, from left to right are the Leu of the L-motif, the Trp of the W-motif, the residues from α₃ (AVR3a11) that pack into the hydrophobic core (Val and Leu), and the Tyr of the Y-motif (and the conserved hydrophobic residue two positions before). b, graphical representation of the final HMM model used to screen the Phytophthora and H. arabidopsidis effector repertoires for the presence of the WY-domain. The four most abundant amino acids at each position in the motif are labeled with their one-letter code, and those that represented ≥20% are shaded in gray. c, distribution of WY-domains in RXLR effectors (top) and the non-RXLR proteome (bottom, stacked bar chart) following sequence database searches with the WY-domain HMM. The number of proteins in each HMM score bin is shown. The positions of AVR3a and PexRD2 are marked, and the dashed line shows the position of the cut-off used to describe WY-domain like sequences. The estimated false positive rate of WY-domain discovery based on the Phytophthora non-RXLR secretome is also shown (bottom, line graph).