A blast fungus zinc-finger fold effector binds to a hydrophobic pocket in host Exo70 proteins to modulate immune recognition in rice

Abstract

Exocytosis plays an important role in plant-microbe interactions, in both pathogenesis and symbiosis. Exo70 proteins are integral components of the exocyst, an octameric complex that mediates tethering of vesicles to membranes in eukaryotes. Although plant Exo70s are known to be targeted by pathogen effectors, the underpinning molecular mechanisms and the impact of this interaction on infection are poorly understood. Here, we show the molecular basis of the association between the effector AVR-Pii of the blast fungus Maganaporthe oryzae and rice Exo70 alleles OsExo70F2 and OsExo70F3, which is sensed by the immune receptor pair Pii via an integrated RIN4/NOI domain. The crystal structure of AVR-Pii in complex with OsExo70F2 reveals that the effector binds to a conserved hydrophobic pocket in Exo70, defining an effector/target binding interface. Structure-guided and random mutagenesis validates the importance of AVR-Pii residues at the Exo70 binding interface to sustain protein association and disease resistance in rice when challenged with fungal strains expressing effector mutants. Furthermore, the structure of AVR-Pii defines a zinc-finger effector fold (ZiF) distinct from the MAX (Magnaporthe Avrs and ToxB-like) fold previously described for a majority of characterized M. oryzae effectors. Our data suggest that blast fungus ZiF effectors bind a conserved Exo70 interface to manipulate plant exocytosis and that these effectors are also baited by plant immune receptors, pointing to new opportunities for engineering disease resistance.

Keywords: Exocyst; NLR; effector; plant immunity.

Fig. 1.

AVR-Pii binds specifically to rice Exo70F2 and Exo70F3 in yeast and in vitro. (A) Y2H assay of AVR-Pii with rice OsExo70B1, OsExo70F2, and OsExo70F3. Left, control plate for yeast growth. Right, quadruple-dropout media supplemented with X-α-gal and increasing concentrations of aureobasidine A (Au A). Growth and development of blue coloration in the selection plate are both indicative of protein-protein interactions. OsExo70 proteins were fused to the GAL4 DNA binding domain and AVR-Pii to the GAL4 activator domain. Each experiment was repeated a minimum of three times, with similar results. (B) Binding of AVR-Pii to OsExo70 proteins determined by ITC. Upper, heat differences upon injection of AVR-Pii into the cell containing the respective OsExo70 allele. Middle, integrated heats of injection (dots) and best fit (solid line) to a single-site binding model calculated using AFFINImeter ITC analysis software (78). Bottom, difference between the fit to a single-site binding model and the experimental data; closer to zero indicates stronger agreement between the data and the fit. The experiments shown are representative of three replicates. Other replicates for each experiment are shown in SI Appendix, Fig. S5. The thermodynamic parameters obtained in each experiment are presented in SI Appendix, Table S1.

Fig. 2.

The crystal structure of OsExo70F2 in complex with AVR-Pii reveals hydrophobic residues dominate the interaction interface. (A) Schematic representation of OsExo70F2 in complex with AVR-Pii. Both molecules are represented as cartoon ribbons, with the molecular surface also shown and colored as labeled in green and yellow for OsExo70F2 and AVR-Pii, respectively. (B) Close-up view of the interaction interface between OsExo70F2 and AVR-Pii. OsExo70F2 is presented as a solid surface, with the effector as cartoon ribbons and side chains displayed as a cylinder for AVR-Pii–interacting residues (Asp45, Tyr48, His49, Tyr64, Phe65, and Asn66) in addition to the residues coordinating the Zn²⁺ atom (Cys51, Cys54, His67, and Cys69). (C) OsExo70F2 surface hydrophobicity representation at the AVR-Pii interaction interface; residues are colored depending on their hydrophobicity from light blue (low) to yellow (high). (D) Representation of OsExo70F2 surface electrostatic potential at the AVR-Pii interaction interface; residues are colored depending on their electrostatic potential from dark blue (positive) to red (negative). AVR-Pii residues 20 to 43 were not observed in the electron density used to derive the structure.

Fig. 3.

Mutations at the AVR-Pii binding interface perturb interactions with rice Exo70 proteins. (A) Y2H assay of AVR-Pii mutants Tyr64Arg and Phe65Glu with rice OsExo70F2 and OsExo70F3. Left, control plate for yeast growth. Right, quadruple-dropout media supplemented with X-α-gal and increasing concentrations of aureobasidine A (Au A). Growth and development of blue coloration in the selection plate are both indicative of protein-protein interactions. Wild-type AVR-Pii is included as positive control. Exo70 proteins were fused to the GAL4 DNA binding domain and AVR-Pii to the GAL4 activator domain. Each experiment was repeated a minimum of three times, with similar results. (B) Binding of AVR-Pii mutants Tyr64Arg and Phe65Glu to rice OsExo70F2 and OsExo70F3 determined by ITC. Wild-type AVR-Pii was included as positive control. Upper, heat differences upon injection of AVR-Pii mutants into the cell containing the respective OsExo70 allele. Middle, integrated heats of injection (dots) and best fit (solid line) to a single-site binding model calculated using AFFINImeter ITC analysis software (78). Bottom, difference between the fit to a single-site binding model and the experimental data; closer to zero indicates stronger agreement between the data and the fit. Panels are representative of three replicates. Other replicates for each experiment are shown in SI Appendix, Figs. S18 and S19. The thermodynamic parameters obtained in each experiment are presented in SI Appendix, Table S3.

Fig. 4.

Mutations at the binding interface of AVR-Pii with OsExo70 abrogate recognition by Pii resistance in rice. (A) Rice leaf blade spot inoculation of transgenic M. oryzae Sasa2 isolates expressing AVR-Pii, AVR-Pii Tyr64Arg, or AVR-Pii Phe65Glu in rice cultivars Moukoto (Pii−) and Hitomebore (Pii+). For each experiment, representative images from replicates with independent M. oryzae transformants are shown. Wild-type (WT) M. oryzae isolate Sasa2 is included as control. Images for each replicate of AVR-Pii, AVR-Pii Tyr64Arg, and AVR-Pii Phe65Glu are presented in SI Appendix, Figs. S21–S23. (B) Measurement of vertical length of the disease lesion caused by M. oryzae Sasa2 as well as transgenic M. oryzae Sasa2 isolates harboring AVR-Pii, AVR-Pii Tyr64Arg, or AVR-Pii Phe65Glu in rice cultivars Moukoto (Pii−) and Hitomebore (Pii+). Lesions in rice cultivars Moukoto (Pii−) and Hitomebore (Pii+) are represented by blue and yellow boxes, respectively. For each isolate, a total of four biological replicates were performed, and the data are presented as box plots. The center line represents the median, the box limits are the upper and lower quartiles, and the whiskers extend to the largest value within Q1 − 1.5× the interquartile range (IQR) and the smallest value within Q3 + 1.5× IQR. All the data points are represented as dots with distinct colors for each biological replicate.