The Structural Basis For The Interation Between The RATX1 Domain Of R Protein Pia From Rice And The Effectors From The Rice Blast Fungus

Rice is one of the most important staple food for more than 50%of the world's population.The product and the security is constantly affected by pathogen stress.Rice blast,caused by the ascomycete fungal pathogen Magnaporthe oryzae,is the most devastating disease.And the use of resistance crops represents the economically and ecologically most sustainable strategy for blast diseases control.Therefore,understanding the mechanisms of the molecular interactions between plant receptors and pathogens underpins a great significance to genetic improvement and rational distribution of rice resistance varieties.Previous study has reported that in phytopathogenic fungi,there exists a family of sequence-unrelated but strcturally conserved effectors:the M.oryzae AVRs and ToxB-like(MAX)effectors.AVR-PikD,AVR1-CO039 and AVR-Pia from M.oryzae MAX-effectors are recognized by resiscance proteins Pikp-1 and RGA5 respectively inside rice cells through direct interactions with unconserved heavy metal-associated domain(HMA or RATX1)for triggering ETI response.A recent study reported the crystal structure of the AVR-PikD/Pikp-HMA complex and showed that HMA-effector binding is crucial for Pikp-mediated resistance.To gain further insight into the structural basis for the specific recognition of distinct MAX-effectors by Pia heavy metal-associated domain(RGA5RATX1),the author tried to express recombinant proteins RGA5A_S,AVR1-CO39 and AVR-Pia for structural analysis.Crystals of RGA5A_S/AVR1-CO39 complex was obtained by vapor diffusion and diffracted to a Bragg spacing of 2.1 A.The structure was solved by molecular replacement and showed that unlike AVR-PikD which binds the second monomer of the Pikp-HMA dimer,AVR1-CO39 interacts with a monomer of the RGA5A_S domain.The 47 residues at the C-terminus of RGA5A_S were not visible on the electron density map and are likely flexible or disordered.ITC showed that RGA5RATX1 missing the 47 residues kept the identical affinity of RGA5A_S to AVR1-CO39(Kds of 5.4 ?M and 5.3 ?M).This shows that RGA5RATX1 is a core binding domain for AVR1-CO39.The crystal structure of the AVR1-CO39/RGA5A_S complex enabled design of mutations to alter protein interaction in vitro,and ITC and GF results showed that replacement of T41 from ?2-sheet of AVR1-CO39 abolished the binding affinity to RGA5A_S.These results demonstrate that Threonine at 41 position of AVR1-CO39 is the core residue for RGA5A_S recognizing.We explored the crystallizing conditions of RGA5A_S/AVR-Pia complex,but there is no complex crystal obtained.Homologous structure comparison analysis shows that RGA5RATX1-binding interface in AVR1-CO39 strongly overplays with that in AVR1-Pia previoulsy identified.In a similar spatial position of key residue T41 in AVR1-CO39,F24 subtitution from N-terminal of ?1-sheet in AVR-Pia is crucial for HMA-binding and effector-mediated immune activation.Furthermore,the analyses of SV-AUC and GF display that the molecular weight of two complexes(RGA5A_S was mixed with AVR1-CO39 or AVR-Pia)fits well to a dimer of RGA5A S.These results indicate that like AVR1-CO39,AVR-Pia competes with RGA5A_S self-interaction and the AVR-Pia-binding interface in RGA5RATX1 overlaps with the homo-dimer surface formed by RGA5RATX1.Collectively,our findings indicate two independently HMA/MAX-effector interaction modes.AVR1-CO39 and AVR-Pia,share the similar binding mode to bind to the dimer interface of RGA5A_S,and form a stable complex with a monomer of RGA5A_S to mediate the ETI response.This interaction mode quietly differs from AVR-PikD which binds the second monomer of the Pikp-HMA dimer.Our study establishes a novel theoretical basis of directly recognition of a fungal effector by a plant immune receptor.