Abstract
Unconventional integrated domains in plant intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NLRs) type can directly bind translocated effector proteins from pathogens and thereby initiate an immune response. The rice (Oryza sativa) immune receptor pairs Pik-1/Pik-2 and RGA5/RGA4 both use integrated heavy metal-associated (HMA) domains to bind the effectors AVR–Pik and AVR–Pia, respectively, from the rice blast fungal pathogen Magnaporthe oryzae. These effectors both belong to the MAX effector family and share a core structural fold, despite being divergent in sequence. How integrated domains in NLRs maintain specificity of effector recognition, even of structurally similar effectors, has implications for understanding plant immune receptor evolution and function. Here, using plant cell death and pathogenicity assays and protein–protein interaction analyses, we show that the rice NLR pair Pikp-1/Pikp-2 triggers an immune response leading to partial disease resistance toward the “mis-matched” effector AVR–Pia in planta and that the Pikp–HMA domain binds AVR–Pia in vitro. We observed that the HMA domain from another Pik-1 allele, Pikm, cannot bind AVR–Pia, and it does not trigger a plant response. The crystal structure of Pikp–HMA bound to AVR–Pia at 1.9 Å resolution revealed a binding interface different from those formed with AVR–Pik effectors, suggesting plasticity in integrated domain-effector interactions. The results of our work indicate that a single NLR immune receptor can bait multiple pathogen effectors via an integrated domain, insights that may enable engineering plant immune receptors with extended disease resistance profiles.
Highlights
Unconventional integrated domains in plant intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NLRs) type can directly bind translocated effector proteins from pathogens and thereby initiate an immune response
Using plant cell death and pathogenicity assays and protein–protein interaction analyses, we show that the rice NLR pair Pikp-1/Pikp-2 triggers an immune response leading to partial disease resistance toward the “mis-matched” effector AVR–Pia in planta and that the Pikp–heavy metal-associated (HMA) domain binds AVR–Pia in vitro
The discovery that rice blast pathogen effectors with a common structural fold can be recognized by the same type of integrated domain in rice NLRs raises questions about specificity, and possible plasticity, of recognition
Summary
Rice plants expressing Pikp are partially resistant to M. oryzae expressing AVR–Pia. We used a spot-inoculation assay to infect rice cultivars with a pathogen strain (Sasa2) transformed to express different. To confirm that each protein was expressed, Western blot analysis of extracted leaf tissue was used to assess protein accumulation (Fig. 2A) These results show that the Pikp NLRs can respond to AVR–Pia, the response was limited compared with their “matched” effector AVR–PikD. There was a weak response to the AVR–PikDH46E negative control, as observed previously, due to differences in the AVR–PikD His-46 interface with Pikm–HMA compared with Pikp–HMA [12]. This suggests that the weak cell death response to AVR–Pia is specific for the Pikp allele. These limited intermolecular interactions and small interface area provide an
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