The allelic rice immune receptor Pikh confers extended resistance to strains of the blast fungus through a single polymorphism in the effector binding interface

Abstract

Arms race co-evolution drives rapid adaptive changes in pathogens and in the immune systems of their hosts. Plant intracellular NLR immune receptors detect effectors delivered by pathogens to promote susceptibility, activating an immune response that halts colonization. As a consequence, pathogen effectors evolve to escape immune recognition and are highly variable. In turn, NLR receptors are one of the most diverse protein families in plants, and this variability underpins differential recognition of effector variants. The molecular mechanisms underlying natural variation in effector recognition by NLRs are starting to be elucidated. The rice NLR pair Pik-1/Pik-2 recognizes AVR-Pik effectors from the blast fungus Magnaporthe oryzae, triggering immune responses that limit rice blast infection. Allelic variation in a heavy metal associated (HMA) domain integrated in the receptor Pik-1 confers differential binding to AVR-Pik variants, determining resistance specificity. Previous mechanistic studies uncovered how a Pik allele, Pikm, has extended recognition to effector variants through a specialized HMA/AVR-Pik binding interface. Here, we reveal the mechanistic basis of extended recognition specificity conferred by another Pik allele, Pikh. A single residue in Pikh-HMA increases binding to AVR-Pik variants, leading to an extended effector response in planta. The crystal structure of Pikh-HMA in complex with an AVR-Pik variant confirmed that Pikh and Pikm use a similar molecular mechanism to extend their pathogen recognition profile. This study shows how different NLR receptor alleles functionally converge to extend recognition specificity to pathogen effectors.

Two NLR immune receptors acquired high-affinity binding to a fungal effector through convergent evolution of their integrated domain

“…This NLR pair was initially cloned from Tsuyuake rice (Ashikawa et al, 2008), and has since been shown to occur in allelic variants, which include Pikp, Pikm, Piks, Pikh, and Pik* (Costanzo and Jia, 2010;Jia et al, 2009;Wang et al, 2009;Zhai et al, 2011). Remarkably, the integrated HMA domain is the most sequence-diverse region among Pik-1 variants, consistent with the view that the receptor is under selection imposed by AVR-Pik (Białas et al, 2018;Costanzo and Jia, 2010;De la Concepcion et al, 2020;Zhai et al, 2014). Conversely, AVR-Pik alleles carry only five amino acid replacements, all of which map to regions located at the HMA-binding interface, indicating the adaptive nature of those polymorphisms (Longya et al, 2019).…”

“…figure 33A) (De la Concepcion et al, 2018;Maqbool et al, 2015). We note that the MKANK/EMVKE and IAQVV/LVKIE regions map to two of the three interaction interfaces previously described to underpin binding of AVR-PikD, and other AVR-Pik variants, to Pik-HMAs (De la Concepcion et al, 2020, 2019.…”

“…This revealed that the integration site most likely falls between the KLL and KTV residues (corresponding to residues 161-163 and 284-286 of Pikp-1); however, the exact boundaries of the integration might be slightly different, given the relatively high sequence divergence around this site among the more distantly related orthologues. We further noted that the integration site encompasses a wider region than that of functionally characterised HMA domains (De la Concepcion et al, 2020, with around 20 additional amino acids (23 and 21 in Pikp-1) on each side of the annotated HMA domain. The ML phylogenetic trees of Pik-1 (left) and Pik-2 (right) orthologues.…”

“…As high-affinity binding to the effector is required for the Pik-mediated immune response (De la Concepcion et al, 2020, 2019Maqbool et al, 2015), we hypothesised that the HMA domain of Pikp-1 (Pikp-HMA) evolved towards high-affinity binding to the AVR-PikD effector. To test this hypothesis, we resurrected the six ancHMA variants determined above by synthesising their predicted sequences and incorporating them into the Pikp-1 receptor, generating Pikp-1:I-N2, Pikp-1:I-N6, Pikp-1:II-N11, Pikp-1:II-N12, Pikp-1:III-N11, and Pikp-1:III-N12 fusions ( Figure 3A).…”

“…While the most ancient of the AVR-Pik allelic variants, AVR-PikD, is recognised by a wide range of Pik-1 proteins, the most recent variants, AVR-PikC and AVR-PikF, evade recognition by all known Pik-1 variants (Kanzaki et al, 2012;Longya et al, 2019). These recognition specificities are thought to reflect the ongoing arms race between rice and the rice blast fungus (Białas et al, 2018;Kanzaki et al, 2012;Li et al, 2019) and have been linked to the effector-HMA binding affinity (De la Concepcion et al, 2020Maqbool et al, 2015). Despite the wealth of knowledge about mechanisms governing effector recognition by the Pik-1-integrated HMA domain, we know little about its evolutionary history.…”