The Piks allele of the NLR immune receptor Pik breaks the recognition of AvrPik effectors of the rice blast fungus

Abstract

Arms race co-evolution of plant-pathogen interactions evolved sophisticated recognition mechanisms between host immune receptors and pathogen effectors. Different allelic haplotypes of an immune receptor in host mount distinct recognition against sequence or non-sequence related effectors in pathogens. We report the molecular characterization of the Piks allele of the rice immune receptor Pik against rice blast pathogen, which requires two head-to-head arrayed nucleotide binding site and leucine-rich repeat proteins. Like other Pik genes, both Piks-1 and Piks-2 are necessary and sufficient for Piks-mediated resistance. However, unlike other Pik alleles, Piks does not recognize any known AvrPik variants of M. oryzae. Sequence analysis of the genome of an avirulent isolate V86010 further revealed that its cognate avirulence (Avr) gene most likely has no significant sequence similarity to known AvrPik variants. We conclude that Piks breaks the canonical Pik/AvrPik recognition pattern. Piks-1 and Pikm-1 have only two amino acid differences within the integrated heavy metal-associated (HMA) domain. Pikm-1-HMA interact with AvrPik-A, -D and -E in vitro and in vivo, whereas Piks-1-HMA does not bind any AvrPik variants. Reciprocal exchanges of single amino acid residues between Piks-1 and Pikm-1 further reveal a dynamic recognition mechanism between Piks/Pikm alleles and their respective effectors. Piks-1E229Q/Pikm-1V261A can only activate immunity to AvrPik-D but not to other effectors, indicating that the amino acid change of E to Q at position 229 leads to its gain of a partial recognition spectrum of Pikm. By contrast, Piks-1A261V/Pikm-1Q229E confers immunity to the Piks cognate effector, indicating that the amino acid change of Q to E at position 229 leads to its shifts of the recognition from Pikm to Piks. Intriguingly, binding activities in both Y2H and analytical gel filtration assays are illustrated between Piks-1A261V/Pikm-1Q229E and AvrPik-D. However, it is unable to mount immunity against AvrPik-D, suggesting that biochemical activities based on in vitro and in vivo assays could be insufficient for sustaining biological function of receptor and effector pairs.

Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector

“…The yeast two-hybrid assay showed that both Piks E229Q -HMA and Piks A261V -HMA as bait bound AVR-PikD as prey to similar levels compared to Pikm-HMA binding with AVR-PikD ( Figs 9A and S18 ). This result is consistent with the result of yeast two-hybrid assay in a recent study [ 117 ]. On the other hand, we found that Piks E229Q -HMA and Piks A261V -HMA as prey weakly bound AVR-PikD as bait, compared to Pikm-HMA binding with AVR-PikD ( Figs 9B and S19 ).…”

“…study [117]. On the other hand, we found that Piks E229Q -HMA and Piks A261V -HMA as prey weakly bound AVR-PikD as bait, compared to Pikm-HMA binding with AVR-PikD (Figs 9B and S19).…”

“…These results support our findings in HR cell death assay showing that both E229Q and A261V quantitatively affect the response to AVR-PikD but not to AVR-Mgk1 ( Fig 8 ). A recent study independently showed the quantitative binding affinity of Piks-HMA mutants (Pikm-HMA > Piks A261V -HMA > Piks E229Q -HMA > Piks-HMA) to AVR-PikD by analytical gel filtration [ 117 ]. Overall, both Q229 and V261 residues of the HMA domain of Pikm are essential for the full binding to AVR-PikD ( Figs 8 and 9 ).…”