A genetically linked pair of NLR immune receptors shows contrasting patterns of evolution

Abstract

Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal-associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained transspecies in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to respond to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs.

Keywords: evolution; integrated domains; nucleotide-binding leucine-rich-repeat receptors (NLRs); paired NLR; rice.

Fig. 1.

Pias gene of rice line WRC17 (cultivar Keiboba) encodes a CC-NLR protein and is allelic to Pia. (A) Segregation of the resistance and susceptibility traits among the 58 RILs derived from a cross between WRC17 (cultivar Keiboba) and Hitomebore. Disease symptoms of WRC17, Hitomebore, and six RILs showing a susceptible phenotype after punch inoculation of M. oryzae isolate 2012-1 (leaf photographs) and the frequency distribution of disease lesion areas of the 58 RILs (bar graphs). (B) Linkage maps of candidate NLR genes at the Pi-W17-1 and Pi-W17-2 loci. (C) Both CNL-04 and CNL-05 are required for Pi-W17-1–mediated resistance against M. oryzae 2012-1. HW-RIL7 contains only Pi-W17-1 and is resistant to M. oryzae 2012-1. Knockout of CNL-04 (cnl-04) and CNL-05 (cnl-05) in HW-RIL7 rendered plants susceptible to 2012-1. (D) Gene structures of Pia consisting of RGA4 and RGA5 and Pias consisting of Pias-1 and Pias-2. The positions of protein domains (CC, NB-APAF-1, R-proteins and CED-4 (NB-ARC), LRR, HMA, and DUF761) encoded by the NLRs are indicated. (E) The M. oryzae 2012-1 AVR-Pias knockout mutant became virulent to HW-RIL7. (F) Amino acid sequence of G9532 protein (AVR-Pias). The secretion signal is indicated by red letters and the Toxin18-like motif is indicated by blue letters. The Toxin18-like motif was annotated by Pfam (https://pfam.xfam.org/).

Fig. 2.

Recurrent integration of extraneous domains in Pias/Pia sensor NLRs. (A) A simplified scheme of the structures of the Pias/Pia NLR pairs. Pias-1/RGA4 helper NLRs are shown in green, and Pias-2/RGA5 sensor NLRs are shown in white. The conserved junction sequences are indicated by gray shading. Fragments containing the IDs are shown by different-colored hexagons. (B) A sequence logo showing conserved amino acids of the junction motif. The red lines indicate LRRs. (C) Distribution of ID motifs among Oryza species. The pie charts show the frequencies of different ID motifs in a given species. The colors correspond to the ID colors in A. The numbers below the pie charts indicate the sample numbers. A cladogram showing the phylogenetic relationships of 11 Oryza species and 4 other Poaceae species (S. italica, P. hallii, H. vulgare, and A. tauschii) based on TimeTree, the Timescale of Life web database (www.timetree.org/). The numbers on the branches indicate the estimated time of the splitting of lineages (MYA, million y ago). (D) DNA sequence similarity between the O. punctata Pias-2/RGA5 sensor NLR and the downstream sequence of the O. sativa (Nipponbare) Pias-2/RGA5. (E) Dot-plot analysis of the O. punctata (W1582) Pias-2/RGA5 sensor NLR and O. sativa (Nipponbare) Pias-2/RGA5 NLR downstream sequences using the Dotmatcher tool (emboss.bioinformatics.nl/cgi-bin/emboss/dotmatcher/). (F) Possible evolutionary process of ID replacement that might have occurred between the O. punctata and the O. sativa Pias-2/RGA5 lineages. We still do not know the mode of interaction between the AVR-Pias effector and DUF761-containing protein, so it is indicated by “?.”.

Fig. 3.

Contrasting evolutionary patterns of the helper and sensor NLRs of the Pias/Pia locus. (A) Phylogenetic tree of the Pias-1/RGA4 helper-NLR gene (Left) and Pias-2/RGA5 sensor-NLR (Right) gene based on the full-length amino acid sequence of Pias-1/RGA4 and the sequence in the region CC to the junction region for Pias-2/RGA5. Pias-2/RGA5 sensor NLRs form two major clades (C1 and C2). The numbers indicate bootstrap values. (B) Nucleotide diversity (π) of the CC, NBS, and LRR(-junction region) domains of the Pias-1/RGA4 helper-NLR gene and Pias-2/RGA5 sensor-NLR gene in 22 Oryza samples. (C) Tajima’s D of the CC, NBS, and LRR(-junction region) domains of the Pias-1/RGA4 helper-NLR gene and Pias-2/RGA5 sensor-NLR gene in 22 Oryza samples. (D) Pairwise d_N and d_S values of the CC-NBS-LRR(-junction region), CC, NBS, and LRR domains of the Pias-1/RGA4 helper-NLR gene and Pias-2/RGA5 sensor-NLR gene in 22 Oryza samples.

Fig. 4.

NLR-helper Pias-1 is functionally conserved. (A) Representative images of N. benthamiana leaves after agroinfiltration with Pias-1:HA, Pias-1:HA/FLAG:RGA5, FLAG:RGA5/AVR-Pia, and Pias-1:HA/FLAG:RGA5/AVR-Pia (Left) and RGA4-Ogr:HA derived from O. granulata, RGA4-Ogr:HA/FLAG:RGA5, FLAG:RGA5/AVR-Pia, and RGA4-Ogr:HA/FLAG:RGA5/AVR-Pia (Right). Autofluorescence under UV light is shown. (B) Pias-1 cooperates with RGA5 to recognize AVR-Pia and induces resistance in rice. The rice line HW-RIL7 with Pias (Pias-1 and Pias-2) recognizes the Ao-92-06-2 strain with AVR-Pias (Ao92-06-2+pex22p:AVR-Pias) and induces resistance. However, HW-RIL7 cannot recognize the Ao02-06-2 strain with AVR-Pia (Ao92-06-2+pex22p:AVR-Pia). Two lines (lines 1 and 2) contain the 35S-RGA5 transgene in the HW-RIL7 background. F1-A is a progeny derived from a cross between Sasanishiki with Pia (RGA4 and RGA5) and HW-RIL7. F1-B is a progeny derived from a cross between a Sasanishiki mutant (Sas1493) with pia (rga4 and RGA5) and HW-RIL7. "R" and "S" indicate resistance and susceptibility, respectively.