Intracellular nucleotide-binding domain and leucine-rich repeat-containing (NLR) receptors play crucial roles in immunity across multiple domains of life. In plants, a subset of NLRs contain noncanonical integrated domains that are thought to have evolved from host targets of pathogen effectors to serve as pathogen baits. However, the functions of host proteins with similarity to NLR integrated domains and the extent to which they are targeted by pathogen effectors remain largely unknown. Here, we show that the blast fungus effector AVR-Pik binds a subset of related rice proteins containing a heavy metal-associated (HMA) domain, one of the domains that has repeatedly integrated into plant NLR immune receptors. We find that AVR-Pik binding stabilizes the rice small HMA (sHMA) proteins OsHIPP19 and OsHIPP20. Knockout of OsHIPP20 causes enhanced disease resistance towards the blast pathogen, indicating that OsHIPP20 is a susceptibility gene (S-gene). We propose that AVR-Pik has evolved to bind HMA domain proteins and co-opt their function to suppress immunity. Yet this binding carries a trade-off, it triggers immunity in plants carrying NLR receptors with integrated HMA domains.

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.

Putative transposable elements in the Sclerospora graminicola genome sequence. (XLSX 51 kb)

Gene IDs for nitrogen and sulfur assimilation enzymes in Sclerospora graminicola and related oomycetes. (XLSX 52 kb)

BACKGROUND:Downy mildew, caused by the oomycete pathogen Sclerospora graminicola, is an economically important disease of Gramineae crops including foxtail millet (Setaria italica). Plants infected with S. graminicola are generally stunted and often undergo a transformation of flower organs into leaves (phyllody or witches' broom), resulting in serious yield loss. To establish the molecular basis of downy mildew disease in foxtail millet, we carried out whole-genome sequencing and an RNA-seq analysis of S. graminicola.RESULTS:Sequence reads were generated from S. graminicola using an Illumina sequencing platform and assembled de novo into a draft genome sequence comprising approximately 360 Mbp. Of this sequence, 73% comprised repetitive elements, and a total of 16,736 genes were predicted from the RNA-seq data. The predicted genes included those encoding effector-like proteins with high sequence similarity to those previously identified in other oomycete pathogens. Genes encoding jacalin-like lectin-domain-containing secreted proteins were enriched in S. graminicola compared to other oomycetes. Of a total of 1220 genes encoding putative secreted proteins, 91 significantly changed their expression levels during the infection of plant tissues compared to the sporangia and zoospore stages of the S. graminicola lifecycle.CONCLUSIONS:We established the draft genome sequence of a downy mildew pathogen that infects Gramineae plants. Based on this sequence and our transcriptome analysis, we generated a catalog of in planta-induced candidate effector genes, providing a solid foundation from which to identify the effectors causing phyllody.