RPW8/HR repeats control NLR activation in Arabidopsis thaliana

Abstract

In many plant species, conflicts between divergent elements of the immune system, especially nucleotide-binding oligomerization domain-like receptors (NLR), can lead to hybrid necrosis. Here, we report deleterious allele-specific interactions between an NLR and a non-NLR gene cluster, resulting in not one, but multiple hybrid necrosis cases in Arabidopsis thaliana. The NLR cluster is RESISTANCE TO PERONOSPORA PARASITICA 7 (RPP7), which can confer strain-specific resistance to oomycetes. The non-NLR cluster is RESISTANCE TO POWDERY MILDEW 8 (RPW8) / HOMOLOG OF RPW8 (HR), which can confer broad-spectrum resistance to both fungi and oomycetes. RPW8/HR proteins contain at the N-terminus a potential transmembrane domain, followed by a specific coiled-coil (CC) domain that is similar to a domain found in pore-forming toxins MLKL and HET-S from mammals and fungi. C-terminal to the CC domain is a variable number of 21- or 14-amino acid repeats, reminiscent of regulatory 21-amino acid repeats in fungal HET-S. The number of repeats in different RPW8/HR proteins along with the sequence of a short C-terminal tail predicts their ability to activate immunity in combination with specific RPP7 partners. Whether a larger or smaller number of repeats is more dangerous depends on the specific RPW8/HR autoimmune risk variant.

Fig 1. DM6–DM7 hybrid necrosis cases.

(A) Morphological variation in three independent DM6–DM7 hybrid necrosis cases. (B) Red lines indicate necrosis in F₁ hybrids, grey indicates normal progeny. (C, D) Variation in morphology in two DM6–DM7 cases sharing the same DM6 allele in Lerik1-3. (C) Entire rosettes of four-week-old plants. (D) Abaxial sides of eighth leaves of six-week-old plants. Inset shows Trypan Blue stained leaf of Lerik1-3 x Fei-0 F₁. (E) Summary phenotypes in crosses of Lerik1-3 to 80 other accessions. Red is strong necrosis in F₁, and yellow is mild necrosis in F₁ or necrosis only observable in F₂. Scale bars indicate 1 cm.

Fig 2. Mapping of two DM6 (RPP7 cluster)–DM7 (RPW8/HR cluster) hybrid necrosis cases.

(A) QTL analyses. The QTL on chromosome 1 includes RPP7 from Lerik1-3 and ICE79 (21.37–22.07 and 21.50–21.98 Mb), and the QTL on chromosome 3 RPW8/HR from ICE106/ICE107 and Don-0 (18.59–19.09 Mb, 18.61–19.06 Mb). The horizontal lines indicate 0.05 significance thresholds established after 1,000 permutations. (B) Heat map for two-dimensional, two-QTL model genome scans. Upper left triangles indicate epistasis scores (LODi) and lower right triangles joint two-locus scores (LODf). Scales for LODi on left and for LODf on right. (C) Manhattan plot for a GWAS of necrosis in hybrid progeny of Lerik1-3 crossed to 80 other accessions (see S2 Table). The hit in the RPW8/HR region (red arrow) stands out, but it is possible that some of the other hits that pass the significance threshold (Bonferroni correction, 5% familywise error) identify modifiers of the DM6–DM7 interaction.

Fig 3. Confirmation of causal genes in RPW8/HR cluster.

(A) Rescue of hybrid necrosis in Lerik1-3 x Fei-0 F₁ plants with an amiRNA against HR4. Fei-0 parents were T₁ transformants. PCR genotyping of numbered plants from left shown on the right. Only plant 1, which does not carry the amiRNA, is necrotic and dwarfed. (B) Rescue of hybrid necrosis in Mrk-0 x KZ10 F₁ plants by CRISPR/Cas9-targeted mutagenesis on RPW8.1^KZ10. (C) Recapitulation of hybrid necrosis in Lerik1-3 T₁ plants transformed with indicated genomic fragments from Fei-0 and ICE106. Representative phenotypes on right. Numbers of T₁ plants examined given on top. (D) Summary of rescue and recapitulation experiments. Asterisks refer to published experiments [10]. Scale bars indicate 1 cm.

Fig 4. Structural variation of the RPW8/HR cluster.

(A) The RPW8/HR cluster in different accessions. The extreme degree of recent duplications in TueWa1-2, with the same HR4 hybrid necrosis risk allele as Fei-0, did not allow for closure of the assembly from PCR products; assembly gaps are indicated. Color coding of HR4 alleles according to Fig 6. HR4 and RPW8.1 form a distinct clade from other RPW8s. Tagging SNPs found in GWAS marked in TueWa1-2 RPW8/HR cluster as black vertical lines. (B) Maximum likelihood tree of RPW8/HR genes from three A. thaliana accessions and the A. lyrata and B. rapa reference genomes. Branch lengths in nucleotide substitutions are indicated. Bootstrap values (out of 100) are indicated on each branch.

Fig 5. Necrosis-inducing activity of RPW8.1 and HR4 chimeras.

N-terminal portions indicated with the initial of the accession in italics (“K”, “M”, etc.), complete repeats indicated with regular capital letters (“A”, “B”, etc.), the partial repeat in KZ10 with a lowercase letter (“c”), and the C-terminal tails with Greek letters (“α”, “β”, etc.). Non-repeat portions are semi-transparent. Repeats with identical amino acid sequences have the same letter designation. Numbers indicate amino acid positions. Constructs on the left, and distribution across phenotypic classes in T₁ transformants on the right, with n given on top of each column. Natural alleles labeled in color and bold. RPW8/HR repeats indicated as light grey boxes. (A) RPW8.1 chimeras, driven by the RPW8.1^KZ10 promoter, were introduced into Mrk-0, which carries the corresponding incompatible RPP7-like allele. (B) HR4 chimeras, driven by the HR4^Fei-0 promoter, were introduced into Lerik1-3, which carries the corresponding incompatible RPP7-like allele. Scale bars indicate 1 cm.

Fig 6. Sequence variation of a large collection of RPW8.1 and HR4 alleles.

(A) Repeat polymorphisms in RPW8.1 and HR4 proteins (grey background). N-terminal regions and tails are semi-transparent. (B) Distribution of HR4 types across 113 Sanger sequenced alleles (see S5 Table). (C) Distribution of HR4 allele types in Eurasia and North America. (D) Haplotype network of HR4 alleles, with a 1-bp minimum difference. (E) F₁ progeny of Mrk-0 crossed to accessions with different RPW8.1 alleles. Short RPW8.1 variants do not induce hybrid necrosis. (F) F₁ progeny of Lerik1-3 crossed to accessions with different HR4 alleles. The shortest HR4 alleles (red) cause strong hybrid necrosis, the second shortest HR4 alleles (yellow) cause mild hybrid necrosis. (G) Rosette growth of F₁ progeny from Lerik1-3 and accessions carrying different HR4 alleles. The shortest HR4 allele causes a strong growth reduction, while the second-shortest HR4 allele has a milder effect. Scale bars indicate 1 cm.