NLR Mutations Suppressing Immune Hybrid Incompatibility and Their Effects on Disease Resistance

Abstract

Genetic divergence between populations can lead to reproductive isolation. Hybrid incompatibilities (HI) represent intermediate points along a continuum toward speciation. In plants, genetic variation in disease resistance (R) genes underlies several cases of HI. The progeny of a cross between Arabidopsis (Arabidopsis thaliana) accessions Landsberg erecta (Ler, Poland) and Kashmir2 (Kas2, central Asia) exhibits immune-related HI. This incompatibility is due to a genetic interaction between a cluster of eight TNL (TOLL/INTERLEUKIN1 RECEPTOR-NUCLEOTIDE BINDING-LEU RICH REPEAT) RPP1 (RECOGNITION OF PERONOSPORA PARASITICA1)-like genes (R1-R8) from Ler and central Asian alleles of a Strubbelig-family receptor-like kinase (SRF3) from Kas2. In characterizing mutants altered in Ler/Kas2 HI, we mapped multiple mutations to the RPP1-like Ler locus. Analysis of these suppressor of Ler/Kas2 incompatibility (sulki) mutants reveals complex, additive and epistatic interactions underlying RPP1-like Ler locus activity. The effects of these mutations were measured on basal defense, global gene expression, primary metabolism, and disease resistance to a local Hyaloperonospora arabidopsidis isolate (Hpa Gw) collected from Gorzów (Gw), where the Landsberg accession originated. Gene expression sectors and metabolic hallmarks identified for HI are both dependent and independent of RPP1-like Ler members. We establish that mutations suppressing immune-related Ler/Kas2 HI do not compromise resistance to Hpa Gw. QTL mapping analysis of Hpa Gw resistance point to RPP7 as the causal locus. This work provides insight into the complex genetic architecture of the RPP1-like Ler locus and immune-related HI in Arabidopsis and into the contributions of RPP1-like genes to HI and defense.

Figure 1.

sulki1 mutations mapping to RPP1-like R8 Ler. A, Schematic representation of nonsynonymous substitutions identified in sulki1 mutants. Exon/intron organization and Toll-IL receptor (TIR), nucleotide binding (NB), and Leu-rich repeat (LRR) domains are shown. B, Detailed representation of RPP1-like R8 Ler amino acid sequence, conserved motifs (Meyers et al., 2003), and position of sulki1 mutations.

Figure 2.

Composite image of sulki phenotypes. Five-week-old sulki1, sulki2, and Ler/Kas2 NIL grown at 14°C to 16°C under 12-h-light/12-h-dark cycles and light intensity of 120 µmol m⁻² s⁻¹.

Figure 3.

Expression of SA and oxidative stress marker genes. Quantitative reverse transcription PCR (RT-qPCR) analyses of PR1, EDS1, GST1, RPP1-like Ler R2, R3, R4, and R8 genes in sulki1-1 (s1-1) to sulki 1-10 (s1-10), sulki2-1 (s2-1), Ler, Kas2, Ler/Kas2 NIL, and NIL complemented with SRF3 Ler (cNIL; Alcázar et al., 2010). Values are relative to Ler and are the mean of three biological replicates, each with three technical replicates. Letters indicate values that are significantly different according to Student-Newman-Keuls test at P < 0.05. Error bars indicate sd.

Figure 4.

Growth phenotypes and expression analyses of Cas9 RPP1-like Ler mutants. A, Composite image of 5-week-old Cas9-r2-1, Cas9-r3-1, Cas9-r4-1, Cas9-r8-1 mutants in the Ler/Kas2 NIL background, Ler/Kas2 NIL, and parental lines (Ler and Kas2) grown at 14°C to16°C. The position of stop codons in TIR (T) or NB (N) domains of RPP1-like genes is marked with an asterisk. B, Gene expression analyses of PR1, EDS1, GST1, RPP1-like Ler R2, R3, R4, and R8 in Cas9 r2-1, r2-2, r3-1, r3-2, r4-1, r4-2, r8-1, and r8-2 mutant alleles Ler, Kas2, Ler/Kas2 NIL, and cNIL plants grown at 14°C to 16°C during 5 weeks. Analyses were performed as described in Figure 3.

Figure 5.

Growth of Pst DC3000 and hrcC mutant, 3 d after spray inoculation of sulki1-1 (s1-1), sulki1-7 (s1-7), sulki1-8 (s1-8), sulki1-9 (s1-9), sulki2-1 (s2-1), Cas9- r2-1, r3-1, r4-1, and r8-1 mutants in the Ler/Kas2 NIL background, Ler, Kas2, Ler/Kas2 NIL, and eds1-2 Ler plants grown at 20°C to 22°C (A) or 14°C to 16°C (B). Different letters indicate significant differences (P < 0.01) in a Student-Newman-Keuls test. Error bars indicate sd.

Figure 6.

Venn diagram of genes differentially expressed in the comparisons between (Kas2 versus Ler/Kas2 NIL) and (sulki1-8 versus Ler/Kas2 NIL). Lists of genes and Gene Ontology analyses are included in Supplemental Tables S2-1 to S2-3.

Figure 7.

Principal component analysis (A) and HCA (B) with Pearson’s correlation and average linkage of samples and metabolites from 5-week-old sulki1-1 (s1-1), sulki1-7 (s1-7), sulki1-8 (s1-8), sulki1-9 (s1-9), Ler, Kas2, and Ler/Kas2 NIL plants grown at 14°C to16°C. C, Log₂-normalized responses for some metabolites determined by GC/MS in the above genotypes, and schematic representation of their metabolic pathways. Different letters indicate significant differences (P < 0.01) in a Student-Newman-Keuls test. Error bars indicate sd. A complete list of analyzed metabolites is provided in Supplemental Table S3.

Figure 8.

Transient expression assays in tobacco. A, Transient expression of genomic versions of 35s: RPP1-like Ler R2, R3, R4, R8, and ATR1-δ51 Gw, tagged with C terminus YFP. B, Coinfiltration of RPP1-like Ler R2, R3, R4, and R8 with ATR1-δ51 Gw. Pictures in A and B were taken 48 h after infiltration. Samples for western-blot analyses in A and B were collected 24 h after infiltration. No symptoms of cell death were observed at later time points of coinfiltration in B.

Figure 9.

QTL and GWAS mapping. A, QTL mapping of disease resistance to Hpa isolate Gw in the Ler/Sha RIL population (Clerkx et al., 2004; see Supplemental Table S6). The position of RPP7 on chromosome 1 is indicated. B, Manhattan plot of GWAS mapping for disease resistance to Hpa Gw in 288 accessions (see Supplemental Table S8). The list of most significant gene associations is shown in Supplemental Table S9.