Barley MLA3 recognizes the host-specificity effector Pwl2 from Magnaporthe oryzae

Abstract

Plant nucleotide-binding leucine-rich repeat (NLRs) immune receptors directly or indirectly recognize pathogen-secreted effector molecules to initiate plant defense. Recognition of multiple pathogens by a single NLR is rare and usually occurs via monitoring for changes to host proteins; few characterized NLRs have been shown to recognize multiple effectors. The barley (Hordeum vulgare) NLR gene Mildew locus a (Mla) has undergone functional diversification, and the proteins encoded by different Mla alleles recognize host-adapted isolates of barley powdery mildew (Blumeria graminis f. sp. hordei [Bgh]). Here, we show that Mla3 also confers resistance to the rice blast fungus Magnaporthe oryzae in a dosage-dependent manner. Using a forward genetic screen, we discovered that the recognized effector from M. oryzae is Pathogenicity toward Weeping Lovegrass 2 (Pwl2), a host range determinant factor that prevents M. oryzae from infecting weeping lovegrass (Eragrostis curvula). Mla3 has therefore convergently evolved the capacity to recognize effectors from diverse pathogens.

Figure 1.

Rmo1 is in complete coupling with Mla3. A) The distal end of the short arm of chromosome 1H based on nonredundant KASP markers in the Baronesse × BCD47 population (2,304 gametes). RE, number of recombination events between markers. Twenty additional markers (not shown) are in complete genetic coupling with K_Mla_RGH1_2920 at the Mla locus. B) Phenotype by genotype plot of homozygous F_2:4 recombinants inoculated with M. oryzae isolate KEN54-20 using a spot-based inoculation. Scores 0 and 1 = resistant and 2 to 4 = susceptible. C) Phenotype by genotype plot of homozygous F_2:4 recombinants inoculated with M. oryzae isolate KEN54-20 using a spray-based inoculation. Scores 0 and 1 = resistant and 2 to 4 = susceptible.

Figure 2.

The Mla locus in Baronesse is highly divergent in sequence and structure. A) Sequence alignment of the region encompassing Mla in barley accessions Morex (mla) and Baronesse (Mla3) found high conservation in the flanking intervals (left and right boxes) but no conservation within the Mla locus. The central region of Mla region is a breakpoint in the assembly of chromosome 1H from barley accession Baronesse. RGH1, RGH2, and RGH3 family members are indicated in orange, blue, and green, respectively. KASP and CAPS markers are indicated with “K_” and “C_” prefixes, respectively. B) RenSeq-PacBio identified genomic contigs encompassing Mla3 (15.3 kb) and RGH2/RGH3 (14.0 kb). In the barley accession Baronesse haplotype, RGH2 and RGH3 are in head-to-head orientation. C) In the barley accession Baronesse haplotype, Mla3 and RGH3 encode CC-NB-LRR, whereas RGH2 encodes an CC-NB-LRR with integrated Exo70F1.

Figure 3.

k-mer analysis identifies 4 copies of Mla3 in barley accession Baronesse. A)k-mer coverage of Bpm. Bpm is located proximal to the Mla locus and exists as a single copy with coverage centered at 175. B)k-mer coverage of Mla3. Two coverage bands are observed at 619 and 790 coverage, corresponding to 4 and 5 copies. Reduced coverage in the second intron is due to the presence of low complexity sequence (dinucleotide repeat). C)k-mer coverage of RGH2 and RGH3. Two coverage bands are observed at 162 and 315 coverage, corresponding to 1 and 2 copies, respectively. The additional copies represent fragments of RGH2 and RGH3, which are located in the distal and proximal boundaries of the Mla locus (Fig. 2A). Zero k-mer coverage represents inaccurate sequence calls from RenSeq-PacBio sequencing in contigs encompassing Mla3 and RGH2/RGH3. Gene models are shown below each plot with exons and introns shown as arrows and lines, respectively.

Figure 4.

Coupling of Mla3 and Rmo1 in diverse barley accessions. Disease phenotypes of barley accessions carrying different alleles of the candidate genes RGH1, RGH2, and RGH3 inoculated with Bgh isolate CC148 and M. oryzae isolate KEN54-20. The haplotypes of the barley accessions are listed on the left-hand side with the allele of RGH1, RGH2, and RGH3 indicated. ND, gene was not detected in RNA-seq data.

Figure 5.

Mla3 confers resistance to Bgh isolate CC148 and M. oryzae isolate KEN54-20. A) Transgenic lines of Mla3 (T₁-4), Mla3Δ6 (T₁-7), and RGH2/RGH3 (T₁-9) inoculated with Bgh isolate CC148 carrying AVR_a3 and transgenic lines of Mla3 (T₁-4), Mla3Δ6 (T₁-3 T₂), and RGH2/RGH3 (T₁-5) M. oryzae isolate KEN54-20. Controls include resistant wild-type Baronesse and susceptible wild-type Golden Promise used for transformation. Complete resistance shown to Bgh and M. oryzae by Baronesse and transgenic line Golden Promise + Mla3, whereas wild-type Golden Promise and transgenic lines Golden Promise + Mla3Δ6 and +RGH2/RGH3 are susceptible. Phenotypes are representative of inoculated T₁ families. B) Two independent T₁ families of Golden Promise + Mla3 (T₁-4 and T₁-5) showing resistance with varying copy numbers (0 to 6) inoculated with M. oryzae isolate KEN54-20. Phenotypic scores 0 and 1 = resistant and 2 to 4 = susceptible. The plot shows the total number of individual lines from merged T₁ families with insert copy number and phenotypic scores based on circle size. The smallest circles ≤5 individuals to large circles = 25 individuals).

Figure 6.

Mla3 confers isolate-specific resistance to M. oryzae. Transgenic Golden Promise + Mla3 (T₁-4 T₂) spot inoculated with M. oryzae isolates KEN54-20 (AVR-Rmo1) and Sasa2 (avr-Rmo1). Mla3 confers resistance to isolates carrying AVR-Rmo1. Controls Golden Promise and hypersusceptible Nigrate are susceptible to both Sasa2 and KEN54-20.

Figure 7.

MLA3 recognizes and interacts with Pwl2. A)PWL2 complements M. oryzae avr-Rmo1 mutants. Baronesse (Mla3) leaves spot inoculated with M. oryzae KEN54-20 (AVR-Rmo1) and avr-Rmo1 mutants M43 and M61. M. oryzae isolate KEN54-20 is avirulent on Baronesse, whereas avr-Rmo1 mutants M43 and M61 are virulent. Ectopic integration of PWL2 driven by native promoter (pPWL2:PWL2) complements the phenotype of mutants M43 and M61. Susceptible control, Nigrate. B)Mla3 recognizes PWL2 in transgenic Golden Promise + Mla3. Transgenic Golden Promise + Mla3 (T₁-4 T₂) spot inoculated with M. oryzae isolate KEN54-20 (AVR-Rmo1), avr-Rmo1 mutants M43 and M61, and avr-Rmo1 mutants M43 and M61 transformed with PWL2 driven by its native promoter (pPWL2:PWL2). Susceptible control, Golden Promise. C) Transient expression of MLA3 with Pwl2 in N. benthamiana triggers hypersensitive response. Representative N. benthamiana leaf infiltrated with A. tumefaciens strains carrying the corresponding binary expression constructs. AVR_a10 and empty vector (EV) were used as negative controls. Leaves were photographed 3 d after agroinfiltration. The experiment was independently repeated 3 times with 6 to 9 technical replicates. D, E) MLA3 interacts with Pwl2 in planta. Coimmunoprecipitation experiment of C-terminally 6xHA-tagged MLA3^L11E and MLA10^L11E with C-terminally 3xFLAG-tagged Pwl2 and AVR_a10. Proteins obtained by coimmunoprecipitation with α-HA beads D) or α-FLAG beads E) and total protein extracts (input) were separated by SDS–PAGE and detected by immunoblotting using the appropriate antibodies labeled on the left of each blot. Asterisks indicate protein bands of the corresponding expected size. The experiments were independently performed 3 times with similar results.

Figure 8.

Conserved recognition specificity of PWL2 in barley and weeping lovegrass. A) Weeping lovegrass spray infected with M. oryzae isolates with and without AVR-Rmo1 (PWL2). WT: M. oryzae isolate KEN54-20 (AVR-Rmo1), avr-Rmo1 (pwl2) mutants M43 and M61, and avr-Rmo1 (pwl2) mutants M43 and M61 transformed with PWL2 driven by its native promoter (pPWL2:PWL2). B) Baronesse (Mla3) spot inoculated with 17 M. oryzae isolates representing PWL2 natural variation. The isolates Naga69-150, Ina85-182, and Ina87T-56A carry both PWL2 and pwl2-2. Susceptible control, Nigrate. Phenotypes are representative of 3 biological replicates with 3 technical replicates.