Disruption of barley immunity to powdery mildew by an in-frame Lys-Leu deletion in the essential protein SGT1

Abstract

Barley (Hordeum vulgare L.) Mla (Mildew resistance locus a) and its nucleotide-binding, leucine-rich-repeat receptor (NLR) orthologs protect many cereal crops from diseases caused by fungal pathogens. However, large segments of the Mla pathway and its mechanisms remain unknown. To further characterize the molecular interactions required for NLR-based immunity, we used fast-neutron mutagenesis to screen for plants compromised in MLA-mediated response to the powdery mildew fungus, Blumeria graminis f. sp. hordei. One variant, m11526, contained a novel mutation, designated rar3 (required for Mla6 resistance3), that abolishes race-specific resistance conditioned by the Mla6, Mla7, and Mla12 alleles, but does not compromise immunity mediated by Mla1, Mla9, Mla10, and Mla13. This is analogous to, but unique from, the differential requirement of Mla alleles for the co-chaperone Rar1 (required for Mla12 resistance1). We used bulked-segregant-exome capture and fine mapping to delineate the causal mutation to an in-frame Lys-Leu deletion within the SGS domain of SGT1 (Suppressor of G-two allele of Skp1, Sgt1ΔKL308-309), the structural region that interacts with MLA proteins. In nature, mutations to Sgt1 usually cause lethal phenotypes, but here we pinpoint a unique modification that delineates its requirement for some disease resistances, while unaffecting others as well as normal cell processes. Moreover, the data indicate that the requirement of SGT1 for resistance signaling by NLRs can be delimited to single sites on the protein. Further study could distinguish the regions by which pathogen effectors and host proteins interact with SGT1, facilitating precise editing of effector incompatible variants.

Keywords: Blumeria graminis; SGT1; barley; in-frame deletion; leucine-rich-repeat receptor (NLR); nucleotide binding; resistance signaling.

Figure 1.

Barley line CI 16151 and its fast neutron-derived mutants. (A) Phenotypes of resistant mutants displaying cell death phenotypes, m9450, m9455, m9463, m9467, and m11542. The causal mutation for m11542 was previously cloned as rrp46 (Xi et al. 2009). (B) Phenotypes of CI 16151 and the mutants displaying sporulation phenotypes, m18982, m19029, m11526, and m19089. The causal mutation present in m18982 and m19089 were previously characterized as Mla6 (Halterman et al. 2001) and Bln1 (Meng et al. 2009), respectively. Gene names indicate the presence of normal (black) or mutated (red) alleles of either Mla6, Rar3, Bln1, or RRP46. Pictures show phenotypes at 7 DAI with Bgh isolate 5874 (AVR_a6), with their designated macroscopic phenotype and score for sporulation. An infection type of 0 is resistant (no sporulation), 1–2 is considered resistant, but with minor Bgh colonization, and an infection type of 3–4 is susceptible (abundant sporulation). 1n, few small necrotic flecks (0.5 mm); 1 – 2n, significant small necrotic flecks (1 mm); 2N, abundant cell death (>2 mm); c, limited chlorosis; C, abundant chlorosis. Earlier time points for m9450 and m9455 are in Supplementary Figure S2.

Figure 2.

Percentage elongating secondary hyphae (ESH) from Bgh isolate 5874 measured at six time points on CI 16151 and its derived mutant genotypes. (A) Microscopic images at 20× magnification of four time points (16, 24, 32, and 48 HAI) for each genotype. (B) Time-course graphs representing infection kinetics of Bgh on resistant and susceptible genotypes. The x-axis represents the time points at which measurements were taken (hours after inoculation, HAI) and the y-axis represents the percentage elongating secondary hyphae [which indicate successful colonization events (Ellingboe 1972)]. The percentage elongating secondary hyphae was calculated as 100× (sum of three hyphal indices/total). Total is sum of spore, appressorium, and the three hyphal indices. The dashed and solid lines represent the susceptible and resistant genotypes, respectively. Error bars indicate one standard deviation.

Figure 3.

F2 segregation results from crosses between m11526 (Mla6, rar3) and multiple near-isogenic or diverse Mla-carrying lines. As Bgh isolates with different AVR genes were used, the Bgh AVR genes relevant to a cross are indicated under “Relevant Bgh AVR genotype” and the AVR_a genotypes of each strain are displayed in the box below. Representative images for each plant infection phenotype are shown above the table. Raw percentages of F2 populations are displayed, and a chi-squared test was used to determine which of the potential segregation ratios was most suitable according to the lowest P-value. Ticks and X’s are used to show whether an Mla variant appears to, or not to, require the indicated gene, respectively. * P03 [Pallas background, Mla6 (Kolster et al. 1986)], m9472 (CI 16151-derived fast-neutron mutant, mla6), m100 [Sultan-5 background, Mla12, rar1 (Torp and Jørgensen 1986)]. Raw data are documented in Supplementary Table S6.

Figure 4.

Mapping of the Rar3 cosegregating region to chromosome 3H. (A) Results of the exome capture allele frequencies. Allele frequencies were determined relative to proportion of reads that had SNPs which mapped to the reference genome, out of the total number of reads. Allele frequencies were averaged over 5 Mb for the resistant and susceptible pools prior to plotting. Red and blue lines indicate allele frequencies for susceptible and resistant pools, respectively. (B) Physical map of chromosome 3H, Mb locations of genetic markers is indicated by black vertical lines with numbers. The orientation of the centromere is indicated by an arrow. The number of X’s represents the number of observed crossover events between the flanking markers. The position of Sgt1 is shown with a green dashed line at position 417.32 Mb. (C) Zoomed in location of closest flanking markers to Sgt1. (D) Alignment of wild-type and m11526 Sgt1 cDNA sequences covering the region containing the rar3-m11526 mutation. Protein alignment and structure included for reference, red dashed line indicates location of mutation.

Figure 5.

Rar3 is required for Mla-mediated H₂O₂ accumulation and the hypersensitive reaction (HR). (A) H₂O₂ accumulation evidenced by intense brown coloration produced by 3,3′-diaminobenzidine (DAB) staining in epidermal cells from CI 16151 (Mla6) at 24 HAI with Bgh isolate 5874 (AVR_a6). Bar, 25 μm. (B) Decreased accumulation of H₂O₂, as indicated by fewer, and less intense, DAB-stained (brown) cells, was observed in the m11526 loss-of-function mutant. (C) Consistent with ROS accumulation, whole-cell autofluorescence is observed in epidermal cells from wild-type CI 16151 at 24 HAI with Bgh isolate 5874 (AVR_a6). Bar, 25 μm. (D) Autofluorescence was absent in the m11526 loss-of-function mutant. (E) Transcript accumulation of Sgt1 (HORVU3Hr1G055920) in wild-type CI 16151 (black solid lines) vs. m11526 (dashed green lines) and m18982 (dashed red lines). * Indicates significant difference from CI 16151 at P < 0.001. (F) Transcript accumulation of Mla6 (HORVU1Hr1G012190) in wild-type CI 16151 vs. m11526.