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.

An interolog-based barley interactome as an integration framework for immune signaling

“…We applied a walktrap clustering algorithm on HvInt using distance correlation from expression data as edge weights (Székely et al, 2007). For this purpose we used RNA-Seq expression data from an infection time course with Bgh isolate 5874 (AVR a6 ) [0,16,20,24,32 and 48 hours after inoculation (HAI)] of the resistant barley line CI 16151 (Mla6, Bln1, Sgt1) and four fastneutron derived mutants; one resistant --bln1-m19089 (Mla6, bln1, Sgt1), and three susceptible --mla6-m18982 (mla6, Bln1, Sgt1), rar3-m11526 (Mla6, Bln1, Sgt1 ΔKL308-309 ), and the double mutant m19028 (mla6, bln1, Sgt1), as shown in Figure 1B (Chapman et al, 2020;Hunt et al, 2019). We acquired fifty-four clusters comprising 1 to 2313 proteins, with 97.6% in the top five clusters (2, 8, 11, 13 and 15).…”

“…An infection time course of the wild-type CI 16151 (Mla6, Bln1, Sgt1) and derived fast-neutron mutants bln1-m19089 (Mla6, bln1, Sgt1), mla6-m18982 (mla6, Bln1, Sgt1), rar3-m11526 (Mla6, Bln1, Sgt1 ΔKL308-309 ), and (mla6+bln1)-m19028 (mla6, bln1, Sgt1) was used for RNA-Seq analysis [NCBI-GEO GSE101304 (Chapman et al, 2020;Hunt et al, 2019)]. CI 16151 contains the functional Mla6 R gene and is resistant.…”

“…Rar3 (required for Mla6 resistance3) is required for Mla6-mediated generation of H 2 O 2 and the hypersensitive response (HR). This susceptible mutant consists of a Lys-Leu deletion in the SGS domain of SGT1, which interacts with NLR proteins (Chapman et al, 2020). The (mla6+bln1) double mutant is susceptible as it contains the same Mla6 deletion as in m18982.…”

“…Time-course RNA-Seq data empowers rapid visualization of differential transcript accumulation in wild-type vs. mutant backgrounds, and thus, in what way immune signaling pathways can be impacted by pathogens. To complement the disease module analysis above, we identified immune-active sub-networks under resistant and susceptible disease outcomes by integrating HvInt with our infection-time-course RNA-Seq data (Hunt et al, 2019;Chapman et al, 2020).…”

Characterization of the Roles of SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 in Sw-5b-Mediated Resistance to Tomato Spotted Wilt Virus

“…SGT1 (suppressor of the G2 allele of skp1) has been found to be required for the induction of the immunity mediated by many NLRs [ 22 , 23 ]. As a protein conserved in eukaryotes, SGT1 functions in a variety of biological processes by interacting with different protein complexes [ 24 , 25 , 26 ]. It regulates the protein accumulation levels of NLR immune receptors [ 27 ] and participates in the nucleocytoplasmic distribution of tobacco NLR protein N [ 28 ].…”