Date of Award
Doctor of Philosophy
Plant Pathology and Microbiology
Roger P Wise
The molecular interactions between crops and pathogens are complex and require elucidation if we are to maintain food supplies against the ever-encroaching threat of disease. The resistance gene Mla (Mildew resistance locus a) and its orthologs protect barley (Hordeum vulgare L.) and other cereals from fungal diseases, such as barley powdery mildew caused by Blumeria graminis f. sp. hordei (Bgh). However, there are many missing links in our knowledge of how MLA functions. The HRS complex is necessary for MLA to function, consisting of the proteins HSP90 (Heat shock protein 90), RAR1 (Required for Mla12 Resistance 1), and SGT1 (Suppressor of G-two allele of Skp1).To identify new mechanisms in MLA-mediated immunity, the Wise laboratory had performed fast-neutron mutagenesis of a resistant barley line to identify mutants compromised against Bgh infection and identified a susceptible line with a heritable recessive mutation. I then utilized this line to perform extensive genetic analysis and discovered that the mutation disrupted a subset of Mla alleles. I used exome-capture, bulk-segregant analysis, and fine-mapping to delineate the causal mutation to a two amino acid deletion in the SGS domain of SGT1, Sgt1ΔKL308-309, which does not display the lethality common to most Sgt1 mutants. I proceeded to characterize the SGT1ΔKL308-309 mutation to uncover specifics of MLA-SGT1 interactions. I generated a yeast strain with stably integrated HvRar1 and HvHsp90, to show that SGT1ΔKL308-309 has weakened interactions with both MLA1 and MLA6 in a Y2H system. Additionally, I utilized parallel reaction monitoring mass-spectrometry to show that MLA6 peptide accumulation was significantly less in the Sgt1ΔKL308-309 mutant line. As the function of Mla6 is disrupted by the Sgt1ΔKL308-309 mutation, whereas Mla1 is not, the mutation appears to disproportionately affects MLA variants with inherently weaker interactions with SGT1. To uncover specific molecular mechanisms behind their interactions, I used site-directed mutagenesis to generate specific MLA1, MLA6, and SGT1 mutant constructs and tested these in our Y2H system. I found that two specific sites in MLA1, when mutated to the MLA6 residues at the same position, abolished interaction between MLA1 and SGT1. Moreover, I show that the Sgt1ΔKL308-309 mutation has a less severe disruption than alanine or arginine substitutions at the same sites, which may indicate that the disorder of the SGS domain is paramount for its function. Combining my data with recent work in CC-NLRs, I propose a cyclical model of the MLA-HRS resistosome function and interaction.
Antony Victor Ernest Chapman
Chapman, Antony Victor Ernest, "Biological and molecular insights on MLA-based resistance of barley to powdery mildew derived from investigation of an in-frame Lys-Leu deletion in the essential protein SGT1" (2021). Graduate Theses and Dissertations. 18470.