Abstract
BackgroundRecessively inherited natural and induced mutations in the barley Mlo gene confer durable broad-spectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei. Mlo codes for a member of a plant-specific family of polytopic integral membrane proteins with unknown biochemical activity. Resistant barley mlo mutant alleles identify amino acid residues that are critical for Mlo function in the context of powdery mildew susceptibility.ResultsWe molecularly analyzed a novel set of induced barley mlo mutants and used site-directed mutagenesis in combination with transient gene expression to unravel novel amino acid residues of functional significance. We integrate these results with previous findings to map functionally important regions of the heptahelical Mlo protein. Our data reveal the second and third cytoplasmic loop as being particularly sensitive to functional impediment by mutational perturbation, suggesting that these regions are critical for the susceptibility-conferring activity of the Mlo protein. In contrast, only mutations in the second but not the third cytoplasmic loop appear to trigger the Endoplasmic Reticulum-localized quality control machinery that ensures the biogenesis of properly folded membrane proteins.ConclusionOur findings identify functionally important regions of the polytopic barley Mlo protein and reveal the differential sensitivity of individual protein domains to cellular quality control.
Highlights
Inherited natural and induced mutations in the barley Mildew resistance locus o (Mlo) gene confer durable broadspectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei
While seven of the eight tested accessions obtained from this site showed the hybridization pattern that is typical for the mlo-11 allele, one accession (CGN0524) lacked this signal, suggesting that this line does not harbor the common natural mlo11 allele
Microscopic evaluation revealed that the Blumeria graminis (Bgh) entry rate in this accession was ca. 30% (29.7% ± 7.5%; Figure 1B), considerably below the 50-80% penetration success that is typical for Bgh on susceptible wild type (Mlo genotype) barley lines, but clearly distinct from the near complete penetration resistance seen in mlo null alleles
Summary
Inherited natural and induced mutations in the barley Mlo gene confer durable broadspectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei. Previous molecular analyses of induced barley mlo mutants in combination with site-directed mutagenesis and bioinformatic analysis based on multiple sequence alignments provided a first glimpse on the amino acids of the heptahelical barley Mlo protein that are decisive for its susceptibility-conferring function [19,20,21,22,23]. Amongst these residues are four extracellularly located cysteines that are thought to form two disulfide bridges as well as few amino acids located in various regions of the polytopic membrane protein. Three distinct ERAD pathways can be defined on the basis of the different ubiquitin ligase complexes involved in substrate elimination: The ERAD-L pathway mediates removal of soluble (luminal) ER substrates whilst depending on the presence of either misfolded transmembrane or cytosolic domains, membrane-anchored substrates are removed by either the ERAD-M or ERAD-C pathway [26]
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