Novel induced mlo mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein.
BACKGROUND
Recessively 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.


RESULTS
We 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.


CONCLUSIONS
Our findings identify functionally important regions of the polytopic barley Mlo protein and reveal the differential sensitivity of individual protein domains to cellular quality control.
Reinstädler
Anja
A
Max-Planck Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, Carl-von-Linné-Weg 10, 50829 Köln, Germany.
Müller
Judith
J
Czembor
Jerzy H
JH
Piffanelli
Pietro
P
Panstruga
Ralph
R
D000483:Alleles; D000595:Amino Acid Sequence; D018744:DNA, Plant / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D006239:Haplotypes; D001467:Hordeum / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics*; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA
D018744:DNA, Plant; C104922:MLO protein, Hordeum vulgare; D010940:Plant Proteins