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Single-Cell Complementation of Barley Mlo Mutants Using a PDS-1000/He Hepta System

Ralph Panstruga, Brigitte Schauf, and Paul Schulze-Lefert, Max-Planck-Institut fr Zchtungsforschung, Carl-von-Linn-Weg 10, 50829 Kln, Germany e-mail: panstrug@mpiz-koeln.mpg.de


Abstract
Transient expression via particle bombardment is widely used as a means of gene transfer to bacteria, yeast, animals, and plants. In this study we describe the use of the Hepta adaptor for the PDS-1000/He biolistic system for transformation and transient expression of Mlo in single epidermal cells of detached barley leaves. Barley Mlo is known to dampen plant defense and its expression in single epidermal cells of mlo resistant mutants restores susceptibility to attack from the powdery mildew fungal pathogen, Blumeria graminis. Consistently high transformation efficiencies were obtained upon delivery of a plasmid carrying both Green Fluorescent Protein (GFP) and Mlo. After bombardment and fungal spore inoculation, we found leaf sectors with numerous green fluorescent epidermal cells supporting growth of the pathogen. Likewise, we observed a high transformation efficiency of Arabidopsis leaf cells upon delivery of a GUS (β-glucuronidase) reporter gene construct. Application of the Hepta adaptor reduces the number of biolistic transfers necessary to obtain sufficient numbers of transformed plant cells for quantitative scoring of single-cell traits.

Introduction
Mutation induced recessive alleles (mlo) of the barley Mlo gene confer broad spectrum resistance against Blumeria graminis f. sp. hordei, the causal agent of powdery mildew. Conversely, the presence of wild-type Mlo leads to susceptibility upon attack from this obligate biotrophic fungal pathogen. Mlo encod
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