Cell-autonomous complementation of mlo resistance using a biolistic transient expression system

The barley geneMloencodes a prototype of a novel class of plant proteins. Inmlomutants, absence of the 60 kDa wild-type Mlo protein results in broad-spectrum resistance to the powdery mildew fungus,Erysiphe graminisf. sp.hordei. To directly assess its function,Mlowas transiently expressed with a marker gene encoding a modified green fluorescent protein (GFP) in leaf epidermal cells ofmloresistant barley lines. Fungal inoculation of epidermal cells transfected with wild-typeMloled to haustorium formation and abundant sporulation. Therefore, expression of the wild-typeMlogene, inmloresistant genotypes, is both necessary and sufficient to restore susceptibility to fungal attack. Complementation ofmloresistance alleles was restricted to single host cells, indicating a cell-autonomous function for the wild-type Mlo protein. We discuss our findings with respect to source–sink relationships of plants and biotrophic fungi and the potentially wide-ranging use of the transient complementation assay to analyse host compatibility and defence in response to powdery mildew attack

Genetic engineering of the multicellular green alga Volvox: a modified and multiplied bacterial antibiotic resistance gene as a dominant selectable marker

Hallmann A, Rappel A. Genetic engineering of the multicellular green alga Volvox: a modified and multiplied bacterial antibiotic resistance gene as a dominant selectable marker. Plant J. 1999;17(1):99-109.The green alga Volvox represents the simplest multicellular organism: Volvox is composed of only two cell types, somatic and reproductive. Volvox, therefore, is an attractive model system for studying various aspects of multicellularity. With the biolistic nuclear transformation of Volvox carteri, the powerful molecular genetic manipulation of this organism has been established, but applications have been restricted to an auxotrophic mutant serving as the DNA recipient. Therefore, a dominant selectable marker working in all strains and mutants of this organism is required. Among several gene constructs tested, the most advantageous results were obtained with a chimeric gene composed of the coding sequence of the bacterial ble gene, conferring resistance to the antibiotic zeocin, modified with insertions of two endogenous introns from the Volvox arylsulfatase gene and fused to 5' and 3' untranslated regions from the Volvox beta 2-tubulin gene. In the most suitable plasmid used, the gene dosage was increased 16-fold by a technique that allows exponential multiplication of a DNA fragment. Co-transformation of this plasmid and a non-selectable plasmid allowed the identification of zeocin resistant transformants with nuclear integration of both selectable and non-selectable plasmids. Stable expression of the ble gene and of genes from several non-selectable plasmids is demonstrated. The modified ble gene provides the first dominant marker for transformation of both wild-type and mutant strains of Volvox