1.2.34
A TRANSIENT EXPRESSION SYSTEM TO ASSAY GENE ACTIVATION AND PATHOGENICITY OF POWDERY MILDEW ON BARLEY LEAVES

KA NIELSEN1, O OLSEN2, K SHIRASU3, P SCHULZE-LEFERT3 and RP OLIVER1

1Department of Physiology, and 2Carlsberg Research Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark; 3Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK

Background and objectives
Mildew (Erysiphe graminis f.sp. hordei; Egh) infection of barley (Hordeum vulgare) is a highly specialized and refined interaction, since the pathogen is an obligate parasite and is entirely restricted to epidermal leaf cells. Detailed studies on gene regulation have until now been hampered by the recalcitrance of the barley leaf cells to transformation. Thus the establishment of a fast and efficient genotype-independent protocol for transient expression of genes in leaves susceptible to Egh would be an important tool to assay functional effect(s) of genes encoding putative antifungal factors. Therefore a transient expression system was established. The system has been used to complement mlo resistance in a barley mutant, Carlsberg mlo~, by transient expression of the recently cloned wild-type Mlo gene [1].

Results and conclusions

The transient expression system was efficient on detached leaf sections, which were transformed by particle bombardment with marker genes encoding GUS and GFP and subsequently inoculated with Egh conidiospores. Normal colonies developed on both non-transformed and transgenic epidermal cells. A synthetic gene encoding an improved GFP was as least as sensitive a marker as GUS using the maize ubiquitin promoter. GFP was detectable for at least 10 days on leaf sections even when intensively colonized by the fungus. Accordingly, the transient expression system combines the use of (i) promoters directing strong expression in leaf epidermal cells; (ii) sensitive detection of marker gene expression, and (iii) heavy colonization of the pathogen on the plant tissue.

The mlo resistance is mediated by recessive alleles. To test the complementation of the mlo gene, the wild-type Mlo gene fused to the maize ubiquitin promoter was introduced into resistant leaf sections of the Carlsberg mlo~ mutant. Inoculation with Egh illustrated that susceptible single cells were indeed obtained on such resistant leaf sections.

References
1. Buschges R, Hollricher K, Panstruga R et al., 1997. Cell 88, 695-705.