Department of Plant Sciences, South Parks Road, University of Oxford, OX1 3RB, UK

Background and Objectives
Erysiphe graminis DC. f.sp. hordei Marchal, the causal agent of powdery mildew of barley (Hordeum vulgare), is an obligate biotrophic fungus. The differentiation of the conidium, on a susceptible host, progresses through a well-described sequence of morphological changes, that is, firstly, the emergence of the primary germ tube (PGT), followed by the appressorial germ tube (AGT) and finally in the formation of the haustorium. Far less is known about changes in gene expression during this developmental sequence. Indeed, only two sequences, corresponding to genes expressed by spores on the non-inductive surface, glass, have been published [1]. Our work aims to identify genes which are differentially expressed during germ tube development in E. graminis f.sp. hordei.

Results and conclusions
Subtraction DDRT-PCR was used to identify genes which are differentially expressed during germ tube differentiation in barley powdery mildew. Here, the amount of relevant tissue which can be collected at the different stages of germ tube differentiation is difficult to synchronise and it is very restricted (the conidia are 28x14 Ám, with the PGT reaching 5-10 Ám and the AGT up to 30 Ám in length). One day old E. graminis conidia were inoculated onto H. vulgare cultivar Golden Promise leaves and harvested into cellulose acetate peels at 4 h (PGT-stage) and at 12 h (AGTstage) post-inoculation. Ungerminated conidia were collected directly. Second strand cDNAs from the ungerminated conidia were subtracted from first strand cDNA species from the PGT and AGT stages of spore differentiation and thence the enriched population was subjected to differential display using onebase anchored primers combined with eight arbitrary primers. Over 50 cDNAs have been cloned and sequenced and confirmed by RT-PCR dot-biol analysis to be stage-specific. Of these, five cDNAs share homology with known sequences, notably, an augmenter gene, hema, nusa, formate dehydrogenase and cytochrome C oxidase. One clone, Al 6-2, is AGT stage specific according to RT-DD and RT dot-blot analysis, whilst six unidentified genes have been subjected to more rigorous Northern blot analysis, revealing one to be AGT stage-specific (G64-3) and five to be up-regulated during differentiation (G39-1, A17-1, A21-9, C31-6 and G55-8).

1. Justesen A, Somerville S, Christensen S, Giese H 1996. Gene 170, 131-135.