Differential expression of two Blumeria graminis chitin synthase genes

Abstract

Two Blumeria graminis chitin synthase genes, designated BgChs1 and BgChs2 were cloned and characterized following the synthesis and use of degenerate PCR primers designed to the conserved regions of fungal chitin synthase (Chs) genes. Their sequences revealed high similarity with the Chs genes previously cloned from other fungi and placed BgChs1 and BgChs2 with the classes I and V, respectively. Each gene was present as a single copy within the barley powdery mildew genome. Semi-quantitative RT-PCR assays revealed BgChs1 to be up-regulated at both the primary germ tube (PGT) and appressorial germ tube (AGT) stages of differentiation whilst the BgChs2 transcript was up-regulated at the PGT stage. The B. graminis β-tubulin gene was used as a control for all RT-PCR reactions. The BgChs1 transcript was some 30 fold less abundant than the β-tubulin transcript and BgChs2 was some 30 fold rarer than the BgChs1 transcript. The effects of the chitin substrate analogues nikkomycin Z and polyoxin D on conidial morphogenesis were assessed. These nucleoside peptide inhibitors did not affect germination but both polyoxin D and nikkomycin Z treatment led to a large population of abnormally swollen 'balloon-shaped' AGTs, whilst by 12 h after inoculation polyoxin treatment caused the swollen germ tubes to burst.