Identification ofHeterobasidion annosum(S-type) genes expressed during initial stages of conidiospore germination and under varying culture conditions

Abstract

Expressed sequence tag analysis (EST) is a robust approach for detecting novel and potentially important genes during fungal development. The technique was used in this study to identify and characterise genes induced during spore germination and hyphal morphogenesis under different culture conditions. For this study, a cDNA library was constructed from the germinated spores of S-type of Heterobasidion annosum. In total, 12,288 clones of the H. annosum germinated spores (HAGS) cDNA library were arrayed on a high-density nylon membrane filter. A total of 1248 trimmed ESTs were randomly selected and sequenced. These ESTs were composed of 343 contigs from a total of 883 EST sequences. The BLASTX analysis of these sequenced clones revealed several kinds of genes with diverse functions (e.g., MAP kinase and endoglucanase, etc.). Differential hybridization assay on the HAGS cDNA library using cDNA probes made from H. annosum pre-grown under different culture conditions, such as 1.0% glucose, 0.01% ferulic acid or 0.01% oxalic acid revealed 20, 37, 44 positive hybridizing clones, respectively. Among the positively hybridizing clones 15 gene clones were common to all conditions whereas 4, 9 and 15 clones were unique in glucose, ferulic acid and oxalic acid medium, respectively. BLASTX analysis of the differentially expressed clones led to the identification of genes with significant homologue to cytochrome P450 monooxygenase and transcript antisense to ribosomal RNA. Northern analyses confirmed that expression of both genes were elevated in ferulic acid and oxalic acid culture conditions than in glucose medium. The enhanced accumulation of the gene transcripts at the different culture conditions is discussed with respect to their potential role during fungal morphogenesis and in planta growth.