Identification and expression analysis of regulatory genes induced during conidiation in Exserohilum turcicum

Abstract

Light influences numerous developmental and biochemical processes in fungi. The objectives of this research were to characterize the influence of light on growth and conidiation and associated gene expression in the plant pathogenic ascomycete, Exserohilum turcicum. We found that vegetative growth was more extensive in light/dark cycles than in constant light or darkness as measured by analysis of ergosterol content and genomic DNA. Cultures grown under continuous white light or blue light (approximately 465-480 nm) were developmentally arrested after the formation of conidiophores, whereas those grown in continuous darkness or a light/dark cycle produced mature conidia. Incubation of conidiophore-producing cultures in darkness for a minimum of 2 h was necessary and sufficient to initiate synchronous conidiation. To identify genes that are expressed during dark-induced conidiation, we constructed subtractive cDNA libraries from cultures grown under conidiation-permissive and -repressive conditions. From 816 sequenced EST clones in the conidiation-permissive and 310 in the repressive libraries, 12 putative regulatory genes were chosen for expression analysis by quantitative real-time PCR. The majority of those genes reached maximum expression by 2 h after initiation of the dark period and then declined to initial levels by 4-24 h in darkness. Expression of two dark-induced genes remained elevated after 24 h in darkness but was reset to initial levels if cultures were returned to light. This study revealed several genes whose expression increased rapidly after dark induction of conidiation, suggesting that they encode regulators of asexual development in E. turcicum.