Comparison of transcription of multiple genes at three developmental stages of the plant pathogen Sclerotinia sclerotiorum Adrienne C. Sexton 1 , Anton J. Cozijnsen 1 , Andrew Keniry 2 , Erica Jewell 2 , Christopher G. Love 2 , Jacqueline Batley 2 , David Edwards 2 & Barbara J. Howlett 1 1 School of Botany, The University of Melbourne, Parkville, VIC, Australia and 2 Department of Primary Industries, Plant Biotechnology Centre, La Trobe University, Bundoora, VIC, Australia Correspondence: Adrienne C. Sexton, School of Botany, The University of Melbourne, Parkville, VIC 3010, Australia. Tel.: 1613 8344 5056; fax: 1613 9347 5460; e-mail: sextona@unimelb.edu.au Received 9 November 2005; revised 8 February 2006; accepted 26 February 2006. First published online 24 March 2006. doi:10.1111/j.1574-6968.2006.00212.x Editor: Holger Deising Keywords Sclerotinia sclerotiorum; appressorium; transcription; Brassica napus; fungi; plant disease. Abstract The ascomycete Sclerotinia sclerotiorum is a plant pathogen with a very broad host range. In order to identify and characterize genes involved in S. sclerotiorum infection of Brassica napus (canola), expressed sequence tags (ESTs) were examined from libraries prepared from three tissues: complex appressorium (infection cushions), mycelia grown on agar and lesions formed on leaves of B. napus. A high proportion of genes (68%) had not been previously reported for S. sclerotiorum in public gene or EST databases. The types of novel genes identified in the infection cushion library highlights the functional specificity of these struc- tures and similarities to appressoria in other fungal pathogens. Quantitative real- time PCR was used to analyse tissue specificity and timing of transcription of genes with best matches to MAS3 (appressoria-associated protein from Magnaporthe grisea), cellobiohydrolase I, oxaloacetate acetylhydrolase, metallothionein, pisatin demethylase, and an unknown gene with orthologs in fungal pathogens but not in saprophytic fungi. Introduction The ascomycete Sclerotinia sclerotiorum has a very broad host range, including Brassica species as well as many other crops and weeds, enabling populations to persist and spread easily (Boland & Hall, 1994). Stem rot caused by this fungus is a serious sporadic threat to production of Brassica napus (canola) worldwide. Genetic diversity studies show that both clonal and sexual recombination contribute to differ- entiation of S. sclerotiorum populations isolated from Bras- sica species in North America and Australia (Kohli & Kohn, 1998; Sexton & Howlett, 2004). Effective and economically feasible disease control measures are lacking, and currently sown canola cultivars have no resistance, although some lines with partial resistance have been identified in China (Zhao & Meng, 2003). The infection process of S. sclerotiorum on canola in the field is usually initiated by aerially dispersed ascospores, which germinate on an exogenous source of nutrients such as fallen petals. Infection can also occur directly from myceliogenic germination of sclerotia, provided there is a source of nonliving organic material for saprophytic growth before infection of the plant (Purdy, 1958). Mycelia grow on the petals before forming simple or complex appressoria, known as infection cushions, on the stem or leaf of the canola plant. Infection cushions are multicellular structures formed via dichotomous branching of a single hypha and terminal swelling of the apex, followed by repeated branch- ing and secretion of adhesive mucilage (Tariq & Jeffries, 1984). These specialized structures form a tight interface with the host surface, and may allow the secretion of enzymes and toxins in addition to exerting mechanical force, facilitating penetration of the host surface (Jamaux et al., 1995). Hyphae then colonize the leaf or stem, both inter- and intracellularly and emerge through stomatal apertures. Sclerotinia sclerotiorum produces various polyga- lacturonases and proteases during infection (reviewed by Hegedus & Rimmer, 2005), and large water-soaked lesions develop. Sclerotia often form in the plant stem during late stages of infection, and can survive in the soil for many years, before germinating into mycelia or apothecia, thus repeating the disease cycle. Approaches used in other fungi to identify genes important for the infection process include suppression subtractive hybridization (SSH), which has identified appressorium- FEMS Microbiol Lett 258 (2006) 150–160 c  2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved