Expressed sequences from the basidiomycetous tree pathogen Heterobasidion annosum during early infection of scots pine q Magnus Karlsson, *  Ake Olson, and Jan Stenlid Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, SE-750 07 Uppsala, Sweden Received 13 February 2002; accepted 7 November 2002 Abstract The pattern of gene expression of the basidiomycete Heterobasidion annosum, causal agent of the root rot of conifers, was analysedduringitsinteractionwithpineroots.AcomplementaryDNA(cDNA)librarywasconstructedfromtotalRNAextracted from H. annosum myceliachallengedwithScotspineseedlingrootsfor6and72h.Singlepasssequencingof1148randomlyselected cDNA clones resulted in 923 expressed sequence tags (ESTs). Contig analysis and sequence comparisons identified 318 unigene sequences, of which 62 were repeatedly sampled. A putative cellular function was assigned to 223 contigs (70%) that showed a moderatetohighhomologytoproteinsequencesfrompublicdatabases.Variationsinexpressionlevelsduringtheinfectionprocess weremonitoredonasetof96unigenesbyreversenorthernusingdothybridisation.Sevenunigenes(7%)wereshowntobeeitherup (4)ordown(3)regulatedduringinteractionofthefunguswithpineroots.Fungalgenesdifferentiallyexpressedduringcontactwith roots include genes encoding mitochondrial proteins, a cytochrome P450 and a vacuolar ATP synthase. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Basidiomycete; Heterobasidion annosum; Annosum root rot; Tree pathogen; Forest disease; cDNA sequencing; Expressed sequence tags 1. Introduction The basidiomycete Heterobasidion annosum is the causal agent of annosum root rot, which is the eco- nomically most important disease of coniferous forests in northern temperate regions (Korhonen and Stenlid, 1998). There are three intersterility groups occurring in H. annosum. These intersterility groups have different distribution and are named after their main host pref- erence, Pine, Spruce or Fir (P, S, and F), although the host range is wide and overlapping (Korhonen et al., 1998). In Europe all three groups have been identified (Capretti et al., 1990; Korhonen, 1978) while in North America only the P and S groups have been found (Harrington et al., 1989). H. annosum is a wood- decaying pathogen which can utilize a variety of carbon sources, such as starch, cellulose and pectin, for review see (Asiegbu et al., 1998). It is known to secrete en- zymes that degrade lignin such as manganese peroxid- asesandligninperoxidases(Farreletal.,1989;Higuchi, 1990). Furthermore H. annosum secretes laccases in- volved in detoxifying phenolic compounds (Reinham- mar, 1984). Twelve phytotoxic benzofuran derivates have also been identified as produced by the fungus (Heslin et al., 1983; Sonnenbichler et al., 1989). Plant growth inhibiting compounds related to abscisic acid have also been detected in H. annosum culture filtrates (Hoque, 1984). Although being an important forest pathogen, H. annosum is still poorly studied at the genomic level. Molecular strategies relying on expressed sequence tags (ESTs) can provide information on uncharacterised ge- nomes. This technique was first developed for human research (Adams et al., 1991) and later used in different organisms, including fungal plant pathogens such as Fusarium graminearum (Kruger et al., 2002), Blumeria [Erysiphe] graminis (Thomas et al., 2001), Magnaporthe grisea (Kim et al., 2001; Rauyaree et al., 2001), My- cosphaerella graminicola [Septoria tritici] (Keon et al., 2000)andfungalplantsymbiontssuchasthemycorrhizal basidiomycete Pisolithus tinctorius (Voiblet et al., 2001). Fungal Genetics and Biology 39 (2003) 51–59 www.elsevier.com/locate/yfgbi q Nucleotide sequence data are deposited in the dbEST databank in GenBank with Accession Nos. BM346837–BM347258. * Corresponding author. Fax: +46-18-673599. E-mail address: magnus.karlsson@mykopat.slu.se (M. Karlsson). 1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S1087-1845(02)00586-8