Identification of soluble secreted proteins from appressoria of Colletotrichum higginsianum by analysis of expressed sequence tags

Max-Planck-Institute for Plant Breeding Research, Department of Plant–Microbe Interactions, D-50829 Köln, Germany Correspondence Richard O'Connell oconnel{at}mpiz-koeln.mpg.de The hemibiotrophic ascomycete Colletotrichum higginsianum causes anthracnose disease on brassica crops and the model plant Arabidopsis . Melanized appressoria pierce the host cuticle and cell wall to form specialized biotrophic hyphae inside living epidermal cells. To identify proteins secreted by appressoria that may function as virulence effectors, a cDNA library was prepared from mature appressoria formed in vitro . Bidirectional sequencing of 980 clones generated 1442 high-quality expressed sequence tags (ESTs), comprising 518 unique sequences. BLASTX analysis showed that 353 (68 %) of these had significant similarity to entries in the NCBI non-redundant protein database, of which 49 were also homologous to experimentally verified fungal pathogenicity genes. ORFs were predicted ab initio from the unique sequences and screened for potential signal peptides using SignalP. Fifty-three unique sequences (10 %) were predicted to encode proteins entering the secretory pathway, of which 26 were likely to be soluble secreted proteins. For a selected subset of these, RT-PCR showed that seven genes that encode secreted proteins of unknown function, including two Colletotrichum -specific genes, are upregulated in appressoria and expressed early during plant infection, and therefore represent candidate effectors. Abbreviations: EST, expressed sequence tag; GPI, glycosyl-phosphatidylinositol; polyA RNA, polyadenylated RNA; RACE, rapid amplification of cDNA ends; SEM, scanning electron microscopy The EMBL accession numbers for reported EST sequences and their allocation to unique sequences are shown in Supplementary Table S1 available with the online version of this paper.