Isolation and characterization of genes expressed uniquely during appressorium formation by Colletotrichum gloeosporioides conidia induced by the host surface wax

Abstract

Appressorium formation in germinating Colletotrichum gloeosporioides is induced by the surface wax of the host, the avocado fruit. To elucidate the mechanism by which differentiation of appressorium formation is induced, the fungal genes specifically activated by this host signal were sought. From a cDNA library of the transcripts present in appressorium-forming conidia, the clones representing nongerminating conidia were removed by hybridization with cDNAs synthesized from the nongerminating conidia. From this subtracted library, clones that hybridized with cDNA for transcripts from appressorium-forming conidia and not with cDNA for transcripts from germinating conidia were selected. Three such clones were isolated and sequenced. The genes for these three transcripts were also cloned and sequenced. Northern blot analysis showed that transcripts that hybridized with these three clones were expressed in the conidium only during the process of appressorium formation induced by avocado surface wax, and that these transcripts were not detectable when appressorium formation was prevented even in the presence of avocado wax. Nucleotide sequences of the clones revealed that one clone, cap3, contained an open reading frame (ORF) that would code for a 26-amino acid, cysteine-rich peptide with significant homology to Neurospora crassa copper metallothionein. Another clone, cap5, contained an ORF that would code for a 27-amino acid cysteine-rich peptide with less homology to metallothioneins. Cu²⁺ and Cd²⁺ also induced the expression of these genes at lower levels. The histochemical analysis of transformants containing the cap5 promoter fused to the β-glucuronidase (GUS) gene showed that the cap5 gene promoter caused GUS expression exclusively during appressorium formation and most of the gus activity was in the appressorium. The cap22 clone contained an ORF coding for a 227-amino acid polypeptide of 22 kDa, which did not show significant homology to any known proteins. Recombinant CAP22 protein was produced using a pET-19b expression system in Escherichia coli, purified, and used to prepare rabbit antibodies. Western blot analysis of proteins from the appressorium-forming conidia revealed a major cross-reacting protein at 43 kDa and a minor band at 68 kDa, indicating that the potential glycosylation sites found in the primary translation product were probably glycosylated. Results of immunogold localization showed that CAP22 protein was located on the wall of the appressorium.