Gene structure and regulation of cryparin, a hydrophobin of the fungus Cryphonectria parasitica

Abstract

Cryphonectria parasitica is a filamentous fungus that causes a disease in chestnut trees called chestnut blight. Hypovirulence and decreased sporulation by the fungus are caused by the presence of a unique double-stranded (ds)RNA. Symptoms of dsRNA infection are correlated with the down-regulation of several host polypeptides and poly(A)+ RNAS. One of the regulated host proteins (cryparin) that has been purified has a glycine-serine repeating sequence near the amino-terminal end that is typical of structural proteins, and it has the properties of a lectin (8,19,39). Antibody staining showed that this protein is specific to aerial hyphae and fruiting bodies and accumulates in large amounts on hyphal cell surfaces (14). The gene for cryparin has been isolated. Structural analysis was conducted by comparison of the sequence data from the subgenomic DNA clone and PCR amplified CDNA product. Putative 5'and 3' regulatory sequences were identified as well as intron splicing sites. The transcription initiation site was identified by primer extension of RNA from a dsRNA-free wild type strain EP155/2. Northern blot analysis was performed on MRNA extracted from both EP155/2 and the isogenic strain UEP1, which contains the dsRNA, to determine steady state levels of cryparin transcript. SDS-PAGE gels were used to examine protein accumulation over time in liquid grown cultures. Cryparin was found to be developmentally regulated with MRNA being most highly expressed in log phase of liquid cultures (70). In order to investigate gene function, a gene deletion vector was constructed using the subgenomic DNA clone of cryparin. Fungal transformations were performed to attempt to delete the cryparin gene. Mitotically stable transformants were selected on media containing the antibiotic hygromycin B. Southern hybridizations with the labeled hygromycin resistance gene confirmed plasmid integration in nuclear DNA, however, hybridization with labeled cryparin coding sequences revealed no deletion of the cryparin gene.