The mycovirus CHV1 disrupts secretion of a developmentally regulated protein in Cryphonectria parasitica.

Infection of the chestnut blight fungus Cryphonectria parasitica with Cryphonectria hypovirus 1 (CHV1) causes disruption of virulence, pigmentation, and sporulation. Transcriptional downregulation of key developmentally regulated fungal genes occurs during infection, but vegetative growth is unaffected. Previous studies showed that CHV1 utilizes trans-Golgi network (TGN) secretory vesicles for replication. In this study, the fungal cell surface hydrophobin cryparin was chosen as a marker to follow secretion in virally infected and noninfected strains. Subcellular fractionation, cryparin-green fluorescent protein (GFP) fusion, and Western blot studies confirmed that vesicles containing cryparin copurify with the same fractions previously shown to contain elements of the viral replication complex and the TGN resident endoprotease Kex2. This vesicle fraction accumulated to a much greater concentration in the CHV1-infected strains than in noninfected strains. Pulse-chase analysis showed that the rates and amount of cryparin being secreted by the CHV1 containing strains was much lower than in noninfected strains, and the dwell time of cryparin within the cell after labeling was significantly greater in the CHV1-infected strains than in the noninfected ones. These results suggest that the virus perturbs a specific late TGN secretory pathway resulting in buildup of a key protein important for fungal development.

Differential expression of the putative Kex2 processed and secreted aspartic proteinase gene family of Cryphonectria parasitica.

Kex2-silenced strains of Cryphonectria parasitica, the ascomycete causal agent of chestnut blight, show a significant reduction in virulence, reduced sexual and asexual sporulation and reductions in mating and fertility. Due to this and the known involvement of Kex2 in the processing of important proproteins in other systems, we searched the whole C. parasitica genome for putative Kex2 substrates. Out of 1299 open reading frames (ORFs) predicted to be secreted, 222 ORFs were identified as potential Kex2 substrates by this screen. Within the putative substrates we identified cell wall modifying proteins, putative proteinases, lipases, esterases, and oxidoreductases. This in silico screen also uncovered a family of nine secreted aspartic proteinases (SAPs) of C. parasitica. Northern blot analyses of this gene family showed differential expression when exposed to chestnut wood and Cryphonectria hypovirus 1 (CHV1). Due to the reduction in fungal virulence known to be caused upon hypoviral infection of C. parasitica, the differential gene expression observed, and the known involvement of SAPs in virulence in other systems, we conducted deletion analyses of four of these proteinases, representing different expression patterns. Deletion of each of the four SAPs did not affect growth rates, sporulation or virulence, suggesting that none of the considered SAPs is essential for the full development or virulence of C. parasitica under the conditions tested.

Silencing of Kex2 significantly diminishes the virulence of Cryphonectria parasitica.

Cryphonectria parasitica is the causal agent of chestnut blight. Infection of this ascomycete with Cryphonectria hypovirus 1 (CHV1) results in reduction of virulence and sporulation of the fungus. The virus affects fungal gene expression and several of the CHV1 downregulated genes encode secreted proteins that contain consensus Kex2 processing signals. Additionally, CHV1 has been shown to colocalize in infected cells primarily with fungal trans-Golgi network vesicles containing the Kex2 protease. We report here the cloning, analysis, and possible role of the C. parasitica Kex2 gene (CpKex2). CpKex2 gene sequence analysis showed high similarity to other ascomycete kexin-like proteins. Southern blot analyses of CpKex2 showed a single copy of this gene in the fungal genome. In order to monitor the expression and evaluate the function of CpKex2, antibodies were raised against expressed protein and Kex2-silenced mutants were generated. Western blots indicate that the Kex2 protein was constitutively expressed. Growth rate of the fungus was not significantly affected in Kex2-silenced strains; however, these strains showed reduced virulence, reduced sexual and asexual sporulation, and reductions in mating and fertility. The reduced virulence was correlated with reduced Kex2 enzymatic activity and reduced relative mRNA transcript levels as measured by real time reverse-transcriptase polymerase chain reaction. These results suggest that secreted proteins processed by Kex2 are important in fungal development and virulence.

A Hydrophobin of the chestnut blight fungus, Cryphonectria parasitica, is required for stromal pustule eruption.

Hydrophobins are abundant small hydrophobic proteins that are present on the surfaces of many filamentous fungi. The chestnut blight pathogen Cryphonectria parasitica was shown to produce a class II hydrophobin, cryparin. Cryparin is the most abundant protein produced by this fungus when grown in liquid culture. When the fungus is growing on chestnut trees, cryparin is found only in the fungal fruiting body walls. Deletion of the gene encoding cryparin resulted in a culture phenotype typical of hydrophobin deletion mutants of other fungi, i.e., easily wettable (nonhydrophobic) hyphae. When grown on the natural substrate of the fungus, however, cryparin-null mutation strains were unable to normally produce its fungal fruiting bodies. Although the stromal pustules showed normal development initially, they were unable to erupt through the bark of the tree. The hydrophobin cryparin thus plays an essential role in the fitness of this important plant pathogen by facilitating the eruption of the fungal fruiting bodies through the bark of its host tree.