Abstract
The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins (e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance.
Highlights
A single T-DNA insertion was generated in the blistering1 gene which manifested in reduced decay in apple and nearly undetectable levels of the mycotoxin patulin in the T625 mutant
Our results show that this locus encodes a unique 35 kDa polypeptide with a DnaJ domain and that it influences multiple functions involved in proper cell wall degrading enzymes (CWDE) secretion, patulin biosynthesis, protein processing and export, and endocytosis
Scanning electron microscopy revealed that T625 had surface alterations including blisters and protrusions that were widespread across the surface of the hyphae and at the conidiophore base whereas the R19 surfaces were smooth (Fig 2A, 2B)
Summary
A TDNA mutant, in the single copy Blistering locus, was created in Penicillium expansum. Quantitative mass spectrometry of mycelial proteins revealed that the mutant had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles These data reveal that Blistering affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance. Our results show that this locus encodes a unique 35 kDa polypeptide with a DnaJ domain and that it influences multiple functions involved in proper CWDE secretion, patulin biosynthesis, protein processing and export, and endocytosis Because this gene bears little homology to other fungi outside the Aspergillaceae, it is a likely that Blistering has a novel role that facilitates host adaptation in apple through proper regulation of protein secretion
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