The bud emergence (BEM)46 proteins are evolutionarily conserved members of the alpha/beta-hydrolase super family, but their exact role remains unknown. To better understand the cellular role of BEM46 and its homologs, we used the model organism Neurospora crassa in conjunction with bem46 RNAi, over-expression vectors, and repeat induced point mutation analyzes. We clearly demonstrated that BEM46 is required for cell type-specific hyphal growth, which indicates a role for BEM46 in maintaining polarity. Vegetative hyphae, perithecia, and ascospores developed normally, but hyphae germinating from ascospores exhibited a loss-of-polarity phenotype. We also found that the BEM46 protein is targeted to the perinuclear endoplasmic reticulum (ER) and also localizes at or close to the plasma membrane. Our findings show that BEM46 can be used as a new ER marker for filamentous fungi, the first for N. crassa. Our data suggest that BEM46 plays a role in a signal transduction pathway involved in determining or maintaining cell type-specific polarity. This implies a higher degree of fungal hyphae differentiation than previously expected. This work also has implications for higher eukaryotic cells with polarized growth, such as pollen tubes or neuronal cells.

Endoscopically directed mucosal brushings of the maxillary sinus provide equivalent concentrations of total DNA to mucosal biopsy specimens but possess greater concentrations of bacterial DNA, likely because of the greater surface area sampled by this method. Given the additional advantage of lower risk associated with obtaining brush samples, we suggest they represent the preferred sampling method for future genomic sinus studies.

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In this study, we investigated the ability of the fungus Neurospora crassa to produce and secrete two ribonucleases: the heterologous bovine RNase A and the endogenous RNase N(1). A set of expression vectors was constructed, each consisting of an RNase A open reading frame under the control of a specific promoter and each with a specific terminator. N. crassa transformants were analyzed at the transcriptional and protein levels. Irrespective of the promoter used, all transformants showed an RNase A-specific transcript in northern hybridization, but transcriptional strengths differed significantly. The strongest transcription was detected in transformants under the control of the cfp promoter. Western blot analysis and ELISA assays of selected transformants showed an effective secretion up to 356 ng/mL of recombinant RNase A protein. However, the highest ribonuclease activity could be detected in transformants carrying the endogenous RNase N(1) under the control of the ccg1 promoter. Expression and secretion of RNase N(1) thus represent an alternative to recombinant expression of RNase A protein. In conclusion, we have created a viable expression system for expression of homologous and heterologous proteins in N. crassa.