The BEM46-like protein appears to be essential for hyphal development upon ascospore germination in Neurospora crassa and is targeted to the endoplasmic reticulum
The bud emergence (BEM)46 proteins are evolutionarily conserved members of the α/β-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.
KeywordsCell type-specific growth Ascospore germination Polarity ER localization Neurospora crassa Bud emergence 46-like (bem46)
The authors thank Hanna Schmidt for excellent technical assistance. We thank the Zentrum für Molekularbiologie und Biochemie, Kiel for assistance with the Nanodrop apparatus and Ruth Schmitz-Streit for providing access to her 7,300 real-time PCR system. English language editing was performed by San Francisco Edit.
- Borges MI, Azevedo MO, Bonatelli R, Felipe MSS, Astolfi-Filho S (1990) A practical method for preparation of total DNA from filamentous fungi. Fungal Genet Newsl 37:269–276Google Scholar
- Margolin BS, Freitag M, Selker EU (1997) Improved plasmids for gene targeting at the his-3 locus of Neurospora crassa by electroporation. Fungal Genet Newsl 44:34–36Google Scholar
- Murray JC, Srb AM (1961) A mutant locus determining abnormal morphology and ascospore lethality in Neurospora. J Hered 52:149–153Google Scholar
- Perkins DD, Margolin BS, Selker EU, Haedo SD (1997) Occurence of repeat-induced point mutations in long segmental duplications of Neurospora. Genetics 141:125–136Google Scholar
- Perkins DD, Radford A, Sachs MS (2001) The Neurospora compendium chromosomal loci. Academic Press, San DiegoGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Lab Press, New YorkGoogle Scholar
- Valencik ML, Pringle JR (1995) Schizosaccharomyces pombe bem1/bud5 suppressor (bem46) mRNA. In. EMBL database, accession number U29892Google Scholar
- Vogel HJ (1956) A convenient growth medium for Neurospora (medium N). Microbiol Genet Bull 13:42–43Google Scholar