Characterization of the Temperature-Sensitive Mutations un-7 and png-1 in Neurospora crassa

The model filamentous fungus Neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. While most of these have been mapped genetically, many remain anonymous. The mutation in the N. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated NCU00651 on linkage group I. Other mutations in this gene have been identified that lead to a temperature-sensitive morphological phenotype called png-1. The mutations underlying un-7 result in a serine to phenylalanine change at position 273 and an isoleucine to valine change at position 390, while the mutation in png-1 was found to result in a serine to leucine change at position 279 although there were other conservative changes in this allele. The overall morphology of the strain carrying the un-7 mutation is compared to strains carrying the png-1 mutation and these mutations are evaluated in the context of other temperature-sensitive mutants in Neurospora

Neurospora TS lethal genes involved in protein production, transport, or quality control.

<p>Neurospora TS lethal genes involved in protein production, transport, or quality control.</p

Temperature shift of growing tips of wild-type, <i>un-7</i> and <i>png-1</i> strains to 37°C.

<p>Panels A–C were taken after shifting overnight slide cultures incubated at room temperature to 37°C for 4 hours and panels D–F show the cultures grown on slides at room temperature. (A,D) Wild type strain 2489. (B, E) <i>png-1</i> strain 9860. (C, F) <i>un-7</i> strain 2176.</p

Selected cosmids spanning the genomic region including NCU00651.

<p>Open reading frames are indicated above the line representing the genome sequence while the cosmids are indicated below the line.</p

Cumulative growth of wild type and mutant strains at 37°.

<p>Values are the average of three measurements plus or minus the standard deviation.</p

Alignment of the putative amino acid sequence from the mutated region of the NCU00651 protein from wild type and two mutants as well as select fungi.

<p>The altered amino acid residues in the UN-7 and PNG-1 proteins are indicated by grey shading and amino acid residue 273 is indicated with an “*” above the sequence. Amino acid residue 279 is indicated with a “∧” above the sequence. The locus designations for other species are as follows: <i>Sordaria macrospora</i> CBI51252; <i>Aspergillus fumigatus</i> EDP54057; <i>Coccidioides immitis</i> CIMG_08062; <i>Magnaporthe grisea</i> MGG_03598; <i>Saccharomyces cerevisiae</i> EE09111; <i>Phycomyces blakesleeanus</i> 178891.</p

Polarisome Meets Spitzenkörper: Microscopy, Genetics, and Genomics Converge

The impact of filamentous fungi on human welfare has never been greater. Fungi are acknowledged as the most economically devastating plant pathogens (1) and are attaining increasing notoriety for their ability to cause life-threatening infections in humans (57, 71), and fungal products sustain a billion dollar manufacturing industry (70). The tools available to study filamentous fungi are more sophisticated than ever and include the complete annotated genome sequences of multiple filamentous fungi (12), resources being made available through various functional genomics projects, and advanced bioimaging methods, including high-resolution live-cell imaging (20, 32) and electron tomography (19, 50). The increasing impact of filamentous fungi, along with the rediscovery of pseudohyphal growth in yeast (22), has focused attention on the molecular mechanisms underlying hyphal morphogenesis. Attempts to understand hyphal morphogenesis have historically followed two different lines of investigation. Microscopists have defined, with increasing detail, the subcellular organization of the hyphal tip. This led to the description of the Spitzenkö̈rper, an apical cluster of vesicles, cytoskeletal elements, and other proteins, which plays a crucial role in hyphal extension (4). Geneticists have identified gene products required for hyphal morphogenesis by characterizing morphological mutants (51, 52). Initial studies in the laboratories of Beadle, Tatum, and colleagues attempted to link morphogenesis to specific biochemical pathways. More recent screens have identified a multitude of signaling and cytoskeletal functions required for hyphal extension (62, 72). In the past few years, comparative genomics efforts have allowed fungal biologists interested in hyphal morphogenesis to exploit the wealth of knowledge about polarized growth in the yeast Saccharomyces cerevisiae. Many informative homologies between filamentous fungi and yeast have been uncovered. Notably, this includes several components of a multiprotein complex termed the polarisome (28), which regulates microfilament formation at polarized growth sites in yeast (61). Perhaps more importantly, several gene products involved in hyphal morphogenesis have been shown to have no homologue outside of the filamentous fungi. This emphasizes the potential novelty of the mechanisms underlying hyphal morphogenesis. In this review, we summarize past efforts to understand hyphal morphogenesis and pose a series of questions designed to focus future efforts in this area