The genetic basis of cellular morphogenesis in the filamentous fungus Neurospora crassa

Abstract

Cellular polarity is a fundamental property of every cell. Due to their extremely fast growth rate (>/=1 microm/s) and their highly elongated form, filamentous fungi represent a prime example of polarized growth and are an attractive model for the analysis of fundamental mechanisms underlying cellular polarity. To identify the critical components that contribute to polarized growth, we developed a large-scale genetic screen for the isolation of conditional mutants defective in this process in the model fungus Neurospora crassa. Phenotypic analysis and complementation tests of ca. 950 mutants identified more than 100 complementation groups that define 21 distinct morphological classes. The phenotypes include polarity defects over the whole hypha, more specific defects localized to hyphal tips or subapical regions, and defects in branch formation and growth directionality. To begin converting this mutant collection into meaningful biological information, we identified the defective genes in 45 mutants covering all phenotypic classes. These genes encode novel proteins as well as proteins which 1) regulate the actin or microtubule cytoskeleton, 2) are kinases or components of signal transduction pathways, 3) are part of the secretory pathway, or 4) have functions in cell wall formation or membrane biosynthesis. These findings highlight the dynamic nature of a fungal hypha and establish a molecular model for studies of hyphal growth and polarity.

Figure 1.

Wild-type and mutant morphologies. (A) Germination of wild-type conidia. Conidia from a wild-type strain were plated on minimal agar medium, incubated at 30°C, and photographed at various times. (B) Identification of hyphal morphogenesis mutants on the basis of abnormal colony morphology (see MATERIAL AND METHODS for details). (C) Characterization of mutants based on the morphological alterations observed when hyphae or conidia were transferred to restrictive temperature. Bars, 20 μm (A and C) and 500 μm (B).

Figure 2.

Representative hyphal morphology mutants. The wild-type parent (A) and various mutants (B-BB) were grown at 25°C on minimal agar medium without added osmotic stabilizer and then shifted to restrictive temperature (39°C) for 10 h. Pictures were taken of hyphae growing embedded in agar with the exception of panel X, which shows hyphae growing on an agar surface. (The pod-9 mutant phenotype was similar, but more difficult to photograph, when the hyphae were growing embedded in agar.) All mutants shown here displayed wild-type or nearly wild-type growth at 25°C. Other mutants had phenotypes more-or-less similar to those shown here (see Table 1). Bars, 20 μm.

Figure 3.

Model for hyphal branching. The phenotypes of several of the isolated mutants suggest that branching and the generation of new hyphal tips is a genetically separable process consisting of at least four discrete steps: (1) the selection of a new branch site; (2) the broadening of the marked spot into a zone of growth; (3) the production of a small stalk-like branch; and (4) a microtubule dependent maturation step to form a functional tip. On the left, the proposed steps and the gene(s) involved are indicated; on the right are references to the corresponding images of mutant phenotypes in Figure 2. See text and Table 1 for detailed descriptions of the mutants. Bars, 20 μm.