In budding yeast, new sites of polarity are chosen with each cell cycle and polarization is transient. In filamentous fungi, sites of polarity persist for extended periods of growth and new polarity sites can be established while existing sites are maintained. How the polarity establishment machinery functions in these distinct growth forms found in fungi is still not well understood. We have examined the function of Axl2, a transmembrane bud site selection protein discovered in Saccharomyces cerevisiae, in the filamentous fungus Ashbya gossypii. A. gossypii does not divide by budding and instead exhibits persistent highly polarized growth, and multiple axes of polarity coexist in one cell. A. gossypii axl2⌬ (Agaxl2⌬) cells have wavy hyphae, bulbous tips, and a high frequency of branch initiations that fail to elongate, indicative of a polarity maintenance defect. Mutant colonies also have significantly lower radial growth and hyphal tip elongation speeds than wild-type colonies, and Agaxl2⌬ hyphae have depolarized actin patches. Consistent with a function in polarity, AgAxl2 localizes to hyphal tips, branches, and septin rings. Unlike S. cerevisiae Axl2, AgAxl2 contains a Mid2 homology domain and may function to sense or respond to environmental stress. In support of this idea, hyphae lacking AgAxl2 also display hypersensitivity to heat, osmotic, and cell wall stresses. Axl2 serves to integrate polarity establishment, polarity maintenance, and environmental stress response for optimal polarized growth in A. gossypii.The establishment and maintenance of cell polarity are essential for proper cell morphology and growth in both unicellular and multicellular eukaryotes (25, 39). Directional growth, neuronal development and function, membrane trafficking in epithelial cells, and whole-cell motility by chemotaxis all rely on the generation of cell polarity (5, 88, 97). Polar growth has been linked to fungal virulence in human pathogens such as Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, and Paracoccidioides brasiliensis (3,6,67,80). Many mechanisms governing cell polarity and polarized growth were discovered in the budding yeast, Saccharomyces cerevisiae, and are highly conserved from unicellular eukaryotes to humans (62).In S. cerevisiae, budding can occur in either an axial or a bipolar manner, which is determined by specific landmark proteins. The axial landmark consists of the septin complex, S. cerevisiae Bud3 (ScBud3), ScBud4, ScAxl1, and ScAxl2 and functions by recruiting members of the Cdc42 signaling pathway (1,14,30,31,38,44,45,69,72,98). The localization and activation of Cdc42 at sites specified by the landmarks then direct bud formation through polarization of the actin cytoskeleton (2, 99). Polarized actin cables form tracks on which myosins transport secretory vesicles to the growing bud (11,29,37). Polarized cortical actin patches are sites of endocytosis and as such can function to internalize any polarity factors that diffuse beyond the region of polarized growth (36,57,61).ScA...