SummaryGrowth-speed-correlated localization of exocyst and polarisome components in growth zones of Ashbya gossypii hyphal tips

Morphogenesis of filamentous ascomycetes includes continuously elongating hyphae, frequently emerging lateral branches, and, under certain circumstances, symmetrically dividing hyphal tips. We identified the formin AgBni1p of the model fungus Ashbya gossypii as an essential factor in these processes. AgBni1p is an essential protein apparently lacking functional overlaps with the two additional A. gossypii formins that are nonessential. Agbni1 null mutants fail to develop hyphae and instead expand to potato-shaped giant cells, which lack actin cables and thus tip-directed transport of secretory vesicles. Consistent with the essential role in hyphal development, AgBni1p locates to tips, but not to septa. The presence of a diaphanous autoregulatory domain (DAD) indicates that the activation of AgBni1p depends on Rho-type GTPases. Deletion of this domain, which should render AgBni1p constitutively active, completely changes the branching pattern of young hyphae. New axes of polarity are no longer established subapically (lateral branching) but by symmetric divisions of hyphal tips (tip splitting). In wild-type hyphae, tip splitting is induced much later and only at much higher elongation speed. When GTP-locked Rho-type GTPases were tested, only the young hyphae with mutated AgCdc42p split at their tips, similar to the DAD deletion mutant. Two-hybrid experiments confirmed that AgBni1p interacts with GTP-bound AgCdc42p. These data suggest a pathway for transforming one axis into two new axes of polar growth, in which an increased activation of AgBni1p by a pulse of activated AgCdc42p stimulates additional actin cable formation and tip-directed vesicle transport, thus enlarging and ultimately splitting the polarity site.

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)-and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.

AgRho1a. Furthermore, it enables the mutated allele to complement the lethality of an AgRHO1b deletion. In summary, our findings show that a simple mutation in the switch I region of a GTP-binding protein can change its affinity towards its GAPs, which finally leads to a decoupling of very similar protein function without impairing effector interaction.Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/7/1065/DC1 Journal of Cell Science 1066 organization of the AgRHO1 genes together with the neighboring genes in A. gossypii and S. cerevisiae is shown in Fig. 1A. The two copies very probably originate from a tandem-duplication event.Little is known about the function of ABR182W. Deletion mutants have been reported to display a weak lysis phenotype and a weak sensitivity to the chitin-binding dye Calcofluor White (Walther and Wendland, 2005). Here, we studied the differences in function of a pair of duplicated RHO-type GTP-binding proteins in A. gossypii and compared them with their single homolog in budding yeast. Surprisingly, we found that a single residue in AgRho1a is altered in the highly conserved switch I region. This residue contributes to divergence of protein function and alters GAP specificity without impairing effector interaction. ResultsThe lytic phenotype of Agrho1a⌬ is temperature-dependent We first characterized Rho1 protein function in A. gossypii to test whether protein function diverged. An alignment of the protein sequences shows that AgRho1a is 73.9% identical to AgRho1b and 75.6% identical to ScRho1, whereas the latter two proteins share the highest number of identical residues (83.3%) (Fig. 1B). As a first step towards defining the functional difference between AgRHO1a and AgRHO1b, we compared the phenotypes of deletion mutants. Although Agrho1aΔ formed a mature mycelium that grew slightly slower than the wild-type control, deletion of AgRHO1b resulted in cells that never reached the state of a mature mycelium and died because of cell lysis (Fig. 1C). Agrho1aΔ cells also lysed but only few hyphae were affected. Fig. 1D shows an example of lysing hyphae taken from Movie 1 (supplementary material). We also monitored the strain deleted for AgRHO1a at different growth temperatures. Lysis of adult mycelia was quantified by an assay that visualizes the release of cytoplasmic alkaline phosphatase upon lysis by a color reaction (Fig. 1E, for details, refer to Materials and Methods). An Agslt2⌬ strain was used as a lysis control. AgSLT2 is the homolog of the S. cerevisiae MAP kinase responsible for maintenance of cellular integrity (Torres et al., 1991). In contrast to Agrho1b deletion mutants, Agslt2 deletions can form a mature mycelium despite extensive lysis. Hyphae at the colony border grow normally and cannot be distinguished microscopically from wild type but the inner, older parts of the mycelium show extensive lysis. At 25°C and 30°C, Agrho1a deletion mutants released about half as much alkaline phosphatase as the Agslt2 deletion mutants. Surprisingly,...

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