Nuclear dynamics during germination, conidiation, and hyphal fusion of Fusarium oxysporum.

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.

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

We use the fungus Ashbya gossypii to investigate how its polar growth machinery is organized to achieve sustained hyphal growth. In slowly elongating hyphae exocyst, cell polarity and polarisome proteins permanently localize as cortical cap at hyphal tips, thus defining the zone of secretory vesicle fusion. In tenfold faster growing hyphae, this zone is only slightly enlarged demonstrating a capacity of hyphal growth zones to increase rates of vesicle processing to reach higher speeds. Concomitant with this increase, vesicles accumulate as spheroid associated with the tip cortex, indicating that a Spitzenkörper forms in fast hyphae. We also found spheroid-like accumulations for the exocyst components AgSec3, AgSec5, AgExo70 and the polarisome components AgSpa2, AgBni1 and AgPea2 (but not AgBud6 or cell polarity factors such as AgCdc42 or AgBem1). The localization of AgSpa2, AgPea2 and AgBni1 depend on each other but only marginally on AgBud6, as concluded from a set of deletions. Our data define three conditions to achieve fast growth at hyphal tips: permanent presence of the polarity machinery in a confined cortical area, organized accumulation of vesicles and a subset of polarity components close to this area, and spatial separation of the zones of exocytosis (tip front) and endocytosis (tip rim).

The function of two closely related Rho proteins is determined by an atypical switch I region.

We show here that the encoded proteins of the two duplicated RHO1 genes from the filamentous fungus Ashbya gossypii, AgRHO1a and AgRHO1b have functionally diverged by unusual mutation of the conserved switch I region. Interaction studies and in vitro assays suggest that a different regulation by the two GTPase activating proteins (GAPs) AgLrg1 and AgSac7 contributes to the functional differences. GAP-specificity and protein function is determined to a large part by a single position in the switch I region of the two Rho1 proteins. In AgRho1b, this residue is a tyrosine that is conserved among the Rho-protein family, whereas AgRho1a carries an atypical histidine at the same position. Mutation of this histidine to a tyrosine changes GAP-specificity, protein function and localization of 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.

From function to shape: a novel role of a formin in morphogenesis of the fungus Ashbya gossypii.

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.