A Ras-like GTPase is involved in hyphal growth guidance in the filamentous fungus Ashbya gossypii

Abstract

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Delta deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.

Figure 1.

Alignment of GTP-binding proteins of the Ras-superfamily. Identical amino acids residues are shaded. Conserved domains are highlighted. Sequence accession numbers are as follows: A. gossypii Rsr1 (AgRsr1), AE016819; S. cerevisiae ScRsr1-ScBud1, P13856; C. albicans CaRsr1, AAB81286; N. crassa Nckrev-1, BAA32410; S. cerevisiae Ras1 (ScRas1), P01119; and S. cerevisiae Ras2 (ScRas2), P01120.

Figure 7.

Functional complementation of Agleu2Δrsr1Δ and localization of AgRsr1p. Agleu2Δ and Agleu2Δrsr1Δ strains were transformed with the indicated plasmids: the control plasmid pRS415 carrying S. cerevisiae ARS and CEN sequences and the LEU2 gene, pAG919 carrying in addition an in-frame GFP-AgRSR1 fusion and pAG919-1 carrying an out-of-frame GFP-AgRSR1 fusion. Left column, fungal colonies grown for 5 d on leucine drop out agar. Middle column, images of colony edges of the respective transformants with tip branching indicated by asterisks and lateral branching by arrows. Right column, fluorescence microscopy images taken under identical conditions of hyphae grown for 16 h in liquid leucine drop out medium. Tip localization of GFP-fluorescence with a cap-like structure at the very tip can be observed for Agleu2Δrsr1Δ(pAG919). Bar, 50 μm (middle); 5 μm (right).

Figure 2.

Development of an A. gossypii wild-type young mycelium monitored by in vivo time-lapse microscopy. Spores were pregrown for 4 h in complete liquid medium at 30°C before mounting for videomicroscopy (see Materials and Methods). Digital images were collected at 2-min intervals (see Movie 1). Representative frames taken at 2-h intervals show the development of wild-type mycelium. Typical landmarks are as follows: isotropic growth of the germ bubble (0 h), formation of the first (2 h) and second germ tube (6 h), and generation of septa and lateral branches (starting at 8–10 h). The time lapse was carried out at room temperature (25°C).

Figure 3.

Development of a Agrsr1Δ young mycelium monitored by in vivo time-lapse microscopy (see Movie 2). Spores were prepared and images taken as described in Figure 2. Arrowheads point to changes in growth direction and the asterisk marks a complete growth arrest of a lateral branch.

Figure 4.

Analysis of hyphal elongation rate in young mycelium of A. gossypii wild type and in Agrsr1Δ. The source of these measurements is Movie 1 and Movie 2, and the elongation of the first germ tube was measured over the entire length of the movies. Time zero corresponds to the last steps of isotropic growth before germination of the first germ tube (see Figures 3 and 4). Arrows mark prolonged periods of pausing in Agrsr1Δ.

Figure 5.

Chitin distribution in wild type (A and B) and in Agrsr1Δ (C and D). Septa and growth regions of hyphal tips are stained with calcofluor. Septa are thicker in Agrsr1Δ and develop at shorter distances compared with wild type, very likely due to frequent pausing events. Arrows mark sites of abortive branch initiations. Bar, 10 μm.

Figure 6.

Organization of the actin cytoskeleton in wild type (A) and in Agrsr1Δ (B). Germinated spores of both strains were grown in liquid complete medium for 12 h at 30°C, fixed with formaldehyde, stained with Alexa 488-phalloidin, and analyzed by fluorescence microscopy. Four representative images of hyphal tips (n = 100 for each strain) are presented. Bar, 10 μm.

Figure 8.

Loss of localization of the polarisome component AgSpa2-GFP during tip growth in Agrsr1Δ. AgSpa2p-GFP distribution in growing hyphal tips as well as during lateral branch initiation was monitored by in vivo time-lapse microscopy in A. gossypii wild type and in Agrsr1Δ. The series of images represent sections of selected frames of Movies 3 and 4. (A) Permanent localization of AgSpa2p-GFP in a growing tip. (B) Polarized AgSpa2p-GFP at a branching site in A. gossypii wild type. (C) Transient disappearance and reappearance of AgSpa2-GFP in Agrsr1Δ at a hyphal tip. (D) Transient assembly of AgSpa2p-GFP at a cortical site. The dashed lines are at constant positions in each image to allow monitoring of growth arrest and resumption of tip growth. The time interval represents minutes in real time. Bar, 10 μm.