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Files in this Data Supplement:
Fig. S1. Comparison of fungal WASP loci. Arrows (not to size) represent the order and transcriptional orientation of genes. Homologues are named according to the systematic yeast nomenclature and with the designated three letter code. Shading corresponds to the yeast chromosomes [chromosome XII (=L) light and chromosome XV (=O) dark]. Sequence information can be obtained from http://www.yeastgenome.org/ for S. cerevisiae and http://www-sequence.stanford.edu/group/candida/ for C. albicans; the accession number for A. gossypii WAL1 locus is AY144115. YLR287c is an uncharacterized yeast ORF coding for a 356 amino acid protein.
Fig. S2. Alignment of fungal WASP homologues. Amino acids corresponding to most fo the aligned sequences are shaded. Sequence identity is particularly high in the N- and C-terminal domains. Sequence origin or accession numbers: A. gossypii AgWal1p, AY144115; C. albicans CaWal1p, http://www-sequence.stanford.edu/group/candida/; S. cerevisiae ScBee1p, NP_014824; S. pombe SpWsp1, AAB92587; N. crassa NcWASP, http://www-genome.wi.mit.edu/annotation/fungi/neurospora/.
Fig. S3. Disruption of AgWAL1. PCR-based methods were used to disrupt AgWAL1. The selectable marker gene GEN3 was amplified with primers S1 and S2 providing the homology region for homologous recombination (black boxes). Upon transformation of the PCR product, homologous recombination resulted in the insertion of GEN3 at the indicated position. Diagnostic PCR with G1-G4 primers was used to verify correct gene targeting. Sporulation of heterokaryotic transformants and clonal selection yielded the Agwal1 mutant strain.
Movie 1. (see Fig.4a). Spore germination of A. gossypii wild type. Freshly germinated spores were imaged over several hours to follow the development of juvenile mycelium. (Time=hours:minutes).
Movie 2. (see Fig. 4b) Spore germination of the A. gossypii wal1 mutant strain. Needle-shaped spores of the heterokaryon were germinated in liquid medium under selective conditions that only allow growth of the mutant spores. Imaging was done under the same settings as with the wild type. (Time=hours:minutes).
Movie 3. (see Fig. 5) Adult mycelium of the A. gossypii wild type. Fast polarized growth and cytoplasmic streaming of wild type hyphae was imaged for several hours using the same settings as in Fig. 4. Note the generation of large vacuoles, the directed movement of vacuoles, the redirection of cytoplasmic streaming upon septation and the reversal of cytoplasmic streaming once tip growth was terminated (also seen in Movie 1). (Time=hours:minutes).
Movie 4. (see Figs 6,7) Timelapse analysis of polarized growth of the A. gossypii wal1 mutant strain. Note the absence of large vacuoles but the ability of the hyphae to promote slow tip growth and apical tip branching. (Time=hours:minutes).
Movie 5. (see Fig. 7) Growth defects of subapical segments in the wal1 hyphae. Enlarged segments of Movie 4 (Fig. 6) show hyphal segments in which new growth results in abnormal swellings. (Time=hours:minutes).
Movie 6. (see Fig. 8a,b) Endocytosis in the A. gossypii wild type (Fig. 8a, left) and wal1 mutant (Fig. 8b, right). Uptake of the lipophilic dye FM4-64 was monitored over several minutes. Note the formation and fusion of endocytotic vesicles in the wild type. Also note the hyphal tip of the wal1 hypha that does not contain endocytotic vesicles. (Time=minutes:seconds).
Movie 7. (see Figs 8e,f) Vacuolar morphology and movement in subapical compartments of the A. gossypii wild type (Fig. 8e, left) and wal1 mutant (Fig. 8f, right). Note the shape changes of wild type vacuoles in comparison to the round shaped wal1 vacuoles. (Time=minutes:seconds).
Movie 8. (Fig. 8c,g) Early endocytosis (Fig. 8c, left) as well as vacuolar morphology and movement (Fig. 8g, right) in the A. gossypii cla4 mutant strain. (Time=minutes:seconds).
Movie 9. (Fig. 8d,h) Early endocytosis (Fig. 8d, left) and vacuolar morphology and movement (Fig. 8h, right) in the A. gossypii bem2 mutant strain. (Time=minutes:seconds).
Movie 10. Vacuolar morphology and movement in the A. gossypii wild type. Hyphae were grown under timelapse conditions in the presence of the lipophilic dye FM4-64. (Time=hours:minutes).
Movie 11. Vacuolar morphology and movement in the A. gossypii wal1 mutant. Hyphae were grown under timelapse conditions in the presence of the lipophilic dye FM4-64. Note the hyphal tips that are empty of endocytosis vesicles as well as the lack of tip directed movement of vacuoles in this strain. (Time=hours:minutes).
Movie 12. (see Fig. 8i,j) Localization and movement of mitochondria in the A. gossypii wild type (Fig. 8i, left) and the wal1 strain (Fig. 8j, right). Note that in both strains mitochondria are localized in the hyphae including the apical compartment which contrasts the localization of endocytotic vesicles in the wal1 strain. (Time= minutes:seconds).
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