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Identification of a motor protein required for filamentous growth in Ustilago maydis

1997; Springer Nature; Volume: 16; Issue: 12 Linguagem: Inglês

10.1093/emboj/16.12.3464

1460-2075

Christiane Lehmler, Gero Steinberg, Karen M. Snetselaar, Manfred Schliwa, Regine Kahmann, Michael Bölker,

Photosynthetic Processes and Mechanisms

Article15 June 1997free access Identification of a motor protein required for filamentous growth in Ustilago maydis Christiane Lehmler Christiane Lehmler Institut für Genetik und Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, D-80638 München, Germany Search for more papers by this author Gero Steinberg Gero Steinberg Adolf Butenandt Institut/Zellbiologie, Universität München, Schillerstrasse 42, D-80336 München, Germany Search for more papers by this author Karen M. Snetselaar Karen M. Snetselaar Department of Biology, St Joseph's University, 5600 City Avenue, Philadelphia, PA, 19131 USA Search for more papers by this author Manfred Schliwa Manfred Schliwa Adolf Butenandt Institut/Zellbiologie, Universität München, Schillerstrasse 42, D-80336 München, Germany Search for more papers by this author Regine Kahmann Regine Kahmann Institut für Genetik und Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, D-80638 München, Germany Search for more papers by this author Michael Bölker Corresponding Author Michael Bölker Institut für Genetik und Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, D-80638 München, Germany Search for more papers by this author Christiane Lehmler Christiane Lehmler Institut für Genetik und Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, D-80638 München, Germany Search for more papers by this author Gero Steinberg Gero Steinberg Adolf Butenandt Institut/Zellbiologie, Universität München, Schillerstrasse 42, D-80336 München, Germany Search for more papers by this author Karen M. Snetselaar Karen M. Snetselaar Department of Biology, St Joseph's University, 5600 City Avenue, Philadelphia, PA, 19131 USA Search for more papers by this author Manfred Schliwa Manfred Schliwa Adolf Butenandt Institut/Zellbiologie, Universität München, Schillerstrasse 42, D-80336 München, Germany Search for more papers by this author Regine Kahmann Regine Kahmann Institut für Genetik und Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, D-80638 München, Germany Search for more papers by this author Michael Bölker Corresponding Author Michael Bölker Institut für Genetik und Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, D-80638 München, Germany Search for more papers by this author Author Information Christiane Lehmler1, Gero Steinberg2, Karen M. Snetselaar3, Manfred Schliwa2, Regine Kahmann1 and Michael Bölker 1 1Institut für Genetik und Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, D-80638 München, Germany 2Adolf Butenandt Institut/Zellbiologie, Universität München, Schillerstrasse 42, D-80336 München, Germany 3Department of Biology, St Joseph's University, 5600 City Avenue, Philadelphia, PA, 19131 USA The EMBO Journal (1997)16:3464-3473https://doi.org/10.1093/emboj/16.12.3464 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The phytopathogenic fungus Ustilago maydis exists in two stages, the yeast-like haploid form and the filamentous dikaryon. Both pathogenicity and dimorphism are genetically controlled by two mating-type loci, with only the filamentous stage being pathogenic on corn. We have identified two genes (kin1 and kin2) encoding motor proteins of the kinesin family. Kin1 is most similar to the human CENP-E gene product, while Kin2 is most closely related to the conventional kinesin Nkin of Neurospora crassa. Deletion mutants of kin1 had no discernible phenotype; Δkin2 mutants, however, were severely affected in hyphal extension and pathogenicity. The wild-type dikaryon showed rapid tip growth, with all the cytoplasm being moved to the tip compartment. Left behind are septate cell wall tubes devoid of cytoplasm. In Δkin2 mutants, dikaryotic cells were formed after cell fusion, but these hyphal structures remained short and filled with cytoplasm. A functional green fluorescent protein (GFP)–Kin2 fusion was generated and used to determine the localization of the motor protein by fluorescence microscopy. Inspection of the hyphal tips by electron microscopy revealed a characteristic accumulation of darkly stained vesicles which was absent in mutant cells. We suggest that the motor protein Kin2 is involved in organizing this specialized growth zone at the hyphal tip, probably by affecting the vectorial transport of vesicles. Introduction The phytopathogenic fungus Ustilago maydis can infect corn plants only in the dikaryotic stage of its life cycle (see Christensen, 1963). Formation of the dikaryon by fusion of yeast-like haploid cells is associated with a morphological switch to filamentous growth. Cell fusion, maintenance of hyphal growth and pathogenicity are controlled by the mating-type loci a and b (see Banuett, 1995). The biallelic a locus encodes a pheromone-based cell recognition system, the multiallelic b locus codes for homeodomain proteins that function as master regulators of filamentous growth and pathogenicity (Bölker et al., 1992; Gillissen et al., 1992; Hartmann et al., 1996). When two haploid cells of different a and b genotype (so-called compatible combinations) are mixed, the secreted pheromones bind to their cognate receptors and induce the formation of mating tubes that orient their growth towards the pheromone source and fuse at their tips (Snetselaar, 1993; Banuett and Herskowitz, 1994; Spellig et al., 1994; Snetselaar et al., 1996). In the common cytoplasm of the dikaryon, the products of the two different b alleles form heterodimers that control the switch to hyphal growth and pathogenicity (Kämper et al., 1995). In the dikaryon, the pheromone genes continue to be expressed, leading to an autocrine stimulation of the pheromone signaling cascade that activates b gene expression (Hartmann et al., 1996; Urban et al., 1996). The dimorphic transition can be followed on solid media containing charcoal (Day and Anagnostakis, 1971; Holliday, 1974) or in microscopic assays (Snetselaar and Mims, 1992; Snetselaar et al., 1997). After cell fusion, a straight hypha emerges and the nuclei migrate into the growing filament. The tip compartment containing the cytoplasm becomes delimited from the older vacuolated part of the hypha by a septum. During hyphal tip growth, new septae are built regularly to separate the growing tip zone from these vacuolated parts left behind (Snetselaar and Mims, 1992). This mode of growth is also characteristic for the early infection stages in planta (Snetselaar and Mims, 1992). Since U.maydis is amenable to classical and molecular genetic analyses, it can be used as a model organism to study the intracellular processes underlying this morphogenetic program. In particular, we have become interested in the function of the cytoskeleton for hyphal tip growth. A characteristic feature of tip growth in fungi is the long distance transport and targeted fusion of vesicles containing cell wall precursors. It has been proposed that motor proteins acting on the cytoskeleton are involved in these processes, but their precise role remains to be elucidated (Heath, 1994). Since microtubules have been shown to play a role in fungal dimorphism (Harris et al., 1989), a participation of kinesin- or dynein-like motor proteins appears likely. In fungi, several members of these protein families have been identified. They affect such diverse processes as karyogamy in Saccharomyces cerevisiae (Meluh and Rose, 1990), nuclear division in Aspergillus nidulans (Enos and Morris, 1990) and nuclear distribution and hyphal morphogenesis Neurospora crassa (Plamann et al., 1994). In this study, we have identified two genes encoding motor proteins of the kinesin family in U.maydis. We demonstrate that one of these proteins is required for hyphal extension and affects fungal virulence. Results Identification of genes encoding motor proteins of the kinesin family We have used degenerate primers to amplify a highly conserved region within the motor domain of kinesins. Two products were identified that were used to isolate corresponding cosmid clones. Sequencing revealed open reading frames whose predicted amino acid sequences were highly similar to other members of the kinesin family. The corresponding genes were designated kin1 and kin2 and have been sequenced in their entirety. For kin1, two in-frame translational start codons, both of which are embedded in a sequence context confined to the fungal consensus (Ballance, 1991), can be found upstream of the region encoding the conserved motor domain. The derived polypeptide sequence of Kin1 consists of 1459 amino acids if the first ATG codon is used for translation initiation (Figure 1A, DDBJ/EMBL/GenBank accession No. U92844). The motor domain of Kin1 (amino acids 237–649) is most similar to that of the human CENP-E protein (Yen et al., 1991) with 40% identity (Figure 1A). The amino acid sequence of the C-terminal domain of Kin1 is predicted to adopt a coiled-coil structure but lacks significant sequence similarity to known proteins in the data base. Figure 1.Deduced amino acid sequences of the kinesin motor proteins Kin1 and Kin2 from U.maydis. (A) Amino acid sequence of U.maydis Kin1. The methionine residue corresponding to the second potential translational start codon is underlined. The motor domain of Kin1 is aligned to that of human CENP-E protein (Swissprot accession No. Q02224). (B) Alignment of the U.maydis kinesin Kin2 with the conventional kinesin Nkin from N.crassa (DDBJ/EMBL/GenBank accession No. L47106). Identical amino acids are shaded, gaps are indicated by dashes. Download figure Download PowerPoint The predicted product of the kin2 gene comprises 968 amino acids (Figure 1B, DDBJ/EMBL/GenBank accession No. U92845). The Kin2 protein has an N-terminal motor domain and displays 52% identity across the entire amino acid sequence to the recently identified Nkin protein from N.crassa (Steinberg and Schliwa, 1995) (Figure 1B). Phenotype of Δkin1 and Δkin2 mutants To analyze the function of the kin1 and kin2 genes, deletion constructs were generated (see Materials and methods). These mutant alleles were introduced by gene replacement into the haploid strains FB1 (a1 b1) and FB2 (a2 b2) which carry different a and b alleles. The absence of the wild-type allele was demonstrated by Southern analysis (Figure 2). Furthermore, this analysis did not reveal additional hybridization signals which could have indicated the existence of genes closely related to kin1 or kin2. Western analysis using the polyclonal Nkc antibody directed against the C-terminus of the related N.crassa Nkin protein (Steinberg and Schliwa, 1995; Steinberg, 1997) identified a single band of the expected size in wild-type cells which is absent in FB1Δkin2 and FB2Δkin2 extracts (Figure 3). Both the Δkin1 and the Δkin2 mutants were viable, and neither cell morphology nor growth of haploid cells were significantly different from the parental strains (data not shown). When compatible mutant strains were mixed and co-spotted on charcoal plates, Δkin1 mutant combinations developed dikaryotic hyphae indistinguishable from those formed by compatible wild-type strains (Figure 4). This suggests that the kin1 gene is not involved in cell fusion and hyphal growth. In crosses between a Δkin2 mutant strain and a compatible wild-type strain, long aerial hyphae were observed (Figure 4), but their appearance was somewhat delayed compared with filaments derived from a cross of compatible wild-type strains (not shown). In a mixture of compatible Δkin2 strains, however, formation of long aerial hyphae was abolished (Figure 4). Instead, colonies were covered with a loose fur of stunted aerial hyphae (Figure 4). This suggests that the Δkin2 mutation affects either the fusion step or the development of dikaryotic hyphae. To distinguish between these possibilities, we performed agar drop matings (Snetselaar et al., 1996) to follow the mating reaction microscopically (Figure 5). When compatible wild-type strains FB1 and FB2 were placed in adjacent drops on water agar, formation of conjugation tubes could be observed already after 2 h. Their numbers had increased dramatically after 3 h, and after 5 h the first fusion events could be observed (Figure 5). After 12 h, straight dikaryotic hyphae could be detected (Figure 5). In mixtures of FB1Δkin2 and FB2Δkin2, the initial events appeared delayed for ∼1 h. After 5 h, fusion events could be observed but the formation of extended straight hyphae did not occur even after 12 h incubation (Figure 5). Figure 2.Southern analysis of Δkin strains. In the lower parts, restriction maps of the wild-type and the Δkin mutant alleles are shown. The motor domains are shaded and the hygromycin (Hyg) and carboxin (Cbx) resistance cassettes are indicated as black bars. Restriction sites are shown for HindIII (H), HincII (Hi), SalI (S), EcoRI (E), RsaI (R) and BamHI (B). (A) DNA was isolated from the strains indicated above each slot, digested with HindIII and probed with the 3.6 kb HindIII fragment comprising the N-terminal portion of the kin1 gene. (B) DNA of the strains indicated above each slot was cleaved with EcoRI and BamHI and probed with the 3.8 kb EcoRI fragment containing the kin2 gene. Download figure Download PowerPoint Figure 3.Immunodetection of Kin2 and GFP–Kin2 fusion proteins. Protein extracts were prepared from the strains indicated above, and 75 μg total protein per slot was separated on a 7.5% SDS–polyacrylamide gel. After blotting, Kin2 proteins were detected using the Nkc antibody. Size markers are indicated on the right. CL42 is FB2Δkin2/psGFP-Kin2#1 and CL53 is FB1Δkin2/pKin2-sGFP#3. Download figure Download PowerPoint Figure 4.Kin2 affects formation of aerial hyphae. Plate mating assays were performed on charcoal-containing media by co-spotting strains indicated on the left and on top of the upper panel. FB1 (a1 b1) and FB2 (a2 b2) were used as wild-type strains. The formation of dikaryotic aerial hyphae is visible as white mycelium. In the lower panels, the edges of colonies are shown at higher magnification for the cross of FB1 and FB2 wild-type strains and the cross between the Δkin2 mutant derivatives. Download figure Download PowerPoint Figure 5.Formation of conjugation tubes and mating reaction between compatible U.maydis wild-type and Δkin2 mutant sporidia. Small droplets of cells were placed on water agar in close proximity and overlaid with liquid paraffin. Mating reactions between the U.maydis cells were observed microscopically, and photographs were taken at the time points indicated on the left. In the schematic drawing on the top, colonies are indicated as shaded circles and the view shown in the micrographs is marked by a rectangle. Arrowheads point to the straight dikaryotic hyphae emerging from mating events between compatible wild-type cells after 20 h incubation. The bar corresponds to 50 μm. Download figure Download PowerPoint Next we have analyzed isolated mating structures for both compatible wild-type and compatible Δkin2 mutant strain combinations. Cells were allowed to mate on water agar after mixing both mating partners, aliquots were removed at different times and analyzed microscopically (Figure 6). Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI). In the combination of compatible wild-type strains FB1 (a1 b1) and FB2 (a2 b2), fused cells that have initiated hyphal development could be observed after 4.5 h (Figure 6). At this stage the length of the hyphae approximated 1–2 times the length of haploid cells, and the nuclei were beginning to invade the filament. After 6 h, the filaments had reached 6–10 times the length of a haploid cell, and most nuclei were found in the hyphal cells (Figure 6). Some of the haploid progenitor cells appeared empty, indicating that the cytoplasm had been translocated to the growing hypha. After prolonged incubation (20 h), the hyphae exceeded the length of the progenitor cells >20-fold. The cytoplasm was restricted to the tip compartment, and empty cell structures divided by regularly spaced septa could be seen at the distal end of the hyphae (Figure 6). Figure 6.DIC (differential interference contrast) and epifluorescence micrographs of DAPI-stained U.maydis wild-type and Δkin2 mating structures. Mixtures of cells were incubated on water agar under paraffin, and aliquots were removed at the time points indicated on the left. For each time point, one micrograph of wild-type cells (left) and two micrographs of Δkin2 mutant cells (right) are shown. The upper and lower panels show DIC and DAPI views, respectively, of the same field. Arrowheads point to regularly spaced septa in the empty cell wall structures of dikaryotic wild-type hyphae. The bar corresponds to 15 μm. Download figure Download PowerPoint When compatible Δkin2 mutant cells were mixed, cell fusion also occurred within 4.5 h (Figure 6). However, the emerging hyphae were much shorter than in the wild-type combination, and nearly all nuclei were still found in the progenitor cells. At 6 h after mixing, the filaments reached approximately twice the length of haploid cells and, in some cases, the nuclei had already migrated into the hyphae (Figure 6). Often, however, the nuclei could be found at the neck of the emerging filament. Even after 20 h the length of the hyphae approximated at most 5 times the length of haploid cells (Figure 6). In general, the hyphae and progenitor cells appeared thicker and more irregular than in mating structures derived from wild-type strains. Nuclei were more rounded and more closely spaced than in wild-type hyphae, and progenitor cells often exhibited irregular projections which were empty in most cases (Figure 6). Most notably, however, long straight filaments with empty compartments at their distal ends could not be detected in the cross between Δkin2 mutant strains. This indicates that the rapid extension of dikaryotic hyphae and, in particular, the cytoplasmic movement to the tip cell compartment is impaired in Δkin2 mutants. Deletion of kin2 affects pathogenicity To test the influence of the kinesin genes kin1 and kin2 on pathogenic development, pairwise combinations of wild-type and mutant strains were co-injected into maize plants. The combination of FB1Δkin1 and FB2Δkin1 was as effective in tumor induction as the respective wild-type combination (Table I). However, when two compatible Δkin2 mutant strains were crossed, tumor development was observed only in 8% of infected plants while for the wild-type combination of strains, 94% of infected plants developed tumors (Table I). To exclude the possibilty that this effect is due to defects in cell fusion or nuclear migration, we have constructed a stable diploid strain CLD1121 (a1 a2 b1 b2 Δkin2-Cbx Δkin2-Hyg) where both alleles of kin2 are disrupted (see Materials and methods). Since CLD1121 is heterozygous for a and b, this strain should be pathogenic without the requirement for cell fusion. Compared with the solopathogenic diploid strain FBD11 (a1 a2 b1 b2), CLD1121 was severely affected in its ability to induce tumors (Table I). This illustrates that Kin2 does not exert its effect during the cell fusion step but must play an important role during fungal development in planta. Table 1. Pathogenicity of Δkin1 and Δkin2 mutants Strain combination No. of infected plants Tumor formation FB1×FB2 19 18 (94%) FB1Δkin1×FB2Δkin1 12 9 (75%) FB1Δkin2×FB2Δkin2 51 4 (8%) FBD11 33 14 (42%) CLD1121 66 3 (5%) Localization of GFP–Kin2 fusion proteins To relate the observed phenotypes to the subcellular localization of Kin2, we have constructed translational fusions between Kin2 and the green fluorescent protein (GFP) from Aequorea victoria (Chalfie et al., 1994). Plasmids that express either an N-terminal (pSGFP-Kin2) or a C-terminal fusion protein (pKin2-SGFP) under the control of constitutive promoters were generated and transformed into haploid Δkin2 mutant strains (Materials and methods). Transformants that carry ectopically integrated plasmids were tested for mating behavior in crosses with compatible wild-type and Δkin2 strains. For both fusion constructs, transformants could be identified which showed a vigorous mating reaction with compatible Δkin2 strains (data not shown). This indicates that both the N- and C-terminal GFP–Kin2 fusion proteins are biologically active. Total protein extracts of transformants were analyzed by Western blotting using the polyclonal Nkc antibody as described before. All transformants expressed levels of GFP–Kin2 which were ∼5- to 20-fold higher than in haploid wild-type cells (two examples are shown in Figure 3). In fluorescence microscopy, such transformants expressing either N- or C-terminal fusion proteins showed evenly distributed slightly granular cytoplasmic staining, with many cells displaying a single intensely stained spot located at either pole of the cell (Figure 7A and B). Because of the overexpression of the fusion proteins, it is likely that these spots represent artefacts. Interestingly, upon treatment of growing cells for 30 min with 100 μM of CCCP (carbonylcyanide-m-chlorophenylhydrazone), a potent uncoupler of oxidative phosphorylation, a completely different staining pattern was observed: most of the green fluorescence was found to be associated with structures characteristic of microtubules (Figure 7C). For comparison, an immunostaining of microtubules in U.maydis cells is shown in Figure 7D. The association of GFP–Kin2 with microtubules is reversible, since the normal staining pattern could be restored within 3 h after removal of CCCP (data not shown). This suggests that depletion of ATP leads to tight binding of Kin2 molecules to cytoplasmic microtubules, as has been observed for conventional kinesins in vitro (see Bloom and Endow, 1995). Figure 7.Intracellular localization of GFP–Kin2 fusion proteins. The left and right panels show DIC and epifluorescence views, respectively, of the same field. (A) FB1Δkin2/pKin2-sGFP#3 (CL53) sporidia expressing C-terminal GFP–Kin2 fusion protein. (B) FB2Δkin2/psGFP-Kin2#1 (CL42) sporidia expressing N-terminal GFP–Kin2 fusion protein. (C) The C-terminal GFP–Kin2 fusion protein expressed in CL53 is bound to microtubules after 30 min incubation with CCCP. (D) Immunostaining of microtubules in FB1Δkin2 cells. (E) Dikaryotic hyphae resulting from mating between FB1Δkin2 and FB2Δkin2/pKin2-sGFP#6 (CL47). (F) Control of untransformed FB1 cells. The bar corresponds to 10 μm. Download figure Download PowerPoint To analyze the distribution of GFP–Kin2 in hyphal cells, the transformant FB1Δkin2/pKin2-SGFP#3 was mixed with FB2Δkin2 and allowed to mate on water agar (Materials and methods). Dikaryotic hyphae were removed after 24 h and subjected to fluorescence microscopy. A cytoplasmic staining similar to that in haploid cells could be observed in the filaments (Figure 7E), and a single bright spot was sometimes visible at either the hyphal tip or the most distal end of the cytoplasm (not shown). To demonstrate that the green fluorescence results from the GFP–Kin2 fusion proteins, an epifluorescence micrograph of untransformed cells is shown in Figure 7F. The even distribution of the GFP–Kin2 fusion proteins suggests that it is localized in the cytoplasm or bound to submicroscopic vesicles. Electron microscopy of hyphal tips To investigate whether the distribution of submicroscopic vesicles is altered in the Δkin2 mutant strain, CLD1121 hyphal tips were analyzed by electron microscopy and compared with hyphal tips of the wild-type strain FBD11. To preserve membraneous structures, KMnO4 was used for post-fixation. Thin sections revealed dramatic differences in the organization of the hyphal apex between wild-type and mutant cells (Figure 8). In wild-type hyphae, an apical zone, comprising ∼1.5 μm, appears only lightly stained and contains an accumulation of darkly stained microvesicles (Figure 8A–C). This zone is absent from mutant hyphae, instead the apical region is indistinguishable from the subapical parts and contains membraneous structures. Microvesicles can be detected in the mutant but they do not accumulate as in wild-type hyphae (Figure 8D–F). Figure 8.Electron micrographs of hyphal tips. Overnight cultures of the diploid strains FBD11 and CLD1121 were transferred to poly-L-lysine-coated coverslips as described in Materials and methods. (A–C) FBD11 wild-type hyphae accumulate darkly stained vesicles in a less densely stained apical region. (D–F) In hyphae of the Δkin2 deletion mutant CLD1121, vesicles are distributed randomly and the organization of the apex is indistinguishable from subapical parts of the hypha. The bar corresponds to 1 μm. Download figure Download PowerPoint Discussion During its life cycle, U.maydis can adopt three well-defined morphologically distinct states: yeast-like growth occurs in haploid cells; upon pheromone stimulation, these cells respond by forming projections (conjugation tubes) that are characterized by polarized growth along a pheromone gradient. After tip fusion, a straight hypha develops into which the two nuclei migrate. In contrast to the short conjugation tubes, the dikaryotic hyphae show accelerated growth with no simultaneous increase in cytoplasmic content. As a consequence, the cytoplasm must be moved along with the growing hypha, as it is found only in the tip cell compartment. To elucidate the underlying molecular mechanism of hyphal tip growth, we have isolated two genes, kin1 and kin2, from U.maydis that belong to the kinesin family of motor proteins. The products of these genes show the highest degree of similarity to the human CENP-E and the N.crassa Nkin protein, respectively. CENP-E is a centromere-binding protein implicated in chromosome movement during prometaphase (Yen et al., 1991). Since the homology between CENP-E and Kin1 is restricted to the motor domain and Δkin1 mutants are viable, we have no evidence that Kin1 has a comparable function. The U.maydis Kin2 protein is highly similar to the N.crassa Nkin protein and thus belongs to the fungal class of conventional kinesins (Steinberg and Schliwa, 1995). The similarities between Kin2 and Nkin extend across the entire amino acid sequence but are most prominent in the motor domain and the C-terminal tail region. The tail regions of kinesins have been proposed to be involved in cargo binding (Bloom and Endow, 1995). Although a wealth of data is available on the function of conventional kinesins in vitro, the complex phenotype of mutants isolated in Drosophila melanogaster and Caenorhabditis elegans did not allow the unambiguous establishment of their biological function in vivo (see Bloom and Endow, 1995). The generation of the kin2 null mutant demonstrates that in U.maydis a conventional kinesin can be deleted without obvious effects on viability and morphology of vegetatively growing cells. In this respect, it is interesting that inspection of the complete sequence of the yeast genome revealed that such a conventional kinesin does not exist in S.cerevisiae, indicating that budding growth in yeast and U.maydis does not require this type of motor molecule. The function of Kin2 became apparent when mutants were analyzed for their potential to undergo morphological transitions. Whereas Δkin2 mutants can form normal-looking conjugation tubes (albeit somewhat delayed compared with wild-type strains), the rapid tip growth and movement of the cytoplasm into the hyphal tip compartment does not occur in the kin2 mutants. This indicates that formation of conjugation tubes and hyphal tip growth are distinct morphological transitions. We envisage that conjugation tubes arise from polarized growth of the cytoplasm, resembling the process of shmoo formation in yeast. In Δkin2 mutants, the beginnings of dikaryotic hyphae are formed, and the nuclei migrate into these structures, but rapid elongation of hyphal tips and concomitant movement of the cytoplasm is not observed. In appearance these structures resemble conjugation tubes. In addition, the two nuclei appear more closely spaced in these structure than in hyphae formed by wild-type cells. Whether the influence of Kin2 on spacing of the nuclei is a direct effect remains to be shown. The distribution of the biologically active GFP–Kin2 fusion protein in haploid cells and dikaryotic filaments does not indicate an association of Kin2 with defined organelles such as mitochondria or the nucleus. Therefore, we consider it unlikely that Kin2 plays an active role in nuclear migration. Furthermore, we have found no differences in the distribution of GFP-labeled mitochondria in wild-type and kin2 mutant dikaryons (data not shown). This is in accordance with the observation that a recently isolated kinesin mutant of N.crassa shows no major alterations of microscopically visible organelle movements [see accompanying manuscript, (Seiler et al., 1997)]. The uniform distribution of GFP–Kin2 fusion protein in U.maydis makes it unlikely that Kin2 is associated with microtubules but rather suggests an association with submicroscopical vesicles. The association of GFP–Kin2 with microtubules after depletion of ATP indicates that the interaction of GFP–Kin2 with microtubules under normal conditions is transient. The availability of a biologically active GFP–Kin2 fusion protein should facilitate the identification of the cargo that is transported by Kin2 in U.maydis by subcellular fractionation and biochemical characterization of vesicles that carry