The Polo-like kinase PLKA in Aspergillus nidulans is not essential but plays important roles during vegetative growth and development

Abstract

The Polo-like kinases (Plks) are conserved, multifunctional cell cycle regulators that are induced in many forms of cancer and play additional roles in metazoan development. We previously identified plkA in Aspergillus nidulans, the only Plk investigated in filamentous fungi to date, and partially characterized its function through overexpression. Here, we report the plkA null phenotype. Surprisingly, plkA was not essential, unlike Plks in other organisms that contain a single homologue. A subset of cells lacking PLKA contained defects in spindle formation and chromosome organization, supporting some conservation in cell cycle function. However, septa were present, suggesting that PLKA, unlike other Plks, is not a central regulator of septation. Colonies lacking PLKA were compact with multibranched hyphae, implying a role for this factor in aspects of hyphal morphogenesis. These defects were suppressed by high temperature or low concentrations of benomyl, suggesting that PLKA may function during vegetative growth by influencing microtubule dynamics. However, the colonies also showed reduced conidiation and precocious formation of sexual Hülle cells in a benomyl- and temperature-insensitive manner. This result suggests that PLKA may influence reproduction through distinct mechanisms and represents the first example of a link between Plk function and development in fungi. Finally, filamentous fungal Plks have distinct features, and phylogenetic analyses reveal that they may group more closely with metazoan PLK4. In contrast, yeast Plks are more similar to metazoan proteins PLK1 to PLK3. Thus, A. nidulans PLKA shows some conservation in cell cycle function but may also play novel roles during hyphal morphogenesis and development.

Fig 1

Phylogenetic analysis of select Plk orthologues in filamentous fungi. A consensus tree including bootstrap values (Phylip, version 3.69) (20) based on full-length sequences of select Plks and uncharacterized orthologues in filamentous fungi. Characterized genes include Homo sapiens Sak (HsSak), PLK1 (HsPlk1), PLK2 (HsPlk2), PLK3 (HsPlk3), and PLK4 (HsPlk4); Drosophila melanogaster Sak (DmSak) and Polo (DmPolo); Candida albicans CDC5 (CaCdc5); Saccharomyces cerevisiae CDC5 (ScCdc5); Schizosaccharomyces pombe plo1 (SpPlo1); and Aspergillus nidulans plkA (AnPLKA). Uncharacterized orthologues are indicated by species initials followed by “Plk” for simplicity, including NcPlk (Neurospora crassa), MoPlk (Magnaporthe oryzae), FvPlk (Fusarium verticillioides), AoPlk (Aspergillus oryzae), AfPlk (Aspergillus flavus), AfuPlk (Aspergillus fumigatus), AniPlk (Aspergillus niger), HsPlk (Histoplasma capsulatum), CnPlk (Cryptococcus neoformans), and UmPlk (Ustilago maydis). Saccharomyces cerevisiae Mek1 (ScMek1) represents an outgroup. Numbers at branch points represent bootstrap values from 100 replicates.

Fig 2

Absence of PLKA results in a temperature-sensitive, compact growth phenotype. (A) Strains KM17 [alcA(p)::plkA riboB⁺], KM5 (plkA riboB⁺), and parental strain TN02A25 (plkA) were spot inoculated onto mmTF or YAG medium and incubated for 72 h at 32° or 37°C. (B) Strains KM14 (ΔplkA pyr4⁺), KM25 (plkA pyr4⁺), and parental strain TN02A25 (plkA) were spot inoculated onto YAG medium and incubated for 72 h at 32 or 37°C.

Fig 3

Absence of PLKA results in hyperbranching and split tips. (A) Strains KM14 (ΔplkA pyr4⁺), KM25 (plkA pyr4⁺), and TN02A25 (plkA) were spot inoculated onto YAG medium and incubated for 72 h at 32°C. (B) Strains KM17 [alcA(p)::plkA riboB⁺], KM5 (plkA riboB⁺), and TN02A25 (plkA) were spot inoculated onto YAG or mmTF plates and incubated for 72 h at 32°C.

Fig 4

Absence of PLKA results in abnormal spindle assembly, chromosome organization, and chromosome segregation in a proportion of cells. Strains KM14 (ΔplkA pyr4+) and KM25 (plkA pyr4⁺) were incubated in YAG medium for 8 h at 32°C. The cells were then fixed, processed for immunolocalization of α-tubulin, and stained with DAPI. (A and C) Normal cytoplasmic microtubules in interphase cells of strains KM25 and KM14, respectively. (B) Normal metaphase mitotic spindles with associated condensed chromosomes in strain KM25. (D to H) Spindle and chromosome organization defects in mitotic cells of strain KM14. Scale bar, 10 μm.

Fig 5

Absence of PLKA results in pleiotropic effects on nuclear distribution. Strains KM14 (ΔplkA pyr4⁺), KM25 (plkA pyr4⁺), and TN02A25 (plkA) were incubated in YAG medium for 7 h at 32°C, fixed, then stained with DAPI. Arrows indicate clustered nuclei. Scale bar, 10 μm.

Fig 6

Cells lacking PLKA can form septa. Strains KM14 (ΔplkA pyr4⁺), KM25 (plkA pyr4⁺), and TN02A25 (plkA) were incubated in YAG medium at 32°C for 9 h (top row) or 12 h (bottom row). Cells were fixed and then stained with calcofluor (top row) or calcofluor and DAPI (bottom row). Scale bar, 10 μm.

Fig 7

Absence of PLKA impairs asexual development and derepresses aspects of sexual development. (A) Strains KM14 (ΔplkA pyr4⁺), KM25 (plkA pyr4⁺), and TN02A25 (plkA) were inoculated into YAG top agar that was poured over standard YAG plates and incubated for 72 h at 32°C. First column, low magnification of plates; second column, high magnification of plate surface; third column, surface cells collected from the plates. (B) Conidiophores collected (36) from strains KM14, KM25, and TN02A25.

Fig 8

Low concentrations of benomyl suppress the compact growth and branching defects of cells lacking PLKA but do not prevent Hülle cell formation or a reduction in pigmentation. (A) A total of 2 × 10⁴ conidia of strains KM14 (ΔplkA pyr4⁺), KM25 (plkA pyr4⁺), and TN02A25 (plkA) were inoculated onto YAG medium containing different concentrations of benomyl and incubated for 72 h at 32°C. Serial dilutions of strains plated on YAG medium with or without 0.6 μg/ml benomyl are shown on the right. (B) Colony edges of strains grown on YAG medium for 72 h at 32°C in the presence or absence of 0.2 μg/ml benomyl. (C) Surface cells collected from plates shown in panel B.