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doi: 10.1371/journal.pone.0058008. Epub 2013 Mar 7.

Functional analysis of the Aspergillus nidulans kinome

Affiliations

Functional analysis of the Aspergillus nidulans kinome

Colin P De Souza et al. PLoS One. 2013.

Abstract

The filamentous fungi are an ecologically important group of organisms which also have important industrial applications but devastating effects as pathogens and agents of food spoilage. Protein kinases have been implicated in the regulation of virtually all biological processes but how they regulate filamentous fungal specific processes is not understood. The filamentous fungus Aspergillus nidulans has long been utilized as a powerful molecular genetic system and recent technical advances have made systematic approaches to study large gene sets possible. To enhance A. nidulans functional genomics we have created gene deletion constructs for 9851 genes representing 93.3% of the encoding genome. To illustrate the utility of these constructs, and advance the understanding of fungal kinases, we have systematically generated deletion strains for 128 A. nidulans kinases including expanded groups of 15 histidine kinases, 7 SRPK (serine-arginine protein kinases) kinases and an interesting group of 11 filamentous fungal specific kinases. We defined the terminal phenotype of 23 of the 25 essential kinases by heterokaryon rescue and identified phenotypes for 43 of the 103 non-essential kinases. Uncovered phenotypes ranged from almost no growth for a small number of essential kinases implicated in processes such as ribosomal biosynthesis, to conditional defects in response to cellular stresses. The data provide experimental evidence that previously uncharacterized kinases function in the septation initiation network, the cell wall integrity and the morphogenesis Orb6 kinase signaling pathways, as well as in pathways regulating vesicular trafficking, sexual development and secondary metabolism. Finally, we identify ChkC as a third effector kinase functioning in the cellular response to genotoxic stress. The identification of many previously unknown functions for kinases through the functional analysis of the A. nidulans kinome illustrates the utility of the A. nidulans gene deletion constructs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Strategy for generation of deletion constructs.
(A) Amplification of 5′ and 3′ gene specific flanking fragments from A. nidulans genomic DNA. Primers 5r and 3f have 5′ extensions complementary to the pyrG Af cassette, whereas 5f and 3r have 5′ extensions complementary to the vector. (B) Co-transformation of yeast with the 5′ flank and 3′ flank together with the pyrG Af cassette and a gapped yeast shuttle vector to generate a plasmid containing the gene specific deletion construct . (C) The final linear deletion construct is generated by PCR from the yeast DNA with primers 5f and 3r.
Figure 2
Figure 2. Phylogenetic analysis of the A. nidulans protein kinases.
Kinase domain alignment generated using ClustalW (http://www.phylogeny.fr) and tree visualization using MEGA version 5 maximum likelihood analyses. Domains of the atypical PIKK, PDHK and histidine kinases were omitted. The major families of kinases are indicated in different colors. The Ffk kinases are indicated in red. Note that FfkB, FfkC, FfkD, FfkE, FfkI, FfkJ and FfkK are more similar to each other than to other kinases.
Figure 3
Figure 3. Identification of essential and non-essential kinases by heterokaryon rescue
. (A) A primary transformation plate for the pkaA kinase deletion construct (left). Conidia from the indicated colonies were tested for their ability to form colonies on selective (YAG) or non-selective (YAGUU) media by either replica streaking or replica plating using a velvet disk. Growth of all 6 tested transformants on selective media suggests that pkaA is non-essential. (B) Diagnostic PCR indicates that 3 tested transformants are haploid nulls confirming that pkaA is non-essential. (C and D) As for (A and B) but showing analysis of an essential gene. Following transformation of the An-mps1 deletion construct, conidia from 4 of 6 tested transformants were unable to form colonies on selective media, strongly suggesting that An-mps1 is essential. Diagnostic PCR indicates that 3 of these heterokaryotic transformants contain both the wild type and deleted allele, confirming that An-mps1 is essential.
Figure 4
Figure 4. Kinase deletion mutants with vegetative growth defects.
Shown is colony formation of the indicated strains after 2 days at 32°, 37° or 42°, or 5 days at 20°. (A) An isogenic wild type control. (B) Kinase deletion mutants with a moderate but reproducible colony growth defect. (C) Kinase deletion mutants with a strong colony growth defect. (D) Septation deficient kinase deletion mutants display a strong growth phenotype characterized by extremely poor conidiation. Colonies are shown as negatives to more clearly visualize the hyphae.
Figure 5
Figure 5. Kinase deletion mutants with developmental defects.
(A) Colony color after 3 days growth of the indicated strains and conditions. * Images for the MMUU series at 37° are also shown from the underside of the colony to more clearly show pigments produced by the colonies. (B) The left column shows colonies point inoculated with 104 spores after 4 days growth at 37°. The ΔplkApolo, ΔAn-mst1 and ΔAn-gin4 strains produce few asexual conidia but show advanced sexual development. The right column shows cells collected from the indicated region of each colony at higher magnification. In contrast to the asexual conidiophores (c) and enormous numbers of asexual conidia of the wild type colony, the ΔplkApolo and ΔAn-mst1 mutant colonies contain numerous Hulle cells (H) surrounding apparent nascent cleistothecia (n). The ΔAn-gin4 mutant also contained Hulle cells and apparent nascent cleistothecia, although more conidiophores were apparent relative to the other mutants. Bar ∼ 100 μm.
Figure 6
Figure 6. Cleistothecia and ascospore formation in the indicated kinase mutants.
Shown are micrographs taken of cells collected from point inoculated colonies after 13 days growth at 37° on minimal media. By this time wild type colonies and the ΔAn-gin4, ΔplkA and ΔAn-Ste20 mutant colonies had formed cleistothecia (CL) containing red pigmented ascospores (a). Contrasting this, ΔAn-mst1 mutant colonies had formed only immature nascent cleistothecia (n) which did not contain ascospores. The insets show the ascospores at higher magnification with the * indicating example ascospores orientated such that their bivalve morphology is apparent. Bar ∼ 100 μm.
Figure 7
Figure 7. A. nidulans kinase deletion mutants with sensitivities to genotoxic agents.
(A) Colony formation of the indicated strains with or without the indicated genotoxic agents. Images were taken after 2 days at 32° except for the 8 mM HU series which is after 3 days. (B) chkC mutants do not enter mitosis prematurely in the presence of HU. Wild type and ΔchkC conidia were inoculated in the presence or absence of 10 mM HU and the chromosome mitotic index (CMI) of DAPI stained cells determined at each time point. Benomyl (2.4 μg/ml) was included in the media to help maintain a mitotic arrest once cells entered mitosis . (C) Schematic diagrams showing the domain structure of the indicated kinases. (D) ClustalW alignment (http://workbench.sdsc.edu/) of the N-terminals of ChkC kinases. Identical (*), conserved strong groups (:), and conserved weak groups (.) are indicated. Di-peptide SQ and TQ motifs are indicated in red. Accession numbers for the sequences are; A. nidulans (AN7563; EAA62143), A. clavatus (ACLA_072560; EAW14223), A. fumigatus (Afu2g14920; XP_755825), A. oryzae (AO090012000405; BAE60587), A. flavus (EED54418), A. terreus (ATEG_07832; EAU32094), A. niger (An15g03280; CAK42403), P. marneffei (EEA23567.1), F. oxysporum (FOXB_01143, EGU88344), F. graminearum (Fg00433), M. oryzae (EHA53924), N. crassa (NCU02751; Mus-59).
Figure 8
Figure 8. Kinase deletion mutants whose growth is reduced or enhanced by osmotic agents.
Shown are the indicated deletion strains grown with or without the indicated concentrations of NaCl or sucrose. Growth is after 3 days at 32° except for the 1.5 M NaCl series which is after 4 days. Mutants were classified as having (A) moderate sensitivity to osmotic stress or (B) strong sensitivity to osmotic stress. (C) Kinase mutants whose colony formation was inhibited by 1.5 M sucrose. (D) Kinase mutants whose defects were partially remediated by sucrose. r = remediation of growth, c = remediation of conidiation.
Figure 9
Figure 9. Null phenotypes of essential kinase mutants.
Uninucleate conidia from heterokaryons grown in media selective for growth of only the kinase deleted cells. Shown are representative cells grown on plates or on coverslips for DAPI staining. Images are micrographs after ∼18 hr growth at 32° unless indicated. (A) An isogenic strain wild type for all kinases. Kinase nulls where classified based on the predominant phenotype as (B) arresting growth as short germlings, (C) arresting growth as swollen cells, (D) undergoing limited polarized growth in the absence of nuclear division to give a cell cycle arrest phenotype, or (E) arresting growth as thin branched germlings displaying an abnormal morphology. Black arrowheads indicate septa. L = lysed cells. Note that An-cdc7 mutants displayed only a single nucleus which in ∼24% of cells appeared mitotic (m). The white arrow distinguishes the single interphase nucleus in the nimXcdk1 mutant from the DAPI staining mitochondria. The white arrowhead indicates DNA stretched between 2 masses of DNA in cells lacking the An-Wee1 kinase. Bars ∼ 50 μm.
Figure 10
Figure 10. The extreme swelling of An-cdk7 mutants correlates with a massive enlargement of vacuoles.
(A and B) Time lapse images of conidia inoculated from the heterokaryon in media selective for growth of only the An-cdk7 deleted cells. Cells separated by septa (arrowheads) are numbered sequentially as they form. Cells initially appear normal but undergo extreme swelling which appears to be potentially mediated by an enlargement of vacuoles (v). Note that septa restrict swelling and following lysis (L) appear to seal the junction between adjacent cells. Bar ∼ 50 μm.
Figure 11
Figure 11. Essential kinase deletion mutants which form microcolonies.
Uninucleate conidia from heterokaryons were streaked on selective media (top 3 rows) or inoculated and fixed for DAPI staining (bottom row). After 4 days growth at 32o these kinase mutants formed microcolonies which could not be propagated as genetically stable haploids by streaking. (A) Wild type pyrG+ and pyrG89 strains. (B) An-pod6 and cotA nulls display an identical brown microcolony phenotype. Insets show over 5 germ tubes emerging from an enlarged spore body indicative of a defect in the establishment of polarized growth. (C) An-cka1 nulls initiate polarized growth normally but arrest as small microcolonies. (D) The An-vps15 and An-vps34 nulls display an identical phenotype. Insets show highly segmented cells at colony edges. (E) The An-ksg1 and pkcB nulls initiate a nearly identical irregular pattern of growth and branching before arresting as microcolonies. (F) The An-stt4 kinase domain deletion mutant displays a range of phenotypes. The arrow indicates a section of a microcolony which appears to have resumed normal growth. The bottom row shows 2 cells from the same field which have grown to different extents and display distinctly different DNA content. Bar ∼ 50 μm.

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