Keywords

1 Introduction

Fungi grow in nature under many environmental stress conditions. To respond to these stresses, fungi have various signal transduction pathways and change their morphology to adapt to their environment. Protein kinases, such as protein kinase A, G, and C (AGC protein kinases), calmodulin-dependent protein kinase (CAMK), and kinases involved in the mitogen-activated protein (MAP) kinase cascade, are speculated to have important roles in these processes (De Souza et al. 2013). The functions of these fungal kinases have been investigated primarily using Saccharomyces cerevisiae, and little is known about their functions in filamentous fungi.

Filamentous fungi belonging to Ascomycota undergo asexual and sexual life cycles and form asexual spores (conidia) and sexual spores (ascospores). In the asexual life cycle of Aspergillus nidulans, conidia grow isotropically at 37 °C for 2 to 3 h and establish cell polarity to form germ tubes. Then, polar growth proceeds with branching followed by hyphae formation. The hyphae differentiate conidiophores, which form conidia after approximately 20 h of cultivation. Finally, autolysis occurs at a late stage of cultivation (Fig. 12.1). These specific structural changes in the life cycle of filamentous fungi are thought to be closely related to both their industrial merits and demerits. Thus, elucidating the mechanisms of conidial germination, hyphal growth, conidiophore, and conidia development at the molecular level is very important for the efficient utilization of filamentous fungi in industry. Fungal cells are surrounded by cell walls, which consist mainly of polysaccharides and proteins and form very rigid structures. The cell wall is crucial for the formation of differentiated structures of filamentous fungi, and the architecture changes during the progression of the life cycle and in response to various cell wall stresses.

Fig. 12.1
figure 1

Asexual life cycle of Aspergillus nidulans. Details described in text

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Protein kinase C (PKC) is a serine/threonine kinase that is ubiquitous from fungi to mammals. There are more than ten isozymes in mammalian cells that function in various signal transduction pathways in these cells (Steinberg 2008). In contrast to mammalian cells, there are only one or a few PKC-encoding genes in the genomes of filamentous fungi and yeasts. Although PKC is known to have a crucial role in the signal transduction pathway in S. cerevisiae under cell wall stress, its functions in filamentous fungi remain poorly understood.

In this chapter, we mainly focus on the protein kinase C (PKC)-related signal transduction pathway of filamentous fungi.

2 PKC Signaling Pathway

Fungal PKCs have a specific domain organization consisting of two HR1 domains, a C2-like domain, a pseudo-substrate sequence, two C1 domains, a protein kinase domain, and a protein kinase C-terminal domain (Fig. 12.2). The organization of these domains is similar to that of the mammalian novel PKC (nPKC) subclass with the exception of the HR1 domain. The HR1 domain is not present in nPKC but is present in the PKC-related kinase, protein kinase N (PKN) (Mukai 2003). In these domains, the HR1 domain is known to bind to the GTPase Rho (Schmitz et al. 2002) and the C2-like domain is a sequence homologous to the C2 Ca2+-binding domain. However, the amino acids required for Ca2+ binding are not conserved in the “C2-like” domain. The C1 domain, a region that binds to diacylglycerol (DAG), is followed by a pseudo-substrate sequence located between C2-like and C1 domains. The pseudo-substrate sequence resembles the amino acid sequence of substrate proteins, with the exception of the phosphoacceptor residue, which is replaced by alanine. The pseudo-substrate sequence is thought to bind to the catalytic subunit of PKC and keep the kinase inactive.

Fig. 12.2
figure 2

Domain organization of the fungal PKC and human nPKC and PKN. Hs Homo sapiens, An A. nidulans

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The phylogenetic relationships among fungal PKCs and human nPKC and PKN are shown in Fig. 12.3. In general, fungi belonging to Ascomycota or Basidiomycota have one or two PKCs, whereas those belonging to Mucoromycotina (formerly known as Zygomycota) (Hibbett et al. 2007) have three or four PKCs.

Fig. 12.3
figure 3

Phylogenetic tree of fungal PKC and human nPKC and PKN. Alignment was performed with ClustalW using the entire amino acid sequence of each PKC and PKN. The tree was drawn using NJprot. Branch length indicates evolutionary distance. PKCs of the fungi belonging to Ascomycota, Basidiomycota, and Mucoromycotina are shown in red, blue, and green, respectively. Hs Homo sapiens, Sc Saccharomyces cerevisiae, An A. nidulans, Tr Trichoderma reesei, Nc N. crassa, Yl Yarrowia lipolytica, Cc Coprinopsis cinerea, Ro Rhizopus delemar. The numbers in parentheses indicate the gene IDs in the genome database of R. delemar

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Saccharomyces cerevisiae has the only PKC-encoding gene, PKC1. The gene product of PKC1, Pkc1, has the typical domain organization of fungal PKC (Fig. 12.2), and its activity is not stimulated by Ca2+ or DAG (Kamada et al. 1996). Amino acids that are thought to be required for binding to DAG are not conserved in the C1 domain of fungal PKCs (Schmitz and Heinisch 2003). The C1 domain of Pkc1 has also been reported to bind Rho1 in S. cerevisiae (Nonaka et al. 1995).

Pkc1 is known to have pivotal function in the cell wall integrity (CWI) signaling pathway. The pkc1 deletion mutant could not form colonies unless an osmotic stabilizer was added to the medium. It has been suggested that PKC1 was involved in various cellular processes, such as the cell wall integrity (CWI) signaling pathway, cell-cycle progression, and phospholipid synthesis (Levin 2005). However, its functions at the molecular level remain poorly understood except for the function in the CWI signaling pathway. In the Aspergillus nidulans genome, there is a PKC-encoding gene, pkcA. pkcA was cloned and its functions have been investigated. Deletion of pkcA caused frequent hyphal lysis and its deletion mutant did not form colonies, indicating that pkcA is essential for hyphal growth (Ichinomiya et al. 2007). This growth defect was not remedied by the addition of osmotic stabilizers to the medium, which suggests that PkcA has other functions besides the function in the CWI signaling pathway (see following). It was reported that pkcA was involved in penicillin biosynthesis and was crucial for the nuclear localization of the transcription factor AnBH1 (Herrmann et al. 2006).

Pkc1 of S. cerevisiae localizes to polarized growth sites. However, Denis and Cyert showed that the deletion of the HR1 domain causes the relocalization of Pkc1 to the mitotic spindle. They also determined a nuclear localization signal (NLS) and a nuclear export signal (NES) in Pkc1. These results suggest that the small portion of Pkc1 is shuttled between the nucleus and cytoplasm (Denis and Cyert 2005). Recently, Pkc1 has been reported to disappear at the polarized growth site and to accumulate in damage sites caused by lasers (Kono et al. 2012).

PkcA of A. nidulans mainly localizes to polarized growth sites, such as hyphal tips, forming septa, and tips of phialides (Teepe et al. 2007). The PKC of Neurospora crassa also localizes at some hyphal tips and subapical membranes in actively growing hyphae and forming septa. The PKC in the cytoplasm accumulates in the plasma membrane after treatment with the phorbol ester, 12-myristate 13-acetate (Khatun and Lakin-Thomas 2011).

2.1 Function of PKC in the Cell Wall Integrity Signaling Pathway

Fungal cells sense cell wall stresses and transduce signals inside the cells. The signals are transmitted to transcription factors that induce the expression of certain genes. This signal transduction pathway is called the CWI signaling pathway. In this pathway, transmembrane sensor proteins, Wsc1 – 3, Mid2, and Mtl1, which localize in the plasma membrane, sense cell wall stresses and transmit signals to the downstream effectors Rom1 and 2. Rom1 and 2 are GDP/GTP exchange factors (GEFs) for the GTPase Rho1, a small G protein that is active when it binds to GTP. The GTP-bound form of Rho1 interacts with Pkc1 and activates it, and Pkc1 phosphorylates MAP kinase kinase kinase Bck1. Signals are transduced to transcription factors Rlm1 and the Swi4/Swi6 SBF complex through the MAP kinase cascade. Because the genes encoding almost all the components involved in the CWI pathway are conserved in the genomes of Ascomycota, Basidiomycota, and Mucoromycotina fungi (Table 12.1), signal transduction pathways are speculated to be present in filamentous fungi. The functions of upstream components in this signaling pathway have been analyzed and are described in Chap. 10. In A. nidulans, MpkA and RlmA, a MAP kinase and a transcription factor in the CWI signaling pathway, respectively, are involved in the regulation of the expression of the genes encoding alpha-glucan synthases (agsA and agsB) and a glutamine-fructose-6-phosphate amidotransferase (gfaA) when the cells are treated with caspofungin, a β-1,3 glucan synthase inhibitor (Fujioka et al. 2007). RlmA of A. niger is also involved in the expression of agsA and gfaA (Damveld et al. 2005).

Table 12.1 Presence of orthologous genes involved in the cell wall integrity (CWI) pathway in fungi
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When pkcA expression is reduced in A. nidulans, the growth of the pkcA- conditional mutant is sensitive to a β-1,3 glucan synthase inhibitor, caspofungin, the chitin-binding dyes Congo red and Calcofluor white, and a PKC inhibitor, staurosporine. Abnormalities in the cell wall organization of this mutant have been observed under the pkcA-downregulating conditions (Ronen et al. 2007). The overexpression of pkcA has also been observed to induce growth sensitivity to a β-1,3-glucan synthase inhibitor, a chitin-binding dye, and chitin synthase inhibitors (Ichinomiya et al. 2007), suggesting that PkcA has a critical role in the CWI signaling pathway. To investigate the function of PkcA in this pathway, we constructed a strain that produces a constitutively active form of PkcA and analyzed the expression patterns of the genes encoding chitin synthases and alpha- and beta-glucan synthases. The results showed that most of these genes, including chsB, csmA, and csmB, are upregulated. Moreover, this upregulation was completely or partially dependent on RlmA (Katayama et al. 2015). These results suggest that chitin synthesis and glucan synthesis are also regulated by the CWI signaling pathway through PKC and Rlm1 orthologues in filamentous fungi. The gene chsB encodes a class III chitin synthase, and its gene product, ChsB, tagged with EGFP (EGFP-ChsB), was observed to localize at hyphal tips and forming septa (Fukuda et al. 2009; Horiuchi 2009). ChsB is crucial in hyphal tip growth (Borgia et al. 1996; Ichinomiya et al. 2002). The genes csmA and csmB encode chitin synthases with myosin motor-like domains, and they are located in a head-to-head configuration in the genome. There are five putative RlmA-binding sites in the promoter region between them. Their gene products, CsmA and CsmB, colocalize at hyphal tips, forming septa, and have compensatory essential functions for tip growth (Takeshita et al. 2006). CsmA is suggested to be involved in repairing cell wall damage (Yamada et al. 2005), and both CsmA and CsmB have similar characteristics (Takeshita et al. 2005; Tsuizaki et al. 2009). It is reasonable to regulate the expression of csmA and csmB by the signaling pathway through PkcA and RlmA.

Recently, RlmA has been shown to be involved in the regulation of brlA expression (Kovács et al. 2013). Because BrlA is a transcription factor that has a crucial role in asexual development (see following), the CWI signaling pathway and asexual development signaling pathway are suggested to be somewhat related.

2.2 Functions of PKC Under Heat Stress

2.2.1 Germination

As described in the former section, pkcA is essential for the growth of A. nidulans. In contrast, bckA encoding a MAP kinase kinase kinase of the CWI signaling pathway and mpkA (Table 12.1) are not essential for growth at 37 °C, an optimal growth temperature for A. nidulans (Katayama et al. 2012), which suggests that the PkcA has other function(s) essential for the growth of A. nidulans. To investigate these functions, we constructed and characterized a temperature-sensitive mutant of pkcA. The resulting pkcA-ts mutant grew as well as the wild-type strain below 30 °C, although it showed a partial growth defect and a severe conidiation defect at 37 °C. The pkcA-ts mutant did not form colonies at 42 °C. Furthermore, the conidiation defect at 37 °C were partially remedied by the addition of an osmotic stabilizer in the medium, whereas the growth defect at 42 °C was not remedied even in the presence of an osmotic stabilizer. Although the growth defect of the S. cerevisiae pkc1-ts mutant at a restrictive temperature was suppressed when Ca2+ was added to the medium, that of the A. nidulans pkcA-ts mutant was not suppressed on the addition of Ca2+.

During germination, the conidia of A. nidulans swell isotropically for a period of time, then the growth polarity is established and germ tubes form (Fig. 12.1). The conidia of the pkcA-ts mutant swelled slightly and stopped growing at 42 °C. Reactive oxygen species (ROS) and DNA-strand breaks, common characteristics that are phenotypes of apoptosis, accumulated in swollen conidia, suggesting that apoptosis was induced in the pkcA-ts mutant at 42 °C and that PkcA suppresses the induction of apoptosis under heat stress. In contrast, induction of apoptosis was not observed in the pkcA-ts mutant grown at 37 °C. The bckA and mpkA deletion mutants did not form colonies, and the conidia of these mutants swelled and stopped growing at 42 °C. The accumulation of ROS and DNA-strand breaks were also observed in the swollen conidia of both mutants. These results suggest that suppression of apoptosis induction is required for the function of BckA and MpkA. On the other hand, the rlmA deletion mutant grew well at 42 °C, suggesting that RlmA is not required for suppression. Taken together, the suppression of apoptosis induction under heat stress conditions would occur through PKC and the downstream MAP kinase cascade but not through RlmA.

The bckA and mpkA deletion mutants formed colonies at 42 °C when osmotic stabilizer was added to the medium, although the pkcA-ts mutant did not form colonies under the same condition, suggesting that PkcA has another function in the growth of A. nidulans under heat stress conditions. To clarify the function of PkcA, we analyzed the terminal phenotype of the pkcA-ts mutant in the presence of the osmotic stabilizer at 42 °C. Under this condition, apoptosis was not induced in the pkcA-ts mutant. However, the mutant formed extremely swollen conidia and did not form germ tubes 8 h after incubation at 42 °C. The DNA content in the swollen conidia of the mutant increased as well as that of the wild-type strain, and polar localization of actin filaments was not observed in the mutant. These results suggest that the cell cycle progressed regularly, but polarity was not established in the mutant. In contrast, the bckA deletion mutant established cell polarity and formed germ tubes under the same condition (Katayama et al. 2012), suggesting that PkcA is required to establish polarity in the swollen conidia, although BckA and downstream factors of the CWI signaling pathway are not involved.

2.2.2 Hyphal Growth

The role of PkcA in hyphal tip growth under heat stress conditions was investigated. When the pkcA-ts mutant was cultivated for 16 h to induce hyphal growth at a permissive temperature (30 °C) and shifted to a restrictive temperature (42 °C), the growth of the pkcA-ts mutant stopped and its hyphal tips swelled. These observations suggest that the hyphal tips were depolarized. Actin filaments are usually observed at the hyphal tips in A. nidulans. When hyphae were exposed to heat stress, actin filaments at the hyphal tips rapidly disappeared in both the pkcA-ts mutant and the wild-type strain. In the wild-type strain, actin filaments at the hyphal tips reappeared within 60 min after the shift to 42 °C, whereas they did not reappear 120 min after the sift in the pkcA-ts mutant. The reappearance of actin filaments at the tips was observed in the bckA deletion mutant as well as the wild-type strain (Katayama et al. 2012), suggesting that the repolarization of hyphae does not depend on the function of BckA. As already mentioned, EGFP-ChsB localizes at the hyphal tips. After the shift to 42 °C, EGFP-ChsB disappeared from hyphal tips in the pkcA-ts mutant and the wild-type strain. EGFP-ChsB was observed again at the hyphal tips a few hours after the shift in the wild-type strain, whereas it was not observed at the hyphal tips 5 h after the shift in the pkcA-ts strain. The localization of microtubules was not affected in the pkcA-ts mutant and wild-type strain when exposed to heat stress (Takai et al., unpublished results). These results suggest that the redistribution of EGFP-ChsB to hyphal tips depends on the actin filament repolarization through the function of PkcA. A model for the PKC functions during germination and hyphal tip growth is shown in Fig. 12.4.

Fig. 12.4
figure 4

A model for the function of PkcA under heat stress. “?” in yellow panels means the possible presence of unknown factors. (Modified from fig. 10 in a previous paper by Katayama et al. 2012

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2.2.3 Asexual Development

As described in the former section, induction of the expression of brlA is an essential step for conidiation in Aspergillus nidulans. brlA encodes a C2H2 transcription factor and controls the expression of genes involved in regulating the early stages of conidiation. The expression of brlA was not induced in the pkcA-ts mutant at 40 °C, suggesting that PkcA is required to induce the expression of brlA (Katayama and Horiuchi, unpublished results). The expression of brlA is induced by the upstream activators FluG, FlbA, FlbB, FlbC, FlbD, and FlbE (Fig. 12.5). FluG is a key activator of the initiation of conidiation and induces conidiation via two independent processes (Etxebeste et al. 2010; Park and Yu 2012). First, FluG inhibits the vegetative growth via the activation of FlbA, which is a key regulator of the G-protein signaling pathway that regulates the balance between hyphal proliferation and conidiophore differentiation. Second, FluG induces the expression of brlA by activating the two separate pathways. One pathway contains FlbC, which is a C2H2 transcription factor and induces the expression of brlA by directly binding to the promoter of brlA. Another pathway consists of FlbE, FlbB, and FlbD. In this pathway, FlbB functions as a bZIP-type transcription factor and forms a complex with FlbE. This complex induces the expression of flbD, which encodes a cMyc-type transcription factor. FlbE interacts and colocalizes with FlbB at the hyphal tips, suggesting that FlbE ensures the proper localization and function of FlbB (Garzia et al. 2009). Both FlbB and FlbD induce the expression of brlA by directly binding to the promoter of brlA. The expression levels of brlA significantly decrease in mutants of the genes encoding these activators, and the mutants show the fluffy phenotype (Wieser et al. 1994). Negative regulators of conidiation have also been identified. SfgA is a putative transcription factor with a Gal4-type Zn(II)2Cys6 binuclear cluster DNA-binding domain. The deletion of sfgA suppressed the fluffy phenotype of the fluG deletion mutant but not the deletion mutants of flbA, flbB, flbC, and flbD, indicating that SfgA acts as a negative regulator of conidiation by functioning downstream of FluG but upstream of FlbA, FlbB, FlbC, and FlbD (Seo et al. 2006). The deletion of sfgA did not suppress the decrease in conidiation efficiency of the pkcA-ts mutant at 37 °C, suggesting that PkcA regulates the expression of brlA downstream of SfgA (Katayama and Horiuchi, unpublished results). The velvet protein VosA is suggested to repress conidiation during vegetative growth (Bayram and Braus 2012; Park et al. 2012). In addition, VosA binds to the promoter of brlA and represses the expression of brlA (Park et al. 2012). The expression of vosA is induced by AbaA, which is a TEA/ATTS transcription factor that functions downstream of BrlA, suggesting that VosA plays a role in the negative feedback regulation of brlA (Park and Yu 2012). The deletion of vosA did not suppress the decrease in the conidiation efficiency of the pkcA-ts mutant at 37 °C, suggesting that VosA is not involved in the PkcA regulation of brlA expression (Katayama and Horiuchi, unpublished results). These results are summarized in Fig. 12.5.

Fig. 12.5
figure 5

Involvement of PkcA in conidiation. Details discussed in text

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Because conidiophores of Aspergillus have distinctive structural features and those are not conserved in the filamentous fungi other than Ascomycota, there are no orthologues of the genes involved in the formation of conidiophores and conidia in filamentous fungi belonging to Basidiomycota and Mucoromycotina. In Ascomycota, only the orthologous genes of brlA are conserved in the genomes belonging to the genus Aspergillus or Penicillium. Future studies are required for understanding the role of PKC in asexual development of filamentous fungi of other genera.

2.3 Other Functions of PKC in Filamentous Fungi

PKCs in filamentous fungi have also been reported to have other functions, as described next.

Unfolded protein response (UPR) is a stress response caused by endoplasmic reticulum stresses. Farnesol induces apoptosis in filamentous fungi, and farnesol treatment of Aspergillus nidulans has been reported to induce UPR via the PKC signaling pathway, suggesting that PkcA is involved in the induction of UPR (Colabardini et al. 2010). In Neurospora crassa, PKC is known to regulate light responses via phosphorylating the WC-1 protein, which is a blue light photoreceptor (Franchi et al. 2005). In Aspergillus oryzae, the assembly of a structural protein of Woronin body, AoHex1, has been suggested to be regulated by PKC activity (Juvvadi et al. 2007) (see Chap. 11). Because the orthologues of the genes encoding WC-1 and AoHex1 are present in the genome of filamentous fungi belonging to Ascomycota, PKCs in these fungi likely have similar functions.

Recently, transcriptome analysis of A. niger was performed under carbon starvation conditions. Carbon starvation induces hyphal morphological changes and asexual development. The expression of a PKC-encoding gene was slightly upregulated under this starvation condition (Nitsche et al. 2012). This observation may suggest a novel function of PKC for morphogenesis in filamentous fungi.

In this chapter, we described the functions of PKC in the morphogenesis of filamentous fungi by focusing on the A. nidulans PkcA under cell wall stress or heat stress. The factors that function upstream of PKC are currently unknown under these conditions, except for MtlA of A. nidulans (Futagami et al. 2014) (see Chap. 10).

Although PKC signaling pathways in S. cerevisiae are activated by many environmental stresses, such as cell wall stress, heat stress, oxidative stress, hypo-osmotic stress, and endoplasmic reticulum stress (Levin 2005, 2011), these functions remain largely uncharacterized in filamentous fungi, partly because of the difficulties of handling filamentous fungi at the molecular level. Because filamentous fungi encounter various stresses in the culture conditions used in industry, it is very important to understand their responses to these stresses. This information enables us to improve the culture conditions and how to breed strains at the molecular level. PKC is probably pivotal in many aspects of these conditions. Because molecular biological techniques have much improved recently in filamentous fungi, other functions of PKCs related to the stresses affecting morphogenesis and conidiation will be elucidated in the near future.