External Signal-Mediated Overall Role of Hormones/Pheromones in Fungi

Abstract

The communication via signaling of chemicals is perhaps one of the earliest forms of communications. The most commonly known interspecific chemical substance such as pheromones is often known to engage in the attraction of mates in insects. Hence, the sensing of environmental and interindividual communication via pheromone systems is fundamental to most organisms that help in guiding the interactional behavior, development, and overall physiological activities. Likewise, the role of pheromones is revealed in fungal species in terms of their role in several cellular activities. The role of pheromones in fungi has been largely unexplored. However, there are few fungal hormones/pheromones such as sirenin, trisporic acid, antheridiol, oogoniol, and peptide hormone in yeast that were documented. Further studies are still underway for their significance in the biology of fungi as a whole and implications they might have on the overall ecosystem. In this chapter, we discuss various progresses made in understanding pheromone related to mating in kingdom fungi and the role of pheromone receptors.

1. Introduction

One of the largest and diverse kingdoms in eukaryotes is kingdom fungi, which may consist of 2.2–3.8 million species (approx.), and most importantly of which many are undergoing characterization, and it is well known to be present in several phyla that show important traits morphologically [1]. The fungal kingdom exhibits ubiquitous nature in our environment that plays a key role in the existence of life on earth as many species are directly or indirectly linked to in terms of several fields such as agricultural or industrial, and therefore, the implication is negatives as well positive for overall well-being of human and plant health [1]. The kingdom fungi are considered to be an ancient group of approximately 3.5 billion years old, which are comprised of large and diversely grouped organisms continuously updated with the discovery of new species yearly but the estimated number is around 1.5 and 7.1 million species [2, 3, 4], of which certain groups of fungi have been associated as pathogens for their ability to grow on humans, animals, or plants but there exists a helpful beneficial or mutual role also [5, 6, 7, 8, 9]. So far, the species that belong to this kingdom may include rusts, molds, lichens, smuts mushrooms, and yeasts.

By the large, the fungi have been relevant to their ability for undergoing the phenomena of secondary metabolism where secondary metabolites (SMs) as bioactive compounds are synthesized which demonstrates properties such as mutagenic, cytotoxic, immune-suppressants, antibiotics, and carcinogenic. In addition, various other SMs also have been shown to contribute to the interaction between host-plant and in resistances such as induced systemic and systemic acquired [10]. Both beneficial and harmful SMs have been reported from fungal species such as well-known antibiotic penicillin from Penicillium, sterigmatocystin, aflatoxin, and gliotoxin from A. nidulans, A. flavus, and A. fumigates [11, 12].

As far as sexual development in fungi is concerned, it is considered to be initiated by the fusion of haploid cells that are morphologically not distinguishable at all. de Bary (1981) was the first one who reported the occurrence of sex hormones in fungi such as Achlya. Later on, gradually various works on it revealed diffusible substance that plays a specific role during sexual reproduction in fungi.

Nonetheless, they can fuse due to differences in mating types. In most cases, under mating types, it undertakes overall particular activities of the cell that are essential for conversion from haploid to the diploid stage and meiosis. The genetic information determines the mating type and is supposedly present at the mating-type locus. So far, various systems have been evolved to make certain continuation of different sexes of either two or multiple mating types. In various fungi, diffusible peptide mating factors are mediated by particular cell recognition and fusion. These peptides act in a very similar way with the secreted chemical substances of insects and mammals especially in very low doses that elicit certain responses related to mating. Hence, it can be termed pheromones in a very similar way as in insects and mammals. The very first evidence of peptide pheromones came into existence from the observations when it was found that a certain diffusible substance was acting from a distance with an effect on cell-type specificity [13]. Hence, the occurrence and study of these pheromones have paved the way to study the varied fields of protein modification and their trafficking, signal transduction, ligand-receptor interactions, and cell cycle regulation. For this, the yeast Saccharomyces cerevisiae proved to be the best model organism due to its genetic, pheromone production response and molecular mechanisms involved in it. Furthermore, several studies have been undertaken in several species of fungi by use of several methods that may include the study of genetics, plant secondary metabolites, etc. for the occurrence of sexual reproduction and its related signaling cascades [10, 14].

Because the majority of fungi are non-motile, therefore, the property of responding to external cues such as signals helps in polarized growth especially in filamentous fungi for facilitating them in search for a continuous supply of essential nutrients and mating partners that prove to be crucial for their survival and abundance, while the mechanism in which external signals mediate in the process of polarization has marked commonalities with the polarization event during the processes such as mitotic division, budding, or fission. Thus, if any new substance is introduced into the outer membrane, then it may result in the growth in the polarized manner in the case of fungi using the secretory pathway and associated re-modeling of the cell wall. This type of growth had been reported to occur via internal as well as external signals such as cell cycle progression and environmental changes or probable occurrence of peptide mating pheromone, respectively. Most importantly, when opposite mating partners are exposed to the effects of pheromone, it has been found that several expressions in genes are observed along with the arrest of the cell cycle [15].

It has been extensively documented that polarized growth exists in several fungal species [16, 17]. Therefore, the biology-related mechanism present in pheromone-mating types of interactions in fungi kingdom may present helpful insights into the evolution of mating mechanisms, which will ultimately serve in understanding the not-so-studied models for sexual eukaryotes. In this chapter, we will not concentrate our views on chemotropism or cell fusion, which already have been extensively reviewed recently.

2. Sex hormone types

As far as endogenous hormone systems are concerned in fungi, it has very significant homologies for animals [18]. According to Machlis in 1972 [19], the sex hormones are may be classified into three following types viz., erotactins, erotropins, and erogens where erotactin functions as a sexual hormone for attracting motile gametes, erotropin plays role in the induction of chemotropic growth of sexual structures, and lastly erogen role is to control the induction and differentiation of sexual structures. Nonetheless, the sex hormone may carry out more than one function for instance in Achlya, where it was found that it not only helps in controlling the overall development of sexual structures but also helps in determining the direction of the sexual organs.

3. Some common fungal sex hormones

Even though a large number of sex hormones have been investigated, but only a few of them have been characterized chemically or widely investigated, which are viz., sirenin, trisporic acid, antheridiol, and oogoniol.

3.1 Sirenin

Among many fungal sex hormones, sirenin became the first known fungal sex hormone that was a sperm-attracting hormone and later on, it was classified according to its chemical composition. It has the basic property of female gamete that helps in attracting male gametes in genus Allomyces. It was first demonstrated in 1958 by Leonard Machlis [20], and organic chemists helped in its purification for its structural determination by 1968 as empirical formula C₁₅ H ₂₄O₂ with a molecular weight of 236. Consequently, further research has been carried out single-handedly on sirenin almost entirely by Jeffrey Pommerville. In his works, it was shown that male gametes helped in releasing a hormone that complements to sirenin, parisin which is attributed to attracting female gametes [21]. It resulted in the demonstration that there are parallels in the system of Allomyces hormone for several others that are present due to specific male as well as female hormones. But, unfortunately thereafter hardly any significant work has been carried out on Allomyces.

3.2 Trisporic acid

After the report of the first female sex hormone in form of sirenin in 1958, the last decade of the twentieth century saw a discovery of metabolite as trisporic acid. Trisporic acid is reported to be a sex hormone that has been isolated from Blakeslea trispora and Mucor mucedo, and it was shown to play an active role in sexual reproduction of various members of the order Mucorales. Also, it was found that trisporic acid caused significant carotene upregulation in the species of B. trispora. Afterward, in the sexual reproduction of Mucor mucedo, it was found that the hormone that was responsible for the process of gametangial conjugation that results in the production of zygospore was trisporic acid. Trisporic acid is an unsaturated and oxygenated form of trimethyl cyclo-hexane and there are three kinds of trisporic acid such as A, B, or C among them C is known to play chief role as a sex-hormone, afterward, trisporic acid B comes in terms of activity, followed by trisporic acid A with least activity. In the case of heterothallic mycelia, trisporic acid B and C have been found to stimulate the zygosphore developments and this particular hormone is produced only when the mycelia of (+) and (−) strains grow in a normal continuous diffusable medium. The trisporic acid hormone synthesized in (−) strain encourages the development of pro-gametangium in (−) strains or the other way round. The empirical formula of trisporic acid is C₁₈ H₂₆ O₄ with a molecular weight of 306. The revelation where sexual involvements are concerned which is under the control hormones in Mucorales group extends comprehensively over many years and workers from various countries are actively got involved.

3.3 Antheridiol and Oogoniol

During the early nineteenth century, John Raper discovered the hormone known as antheridiol which was initially termed as hormone A when he was studying the mode of mating in the oomycete water mold Achlya. It is demonstrated that the hormone antheridiol is responsible for several types of reactions such as antheridial hyphae initiation on the male plant, stimulation of antheridial hyphae chemotropic way, male hyphae stimulation for the oogoniol production, and their role in antheridia delimitation. The hormone was retrieved from the female was obtained by Raper and the chemist Haagen-Smit in a highly concentrated state was earlier shown to induce the development of antheridia in the male by the early twentieth by Raper. The hormone oogoniol is synthesized by male hyphae of Achlya ambisexualis but only when antheridiol is present. However, it was reported that oogoniol may be synthesized as well by some hermaphrodite strains exclusive of antheridiol stimulus [22]. McMorris [23] and his coworkers found that two crystalline compounds that possessed hormone B activity have been isolated from culture filtrates of Achlya heterosexualis which received the name of oogoniol-1 and oogoniol-2. Hence, the hormone that stimulates the development of oogonium on female hyphae came into existence as oogoniol that is a crystalline steroid with 500 as molecular weight.

3.4 Yeast a-factor and alpha-factor

In S. cerevisiae, mating-type factors are peptide hormones called a and alpha pheromones and they have specific receptors on the cell surface. These pheromones binding to specific receptors on opposite mating-type cause G1 arrest in the cell cycle which is the same stage that is required for nuclear fusion. Investigating the effects on the cell cycle by the responses generated by signal transduction to the nucleus via extracellular pheromones seems to be a better prospect. In S. cerevisiae, the receptor family that it belongs to is the large family of receptors known as G-protein-coupled serpentine seven-trans-membrane (GPCR) receptor and it has been involved in studying these receptors as a useful model system for the investigation of complex signal transduction cascade. And the enzymes that are required for this signal transduction have very significant implication for the eukaryotic protein such as RAS oncoproteins because it is proofing useful in various forms of cancer. Hence, studying yeast mating has therapeutic value as it may provide novel tumor-suppressing agents. Nevertheless, the marked evidences of fungal pheromones seem to be widespread and it not only have a significant role in cell-to-cell recognition but also in post-fusion events viz., induction of meiosis and maintenance of the filamentous state in some species of fungi [24].

4. Basidiomycetes pheromone signaling

If we have a look at the system of mating in basidiomycetes, it consists of haploid monokaryotic that induces dikaryotic stages. The monokaryotic mycelium consists of nuclei with one genetic type; therefore, the terminology homokaryon is derived. From the haploid spores, the mycelium grows, which may contain one nucleus. And when genetically different types of homokaryons mate, then dikaryon is formed. Generally, the tetrapolar system regulates the mating in the mushroom-forming species, which consists of genetic complexes namely A and B, which may not be linked with each other. The condition of tetrapolar reveals that there may be possibilities that are four in number as far as mating interactions are concerned involving haploid strains. And full compatible interaction may take place between mates when both genetic complexes have different specificities as compared to different allelic specificities and two semi-compatible interactions have been observed to take place when development is regulated by A- or B due to differences present in either complex. As far as basidiomycetes are concerned, both kinds of mating behavior are observed. The induction of sexual development during mating has to be responsive to binding of ligand on pheromone receptor followed by an ensuing signal transduction pathway, which will ultimately leads to dikaryotization. Hence, one of the chief functions in a system where pheromone and receptors are involved is to bring about the gene expressions of encoded proteins that are involved in attracting and subsequently directing mates to grow toward each other.

Hence, the interaction between pheromone and receptor is believed to be significantly appropriate for proceedings of fusion and most particularly when there is the presence of shared exchanges and migration of nuclei among the mating partners [25]. In addition, the evidences suggest that there is hardly any significant correlation between the strength of responses in species or its genetic distance from pheromone source sequence but due to influencing conditions or differences in development a species may be either weak or strong responder [26].

So, during a response to pheromone and nuclear migration in the fungal species of S. cerevisiae, it has been observed that a mating-specific Gα, Gpa1, has been found to interact with kinesin-14 (Kar-3) that is a minus-end-directed microtubule-associated motor [27]. In addition, due to this interaction nuclear migration induced by pheromone is regulated toward shmoo tip. After pheromone treatment, Kar3 immunoprecipitates the Gpa1, thereby demonstrating interactions among protein–protein complexes. Finally, at the shmoo tip visualization of Gpa1 and Kar3 occurs. It was regarded that the positive association of Kar3 with Gpa1 gets affected when utilization of mutant Gpa1 is undertaken to the shmoo tip. The dynamics and orientation of microtubules also have a significant association with Gpa1. It was concluded that Gpa1 was considered to provide an externally regulated position determinant for the anchorage of Kar3 [27]. Even though, the conserved pheromone/receptor system and the presence of different regulations, the de novo in silico discovery of proteins similar to a receptor, and interactions of various intracellular signal transduction pathways build the perceptive of the origin as well as the functionality of the pheromone receptor system in highly complex systems of agaricomycetes a challenge to undertake [28].

As far as detection of external stimuli is concerned in eukaryotic organisms, there are arrangements in terms of signaling transduction pathways that are employed for detections [29]. The signal transduction pathway occurs via mitogen-activated protein kinases (MAPKs) that comprises of Ser/Thr protein kinases which helps in converting the extracellular stimuli into several downstream cellular responses. And it has been reported that since ancient times the MAPKs signal transduction pathway is extensively utilized in many physiological processes throughout evolution.

Hence, we can mention that MAPK pathway involves well-conserved signal transduction cascade in eukaryotes [30]. The signaling pathway plays a significant role in response to several factors such as growth factors, mitosis, gene expression, cytokine regulation, motility, metabolism, cell death, cellular stressors, differentiation, and pheromones [29]. In mammals, MAPKs are 14 in number and have been characterized into 7 groups. Generally, MAPK pathways consist of kinases that are MAP3K (MAPKKK), MAP2K (MAPKK), and MAPK that upon stimulus detection become co-localized and subsequently allow sequential phosphorylation and activation. The MAPKKKs are Ser/Thr protein kinases that get activated due to phosphorylation results into their interaction with small proteins that are GTP-binded that belongs to Ras/Rho family. Hence, activation of MAPKKK happens and it directs phosphorylation, which brings about the MAPK activity via phosphorylation of Thr and Tyr residues within a conserved motif known as Thr-X-Tyr located in the kinase domain [29]. Thus, this pathway consists of several described adaptors, docking, and scaffold proteins that are occupied in the overall regulation of MAPK cascade of signaling. In these, the scaffolds are considered to be the largest, a multi-domain protein. The scaffold protein is provided as a physical platform that has the capability of binding several members of a MAPK pathway that regulates in allocating in the regulation of localization of kinase, complex assembly, and transcriptional factors for signal propagation toward nucleus for respective expression [31].

Nevertheless, in fungi, the growth and development and several other processes require a wide range of signal transduction pathways [31, 32, 33]. Therefore, various MAPK-involved pathways have been reported in the biological regulations concerned in fungal species. So far, all MAPK pathways in S. cerevisiae have been defined genetically. But the pathway related to mating was the first MAPK module to be defined. S. cerevisiae has been reported to have five MAPK pathways (Fus3, Kss1, Hog1, Mpk1, and Mpl1) wherein each regulates separate cellular processes [34].

Despite very few information available on the signaling mechanisms that are involved in fungal development, still among various known pathways, especially in eukaryotes sexual development occurs via the MAPK pathway, which is widely studied during pheromone signaling [35]. In addition, since it came into existence, it is known to be highly conserved in the fungi in terms of orthologous pheromone module MAPK pathways. In the review of Frawley and Bayram [31], where they specifically mentioned the role of a module that involves pheromone as a foremost signaling in filamentous fungal species viz., A. nidulans, A. flavus, and A. fumigates.

For instance, it has been shown in the case of filamentous growth in S. cerevisiae via MAPK pathway in response to pheromones where Ste7, Ste11, Ste20, and Kss1 kinases and the Ste12 transcription factor acted as several components [36, 37]. However, in diploid cells, for the filamentous growth pheromones, pheromone receptors and subunits of the pheromone-activated heterotrimeric G protein are not necessarily expressed in case of diploid cells (161). The signaling, in particular, involves different specializations viz.,

1. Activation of the MAP kinase cascade by the beta-gamma subunits of the pheromone-activated heterotrimeric G protein during mating of haploid cells. There exist various mechanisms that involve Cdc42, Ras2, and 14–3-3 proteins Bmh1 and Bmh2 during growth in filamentous way and this ultimately leads to the activation of the MAP kinase pathway [38, 39].

2. During mating the scaffold protein Ste5 helps in securing the components of the MAP kinase cascade; however, during filamentous growth Ste5 is not vital and the role of scaffolding may be played by another protein. In addition, Spa2 protein is suggested to interact with the MAP kinase cascade components as the scaffold during filamentous growth [40].

3. The Fus3 and Kss1 kinases differ in the MAP kinase of S. cerevisiae where Fus3 specifically regulates mating and invasive growth is inhibited while Kss1 is comprehensively assisted in the regulation of invasive and filamentous growth [41]. Furthermore, during filamentous growth Kss1 role has been positive and negative regulation and it helps in preventing repression of Ste12 by the proteins Dig1 and Dig2 [42, 43, 44].

4. In the last specialization, Ste12 interacts with the Mcm1 protein during mating and transcription is activated, which eventually leads to gene expression of pheromone-responding features, while heterodimer formation occurs in diploid cells by Ste12 accompanied by Tec1 that leads to activation of transcription related genes along with filamentation-responding features [37]. Thus, in such a way, Ste12 protein may give way for two different patterns of suitable transcriptional responses in haploid and diploid cells.

In the case of A. flavus pheromone module, there is the presence of kinases and SteD and the process of dimerization occurs at hyphal tip as MkkB-MpkB where it interacts in the cytoplasm as SteC-SteD dimer and a tetrameric complex is formed. From this tetrameric complex, phosphorylation of MpkB occurs and it enters the nucleus and respective regulation of transcriptional factors occurs. Furthermore, at the hyphal tip HamE localization is also observed for respective regulations. However, the exact mechanisms that are involved in its regulation are still subjected to further research.

5. Conclusions

The signal transduction cascade in the fungal kingdom which is highly conserved occurs not only by the element of pheromone but also by both secondary metabolism and pathogenicity present in various fungal pathogens. But still very few investigations have been carried out about the pathways that involve the protein complexes for signaling to happen in the fungal kingdom.

The fungal pheromones are generally secreted by the opposite mating cells to stimulate the production of the opposite sex organs. The fungal pheromone such as sirenin is produced by female gametes of Allomyces is used for attraction of male gametes and fusion. Trisporic acid reported from Zygomycetes fungi is responsible for zygotrophism and development of progametangium. Antheridiol and oogoniol hormones are produced by Achlyabisexualis, and vegetative hyphae of female strain-produced antheridiol are responsible for the development of antheridial hyphae on male thallus. Then, the male hyphae-produced oogoniol causes the initiation of oogonial hyphae on female thallus. In yeast, peptide hormone a and alpha-factor have specific receptors present on the opposite mating types. These peptide hormones bind to receptors that are specific to the cell surface of opposite mating type through GTP-binding protein causes the production of agglutinin of recipient cell and stops the G1 stage of the cell cycle. In basidiomycetes fungi different mating homokaryons are having either A or B genetic alleles and also have two semi-compatible interactions been observed in it leading to dikaryotization.

Even though there are evidences of the signaling pathway taking part in the regulation of various fungal progression that may be vegetative in nature, sporulation asexually, and sexual reproduction but the evidences concerning the requisite stimulation to activate these pathways or transcriptional regulation in the nucleus of filamentous fungi especially are meager. Therefore, there are still more prospects in the field of complex signal transduction pathways present in fungi and the mechanisms via in which certain genes are regulated utilizing the pheromone module. Hence, if in near future our researchers are able to characterize the vital regulators in the development of the fungal kingdom, then it could help in the sustenance of the global population by reducing food production loss by preventing various fungal crop diseases.

Conflict of interest

On behalf of all authors, the authors declare no competing and conflict of interest.