Autophagy is an evolutionally conserved self-eating process in which cellular components such as organelles, aggregated proteins, invading microorganisms, and other cytoplasmic material are sequestered by a double-membrane structure called autophagosome and delivered to the degradative organelle for breakdown and recycling^[1]. So far there are at least 42 autophagy-related genes (ATGs) have been identified in the model yeast Saccharomyces cerevisiae by genetic screening and many of them are conserve in fungi, plants and mammals^[2]. There are three well-defined autophagy processes: macroautophagy, microautophagy and chaperone mediated autophagy^{[1, 3-4]}. Macroautophagy is the main pathway that involves delivery of cytosolic components to the vacuole/lysosome for degradation by double membrane vesicles known as autophagosome. Microautophagy, on the other hand involves direct engulfment of cytosolic material into the vacuole/lysosome. Chaperone mediated autophagy (CMA) is a complex and specific pathway for proteolysis of specific cytosolic proteins with the aid of chaperone molecules. Macroautophagy (referred to hereafter as autophagy) plays important roles to protect organism against diverse pathologies including infections, cancer, neurodegeneration, aging, and heart diseases^[5]. Autophagy also appears to play a critical role in fungi, impacting growth, morphology, pathogenicity and development^[6].

Pathogenic fungi include fungal species that can cause disease in plants, animals and human and have a great impact on agriculture and health care. Most pathogenic fungi undergo a life cycle composed of two important stages: asexual/sexual reproduction and invasive growth in the host tissues. Molecular mechanisms of autophagy have been extensively studied in many model pathogenic fungi such as Magnaporthe oryzae and Fusarium graminearum (Table 1). A number of reviews have summarized the role of autophagy in fungal development and pathogenicity^[7-8]. In this review, we focus on the recent advances in our understanding of autophagy in fungal asexual and sexual reproduction.

1 The process of autophagy

Autophagy is a highly conserved cellular degradation process in which the cytoplasmic components can be sequestered by a double-membraned organelle called autophagosome and are subsequently transferred to vacuole or lysosome for cytoplasmic contents degradation and recycling^[1]. Through this basic mechanism, autophagy plays a crucial role in cellular homeostasis as dysregulation of autophagy is associated with a wide range of diseases such as neurodegeneration, cancer, myopathies, and diabetes and so on^[9]. Autophagy can be triggered by various types of stress including starvation, hypoxia and hormonal stimuli^[9]. Autophagy pathways can be broken down into the following five sequential steps: Induction and autophagosome nucleation, autophagosome expansion and completion, autophagosome fusion with the vacuole/lysosome, and cargo breakdown and recycling (Figure 1).

Fungal autophagy is typically induced by nutrient (e.g. carbon, nitrogen) starvation or in response to treatment with rapamycin. Under autophagy-inducing conditions, a membranous cistern called the phagophore begins to form. The phagophore is generated from the phagophore assembly site (PAS) that is a putative early autophagosome precursor. The second step of autophagy is autophagosome expansion. In this step, acquisition of extra lipids permits the expansion of the phagophore and subsequent engulfment of the cytoplasmic content for degradation. After that the inner and outer bilayer of the autophagosome fuses to form two distinct membranes, forming the complete double-layered autophagosome. The autophagosome then docks with the surface of a vacuole and its outer membrane fuses with the vacuole (in yeast or plants) or lysosome (in mammals) membrane to form autolysome. Hydrolytic enzymes from the vacuole or lysosome degrade the inner membrane of the autophagosome, gaining access to the cargo of the inner vesicle. In the last step, all the contents of the autophagosome are degraded, yielding basic metabolites that are transported into the cytoplasm to be reused as a source of energy or building blocks for new proteins and lipids^[10].

2 Fungal asexual and sexual reproduction

Fungi produce spores, which may be asexual or sexual. The asexual spores are produced by mitosis having the genetic material inside, which allows them to make a whole new organism identical to its parent. Sexual reproduction enables genetic exchange in fungi and accelerates adaptation to a new environment. Fungal sexual reproduction is a complicated process dominated by two mating types. The process of fungal sexual reproduction involves mate recognition, cell-cell fusion yielding a zygote, generation of gametes via meiosis, and ploidy changes. Sexual reproduction has been studied to occur more predominantly in the Ascomycota and Basidiomycota phyla. There are two main types of sexual reproductions in fungi: homothallism, when mating occurs within a single individual, or in other words each individual is self-fertile; and heterothallism, when hyphae from a single individual is self-sterile and needs to interact with another compatible individual for mating to take place. Here we discuss fungal sexual reproduction using C. neoformans as an example.

C. neoformans is a basidiomycete yeast that can cause fungal meningoencephalitis in mostly immunocompromised individuals. As a heterothallic basidiomycete, C. neoformans has two mating type, α and a. Although C. neoformans is most commonly isolated as a budding yeast from patients and the environment, it can undergo a dimorphic transition to a filamentous growth form by two distinct differentiation pathways: mating (heterothallism) and monokaryotic fruiting (homothalism) (Figure 2). After fusion of haploid cells of α and a opposite mating types, dikaryotic filaments are produced and a basidia eventually formed in C. neoformans. Following the completion of meiosis finished inside a basidium, four chains of readily aerosolized basidiospores are produced on top of the basidium. Under laboratory conditions, C. neoformans var. neoformans strains can also differentiate and undergo monokaryotic fruiting to produce filaments and basidiospores^[11]. Although monokaryotic fruiting was originally thought to be strictly haploid, mitotic and asexual, fruiting has recently been recognized to be a modified form of sexual reproduction occurring between strains of the same mating type^[12] (Figure 2). Although mating and monokaryotic fruiting have similar morphological features, the hyphal cells that are produced during fruiting are mononucleate with unfused clamp connections, whereas those produced during mating contain two nuclei and are linked by fused clamp connections^[11] (Figure 2).

In C. neoformans, sexual reproduction contributes to fungal virulence via the production of infectious spores, in that α isolates can be more virulent than congenic a isolates^[13].Spores and desiccated yeast cells are thought to be the initial infectious propagules to cause infection by Cryptococcus because they are small enough to fit down into the deep alveoli of the lung^[14].Spores are also documented to be infectious propagules in a serious of studies via inhalation and direct intracerebral infection^[15].

Sexual reproduction enables the pathogenic fungi to proliferate and undergo genetic exchange in response to new environmental conditions such as stressful conditions, different host organisms, or changes in the host such as antimicrobial therapy. Further research of the sexual nature of pathogenic fungi will help to elucidate how fungi have evolved into successful pathogens.

3 Autophagy in fungal reproduction of model pathogenic fungi

Autophagy has important roles in various cellular functions including sporulation in various fungi. Natural induction of autophagy was reported in fungal asexual or sexual sporulation in numerous model pathogenic fungi. During fungal reproduction, autophagy may help to provide materials or energy source to build up new intracellular structures by breaking down of cellular components. This is particularly important because nutrient scarcity may commonly occur during fungal morphogenesis and development.

So far there are at least 42 autophagy-related genes (ATGs) have been identified in the model yeast S. cerevisiae by genetic screening and many of them are conserve in fungi, plants and mammals. In model filamentous fungi, ATG1, 2, 4, 5, 7, 8, 9, 15, 17, 18 and the phosphatidylinositol 3-kinase encoding gene VPS34 are conserved with their orthologs in yeasts or animals^[16]. Functional requirement of autophagy for fungal asexual or sexual development was confirmed by characterization of autophagy-deficient mutants in diverse model fungi as removing one of the conserved ATGs resulted in defects in asexual and/or sexual sporulation. In the next couple of sections, we will discuss the role and mechanism of autophagy in fungal reproduction of pathogenic fungi causing diseases in plants, insects and human.

3.1 Autophagy regulates nutrient metabolism for fungal reproduction

During fungal development and morphogenesis, nutrient deprivation may commonly occur and autophagic degradation may need to produce abundant nutrients and small molecules for energy source or materials for building up new intracellular structures. Functional requirement of autophagy for fungal reproduction was investigated by characterization of autophagy-deficient mutants in diverse fungal systems in which autophagy-deficient fungal mutants showed defects in conidiation and/or sporulation. Studies on model pathogenic fungi such as M. oryzae have shown that autophagic degradation helps to utilize the cellular carbohydrate and nitrogen storages as a source of nutrients for fungal reproduction.

M. oryzae, also known as rice blast fungus, is a plant-pathogenic fungus that causes devastating blast disease in rice, wheat and barley. The asexual spores called conidia produced by M. oryzae are responsible for the spread of blast disease. Involvement of autophagy in conidiation in M. oryzae was relatively well studied and there are at least 5 ATG genes (ATG1, ATG4, ATG5, ATG8, ATG14) are confirmed to be essential for conidiation^[17-21]. Among them, ATG4 and ATG5 were further confirmed to be necessary for perithecia formation in M. oryzae^[18-21]. Conidiation defects in autophagy deficient strains could be restored by addition of carbon sources such as glucose or sucrose in M. oryzae^[17]. A comparative proteomics study showed that glycogen phosphorylase was differentially expressed in the atg8Δ mutant, and further detailed analysis on glycogen catabolism indicated that autophagy-assisted glycogen homeostasis is important for M. oryzae conidiation^[17-22]. These results suggest that autophagy plays an important role in carbon source utilization during M.. oryzae conidiation.

Besides M.. oryzae, autophagy assisted carbon utilization was also reported in F. graminearum, M. robertsii and U. maydis. So far, 10 autophagy-related genes, ATG1, ATG5, ATG8, ATG9, ATG13-16, ATG20 and ATG24^[34-36], were analyzed and shown to be essential for fungal asexual reproduction in F. graminearum. Among them, ATG1 and ATG5 were also found to be necessary for sexual reproduction in F. graminearum as deletion either ATG1 or ATG5 resulted in reduced Perithecia formation^[35]. Functional analysis showed that the FgATG15 disruptants were reduced in storage lipid degradation under starvation conditions, implicating autophagy's involvement in lipid turnover in F. graminearum^[36]. Involvement of autophagy in lipid metabolism was also found in the entomopathogen M. robertsii. M. robertsii autophagy-deficient mutant Mratg8Δ in which MrATG8, an ortholog of yeast ATG8, was deleted failed to produce conidia either on a nutrient-poor minimum medium or a nutrient-rich potato dextrose agar, indicating autophagy is indispensable for M. robertsii to form conidiophores^[40]. TEM analysis also found that the accumulation of lipid droplets in conidia was also significantly impaired in Mratg8Δ, indicating that autophagy is liked with lipid metabolism in M. robertsii. The role of autophagy in the development and virulence of U. maydis, a basidiomycetous fungus that causes smut on maize, was investigated using a reverse genetic approach. Deletion of the ATG8 orthologue in U. maydis resulted in the formation of very few teliospores^[42]. The reduced conidiation or teliospores formation of the atg8 mutant in F. graminearum^[36] and U. maydis^[42] could potentially be explained by a lack of autophagic activity, which leads to a defect in glycogen metabolism.

Autophagy was also suggested to play role in recycling nitrogen sources as nutrient source. In A. fumigatus, one of the most common Aspergillus species causing disease in immunocompromised individuals, an autophagy-deficient strain of A. fumigatus constructed by disrupting the Afatg1 gene failed to produce conidia normally unless the nitrogen content of the medium was increased, suggesting that starvation-associated conidiation relies upon autophagy to provide sufficient nitrogen to support conidiophore development^[24].

Hence, based on the functions of autophagy for nutrient catabolism in fungal reproduction of the model pathogenic fungi discussed above, we can appreciate that autophagy-assisted nutrient catabolism plays an essential role in fungal differentiation and development, including fungal asexual and sexual reproduction.

3.2 Autophagy contributes to nuclear degradation and/or distribution for fungal reproduction

Fungi produce asexual or sexual spores during the process of fungal reproduction. This process involves a series of nuclear events such as nuclear replication, migration, fusion, division and even the degradation of nuclei. Over the past decades, there are a large number of reports on the requirement of autophagy in fungal differentiation. However, the actual regulatory mechanism of autophagy in fungal reproduction remains largely unknown. Recently, autophagy-deficient mutants in A. oryzae showed defects in aerial hyphae development and asexual sporulation^[43-46], indicating that autophagy is essential for fungal asexual reproduction in A. oryzae. The deduced function of autophagy involved in fungal asexual reproduction in A. oryzae might be related to the nuclei degradation mediated by autophagy^[45-47].

Autophagy was also found to mediate nuclear degradation after hyphal fusion in the plant and human pathogen F. oxysporum. The Foatg8Δ strain in which the F. oxysporum ATG8 gene was deleted displayed reduced rates of conidiation and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. The hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with two or more nuclei while wild-type hyphae are almost exclusively composed of uninucleated hyphal compartments. Timelapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants, suggesting that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum^[38].

C. neoformans is an encapsulated yeast-like fungal pathogen causing cryptococcal pneumonia or meningitis predominantly in immunocompromised individuals. Autophagy is required for successful infection in C. neoformans. The upstream inducer of autophagy Vps34 was proved to be essential for pathogenesis in C. neoformans because the vps34Δ mutant in which autophagy pathway was blocked showed reduced viability under starvation and fast clearance from the infected host tissue^[29]. Similar results were obtained from the autophagy-deficient strain CnATG8 RNAi strain and the atg7Δ mutants strain^[29-31]. The above results of study showed that autophagy plays an important role in adapting to nutrient starvation conditions and fungal virulence in C. neoformans. However, the influence of autophagy on Cryptococcus mating or sexual reproduction has not yet been studied. Recently we examined three ubiquitin-like autophagy proteins, Atg5, Atg8 and Atg12, and the results showed that basidiospore production was completely blocked even though the autophagy-deficient mutants (atg5Δ, atg5Δ, and atg12Δ) can still form dikaryotic hyphae and basidia in the bilateral mating assay. Further study showed that the two haploid nuclei inside the dikaryotic hyphae cell can fuse but failed to separate resulting block of basidiospore formation at the stage of basidium development, indicating autophagy regulates meiosis and basidiospore formation in C. neoformans (data not published). Functions of autophagy in fungal reproduction in human fungal pathogens were also summarized in Table 1. Taken together, autophagy plays an important role in autophagic degradation or distribution of the nuclei in fungal pathogens, which may physiologically contribute to the asexual and/or sexual reproduction in pathogenic fungi.

3.3 Autophagy in other fungal pathogens

Autophagy was also found to be essential for fungal reproduction in other model fungal pathogens as autophagy-deficient mutants showed defects in conidiation and/or sporulation in these fungi. Examples include: B. cinerea Bcatg1Δ^[28], B. bassiana Bbatg1Δ^[26], Bbatg5Δ^[27], Bbatg8Δ^[26], and C. orbiculare atg8Δ and atg26Δ mutants^[32-33]. Similarly, The autophagy-related gene TrATG5, a homolog of S. cerevisiae ATG5, was found to be essential for autophagy, conidiophore formation and asexual sporulation in T. reesei^[48]. However, the mechanistic role of autophagy in fungal reproduction remains unknown and further mechanistic studies are needed to determine how autophagy regulates the reproduction in these fungi.

4 Conclusions

In the past two decades, functions of autophagy have been extensively studied in various model fungi and much progress has been made in our understanding of the disease mechanisms of pathogenicity of fungal pathogens. However, our knowledge about specific functions of autophagy in fungal development especially in fungal sexual and asexual reproduction remains limited. Current and relevant studies focused mainly on the phenotypic analysis of the autophagy-deficient mutants and there has been very little research on how autophagy regulates fungal reproduction. During fungal growth, development and reproduction, autophagy helps the fungi to transport the nutrients to the new cells by regulating the utilization of their own cellular nutrient storage. Thus, the deeper and more extensive researches are needed to further enrich our knowledge on the regulation of autophagy during fungal development and reproduction.

In addition, fungal development often linked to pathogenicity of pathogenic fungi as most of the autophagy-deficient mutants having developmental defects reduce or lose pathogenicity in pathogenic fungi. During the infection of a pathogen, autophagy helps the fungus to adapt to the adverse conditions and better infect and colonize the host. Considering the importance of pathogenic fungi in health care and agricultural production, key autophagy-related proteins need to be taken into consideration as potential antifungal targets for pharmaceutical development. Therefore, further studies on the roles of autophagy in fungal development and pathogenesis are warranted, and may aid to the future development of new fungicides and control measures of fungal diseases in plants and humans.

Acknowledgments

We thank Dr. Chaoyang Xue at Rutgers University for critical reading of the manuscript and valuable comments for the study.