A cyclin protein governs the infectious and sexual life cycles of Cryptococcus neoformans.

Cell cycle is a fundamental process underlying growth and development in evolutionarily diverse organisms, including fungi. In human fungal pathogens, cell cycle control generally determines their life cycles, either in the environment or during infections. Thus, cell cycle components can potentially serve as important targets for the development of antifungal strategy against fungal infections. Here, in Cryptococcus neoformans, the most common cause of fatal fungal meningitis, we show that a previously uncharacterized B-type cyclin named Cbc1 is essential for both its infectious and sexual cycles. We reveal that Cbc1 coordinates various sexual differentiation and molecular processes, including meiosis. Especially, the absence of Cbc1 abolishes formation of sexual spores in C. neoformans, which are presumed infectious particles. Cbc1 is also required for the major Cryptococcus pathogenic attributes. Virulence assessment using the murine model of cryptococcosis revealed that the cbc1 mutant is avirulent. Together, our results provide an important insight into how C. neoformans employs shared cell cycle regulation to coordinate its infectious and sexual cycles, which are considered crucial for virulence evolution and the production of infectious spores.

Polyphasic Discrimination of Trichophyton tonsurans and T. equinum from Humans and Horses.

The anthropophilic dermatophyte Trichophyton tonsurans and its zoophilic counterpart T. equinum are phylogenetically closely related. The barcoding marker rDNA internal transcribed spacer (ITS) shows limited variation between these two species. In the current study, we combined molecular approaches with phenotypic data to determine the species boundaries between T. tonsurans (n = 52) and T. equinum (n = 15) strains originating from humans (n = 40), horses (n = 26), and a mouse (n = 1). Culture characteristics and physiology on Trichophyton agar media 1 and 5 were evaluated. Multi-locus sequencing involving ITS, partial large rDNA subunit (LSU), ß-tubulin (TUB), 60S ribosomal protein (RPB), and translation elongation factor-3 (TEF3) genes, and the mating-type (MAT) locus was performed. Amplified fragment length polymorphism data were added. None of the test results showed complete mutual correspondence. With the exception of strains from New Zealand, strains of equine origin required niacin for growth, whereas most strains from human origin did not show this dependence. It is concluded that T. tonsurans and T. equinum incompletely diverged from a common lineage relatively recently. MAT1-1 and MAT1-2 are the main distinguishing genes between the two species.

Mating-type switching and mating-type gene array expression in the methylotrophic yeast Ogataea thermomethanolica TBRC656.

The methylotrophic yeast, Ogataea thermomethanolica TBRC656, is an attractive host organism for heterologous protein production owing to the availability of protein expression vectors and a genome-editing tool. In this study, we focused on mating-type switching and gene expression in order to elucidate its sexual life cycle and establish genetic approaches applicable for the strain. A putative mating-type gene cluster was identified in TBRC656 that is syntenic to the cluster in Ogataea parapolymorpha DL-1 (previously named Hansenula polymorpha). Like DL-1, TBRC656 possesses two mating loci, namely MATa and MATα, and also shows flip-flop mating-type switching. Interestingly, unlike any other methylotrophic yeast, TBRC656 robustly switched mating type during late growth in rich medium (YPD). Under nutrient depletion, mating-type switching was observed within one hour. Transcription from both MATa and MATα mating loci was detected during growth in YPD, and possibly induced upon nitrogen depletion. Gene expression from MATα was detected as a single co-transcript from a three-gene array (α2-α1-a1S). Deletion of a putative a1S ORF at the MATα locus had no observed effect on mating-type switching but demonstrated significant effect on mating-type gene expression at both MATa and MATα loci.

She Loves Me, She Loves Me Not: On the Dualistic Asexual/Sexual Nature of Dermatophyte Fungi.

Dermatophytes are ascomycetous fungi whose sexuality is greatly influenced by their ecology. Sexual reproduction is ubiquitous among soil-related geophiles and some animal-associated zoophiles. In contrast, anthropophiles are generally present as a single mating type in the population and appear to reproduce asexually. In this article, the current knowledge on the sexuality of dermatophytes including reproduction modes, mating conditions, mating type distributions and the mating type (MAT) locus is presented in the context of revised taxonomy and discussed from an evolutionary perspective.

Frequency of the Mating-Type (MAT1) in Histoplasma capsulatum Isolates from Buenos Aires, Argentina.

Sex is genetically determined in Histoplasma capsulatum, governed by a sex-specific region in the genome called the mating-type locus (MAT1). We investigate the distribution of isolates of two H. capsulatum mating types in the clades circulating in Buenos Aires, Argentina. Forty-nine H. capsulatum isolates were obtained from the culture collection of the Mycology Center. The MAT1 locus was identified by PCR from the yeast suspension. The analysis of forty-eight isolates from clinical samples exhibited a ratio of 1.7 (MAT1-1:MAT1-2) and the only isolate from soil was MAT1-1. Forty-five H. capsulatum isolates belonged to the LAm B clade (H. capsulatum from Latin American group B clade) and showed a ratio of 1.8 (MAT1-1:MAT1-2). These results suggest an association between the mating types in isolates belonging to the LAm B clade. It remains to be defined whether a greater virulence should be attributed to the differences between the strains of the opposite mating type of the LAm B clade.

Convergent evolution of linked mating-type loci in basidiomycete fungi.

Sexual development is a key evolutionary innovation of eukaryotes. In many species, mating involves interaction between compatible mating partners that can undergo cell and nuclear fusion and subsequent steps of development including meiosis. Mating compatibility in fungi is governed by the mating type (MAT) loci. In basidiomycetes, the ancestral state is hypothesized to be tetrapolar, with two genetically unlinked MAT loci containing homeodomain transcription factor genes (HD locus) and pheromone and pheromone receptor genes (P/R locus), respectively. Alleles at both loci must differ between mating partners for completion of sexual development. However, there are also basidiomycetes with bipolar mating systems, which can arise through genomic linkage of the HD and P/R loci. In the order Tremellales, bipolarity is found only in the pathogenic Cryptococcus species. Here, we describe the analysis of MAT loci from 24 species of the Trichosporonales, a sister order to the Tremellales. In all of the species analyzed, the MAT loci are fused and a single HD gene is present in each mating type, similar to the organization in the pathogenic Cryptococci. However, the HD and P/R allele combinations in the Trichosporonales are different from those in the pathogenic Cryptococci. This and the existence of tetrapolar species in the Tremellales suggest that fusion of the HD and P/R loci occurred independently in the Trichosporonales and pathogenic Cryptococci, supporting the hypothesis of convergent evolution towards fused MAT regions, similar to previous findings in other fungal groups. Unlike the fused MAT loci in several other basidiomycete lineages though, the gene content and gene order within the fused MAT loci are highly conserved in the Trichosporonales, and there is no apparent suppression of recombination extending from the MAT loci to adjacent chromosomal regions, suggesting different mechanisms for the evolution of physically linked MAT loci in these groups.

Unisexual reproduction promotes competition for mating partners in the global human fungal pathogen Cryptococcus deneoformans.

Courtship is pivotal for successful mating. However, courtship is challenging for the Cryptococcus neoformans species complex, comprised of opportunistic fungal pathogens, as the majority of isolates are α mating type. In the absence of mating partners of the opposite mating type, C. deneoformans can undergo unisexual reproduction, during which a yeast-to-hyphal morphological transition occurs. Hyphal growth during unisexual reproduction is a quantitative trait, which reflects a strain's ability to undergo unisexual reproduction. In this study, we determined whether unisexual reproduction confers an ecological benefit by promoting foraging for mating partners. Through competitive mating assays using strains with different abilities to produce hyphae, we showed that unisexual reproduction potential did not enhance competition for mating partners of the same mating type, but when cells of the opposite mating type were present, cells with enhanced hyphal growth were more competitive for mating partners of either the same or opposite mating type. Enhanced mating competition was also observed in a strain with increased hyphal production that lacks the mating repressor gene GPA3, which contributes to the pheromone response. Hyphal growth in unisexual strains also enables contact between adjacent colonies and enhances mating efficiency during mating confrontation assays. The pheromone response pathway activation positively correlated with unisexual reproduction hyphal growth during bisexual mating and exogenous pheromone promoted bisexual cell fusion. Despite the benefit in competing for mating partners, unisexual reproduction conferred a fitness cost. Taken together, these findings suggest C. deneoformans employs hyphal growth to facilitate contact between colonies at long distances and utilizes pheromone sensing to enhance mating competition.

Multiple Reinventions of Mating-type Switching during Budding Yeast Evolution.

Cell type in budding yeasts is determined by the genotype at the mating-type (MAT) locus, but yeast species differ widely in their mating compatibility systems and life cycles. Among sexual yeasts, heterothallic species are those in which haploid strains fall into two distinct and stable mating types (MATa and MATα), whereas homothallic species are those that can switch mating types or that appear not to have distinct mating types [1, 2]. The evolutionary history of these mating compatibility systems is uncertain, particularly regarding the number and direction of transitions between homothallism and heterothallism, and regarding whether the process of mating-type switching had a single origin [3-5]. Here, we inferred the mating compatibility systems of 332 budding yeast species from their genome sequences. By reference to a robust phylogenomic tree [6], we detected evolutionary transitions between heterothallism and homothallism, and among different forms of homothallism. We find that mating-type switching has arisen independently at least 11 times during yeast evolution and that transitions from heterothallism to homothallism greatly outnumber transitions in the opposite direction (31 versus 3). Although the 3-locus MAT-HML-HMR mechanism of mating-type switching as seen in Saccharomyces cerevisiae had a single evolutionary origin in budding yeasts, simpler "flip/flop" mechanisms of switching evolved separately in at least 10 other groups of yeasts. These results point to the adaptive value of homothallism and mating-type switching to unicellular fungi.

Modular and tunable biological feedback control using a de novo protein switch.

De novo-designed proteins1-3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4-7. One such designer protein-degronLOCKR, which is based on 'latching orthogonal cage-key proteins' (LOCKR) technology8-is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here we leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signalling pathways and synthetic gene circuits. We first generate synthetic negative and positive feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signalling molecules, illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. We next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behaviour in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biology10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnological and therapeutic applications.

Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1-Hsp90 pathway.

While sexual reproduction is pervasive in eukaryotic cells, the strategies employed by fungal species to achieve and complete sexual cycles is highly diverse and complex. Many fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, are homothallic (able to mate with their own mitotic descendants) because of homothallic switching (HO) endonuclease-mediated mating-type switching. Under laboratory conditions, the human fungal pathogen Candida albicans can undergo both heterothallic and homothallic (opposite- and same-sex) mating. However, both mating modes require the presence of cells with two opposite mating types (MTLa/a and α/α) in close proximity. Given the predominant clonal feature of this yeast in the human host, both opposite- and same-sex mating would be rare in nature. In this study, we report that glucose starvation and oxidative stress, common environmental stresses encountered by the pathogen, induce the development of mating projections and efficiently permit same-sex mating in C. albicans with an "a" mating type (MTLa/a). This induction bypasses the requirement for the presence of cells with an opposite mating type and allows efficient sexual mating between cells derived from a single progenitor. Glucose starvation causes an increase in intracellular oxidative species, overwhelming the Heat Shock transcription Factor 1 (Hsf1)- and Heat shock protein (Hsp)90-mediated stress-response pathway. We further demonstrate that Candida TransActivating protein 4 (Cta4) and Cell Wall Transcription factor 1 (Cwt1), downstream effectors of the Hsf1-Hsp90 pathway, regulate same-sex mating in C. albicans through the transcriptional control of the master regulator of a-type mating, MTLa2, and the pheromone precursor-encoding gene Mating α factor precursor (MFα). Our results suggest that mating could occur much more frequently in nature than was originally appreciated and that same-sex mating could be an important mode of sexual reproduction in C. albicans.

Asymmetric diversification of mating pheromones in fission yeast.

In fungi, mating between partners depends on the molecular recognition of two peptidyl mating pheromones by their respective receptors. The fission yeast Schizosaccharomyces pombe (Sp) has two mating types, Plus (P) and Minus (M). The mating pheromones P-factor and M-factor, secreted by P and M cells, are recognized by the receptors mating type auxiliary minus 2 (Mam2) and mating type auxiliary plus 3 (Map3), respectively. Our recent study demonstrated that a few mutations in both M-factor and Map3 can trigger reproductive isolation in S. pombe. Here, we explored the mechanism underlying reproductive isolation through genetic changes of pheromones/receptors in nature. We investigated the diversity of genes encoding the pheromones and their receptor in 150 wild S. pombe strains. Whereas the amino acid sequences of M-factor and Map3 were completely conserved, those of P-factor and Mam2 were very diverse. In addition, the P-factor gene contained varying numbers of tandem repeats of P-factor (4-8 repeats). By exploring the recognition specificity of pheromones between S. pombe and its close relative Schizosaccharomyces octosporus (So), we found that So-M-factor did not have an effect on S. pombe P cells, but So-P-factor had a partial effect on S. pombe M cells. Thus, recognition of M-factor seems to be stringent, whereas that of P-factor is relatively relaxed. We speculate that asymmetric diversification of the two pheromones might be facilitated by the distinctly different specificities of the two receptors. Our findings suggest that M-factor communication plays an important role in defining the species, whereas P-factor communication is able to undergo a certain degree of flexible adaptation-perhaps as a first step toward prezygotic isolation in S. pombe.

Candida albicans White-Opaque Switching Influences Virulence but Not Mating during Oropharyngeal Candidiasis.

The capacity of Candida albicans to switch reversibly between the white phenotype and the opaque phenotype is required for the fungus to mate. It also influences virulence during hematogenously disseminated candidiasis. We investigated the roles of the mating type loci (MTL) and white-opaque switching in the capacity of C. albicans to mate in the oropharynx and cause oropharyngeal candidiasis (OPC). When immunosuppressed mice were orally infected with mating-competent opaque a/a and α/α cells either alone or mixed with white cells, no detectable mating occurred, indicating that the mating frequency was less than 1.6 × 10-6 Opaque cells were also highly attenuated in virulence; they either were cleared from the oropharynx or switched to the white phenotype during OPC. Although there were strain-to-strain differences in the virulence of white cells, they were consistently more virulent than opaque cells. In vitro studies indicated that relative to white cells, opaque cells had decreased capacity to invade and damage oral epithelial cells. The reduced invasion of at least one opaque strain was due to reduced surface expression of the Als3 invasin and inability to activate the epidermal growth factor receptor, which is required to stimulate the epithelial cell endocytic machinery. These results suggest that mating is a rare event during OPC because opaque cells have reduced capacity to invade and damage the epithelial cells of the oral mucosa.

Regulation of mating type switching by the mating type genes and RME1 in Ogataea polymorpha.

Saccharomyces cerevisiae and its closely related yeasts undergo mating type switching by replacing DNA sequences at the active mating type locus (MAT) with one of two silent mating type cassettes. Recently, a novel mode of mating type switching was reported in methylotrophic yeast, including Ogataea polymorpha, which utilizes chromosomal recombination between inverted-repeat sequences flanking two MAT loci. The inversion is highly regulated and occurs only when two requirements are met: haploidy and nutritional starvation. However, links between this information and the mechanism associated with mating type switching are not understood. Here we investigated the roles of transcription factors involved in yeast sexual development, such as mating type genes and the conserved zinc finger protein Rme1. We found that co-presence of mating type a1 and α2 genes was sufficient to prevent mating type switching, suggesting that ploidy information resides solely in the mating type locus. Additionally, RME1 deletion resulted in a reduced rate of switching, and ectopic expression of O. polymorpha RME1 overrode the requirement for starvation to induce MAT inversion. These results suggested that mating type switching in O. polymorpha is likely regulated by two distinct transcriptional programs that are linked to the ploidy and transmission of the starvation signal.

Sex and the Imperfect Fungi.

Approximately 20% of species in the fungal kingdom are only known to reproduce by asexual means despite the many supposed advantages of sexual reproduction. However, in recent years, sexual cycles have been induced in a series of emblematic "asexual" species. We describe how these discoveries were made, building on observations of evidence for sexual potential or "cryptic sexuality" from population genetic analyses; the presence, distribution, and functionality of mating-type genes; genome analyses revealing the presence of genes linked to sexuality; the functionality of sex-related genes; and formation of sex-related developmental structures. We then describe specific studies that led to the discovery of mating and sex in certain Candida, Aspergillus, Penicillium, and Trichoderma species and discuss the implications of sex including the beneficial exploitation of the sexual cycle. We next consider whether there might be any truly asexual fungal species. We suggest that, although rare, imperfect fungi may genuinely be present in nature and that certain human activities, combined with the genetic flexibility that is a hallmark of the fungal kingdom, might favor the evolution of asexuality under certain conditions. Finally, we argue that fungal species should not be thought of as simply asexual or sexual, but rather as being composed of isolates on a continuum of sexual fertility.

Fungal Sex: The Basidiomycota.

Fungi of the Basidiomycota, representing major pathogen lineages and mushroom-forming species, exhibit diverse means to achieve sexual reproduction, with particularly varied mechanisms to determine compatibilities of haploid mating partners. For species that require mating between distinct genotypes, discrimination is usually based on both the reciprocal exchange of diffusible mating pheromones, rather than sexes, and the interactions of homeodomain protein signals after cell fusion. Both compatibility factors must be heterozygous in the product of mating, and genetic linkage relationships of the mating pheromone/receptor and homeodomain genes largely determine the complex patterns of mating-type variation. Independent segregation of the two compatibility factors can create four haploid mating genotypes from meiosis, referred to as tetrapolarity. This condition is thought to be ancestral to the basidiomycetes. Alternatively, cosegregation by linkage of the two mating factors, or in some cases the absence of the pheromone-based discrimination, yields only two mating types from meiosis, referred to as bipolarity. Several species are now known to have large and highly rearranged chromosomal regions linked to mating-type genes. At the population level, polymorphism of the mating-type genes is an exceptional aspect of some basidiomycete fungi, where selection under outcrossing for rare, intercompatible allelic variants is thought to be responsible for numbers of mating types that may reach several thousand. Advances in genome sequencing and assembly are yielding new insights by comparative approaches among and within basidiomycete species, with the promise to resolve the evolutionary origins and dynamics of mating compatibility genetics in this major eukaryotic lineage.

Efficient Mating-Type Switching in Candida glabrata Induces Cell Death.

Candida glabrata is an apparently asexual haploid yeast that is phylogenetically closer to Saccharomyces cerevisiae than to Candida albicans. Its genome contains three MAT-like cassettes, MAT, which encodes either MATa or MATalpha information in different strains, and the additional loci, HML and HMR. The genome also contains an HO gene homolog, but this yeast has never been shown to switch mating-types spontaneously, as S. cerevisiae does. We have recently sequenced the genomes of the five species that, together with C. glabrata, make up the Nakaseomyces clade. All contain MAT-like cassettes and an HO gene homolog. In this work, we express the HO gene of all Nakaseomyces and of S. cerevisiae in C. glabrata. All can induce mating-type switching, but, despite the larger phylogenetic distance, the most efficient endonuclease is the one from S. cerevisiae. Efficient mating-type switching in C. glabrata is accompanied by a high cell mortality, and sometimes results in conversion of the additional cassette HML. Mortality probably results from the cutting of the HO recognition sites that are present, in HML and possibly HMR, contrary to what happens naturally in S. cerevisiae. This has implications in the life-cycle of C. glabrata, as we show that efficient MAT switching is lethal for most cells, induces chromosomal rearrangements in survivors, and that the endogenous HO is probably rarely active indeed.

Structural and functional conservation of fungal MatA and human SRY sex-determining proteins.

Sex determination in animals and fungi is regulated by specific sex-determining genes. The Aspergillus nidulans mating type gene matA and the human SRY (Sex-Determining Region Y) encode proteins containing a single HMG (high-mobility group) domain. Analysis of the amino-acid sequence of MatA and SRY transcription factors revealed significant structural similarity. The human SRY protein is able to functionally replace MatA and drives the sexual cycle in the fungus A. nidulans. Functional studies indicate that SRY drives early fruiting body development, and hybrid MatA protein carrying the SRY HMG box is fully capable of driving both early and late stages of sexual development, including gametogenesis. Our data suggest that SRY and MatA are both structurally and functionally related and conserved in regulating sexual processes. The fundamental mechanisms driving evolution of the genetic pathways underlying sex determination, sex chromosomes and sexual reproduction in eukaryotes appear similar.

Crosses between monokaryons of Pleurotus sapidus or Pleurotus florida show an improved biotransformation of (+)-valencene to (+)-nootkatone.

Several hundred monokaryotic and new dikaryotic strains derived thereof were established from (+)-valencene tolerant Pleurotus species. When grouped according to their growth rate on agar plates and compared to the parental of Pleurotus sapidus 69, the slowly growing monokaryons converted (+)-valencene more efficiently to the grapefruit flavour compound (+)-nootkatone. The fast growing monokaryons and the slow×slow and the fast×fast dikaryotic crosses showed similar or inferior yields. Some slow×fast dikaryons, however, exceeded the biotransformation capability of the parental dikaryon significantly. The activity of the responsible enzyme, lipoxygenase, showed a weak correlation with the yields of (+)-nootkatone indicating that the determination of enzyme activity using the primary substrate linoleic acid may be misleading in predicting the biotransformation efficiency. This exploratory study indicated that a classical genetics approach resulted in altered and partly improved terpene transformation capability (plus 60%) and lipoxygenase activity of the strains.

White-opaque switching in natural MTLa/α isolates of Candida albicans: evolutionary implications for roles in host adaptation, pathogenesis, and sex.

Phenotypic transitions play critical roles in host adaptation, virulence, and sexual reproduction in pathogenic fungi. A minority of natural isolates of Candida albicans, which are homozygous at the mating type locus (MTL, a/a or α/α), are known to be able to switch between two distinct cell types: white and opaque. It is puzzling that white-opaque switching has never been observed in the majority of natural C. albicans strains that have heterozygous MTL genotypes (a/α), given that they contain all of the opaque-specific genes essential for switching. Here we report the discovery of white-opaque switching in a number of natural a/α strains of C. albicans under a condition mimicking aspects of the host environment. The optimal condition for white-to-opaque switching in a/α strains of C. albicans is to use N-acetylglucosamine (GlcNAc) as the sole carbon source and to incubate the cells in 5% CO2. Although the induction of white-to-opaque switching in a/α strains of C. albicans is not as robust as in MTL homozygotes in response to GlcNAc and CO2, opaque cells of a/α strains exhibit similar features of cellular and colony morphology to their MTL homozygous counterparts. Like MTL homozygotes, white and opaque cells of a/α strains differ in their behavior in different mouse infection models. We have further demonstrated that the transcriptional regulators Rfg1, Brg1, and Efg1 are involved in the regulation of white-to-opaque switching in a/α strains. We propose that the integration of multiple environmental cues and the activation and inactivation of a set of transcriptional regulators controls the expression of the master switching regulator WOR1, which determines the final fate of the cell type in C. albicans. Our discovery of white-opaque switching in the majority of natural a/α strains of C. albicans emphasizes its widespread nature and importance in host adaptation, pathogenesis, and parasexual reproduction.

The mitochondrial Dnm1-like fission component is required for lgA2-induced mitophagy but dispensable for starvation-induced mitophagy in Ustilago maydis.

Selective elimination of mitochondria by autophagy (mitophagy) is a crucial developmental process to dispose of disintegrated or superflous organelles. However, little is known about underlying regulatory mechanisms. We have investigated mitophagy in response to conditional overexpression of the a2 mating-type locus gene lga2, which encodes a small mitochondrial protein critically involved in uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. In this study, we show that conditional overexpression of lga2 efficiently triggers mitophagy that is dependent on atg8 and atg11, consistent with selective autophagy. lga2-triggered mitophagy is preceded by mitochondrial dysfunction, including depletion of mitochondrial RNA transcripts, and is mechanistically distinct from starvation-induced mitophagy despite a common requirement for atg11. In particular, lga2-triggered mitophagy strongly depends on the mitochondrial fission factor Dnm1, but it is only slightly affected by N-acetylcysteine, which is an inhibitor of starvation-induced mitophagy. To further delineate the role of mitochondrial fission, we analyzed lga2 effects in Δfis1 mutants. This revealed that mitochondrial fragmentation was only attenuated and mitophagy was largely unaffected. In further support of a Fis1-independent role for Dnm1, mitochondrial association of green fluorescent protein-tagged Dnm1 as well as Dnm1-opposed mitochondrial fusion during sexual development were fis1 independent. In conclusion, our results specify the role of the mitochondrial fission factor Dnm1 in mitophagy and uncover differences between mitophagy pathways in the same cellular system.