Bacterial endosymbionts control host sexuality and reveal reproductive genes of early divergent fungi
Ontology highlight
ABSTRACT: Through transcriptome profiling using RNA-seq, we investigated the mechanisms behind bacterial endosymbiont (Burkholderia rhizoxinica) control over host (Rhizopus microsporus) reproductive biology. By analyzing differential expression across six different conditions, including fungal opposite mates growing independently with or without endosymbionts, as well as opposite mates growing together with endosymbionts (mating) or without endosymbionts (no mating), we were able to identify that endosymbionts control expression of a Ras signaling protein critical for sexual reproduction in many fungi (Ras2). As little is known regarding sexual reproduction in Mucoromycotina, we also used these data to investigate conservation of sex-related genes across all fungi, as well as predict potential genes involved in sensing of trisporic acid, the mating pheromone used by these fungi.
Project description:Through transcriptome profiling using RNA-seq, we investigated the mechanisms behind bacterial endosymbiont (Burkholderia rhizoxinica) control over host (Rhizopus microsporus) reproductive biology. By analyzing differential expression across six different conditions, including fungal opposite mates growing independently with or without endosymbionts, as well as opposite mates growing together with endosymbionts (mating) or without endosymbionts (no mating), we were able to identify that endosymbionts control expression of a Ras signaling protein critical for sexual reproduction in many fungi (Ras2). As little is known regarding sexual reproduction in Mucoromycotina, we also used these data to investigate conservation of sex-related genes across all fungi, as well as predict potential genes involved in sensing of trisporic acid, the mating pheromone used by these fungi. 6 different conditions were analyzed, each consisting of two biological replicates. These included Rhizopus microsporus ATCC52813 (sex +) growing alone with endosymbionts, R. microsporus ATCC52814 (sex -) growing alone with endosymbionts, ATCC 52813 growing alone without endosymbionts, ATCC52814 growing alone without endosymbionts, ATCC52813 and ATCC52814 growing together with endosymbionts (successfully mating), and ATCC52813 and ATCC52814 growing together without endosymbionts (failure to mate). In each condition, fungi were cultivated on half-strength PDA and plugs of mycelium were placed at the edge of the plate. After 6 days, approximately 2.5 cm of tissue were harvested from the center of the plate. Each biological replicate consists of 5 plates which were pooled prior to RNA extraction to ensure sufficient tissue was collected.
Project description:Modes of sexual reproduction in eukaryotic organisms are highly diversified. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally and morphologically differentiated white and opaque cells show a coordinating behavior in the process of mating. Although white cells are mating-incompetent, they are induced to produce sexual pheromones when treated with opposite pheromones or interacted with opaque cells of an opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections and thus facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTLa) in sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling and thus create an environment conducive to sexual mating. This coordination behavior of the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans. total RNA profiles of white cell treated with pheromone
Project description:Modes of sexual reproduction in eukaryotic organisms are highly diversified. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally and morphologically differentiated white and opaque cells show a coordinating behavior in the process of mating. Although white cells are mating-incompetent, they are induced to produce sexual pheromones when treated with opposite pheromones or interacted with opaque cells of an opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections and thus facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTLa) in sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling and thus create an environment conducive to sexual mating. This coordination behavior of the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.
Project description:Pseudo-nitzschia multistriata is a planktonic diatom species with a diplontic life cycle comprising a short sexual phase during which gametes are produced from the encounter of two diploid cells of opposite mating type (MT). Gene expression studies have highlighted the presence of substantial changes occurring at the onset of sexual reproduction. Collectively, there is clear evidence of a chemical cross talk which mediates the mating step.Herein, we have hypothesized that these major transcriptomic changes are associated to variations in the amount and nature of the metabolites produced by the cells. In order to investigate such chemical diversity in an unbiased manner, we undertook an untargeted metabolomics approach. Liquid chromatography – tandem mass spectrometry was applied to investigate the differences in the metabolic profiles between control cells in the vegetative phase (MT+ and MT-) and mixed strains of opposite MTs (cross) undergoing sexual reproduction. Of the 2408 high-quality features obtained, 70 known metabolites could be identified based on in-house libraries and online databases; molecular networking proved valuable to classify additional 47 non-identified features.The present exploratory study provides an overview of the chemical diversity detectable in P. multistriata, presenting a dynamic picture of the changes occurring during sexual reproduction. In particular, the reduction of phytol detected in the cross can be linked to the general downregulation of photosynthesis during sexual reproduction observed elsewhere. The dysregulation of other metabolites stands as a starting point for the understanding of the mechanisms regulating sexual reproduction: a metabolite that was tentatively identified as 7-dehydrodesmosterol exhibited the highest upregulation in the comparison between the two MTs of opposite sex, as well as between MT+ vs cross.The effects of extraction solvents and harvesting times on the ensemble of endo-metabolites were also analyzed.
Project description:Phytophthora infestans is a filamentous plant pathogen. It belongs to the class Oomycota within the Stramenopiles. Despite the importance of sexual reproduction in P. infestans, many aspects of the mating process remain unknown. In this study we are investigating its mating mechanisms using different molecular techniques. Isolates with A1 and A2 mating types from Sweden, the Netherlands and the UK, were used for this purpose because mating frequencies are known to differ among European countries. RNA was prepared from the “mating-zone” between the 4 Swedish, 1 Dutch and 1 British pairs. RNA-seq analysis was performed on an Illumina HiSeq 2500 platform and data normalized to parental isolates growing individually.
Project description:Sexual reproduction facilitates infection by the production of both a lineage advantage and infectious sexual spores in the ubiquitous human fungal pathogen Cryptococcus deneoformans. However, the regulatory determinants specific for initiating mating remain poorly understood. Here, we identified a velvet family regulator, Cva1, that strongly promotes sexual reproduction in C. deneoformans. This regulation was determined to be specific, based on a comprehensive phenotypic analysis of cva1 under 25 distinct in vitro and in vivo growth conditions. We further revealed that Cva1 plays a critical role in the initiation of early mating events, especially sexual cell-cell fusion, but is not important for the late sexual development stages or meiosis. Thus, Cva1 specifically contributes to mating activation. Importantly, a novel mating-responsive surface protein, Cfs1, serves as the key target of Cva1 during mating, since its absence nearly blocks cell-cell fusion in C. deneoformans and its sister species C. neoformans. Together, our findings provide insight into how C. deneoformans ensures regulatory specificity of mating.
Project description:The human fungal pathogen Candida albicans can switch stochastically and heritably between a “white” phase and an “opaque” phase. Opaque cells are the mating-competent form of the species whereas white cells are essentially “sterile”. Here, we report that glucose depletion, a common nutrient stress, enables C. albicans white cells to undergo efficient sexual mating. The relative expression levels of pheromone-sensing and mating-associated genes (including STE2/3, MFA1, MFalpha1, FIG1, FUS1, and CEK1/2) were increased under glucose depletion conditions, while expression of mating repressors TEC1 and DIG1 was decreased. We show that Cph1 and Tec1, factors that act downstream of the pheromone MAPK pathway, play opposite roles in regulating white cell mating as TEC1 deletion or CPH1 overexpression promoted white cell mating. Moreover, inactivation of the Cph1 repressor Dig1 increased white cell mating ~4,000 fold in glucose-depleted medium relative to that in the presence of glucose. These findings reveal that the white-to-opaque epigenetic switch may not be a prerequisite for sexual mating in C. albicans in nature. Given parallels between C. albicans white cell mating to that of other yeast species, this mechanism of mating could represent a more ancient strategy of sexual reproduction in C. albicans.
Project description:Sexual reproduction can promote genetic diversity in eukaryotes, and yet many pathogenic fungi have been labeled as obligate asexual species. It is becoming increasingly clear, however, that cryptic sexual programs may exist in some species, and that efficient mating requires the necessary developmental switch to be triggered. In this study we investigate Candida tropicalis, an important human fungal pathogen that has been reported to be asexual. Significantly, we demonstrate that C. tropicalis uses a phenotypic switch to regulate a cryptic program of sexual mating. Thus, diploid a and α cells must undergo a developmental transition to the mating-competent form, and only then does efficient cell-cell conjugation take place resulting in the formation of stable a/α tetraploids. We show that both the phenotypic switch and sexual mating depend on the conserved transcriptional regulator Wor1, which is regulated by temperature in other fungal species. In contrast, C. tropicalis mating occurs efficiently at both 25 °C and 37 °C, suggesting that it could occur in the mammalian host and have direct consequences for the outcome of an infection. Transcriptional profiling further reveals that ≈400 genes are differentially expressed between the two phenotypic states, including the regulatory factor Wor1. Taken together, our results demonstrate that C. tropicalis has a unique sexual program, and that entry to this program is controlled via a Wor1-mediated, metastable switch. These observations have direct implications for the regulation and evolution of cryptic sexual programs in related fungal pathogens.
Project description:Pseudo-nitzschia multistriata is a planktonic diatom species with a diplontic life cycle comprising a short sexual phase during which gametes are produced from the encounter of two diploid cells of opposite mating type (MT). Gene expression studies have highlighted the presence of substantial changes occurring at the onset of sexual reproduction. Collectively, there is clear evidence of a chemical cross talk which mediates the mating step.
Herein, we have hypothesized that these major transcriptomic changes are associated to variations in the amount and nature of the metabolites produced by the cells. In order to investigate such chemical diversity in an unbiased manner, we undertook an untargeted metabolomics approach. Liquid chromatography – tandem mass spectrometry was applied to investigate the differences in the metabolic profiles between control cells in the vegetative phase (MT+ and MT-) and mixed strains of opposite MTs (cross) undergoing sexual reproduction. Of the 2408 high-quality features obtained, 70 known metabolites could be identified based on in-house libraries and online databases; molecular networking proved valuable to classify additional 47 non-identified features.
The present exploratory study provides an overview of the chemical diversity detectable in P. multistriata, presenting a dynamic picture of the changes occurring during sexual reproduction. In particular, the reduction of phytol detected in the cross can be linked to the general downregulation of photosynthesis during sexual reproduction observed elsewhere. The dysregulation of other metabolites stands as a starting point for the understanding of the mechanisms regulating sexual reproduction: a metabolite that was tentatively identified as 7-dehydrodesmosterol exhibited the highest upregulation in the comparison between the two MTs of opposite sex, as well as between MT+ vs cross.
The effects of extraction solvents and harvesting times on the ensemble of endo-metabolites were also analyzed.
Project description:Sexual reproduction can promote genetic diversity in eukaryotes, and yet many pathogenic fungi have been labeled as obligate asexual species. It is becoming increasingly clear, however, that cryptic sexual programs may exist in some species, and that efficient mating requires the necessary developmental switch to be triggered. In this study we investigate Candida tropicalis, an important human fungal pathogen that has been reported to be asexual. Significantly, we demonstrate that C. tropicalis uses a phenotypic switch to regulate a cryptic program of sexual mating. Thus, diploid a and α cells must undergo a developmental transition to the mating-competent form, and only then does efficient cell-cell conjugation take place resulting in the formation of stable a/α tetraploids. We show that both the phenotypic switch and sexual mating depend on the conserved transcriptional regulator Wor1, which is regulated by temperature in other fungal species. In contrast, C. tropicalis mating occurs efficiently at both 25 °C and 37 °C, suggesting that it could occur in the mammalian host and have direct consequences for the outcome of an infection. Transcriptional profiling further reveals that ≈400 genes are differentially expressed between the two phenotypic states, including the regulatory factor Wor1. Taken together, our results demonstrate that C. tropicalis has a unique sexual program, and that entry to this program is controlled via a Wor1-mediated, metastable switch. These observations have direct implications for the regulation and evolution of cryptic sexual programs in related fungal pathogens. 4 biological replicates of both the white (CAY1504) and opaque (CAY2275) states of C. tropicalis a cells are included on this array. All are hybridized against a universal reference sample, which consists of the combined RNA from all 8 replicates used on this array.