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The chytrids (phylum Chytridiomycota) are a major fungal lineage of ecological and evolutionary importance. Despite their importance, many fundamental aspects of chytrid developmental and cell biology remain poorly understood. To address these knowledge gaps, we combined quantitative volume electron microscopy and comparative transcriptome profiling to create an ‘atlas’ of the cellular and molecular basis of the chytrid life cycle, using the model chytrid Rhizoclosmatium globosum . From our developmental atlas, we describe the transition from the transcriptionally inactive free-swimming zoospore to the more biologically complex germling, and show that lipid processing is multifaceted and dynamic throughout the life cycle. We demonstrate that the chytrid apophysis is a compartmentalised site of high intracellular trafficking, linking the feeding/attaching rhizoids to the reproductive zoosporangium, and constituting division of labour in the chytrid cell plan. We provide evidence that during zoosporogenesis, zoospores display amoeboid morphologies and exhibit endocytotic cargo transport from the interstitial maternal cytoplasm. Taken together, our results reveal insights into chytrid developmental biology and provide a basis for future investigations into non-dikaryan fungal cell biology.

Compared to multicellular fungi and unicellular yeasts, unicellular fungi with free-living flagellated stages (zoospores) remain poorly known and their phylogenetic position is often unresolved. Recently, rRNA gene phylogenetic analyses of two atypical parasitic fungi with amoeboid zoospores and long kinetosomes, the sanchytrids Amoeboradix gromovi and Sanchytrium tribonematis , showed that they formed a monophyletic group without close affinity with known fungal clades. Here, we sequence single-cell genomes for both species to assess their phylogenetic position and evolution. Phylogenomic analyses using different protein datasets and a comprehensive taxon sampling result in an almost fully-resolved fungal tree, with Chytridiomycota as sister to all other fungi, and sanchytrids forming a well-supported, fast-evolving clade sister to Blastocladiomycota. Comparative genomic analyses across fungi and their allies (Holomycota) reveal an atypically reduced metabolic repertoire for sanchytrids. We infer three main independent flagellum losses from the distribution of over 60 flagellum-specific proteins across Holomycota. Based on sanchytrids’ phylogenetic position and unique traits, we propose the designation of a novel phylum, Sanchytriomycota. In addition, our results indicate that most of the hyphal morphogenesis gene repertoire of multicellular fungi had already evolved in early holomycotan lineages. Unicellular fungi with free-living flagellated stages (zoospores) remain poorly known. Here, Galindo et al . sequence single-cell genomes for two atypical parasitic fungi with amoeboid zoospores, and re-evaluate the branching order of early-diverging fungi and the evolution of fungal multicellularity and flagellum-mediated motility.

Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi,which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed “fungal shunt” can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton–bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles.

Seaweed aquaculture, which takes place mostly in Asia, is a lucrative industry that is valued > US $9 billion. However, technological modifications are needed to ensure economic viability and growth of the seaweed aquaculture industry throughout Europe. While current research is investigating the use of certain mechanised processes in seaweed aquaculture, the impact of pressurised spraying of macroalgal cultures on subsequent growth remains unknown. Here, we aimed to determine the efficacy of a future mechanised seeding procedure by investigating how differing pressure treatments impact upon the growth and percentage cover of zoospores seeded onto twine in the hatchery, using the kelp Saccharina latissima as a model species. Zoospore solutions were subjected to pressures of 1, 2, 3, 4, and 5 bar, before being seeded on hatchery twine and left to grow for 7 weeks. We demonstrate that both percentage cover and sporophyte lengths for S. latissima are significantly reduced by ~ 22% and ~ 61%, respectively, when juvenile zoospores are subjected to increasing pressure from 1 to 5 bar. This indicates that minimal pressure in the use of mechanised hatchery techniques is optimal for growth of seaweed.

There has been limited research into the role of the Pythium insidiosum antigen (PIA) in modulating immune response in patients with pythiosis. This study investigated the balance of T helper type 2 (Th2) and T helper type 1 (Th1) responses after receiving PIA immunotherapy in patients with pythiosis. Next, the phagocytic activity and phagocytic index of IFN-γ primed PIA-treated macrophages were examined. Furthermore, the phagocytosis of infective P. insidiosum zoospores by macrophages was investigated. This work showed that the PIA vaccine induced Th1 response and M1 macrophages in patients with vascular pythiosis who survived and those with localized pythiosis. Phagocytic activity and phagocytic index were increased considerably in localized pythiosis patients compared to vascular pythiosis patients with hematological diseases. IFN-γ priming of PIA-treated macrophages against P. insidiosum zoospores enhanced the phagocytic activity and phagocytic index in vascular and localized pythiosis patients. Macrophages engulfed P. insidiosum zoospores, but the zoospores continued germination, resulting in macrophage death. Overall, our results suggest that PIA can modulate the immune responses, contributing to higher levels of Th1-type cytokine and potentially improving the survival of patients with vascular pythiosis. This study is the first to uncover that P. insidiosum zoospores can survive within macrophages.

Background The oomycete Phytophthora infestans causes the devastating late blight diseases of potato and tomato. P. infestans uses spores for dissemination and infection , like many other filamentous eukaryotic plant pathogens. The expression of a subset of its genes during spore formation and germination were studied previously, but comprehensive genome-wide data have not been available. Results RNA-seq was used to profile hyphae, sporangia, sporangia undergoing zoosporogenesis, motile zoospores, and germinated cysts of P. infestans. Parallel studies of two isolates generated robust expression calls for 16,000 of 17,797 predicted genes, with about 250 transcribed in one isolate but not the other. The largest changes occurred in the transition from hyphae to sporangia, when >4200 genes were up-regulated. More than 1350 of these were induced >100-fold, accounting for 26% of total mRNA. Genes encoding calcium-binding proteins, cation channels, signaling proteins, and flagellar proteins were over-represented in genes up-regulated in sporangia. Proteins associated with pathogenicity were transcribed in waves with subclasses induced during zoosporogenesis, in zoospores, or in germinated cysts. Genes involved in most metabolic pathways were down-regulated upon sporulation and reactivated during cyst germination, although there were exceptions such as DNA replication, where transcripts peaked in zoospores. Inhibitor studies indicated that the transcription of two-thirds of genes induced during zoosporogenesis relied on calcium signaling. A sporulation-induced protein kinase was shown to bind a constitutive Gβ-like protein, which contributed to fitness based on knock-down analysis. Conclusions Spore formation and germination involves the staged expression of a large subset of the transcriptome, commensurate with the importance of spores in the life cycle. A comparison of the RNA-seq results with the older microarray data indicated that information is now available for about twice the number of genes than before. Analyses based on function revealed dynamic changes in genes involved in pathogenicity, metabolism, and signaling, with diversity in expression observed within members of multigene families and between isolates. The effects of calcium signaling, a spore-induced protein kinase, and an interacting Gβ-like protein were also demonstrated experimentally. The results reveal aspects of oomycete biology that underly their success as pathogens and potential targets for crop protection chemicals.

The current biodiversity crisis encompasses a sixth mass extinction event affecting the entire class of amphibians. The infectious disease chytridiomycosis is considered one of the major drivers of global amphibian population decline and extinction and is thought to be caused by a single species of aquatic fungus, Batrachochytrium dendrobatidis. However, several amphibian population declines remain unexplained, among them a steep decrease in fire salamander populations (Salamandra salamandra) that has brought this species to the edge of local extinction. Here we isolated and characterized a unique chytrid fungus, Batrachochytrium salamandrivorans sp. nov., from this salamander population. This chytrid causes erosive skin disease and rapid mortality in experimentally infected fire salamanders and was present in skin lesions of salamanders found dead during the decline event. Together with the closely related B. dendrobatidis, this taxon forms a well-supported chytridiomycete clade, adapted to vertebrate hosts and highly pathogenic to amphibians. However, the lower thermal growth preference of B. salamandrivorans, compared with B. dendrobatidis, and resistance of midwife toads (Alytes obstetricans) to experimental infection with B. salamandrivorans suggest differential niche occupation of the two chytrid fungi.

Purpose: The aim of this work was to study the demographic profile, clinical diagnostic features, challenges in management, treatment outcomes, and ocular morbidity of microbiological culture-proven Pythium keratitis in a tertiary eye care hospital in South India. Methods: Retrospective analysis of microbiologically proven Pythium keratitis patients was performed at a tertiary eye center from October 2017 to March 2020. Demographic details, risk factors, microbiological investigations, clinical course, and visual outcomes were analyzed. Results: Thirty patients were analyzed. The mean age was 43.1±17.2 years. Most common risk factors were history of injury in 80% and exposure to dirty water in 23.3%. Visual acuity at baseline was 20/30 to perception of light (PL). The most common clinical presentation was stromal infiltrate and hypopyon in 14 (46.6%) patients each. The microbiological confirmation was based on culture on blood agar and vesicles with zoospores formation with incubated leaf carnation method. Seven (23.3%) patients improved with topical 0.2% Linezolid and topical 1% Azithromycin, 19 (63.3%) patients underwent Therapeutic keratoplasty (TPK) and 4 were lost to follow-up. Seven (23.3%) patients had graft reinfection, and 3 (10%) developed endophthalmitis. The final visual acuity was 20/20- 20/200 in 6 (20%) patients, 20/240-20/1200 in 5 (16.6%) patients, hand movement to positive perception of light in 16 patients and no perception of light (Pthisis Bulbi) in 3 (10%) patients. Conclusion: P. insidiosum keratitis is a rapidly progressive infectious keratitis with prolonged and relapsing clinical course. It usually results in irreparable vision loss in majority of the patients. Prompt diagnosis, clinical awareness, and specific treatment options are needed for successfully managing this devastating corneal disease.

Dispersal by means of zoospores is a common feature in the life cycle of many lower eukaryotes, including chytrid fungi, oomycetes, and some bacteria of actinomycetes. However, the molecular mechanisms underlying zoospore release from (zoo)sporangia remain largely unknown. Here, we revealed that two paralogous glycoside hydrolases (GimA and GimB) play essential roles in the release of spores, which can swim in aquatic environments as zoospores, from sporangia in Actinoplanes missouriensis , a filamentous soil-inhabiting bacterium. During sporangium dehiscence, GimA and GimB are produced and secreted extracellularly to hydrolyze the polysaccharide component of the sporangium matrix that encapsulates the spores. This study clarifies an unprecedented molecular mechanism in the process of zoospore release from the sporangia.

The Rhizoclosmatium globosum genome encodes three rhodopsin-guanylyl cyclases (RGCs), which are predicted to facilitate visual orientation of the fungal zoospores. Here, we show that RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins ( λ max  = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light-activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared-sensitive (NIR, λ max  = 690 nm) highly fluorescent state ( Q F  = 0.2) and a UV-sensitive non-fluorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high fluorescence yield. Site-specific mutagenesis and hybrid quantum mechanics/molecular mechanics simulations support the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad composed of two glutamic and one aspartic acids. These findings substantially expand our understanding of the natural potential and limitations of spectral tuning in rhodopsin photoreceptors. Rhizoclosmatium globosum contains three rhodopsin-guanylyl cyclases (RGCs) predicted to enable visual orientation of zoospores. Here authors show that RGC1 and 2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR), a near-infrared absorbing, highly fluorescent rhodopsin.