Characterization of the architecture, composition and evolution of a novel light perception organelle in an emerging model fungus.

Project Information

FungEye

Grant agreement ID: 101022101

DOI

10.3030/101022101

Project closed

EC signature date 30 March 2021

Start date 7 September 2021

End date 6 September 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
United Kingdom

Periodic Reporting for period 1 - FungEye (Characterization of the architecture, composition and evolution of a novel light perception organelle in an emerging model fungus.)

Reporting period: 2021-09-07 to 2023-09-06

Summary of the context and overall objectives of the project

Light perception is one of the most important sources of information for life on Earth. In animals, a diverse range of sophisticated light-sensing organs (e.g. eyes) have evolved. However, animals are not the only organisms to evolve such structures, with sub-cellular analogues found in several eukaryotic microbes. In Fungi, species from most major lineages have been shown to use light as a source of information using a diversity of photo-responsive proteins (photoreceptors) conserved across multiple groups and responding to important processes. These photoreceptors are not localized to an eyespot-like organelle, with one key exception. A light sensing eyespot composed of lipid-filled organelles has been described in the early diverging zoosporic fungus Blastocladiella emersonii (Be).
This structure is called the ‘CyclOp-organelle’, due to its association with the ‘CyclOp’ protein. The CyclOp-organelle is an ‘eye-like’ organelle closely associated to the flagellum, although the precise cellular-architecture of this system is unknown. The CyclOp protein controls phototaxis behaviour of Be zoospores by generating a biochemical response from the function of a type 1 rhodopsin, which forms part of a unique gene-fusion protein with a guanylyl cyclase enzyme domain.
Recently, the CyclOp protein of Be has been identified as a tool for optogenetic control, providing a means to control localized action potentials via a tuneable light signal. Optogenetics has important implications for society, for example, using light signalling to control individual neurons (precisely and rapidly) within a brain a useful application for deciphering the neural circuitries underlying behaviour and/or disease outcomes. There are still many questions surrounding how the CyclOp system functions in its native fungal cell, specifically what is the architecture and evolutionary origin of this sub-cellular system?

The overall aim of the FungEye project is to characterize the cellular structure, proteome and evolutionary ancestry of the CyclOp-organelle. Such information is vital for understanding how this system evolved and for further optogenetic/synthetic cell biological utilization.

The three main objectives of the proposed project are:
1) Use microscopy to understand the architecture and relationship between the rhodopsin surface, lipid droplets, flagellum root, and mitochondrion of the CyclOp-system.
2) Explore the proteome network associated with the CyclOp protein and the wider organelle.
3) Identify evolutionary origins of the CyclOp-organelle protein-network.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Regarding the architecture of the CyclOp organelle we first obtained cultures of several flagellated fungi from culture collections to grow them and induce zoospores sporulation. Zoospores where then prepared for immunostaining of the lipid and cytoskeletal components for confocal imaging. We then generated images with unprecedented resolution in order to study the interaction between these two components of the CyclOp organelle.
Regarding the protein network of the CyclOp organelle we first extracted high-molecular weight DNA from the zoospores of the fungus Blastocladiella emersonii. After sequencing using long-read PacBio technology, we assembled and annotated the genome. The resulting genome is one of the most complete and contiguous genomes of any flagellated fungus. The genome itself is a valuable resource for the community but our analyses also characterised the repertoire of light-sensing protein genes.
For our work focusing on the evolutionary history of the CyclOp organelle we first screened public databases of eukaryotic genomes, searching for CyclOp system components. After identifying several candidates, we created a phylogenetic dataset and reconstructed the evolutionary history of the CyclOp fusion protein and the BeCNG1 ion-channel. We then broaden our search for photoreceptors to understand the entire evolution of light-sensing in fungi. Our results showed how the CyclOp system and photoreceptors for several additional wavelengths of light are distributed in early fungi, indicating that the ancestor of all fungi sensed a range of light signals.
Overall results:
1) The description of a wide diversity of flagellated fungi that possess the CyclOp light response circuit. Thus, showing that the CyclOp system is a highly diversified function present in all three early flagellated fungi phyla (Blastocladiomycota, Sanchytriomycota and Chytridiomycota).
2) The same fungi possess the subcellular equipment to build lipid-based eyespots. Our confocal microscopy analyses and bibliography review of the flagellated zoospores of these fungi showed that lipid-based organelles were present in all the species that also had the CyclOp protein.
3) The last common ancestor of fungi possessed the CyclOp eyespot system. Our phylogenetic analyses reconstructed the evolutionary history of the CyclOp protein and the BeCNG1 channel, showing that these are conserved genes that were present in the ancestor of all fungi.
4) The ancestral fungus could see a rainbow of light wavelengths. Our comparative phylogenetic analyses of other photoreceptor proteins for blue (cryptochrome) and red (phytochrome) light proved that the ancestor of all fungi could sense several wavelengths of light.
5) The generation of the most complete and near chromosome-level (contiguous) genome assembly and annotation of any other flagellated fungi so far. This genome is a valuable resource for the overall community and holds the key to finding new optogenetically-useful proteins and to the reconstruction of the protein network associated with the CyclOp organelle.
6) The description of a newly discovered and diverse repertoire of both sensorial and (specifically) light-sensitive genes. These new genes may be involved in other (still unknown) sensing pathways of this fungi and may be good targets for the development of new optogenetic proteins.
These results have been published in high-ranking journals as Current Biology (https://doi.org/10.1016/j.cub.2022.05.034) and Genome Biology and Evolution (https://doi.org/10.1093/gbe/evac157). These results have also been presented several international conferences including an EMBO conference in 2022 (Spain), the 16th European Conference on Fungal Genetics in 2022 (Austria), and as an invited speaker in the next Molecular Biology of Fungi conference in 2024 (Germany). All public datasets of this project are available at: https://doi.org/10.6084/m9.figshare.19182086.v5 and https://doi.org/10.6084/m9.figshare.c.6221042.v1

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We characterised the CyclOp light sensing system with an unprecedented level of detail. We expect that our results will contribute to the continuous generation of knowledge on this unique system, and it is our goal to carry on research in this direction.
Regarding the widder societal implications of FungEye, we hope that our findings on the evolution, and widder architecture of this organelle will contribute to improving the already established biotechnological optogenetic protein, CyclOp. On the other hand, the new genome of Blastocladiella emersonii may hold the key to new useful optogenetic systems still wating to be discovered and characterized. Thus, in the future the FungEye may help creating new optogenetic tools to better understand behaviour and/or diseases.

Phylogenetic position, CyclOp-organelle, and light-sensing pathway of Blastocladiella emersonii