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Activity, regulation and evolution of carbohydrate-active enzymes used in cell wall penetration by unicellular predators and parasitoids of microalgae

There is a diversity of parasitoid microbes that attack microalgae and fungi in aquatic and terrestrial ecosystems, forming an integral part of the microbial food web. As suggested by environmental sequencing, many of these highly-specialised interactions are still poorly known. This applies in particular to algivorous protoplast feeders, phagotrophic microeukaryotes that perforate the cell walls of algae and consume the cell contents. During the past years we made progress in understanding the phylogenetic diversity, cellular structure and ecology of protoplast feeders. However, it is still unclear how exactly protoplast feeders recognise their prey and perforate the prey cell wall. Transcriptomic data about the viridiraptorid amoeboflagellate Orciraptor agilis (Rhizaria) suggest that carbohydrate-active enzymes (CAZymes) mediate binding to the prey cell wall and cell wall dissolution. The protein sequences of these CAZymes represent an ideal basis for taking the next experimental steps, to establish a refined model of how protoplast feeders recognise and perforate the cell walls of their prey. Employing heterologous expression systems, we will produce recombinant candidate CAZymes and assay their activity on artificial and natural substrates. To establish the cellular localisation of these proteins in Orciraptor, antibodies will be raised and used for immunofluorescence microscopy. Furthermore, we will investigate the evolution of protoplast feeders by exploring the transcriptomes of the bacterivorous relatives of Orciraptor, specifically screening for cellulases and carbohydrate-binding proteins. Finally, we will expand our research to the vampyrellid amoebae to study how these versatile predatory amoebae adapted to diverse prey types. We will compare the feeding ecology and CAZyme expression in a wide phylogenetic framework and test with differential expression analyses if generalist vampyrellids regulate CAZyme expression in response to the prey cell wall biochemistry. By comparing the molecular toolkit of protoplast feeders of several distant phylogenetic lineages, we will finally understand how this fascinating feeding strategy evolved several times independently in the tree of life.

  • Methods: Transcriptomics, cloning, protein biochemistry, enzymatic assays, immunocytochemistry, electron microscopy
  • Relevant publications: Hess and Melkonian 2014, Busch and Hess 2017

Diversity and systematics of microbial protoplast feeders

In aquatic and terrestrial ecosystems, microalgae and fungi represent a large fraction of the biologically available carbon. In consuming much of this biomass, phago­trophic microeukaryotes are an integral part of aquatic and terrestrial food webs. To exploit the nutrient-rich cell content of algal and fungal cells, these phagotrophs have to defeat the physical protection (cell wall) of their prey. This led to the evolution of highly specialised feeding strategies and a diversity of species-specific trophic interactions, most of which are still poorly understood.

‘Protoplast feeders’ are necrophagous or parasitoid microeukaryotes widespread in marine, freshwater and terrestrial ecosystems. They perforate foreign cell walls to exclusively feed on the cell contents of their prey. In fact, there is a wealth of protoplast feeding microeukaryotes targeting diverse cell walls and extracellular polymers, which evolved in several distant evolutionary lineages in the tree of life. In the Hess laboratory, we explore a widespread group of predatory amoebae, the Vampyrellida (Rhizaria), which are relatively close relatives of the plasmodiophorid plant pathogens (e.g. Hess et al. 2012). These vampyrellid amoebae are surprisingly diverse, comprising several family-level clades, and common in aquatic and terrestrial ecosystems. As far as we know, they feed exclusively on eukaryotic prey, devouring algae of diverse phylogenetic affiliation and structure (e.g. streptophytes, chlorophytes, euglenophytes, diatoms), fungi and even micrometazoa such as nematodes. Furthermore, different vampyrellid species show varying degree of prey specificity, ranging from versatile predators feeding by “typical” phagocytosis to highly specialised protoplast feeders (Hess et al. 2012, Hess 2017a, b).

Another group of microbial protoplast feeders studied in the Hess laboratory are the viridiraptorid flagellates (Viridiraptoridae, Rhizaria). Although they differ fundamentally from vampyrellid amoebae in cellular structure and evolutionary origin their feeding processes show remarkable similarities (Hess and Melkonian 2013).

  • Methods: Light microscopy, electron microscopy, molecular phylogenetics, taxonomy
  • Relevant publications: Hess et al. 2012, Hess and Melkonian 2013, Hess 2017a, Hess 2017b, More et al. 2018

The rotary eukaryotic flagellum of Idionectes vortex (Amoebozoa)

  • Methods: Video microscopy, motion tracking experiments, transmission electron microscopy, immunocytochemistry, fluorescence microscopy
  • Relevant publications: Hess et al. 2019

Protist-bacteria endosymbioses

  • Methods: Transmission electron microscopy, design of molecular probes, fluorescence in situ hybridisation (FISH), confocal laser scanning microscopy, molecular phylogenetics, taxonomy of prokaryotes
  • Relevant publications: Hess et al. 2016, Hess 2017a, Muñoz-Gómez et al. 2019

Diversity of saccoderm desmids (Conjugatophyceae, Streptophyta)

  • Methods: Light microscopy incl. fluorescence microscopy, molecular phylogenetics, taxonomy