Comparative transcriptomics reveals the molecular toolkit used by an algivorous protist for cell wall perforation

Gerbracht, Jennifer V.; Harding, Tommy; Simpson, Alastair G. B.; Roger, Andrew J.; Hess, Sebastian

doi:10.1016/j.cub.2022.05.049

Cited by 7 publications

(14 citation statements)

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“…1 A). In contrast to the filamentous alga Mougeotia sp., which was used in previous experiments [ 27 , 28 ], the Actinotaenium cells have a relatively high sinking rate and can be easily counted. Furthermore, the cell wall of Actinotaenium cf.…”

Section: Resultsmentioning

confidence: 99%

“…Based on transcriptome assemblies [ 27 ], the relatively large hypothetical protein (2248 amino acids; approx. 226 kDa) was predicted to contain an N-terminal Sec/SPI signal peptide, a GH5_5 catalytic domain, internal repeats of unknown function (weakly annotated as CBM2) and a C-terminal transmembrane domain (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Here, we corroborated the gene structure of GH5_5A by PCR (the ORF was not fully assembled in the first place, [ 27 ]) and studied the activity, substrate specificity and cellular localisation of the GH5_5 catalytic domain. For this, we established an optimised experimental system with Orciraptor agilis and the unicellular desmid Actinotaenium cf.…”

Section: Introductionmentioning

confidence: 92%

“…Based on light and electron microscopic observations on vampyrellid amoebae (Vampyrellida, Rhizaria), some authors suggested the action of lytic enzymes [ 9 , 12 , 25 , 26 ], but there is no experimental evidence. More recently, comparative transcriptomics applied to the necrophagous protoplast feeder Orciraptor agilis (Viridiraptoridae, Rhizaria) revealed a hypothetical protein with a domain annotated as glycoside hydrolase of family GH5_5 [ 27 ]. This candidate protein (referred to as GH5_5A) was the highest expressed carbohydrate-active enzyme (CAZyme) of Orciraptor , strongly upregulated during attack on the green alga Mougeotia sp.…”

Section: Introductionmentioning

confidence: 99%

“…The degradation of the plant-like, cellulosic walls of zygnematophytes likely requires glycoside hydrolases that cleave β-1,4-glycosidic bonds, a function that was predicted for Orciraptor’s GH5_5A protein. The open reading frame (ORF) of GH5_5A is relatively long (2248 amino acids) and further contains an N-terminal signal peptide, a C-terminal transmembrane domain, and a series of seven related sequence motifs (repeats), some of which were weakly annotated as cellulose-binding domain CBM2 [ 27 ]. This points to a dual function (catalytic and binding) of GH5_5A and suggests an extracellular localisation, which would be required for dissolving the algal cell wall.…”

Section: Introductionmentioning

confidence: 99%

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Experimental evidence for enzymatic cell wall dissolution in a microbial protoplast feeder (Orciraptor agilis, Viridiraptoridae)

2022

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Background Several protists have evolved the ability to perforate the cell walls of algae and fungi to specifically feed on their cell contents. These phagotrophic “protoplast feeders” represent an interesting mechanistic intermediate between predators and parasites and pose a number of cell biological questions. Although their fascinating feeding behaviour has been observed for the last 150 years, it is still unknown how protoplast feeders produce the well-defined and species-specific perforations in biochemically diverse cell walls. Differential expression analyses of the algivorous flagellate Orciraptor agilis (Viridiraptoridae, Cercozoa, Rhizaria) suggested the involvement of a highly expressed putative glycoside hydrolase of family GH5_5. To assess the importance of this carbohydrate-active enzyme in the feeding act of Orciraptor, we recombinantly produced its catalytic domain and studied the enzymatic activity, cellular localisation and function. Results The GH5_5 catalytic domain from Orciraptor showed pronounced activity on soluble cellulose derivatives and mixed-linkage glucans, with reaction optima comparable to known GH5_5 representatives. Crystalline cellulose was not digested by the enzyme, which suggests a typical endocellulase activity. Immunocytochemistry with a polyclonal antibody raised against the GH5_5 domain revealed that the native endocellulase localises to the contact zone of Orciraptor and the algal cell wall (= perforation zone) and to intracellular granules, which were enriched during attack. Furthermore, the anti-GH5_5 antibody applied to live cells significantly reduced the feeding success of Orciraptor. The cells attacked the algae, which, however, resulted in numerous incomplete perforations. Conclusions Our experimental data from enzymatic assays, immunocytochemistry and inhibition experiments strongly suggest a key role of the GH5_5 endocellulase in cell wall dissolution by Orciraptor agilis. With that, we provide evidence that the well-defined perforations produced by protoplast feeders are caused by extracellular carbohydrate-active enzymes and made a first step towards establishing the molecular basis of a fascinating, yet poorly understood microbial feeding strategy.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Experimental evidence for enzymatic cell wall dissolution in a microbial protoplast feeder (Orciraptor agilis, Viridiraptoridae)

2022

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Unveiling the Biomass Valorization: The Microbial Diversity in Promoting a Sustainable Socio-economy

Monclaro,

Gomes,

Duarte

et al. 2024

Bioenerg. Res.

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Comparative transcriptomics elucidates the cellular responses of an aeroterrestrial zygnematophyte to UV radiation

Busch,

Gerbracht,

Davies

et al. 2024

Journal of Experimental Botany

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The zygnematophytes are the closest relatives of land plants and comprise several lineages that adapted to a life on land. Species of the genus Serritaenia form colorful, mucilaginous capsules, which surround the cells and block harmful solar radiation, one of the major terrestrial stressors. In eukaryotic algae, this “sunscreen mucilage” represents a unique photoprotective strategy, whose induction and chemical background are unknown. We generated a de novo transcriptome of Serritaenia testaceovaginata and studied its gene regulation under moderate ultraviolet radiation (UVR) that triggers sunscreen mucilage under experimental conditions. UVR induced the repair of DNA and the photosynthetic apparatus as well as the synthesis of aromatic specialized metabolites. Specifically, we observed pronounced expressional changes in the production of aromatic amino acids, phenylpropanoid biosynthesis genes, potential cross-membrane transporters of phenolics, and extracellular, oxidative enzymes. Interestingly, the most upregulated enzyme was a secreted class III peroxidase, whose embryophyte homologs are involved in apoplastic lignin formation. Overall, our findings reveal a conserved, plant-like UVR perception system (UVR8 and downstream factors) in zygnematophyte algae and point to a polyphenolic origin of Serritaenia’s sunscreen pigment, whose synthesis might be extracellular and oxidative, resembling that of plant lignins.

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Comparative transcriptomics reveals the molecular toolkit used by an algivorous protist for cell wall perforation

Cited by 7 publications

References 78 publications

Experimental evidence for enzymatic cell wall dissolution in a microbial protoplast feeder (Orciraptor agilis, Viridiraptoridae)

Experimental evidence for enzymatic cell wall dissolution in a microbial protoplast feeder (Orciraptor agilis, Viridiraptoridae)

Unveiling the Biomass Valorization: The Microbial Diversity in Promoting a Sustainable Socio-economy

Comparative transcriptomics elucidates the cellular responses of an aeroterrestrial zygnematophyte to UV radiation

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