DOI: 10.5061/dryad.bk3j9kdjk
title: Ectopical expression of bacterial collagen-like protein supports its role as adhesin in host-parasite coevolution

description: For a profound understanding of antagonistic coevolution, it is necessary
 to identify the coevolving genes. The bacterium Pasteuria and its host,
 the microcrustacean Daphnia, are a well-characterized paradigm for
 co-evolution, but the underlying genes remain largely unknown. A
 genome-wide association study suggested a Pasteuria collagen-like protein
 7 (Pcl7) as a candidate mediating parasite attachment and driving its
 coevolution with the host. Since Pasteuria ramosa cannot currently be
 genetically manipulated, we used Bacillus thuringiensis to express a
 fusion protein of a Pcl7 carboxy- terminus from P. ramosa and the
 amino-terminal domain of a B. thuringiensis collagen-like protein (CLP).
 Mutant B. thuringiensis (Pcl7-Bt) spores but not wild-type B.
 thuringiensis (WT-Bt) spores, attached to the same site of susceptible
 hosts as P. ramosa. Furthermore, Pcl7-Bt spores attached readily to
 susceptible host genotypes, but only slightly to resistant host genotypes.
 These findings indicated that the fusion protein was properly expressed
 and folded and demonstrated that indeed the C-terminus of Pcl7 mediates
 attachment in a host genotype-specific manner. These results provide
 strong evidence for the involvement of a CLP in the coevolution of Daphnia
 and P. ramosa and open new avenues for genetic epidemiological studies of
 host–parasite interactions.
description: Daphnia were
 individually placed into a 96-well plate containing 150 μl of ADaM per
 well. A 10 μl of spore solution containing ~500 fluorescently labelled P.
 ramosa spores were added to each well and incubated in the dark for 5 min.
 For B. thuringiensis, 10 μl of spore solution containing ~50 000 labelled
 B. thuringiensis spores were added to each well and incubated in the dark
 for 5 min. The entire liquid volume in each well was removed and replaced
 with 150 μl fresh ADaM. This washing step was repeated twice, after which
 the entire liquid volume in each well was removed. The Daphnia were placed
 individually on a microscopy slide using a toothpick. A glass cover slide
 was applied to the Daphnia gently to avoid crushing it. Spore attachment
 was assessed by carefully assessment the entire depth of the field of
 vision in transparent animal, while the animal was alive.
 To demonstrate the different phenotypes, we took pictures through
 the transparent body wall of the Daphnia. Using living animals is
 necessary to see the spores in the oesophagus, but puts limits on the
 quality of the pictures, as animals move and the body tissues surrounding
 the oesophagus blur the picture. Images were taken using Leica Application
 Suite (v. 4.12, using package ‘montage’) with a Leica DM6 B (Leica
 Microsystems, Wetzlar, Germany) microscope fitted with a Leica DFC 7000T
 camera and a GFP Filter cube (Excitation Filter BP 470/40).
description: # Ectopical expression of bacterial collagen-like protein supports its
 role as adhesin in host-parasite coevolution
 [https://doi.org/10.5061/dryad.bk3j9kdjk](https://doi.org/10.5061/dryad.bk3j9kdjk) ## Description of the data and file structure The data associated with this work are organized in an excel file with three sheets. Each sheet refers to part of the entire dataset and are linked to the figures in the manuscript. Each sheet also includes explanations to the names of the variables and abbreviations. ### Description of variables: **Fig. 3**: * Attachment: Indicates whether spores attached (1) or no attachment (0) was observed * Isolate: Indicates which spores were used for the attachment test C1 C1 P. ramosa clone WT-Bt B. thuringiensis wild-type spores Pcl7-Bt Mutant B. thuringiensis wild-type spores expressing Pcl7 * Resistotype: Indicates the resistance phenotype to the C1 P. ramosa clone R Resistant, no attachment of C1 S Suscpetible, attachment of C1 * Daphnia_clone: Indicates the Daphnia magna clone used for the test. **Fig. 4**: Competitive attachment assay for C1 P. ramosa * Count: Indicates the number of attached C1 P. ramosa spores * Isolate: Indicates which spores were used for the attachment test C1 Individuals were exposed to 50 labelled C1 P. ramosa spores WT-Bt Individuals were exposed to ~20’000 B. thuringiensis WT-Bt spores prior to the addition of 50 C1 P. ramosa spores Pcl7-Bt Individuals were exposed to ~20’000 Pcl7-Bt spores prior to the addition of 50 C1 P. ramosa spores * Clone: Indicates the D. magna clone used for the assay **Fig. 5**: Competitive attachment assay for C19 P. ramosa * Count Indicates the number of attached C19 P. ramosa spores * Isolate Indicates which spores were used for the attachment test C19 Individuals were exposed to 50 labelled C19 P. ramosa spores WT-Bt Individuals were exposed to ~20’000 B. thuringiensis WT-Bt spores prior to the addition of 50 C19 P. ramosa spores Pcl7-Bt Individuals were exposed to ~20’000 Pcl7-Bt spores prior to the addition of 50 C19 P. ramosa spores * Clone Indicates the D. magna clone used for the assay