DOI: 10.5061/dryad.ttdz08kw2
title: Identification of scavenger receptors and thrombospondin type 1 repeat proteins potentially relevant for plastid recognition in Sacoglossa

description: Functional kleptoplasty is a photosymbiotic relationship, in which
 photosynthetically active chloroplasts serve as an intracellular symbiont
 for a heterotrophic host. Among Metazoa, functional kleptoplasty is only
 found in marine sea slugs belonging to the Sacoglossa and recently
 described in rhabdocoel worms. Although functional kleptoplasty has been
 intensively studied in Sacoglossa, the fundamentals of the specific
 recognition of the chloroplasts and their subsequent incorporation are
 unknown. The key to ensure the initiation of any symbiosis is the ability
 to specifically recognize the symbiont and to differentiate a symbiont
 from a pathogen. For instance, in photosymbiotic cnidarians, several
 studies have shown that the host innate immune system, in particular
 scavenger receptors (SRs) and thrombospondin type 1 repeat protein
 superfamily (TSRs), is playing a major role in the process of recognizing
 and differentiating symbionts from pathogens. In the present study, SRs
 and TSRs of three Sacoglossa sea slugs, Elysia cornigera, Elysia timida,
 and Elysia chlorotica, were identified by translating available
 transcriptomes into potential proteins and searching for receptor specific
 protein and/or transmembrane domains. Both receptors classes are highly
 diverse in the slugs, and many new domain arrangements for each receptor
 class were found. The analyzes of the gene expression of these three
 species provided a set of species-specific candidate genes, i.e. SR-Bs,
 SR-Es, C-type lectins, and TSRs, that are potentially relevant for the
 recognition of kleptoplasts. The results set the base for future
 experimental studies to understand if and how these candidate receptors
 are indeed involved in chloroplast recognition.
description: Publicly available assembled transcriptomes of the Sacoglossa
 <i>Elysia cornigera</i>: NCBI TSA version GBRW00000000.1;
 <i>E. timida</i>: TSA version GBRM00000000.1; and <i>E.
 chlorotica: </i>http://cyanophora.rutgers.edu/Elysia-expression/)
 were first clustered using CD-HIT v4.6.8 with default parameters (Fu, Niu,
 Zhu, Wu, & Li, 2012; Li & Godzik, 2006) and then
 translated into the longest open reading frame to retrieve potential
 proteins using TransDecoder v3.0.1 (Haas & Papanicolaou, 2015)
 with default settings. The datasets were subsequently subjected to a
 BLASTP search against the UniProt database version 11/13/19 (The UniProt
 Consortium, 2019) setting the E-value to 1e<sup>-10</sup>.
 Taxonomic assignment for each protein sequence was performed using the
 UniProt taxonomic database and sequences were subsequently filtered for
 Metazoa annotations (Supporting Information 1). Respective short reads
 were downloaded for each species from the short read archive deposited in
 GenBank (<i>Elysia chlorotica</i>; NCBI SRA sample accession
 SRS3101883),<i> Elysia timida</i> SRS706683, and
 <i>Elysia cornigera</i> SRS706681). Reads were then mapped
 using Bowtie2 v2.3.4.3 (Langmead & Salzberg, 2012) onto the
 clustered transcriptomes. Transcript abundance of sequences with a raw
 read count of at least 100 raw counts in any two samples tested was
 estimated using RSEM (Li & Dewey, 2011) implemented in Trinity
 v.2.9.0 (Grabherr et al., 2011). Differential gene expression analyzes
 were performed using edgeR v3.30.3 (Robinson, McCarthy, & Smyth,
 2010). For feeding juveniles of <i>E. chlorotica</i>,
 specimens fed for five days were compared to the apo-symbiotic state
 (initial); specimens fed for seven days with specimens fed for five days
 (transient); and specimens fed for 10 days with specimens fed for seven
 days (stable). For <i>E. timida </i>and <i>E. cornigera
 </i>the freshly fed animals were compared to the different
 starvation periods. Only genes with a log<sub>2</sub> fold
 change (L2FC) &gt; 1 or &lt; -1 were considered as significantly
 differentially expressed, because for <i>E. cornigera </i>and
 <i>E. timida </i>no biological replicates were available. The
 domain architecture of the filtered protein sequences were characterized
 by using HMMER v.3.1b2 (Eddy, Wheeler, & the HMMER development
 team, 2015) with default settings against the protein database PfamA 31.0
 (Finn et al., 2016). Transmembrane regions (TM) were identified using the
 TMHMM server v.2.0 (Sonnhammer, Von Heijne, & Krogh, 1998; Krogh,
 Larsson, Von Heijne, & Sonnhammer, 2001). Sequences were then
 filtered for the different receptor class specific domains, as defined in
 PrabhuDas et al. (2014, 2017).