NOD-like receptor repertoire in the chromosome-level genome of the demosponge Dysidea avara (Schmidt, 1862)

Abstract

Porifera, one of the earliest diverging metazoans, have shown a surprisingly complex immune repertoire. However, most information to date is based on de novo transcriptome assemblies, limiting our knowledge regarding the presence and evolution of poriferan immune repertoire. Here, we generated the chromosome-level genome of the demosponge Dysidea avara, a target species in studies on symbiosis and differential expression of immune genes. We examined the presence and the number of common immune protein domains in the annotated genome of D. avara, and we further focused on NOD-like Receptors (NLRs), which are one of the most expanded immune receptors in Porifera according to previous reports on draft genomes and transcriptome assemblies. Dysidea avara has a 575 Mb genome with N50 41Mb, 162 scaffolds, and 15 chromosomes. We additionally recovered 37 sequences corresponding to microbial genomes, including complete bacterial and viral genomes. Based on the presence of conserved domains, we detected a large number of immune receptors and other immune genes in D. avara genome, such as 14 TIR, 39 CARD, 128 DEATH, and 230 NACHT domain-containing genes. Based on their architecture, we identified a large expansion of bona fide NLRs (i.e., 126 NACHT+LRR domain-containing genes); of which, 20 included a N-terminal CARD domain (NLRC), and 25 included a N-terminal DEATH domain (NLRD). In D. avara, the different NLR categories (i.e., NLRX, NLRC and NLRD) formed distinct phylogenetic clusters, while the NLR phylogenetic analysis across sponge chromosome-level genomes indicated that NLRs were mainly grouped by species rather than category. The NLRX category was the most expanded, while the NLRC category was absent in 7 out of 11 studied sponge genomes. These observations indicate that the diversification of NLRs in sponges, most likely derived from the ancestor NLRX, responds to species-specific selective pressures related to their immunity. This is the first study characterizing sponge NLR diversity in a chromosome-level genome, enhancing our knowledge of NLR evolution in the ancient phylum Porifera.

Keywords: NOD-Like receptors; Porifera; chromosome-level genome assembly; comparative genomics; innate immunity.

Figure 1

Dysidea avara species and its genome statistics. (A) Underwater photograph of the sponge D avara. (B) BUSCO scores indicating the quality of the genome and its annotation, taking into account the eukaryota_odb10 and metazoa_odb10 databases. The BUSCO analysis was run with Busco v.5 version.

Figure 2

NOD-Like Receptors (NLRs) in Dysidea avara genome. (A) Domain architecture of proteins annotated to the different categories of bona fide NLRs (NLRD, NLRC, NLRX) in the genome assembly of D avara. The number indicates the annotated genes assigned to each NLR category. The graph was generated in Biorender.com. (B) Genes coding for bona fide NLRs were depicted in the chromosomes they are located in the genome. The graph was created with RIdeogram. NLRC, CARD-NACHT-LRR; NLRD, DEATH-NACHT-LRRs; and NLRX, NACH-LRR. CARD, Caspase recruitment domain; LRR, Leucine-rich repeat domain; Kix, kinase-inducible domain (KID) interacting domain; TNFR, tumor necrosis factor receptor; RPT, internal repeat; CASc, caspase; UBQ, ubiquitin; Litaf, LPS-induced TNF-activating factor; COR, C-terminal of Ras of Complex domain.

Figure 3

Phylogenetic analysis of NLRs in Dysidea avara. The NACHT domain alignment from NLR sequences was used for the analysis (Supplementary File 1B; Supplementary File 2). Different clusters were shaped, depending on the NLR category NLRD or NLRC, while NLRX was grouped with the two other groups. Within the NLRX category, the number of C-terminal LRR repeats also determined different clusters. The ML tree was constructed with GTR Bootstrap expectation model and an estimated gamma shape parameter and 100 independent searches with RAxML. NLR, NOD-Like Receptor; LRR, Leucine Rich Repeat; NLRC, CARD-NACHT-LRR; NLRD, DEATH-NACHT-LRR; and NLRX, NACHT-LRR.

Figure 4

Phylogenetic analysis of NLRs from 8 sponge species based on the alignment of the NACHT domain. A phylogenetic analysis of NLRs was derived from the NACHT domain alignment in 8 sponge species, including our species (Supplementary File 1C; Supplementary File 2). As an outgroup, the NACHT domain from NLRs in the cnidarian Nematostella vectensis was used. The tree shows that NLRs are mainly grouped by species rather than by category. However, within the D. avara cluster, we see different clusters based on NLRD or NLRC category. The ML tree was constructed with GTR Bootstrap expectation model and an estimated gamma shape parameter and 100 independent searches with RAxML. NLRC, CARD–NACHT-LRR; NLRD, DEATH-NACHT-LRR; and NLRX, NACHT- LRR.