The symbiotic interaction between cnidarians, such as corals and sea anemones, and the unicellular algae Symbiodinium is regulated by yet poorly understood cellular mechanisms, despite the ecological importance of coral reefs. These mechanisms, including host-symbiont recognition and metabolic exchange, control symbiosis stability under normal conditions, but also lead to symbiosis breakdown (bleaching) during stress. This study describes the repertoire of the sterol-trafficking proteins Niemann-Pick type C (NPC1 and NPC2) in the symbiotic sea anemone Anemonia viridis. We found one NPC1 gene instead of two in vertebrates. While only one NPC2 gene is present in most metazoans, this gene has been duplicated in cnidarians and we detected four NPC2 genes in A. viridis. However, only one gene (AvNPC2-d) was upregulated in symbiotic sea anemones and displayed higher expression in the gastrodermis (symbiont-containing tissue) than in the epidermis. We performed immunolabeling experiments on tentacle cross sections and demonstrated that the AvNPC2-d protein was closely associated with symbiosomes. In addition, AvNPC1 and AvNPC2-d gene expression was strongly downregulated during stress, especially at the onset of symbiosis breakdown. These data suggest that AvNPC2-d is involved in both the stability and dysfunction of cnidarian-dinoflagellate symbioses.
Alignment file of NPC2 protein sequences_Figure1A
Sequences of NPC2 proteins aligned using the MAFFT program. This alignment was used to construct a phylogenetic tree using the maximum likelihood method (phyML3.0 software) and the WAG+I+G substitution model (I=0.057 and gamma factor=5.831). Accession number of sequences: AvNPC2-d,HG670301; AvNPC2-c, HG670300; NvNPC2-2, XP_001627355; NvNPC2-3, XP_001635502; AdNPC2-2, adi_v1.06781; AdNPC2-3, adi_v1.23434;
AdNPC2-4, adi_v1.10342; AvNPC2-b, HG670299;
AdNPC2-1, adi_v1.14987 + contig adi_v1.08570; AdNPC2-5, aug_v2a.08569; AdNPC2-6, aug_v2a.08570;
AvNPC2-a, HG670298; NvNPC2-1, XP_001622874;
HsNPC2, NP_006423.1; DrNPC2-a, NP_001122191.1;
DrNPC2-b, NP_775331.1; CiNPC2-1, XP_002127695.1;
TaNPC2, XP_002109765.1; HvNPC2-3, XP_002166396.2;
Hv NPC2-4, XP_002166374.2; Hv NPC2-2, XP_002164043.1; Hv NPC2-1, XP_002156875.1;
AqNPC2, CL5422Contig1 from AQUE_CAP3_100104 assembly from compagen
Alignment file of NPC1 protein sequences_Figure2
Sequences of NPC1 proteins aligned using the MAFFT program. This alignment was used to generate a phylogenetic tree using the phyML 3.0 sofware, the maximum likelihood method and the LG+I+G substitution model (I=0.1 and gamma factor=1.266). Accession numbers: HsNPC1L1, NP_001095118.1; BtNPC1L1, XP_588051; DrNPC1L1, XP_002663230.2; CiNPC1-b, XP_002122922; DmNPC1-a, NP_609357; DmNPC1-b, NP_608417; HsNPC1, AAK25791.1; BtNPC1, AAI51277; DrNPC1, XP_001919958.3; CiNPC1-a, XP_002120129;
SpNPC1, XP_780036; AvNPC1, HG670297;
NvNPC1, XM_001634881.1; AdNPC1, adi_v1.19271 from ADIG_G-PEP_111201 database; HmNPC1, XM_002164383.1; AqNPC1, XP_003384854.1;
PrimersEfficiency
PCR efficiency of primers used in this study (DNA quantification and heat stress experiment)
PrimerEfficiency.xlsx
qPCR_QuantifZxAv
Symbiodinium quantification by real-time quantitative PCR measurement of the relative nuclear gene copy number (symbiont to host nuclei) during hyperthermal stress.
qPCR_HeatStress
Relative gene expression data analysis (raw Ct and normalized expression data) for the heat stress experiment
Normalizationfactor_HeatStress
Normalization factor was calculated based on the expression level of RCC2 and COP-ɣ genes using the GeNorm software, for the heat stress experiment.
TreeFile_NPC2_Figure1
Tree data generated using the maximum likelihood method and the PhyML3.0 software. Compute these data with the SeaView program to display the NPC2 phylogenetic tree.
TreeFile_NPC1_Figure2
Tree data generated using the maximum likelihood method and the PhyML3.0 software. Compute these data with the SeaView program to display the NPC1 phylogenetic tree.