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Coral–algal endosymbiosis characterized using RNAi and single-cell RNA-seq

Nature Microbiology volume 8, pages 1240–1251 (2023)Cite this article

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Abstract

Corals form an endosymbiotic relationship with the dinoflagellate algae Symbiodiniaceae, but ocean warming can trigger algal loss, coral bleaching and death, and the degradation of ecosystems. Mitigation of coral death requires a mechanistic understanding of coral–algal endosymbiosis. Here we report an RNA interference (RNAi) method and its application to study genes involved in early steps of endosymbiosis in the soft coral Xenia sp. We show that a host endosymbiotic cell marker called LePin (lectin and kazal protease inhibitor domains) is a secreted Xenia lectin that binds to algae to initiate phagocytosis of the algae and coral immune response modulation. The evolutionary conservation of domains in LePin among marine anthozoans performing endosymbiosis suggests a general role in coral–algal recognition. Our work sheds light on the phagocytic machinery and posits a mechanism for symbiosome formation, helping in efforts to understand and preserve coral–algal relationships in the face of climate change.

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Fig. 1: shRNA knockdown of genes in Xenia.
Fig. 2: LePin aids the formation of new alga-containing Xenia cells and impacts gene expression in the endosymbiotic cell lineage.
Fig. 3: LePin facilitates alga attachment to the stalk_mouth region of the gastrodermis.
Fig. 4: LePin in alga binding and recognition.
Fig. 5: Scavenger receptors and actin regulators for endosymbiosis.

Data availability

We have uploaded all raw data to NCBI (PRJNA869069). The Xenia genome is also available at NCBI (Genebank accession: JAJSDR000000000.1, https://www.ncbi.nlm.nih.gov/assembly/GCA_021976095.1). Select intermediate RDS objects are available at figshare (https://figshare.com/articles/dataset/Processed_R_objects_for_LePin_RNAi_/20481900). The Protein sequence for phylogenetic tree-building were downloaded from different sources: Acropora digitifera, A. millepora, A. hyacinthus, A. palmata62 (no genome version was available for these four genomes, data were downloaded in April 2019), Aiptasia genome (v1.0)63, Stylophora pistillata (v1.0)64, Fungia sp. (v1.0),Galaxea fascicularis (v1.0), Goniastrea aspera (v1.0), from reef genomics (http://reefgenomics.org/), Nematostella vectensis (ASM20922v1)65 from JGI, Orbicella faveolata (v1.0, GCF_002042975.1), Dendronephthya gigantea(DenGig_1.0, GCF_004324845.1) from NCBI, Renilla reniformis(v1) from http://ryanlab.whitney.ufl.edu/genomes/Renilla_reniformis/, Hydra viridissima66 (v1) from https://marinegenomics.oist.jp/hydra_viridissima_a99/viewer/download?project_id=82 and Hydra magnipapillata (v2)67 from https://research.nhgri.nih.gov/hydra/. Source data are provided with this paper.

Code availability

All analysis codes for scRNA-seq and alga counting are available in GitHub at https://github.com/MinjieHu/Xenia_RNAi.

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Acknowledgements

We thank F. Tan and A. Pinder for assistance with all the sequencing and initial processing of raw reads; N. Marvi for the model sketch; L. Hugendubler and M. Watts for maintaining the coral aquarium; and R. Pedersen and J. Tran for critical comments. This work was supported by the Gordon and Betty Moore Foundation, Aquatic Symbiosis no. GBMF9198 (https://doi.org/10.37807/GBMF9198, Y.Z.).

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  1. Minjie Hu

    Present address: College of Life Sciences, Zhejiang University, Hangzhou, China

Authors and Affiliations

  1. Department of Embryology, Carnegie Institution for Science, Baltimore, MD, USA

    Minjie Hu, Yun Bai, Xiaobin Zheng & Yixian Zheng

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Contributions

M.H.and Y.Z. conceived the project. M.H. and Y.Z. designed experiments. M.H. and Y.B. performed the experiments. M.H. and X.Z. analysed the data. M.H., Y.B., X.Z. and Y.Z. interpreted the data. M.H. and Y.Z. wrote the manuscript.

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Correspondence to Minjie Hu or Yixian Zheng.

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Extended data

Extended Data Fig. 1 Phylogenetic tree and domain organization of RNAi components in different cnidarians.

Phylogenetic tree and domain organization of Argonaute (a) and Dicer proteins (b). The bootstrap value is indicated at each branch of the trees. Abbreviations: PAZ, PAZ or PAZ_argonaute_like domain; Piwi, Piwi-like or Piwi_ago-like domain; DEXHc_dicer, DEXH-box, helicase domain of endoribonuclease Dicer; SF2_C_dicer, C-terminal helicase domain of the endoribonuclease Dicer; MPH1, ERCC4-related helicase domain; RIBOc, Ribonuclease III C terminal domain; Rnc, dsRNA-specific ribonuclease.

Extended Data Fig. 2 Illustration for proteins studied.

Illustrations of domains and target regions of shRNA and peptides (for antibodies) for the proteins studied in this report. One single shRNA targets the four heavily repeated regions in DMBT1 (d).

Extended Data Fig. 3 Domain organization of the predicted LePin-like proteins from sequenced marine anthozoans plotted with the phylogenetic tree based on LePin sequences.

CLECT: C type Lectin domain; EGF/EGF_Ca/EGF_3/cEGF: EGF, EGF-Cacium binding and EGF like domains (EGF_3 or cEGF); H_lectin: H type lectin domain; Kazal: kazal domain. Xenia LePin is highlighted by a dashed blue rectangle. The two lectins in Nematostella vectensis (highlighted by a dashed magenta rectangle) that are most similar to Xenia LePin miss a few domains and appear as an outgroup. The bootstrap value is indicated at each branch of the trees.

Extended Data Fig. 4 Additional analyses of scRNA-seq for RNAi-treated samples.

a, The integrated UMAP of all cells from the control and LePin RNAi samples. b, c, Distributions of the detected UMI (Unique Molecular Identifier) numbers (b) and gene numbers (c).

Extended Data Fig. 5 Heat map of gene expression along the host endosymbiotic cell developmental progression as measured by scRNA-seq in this study.

Most previously defined genes expressed in the host progenitor endosymbiotic cells (not carrying algae) have higher expression during the early stages of lineage progress than later stages in the new trajectory analysis.

Extended Data Fig. 6 Relative expression of other GEPs identified by NMF analysis along the trajectory in control and LePin RNAi treated samples.

The box plot indicates the GEP expression distribution for the cells within one pseudotime unit. The box represents the middle 50% of the data. The line in the box is the median. The upper and lower edges of the box represent the upper and lower quartiles, respectively. The whiskers extend to 1.5 times the interquartile range. n= 314 cell for control and 217 cell for LePin RNAi.

Extended Data Fig. 7 Unbiased high throughput quantification of algae attached to the surface of Xenia gastrodermis.

a, An example of an epifluorescence image from a tissue section. Blue, DAPI staining of nuclei. Red, auto-fluorescence from the alga. b, Labeling of all cells by the DAPI signal. Each nucleus was pseudo colored to enable counting. c, Labeling of alga cells by the auto-fluorescence signal. Each alga was pseudo colored to enable counting. d, Tissue mask (green) is generated with a lower threshold of the DAPI signal and overlaid with the algal autofluorescence channel. The algae within the tissue mask are labeled blue. The algae outside or not completely inside the tissue mask are labeled pink. Red arrows point to the algae protruding from the tissue surface and are counted as algae attached to the surface of the gastrodermis. Scale bar, 50μm. . Four independent experiments were performed and quantified.

Extended Data Fig. 8 Prediction of signal peptide and transmembrane sequences in LePin.

a, the signal peptide is predicted by SignalP 5.0 with the Eukarya model. OTHER: no signal peptide predicted. Only the first 70 amino acids of LePin are plotted. b, LePin is predicted as a protein without transmembrane domain. TMHMM (v2.0) was used for the prediction. The full length LePin sequence is plotted.

Extended Data Fig. 9 Quantification of LePin signal on the isolated free algae in Xenia by FACS.

a, b, Gating strategy for the free algae in Xenia. The algae are first gated based on the DAPI staining of the nuclei and algae autofluoresce (Cy5.5 signal) (a, free algae gate1). The algae are further gated based on the forward scatter (FSC) and side scatter (SSC) signals to further exclude those algae that are inside the Xenia cells (b, free algae gate2). c-e, LePin signal distribution on free algae in control (c) and LePin knocking down samples (d,e). Color codes for individual animals. The percentage of algae with high LePin signals (as indicated by the brackets) are quantified and labeled.

Extended Data Fig. 10 The pairwise alignment of DMBT1 from Xenia and mouse.

The conserved SR domains are enclosed with red rectangles.

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Tables

Supplementary Data

Fasta file for NMF analysis related protein sequence

Source data

Source Data Fig. 1

Raw data for Fig. 2c.

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Hu, M., Bai, Y., Zheng, X. et al. Coral–algal endosymbiosis characterized using RNAi and single-cell RNA-seq. Nat Microbiol 8, 1240–1251 (2023). https://doi.org/10.1038/s41564-023-01397-9

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