Using Transcriptomics and Reverse Genetics to Understand Cnidarian-Dinoflagellate Symbiosis

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1 Using Transcriptomics and Reverse Genetics to Understand Cnidarian-Dinoflagellate Symbiosis Phillip Cleves Pringle Lab Department of Genetics, Stanford University

2 Corals differ in bleaching tolerance What coral genes help confer this tolerance?

3 Corals can be difficult to study in the lab. Inconvenient sizes Slow growing Difficult to maintain Hard calcareous skeleton Only seasonal access to larvae

4 Aiptasia as a model organism Aiptasia advantages Convenient sizes for experimentation Soft and hardy Large clonal populations Transcriptomic and genomic resources Symbiotic with similar types of Symbiodinium as corals Culturable in symbiotic and aposymbiotic states

5 Aiptasia bleach upon thermal stress Red Chlorophyll Fluorescence

6 Aiptasia bleach gradually at 34 o C Mean algal cells per µg protein (% of Day 0 Means) CC7 - SSB Days at 34 o C Clonal Aiptasiastrain CC7 harboring an axenic clade B Symbiodinium (SSB01).

7 What Aiptasia genes are involved in symbiosis and bleaching? We performed RNA- seq time courses on CC7- SSB01 and aposymbiotic CC7 anemones during heat stress.

8 A early heat- responsive gene cluster is up- regulated early in symbiotic and aposymbiotic anemones CC7 - APO CC7 - SSB01 Log 2 Fold Change Heat shock cognate 71 kda protein Heat shock 70 kda protein 1A Heat shock 70 kda protein XIAP associated factor 1 DnaJ homolog subfamily B member 4 DnaJ protein homolog 1 Predicted protein Predicted protein CD9 antigen Predicted protein Predicted protein Interferon regulatory factor 8 Caspase 7 predicted protein ETS domain containing protein Elk 1 NA Retrovirus related Pol polyprotein Retrovirus related Pol polyprotein Retrovirus related Pol polyprotein Krueppel like factor 5 Neuropeptide FF receptor 2 NA Uncharacterized protein Time (h)

9 Symbiosis genes change overtime as bleaching occurs in symbiotic animals Log 2 Fold Change Symbiotic - Aposymbiotic Transtition Genes CC7 - APO CC7 - SSB NPC2 Epididymal secretory protein E1 NA Equinatoxin 2 predicted protein Putative protein methyltransferase NA Vascular endothelial growth factor Sodium and chloride dependent GABA Ammonium transporter Rh type B Uncharacterized protein Cation transport regulator like protein Protransforming growth factor alpha Reticulocyte binding protein 2 homo

10 Which genes matter for symbiosis and bleaching? Establish genetic tools in Aiptasia and coral to functionally test candidate genes

11 Microinjection at the 1- cell stage yields viable larvae

12 Establish over-expression tools to functionally test candidate genes Microinject mrna into 1-cell zygotes Kaede mrna AAAAAAAA Photoconversion (358 nm) Kaede protein

13 Expression of Kaede protein from injected mrna into Aiptasia

14 How can we identify genes that are good targets for knock-down and knock-out?

15 FGF signaling is required for apical tuft formation in Nematostella (Rentzsch Technau, 2008)

16 FGF inhibitor (SU5402) blocks apical tuft formation in Aiptasia Larvae Anti- tubulin Phalloidin Wild- type 20 µm SU5402

17 FGF1a is expressed at the base of the apical tuft during development N = 88

18 Putative knockdown of FGF1a by morpholino microinjection Translation-blocking Morpholino Exon 1 FGF1a Exon 2

19 Putative knockdown of FGF1a by morpholino microinjection N = 88 N = 30 P < Percent larvae with apical tuft 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Not Fluorescent Fluorescent

20 Can we modify the genomes of corals?

21 Microinjecting CRISPR/Cas9 complexes into Acropora millepora 1- cell zygotes

22 In vivo genome editing

23 Efficient genome editing

24 In vivo genome editing

25 Within larvae mutation rate

26 Genome editing in two paralogs of GFP with single sgrna Cleves et al, 2018 PNAS

27 Unified Approach to Understanding Cnidarian-Dinoflagellate Symbiosis Aiptasia + Coral Reverse Genetics

28 Thank You! Cory Krediet Postdoc Olivia Barry Lab Tech Ben Mason Postdoc John Pringle PI CRISPR/Cas9 in Coral Marie Strader Misha Matz Line Bay Arthur Grossman Steve Palumbi Mani Aranda