1. Evolutionary Biology
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Metabolic co-dependence drives the evolutionarily ancient Hydra–Chlorella symbiosis

  1. Mayuko Hamada
  2. Katja Schröder
  3. Jay Bathia
  4. Ulrich Kürn
  5. Sebastian Fraune
  6. Mariia Khalturina
  7. Konstantin Khalturin
  8. Chuya Shinzato
  9. Nori Satoh
  10. Thomas CG Bosch  Is a corresponding author
  1. Okinawa Institute of Science and Technology Graduate University, Japan
  2. Okayama University, Japan
  3. Kiel University, Germany
  4. The University of Tokyo, Japan
Research Article
Cite this article as: eLife 2018;7:e35122 doi: 10.7554/eLife.35122
8 figures, 8 tables, 11 data sets and 8 additional files

Figures

Figure 1 with 3 supplements
Hydra growth and differential expression of Hydra genes resulting from symbiosis.

(A) Hydra viridissima strain A99 used for this study. Scale bar, 2 mm. (B) Growth rates of polyps grown with native symbiotic Chlorella A99 (Hv_Sym, dark green), Aposymbiotic polyps from which Chlorella were removed (Hv_Apo, orange) and aposymbiotic polyps reinfected with Chlorella variabilis NC64A (Hv_NC64A, light green). Average of the number of hydra in each experimental group (n = 6) is represented. Error bars indicate standard deviation. (C) Graphic representation of differentially expressed genes identified by microarray. The transcriptome of Hv_Sym is compared with that of Hv_Apo and Hv_NC64A with the number of down-regulated contigs in Hv_Sym shown in red and those up-regulated in green. Genes differentially expressed in Hv_Sym compared to both Hv_Apo and Hv_NC64A are given as ‘A99-specific’, those differentially expressed between Hv_A99 and Hv_Apo but not Hv_NC64A as ‘Symbiosis-regulated’. (D) GO distribution of Biological Process at level two in all contigs (All), up-regulated contigs (Hv_Sym > Hv_Apo) and down-regulated contigs (Hv_Sym < Hv_Apo) in Hv_Sym. (E) Overrepresented GO terms in up-regulated contigs (Hv_Sym > Hv_Apo) and down-regulated contigs (Hv_Sym < Hv_Apo). Category, F: molecular function, C: cellular component, P: biological process. P-values, probability of Fisher’s exact test. #Test, number of corresponding contigs in differentially expressed contigs. #Ref, number of corresponding contigs in all contigs.

https://doi.org/10.7554/eLife.35122.003
Figure 1—source data 1

GO distribution of Biological Process in all contigs (All), up-regulated contigs (up: Hv_Sym > Hv_Apo) and down-regulated contigs (down: Hv_Sym < Hv_Apo) in Hv_Sym.

https://doi.org/10.7554/eLife.35122.007
Figure 1—figure supplement 1
Chlorella sp. A99 and Chlorella variabilis NC64A in Hydra viridissima A99.

(A) Average number of algae per Hydra cell, for native Chlorella sp. A99 (Hv_Sym) and aposymbiotic Hydra re-infected with Chlorella variabilis NC64A (Hv_NC64A). P: p-value of student t-test. (B) Endodermal epithelial cells of Hv_Sym showing intracellular algae (C) Endodermal epithelial cells of Hv_NC64A. Scale bar, 20 µm.

https://doi.org/10.7554/eLife.35122.004
Figure 1—figure supplement 2
Conserved genes and species-specific genes differentially expressed in symbiotic Hydra.

Distribution of well-conserved Hydra viridissma genes (pink), Hydra viridissima-specific genes (green) and other genes (shared by some but not all metazoans, gray) among eight metazoans: Hydra magnipapillata, Acropora digitifera, Nematostella vectensis, Strongylocentrotus pupuratus, Branchiostoma floridae, Homo sapiens and Drosophila melanogaster and Hydra viridissima A99. Pie charts are shown for all contigs (All), up-regulated contigs (Hv_Sym > Hv_Apo) and down-regulated contigs (Hv_Sym < Hv_Apo).

https://doi.org/10.7554/eLife.35122.005
Figure 1—figure supplement 3
Glutamine synthetase (GS) genes in Cnidarians.

(A) Phylogenetic tree of the GS gene of four species in Cnidarians. While anthozoans (Nematostella vectensis, Acropora digitifera) have a single GS gene, Hydra magnipappilata (Hma) has five genes and Hydra viridissima A99 has three genes, Hv_1046 (GS-1), Hv_315 (GS-2) and Hv_4671 (GS-3). (B) Average of relative expression level of the three GS genes in Hv_Sym, Hv_NC64A and Hv_Apo as determined by microarray analysis. Error bars indicate standard deviation. P-value of t-test, *<0.05.

https://doi.org/10.7554/eLife.35122.006
Figure 2 with 1 supplement
Differential expression of Hydra genes under influence of Chlorella photosynthesis.

(A) Sampling scheme. Hv_Sym (green) and Hv_Apo (orange) were cultured under a standard light-dark regime (Light: L) and in continuous darkness (Dark: D), and RNA was extracted from the polyps at the days indicated by red arrows. (B) Expression difference of five A99-specific genes in Hv_Sym (green bars) and Hv_Apo (orange bars) between the light-dark condition and darkness. The vertical axis shows log scale (log2) fold changes of relative expression level in Light over Dark. (C) Sampling scheme of inhibiting photosynthesis. (D) Differential expression of the five A99-specific genes under conditions allowing (Control) or inhibiting photosynthesis (DCMU). The vertical axis shows log scale (log2) fold changes of relative expression level in Control over DCMU treated. T-tests were performed between Light and Dark (B), and DCMU and Control (D). For each biological replicate (n = 3) 50 hydra polyps were used for total RNA extraction. Error bars indicate standard deviation. P-value of t-test, *<0.05, **<0.01.

https://doi.org/10.7554/eLife.35122.012
Figure 2—figure supplement 1
Differential expression of symbiosis-dependent Hydra genes grown under light/dark condition and in darkness.

(A) Sampling scheme. Hv_Sym was cultured in the light-dark condition (Light: L) and in the continuous dark (Dark: D). Gene expression levels were examined by qPCR at 1, 2, 4 days for each condition (red arrows). (B) Expression difference of the genes in Hv_A99 between the two conditions. DEATH-1 and DEATH-2: Death domain containing proteins (gene ID: 6508 and rc_2417), TIR: Toll/interleukin-1 receptor domain containing protein (gene ID: 5168), PRKRIR: protein-kinase interferon-inducible double stranded RNA dependent inhibitor, repressor of (p58 repressor) (gene ID: rc_9398), ephrinR: ephrin receptor (gene ID: 26108), CLEC: C-type mannose receptor (gene ID: 11411), PRRT1: proline-rich transmembrane protein 1 (gene ID: rc_24563). For each biological replicate (n = 3) 50 hydra polyps were used for total RNA extraction. The vertical axis shows log scale (log2) fold change of relative expression levels in the light condition over the dark condition. Error bars indicate standard deviation. Pvalue of t-test, *<0.05, **<0.01.

https://doi.org/10.7554/eLife.35122.013
Figure 2—figure supplement 1—source data 1

Hydra genes under influence of Chlorella photosynthesis examined by qPCR.

https://doi.org/10.7554/eLife.35122.014
Figure 3 with 2 supplements
Spatial expression patterns of genes coding for glutamine synthetase, Spot 14 and Na/Pi-transporter.

(A-F); Whole mount in situ hybridization using antisense (A–C) and sense probes (D-F; negative controls) for glutamine synthetase-1 (GS-1; left), Spot 14 (center) and Na/Pi-transporter (NaPi; right). Inserts show cross sections of the polyp’s body. (G–I) Relative expression levels of whole animal (whole), isolated endoderm (End) and isolated ectoderm (Ect) tissue of Hv_Sym (green bars) and Hv_Apo (orange bars). For each biological replicate (n = 3) 10–20 hydra polyps were used for total RNA extraction of endodermal and ectodermal tissue. T-test was performed between Hv_Sym and Hv_apo. Pvalue, *<0.05, **<0.01. (J) Expression change of genes GS-1, Spot14, NaPi, Sym-1 and Sym-2 following exposure to 25, 50 and 100 mM maltose in Hv_Apo. For each biological replicate (n = 3) 50 hydra polyps were used for total RNA extraction The vertical axis shows log scale (log2) fold changes of relative expression level of maltose-treated over the untreated Hv_Apo control. T-test was performed between maltose-treated in each concentration and control (*: p value <0.05) and Kruskal-Wallis test (†: p value <0.05) in the series of 48 hr treatment were performed. Error bars indicate standard deviation.

https://doi.org/10.7554/eLife.35122.017
Figure 3—source data 1

Expression change of genes GS-1, Spot14, NaPi, Sym-1 and Sym-2 following exposure to 25, 50 and 100 mM maltose in Hv_Apo examined by qPCR.

https://doi.org/10.7554/eLife.35122.021
Figure 3—figure supplement 1
Tissue isolation of green hydra.

(A) Isolated endoderm (left) and isolated ectoderm (right). Scale bar, 1 mm. Expression levels of an endoderm-specific gene finalASM_15403 (B) and that of an ectoderm specific gene finalASM_344 (C) in whole hydra (Whole) and isolated endoderm (End) and ectoderm (Ect) were examined to confirm whether tissue isolation had performed properly. For each biological replicate (n = 3) 10–20 hydra polyps were used for total RNA extraction of endodermal and ectodermal tissue. Error bars indicate standard deviation.

https://doi.org/10.7554/eLife.35122.018
Figure 3—figure supplement 2
Effects of sugars on Hydra growth.

Effects of growth in presence of maltose (A), glucose (B), sucrose (C) and galactose (D) on gene expression of GS-1, Spot14 and NaPi. Hv_Apo were cultured in medium containing 10, 25, 50 or 75 mM of each sugar for 48 hr, and 75 mM maltose (orange) and glucose (blue) for 6 hr (E). RNA was extracted from the polyps in the light condition. Expression difference of the genes was examined by qPCR. For each biological replicate (n = 3) 50 hydra polyps were used for total RNA extraction. The vertical axis is log scale (log2) fold change of relative expression level of sugar-treated hydras over controls. T-test (*: p-value<0.05) in each concentration and Kruskal-Wallis test (†: pvalue <0.05) in the series of 48 hr treatment were performed. Error bars indicate standard deviation.

https://doi.org/10.7554/eLife.35122.019
Figure 3—figure supplement 2—source data 1

Effects in presence of maltose, glucose, sucrose and galactose on gene expression of GS-1, Spot14 and NaPi in Hv_Apo examined by qPCR.

https://doi.org/10.7554/eLife.35122.020
Comparison of key features deduced from the Chlorella A99 genome with other green algae.

(A) Phylogenetic tree of eight genome sequenced chlorophyte green algae including Chlorella sp. A99. The NJ tree is based on sequences of the 18S rRNA gene, ITS1, 5.8S rRNA gene, ITS2 and 28S rRNA gene. (B) Genomic features and taxonomy of the sequenced chlorophyte green algae. Hel: Helicosporidium sp. ATCC50920. (C) The proportion of similarity of Chlorella A99 gene models to those of other organisms.

https://doi.org/10.7554/eLife.35122.022
Genes missing in the genome of Chlorella A99.

Functional categorization of genes present in Coccomyxa subellipsoidea C169 (A, C) and genes missing in Chlorella A99 (B, D) by GO terms using Bast2GO. Multilevel pie charts show enrichment of GO’ Biological Process’ terms (A, B) and GO ‘Molecular Function’ terms (C, D) on the lowest level, which cover at least 10% of the total amount of annotated sequences.

https://doi.org/10.7554/eLife.35122.025
Figure 5—source data 1

Functional categorization of genes present in Coccomyxa subellipsoidea C169 (C169_all) and genes missing in Chlorella A99 (A99 gene loss) by GO terms’ Biological Process’ terms and ‘Molecular Function’ on the lowest level, which cover at least 10% of the total amount of annotated sequences.

https://doi.org/10.7554/eLife.35122.026
Figure 6 with 1 supplement
Nitrogen assimilation pathways in Chlorella A99.

(A) Schematic diagram of the nitrogen assimilation pathway in plants showing the function of nitrate transporters NRT1 (peptides/nitrate transporter) and NRT2 (nitrate/nitrite transporter), nitrate assimilation-related components NAR1 and NAR2, nitrate reductase NR, nitrite reductase NiR, ammonium transporter AMT, glutamate synthetase GOGAT and glutamine synthetase GS. Genes shown in red boxes (NRT2, NAR2 and NiR) were not found in the Chlorella sp. A99 genome. (B) Table showing the number of nitrogen assimilation genes in Chlorella sp. A99 (A99), Chlorella variabilis NC64A (NC64A), Coccomyxa subellipsoidea C169 (C169), Volvox carteri f. nagariensis (Vc), Chlamydomonas reinhardtii (Cr), Ostreococcus tauri (Ot) and Micromonas pusilla (Mp). (C) Gene clusters of nitrate assimilation genes around the shared NR genes (blue) in the genomes of NC64A, C169 and A99. Red boxes show nitrate assimilation genes absent in A99 and gray boxes depict other genes. Numbers below the boxes are JGI protein IDs of NC64A and C169. Numbers below the genes of A99 are JGI protein IDs of the best hit genes in NC64A and C169 and their gene name.

https://doi.org/10.7554/eLife.35122.028
Figure 6—figure supplement 1
PCR of nitrate assimilation genes.

PCR amplification of genomic DNA corresponding to the genes NRT2, NiR and NR (positive control) was performed in Chlorella sp. A99 (A99), Chlorella variabilis NC64A (NC64A), Coccomyxa subellipsoidea C169 (C169) and Chlamydomonas reinhardtii (Cr).

https://doi.org/10.7554/eLife.35122.029
Figure 7 with 1 supplement
Growth of green algae in presence of various nitrogen sources.

The growth rate of Chlorella A99 (A99), Chlorella variabilis NC64A (NC64A) and Coccomyxa subellipsoidea C-169 (C169) by in vitro culture was assessed for different nitrogen sources with casamino acids (blue), glutamine (orange), ammonium (gray) and nitrate (yellow). Mean number of algae per ml were determined at 4, 8, 12 days after inoculation with 106 cell/ml. Error bars indicate standard deviation.

https://doi.org/10.7554/eLife.35122.030
Figure 7—figure supplement 1
PCR of 18S rRNA genes in cultured algae.

PCR amplification of genomic DNA of the 18S rRNA gene was performed in Chlorella A99 shortly after isolation from H. viridissima A99 (Isolated A99), cultured in medium containing glutamine (Glu) and in medium with casamino acids for 12 days, with cultured NC64A and C169 added for comparison.

https://doi.org/10.7554/eLife.35122.031
Molecular interactions in the symbiosis of cnidarians.

(A) Summary of symbiotic interactions between Hydra and Chlorella A99. During light conditions, Chlorella A99 performs photosynthesis and produces maltose (Mal), which is secreted into the Hydra symbiosome where it is possibly digested to glucose (Gluc), shown in red. The sugar induces expression of Hydra genes encoding glutamine synthetase (GS), Na/Pi transporter (NaPi) and Spot14. GS catalyzes the condensation of glutamate (Glu) and ammonium (NH4+) to form glutamine (Gln), which is used by Chlorella as a nitrogen source. Since the sugar also up-regulates the NaPi gene, which controls intracellular phosphate levels, it might be involved in the supply of phosphorus to Chlorella as well (blue broken line). The sugar is transported to the ectoderm (red broken line) and there induces the expression of GS and Spot14. In the Chlorella A99 genome, degeneration of the nitrate assimilation system and an increase of amino acid transporters was observed (green balloon). (B, C) Comparison between Hydra-Chlorella symbiosis and coral-Symbiodinium symbiosis. Red indicates transfer of photosynthesis products from the symbiont to the host, and blue indicates transfer of nitrogen sources from the host to the symbiont. While the host organisms Hydra and coral can assimilate NH4+ to Gln (B, C), assimilation of inorganic nitrogen by Symbiodinidium plays an important role for the symbiotic system in coral (C).

https://doi.org/10.7554/eLife.35122.032

Tables

Table 1
List of differentially expressed genes between Hv_Sym and Hv_Apo, which are likely to be involved in symbiotic relationship
https://doi.org/10.7554/eLife.35122.008
ProbenameFold changeHuman_BestHitblast2GO_Description
Hv_Sym
/Hv_Apo
Hv_Sym_sexy
/Hv_Apo
Hv_NC64A
/Hv_Sym
Localization and Transport
Hv_Sym > Hv_Apo
rc_67889.878.001.01helicase conserved c-terminal
domain containing protein
rc_102468.265.151.82protein
rc_62987.104.730.99hypothetical protein LOC220081protein fam194b
22686.963.581.26protein Dapleviral a-type inclusion protein
105486.746.890.73transient receptor potential
cation channel subfamily M
member three isoform d
transient receptor potential cation
channel subfamily m member 3-like
rc_12906.447.180.99tetratricopeptide repeat protein
eight isoform B
tetratricopeptide repeat protein 8
187366.046.341.03BTB/POZ domain-containing
protein KCTD9
btb poz domain-containing protein
kctd9-like; unnamed protein product
rc_92705.9610.031.37PREDICTED: hypothetical
protein LOC100131693
eukaryotic translation initiation factor 4e
NPNHRC_156973.852.740.62major facilitator superfamily domain-
containing protein 1
2903.683.731.32splicing factor, arginine/
serine-rich 6
splicing arginine serine-rich 4
rc_95963.564.191.62BTB/POZ domain-containing
protein KCTD10
btb poz domain-containing adapter for
cul3-mediated degradation protein 3
rc_67743.343.321.31solute carrier family 43,
member 2
large neutral amino acids transporter
small subunit 4
rc_262183.292.910.41sodium-dependent phosphate
transport protein 2A isoform 1
sodium-dependent phosphate transport
protein 2b
NPNHRC_260943.203.981.31SPE-39 proteinid="T5"spe-39 protein
90963.102.200.69otoferlin isoform dotoferlin
rc_213492.894.250.785'-AMP-activated protein kinase
catalytic subunit alpha-2
5 -amp-activated protein kinase catalytic
subunit alpha-2
npRC_144882.882.650.71solute carrier family 2, facilitated
glucose transporter member 8
solute carrier family facilitated glucose
transporter member 8-like
88632.752.700.81ATP-binding cassette, sub-family B,
member 10 precursor
abc transporter b family protein
rc_118962.492.561.52ATP-binding cassette, sub-family B,
member 10 precursor
abc transporter b family member 25-like
rc_68422.413.351.59hypothetical protein LOC112752
isoform 2
intraflagellar transport protein 43 homolog
52422.363.351.22growth arrest-specific protein 8growth arrest-specific protein 8
58152.232.470.78plasma membrane calcium-
transporting ATPase 4 isoform 4a
plasma membrane calcium atpase
87652.223.250.91growth arrest-specific protein 8growth arrest-specific protein 8
NPNH_140522.192.170.79V-type proton ATPase 21 kDa
proteolipid subunit isoform 2
v-type proton atpase 21 kda proteolipid
subunit-like
rc_24992.182.031.47endoplasmic reticulum-Golgi
intermediate compartment
protein three isoform a
endoplasmic reticulum-golgi intermediate
compartment protein 3 isoform 2
rc_139692.083.090.97major facilitator superfamily
(IPR023561) Carbonic anhydrase, alpha-class
rc_248252.492.380.83protein tyrosine phosphatase,
receptor type, G precursor
receptor-type tyrosine-protein phosphatase
gamma
Cell Adhesion and extracelluar matrix
Hv_Sym > Hv_Apo
79154.015.090.94fibrillin-2 precursorfibrillin-1- partial
npRC_24163glutamate3.693.591.32semaphorin 5A precursorrhamnospondin 1
Immunity, apoptosis and recognition
Hv_Sym > Hv_Apo
(IPR000157) Toll/interleukin-1 receptor homology (TIR) domain
51689.284.920.61protein; PREDICTED: uncharacterized
protein LOC100893943
127495.133.351.26PREDICTED: uncharacterized protein
LOC100893943 [Strongylocentrotus
purpuratus]
(IPR011029) DEATH-like
65086.705.100.64PREDICTED: hypothetical protein
[Hydra magnipapillata]
rc_24175.392.701.01nod3 partial; PREDICTED: uncharacterized
protein LOC100206003
(IPR002398) Peptidase C14, caspase precursor p45
NPNH_212752.363.531.18caspase seven isoform alpha
precursor
caspase d
(IPR016187) C-type lectin fold
114112.932.980.75C-type mannose receptor 2PREDICTED: similar to predicted protein,
partial [Hydra magnipapillata]
Hv_Sym < Hv_Apo
(IPR000488) Death
73190.450.311.10probable ubiquitin carboxyl-
terminal hydrolase CYLD isoform 2
ubiquitin carboxyl-terminal hydrolase cyld
(IPR001875) Death effector domain
RC_FV81RT001CSTY0.310.390.93astrocytic phosphoprotein PEA-15fadd
Chitinase
Hv_Sym > Hv_Apo
(IPR001223) Glycoside hydrolase, family 18, catalytic domain
rc_44502.783.830.66chitinase 2
Hv_Sym < Hv_Apo
(IPR000726) Glycoside hydrolase, family 19, catalytic
FPVQZVL01EAWBY0.210.161.78endochitinase 1-like
10280.230.181.47endochitinase 1-like
Oxidative Stress Response
Hv_Sym > Hv_Apo
np_12765.997.160.78glutaredoxin-2, mitochondrial
isoform 2
cpyc type
109263.92.30.8hydroxysteroid dehydrogenase-
like protein 2
hydroxysteroid dehydrogenase-like
protein 2
4692.973.530.76cytochrome P450 3A7cytochrome p450
FV81RT001DCTAQ2.692.500.75oxidoreductase NAD-binding
domain-containing protein
one precursor
oxidoreductase nad-binding domain-
containing protein 1
6962.303.240.69methionine-R-sulfoxide
reductase B1
selenoprotein 1; methionine-r-sulfoxide
reductase b1-a-like
65722.232.151.06L-xylulose reductasel-xylulose reductase
132982.103.490.64eosinophil peroxidase
preproprotein
peroxidase
npRC_69752.042.771.42methionine-R-sulfoxide
reductase B1
selenoprotein 1; methionine-r-sulfoxide
reductase b1-a-like
(IPR024079) Metallopeptidase, catalytic domain
Hv_array_49524.7713.310.72meprin A subunit beta
precursor
zinc metalloproteinase nas-4-like
Hv_array_rc_39922.662.231.27matrix metalloproteinase
seven preproprotein
matrix metalloproteinase-24-like
Hv_Sym < Hv_Apo
RC_FWZAEML02HKSC0.2550.1531.444ascorbate peroxidase
np_149620.2930.4551.390tryptophan 5-hydroxylase 2phenylalanine hydroxylase
rc_41510.3180.4631.693phenylalanine-4-hydroxylasephenylalanine hydroxylase
28350.3840.3441.787u1 small nuclear ribonucleoprotein 70 kda
rc_114260.4130.4581.591short-chain dehydrogenase/
reductase family 9C member 7
uncharacterized oxidoreductase -like
FWZAEML02IC34R0.4270.4481.159aldehyde dehydrogenase 5A1
isoform two precursor
succinate-semialdehyde mitochondrial-like
FWZAEML02HKSCO0.4540.3070.833ascorbate peroxidase
(IPR004045) Glutathione S-transferase, N-terminal
RC_FWZAEML02GGHN0.090.071.81hematopoietic prostaglandin
D synthase
glutathione s-transferase family member
(gst-7)
(IPR024079) Metallopeptidase, catalytic domain
rc_112700.140.201.33meprin A subunit beta precursorprotein; zinc metalloproteinase nas-4-like
rc_RSASM_150590.220.291.42---NA---
21110.370.431.74meprin A subunit beta precursorzinc metalloproteinase nas-4-like
124510.500.390.78meprin A subunit alpha precursorzinc metalloproteinase nas-13- partial
(IPR013122) Polycystin cation channel, PKD1/PKD2
288540.370.280.94polycystin-2receptor for egg jelly partial
157740.400.260.76polycystic kidney disease protein
1-like two isoform a
protein
Table 2
List of genes differentially expressed in Hv_Sym compared to both Hv_Apo and Hv_NC64A (‘A99-specific’)

Fold change of expression level determined by microarray analysis and qPCR analysis

https://doi.org/10.7554/eLife.35122.009
Hv_Sym > Hv_Apo, Hv_NC64A
Probe name (gene ID)MicroarrayqPCRGene annotationInterProScan
Sym/ApoSym/NC64ASym/ApoSym/NC64A
rc_1357912.84.011.24.0(Hydra specific)
rc_128919.02.914.66.9(Hydra viridis specific)
274174.54.83.03.0IPR009786 Spot_14
rc_262183.32.42.52.3sodium-dependent phosphate
transport protein
PTHR10010 Sodium-dependent
phosphate transport protein 2C
10463.12.12.21.6glutamine synthetase
Hv_Sym < Hv_Apo, Hv_NC64A
Probe name (gene ID)MicroarrayqPCRGene AnnotationInterProScan
Apo/SymNC64A/SymApo/SymNC64A/Sym
NPNHRC_2685983.29.7(Hydra viridis specific)
RC_FVQRUGK01AXSJ13.72.62.11.5acetoacetyl-CoA synthetase
rc_147937.24.19.44.82-isopropylmalate synthaseIPR013785 Aldolase_TIM,
FV81RT002HT2FL2.82.03.11.8histidine ammonia-lyaseIPR001106 Aromatic_Lyase
IPR008948 L-Aspartase-like
NPNHRC_122012.7glutamate2.32.62.5(Hydra viridis specific)
Table 2—source data 1

Expression level of ‘A99-specific’ genes and ‘Symbiosis related’ genes examined by microarray and qPCR.

https://doi.org/10.7554/eLife.35122.010
Table 3
List of annotated genes up-regulated in Hv_NC64A compared to Hv_Sym
https://doi.org/10.7554/eLife.35122.011
ProbenameHv_NC64A/
Hv_Sym
Hv_Apo/
Hv_Sym
Hv_Sym_sexy/
Hv_Sym
Blast2GO description
rc_16234.571.645.98methylase involved in ubiquinone
menaquinone biosynthesis
289473.521.590.63non-ribosomal peptide synthetase
13533.131.630.10nuclear protein set
143472.692.400.54n-(5-amino-5-carboxypentanoyl)-l
-cysteinyl-d-valine synthase
SSH_3972.672.390.50n-(5-amino-5-carboxypentanoyl)-l
-cysteinyl-d-valine synthase
RC_FWZAEML01C7BP2.280.820.41ubiquitin carboxyl-terminal
hydrolase family protein
RC_FVQRUGK01EOXS2.251.520.53ubiquitin carboxyl-terminal
hydrolase family protein
rc_117102.151.260.31ubiquitin carboxyl-terminal
hydrolase family protein
16772.101.190.38ubiquitin carboxyl-terminal
hydrolase family protein
rc_3632.211.040.76gcc2 and gcc3 family protein
Table 4
List of the genes differentially expressed between Hv_Sym and Hv_Apo

Fold change of expression level determined by microarray analysis and qPCR

https://doi.org/10.7554/eLife.35122.015
Hv_Sym > Hv_Apo
Probe name
(gene ID)
MicroarrayqPCRGene annotationInterProScan
Sym/ApoSym/Apo
51689.37.4IPR000157 TIR_dom
PTHR23097 Tumor necrosis factor
receptor superfamily member
65086.72.9IPR011029:DEATH-like_dom
114112.92.0C-type mannose receptor 2IPR000742 EG-like_dom
IPR001304 C-type_lectin
261087.27.2ephrin type-A receptor six isoform a
rc_24175.43.5IPR000488 Death_domain
rc_245636.16.7Proline-rich transmembrane protein 1IPR007593 CD225/Dispanin_fam
PTHR14948 NG5
rc_93986.25.4protein-kinase, interferon-inducible
double stranded RNA dependent inhibitor,
repressor of (P58 repressor)
PTHR11697 general transcription factor
2-related zinc finger protein
Hv_Sym < Hv_Apo
Probe name
(gene ID)
MicroarrayqPCRGene AnnotationInterProScan
Apo/SymApo/Sym
rc_107892.53.7endoribonuclease DicerIPR000999 RNase_III_dom
PTHR1495 helicase-related
rc_128263.02.3interferon regulatory factor 1IPR001346 Interferon_reg_fact_DNA-bd_dom;
IPR011991 WHTH_DNA-bd_dom
PTHR11949 interferon regulatory factor
rc_88986.14.1leucine-rich repeat-containing protein 15
isoform b
IPR001611 Leu-rich_rp
PTHR24373 Toll-like receptor 9
FV81RT001CSTY3.22.0astrocytic phosphoprotein PEA-15IPR001875 DED, IPR011029 DEATH-like_dom
RSASM_177524.02.1CD97 antigen isoform two precursorIPR000832 GPCR_2_secretin-like
PTHR12011 vasoactive intestinal polypeptide
receptor 2
Table 4—source data 1

Expression level of 'Symbiosis related' genes examined by microarray and qPCR.

https://doi.org/10.7554/eLife.35122.016
Table 5
Summary of sequence data for assembling Chlorella sp. A99 genome sequences
https://doi.org/10.7554/eLife.35122.023
Number of reads85469010
Number of reads assembled61838513
Number of bases17398635102
ScaffoldsContigs
Total length of sequence4093403740687875
Total number of sequences827455
Maximum length of sequence4003385171868
N50172741912747
GC contents (%)68.07%69.95%
Table 6
Amino acid transporter genes in Chlorella sp. A99 (A99), Chlorella variabilis NC64A (NC64A), Coccomyxa subellipsoidea C-169 (C169), Volvox carteri (Vc), Micromonas pusilla (Mp) and Ostreococcus tauri (Ot) and Chlamydomonas reinhardtii (Cr)
https://doi.org/10.7554/eLife.35122.024
A. The number of Pfam domains related to amino acids transport
Pfam domain nameA99NC64Ac169CrVcMpOt
Aa_trans3038219798
AA_permease46155611
B. Ortholog groups including Aa_trans domain containing genes
overrepresented in symbiotic Chlorella
Ortholog group ID: Gene annotationA99NC64Ac169CrVcMpOt
OG0000040: amino acid permease 2121263100
OG0000324: transmembrane amino acid transporter
family protein (solute carrier family 38, sodium-coupled neutral amino acid transporter)
6712100
Table 7
List of Coccomyxa subellipsoidea C169 (C169) genes, which are present in Chlamydomonas reinhardtii and Volvox carteri, but missing in the genome of Chlorella A99
https://doi.org/10.7554/eLife.35122.027
UniProt ID in C169Description
F1DPL8_9CHLOATP synthase F0 subunit 6 (mitochondrion)
F1DPL7_9CHLOcytochrome c oxidase subunit 3 (mitochondrion)
I0YZU4_9CHLOequilibrative nucleoside transporter 1
I0Z311_9CHLOequilibrative nucleoside transporter family
I0YZC9_9CHLOhigh affinity nitrate transporter
I0Z2L2_9CHLOhypothetical protein COCSUDRAFT_28432
I0YJ99_9CHLOhypothetical protein COCSUDRAFT_34498
I0YKQ1_9CHLOhypothetical protein COCSUDRAFT_45098
I0YYD3_9CHLOhypothetical protein COCSUDRAFT_65897
I0YYP5_9CHLOimportin-4 isoform X1
I0YQQ1_9CHLOlow-CO2-inducible membrane
I0YJD4_9CHLOMFS transporter
I0YTY0_9CHLOmolybdate transporter 2
F1DPM0_9CHLONADH dehydrogenase subunit 3 (mitochondrion)
F1DPM4_9CHLONADH dehydrogenase subunit 6 (mitochondrion)
F1DPM8_9CHLONADH dehydrogenase subunit 9 (mitochondrion)
I0Z357_9CHLOplasma membrane phosphate transporter Pho87
I0Z9Y1_9CHLOpre translocase subunit
I0YPT2_9CHLOtranscription and mRNA export factor ENY2-like
I0Z976_9CHLOtransport SEC23
I0Z3Q6_9CHLOtyrosine-specific transport -like isoform X1
I0YXU9_9CHLOurea active transporter
I0YRT0_9CHLOurea active transporter
I0YRL4_9CHLOurea-proton symporter DUR3
I0YUF9_9CHLOurea-proton symporter DUR3
I0YJS6_9CHLOurea-proton symporter DUR3
I0YQ78_9CHLOurea-proton symporter DUR3-like
I0YIH7_9CHLOZip-domain-containing protein
Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background
(Hydra viridissima A99)
Hydra viridissima A99PMID: 16351895
Strain, strain background
(Chlorella sp. A99)
Chlorella sp. A99PMID: 16351895NCBI BioProject ID:
PRJNA412448
Strain, strain background
(Chlorella variabilis NC64A)
Chlorella variabilis NC64AMicrobial Culture Collection
at the National Institute
for Environmental Studies
NIES-2541
Strain, strain background
(Coccomyxa subellipsoidea C-169)
Coccomyxa subellipsoidea
C-169
Microbial Culture Collection
at the National Institute
for Environmental Studies
NIES-2166
Strain, strain background
(Chlamydomonas reinhardtii)
Chlamydomonas reinhardtiiMicrobial Culture Collection
at the National Institute
for Environmental Studies
NIES-2235
Commercial assay or kitTruSeq DNA LT Sample Prep
Kit
IlluminaFC-121–2001
Commercial assay or kitNextera Mate Pair Sample
Preparation Kit
IlluminaFC-132–1001
Commercial assay or kitMiseq reagent kit v3IlluminaMS-102–3003
Commercial assay or kitHiSeq SBS kit v4IlluminaFC-401–4003
Commercial assay or kitBigDye Terminator v3.1 Cycle
Sequencing Kit
Thermo Fisher Scientific4337454
Commercial assay or kit4 × 44K Hydra viridissima A99
Custom-Made Microarray
Agilent TechnologiesNCBI GEO Platform
ID: GPL23280
Commercial assay or kitGE Hybridization Kit and GE
Wash Pack
Agilent Technologies5188–5242, 5188–5327
Commercial assay or kitHigh Sensitivity DNA KitAgilent Technologies5067–4626
Commercial assay or kitRNA6000 nano kitAgilent Technologies5067–1511
Commercial assay or kitLow Input Quick Amp
Labeling Kit
Agilent Technologies5190–2305
Commercial assay or kitPureLink RNA Mini KitThermo Fisher Scientific12183018A
Commercial assay or kitFermentas First Strand
cDNA Synthesis Kit
Thermo Fisher ScientificK1621
Chemical compound,
drug
Trizol reagentThermo Fisher Scientific15596026
Chemical compound,
drug
AmpliTaq Gold 360
Master Mix
Thermo Fisher Scientific4398901
Chemical compound,
drug
ISOPLANT IINippon Gene316–04153
Chemical compound,
drug
GoTaq qPCR Master MixPromegaA6002
Chemical compound,
drug
KOD FX NeoTOYOBOKFX-201
Software, algorithmFeature Extraction
Software
Agilent TechnologiesRRID:SCR_014963
Software, algorithmNewbler454 Life Sciences,
Roche Diagnostics
RRID:SCR_011916
Software, algorithmSSPACEPMID: 21149342RRID:SCR_005056
Software, algorithmGapCloserPMID: 23587118RRID:SCR_015026
Software, algorithmNCBI BLASTPMID: 2231712RRID:SCR_004870
Software, algorithmCEGMAPMID: 17332020RRID:SCR_015055
Software, algorithmAugustus: Gene PredictionPMID: 16845043RRID:SCR_008417
Software, algorithmBlast2GOPMID: 16081474RRID:SCR_005828
Software, algorithmHmmerPMID: 9918945RRID:SCR_005305
Software, algorithmCLUSTALX2PMID: 17846036RRID:SCR_002909
Software, algorithmBioEditNucleic Acid Symposium
Series 41, 95–98
RRID:SCR_007361
Software, algorithmNjplotBiochimie 78, 364–369NA
Software, algorithmOrthoFinderPMID: 26243257NA

Data availability

Microarray information and the data series are accessible at NCBI GEO under accession number GPL23280 and GSE97633 respectively. All the results of microarray analysis are included in Supplementary Table 1. The Whole Genome Shotgun project of Chlorella sp. A99 has been deposited at DDBJ/ENA/GenBank under the accession PCFQ00000000 (BioProject ID: PRJNA412448). Genome sequences and gene models are also accessible at the website of OIST Marine Genomics Unit Genome Project (http://marinegenomics.oist.jp/chlorellaA99/viewer/info?project_id=65). All data generated by qPCR are included in Source Data: Figure2, Figure2 - Figure supplement 1, Source Data: Figure3, Source Data: Figure3 - Figure Supplement 2 and Source Data: Table 2, Table 4

The following data sets were generated
  1. 1
    Chlorella sp. A99 genome sequence and gene models
    1. Mayuko Hamada
    (2018)
    Publicly available at OIST Marine Genomics Unit (Chlorella sp. A99).
  2. 2
    Chlorella sp. A99 genome sequence
    1. Mayuko Hamada
    (2018)
    Publicly available at NCBI BioProject (Accession no: PCFQ00000000).
  3. 3
    Agilent-029560 Hydra viridissima transcriptome-based custom microarray
    1. Fraune S
    2. Bosch TC
    (2017)
    Publicly available at the NCBI Gene Expression Omnibus (accession no: GPL23280).
  4. 4
    Identification of genes involved in symbiosis of green hydra and Chlorella
    1. Fraune S
    2. Kürn U
    3. Bosch TC
    (2017)
    Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE97633).
The following previously published data sets were used
  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7

Additional files

Supplementary file 1

Results of microarray analysis and list of differentially expressed genes.

Gene expression of green hydra with native symbiotic Chlorella A99 (Hv_Sym), that in sexual phase (Hv_Sym_sexy), aposymbiotic polyps from which symbiotic Chlorella were removed (Hv_Apo) and aposymbiotic polyps reinfected with Chlorella variabilis NC64A (Hv_NC64A) were compared.

https://doi.org/10.7554/eLife.35122.033
Supplementary file 2

(A) Ortholog groups of Aa_trans containing protein in Chlorella variabilis NC64A (NC64A), Coccomyxa subellipsoidea C-169 (C169), Chlamydomonas reinhardtii (Cr), Volvox carteri (Vc), Micromonas pusilla (Mp) and Ostreococcus tauri (Ot).

(B) Blast best hit genes of Arabidopsis thaliana in Chlorella sp. A99 genes belonging to OG0000040 and OG0000324.

https://doi.org/10.7554/eLife.35122.034
Supplementary file 3

List of Coccomyxa subellipsoidea C169 (C169) and their BLAST best hit genes in Chlamydomonas reinhardtii (Cr), Volvox carteri (Vc) and Chlorella A99 (A99) gene model and genome scaffolds.

https://doi.org/10.7554/eLife.35122.035
Supplementary file 4

Sequence ID of nitrogen assimilation genes in Symbiodinium.

https://doi.org/10.7554/eLife.35122.036
Supplementary file 5

Sequence ID of nitrogen assimilation genes in Chlorella variabilis NC64A (NC64A), Coccomyxa subellipsoidea C-169 (C169), Volvox carteri (Vc), Micromonas pusilla (Mp) and Ostreococcus tauri (Ot) and Chlamydomonas reinhardtii (Cr).

https://doi.org/10.7554/eLife.35122.037
Supplementary file 6

Primers used in this study, for quantitative real time RT-PCR. (A), in situ hybridization probes (B), PCR amplification of nitrogen assimilation genes in green algae (C) and PCR amplification of 18S ribosomal DNA gene in green algae (D).

https://doi.org/10.7554/eLife.35122.038
Supplementary file 7

Composition of modified Bold’s Basal Medium for one liter (pH. 7).

https://doi.org/10.7554/eLife.35122.039
Transparent reporting form
https://doi.org/10.7554/eLife.35122.040

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