Isolation of Symbiotically Expressed Genes From the Dinoflagellate Symbiont of the Solitary Radiolarian Thalassicolla nucleata

5 Pages·2003·2.1 MB·English

by  Rebecca J Gast

Reference: Biol. Bull. 204: 210-214. (April 2003)
2003 Marine Biological Laboratory
Isolation of Symbiotically Expressed Genes From
the Dinoflagellate Symbiont of the Solitary
Radiolarian Thalassicolla nucleata
REBECCA J. CAST1'*, DAVID J. BEAUDOIN2. AND DAVID A. CARON'
1Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543; 2Marine Biological
Laboratory, Woods Hole, Massachusetts 02543; and ^University ofSouthern California,
Los Angeles, California
Symbiotic associations arefundamental to the survival of both terrestrial and aquatic environments and depend upon
many organisms on Earth. The ability of the symhiont to regulated genomic communication between the interacting
perform ke\ biochemical functions often allows the host to organisms. We are particularly interested in the photosym-
occupy environments that it would otherwise find inhos- biotic relationships that involve microscopic (protistan) al-
pitable. This can have profound impacts upon the diversi- gae in association with a variety of "invertebrate" hosts
fication and distribution of the host. Cellular organelles (protistan and metazoan); these relationships are fairly com-
(chloroplasts and mitochondria} represent thefined stages mon in the marine environment and contribute significantly
ofintegration ofendosymbionts. These organelles were of to nutrient flow in the nutrient-poor regions of the ocean
critical importance to the evolution and success ofeukary- where they occur. The symbiosis is essential to the survival
otic lineages on our planet because they allowed the host ofthe host organisms, although the algal symbiont can exist
cells to harness lightenergyandto thrive in thepresence of in the free-living state.
oxvgen. The marine photosymbiotic associations that we Besides the conspicuous invertebrate photosymbioses
study represent an earlier stage in the process ofsymbiont that most people are familiar with (anemones and corals), a
integration one in wliich the photobiont can still be re- variety of protistan species also harbor cytoplasmic algal
movedfrom the host andexiston its own. These systems are symbionts. Most notably, protozoa within the subphylurn
ofinterest to usfortwo reasons. First, theyare ecologically Sarcomastigophora (the foraminifera, radiolaria, and acan-
important in the marine environment where they occur. tharia; collectively referred toas "sarcodine protozoa") exist
Theseorganismsformzonesofphotosyntheticproduction in in symbioses with a wide array of algae (Anderson, 1983;
oceanic regions typically low in nutrients. Second, investi- Lee and McEnery, 1983; Caron and Swanberg, 1990;
gation ofthese interactionsmayshedlighton the molecular Michaels, 1991). These sarcodine protozoa are often large
and evolutionary mechanisms involved in the integration of for single-celled organisms. Solitary cells often attain sizes
cells and their genomes. more than 1 mm in diameter (some benthic foraminifera
Associations between microbial symbionts and their form discs more than 1 cm in diameter), and the presence of
hosts are fundamental trophic relationships that occur in complex pseudopodial networks can increase the effective
size of these organisms up to a few centimeters (Fig. 1).
*To whom correspondence should be addressed. E-mail: Some colonial species of radiolaria even form gelatinous
[email protected] pseudopodial matrices several centimeters in diameter and
The paper was originally presented at a workshop titled Outcomes of more than I m in length. These organisms are important
Genome-Genome Interactions. The workshop, which was held at the J. contributors to the biological assemblages in all tropical and
ErikJonsson Center of the National Academy ofSciences, Woods Hole, subtropical oceans of the world. Sarcodines form conspic-
AMadsvsaanccheusdetSttsu,difesromin 1t-he3 SMpaayce2L0i0f2e,Swcaisencsepsonastortehde MbyarithneeCBeinotleorgicfaolr uous zooplankton assemblages in oligotrophic surface wa-
Laboratory, and funded by the National Aeronautics and Space Adminis- ters, where they contribute significantly to primary produc-
tration underCooperative Agreement NCC 2-1266. tivity (by virtue of their intracellular symbionts), predation.
210
SYMBIOTICALLY EXPRESSED GENES IN DINOFLAGELLATE SYMBIONTS
We chose to begin our work of isolating algal genes
expressed in the symbiotic state by studying the dinoflagel-
late symbiont (Scrippsiella niitriciila) of the solitary radio-
larian T. nucleata (Fig. 1). To identify genes being ex-
pressed in the symbiosis, we required RNA from both the
We
free-living and symbiotic states of the alga. currently
have the different algal symbionts as free-living laboratory
cultures, but the intact association must be collected from
the environment. T. nucleata generally harbors thousands of
symbiotic algae, it can be collected from the environment
fairly easily, and, most importantly, the host and symbiont
can be separated from each other with minimal host RNA
contamination of the sample. The host nucleus, organelles.
Figure 1. Solitary planktonic radiolurian Tlicilu.ixicolUi niiclcutu. The and ribosomes are separated from the symbionts by the
cseunrtrroaulndceadpsbuylethoefrhtihzeopoordgiaanicosnmtaiisnitnhgetdhearskymcbiircolnetsin(stmhaellcdeonttsera;roitunids central capsule (pp.m9R0-N9A4, Anderson, 1983). We believe
thecentralcapsule).Theentireorganism isaboutamillimeterindiameter. that very little host will be present in the extracap-
sular material, and we are careful not to destroy the central
capsule when stripping away the desired extracapsular ma-
and the vertical flux of organic material and crystal miner- trix.
als, such as calcium carbonate and strontium sulfate (Caron Thalassicolla nucleata was collected by plankton net
and Swanberg. 1990; Swanberg and Caron. 1991; Caron et tows in the Sargasso Sea and the San Pedro Channel.
Hi, 1995; Michaels et al.. 1995). Individuals were transferred to sterile seawater in multiwell
Our interest in the regulation of photosymbiotic relation- culture dishes and placed in lighted incubators or on work-
ships arose while working on the identification of the sym- benches at ambient temperatures. This allowed the hosts to
bionts in planktonic foraminifera and radiolaria to deter- clear themselves of prey items prior to recovery of the
mine whether co-evolution of host and symbiotic alga symbiont. All hosts were examined for prey before the
occurred (Cast and Caron, 1996; Gast et al., 2000). We extracapsular material was stripped away from the central
molecularly identified several taxonomically different algae capsule. Studies involving host incorporation of I4C through
that form symbioses in the planktonic sarcodines. the uptake of symbiont-produced photosynthate indicated
Dinoflagellates are the most common of these, but we also that the interaction continued to function normally when the
found prymnesiophyte and prasinophyte algae. Further- host was held in the laboratory (Anderson. 1978; Gast.
more, it appears that most algal symbionts are actually unpubl. results). We believe that the symbiotic pattern of
genetically more similar to non-symbiotic algae than they gene expression is also maintained. Stripped extracapsular
are to other symbiotic algal lineages. This led us to wonder material was immediately placed in RNALater (Ambion)
about what made very different algae suitable as symbionts, until processed for RNA extraction (RNAqueous, Ambion).
sometimes in the same host (Gast and Caron, 2001 ), and to At least 200 individual hosts were collected and processed
hypothesize that symbiotic algae have something in com- for each subtraction analysis.
mon, which can be identified genetically, that makes them Due to the relatively small amount of symbiotic-state
acceptable as symbionts. We have undertaken comparative RNA available for our project, we used a subtraction
studies ofgene expression in a symbiotic alga to investigate method that would permit the analysis of very small
the mechanisms of symbiotic communication. amounts of starting material. Through the use of two PCR
We are studying the symbiont-host relationship between enrichment amplifications, suppression subtractive hybrid-
thedinoflagellate algaScrippsiella niitricula and the solitary ization (SSH; Diatchenko et al.. 1996) allows as little as 25
planktonic radiolarian Thalassicolla nucleata. The radiolar- ng of mRNA (or 50 ng of total RNA) to be analyzed. We
ian system allows us to compare the genes expressed in the used the Clontech PCR-Select cDNA Subtraction Kit to
symbiotic state of the algal symbiont with those expressed analyze both total RNA and mRNA from the dinoflagellate
in the free-living state of the alga in culture. The informa- symbionts (Fig. 2). To date, we have completed five sub-
tion that we obtain from this system can be used to identify tractions one from total RNA that failed, three from total
the genes necessary for maintaining the interaction, deter- RNA that were successful but yielded mostly host and
mine the timing of gene expression, examine the genomic bacterial ribosomal genes, and one from mRNA that was
characteristics associated with symbiotic-competence (why successful. We switched to analyzing mRNA because we
one alga is a good symbiont and another is not), and recovered as many ribosomal clones as symbiosis clones
examine the mechanisms of secondary and higher level when we analyzed total RNA. Contaminating ribosomal
endosymbiotic events. fragments were not unexpected since they would not be
212 R J. CAST ET AL
B
Figure2. Results from suppression subtractive hybridization. (A)cDNA synthesis fromsymbiotic (lanes 1
& 2). free-living (lanes 3 & 4). and control (lane 5) RNA. M = 1-kh ladder. (B) Final PCR products from
subtracted samples. Lane 1 symbiotic, subtracted: lane 2 symbiotic unsubtracted; lane 3 free-living sub-
tracted; lane4 free-living unsubtracted; lane 5 control subtracted; lane6 control unsubtracted. M = l-kb
ladder. cDNAs were generated and subtracted by using the PCR-Select cDNA Subtraction kit from Clontech.
Total RNA was isolated from the algae in the symbiotic state and the free-living state usingeither the Ambion
RNAEasy kit or the Epicentre MasterPure RNA Purification kit. and mRNA was recovered using the Qiagen
Oligotex mRNA kit. In both instances, the RNA from each state was separately reverse transcribed using
MMLV-reverse transcriptase (Superscript II) to generate double-stranded cDNAs. Both sets ofcDNAs were
restrictiondigestedwithRsa\togeneratefragmentswithbluntends,andthedigestedcDNAsfromthesymbiotic
state were divided into two aliquots. Each aliquot was ligated to one oftwo different adaptors supplied in the
kit. The strategy ofthe forward subtraction is to identify cDNAs up-regulated or unique to the symbiotic state
ofthealga. In thisprocess, anexcessofcDNAsfrom thefree-living state (without ligatedadapters) wasmixed
in two separate reactions with the adaptor-ligated cDNAs from the symbiotic state. The two tubes were
separately heated to95C todenaturethecDNAs,andtheneachwasallowedtohybridizeat68Cfor8h.This
step promotes hybridization between the cDNAs expressed in common in the symbiotic and free-living states.
To furtherenhance the subtraction ofcommon cDNAs, thecontentsofthe twotubes were then mixedtogether
without a second denaturation step, combined with an additional excess of heat-denatured cDNAs from the
free-living state, and hybridized overnight at 68C. PCR primers specific to the two adapters (Clontech) were
used toamplify cDNAs createdduring the subtraction that possessed one DNA strand ligated to adapter 1 and
the other DNA strand ligated to adapter 2R. Only cDNAs from the symbiotic state ofthe alga should be able
to amplify in this step, so we are enriching for the differentially regulated transcripts. The subtracted and
amplified cDNAs were cloned into pGEM-T (Promega) and used to transform JM109 competent cells (Pro-
mega). Areverse subtraction wasalsoperformedinwhichthe adaptor-freecDNA was fromthe symbiotic state
and the adaptor-ligated cDNAs had been isolated from the free-living state. These subtracted cDNAs are
therefore enriched in genes specific to the free-living state.
removed by subtraction with cultured dinoflagellate RNA. that are also present in the gene. This 30-base repeat seems
We identified 23 clones as being potentially up-regulated in to occur in other proteins as well, and may represent a
the symbiotic state of the alga. shared structural region. There are generally very few algal
Nucleotide Blast searches (Altschul et /., 1997) were or protist cDNA sequences in GenBank, so the overall lack
conducted for sequences from each of the putatively up- of homology to sequences in the database is disappointing
regulated clones that were isolated. Of the 23 selected but not unexpected. We are continuing to screen clones
clones, 8 turned out to be ribosomal genes from the host, from the final PCR products of our mRNA subtraction to
bacteria, or fungi. Twelve of the remaining clones (C4 obtain more genes unique to the symbiotic state ofthe alga.
group) were very similar toeach other, varying primarily by Because ofthe small amount of RNA available from our
small differences in length. These clones had no significant symbiotic state, it was not possible to confirm differential
sequence match in the database (Table 1). Ofthe remaining gene expression using northern blot. Instead, we used
three clones, only F7 showed significant homology to data- cDNA generated from the free-living and symbiotic state
base sequences. The homology between F7 and Cyplasin S RNAs in a dot blot format (Fig. 3). Clone F9 was included
is the result of three 30-base repeats in the clone sequence as a control for positive hybridization to the free-living
SYMBIOTICALLY EXPRESSED GENES IN DINOFLAGELLATE SYMBIONTS 213
Table 1
Listofclones recoveredandanalyzed with suppression subtractive hybridization
Clone
214 R. J. CAST ET AL.
Literature Cited ization: A method for generating differentially regulated or tissue-
specilic cDNA probes and libraries. Pnn: Null. Actiil. Sci. 93: 6025-
Altschul.S. F.,T. L. Madden,A. A. Schaffer.J. Zhang, Z. Zhang, VV. 6030.
RaMeisnl.leew2r5,g:ean3ne3dr8a9Dt-.i3oJ4n.0o2Lf.ipprmoatne.in1d9a9t7a.basGeaspepaerdchBpLrAogSrTamsa.ndNuPcSlIe-iBcLAAcSiTd:s Castd,inRo.flJa.g,elalnadtesD.frAo.mCFaorroanm.in1i9f9e6r.a anMdolReacduiloalranpah.ylMougl.enByino/f.sEynmibli.ot1i3c:
1192-1197.
Anderardsioolna,riOan.. RC.ol1l9o7s8p.haeFrianeglsotbruulcatruirse aonfda1s4yCmbiiosnotto-pbiecareivnigdecnocleonifaolr Cast, R. J., and D. A. Caron. 2001. Photosymbiotic associations in
assimilation of organic substances from its zooxanthellae. J. Ultra- planktonic foraminifera and radiolaria. Hvdrobiologiu 461: 1-7.
struct. Res. 62: 181-189. Cast, R. J., T. A. McDonnell, and D. A. Caron. 2000. srDNA based
Anderson, O. R. 1983. Radiolaria. Springer-Verlag, New York. taxonomic affinities ofalgal symbionts from a planktonic foraminifer
Armbrust, E. V. 1999. Identification of a new gene family expressed and a solitary radiolarian. J. Plivcol. 36: 172-177.
during the onset of sexual reproduction in the centric diatom TluiUis- Lee, J. J., and M. E. McEnery. 1983. Symbiosis in foraminifera. Pp.
siosira weissflogii. Appl. Environ. Microbiol. 65: 3121-3128. 37-68 inAlgalSymbiosis. E. L.Goff,ed.CambridgeUniversityPress.
Caron. D. A., and N. R. Swanberg. 1990. The ecology of planktonic Cambridge.
sarcodines. Rev. Aqitat. Sci. 3: 147-180. Michaels,A.F. 1991. Acanthananabundanceandsymbiontproductivity
Caron.D. A.,A. F. Michaels.N. R.Swanberg,and F. A. Howse. 1995. at the VERTEX seasonal station. J. Plankton Res. 13: 399-418.
Primary productivity by symbiont-bearing planktonic sarcodines Michaels,A. F., D. A.Caron,N. R.Swanberg,F.A.Howse,andC.M.
(Acantharia,Radiolaria,Foraminifera)insurfacewatersnearBermuda. Michaels. 1995. Planktonic sarcodines (Acantharia. Radiolaria. Fo-
J. Plankton Res. 17: 103-129. raminifera) in surface waters near Bermuda: abundance, biomass and
Diatchenko, L., C. Y.-F. Lau, A. P. Campbell, A. Chenchik, F. Mo- vertical flux. J. Plankton Res. 17: 131-163.
qadam. B. Huang,S. Lukyanov, K. Lukyanov, N. Gurskaya, E. D. Suanherg,N. R.,and D. A. Caron. 1991. Patternsofsarcodinefeeding
Sverdlov, and P. D. Siebert. 1996. Suppression subtractive hybrid- in epipelagic oceanic plankton. J. Plankton Res. 13: 287-312.