Elsevier

Protist

Volume 164, Issue 4, July 2013, Pages 510-527
Protist

Original Paper
Proof that Dinoflagellate Spliced Leader (DinoSL) is a Useful Hook for Fishing Dinoflagellate Transcripts from Mixed Microbial Samples: Symbiodinium kawagutii as a Case Study

https://doi.org/10.1016/j.protis.2013.04.002Get rights and content

The ability to analyze dinoflagellate lineage-specific transcriptomes in the natural environment would be powerful for gaining understanding on how these organisms thrive in diverse environments and how they form harmful algal blooms and produce biotoxins. This can be made possible by lineage-specific mRNA markers such as the dinoflagellate-specific trans-spliced leader (DinoSL). By constructing and sequencing a 5′-cap selective full-length cDNA library for a monoculture of the coral reef endosymbiotic dinoflagellate Symbiodinium kawagutii and a DinoSL-based cDNA library for a mixture of S. kawagutii and other phytoplankton, we found DinoSL in essentially all full-length cDNAs in the 5′-cap selective library. We also discovered that the DinoSL-based library contained functionally diverse transcripts all belonging to dinoflagellates with no evidence of biases toward certain groups of functional genes. The results verified that DinoSL is specific to dinoflagellate mRNAs and is ubiquitous in the dinoflagellate transcriptomes. Annotation of the unigene dataset generated from the two libraries combined indicated high functional diversity of the transcriptome and revealed some biochemical pathways previously undocumented in Symbiodinium such as an mRNA splicing machinery potentially serving both cis- and trans-splicing. The protocol will be useful for transcriptomic studies of Symbiodinium in hospite and other dinoflagellates in natural environments.

Introduction

The ability to analyze lineage-specific transcriptomes for microbes in the natural environment would be extremely useful for gaining understanding on how these microbes interact with the environment. Knowledge on in situ transcriptomics of key players in the microbial community will provide insights into how the microbial community functions in facing the environmental changes. Dinoflagellates are one of the most abundant and important groups of primary producers in the marine ecosystem. The photosynthesis of a symbiotic lineage (Symbiodinium) is essential to the growth of reef-building corals (e.g. Rowan et al. 1996), while the mixotrophic/heterotrophic dinoflagellates are important micrograzers in the coastal waters (Burkholder et al., 2008, Lin et al., 2004, Nakamura, 1999). Dinoflagellates are also the most important contributors of harmful algal blooms and algal toxins in the marine ecosystem, exerting profound impacts on fisheries industry, recreational values of coastal zones, and public health (e.g. Anderson et al. 1994). Although extensive research has been conducted on dinoflagellates, our knowledge of dinoflagellate physiology and interaction with the biotic and abiotic factors in the natural marine environment is still limited and often largely unverified, because most of the previous research had to be based on manipulation experiments (culturing in the laboratory and field) which often do not represent their natural conditions. The ability to measure in situ gene expression for a natural dinoflagellate assemblage amidst other co-existing organisms would enable us to gain understanding on the dinoflagellate-associated biological oceanographic processes in situ. For instance, analysis of expressed genes in Symbiodinium spp. living endosymbiotically in reef-building corals would provide clues as to how these coral- endosymbiotic dinoflagellates support the life of the host corals and how global warming causes coral bleaching. Similarly, through analysis of expressed genes, we can gain insights into how carbon fixation, growth, and interaction with other organisms respond to environmental changes.

The unique spliced leader found at the 5′-end of nucleus-encoded mRNAs in dinoflagellates (Lidie and Van Dolah, 2007, Zhang et al., 2007) offers a potential tool to enable dinoflagellate-specific transcriptomic studies in situ (metatranscriptomics). This 22-nucleotide (nt) spliced leader sequence (DinoSL), DCCGUAGCCAUUUUGGCUCAAG (D=U, A, or G), is transplanted from the 5′-end of a small non-coding RNA (SL RNA) to the 5′-end of each mRNA molecule. Examination of 15 representative species indicates existence of SL trans-splicing in all the species; detection of DinoSL in the transcripts of various genes from individual species indicates that trans-splicing is widespread within each dinoflagellate transcriptome (Zhang et al. 2007). Following this, DinoSL has been used to obtain full-length cDNAs from various dinoflagellates (e.g. Stüken et al., 2011, Zhang and Lin, 2009). The results so far suggest that the presence of DinoSL can be used as a demarcating criterion to distinguish dinoflagellates from non-dinoflagellate organisms (Zhang and Lin 2008). The utility of DinoSL in studying dinoflagellate gene expression in mixed samples (metatranscriptomics) has since been explored in Pfiesteria piscicida-Rhodomonas sp. grazer-prey system (Lin and Zhang 2010) and natural aquatic ecosystems (Lin et al. 2010). In both cases, results show that the cDNA prepared using DinoSL as the selective 5′- primer is highly dinoflagellate specific and functionally diverse. The metatranscriptomics study (Lin et al. 2010) resulted in the uncovering of the long missed histones, an apparently complete set of ribosomal proteins, and unsuspected proteorhodopsin.

Despite the initial success in employing DinoSL in dinoflagellate metatranscriptomics, the potential of the DinoSL-based technique remains to be systematically evaluated. The usefulness of the technique hinges on two critical requirements, i.e. that DinoSL should be 1) universal for all dinoflagellate nuclear gene transcripts and 2) specific for dinoflagellates. To address these two issues, in this study, we constructed and sequenced a full-length cDNA library using a non-PCR based, 5′-cap selective method for a monoculture of coral-endosymiont Symbiodinium kawagutii originally isolated from a reef building coral (SymkaFL library). We also constructed a cDNA library for mixed RNA from S. kawagutii and other phytoplankton using DinoSL-based method (SymkaSLR3 library). Results from cloning, sequencing, and bioinformatics analysis of both libraries verified the ubiquity of DinoSL in nucleus-encoded mRNAs of S. kawagutii and its specificity for retrieving dinoflagellate mRNAs from multi-species plankton community RNA.

Section snippets

DinoSL Distribution in SymkaFL Library

From the test sequencing of the SymkaFL library, we got 178 good-quality cDNA sequences. From the high-throughput Sanger sequencing of the ∼11,000 cDNA clones, we obtained 15,479 reads with read-length >100 bp. The 5′-end reads of the cDNAs generally had better quality than the 3′-end ones, largely due to the existence of long polyA tails (25 to >100 bp) in the latter. After quality filtration, we obtained 7,358 cDNA sequences with good-quality 5′-end, which along with the 178 clones from the

Discussion

The kit we used to generate the full-length cDNA library SymkaFL has been proven to be effective and efficient. Many other reported full-length cDNA construction methods usually involve PCR amplification and adaptor ligation (Sugahara et al. 2001 and references therein), or have relatively complicated steps (e.g. Carninci et al., 1996, Carninci et al., 1998, Carninci et al., 2000), which often yield the full-length cDNAs missing the first several base pairs of the very 5′-end of the transcripts

Methods

Algal cultures: All algal cultures used in this study were purchased from the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA; formerly the Provasoli-Guillard National Center for Culture of Marine Phytoplankton, CCMP). Symbiodinium kawagutii strain CCMP2468 was chosen because it was isolated from the scleractinian (stony) coral and its host species (Montipora verrucosa) was clearly identified, and the location of source of this alga was also clear: 21.25000N 158.0000W,

Acknowledgement

This project was supported by the US National Science Foundation Small Grant for Exploratory Research (SGER) grant OCE-0854719 and Microbial Genome Sequencing Program (MGSP) grant EF-0626678.

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    1

    Corresponding authors; fax +1 860 405-9153

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    Current address: cSynthetic Genomics, Inc. 11149 North Torrey Pines Road, La Jolla, California 92037, USA.

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