Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum

7/25/2019 Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum

1/131SCIENTIFICREPORTS| 5:18708 | DOI: 10.1038/srep18708

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Characterization of marinediatom-infecting virus promoters inthe model diatom PhaeodactylumtricornutumTakashi Kadono1, Arisa Miyagawa-Yamaguchi1,, Nozomu Kira2, Yuji Tomaru3,

Takuma Okami

1

, Takamichi Yoshimatsu

1

, Liyuan Hou

4

, Takeshi Ohama

4

, Kazunari Fukunaga

1

,Masanori Okauchi5, Haruo Yamaguchi1, Kohei Ohnishi6, Angela Falciatore7& Masao Adachi1

Viruses are considered key players in phytoplankton population control in oceans. However,

mechanisms that control viral gene expression in prominent microalgae such as diatoms remain largely

unknown. In this study, potential promoter regions isolated from several marine diatom-infecting

viruses (DIVs) were linked to the egfpreporter gene and transformed into the Pennales diatom

Phaeodactylum tricornutum. We analysed their activity in cells grown under dierent conditions.

Compared to diatom endogenous promoters, novel DIV promoter (ClP1) mediated a signicantly

higher degree of reporter transcription and translation. Stable expression levels were observed in

transformants grown under both light and dark conditions, and high levels of expression were reported

in cells in the stationary phase compared to the exponential phase of growth. Conserved motifs in the

sequence of DIV promoters were also found. These results allow the identication of novel regulatory

regions that drive DIV gene expression and further examinations of the mechanisms that control virus-

mediated bloom control in diatoms. Moreover, the identied ClP1 promoter can serve as a novel tool formetabolic engineering of diatoms. This is the rst report describing a promoter of DIVs that may be of

use in basic and applied diatom research.

Diatoms are one o the most effective and diverse unicellular photosynthetic eukaryotes, with perhaps as many as200,000 extant species ound in aquatic environments1. Tere is broad interest in diatoms due to their substantialcontributions to global carbon cycling and oxygen production2, their complex evolutionary background as second-ary endosymbionts3, and their unique capacities to produce silica-based cell walls 4. Moreover, diatoms present greatpotential as a source o beneficial chemicals or use in human activities because they produce biouel precursorssuch as atty acids and hydrocarbons that may prove useul in solving ecological problems such as the energy crisis 5.o understand diatom biology and the means o diatom exploitation in biotechnology, novel genomic resourcesand molecular tools have been developed in recent years. Databases o genome sequences and the expressedsequence tag (ES) o the model diatoms Pennales Phaeodactylum tricornutumand Centrics Talassiosira pseu-donanahave been released or public use, allowing the identification o distinct metabolic eatures in diatoms69.Recently, the genomic sequences o other diatoms such as Pseudo-nitzschia multiseries(http://genome.jgi-ps.org/Psemu1/Psemu1.home.html ) and Fragilariopsis cylindrus(http://genome.jgi-ps.org/Fracy1/Fracy1.home.html)

1Laboratory of Aquatic Environmental Science (LAQUES), Faculty of Agriculture, Kochi University, Otsu-200,Monobe, Nankoku, Kochi 783-8502, Japan. 2The United Graduate School of Agricultural Sciences, Ehime University,3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan. 3National Research Institute of Fisheries and Environmentof Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan. 4School ofEnvironmental Science and Engin eering, Kochi University of Technology, Tosayamada, Kami, Kochi 782-8502,Japan. 5National Research Institute of Aquaculture, Fisheries Research Agency, 422-1 Nakatsuhamaura, Minami-ise,Mie 516-0193, Japan. 6Research Institute of Molecular Genetics, Kochi University, Otsu-200, Nankoku, Kochi 783-8502, Japan. 7Sorbonne Universits, UPMC Univ Paris 06, Institut de Biologie Paris-Seine, UMR 7238, F-75006 Paris,France; CNRS, UMR 7238, F-75006 Paris, France. Present address: Center for Innovative and Translational Medicine,Kochi University Medical School, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan. Correspondence and requestsfor materials should be addressed to M.A. (email: [email protected])

Received: 30 June 2015

Accepted: 24 November 2015

Published: 22 December 2015

OPEN
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2SCIENTIFICREPORTS| 5:18708 | DOI: 10.1038/srep18708

have also been made available on the U.S. Department o Energy Joint Genome Institutes website (http://jgi.doe.gov/). In addition, methods o genetic transormation via biolistic bombardment and/or electroporation havebeen developed or several diatom species including Pennales P. tricornutum1016, Cylindrotheca fusiformis17,18,Navicula saprophila19, Pseudo-nitzschia arenysensis20, Pseudo-nitzschia multistriata20, Centrics Cyclotella cryp-tica19, Talassiosira pseudonana21, Talassiosira weissflogii22, Chaetocerossp.23, Cha. gracilis24, and Fistuliferasp.25.Moreover, episomal vector technology based on yeast-derived sequences has recently been developed or P. tri-cornutumand Talassiosira pseudonana26.

However, many o the processes that control diatom biology and growth in marine environments remain to beelucidated. Recently, it has been proposed that marine viruses play a key role in controlling the blooms o diatom

populations

27,28

. Viruses may also influence the compositions o marine communities and may act as a majororce behind biogeochemical cycles29,30. Tus ar, several marine diatom-inecting DNA/RNA viruses that causethe lysis o host diatoms have been isolated rom both Centrics and Pennales diatoms28, or example, the Cha.debilis-inecting DNA virus (CdebDNAV)31, the Cha. lorenzianus-inecting DNA virus (ClorDNAV)32, and theTalassionema nitzschioides-inecting DNA virus (nitDNAV)33. In silicoanalysis o these genomes has revealedthe presence o putative open reading rames (ORFs) such as the replication-associated protein (VP3) gene, thestructural protein (VP2) gene, and genes o unknown unction28,32.

o determine the contributions o marine diatom-inecting viruses (DIVs) to phytoplankton biology and ecol-ogy, it is important to urther examine the DIV genome and to identiy mechanisms that control viral gene expres-sion through the characterization o viral promoters. Moreover, DIV promoter characterization may also advancethe manipulation and modulation o gene expression in diatoms. Tus ar, this strategy is used primarily throughendogenous promoters such as the gene-encoding ucoxanthin chlorophyll a/c-binding protein (FCP) gene (fcp,now called Lhcf), which encodes members o the light harvesting complex superamily10,18,21,24, rustulin17, nitratereductase (NR)18,21,24, acetyl-CoA acetyltranserase24, acetyl-CoA carboxylase19, -carbonic anhydrase 1 (CA1)34,long-chain atty acyl-CoA synthetase24, -tubulin24, AP synthase subunit C24, and elongation actor 2 (EF2)16.

O the endogenous promoters, thefcppromoter has been requently used in biotechnological applicationsinvolving diatoms3539. However, reports also suggest thatfcppromoter activity may not be strong enough to over-express introduced genes and may promote significant increases in target products35,39. Other studies report thattransgene expression ollows a circadian pattern due to the presence o light-responsive cis-regulatory elementsin thefcppromoter16,40,41. In addition, in some cases, diatom endogenous gene promoters drive species-specifictransgene expression and do not unction in different diatom species18,20,22. Tese issues considerably limit theapplications o diatom transormation techniques, potentially reducing the utility o diatoms in basic and appliedresearch. o address these problems, the development o promoters that stably induce high-level gene expressionis anticipated, which should result in the creation o large accumulations o introduced gene transcripts and pro-teins. Promoters rom viruses that inect plants or mammals have generally been used to transorm a wide range ohigher plants and mammals, allowing the constitutive and strong expression o introduced genes. For example, thecauliflower mosaic virus 35S (CaMV 35S) and cytomegalovirus (CMV) promoters are known to efficiently acilitateplant and mammalian transormations, respectively42,43. However, only a ew reports ocus on the transormationo diatoms through the use o CaMV 35S and CMV promoters19,44in P. tricornutum,and the same promoters donot seem to be effective in other species, such as the Centrics diatom Cyc. cryptica19.

DIV promoters may enable us to solve the above-described limitations in the bioengineering o diatoms aswell as to iner the ecological roles o DIVs in marine ecosystems. Tus, in this study, potential promoter regionslocated upstream o ORFs, such as the replication-associated protein (VP3) gene and the structural protein(VP2) gene in the DIV genome (Fig. 1a), were isolated. DIV promoter-driving gene expression capacities in thePennales diatom P. tricornutumwere evaluated and compared to those o the endogenousfcppromoter (Fig. 1b).We then examined the stability o DIV promoters in cells that were cultured under various growth conditions.Te effects o the growth stage, the nutrient concentrations in media, and the photoperiods on viral promoteractivity were investigated. Te application o viral promoters to a Centrics diatom and to unicellular green algawas also examined. We then discussed the structure o a DIV promoter to show how gene expression mechanismsmay play a key ecological role in the control o diatom blooms and how the DIV promoter may be used in appliedresearch on diatoms.

ResultsIsolation of potential DIV promoter regions. Potential DIV promoter regions were determined rom thegenome sequences o CdebDNAV and nitDNAV via in silicoanalyses. Te ORFs o the two DIVs were identified

using an ORF finder (National Center or Biotechnology Inormation, http://www.ncbi.nlm.nih.gov/gor/). Tree(VP1, VP2, and VP3) and two (VP2 and VP3) ORFs were identified in the CdebDNAV and nitDNAV genomes,respectively. In regard to ClorDNAV, the our ORFs (VP1, VP2, VP3, and VP4) were previously described byomaru et al.32(Fig. 1a). Regions (approximately 500 bases long) upstream o the translation initiation codon(AG) o a putative ORF in the DIV genome were predicted to be potential promoter regions (able 1, Fig. 1a,and Supplementary Fig. 1).

Construction of transformation vectors. Te potential promoter region o each ORF was amplified viapolymerase chain reaction (PCR) using DIV genomic DNA and specific primers (Supplementary able 1). Inaddition to the potential DIV promoters, the endogenousfcppromoter o P. tricornutumand some extrinsic pro-moters, such as CaMV 35S, CMV, and the nopaline synthase gene (nos) promoter oAgrobacterium tumefaciens,were amplified rom the genomic DNA o P. tricornutumand rom commercially available vectors, respectively,

via PCR amplification (Supplementary able 1). o investigate the activity o each tested promoter, we constructeda double-cassette transormation vector (Fig. 1a) that contained the enhanced green fluorescence protein (eGFP)gene (egfp) driven by each tested promoter and the antibiotic-resistant gene Sh bledriven by the Cyl. fusiformisFCP
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A-1A gene promoter (termed CffcpA pro.). We also constructed a single-cassette transormation vector containingSh bledriven by each tested promoter (Supplementary Fig. 2) and natdriven by DIV promoters, such as CdP1 andClP1 (Supplementary Fig. 3), to investigate transormation efficiency.

Figure 1. Schematic diagram for the evaluation of promoter activity. (a) Outline o the construction otransormation vectors and transormations. Afer predicting putative ORF positions32, upstream regions othe ORFs were determined as potential promoter regions. Potential promoter regions amplified by PCR wereused to construct the transormation vectors. Te double-cassette vector containing the reporter gene egfpdriven by each tested promoter and the antibiotic-resistant gene Sh bledriven by the promoter region o theucoxanthin chlorophyll a/c-binding protein (FCP) A-1A gene derived rom Cyl. fusiformis(termed CffcpApro.) were constructed. (b) Assessment o promoter activity. Promoter activity was determined by averaging theratios o egfpmRNA transcript levels to those o Sh blemRNA transcripts in ten transormants to minimize theeffects o copy numbers on the expression o transgenes. Tese transormants were also used to investigate eGFPprotein expression patterns. CffcpA ter.: terminator region o the FCP A-1A gene derived rom Cyl. fusiformis.Te structure o the ClorDNA genome was modified rom omaru et al.32. *For the transormation vector o thenitrate reductase gene promoter, we used pNICgp18(Supplementary Fig. 5a).
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Conrmation of the introduced gene and promoter in the transformants. Colonies were obtainedrom a solid /2 medium containing 500 g ml1antibiotics afer its transormation via microparticle bombardmentusing the double-cassette transormation vectors. o confirm the introduction o the two cassettes, a genomic PCRanalysis was perormed using different primer sets (Supplementary ables 1 and 2 and Supplementary Figs 4a and5a) and using the colony-orming cells as templates. We obtained more than ten transormants with the expectedDNA ragment length and containing egfpwith the tested promoter, with the exception o transormants that were

transected with pNICgp18. Six transormants that had been transected with pNICgp18possessed DNA rag-ments o the expected length, including egfpunder the control o the NR gene (nr) promoter rom Cyl. fusiformis(termed Cnr pro.) and the Sh blegene under the CffcpA pro. A typical electrophoretogram o the amplicons ineach transormant is shown in Supplementary Figs 4b and 5b. en transormants with each o the tested promotersand six transormants with Cnr pro. were analysed urther.

Quantitative analysis of promoter activity. Variations in the levels o egfpmRNA transcripts normalizedto the internal standard gene (ribosomal protein small subunit 30S gene, rps) mRNA transcripts were detected usingquantitative reverse transcription-PCR (qR-PCR) in 610 transormants with the egfpdriven by each o the testedpromoters (Supplementary Fig. 6). Variations in the levels o Sh blemRNA transcripts driven by the diatom endog-enous promoter (CffcpA pro.) that had been normalized to rpswere also ound amongst the 610 transormants(Supplementary Fig. 6). Beore determining the activity o each promoter, the egfpmRNA transcript levels in eachtransormant were divided by those o the Sh blemRNA transcripts. Tis method has been used to compare geneexpression levels in independent lines and to prevent misinterpretation due to the presence o different transgenecopy numbers and possible site integration effects. Figure 2shows averages and ranges o promoter activity in the

610 independent transormants with egfpdriven by each tested promoter. We compared the activity o each testedpromoter with that o an endogenous promoter PtcpA pro. because the PtcpA pro. has generally been used orthe transormation o the marine diatom P. tricornutum10,11,13,39. Average promoter activities in the transormantswith Cnr pro. (P=0.010), nP1 (P=0.012), nP2 (P=0.016), the CaMV 35S promoter (P=0.014), the CMVpromoter (P=0.021), and the nospromoter (P=0.034) (average: 0.08330.499) were ound to be significantlylower than those in the transormants with PtcpA pro. (average: 2.08, range: 0.02965.97) (Fig. 2). By contrast,the average promoter activity in ClP1 (average: 10.3, range: 2.0625.1, P=0.0058) was ound to be approximatelyfive times higher than that in PtcpA pro. (Fig. 2). Te average CdP1 (average: 1.99, range: 0.09177.34, P=0.93)and ClP2 activity (average: 3.09, range: 0.35221.4, P=0.64) levels showed no significant differences rom theaverage PtcpA pro. activity level (Fig. 2). Te maximum activity levels in the ten transormants o CdP1, ClP1,and ClP2 were higher than that ound in the transormants with PtcpA pro. (Fig. 2).

Eect of culture conditions on DIV promoter activity. Promoter activities are known to respond tochanges in environmental and intracellular conditions. o explore the effects o culture conditions and growthphases on the activity o the DIV promoter (ClP1) that showed the highest levels o activity (Fig. 2), the abundance

o egfpmRNA levels in two independent ClP1 transormants was analysed using qR-PCR under various cultureconditions (Fig. 3). Under a low nutrient culture condition (/10 medium), the abundance o egfpmRNA transcriptsdriven by ClP1 was almost identical to that reported or the standard nutrient culture condition (/2 medium)(Fig. 3b). Te abundance o egfpmRNA transcripts in the stationary phase seemed to be higher than that oundor the log phase under both low and standard nutrient culture conditions ( Fig. 3b). ClP1 can effectively drivethe expression o egfpmRNA transcripts in both light and dark conditions during the stationary phase (Fig. 3b).

Levels of eGFP uorescence. Te fluorescence o the eGFP reporter in the different transgenic linesdescribed above was quantified via flow cytometry. Figure 4shows normalized eGFP fluorescence levels or cell sizesin the 910 independent transormants with egfpdriven by each tested promoter. Te average eGFP fluorescencelevels in the transormants containing nP1 (P=0.0071), nP2 (P=0.012), the CaMV 35S promoter (P=0.0063),the CMV promoter (P=0.021), and the nospromoter (P=0.012) (average: 2.533.24) were ound to be signifi-cantly lower than those o the transormants containing PtcpA pro. (average: 6.10, range: 2.2111.6) (Fig. 4). Testatistical analysis results show that the average eGFP fluorescence levels in the transormants containing ClP1

Promoter Source organism/virus Promoter associated geneAmplifiedsize (bp) Ref.

CdP1 Chaetoceros debilis- inecting DNA virus (CdebDNAV) Putative repl ication-associated protein (VP3) gene 477 31

ClP1 Chaetoceros lorenzianus-inecting DNA virus (ClorDNAV) Putative replication-associated protein (VP3) gene 502 32

ClP2 Chaetoceros lorenzianus-inecting DNA virus (ClorDNAV) Putative structural protein (VP2) gene 474 32

nP1 Talassionema nitzschioides-inecting DNA virus (nitDNAV) Putative replication-associated protein (VP3) gene 424 33

nP2 Talassionema nitzschioides-inecting DNA virus (nitDNAV) Putative structural protein (VP2) gene 424 33

PtcpA pro. Phaeodactylum tricornutum Fucoxanthin chlorophylla/c-binding protein A gene 444 10

Cnr pro. Cylindrotheca fusiformis Nitrate reductase gene 774 18

CaMV 35S pro. Cauliflower mosaic virus 35S gene 454 44,81

CMV pro. Cytomegalovirus Immediate early promoter regulatory gene 742 44,82

nospro. Agrobacterium tumefaciens Nopaline synthase gene 338 81

Table 1. Promoters used in this study.
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(average: 13.2, range: 2.5225.0, P=0.020) were significantly higher than that o the transormants containingPtcpA pro. Te average eGFP fluorescence levels in the transormants with CdP1 (average: 5.27, range: 2.3816.7,P=0.63) and ClP2 (average: 7.45, range: 2.3731.8, P=0.67) showed no significant differences rom that in thetransormants with PtcpA pro. (Fig. 4). Te maximum levels o eGFP fluorescence in the transormants o CdP1,ClP1, and ClP2 were ound to be higher than that in the transormants o PtcpA pro. (Fig. 4). Te eGFP fluores-cence levels without normalization were similar to those observed by normalizing fluorescent signals with cellsizes (Supplementary Fig. 7).

Figure 2. Relative activities of various promoters including DIV promoters in transformants. enindependent transormants or each promoter were analysed. Te promoter activity levels were determinedby dividing egfpmRNA levels by Sh blemRNA levels. Te circles indicate the mean triplicate measurements othe independent transormants. Te diamonds denote the average values o the six transormants rom Cnrpro. or o the ten transormants rom the other promoters. Pro.-less denotes cases in which no promoter was

linked to the egfpgene in the transormation vector (negative control). PtcpA pro. shows the positive controlin the transormation vector egfpgene driven by PtcpA pro. Asterisks indicate the presence o a statisticallysignificant difference derived rom the PtcpA pro. transormants (**P


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Transformation eciency using DIV promoters. We also assessed whether the different promotersidentified in this study may affect transormation efficiency levels in P. tricornutum. o this end, single-cassette

vectors containing the selective marker, Sh ble, driven by PtcpA pro. or ClP1 (Supplementary Fig. 2), were intro-duced into P. tricornutumvia microparticle bombardment. Te average transormation efficiencies o PtcpApro. and ClP1 reached 13 and 13.5 transormants per 108cells (n=4), respectively. Statistical analysis based onthe number o colonies ormed showed that the transormation efficiency using PtcpA pro. and ClP1 were notsignificantly different.

Application of diatom-infecting DIV promoters to other algae. We investigated whether the DIVpromoter may also be applied to a species o Centrics diatoms such as Chaetocerossp. Strain CCK09. o this end,we assayed transormation efficiency levels in this species using single-cassette vectors with the antibiotic-resistantgene natdriven by the DIV promoters CdP1 and ClP1 (Supplementary Fig. 3). Te average transormation efficien-cies o CdP1 and ClP1 transected to Chaetocerossp. were 4 and 2 transormants per 108cells (n=3), respectively.

Te activity levels o the DIV promoters in the unicellular green alga Chlamydomonas reinhardtii, a speciesthat is phylogenetically different rom the diatoms, were also investigated. Single-cassette vectors, in which Sheblewas driven by a DIV promoter (or example, CdP1 and ClP1) or the Chlamydomonas endogenous promoterrbcS2(pSP108)45, were used or the assay. A standard electroporation method46and wall-less Chlamydomonasstrain CC503 were used or the transormation. Te transormation efficiency o pSP108 was recorded at 168transormants per 2.5107cells (n=14). By contrast, the average transormation efficiency ound via CdP1and ClP1 were recorded at 0 and 0.14 transormants per 2.5107cells (n=14), respectively. Te transormationefficiency levels obtained using viral promoters were not significantly different rom those o the promoter-less

vector (average: 0 transormant, n=14).

In silicoanalyses of DIV promoter regions. We searched or potential cis-regulatory elements in the DIVpromoters using the PLACE47and PlantCARE48programs. Both programs revealed the existence o cis-regulatoryelements o Myb49, bZIP50, CCAA-binding51, homeobox52, and E2F-DP53-related transcription actors (Fs)(which have already been ound in genomic sequences o the Pennales P. tricornutumand the Centrics Talassiosira

pseudonana54) in the DIV promoters (Supplementary Fig. 1). Te PlantCARE program 48also showed that all theDIV promoter regions include a plant-type light-responsive cis-regulatory element (Supplementary Fig. 1). oidentiy other regulatory sequences that are conserved amongst DIV and endogenous promoters, we examinedthe requency o consensus F binding sites (FBSs), which are available in the JASPAR database55, throughpromoterome analyses41using oPOSSUM version 356. Te single site analysis (SSA) algorithm o oPOSSUM ver-sion 3 ailed to detect any FBS consensus between the DIV promoters and the potential promoter regions o P.tricornutum. Using the FBS cluster analysis (CA) algorithm available in oPOSSUM version 3, sequence motiso some insect- and vertebrate-type homeobox binding sites were identified in the five DIV promoters and in the99.9% potential promoter region o P. tricornutum(12,234 o 12,237 sequences, Z-score: 14.406, Fisher score:0.001) (Supplementary Fig. 1 and Supplementary data). o obtain inormation on specific mechanisms that driveexpression rom DIV promoters, we attempted to find conserved motis among the DIV promoter regions (CdP1,ClP1, and ClP2) using a consensus moti-finding algorithm (CONSENSUS) available through Melina II57and using

Figure 4. Relative abundance of eGFP protein translated from transcripts driven by various promotersin transformants using flow cytometry. en independent transormants derived rom various promoterswith the exception o the CaMV 35S promoter were analysed. For the CaMV 35S promoter, nine independenttransormants were analysed. For normalization purposes, the eGFP fluorescence was divided by the cell size,which was estimated based on values o orward scatter areas (FSC-A) obtained via flow cytometry. Te circlesdenote the mean value or approximately 10,000 cells o independent transormants. Te diamonds denote

the average values o the transormants. Asterisks indicate statistically significant differences derived rom thePtcpA pro. transormants (**P


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deault parameters. Te GGCAGGCG sequence was detected in CdP1, ClP1, and ClP2 through the CONSENSUSalgorithm. In the sense strand o ClP1, there are three tandem repeats o the GGCAGGCG moti ordered rom5to 3(Supplementary Fig. 1). In ClP2, there are three tandem repeats, with the direction o the motis orderedrom 3to 5. Only one moti o the 5to 3direction was ound in the sense strand o CdP1 (Supplementary Fig. 1).

DiscussionTis is the first report describing the unctional characterization o various promoters identified and isolated romDIVs. We tested viral promoter activity levels in diatom cells transormed with specific vectors, in which the viralpromoter drove the expression o an eGFP reporter gene. Te activity o each tested promoter was estimated rom

the relative abundance o the eGFP gene transcript relative to that o the antibiotic-resistant gene driven by theendogenousfcppromoter (Fig. 1b). In turn, we identified the strongest promoters and overcame issues o geneexpression variability between independent transgenic lines that can be due to differences in the copy numberso transgenes integrated in a host genome58and rom the positioning o the introduced vector in the genome, i.e.,the position effect59. Similar variations have been reported during the characterization o several promoters 60.Moreover, variations in fluorescence levels o the eGFP protein were used to assess possible variations in transla-tional efficiency levels in the different transgenic lines. Tese analyses allowed us to show or the first time that theisolated DIV promoters are unctional in P. tricornutum. In particular, we ound that the ClP1 promoter o a putativereplication-associated (VP3) gene derived rom ClorDNAV32drives the stable expression o the reporter in cellsgrown in both light and dark conditions, and it shows higher levels o activity than a diatom endogenous promoter.

Interestingly, the activity levels o viral promoters may be heavily modulated in the growth phase. InSchizosaccharomyces pombe, gene expression mediated by the CaMV 35S promoter is induced during the log phaserather than during the stationary phase61. In our study, reporter expression controlled by ClP1 was induced at thestationary phase rather than at the log phase o P. tricornutumgrowth. In accordance with our data, a study on therelationship between the Cha. tenuissimus-inecting DNA virus (CtenDNAV) type II and the growth phase o Cha.

tenuissimusshowed that when CtenDNAV type II is inoculated into host cells at the stationary phase, a decrease inhost cell quantities occurred one day afer inoculation28. However, in host cells at the log phase, a decrease in hostcell quantities occurred only afer the cells reached the stationary phase28. Te inectivity o CtenDNAV type I wasalso considerable when the host was at a stationary phase62. Moreover, an active replication o the viral genomeo CtenDNAV type I was observed afer the host cells reached the stationary phase62. aken together, these resultssuggest that DIV promoter activation in a host diatom at the stationary phase may acilitate diatom lysis. Althoughgene expression regulation by DIV promoters in P. tricornutumis not ully understood, these findings suggest thata stronger understanding o the mechanisms that control DIV gene expression may lead to the development o astronger understanding o diatom population control processes by DIVs in oceans.

Our assessment o viral promoter activity also allowed us to identiy ClP1 as a novel DIV promoter thatdrives strong and stable gene expression in diatoms. ClP1 serves as a useul tool that may be exploited in diatombiotechnology applications or the high-level production o useul materials and or gene unctional studies ingenetically engineered diatoms63,64. Te activity o the stable endogenous EF2 gene promoter recently isolatedrom P. tricornutum16drove the expression o a transgene 1.2-old stronger than that driven by thefcppromoterin light conditions16. In comparison, egfpmRNA levels normalized to rpsin ClP1 transormants were ound to

be 3.3 times stronger than those o PtcpA pro. transormants (Supplementary Fig. 6). Tis result suggests thatClP1 may induce a stronger expression o transgenes than the EF2 gene promoter throughout a photoperiod. Inaddition, ClP1 activity remains stable in low nutrient culture conditions. A combination o stable ClP1 activitylevels in light/dark cycles under low nutrient conditions and high-level ClP1 activity levels in the stationary phasemay have value in biotechnological applications.

In discussing whether DIV promoters may be applied to various diatom species, it is important to considerthe phylogeny o diatoms composed o the two orders Pennales and Centrics1,65. In this study, the finding thatClP1 can be applied to both the Centrics diatom Chaetocerossp. and the Pennales diatom P. tricornutumsuggeststhat the DIV promoter region contains conserved regulatory elements that may be recognized via conserved Fsidentified in Pennales and Centrics species54. Considering the phylogenetic relationship, conservation levels inF amilies between the Pennales and Centrics diatoms, and our results, DIV promoters including ClP1 may beapplied to various diatom species.

Both DIV promoters CdP1 and ClP1 are derived rom Chaetocerosspp., although the transormation efficiencyo P. tricornutumwas ound to be higher than that o Chaetocerossp. It was reported that transormation efficienciesobtained by multi-pulse electroporation using the endogenousfcppromoter in P. tricournutum13were approx-

imately 10 times higher than those in Chaetoceros gracilis24, suggesting that the transormation efficiency levelsin Chaetocerosare lower than those o Phaeodactylum, independent o the promoter used to drive the transgeneexpression. By contrast, ClP1 cannot unction in green algae Chl. reinhardtii, suggesting that regulatory elementsin promoter regions and transcriptional regulators differ between the Heterokont and Plantae lineages.

Via an in silicoanalysis o the DIV promoters, we examined conserved motis that may be involved in the ini-tiation o diatom virus gene expression. opically, eukaryotic promoters consist o two regions: a core region andan upstream regulatory region66. Te core region consists o an approximately 50100 base pair (bp) sequence. Itincludes the transcription start site, known as an initiator (Inr), and flanking sequences such as the AA box67.Te regulatory region includes cis-regulatory elements that control gene expression through interaction withFs66. Cis-regulatory elements mediate inducible, tissue specific, or development stage-specific gene expression66.In terms o Inr sequences, an Inr consensus has been ound or some eukayotes6769. Based on the transcriptionstart site o P. tricornutum, the conserved sequence flanking the transcription start sites (CAY+1A, degeneratebases are described according to the IUPAC nucleotide code) is reported rom the upstream regions o some FCPgenes70. In addition, 2 to +2 sequences relative to the transcription start site o the CA1 gene (ca1) in P. tricor-nutumwere ound to be CACA34. We ocused on sequences surrounding CAYA located in the upstream DNA
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sequence o FCP genes, and other endogenous genes o diatoms were examined via visual observation. We ounda CAH1W Inr-like sequence located between bp 2661 upstream o the translation site in P. tricornutumFCPgenes such as thefcpC,fcpDandfcpE, and P. tricornutumCA1 genes (Supplementary able 3). In the tested DIVpromoter regions, we also ound this Inr-like sequence between bp 1465 upstream o the proposed translationsite (CdP1: 14 and 35 upstream, ClP1: 36 and 61 upstream, ClP2: 40 upstream, nP1: 35 upstream, and nP2: 65upstream) (Supplementary able 3 and Supplementary Fig. 1). o determine the universality o CAH+1W inthe 5-flanking sequences (80 bases) o the P. tricornutumgenes, we searched or CAH+1W in 12,237 o the5-flanking sequences and ound CAH+1W in 8,306 o the 12,237 sequences (67.9%) o the potential promoterregion o the P. tricornutumgenes. From these findings, we present a new consensus moti CAH+1W, which

serves as a potential Inr-like sequence in the diatom.While identiying other regulatory sequences that are conserved amongst DIV and endogenous promoters,we ound sequence motis o some insect- and vertebrate-type homeobox binding sites in the five DIV promotersand in the 99.9% potential promoter region o P. tricornutumvia a promoterome analysis41. Tis finding suggeststhat these motis may play a role in P. tricornutumtranscription.

In addition to revealing the existence o canonical cis-regulatory elements, several studies have reported on theexistence o other cis-regulatory elements in diatom endogenous gene promoters. Tree putative cis-regulatoryelements rom the ca1promoter o P. tricornutum,which responds to changes in CO2and cAMP concentrations,have been ound34,71. Te two iron-responsive conserved cis-regulatory elements were ound in upstream DNAsequences o iron starvation-induced protein genes in P. tricornutum72. Te expression levels o FCP genes wereound to oscillate in a circadian manner in response to a plant-type light-responsive cis-regulatory element inthefcppromoter40,41,7375. Cis-regulatory elements o CO2/cAMP

71or iron72were not ound in any o the DIVpromoter regions by visual observation. Te PlantCARE program48showed that all the DIV promoter regionspossess a plant-type light-responsive cis-regulatory element. However, ClP1 activity was unaffected by light anddark cycles (Fig. 3), suggesting that the identified elements do not respond to light. EF2 gene promoter activity was

also ound to be stable in P. tricornutumthroughout light and dark cycles. In the sequence o the promoter regiono the EF2 gene, a plant-type light-responsive cis-regulatory element was also detected through the PlantCAREprogram48, supporting the notion that plant-type light-responsive cis-regulatory elements cannot respond to lightin P. tricornutum.

Te GGCAGGCG sequence was detected in CdP1, ClP1, and ClP2 via the CONSENSUS algorithm. Tequantity, direction, and proximity o this consensus moti varied among these promoters. ClP1, which showed thehighest degree o promoter activity (Fig. 2), possessed three tandem repeats o the GGCAGGCG moti in thesense strand in the 5-to-3direction. For ClP2, which showed modest activity levels (Fig. 2), a 3-to-5directionwas ound, even when three tandem repeats were present in the promoter region (Supplementary Fig. 1). Only onemoti was ound in the sense strand o CdP1, which also showed modest activity levels (Fig. 2and SupplementaryFig. 1). However, one and three GGCAGGCG sequences were ound in the antisense strands o the nosand CMVpromoters, respectively, which showed their very weak activity levels (Fig. 2). In the latter case, the three motis arescattered across the promoter region (Supplementary Fig. 8). Tese findings suggest that the quantity, direction,and proximity o the novel consensus moti may heavily affect the stability o promoter activity levels though light/dark cycles and o high-level promoter activity levels during the stationary phase in P. tricornutum. In addition

to the motis described above, unknown novel motis may exist in the promoter regions o DIVs such as ClP1.In summary, we have isolated promoters rom several marine DIVs or the first time. Te activity o the DIVpromoter o ClP1 in the transormants was higher in the stationary phase than it was in the log phase. Comparedto a diatom endogenous promoter, the mediated ClP1 showed a significantly higher level o reporter transcriptionand translation. Conservative motis in sequences o the DIV promoters were ound. Finally, our findings mayhelp elucidate the mechanism o DIV gene expression, which may play a key role in the ormation/decay o diatomblooms in oceans. In addition, the stable and high ClP1 promoter activity levels reported should prove useul orthe metabolic engineering o diatoms.

MethodsAlgal culture. Te Pennales P. tricornutumBohlin (National Research Institute o Aquaculture, FisheriesResearch Agency, Japan, strain NRIA-0065), the Centrics Chaetocerossp. (Japan Marine Science and echnologyCenter, Japan, strain CCK09)23, and unicellular green alga Chl. reinhardtii(strain CC-503) were used in this study.P. tricornutumwas grown at 20 C under a light/dark cycle o 12 h light and 12 h dark with ca. 90mol photons m2s1in liquid /2 medium76. Chaetocerossp. was grown at 20 C under continuous illumination at 300 mol photons

m2s1in SWM3 medium77. Te Chl. reinhardtiistrain CC-503 was provided by the ChlamydomonasResourceCenter (University o Minnesota). Te cells were cultivated mixotrophically at 25 C in ris-acetate phosphate(AP) medium78under constant white fluorescent light conditions (84mol photons m2s1) with gentle shaking.

Isolation of DIV promoter regions. DIVs and the sequences o the viral genome were isolated ollowingthe method described by omaru et al.32. In brie, a 450 ml exponentially growing diatom culture was inoculatedwith 5 ml o DIV suspension and then lysed. Te lysate was filtrated through a 0.4 m pore size polycarbonatemembrane filter (Whatman; GE Healthcare UK Ltd., Buckinghamshire, England) or the removal o cellulardebris. Te filtrated lysate was mixed with polyethylene glycol 6000 (final concentration: 10% wt/vol; Wako PureChemical Industries, Ltd., Osaka, Japan), and the mixture was stored at 4 C in the dark overnight. o collect the

virus particles, afer centriugation at 57,000gat 4 C or 1.5 h, the pellet was washed with 10 mM phosphatebuffer (pH 7.2) and centriuged at 217,000gand 4 C or 4 h. Viral genomic DNA was extracted rom the pelletusing a DNeasy Plant Mini Kit (QIAGEN Inc., CA) according to the manuacturers instructions. We isolatedthree viral genomes: the CdebDNAV31genome, the ClorDNAV32genome, and the nitDNAV33genome. ORFs oClorDNAV32have been identified by omaru et al.32. We identified ORFs o CdebDNAV31and nitDNAV33using
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an ORF finder ollowing the method reported by omaru et al.32. Fragments o upstream regions o the ORFs,such as the replication-associated protein (VP3) gene and the structural protein (VP2) gene as promoter regions(Fig. 1a), were amplified using PCR. Te isolated viral promoters and the source o the promoters are summarizedin able 1. Te primers used in this study are shown in Supplementary able 1. All the DNA ragments, includingthose in the promoter regions, were amplified using PrimeSARGXL DNA Polymerase (akara Bio Inc., Otsu,Japan) according to the manuacturers instructions.

Amplication of DNA fragments for vector construction. o construct transormation vectors (Fig. 1and Supplementary Figs 2 and 3), DNA ragments containing various promoter regions, a reporter gene (egfp),

antibiotic-resistant genes such as the bleomycin-resistant gene (Sh ble) and the nourseothricin-resistant gene(nat), and a terminator region were cloned into GatewayPro Donor vectors using a Gatewaycloning systemaccording to the instructions o MultiSite GatewayPro (Lie echnologies Corporation, CA) or the preparationo entry vectors containing each ragment. DNA ragments containing PtcpA pro., a CaMV 35S promoter, a CMVpromoter, and a nospromoter were amplified rom the genomic DNA o the P. tricornutumUEX 646 strain (TeCulture Collection o Algae (UEX, Te University o exas at Austin)), pCAMBIA2301 (CAMBIA: Center orApplication o Molecular Biology or International Agriculture, Canberra, Australia), phMGFP (Promega, WI),and pBI101.2 (Clontech, CA); these were used as templates. Te egfpragment was amplified rom pNICgp18as atemplate. Te terminator region o the FCP A-1A gene derived rom Cyl. fusiformis(termed CffcpA ter.) was alsoamplified rom pNICgp18as a template. Te terminator region o the FCP gene derived rom Talassiosira pseu-donana(termed pcp ter.) was amplified rom ppcp/nat21as a template. Antibiotic genes, Sh ble, and natwereamplified rom pNICgp18and ppcp/nat21as templates. All the DNA ragments were amplified using PrimeSARGXL DNA Polymerase (akara Bio Inc., Otsu, Japan), with the primer added to the attsites in the Gatewaysystem(Supplementary able 1) according to the manuacturers instructions. Te amplicons were purified via agarosegel electrophoresis using a QIAquickGel Extraction Kit (QIAGEN Inc., CA).

Construction of double-cassette vectors. o evaluate promoter activity levels based on different trans-gene copy quantities and possible site integration effects, we prepared double-cassette vectors as shown in Fig. 1using the Gatewaycloning system according to the instructions or MultiSite GatewayPro (Lie echnologiesCorporation, CA). o construct entry vectors, DNA ragments with various promoter regions, a reporter gene(egfp), and CffcpA ter. were cloned into pDONR221P1-P4, pDONR221P4r-P3r, and pDONR221P3-P2, respec-tively, using the GatewayBP ClonaseII enzyme mix (Lie echnologies Corporation, CA) according to theinstructions designated or MultiSite GatewayPro (Lie echnologies Corporation, CA). In cases involvingthe construction o the promoter-less vector, the egfpgene and CffcpA ter. were cloned into pDONR221P1-P5rand pDONR221P5-P2, respectively. o construct double-cassette vectors, a destination vector was prepared bycloning the Gatewaycloning cassette and Reading Frame Cassette A (Lie echnologies Corporation, CA) intothe KpnI site o pCcp-ble18that contained the antibiotic gene Sh bledriven by CffcpA pro. Te LR recombina-tion reactions, which allow the recombination o three (promoter-reporter gene-terminator) or two ragments(reporter gene-terminator) rom the entry vector into the destination vector, were conducted using the GatewayLR ClonaseII PLUS enzyme mix (Lie echnologies Corporation, CA) according to the instructions designatedor MultiSite GatewayPro (Lie echnologies Corporation, CA). Finally, we constructed the transormationvector described as pested Pro/EGFP/Cffcp er (P-ble) (Fig. 1a). o test the activity o the promoter rom Cnrpro., we used pNICgp18(Supplementary Fig. 5a).

Construction of single-cassette vectors. o determine transormation efficiency, the single-cassettevectors shown in Supplementary Figs 2 and 3 were constructed ollowing the method or the construction odouble-cassette vectors. Te antibiotic genes (Sh bleand nat) and terminator regions (CffcpA ter. And pcpter.) were cloned into pDONR221P4r-P3r and pDONR221P3-P2, respectively, according to the instructions orMultiSite GatewayPro (Lie echnologies Corporation, CA). o construct the promoter-less vector, the anti-biotic gene and terminator were cloned into pDONR221P1-P5r and pDONR221P5-P2, respectively. o con-struct single-cassette vectors, the destination vector was prepared by cloning the Gatewaycloning cassette,Reading Frame Cassette A (Lie echnologies Corporation, CA), into the EcoRI site o the pBluescript SK(-)(Agilent echnologies, CA). Te LR recombination reactions, which allow the recombination o three ragments(promoter-antibiotic gene-terminator) or two ragments (antibiotic gene-terminator) rom the entry vector intothe destination vector, were perormed using the Gateway LR Clonase II PLUS enzyme mix (Lie echnologies

Corporation, CA) according to the instructions or MultiSite GatewayPro (Lie echnologies Corporation, CA).Finally, we constructed the single-cassette vectors (Supplementary Figs 2 and 3). For the transormation o P.tricornutum, PtcpA pro. and ClP1 were examined. For the transormation o Chaetocerossp. and Chl. reinhardtiiCC-503, CdP1 and ClP1 were examined.

Transformation of P. tricornutum. ransormation vectors were introduced into P. tricornutumcells usingthe Biolistic PDS-1000/He particle delivery system (Bio-Rad Laboratories, CA) according to a method previouslydescribed10,12,23. Approximately 1108cells were spread on central areas covering one-third o a plate o solid /2medium containing 1% agarose HGS (Nacalai esque, Kyoto, Japan) ca.1 h prior to bombardment. Te plate waspositioned on the second level (6 cm rom the stopping screen) within the Biolistic chamber or bombardment.ungsten particles M17 (3 mg, 1.1m diameter, Bio-Rad Laboratories, CA) were coated with 5g o the transor-mation vector in the presence o CaCl2and spermidine or five shots, as instructed by the manuacturer. Te cellswere then bombarded with 600 ng o DNA-coated tungsten particles using a two-ply 650 psi (1300psi) rupture disk.Afer bombardment, the cells were recovered rom the plates and incubated in liquid /2 medium under standardgrowth conditions or 24 h. Cultures o P. tricornutumwere concentrated by centriugation and resuspended in
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1 0SCIENTIFICREPORTS| 5:18708 | DOI: 10.1038/srep18708

100l o /2 medium. Te cell suspensions were spread onto plates o solid /2 medium containing 0.5% agaroseHGS (Nacalai esque, Kyoto, Japan) supplemented with 500 g ml1ZeocinM(InvivoGen, CA). Afer 5 weeks oselective incubation under standard growth conditions, individual colonies that had ormed on the plates wereextracted using a platinum loop and suspended into liquid media with 300g ml1ZeocinM.

Transformation of Chaetocerossp. Single-cassette vectors or the transormation o Centrics diatoms(Supplementary Fig. 3) were introduced into Chaetocerossp. cells using the Biolistic PDS-1000/He particle deliverysystem (Bio-Rad Laboratories, CA) according to a method previously described23. Te cells were bombarded usinga two-ply 650 psi (1300psi) rupture disk.

Transformation of Chl. reinhardtiiCC-503. Nuclear transormation was perormed using the electropo-ration method46. In brie, the cells were grown to 4106cells ml1in AP medium. Subsequently, 2.5107cellswere harvested by centriugation and suspended in 250 l o AP medium supplemented with 50 mM sucrose(AP/sucrose). Electroporation was perormed by applying an exponential electric pulse o 0.7 kV at a capacitancelevel o 50F (Electro Cell Manipulator600; BXHarvard Apparatus, MA) and using 300 ng o non-linearizedplasmids according to the manuacturers instructions. ransgenic strains were selected directly rom AP/agarplates containing ZeocinM(10g ml1), and the plates were incubated under continuous fluorescent light con-ditions (20mol m2s1) at 25 C.

PCR analysis of the introduced genes and of their promoters in the transformed cells. o deter-mine whether the egfpgene with the tested promoters and the Sh blegene with CffcpA pro. were introducedinto colony-orming cells that showed resistance to ZeocinM, regions containing the promoters and genes wereamplified using the cells as a template. Genomic PCR analyses were conducted using ks Gflex MDNA Polymerase(akara Bio Inc., Otsu, Japan). Te amplicons were analysed using agarose gel electrophoresis. Te primers used

in the genomic PCR analysis are listed in Supplementary ables 1 and 2.

RNA isolation. otal RNA was isolated rom approximately 5107transormed cells using an RNeasy PlantMini Kit (QIAGEN Inc., CA) coupled with a RNase-Free DNase Set (QIAGEN Inc., CA). Reverse transcription(R) was perormed using a PrimeScriptR reagent Kit (Perect Real ime) (akara Bio, Otsu, Japan). We alsoused a SuperPrepMCell Lysis & R Kit or qPCR (OYOBO, Osaka, Japan) to isolate total RNA and R romsome o the transormed cells.

Assessment of relative promoter activities. qR-PCR experiments to quantiy egfpand Sh bletran-scripts in the transormed cells were perormed in triplicate on a Termal Cycler DiceReal ime System SingleMRQ (akara Bio Inc., Otsu, Japan) using 2 l o the cDNA mixture added as a template and SYBRPremixEx aqMII (li RNaseH Plus) (akara Bio Inc., Otsu, Japan). Te cycling conditions involved denaturing or30 s at 95 C and 40 cycles o melting (5 s at 95 C) and annealing coupled with an extension (30 s at 60 C). odetermine promoter activities, each qR-PCR measurement was perormed in triplicate using primers to iden-tiy the abundance o Sh blemRNA and egfpmRNA (shown in Supplementary able 1). Te egfpmRNA levels

were normalized by dividing by the Sh blemRNA levels to minimize variations in transgene expression due tomultiple insertions (Fig. 1b).

Eect of culture conditions on DIV promoter activity. o explore the effects o nutrient concentra-tions in media, growth phases o the tested strain, and light cycles on DIV promoter activity, two transormantswith ClP1 were cultured in a low nutrient medium (/10 medium) and standard nutrient medium (/2 medium).ransormants at the log and stationary growth phases were collected at approximately 13:30. ransormantscultured in /2 medium at the stationary growth phase were collected during various photoperiods. otal RNAsamples rom approximately 5107transormed cells were isolated using the above methods. Using qR-PCR,we determined the abundance o egfpmRNA and that o rpsmRNA. Te transcript number o egfpwas dividedby that o rps, the expression o which was ound to be constitutive under various growth conditions74and whichwas used as an internal control. Te primers or detecting rpsmRNA are listed in Supplementary able 1.

Flow cytometry. o measure the eGFP fluorescence or the transormed cells, approximately 1104cellswere analysed using a BD LSRFortessaX-20 flow cytometer system equipped with BD FACS Divasofware(BD Biosciences, CA). Te eGFP fluorescence was analysed using a 488 nm laser and a 530/30 nm bandpass filter.Endogenous chlorophyll fluorescence was analysed using a 488 nm laser and a 610/20 nm bandpass filter. Weevaluated cell sizes by measuring the mean orward scatter area (FSC-A) using a 488 nm laser and a 488/10 nmbandpass filter. Te data were analysed using FlowJo sofware (FlowJo, LLC, OR). Te cells were gated on endoge-nous chlorophyll fluorescence so that debris could be eliminated rom the analysis conditions. Te average valueso approximately 1104cells or each sample were used or the statistical analysis.

Statistical analyses. Statistical analyses were perormed on the activity levels o the promoters at the tran-scriptional and translational levels using Students t-test or two group mean values (the PtcpA pro. transormantsand the other promoter transormants), and statistical significance was achieved when P


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In silicoanalyses. Putative cis-regulatory elements in potential DIV promoter regions and other extrinsicpromoters (the CaMV 35 S, CMV, and nospromoters) were identified by searching the PLACE47and PlantCARE48databases. Te promoterome analysis was perormed ollowing the method described by Russo et al.41. In brie, weobtained 12,237 o the 5-flanking sequences o the P. tricornutumgenes rom Ensembl Protists BioMart (Dataset:ASM15095v2 (2013-07-EBI-Phatr3))79,80. Using the oPOSSUM version 356program, SSA and CA tests wereperormed using deault parameters. Te FBS profile matrix file or vertebrates, insects, nematodes, and ungiwas used in the SSA. For the CA, the FBS profile matrix file or vertebrates, insects, and nematodes was used.A consensus sequence or the amplified regions o the tested promoters was also analysed using the consensusmoti-finding algorithms CONSENSUS rom Melina II57and using deault parameters amongst potential DIV

and extrinsic promoters (CaMV 35S, CMV, and nospromoters).

References1. Mann, D. G. & Droop, S. J. M. Biodiversity, biogeography and conservation o diatoms. Hydrobiologia336,1932 (1996).2. Nelson, D. M. et al.Production and dissolution o biogenic silica in the ocean: revised global estimates, comparison with regional

data and relationship to biogenic sedimentation. Global Biogeochem. Cycle9,359372 (1995).3. Falowsi, P. G. et al.Te evolution o modern euaryotic phytoplanton. Science305,354360 (2004).4. Martin-Jzquel, V., Hildebrand, M. & Brzezinsi, M. A. Silicon metabolism in diatoms: Implications or growth.J. Phycol.36,

821840 (2000).5. Wijffels, . H. & Barbosa, M. J. An outloo on microalgal biouels. Science329,796799 (2010).6. Armbrust, E. V. et al.Te genome o the diatom Talassiosira pseudonana: ecology, evolution, and metabolism. Science306,7986

(2004).7. Bowler, C. et al.Te Phaeodactylum genome reveals the evolutionary history o diatom genomes. Nature456,239244 (2008).8. Fabris, M. et al.Te metabolic blueprint o Phaeodactylum tricornutumreveals a euaryotic Entner-Doudoroff glycolytic pathway.

Plant J.70,10041014 (2012).9. Maheswari, U., Moc, ., Armbrust, E. V. & Bowler, C. Update o the Diatom ES Database: a new tool or digital transcriptomics.

Nucleic Acids es.37,D10011005 (2009).10. Apt, . E., roth-Pancic, P. G. & Grossman, A. . Stable nuclear transormation o the diatom Phaeodactylum tricornutum.Mol. Gen.

Genet.252,572579 (1996).11. Zaslavsaia, L. A., Casey Lippmeier, J., roth, P. G., Grossman, A. . & Apt, . E. ransormation o the diatom Phaeodactylum

tricornutum(Bacillariophyceae) with a variety o selectable marer and reporter genes.J.Phycol.36,379386 (2000).12. Miyagawa, A. et al.esearch note: High efficiency transormation o the diatom Phaeodactylum tricornutumwith a promoter rom

the diatom Cylindrotheca fusiformis. Phycol. es.57,142146 (2009).13. Miyahara, M., Aoi, M., Inoue-ashino, N., ashino, Y. & Iuu, . Highly efficient transormation o the diatom Phaeodactylum

tricornutumby multi-pulse electroporation.Biosci. Biotechnol. Biochem.77,874876 (2013).14. Weyman, P. D. et al.Inactivation o Phaeodactylum tricornutumurease gene using transcription activator-lie effector nuclease-based

targeted mutagenesis. Plant Biotechnol. J. 13,460470 (2014).15. Daboussi, F. et al.Genome engineering empowers the diatom Phaeodactylum tricornutumor biotechnology. Nat. Commun. 5,3831

(2014).16. Seo, S., Jeon, H., Hwang, S., Jin, E. & Chang, . S. Development o a new constitutive expression system or the transormation o the

diatom Phaeodactylum tricornutum.Algal es. 11,5054 (2015).17. Fischer, H., obl, I., Sumper, M. & rger, N. argeting and covalent modification o cell wall and membrane proteins heterologously

expressed in the diatom Cylindrotheca fusiformis(Bacillariophyceae).J. Phycol.35,113120 (1999).18. Poulsen, N. & rger, N. A new molecular tool or transgenic diatoms: control o mNA and protein biosynthesis by an inducible

promoter-terminator cassette. FEBS J.272,34133423 (2005).

19. Dunahay, . G., Jarvis, E. E. & oessler, P. G. Genetic transormation o the diatoms Cyclotella cryptiaand Navicula saprophila.J.Phycol.31,10041012 (1995).

20. Sabatino, V. et al.Ferrante MI. Establishment o genetic transormation in the sexually reproducing diatoms Pseudo-nitzschiamultistriataand Pseudo-nitzschia arenysensisand inheritance o the transgene.Mar. Biotechnol. (NY)17,452462 (2015).

21. Poulsen, N., Chesley, P. M. & rger, N. Molecular genetic manipulation o the diatomTalassiosira pseudonana(Bacillariophyceae).J. Phycol.42,10591065 (2006).

22. Falciatore, A., Casotti, ., Leblanc, C., Abrescia, C. & Bowler, C. ransormation o nonselectable reporter genes in marine diatoms.Mar. Biotechnol. (NY)1,239251 (1999).

23. Miyagawa-Yamaguchi, A. et al.Stable nuclear transormation o the diatom Chaetocerossp. Phycological es. 59,113119 (2011).24. Iuu, . et al.A stable and efficient nuclear transormation system or the diatom Chaetoceros gracilis. Photosynth. es.123,203211

(2015).25. Muto, M. et al.Establishment o a genetic transormation system or the marine pennate diatom Fistuliferasp. strain JPCC DA0580a

high triglyceride producer.Mar. Biotechnol. (NY)15,4855 (2013).26. aras, B. J. et al.Designer diatom episomes delivered by bacterial conjugation. Nat. Commun.6,6925 (2015).27. Lehahn, Y. et al.Decoupling physical rom biological processes to assess the impact o viruses on a mesoscale algal bloom.Curr. Biol.

24,20412046 (2014).28. imura, . & omaru, Y. Discovery o two novel viruses expands the diversity o single-stranded DNA and single-stranded NA

viruses inecting a cosmopolitan marine diatom.Appl. Environ. Microbiol.81,11201131 (2015).29. Suttle, C. A. Marine virusesmajor players in the global ecosystem. Nat. ev. Microbiol.5,801812 (2007).30. de Vargas, C. et al.Ocean planton. Euaryotic planton diversity in the sunlit ocean. Science348,1261605 (2015).31. omaru, Y., Shirai, Y., Suzui, H., Nagasai, . & Nagumo, . Isolation and character ization o a new single-stranded DNA virus

inecting the cosmopolitan marine diatom Chaetoceros debilis.Aquat. Microb. Ecol.50,103112 (2008).32. omaru, Y. et al.Isolation and characterization o a single-stranded DNA virus inecting Chaetoceros lorenzianusGrunow.Appl.

Environ. Microbio.l77,52855293 (2011).33. omaru, Y. et al.First evidence or the existence o pennate diatom viruses. ISME J.6,14451448 (2012).34. Harada, H., Naatsuma, D., Ishida, M. & Matsuda, Y. egulation o the expression o intracellular beta-carbonic anhydrase in response

to CO2and light in the marine diatom Phaeodactylum tricornutum. Plant Physiol.139,10411050 (2005).35. adaovits, ., Eduao, P. M. & Posewitz, M. C. Genetic engineering o atty acid chain length in Phaeodactylum tricornutum.Metab.

Eng.13,8995 (2011).36. Niu, Y. F. et al.Improvement o neutral lipid and polyunsaturated atty acid biosynthesis by overexpressing a type 2 diacylglycerol

acyltranserase in marine diatom Phaeodactylum tricornutum.Mar. Drugs11,45584569 (2013).37. rentacoste, E. M. et al.Metabolic engineering o lipid catabolism increases microalgal lipid accumulation without compromising

growth. Proc. Natl. Acad. Sci. USA110,1974819753 (2013).38. Hamilton, M. L., Haslam, . P., Napier, J. A. & Sayanova, O. Metabolic engineering o Phaeodactylum tricornutumor the enhanced

accumulation o omega-3 long chain polyunsaturated atty acids.Metab. Eng.22,39 (2014).
http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-


12/13



39. adono, . et al.Effect o an introduced phytoene synthase gene expression on carotenoid biosynthesis in the marine diatomPhaeodactylum tricornutum.Mar. Drugs13,53345357 (2015).

40. Oeltjen, A., Marquardt, J. & hiel, E. Differential circadian expression o genesfcp2 andfcp6 in Cyclotella cryptica. Int. Microbiol.7,127131 (2004).

41. usso, M. ., Annunziata, ., Sanges, ., Ferrante, M. I. & Falciatore, A. Te upstream regulatory sequence o the light harvestingcomplex Lhcf2gene o the marine diatom Phaeodactylum tricornutumenhances transcription in an orientation- and distance-independent ashion.Mar. Genomics24,6979 (2015).

42. Schmidt, E. V., Christoph, G., Zeller, . & Leder, P. Te cytomegalovirus enhancer: a pan-active control element in transgenic mice.Mol. Cell Biol.10,44064411 (1990).

43. Beney, P. N. & Chua, N. H. Te cauliflower mosaic virus 35S promoter: combinatorial regulation o transcription in plants. Science250,959966 (1990).

44. Saaue, ., Harada, H. & Matsuda, Y. Development o gene expression system in a marine diatom using viral promoters o a widevariety o origin. Physiol. Plant.133,5967 (2008).45. Stevens, D. ., ochaix, J. D. & Purton, S. Te bacterial phleomycin resistance gene bleas a dominant selectable marer in

Chlamydomonas.Mol. Gen. Genet.251,2330 (1996).46. Shimogawara, ., Fujiwara, S., Grossman, A. & Usuda, H. High-efficiency transormation o Chlamydomonas reinhardtiiby

electroporation. Genetics148,18211828 (1998).47. Higo, ., Ugawa, Y., Iwamoto, M. & orenaga, . Plant cis-acting regulator y DNA elements (PLACE) database: 1999. Nucleic Acids

es.27,297300 (1999).48. Lescot, M. et al.PlantCAE, a database o plant cis-acting regulatory elements and a portal to tools orin silicoanalysis o promoter

sequences. Nucleic Acids es.30,325327 (2002).49. Ambawat, S., Sharma, P., Yadav, N. . & Yadav, . C. MYB transcription actor genes as regulators or plant responses: an overview.

Physiol. Mol. Biol. Plants19,307321 (2013).50. Jaoby, M. et al.bZIP transcription actors inArabidopsis. rends Plant Sci.7,106111 (2002).51. Laloum, ., De Mita, S., Gamas, P., Baudin, M. & Niebel, A. CCAA-box binding transcription actors in plants: Y so many? rends

Plant Sci.18,157166 (2013).52. ezsohazy, ., Saurin, A. J., Maurel-Zaffran, C. & Graba, Y. Cellular and molecular insights into Hox protein action. Development

142,12121227 (2015).53. Zheng, N., Fraenel, E., Pabo, C. O. & Pavletich, N. P. Structural basis o DNA recognition by the heterodimeric cell cycle transcription

actor E2F-DP. Genes Dev.13,666674 (1999).54. ayo, E., Maumus, F., Maheswari, U., Jabbari, . & Bowler, C. ranscription actor amilies inerred rom genome sequences o

photosynthetic stramenopiles. New Phytol.188,5266 (2010).55. Mathelier, A. et al.JASPA 2014: an extensively expanded and updated open-access database o transcription actor binding profiles.

Nucleic Acids es.42,D142147 (2014).56. won, A. ., Arenillas, D. J., Worsley Hunt, . & Wasserman, W. W. oPOSSUM-3: advanced analysis o regulatory moti over-

representation across genes or ChIP-Seq datasets. G3 (Bethesda)2,9871002 (2012).57. Oumura, ., Maiguchi, H., Maita, Y., Yamashita, . & Naai, . Melina II: a web tool or comparisons among several predictive

algorithms to find potential motis rom promoter regions. Nucleic Acids es.35,W227231 (2007).58. Hobbs, S. L., Warentin, . D. & DeLong, C. M. ransgene copy number can be positively or negatively associated with transgene

expression. Plant Mol. Biol.21,1726 (1993).59. Waimoto, B. . Beyond the nucleosome: epigenetic aspects o position-effect variegation in Drosophila. Cell93,321324 (1998).60. Mitra, A. & Higgins, D. W. Te Chlorellavirus adenine methyltranserase gene promoter is a strong promoter in plants. Plant Mol.

Biol.26,8593 (1994).61. Smerdon, G. ., Aves, S. J. & Walton, E. F. Production o human gastric lipase in the fission yeast Schizosaccharomyces pombe. Gene

165,313318 (1995).62. omaru, Y., imura, . & Yamaguchi, H. emperature alters algicidal activity o DNA and NA viruses inecting Chaetoceros

tenuissimus.Aquat. Microb. Ecol.73,171183 (2014).63. Venaya, N., Anesiadis, N., Cluett, W. . & Mahadevan, . Engineering metabolism through dynamic control. Curr. Opin. Biotechnol.34,142152 (2015).

64. Pulz, O. & Gross, W. Valuable products rom biotechnology o microalgae.Appl. Microbiol. Biotechnol.65,635648 (2004).65. Teriot, E. C., Ashworth, M., uc, E., Naov, . & Jansen, . . A preliminary multigene phylogeny o the diatoms (Bacillariophyta):

challenges or uture research. Plant Ecol. Evol. 143,278296 (2010).66. Dutt, M., Dheney, S. A., Soriano, L., andel, . & Grosser, J. W. emporal and spatial control o gene expression in horticultural

crops. Horticulture esearch1,14047 (2014).67. Juven-Gershon, . & adonaga, J. . egulation o gene expression via the core promoter and the basal transcriptional machinery.

Dev. Biol.339,225229 (2010).68. McLeod, A., Smart, C. D. & Fry, W. E. Core promoter structure in the oomycete Phytophthora infestans. Euaryotic Cell3,9199 (2004).69. Liston, D. . & Johnson, P. J. Gene transcription in richomonas vaginalis. Parasitol oday14,261265 (1998).70. Bhaya, D. & Grossman, A. . C haracterization o gene clusters encoding the ucoxanthin chlorophyll proteins o the diatom

Phaeodactylum tricornutum. Nucleic Acids es.21,44584466 (1993).71. Ohno, N. et al.CO2-cAMP-responsive cis-elements targeted by a transcription actor with CEB/AF-lie basic zipper domain in

the marine diatom Phaeodactylum tricornutum. Plant Physiol.158,499513 (2012).72. Yoshinaga, ., Niwa-ubota, M., Matsui, H. & Matsuda, Y. Characterization o iron-responsive promoters in the marine diatom

Phaeodactylum tricornutum.Mar. Genomics16,5562 (2014).

73. Leblanc, C., Falciatore, A., Watanabe, M. & Bowler, C. Semi-quantitative -PC analysis o photoregulated gene expression inmarine diatoms. Plant Mol. Biol.40,10311044 (1999).

74. Siaut, M. et al.Molecular toolbox or studying diatom biology in Phaeodactylum tricornutum. Gene406,2335 (2007).75. Braemann, ., Fran, B., Peter, . & Erhard, . Structural and unctional characterization o putative regulatory DNA sequences o

fcpgenes in the centric diatom Cyclotella cryptica. Diatom es.23,3149 (2008).76. Guillard, . L. [Culture o phytoplanton or eeding marine invertebrates]. In: Culture of Marine Invertebrate Animals[Smith, W.,

Chanley, M. & Guillard, . L. (eds.)] [2660] (Springer US, New Yor, 1975).77. Chen, L. C.-M., Edelstein, . & McLachian, J.Bonnemaisonia hamiferaHariot in nature and in culture.J. Phycol.5,211220 (1969).78. Gorman, D. S. & Levine, . P. Cytochromefand plastocyanin: their sequence in the photosynthetic electron transport chain o

Chlamydomonas reinhardi. Proc. Natl. Acad. Sci. USA54,16651669 (1965).79. ersey, P. J. et al.Ensembl Genomes 2013: scaling up access to genome-wide data. Nucleic Acids es.42,D546552 (2014).80. Smedley, D. et al.Te BioMart community portal: an innovative alternative to large, centralized data repositories. Nucleic Acids es.

43,W589598 (2015).81. Sanders, P. ., Winter, J. A., Barnason, A. ., ogers, S. G. & Fraley, . . Comparison o cauliflower mosaic virus 35S and nopaline

synthase promoters in transgenic plants. Nucleic Acids es.15,15431558 (1987).82. Boshart, M. et al.A very strong enhancer is located upstream o an immediate early gene o human cytomegalovirus. Cell41,521530

(1985).


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AcknowledgementsWe thank Nicole Poulsen and Nils Krger (echnische Universitt Dresden) or providing the transormation

vectors (ppcp/nat, pNICgp, and pCcp-ble); Keizo Nagasaki (Fisheries Research Agency, Japan) or criticaldiscussions about DIVs; and Yasuumi Hikichi (Kochi University) or kind advice about the nospromoter andpBI101.2 vector. We also thank Sayo Kataoka and Ken-ichi Yagyu (Science Research Center, Kochi University) ortechnical supports on the analysis o eGFP fluorescence. We are grateul to Dennis Murphy (Ehime University) orEnglish correction. Tis study was supported in part by the Strategic Development o Next Generation BioenergyUtilization echnology o New Energy and Industrial echnology Development Organization (NEDO), Japan;in part by a Grant-in-Aid or Scientific Research (Nos. 24580268 and 15K14804) rom the Ministry o Education,

Culture, Sports, Science and echnology (MEX), Japan to M.A.; and in part by the Kochi University PresidentsDiscretionary Grant to M.A.; and in part by the Grand Emprunt Project-European Marine Biological ResourceCentre-France (EMBRC) and ANR DiaDomOil PROGRAMME BIO-MAIERES & ENERGIES to A.F.

Author ContributionsExperiments were conceived and designed by .K., A.M., . Ohama, A.F., N.K. and M.A. Experiments wereperormed by .K., .Y., A.M., . Okami, N.K. and L.H. Data were analyzed by .K., .Y., . Ohama and M.A.Reagents, materials, and analysis tools were contributed by .K., A.M., N.K., Y.., . Okami, K.F., M.O., H.Y. andK.O. Te final orm o the manuscript was written and discussed by .K., A.M., N.K., . Ohama, A.F. and M.A.

Additional InformationSupplementary informationaccompanies this paper at http://www.nature.com/srep

Competing financial interests: Te authors declare no competing financial interests.

How to cite this article: Kadono, . et al.Characterization o marine diatom-inecting virus promoters in themodel diatom Phaeodactylum tricornutum. Sci. Rep.5, 18708; doi: 10.1038/srep18708 (2015).

Tis work is licensed under a Creative Commons Attribution 4.0 International License. Te imagesor other third party material in this article are included in the articles Creative Commons license,

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Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum