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Michael Schroda

RNA silencing in Chlamydomonas: mechanisms and tools

Received: 4 August 2005 / Revised: 19 October 2005 / Accepted: 26 October 2005 / Published online: 25 November 2005 Springer-Verlag 2005

AbstractThe generation of a comprehensive EST libraryand the sequencing of its genome set the stage for reversegenetics approaches in Chlamydomonas reinhardtii.However, these also require tools for the specific

downregulation of target gene expression. Conse-quently, a large number of diverse constructs weredeveloped aimed at reducing target gene expression inChlamydomonas via the stable expression of antisense orinverted repeat-containing RNA. Double-strandedRNA (dsRNA) generated by the annealing of antisenseand sense RNAs or by hairpin formation of an invertedrepeat, feeds into the RNA silencing pathway. In thispathway, dsRNA is cleaved into 25-bp small interfer-ing RNAs (siRNAs) by the endonuclease Dicer. One ofthe two complementary strands of a siRNA is thenloaded onto an Argonaute-like protein present as corecomponent within larger complexes. Guided by this

single-stranded RNA, the Argonaute-like protein eitherdetects homologous transcripts and cleaves these endo-nucleolytically, or initiates transcriptional gene silenc-ing. This article summarizes current information derivedmainly from the Chlamydomonas genome project oncomponents that are assumed to be involved in RNAsilencing mechanisms in Chlamydomonas. Furthermore,all approaches employed in Chlamydomonas to date todownregulate target gene expression by antisense or in-verted repeat constructs are reviewed and discussedcritically.

Keywords Antisense Argonaute Chlamydomonas

genome sequence

Inverted repeat

PTGS

RNAi

Introduction

Since more than five decades Chlamydomonas reinhardtii

has been used as a model organism to study various as-pects of cell biology (for recent reviews on Chlamydo-monas see for example Grossman et al. 2003; Rochaix2004;Snell et al. 2004). Chlamydomonas is the first pho-tosynthetic eukaryote which allowed stable transforma-tion of the nuclear (Debuchy et al. 1989; Kindle et al.1989), chloroplast (Boynton et al. 1988), and mitochon-drial (Randolph-Anderson et al. 1993) genomes. Forefficient and stable nuclear transformation, a variety oftechniques have emerged, including particle gun bom-bardment (Debuchy et al. 1989; Kindle et al. 1989),agitation with glass beads (Kindle 1990), or electropo-ration (Shimogawara et al. 1998). Moreover, valuablemolecular tools have been developed that facilitatestudying of Chlamydomonas nuclear genes. These toolsinclude reporter genes with optimized codon usage (Fu-hrmann et al. 1999,2004), dominant selectable markers(Kovar et al. 2002; Sizova et al. 2001; Stevens et al. 1996),and strong promoter systems like the HSP70A-RBCS2tandem promoter (Schroda et al.1999,2000,2002)or thePsaD promoter (Fischer and Rochaix 2001).

A major breakthrough for Chlamydomonas as amodel system was the generation of a comprehensiveEST library that currently comprises about 200,000ESTs (Asamizu et al. 1999, 2000; Shrager et al. 2003),and the sequencing of the 100 Mbp nuclear genome bythe Joint Genome Institute (http://www.genome.jgi-psf.org/chlre2/chlre2.home.html). EST library andgenome sequence made reverse genetics approachespossible; for these, however, tools for the targetedknock-out or knock-down of the genes of interest arerequired.

Targeted knock-out of an endogenous gene byhomologous recombination has been demonstrated forChlamydomonas (Nelson and Lefebvre 1995). Unfortu-nately, homologous recombination events occur sorarely in Chlamydomonas that targeted knock-out

Communicated by F.-A. Wollman

M. Schroda (&)Institute of Biology II/Plant Biochemistry,University of Freiburg, Scha nzlestr. 1, 79104 Freiburg, GermanyE-mail: michael.schroda@biologie.uni-freiburg.deTel.: +49-761-2032708Fax: +49-761-2032601

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appeared unsuitable as a standard approach (Gumpelet al. 1994; Nelson and Lefebvre 1995; Sodeinde andKindle 1993). In a recent report, homologous recombi-nation was shown to be strongly favored over nonho-mologous recombination if the transforming DNA issingle-stranded (Zorin et al. 2005). This finding mighteventually lead to the development of efficient tools fortargeted knock-outs in Chlamydomonas, but substantialefforts are still required.

In contrast, knock-down approaches that triggerRNA silencing via constructs that express antisense orinverted repeat-containing RNAs have been successfullyintroduced to Chlamydomonas (Fuhrmann et al. 2001;Schroda et al. 1999) and have been continuously im-proved since then. RNA silencing, also termed post-transcriptional gene silencing (PTGS) in plants, RNAinterference (RNAi) in nematodes, or quelling in fungi,involves double-stranded RNA (dsRNA) intermediatesthat may specifically affect gene expression at the tran-scriptional and/or posttranscriptional levels. The goal ofthis article is (1) to summarize and to discuss theinformation currently available on the molecular players

leading to RNA silencing in Chlamydomonas, and (2) toreview the recent activities of the Chlamydomonas com-munity to develop tools that trigger RNA silencing viaantisense and inverted repeat constructs.

The molecular mechanisms underlying RNA silencing

Injection or stable production of antisense RNA byantisense gene constructs for a long time was the methodof choice for the targeted downregulation of a gene ofinterest (Van der Krol et al.1988a,b). This was until thefinding that dsRNA triggered target gene silencing with

at least two orders of magnitude higher efficiency thanantisense RNA (Fire et al. 1998). Since this finding, themolecular machinery for RNA silencing has been elu-cidated in great detail. Here, I will briefly summarizeonly those features of the RNA silencing machinery thatare required for the introduction of Chlamydomonas-specific aspects. Readers interested in more details ofRNA silencing may refer to the many excellent reviewsthat have recently been published on this topic (Baul-combe 2004; Dykxhoorn et al. 2003; Herr 2004; Mart-ienssen et al.2005; Matzke and Birchler2005; Mello andConte 2004; Sontheimer 2005; Tomari and Zamore2005).

The ultimate trigger for RNA silencing is dsRNA. Invivo, dsRNA may be generated by transcribing theantisense strand of the gene to be targeted; the antisenseRNA may then anneal with complementary transcriptsin the cell to form dsRNA (Fig. 1). Alternatively, bidi-rectional transcription from promoters located 5 and 3from the target gene may produce complementaryRNAs that anneal to dsRNA. Moreover, dsRNA mayalso be generated by the transcription of target genesequences that are arranged as inverted repeats. At last,single-stranded RNA may be converted into dsRNA by

the activity of an RNA-dependent RNA polymerase(RdRP). This enzyme may recognize specific features ofaberrant transcripts and perform synthesis of dsRNA ina primer-independent reaction (Makeyev and Bamford2002; Tang et al. 2003). Alternatively, a small RNAcomplementary to an RNA template may serve to primethe reaction (Makeyev and Bamford 2002). RdRPs sofar have been found in Caenorhabditis elegans, Neuros-pora crassa, fission yeast and plants, but not in Dro-sophila melanogaster and mammals (Tomari andZamore2005). dsRNA may also be delivered artificially,e.g., by injection into worms (Fire et al. 1998) or flyembryos (Kennerdell and Carthew 1998), or by intra-venous injection into mice (Lewis et al. 2002). Also thefeeding ofC. elegans with bacteria that express dsRNAleads to ingestion of the latter, which in turn trigger thesilencing of homologous target genes within the nema-tode (Timmons and Fire1998).

Once present within a cell, dsRNA is recognized by acytoplasmic RNase III-like enzyme called Dicer andcleaved into 25 bp long dsRNA fragments termedsmall interfering RNAs (siRNAs) (Bernstein et al. 2001)

(Fig.1). Characteristic of the latter is that they bear 5-phosphate groups and two-nucleotide 3 overhangs (El-bashir et al.2001). Dicer contains an N-terminal RNA-helicase domain, one domain of unknown function, twoRNase III domains, and frequently also a so-calledPAZ-domain (Zhang et al. 2004). PAZ stands for Piwi/Argonaute/Zwille, three proteins that share this domain(Cerutti et al. 2000). One of the two siRNA strands isthen loaded onto RISC, the RNA-induced silencingcomplex (Hammond et al. 2000), or onto RITS, theRNA-induced initiation of transcriptional gene silencingcomplex (Verdel et al. 2004) (Fig.1). Which of the twostrands will be loaded onto RISC or RITS depends on

their sequence: the strand with the less tightly base-paired 5end will become the guide strand, the one withthe more tightly base-paired 5 end will be the passen-ger strand, which is degraded (Schwarz et al. 2003).siRNA unwinding appears to require ATP hydrolysis(Nykanen et al. 2001), however, it is not yet clear whichenzyme activity performs the unwinding (Tomari andZamore2005).

RNA-induced silencing activities have been detectedin complexes of sizes ranging from 160 kDa to 80S; allof them, including the RITS complex, have in commonthe presence of a member of the Argonaute proteinfamily (Sontheimer 2005; Verdel et al. 2004) (Fig.1).

Characteristics of Argonaute proteins are the PAZ andPIWI domains (Cerutti et al.2000). The PAZ domain issimilar to the OB fold (oligonucleotide/oligosaccharidebinding fold) typical for proteins involved in ssDNA orRNA-binding (Song et al.2003). The PAZ domain bindsthe 3 end of siRNAs with rather low affinity in a non-sequence specific manner (Ma et al. 2004; Song et al.2003). The PIWI domain contains two striking features:First, it adopts an RNase H fold that immediatelyimplicated Argonaute as Slicer; Slicer is the nucleasethat carries out cleavage of target RNAs (Liu et al.2004;

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Parker et al. 2004; Song et al. 2003). Second, PIWIcontains a conserved basic pocket that anchors the 5phosphate and the 5base of the guide siRNA (Ma et al.2005; Parker et al. 2004,2005). The N-terminal, middle,and PIWI-domains of Pyrococcus furiosus Argonaute

form a crescent-shaped base with the PIWI domain inthe center of the crescent (Song et al. 2004). The regionfollowing the N-terminal domain forms a stalk thatholds the PAZ domain above the crescent. The putativeactive site of the RNase H fold is positioned in a cleft inthe middle of the crescent in a groove below the PAZdomain. The groove is lined with positively chargedamino acid residues creating an ideal environment forthe binding of the negatively charged phosphodiesterbackbones of target RNAs (Parker et al. 2004; Songet al.2004).

Hence, the scenario for target RNA cleavage mightbe as follows: the guide siRNA would be tightly locked

into Argonaute via binding of the 5 phosphate and the5 base to PIWI and interaction of its 3 end with PAZ;the interaction between guide siRNA and target RNAwould nucleate at the 5end of the guide and zipper up.Subsequently, the PAZ domain would lock down overthe guide-target complex to orient recognition andcleavage of the RNA substrate by the catalytic site of theRNase H fold within PIWI. After cleavage, the siRNAdeparts intact with RISC and may guide RISC formultiple cleavage reactions (Hutvagner and Zamore2002) (Fig.1).

For the RITS complex, it is assumed that the guidesiRNA bound to Argonaute is required only to targetthe complex to homologous DNA sequences, where thechromodomain protein Chp1 present in the RITScomplex would modify chromatin to initiate hetero-

chromatin formation (Verdel et al.2004) (M in Fig.1).It is not yet clear which features of an siRNA determinewhether it affects target gene expression in RITS at thetranscriptional level, or in RISC at the posttranscrip-tional level.

Potential components of the RNA silencing machineryin Chlamydomonas

The first (and so far the only) experiments that aimed atdissecting the mechanisms leading to PTGS in Chla-mydomonas were performed by Cerutti and coworkers.

The starting point was a transformant that contained asingle copy of the aadA gene conferring resistance tospectinomycin that was controlled by the Chlamydo-monas RBCS2 promoter and terminator sequences.When kept under nonselective conditions, the aadAgenein this transformant was readily silenced. As judged bynuclear run-on assays, the aadA gene was transcribedbut transcripts were degraded posttranscriptionally(Cerutti et al. 1997). Since the aadA gene is of pro-karyotic origin and has a low GC-content (52% com-pared to an average of 65% for Chlamydomonasnuclear

Fig. 1 Possible origins ofdsRNA in the cell andpathways of the RNA silencingmechanism leading totranscriptional or PTGS. Seetext for details

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genes), a likely reason for PTGS are aberrant transcriptsoriginating from AU-rich instability determinants and/or false splice sites (Cerutti et al. 1997).

Wu-Scharf et al. (2000) used this transformant forinsertional mutagenesis and selected for secondarytransformants that had reactivated the originally post-transcriptionally silenced aadA gene. One of the specti-nomycin-resistant transformants obtained contained aninsertion within a gene encoding a DEAH-box RNAhelicase (Wu-Scharf et al. 2000). This mutant (Mut-6) notonly recovered detectableaadAtranscripts, but also hadthreefold higher transposon ofChlamydomonas 1 (TOC1)transcript levels. Accordingly, the transposition fre-quency of the TOC1 and Gulliver transposons was sig-nificantly enhanced. The authors suggested that theaffected RNA helicase might either be a component of themachinery responsible for PTGS (e.g., for the unwindingof siRNAs or for the unwinding of the guide RNA-targetRNA duplex for the regeneration of RISC after cleavage),or part of an RNA surveillance system that recognizesand degrades aberrant RNAs. Evidence for a direct roleof Mut6p downstream of dsRNA is yet missing.

To elucidate the mechanisms leading to transcrip-tional gene silencing in Chlamydomonas, Cerutti andcoworkers again used a strain that contained theRBCS2-aadA transgene. However, as judged by nuclearrun-on assays, this strain was impaired in transgenetranscription. Insertional mutagenesis gave rise to twomutant strains (Mut-9 and Mut-11) that had reactivatedthe silenced aadA transgene. Mut-9 was affected in agene encoding a Ser/Thr kinase and Mut-11 in a geneencoding a WD-repeat protein (Jeong et al.2002; Zhanget al.2002). Mut11p turned out to form (a) complex(es)with histone methyltransferase(s) that mediate(s) meth-ylation of histones H3, H2A, and H4 (van Dijk et al.

2005). Although possible, it is not clear whether Mut9por Mut11p play roles in RNA-mediated transcriptionalgene silencing.

Orthologs for Dicer and Argonaute in Chlamydomonas

In the absence of more experimental data on potentialcomponents required for RNA silencing in Chlamydo-monas, it might be helpful to screen for information thatmay be acquired from the Chlamydomonas genome se-quence. As outlined above, the central activities requiredfor RNA silencing are mediated by RdRP, Dicer, and

Argonaute proteins. Whereas an RdRP ortholog couldnot be found in the current version of the Chlamydo-monas genome sequence, one gene model was identified(C_130110) whose gene product is similar to Dicer andwas therefore annotated as Dicer-like protein 1 (DCL1).Gene models C_130206 and C_1700017 encode Arg-onaute-like proteins and accordingly were annotated asAGO1 and AGO2, respectively.

Interestingly, the genes encoding AGO1 and DCL1are arranged head-to-head with less than 800 bp sepa-rating their respective transcriptional start sites.

Intriguingly, downstream ofDCL1, a partial gene (genemodel C_130015) is located whose putative gene productexhibits significant similarity with reverse transcriptases(RNA-dependent DNA polymerases). A reverse trans-criptase gene is usually indicative of a mobile elementsuch as a retrotransposon, retrovirus, group II intron,bacterial msDNA, hepadnavirus, or caulimovirus. Theclustering ofAGO1,DCL1, and the reverse transcriptasegene fragment might indicate that these genes haveoriginally been pirated from a virus or retroelement;now they may serve to protect the cell against the verymobile elements they originated from (Liu et al. 2004).Clustering of genes that are likely to participate in thesame or related processes has been reported earlier forChlamydomonas. For example, genes that encode theER-lumenal chaperones BIP1, BIP2, and HSP90B forma cluster (Schroda2004).

ESTs are available for AGO1, AGO2, and for thereverse transcriptase gene, however, not for the Dicerortholog. The deduced amino acid sequence of AGO1contains both the PAZ and the PIWI domains, whereasthe sequence of AGO2 due to extended sequence gaps is

partial and contains only part of its PIWI domain. Be-cause of the relatively low level of sequence conservationbetween PAZ domains and frequently inaccurate exon/intron border predictions in Chlamydomonas genemodels, it is not clear whether DCL1 contains a func-tional PAZ domain. However, all other domains char-acteristic of Dicer proteins (Zhang et al. 2004), i.e., theRNA-helicase domain, the domain of unknown func-tion, and the tandem RNAse III domains, are present inDCL1. The detection of siRNAs derived from theexpression of an inverted repeat construct in Chla-mydomonas (Rohr et al.2004) implies the presence of afunctional Dicer protein in this organism, which could

well be DCL1.

The PIWI domains ofChlamydomonasArgonaute proteins

Sequence alignment of the PIWI domains from Chla-mydomonas AGO1 and AGO2 with those of Argonauteorthologs from human, Drosophila, Arabidopsis, andNeurosporareveals a high level of sequence conservation,in particular, within distinct regions (Fig. 2). Accordingto their degree of similarity with the Arabidopsis AGO1or theDrosophila Piwi proteins, Argonaute proteins wereseparated into the Piwi and Ago subclasses (Carmell

et al. 2002). Parker et al. (2004) identified the so-calledclass-switch residues within the PIWI domain that definethe subclass to which a given Argonaute protein belongs.Since bothChlamydomonasAGO proteins share none ofthe seven class-switch residues with neither Piwi nor Agosubfamily (Fig.2), they cannot be assigned to any of thesubclasses.

With the recently published crystal structure of thePIWI domain (Ma et al. 2005; Parker et al. 2004,2005;Song et al. 2003) it is possible to illuminate whethercertain amino acid residues implicated in siRNA-binding

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or Slicer activity are conserved within the Chlamydo-

monas AGO proteins. As outlined above, the duplexbetween siRNA and target RNA lies within a basicchannel spanning the interface of subdomains A and B ofthe PIWI domain (Ma et al. 2005; Parker et al. 2005). The5phosphate of the siRNA is bound in a conserved basicpocket created by subdomain A and the C-terminus ofsubdomain B; binding is achieved via hydrogen bondscontributed by the side chains (in human Ago2) of Y529,K533, Q545, K570 (Fig.2), and by a divalent cation(most likely Mg2+). Moreover, the aromatic ring ofY529 stacks on the unpaired base of the siRNAs

ultimate 5nucleotide in a nonsequence specific manner.

Mutation of either of these four residues leads to lowerRNA binding affinity in Archaeoglobus fulgidus PIWIand reduced target cleavage efficiency of human AGO2(Ma et al.2005). All four residues are invariant in PIWIdomains, including that of Chlamydomonas AGO1(AGO2 sequences are missing in that region) (Fig. 2).

The PIWI subdomain B adopts an RNase H foldcommon to nucleases like RNase H1, RNase HII,transposases, and integrases, which implicated Argona-ute as Slicer (Liu et al. 2004; Parker et al. 2004; Songet al. 2003). The catalytic center of human AGO2

Fig. 2 Sequence alignment of Argonaute PIWI domains. Alignedare amino acid sequences deduced from argonaute genes from

human, accessions Q9UL18 (HsAGO1), NP_036286 (HsAGO2),NP_079128 (HsAGO3), NP_060099 (HsAGO4), D. melanogaster(DmAGO2), accession Q9VUQ5, Arabidopsis thaliana (AtAGO1),accession NP_175274, Neurospora crassa, accessions EAA29350(NcAGO), AAF43641 (NcQDE2), andChlamydomonas reinhardtii,gene models C_130206 (CrAGO1) and C_1700017 (CrAGO2).Dots in CrAGO2 indicate missing sequence data. Residueshighlighted in black are conserved in all sequences, thosehighlighted in grey are conserved in at least seven out of ten.Conserved amino acids were defined as N/Q, D/E, R/K, S/T, F/Y,A/G, and V/I/L/M. The separation into domains A and B wasmade according to Ma et al. (2005). The four amino acid residues

given on top of the alignment as unformatted letters indicateresidues that in the A. fulgidus Piwi protein were shown to mediate

anchoring of the 5 phosphate of siRNAs (Ma et al. 2005). Aminoacid residues given in bold letters represent the DDH motif, whichis required for metal ion coordination in the catalytic center (Rivaset al. 2005). The glutamate given in parentheses is part of thecatalytic DDE motif in RNase H, but upon mutation did notimpair Slicer activity in HsAGO2 (Rivas et al. 2005). Residues initalicized letters have been identified as class-switch residues thatattribute a given protein either to the (p)iwi or to the (a)rgonautesubfamily (Parker et al. 2004). The N-terminal 76 amino acids ofdomain A are not shown because sequence similarity is very low;the N-terminal borders of the high similarity region were adoptedfrom Cerutti et al. (2000) and Carmell et al. (2002)

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contains a divalent metal ion (usually Mg2+), which iscoordinated by two aspartate and one histidine residue(the DDH motif) (Rivas et al. 2005). Mutation of any ofthese three residues generated proteins that could stillbind the siRNA guide, but lost Slicer activity (Liu et al.2004; Rivas et al. 2005). Substitution of the histidineresidue in the active center by arginine in human AGO1and AGO4, and of the second aspartate by glycine inhuman AGO4, might thus explain why these proteinslack Slicer activity (Fig. 2). Note, however, that humanAGO3 also lacks Slicer activity, although it contains theDDH motif. Interestingly, both Chlamydomonas AGOproteins and the Neurospora QDE-2 Argonaute ortho-log contain the aspartate residues of the DDH motif, buthave an aspartate at the position where cleavage-activeArgonautes like human AGO2 and Drosophila AGO2contain a histidine (Fig. 2). So far, only substitutions ofthe histidine residue of the DDH motif by arginine oralanine were tested and shown to abolish Slicer activityin human AGO2 (Rivas et al. 2005). Therefore, onecannot predict whether substitution of histidine byaspartate would also abolish Slicer activity. Since the

second Argonaute protein in Neurospora does contain acomplete DDH motif and, therefore, might account forSlicer activity in this organism, three possibilities may beenvisaged for Chlamydomonas: (1) RNA silencing mightoperate only via RITS at the transcriptional level, henceSlicer would not be required. (2) The DDD motif mayproduce cleavage-active Argonautes. (3) A gene encod-ing an Argonaute ortholog with a DDH motif does existinChlamydomonas, but locates to an extended gap of thegenome sequence.

RNA silencing triggered in Chlamydomonas by antisense

constructs

The first successful application of antisense technologyto Chlamydomonas was reported by Schroda et al.(1999). The authors used the strong HSP70A-RBCS2tandem promoter to drive expression of a 2.5 kbHSP70B cDNA fragment in antisense orientation thatwas terminated by the RBCS2 3 UTR (Fig.3). TheHSP70B gene encodes a plastidic chaperone of theHsp70 family (Schroda2004). In higher plants, this kindof approach, normally using the strong 35S CaMVpromoter to drive expression of the antisense gene, wasintroduced by Van der Krol et al. (1988a,b) as a tool to

downregulate wild-type target genes. Screening of 300transformants at the mRNA level was required toidentify a single transformant affected in HSP70B geneexpression. In this transformant, HSP70B transcriptlevels were reduced by 2040%, protein levels werereduced by 30%; decreased expression was observedmainly after induction of target gene and antisensetransgene by heat stress or light (Schroda et al. 1999;Table1). The underexpression phenotype of this trans-formant was lost after storage of the strain for severalmonths.

Later, the same regulatory sequences, i.e., HSP70A-RBCS2 promoter and RBCS2 terminator, were used todrive expression of the 336-bp DIP13 coding region inantisense orientation (Pfannenschmid et al. 2003;Fig.3). DIP13 encodes a 13 kD microtubule-associatedprotein that is induced by deflagellation. Three of sixteentransformants that contained the DIP13 antisense con-

struct showed a 1050% reduction in DIP13 proteinlevels (Table1). The antisense phenotype was stable forat least 1.5 years (W. Mages, personal communication).

Similar approaches using cDNA fragments in anti-sense orientation terminated by the RBCS23UTR wereemployed to affect expression of the chlamyopsin (COP)and the sulfate permease (SulP) genes, however, theRBCS2 promoter alone was used to drive transgeneexpression. In the case of the COP gene, none of the 48transformants screened was reported to exhibit reducedtarget gene expression (Fuhrmann et al. 2001). In con-trast, about half the transformants containing the SulPantisense construct showed at the maximum a 50%

reduction in SulP transcript levels (Chen and Melis2004; Table1, Fig.3).

Transgene expression in Chlamydomonas was shownto have significantly improved by the presence of intronsin the transgene construct (Lumbreras et al. 1998).Consequently, approaches were launched in which someexons and introns in the 3 part of the opsin genes ofVolvox and Chlamydomonas were inverted (Fig.3). Inboth cases, the native opsin promoters were used. Thisapproach turned out to be highly effective in Volvox,where transformants showed reductions in transcript

Fig. 3 Constructs used in Chlamydomonas to trigger RNA silenc-ing by antisense. Target gene sequences are represented by greyboxes, which in case of genomic sequences contain the exonnumber. The direction of the sequence (sense or antisense) is givenby thearrowheadterminating the boxes. Resistance gene sequencesare drawn as black boxes. Introns are indicated by Vs. Thin-linedarrows designate transcriptional start sites. White boxes indicateheterologous promoter or 3 UTR sequences, native promoters are

given as grey boxes. PR stands for the RBCS2 promoter(Goldschmidt-Clermont and Rahire 1986), PAR for the HSP70A-RBCS2tandem promoter (Schroda et al. 2000). Theasteriskbelowthe second intron of the VOP gene designates a stop codon.Constructs are not drawn to scale

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Table1

Chlamydomonasgenesa

ffectedbyantisenseconstructs

Gene

downregulated

Constructtype

(c)cDNA

(g)genomicDN

A

(s)sense

(as)antisense

Regionof

targetgene

affected

Promoter

driving

construct

Terminator

of

construct

Recipient

strainused

Selection

markerused

(S)ameplasmid

(C)otransformation

%

transformants

showing

downregulation

%

ofWT(R)

mRNA/(P)

proteinleft

inmost

affectedstrains

Stabilityof

phenotype

References

HSP70B

Antisensec(as)

5

2,485bpHSP70A/

RBCS2

RBCS2

cw15302

ARG7(S)

0.3%

(1/300)

608

0%

(R)

70%

(P)

(mainlyafter

induction)

Lost

after

few

months

Schroda

etal.

(1999)

DIP13

Antisensec(as)

336bp

cDNA

HSP70A/

RBCS2

RBCS2

cw15arg7mtARG7(C)

19%

(3/16)

509

0%

(P)

Stablefor

>1.5

years

Pfannenschmid

etal.

(2003)

COP

Antisensec(as)

ex1-8

RBCS2

RBCS2

cw15arg-A

ARG7(C)

0%

(0/48)

Fuhrmann

etal.

(2001)

SULP

Antisensec(as)

3

884bp

RBCS2

RBCS2

cc425

ARG7(C)

50%

(19/31)

50%

(R)

Chenand

Melis(2004)

VOP

Antisenseg(s)/g(as)3

579bp

(ex6-8

)

VOP

VOP

Volvox

carteri

15348

nitA(C)

70%

(P)

Fuhrmann

etal.

(2001)

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levels by more than 90% and in protein levels by morethan 95% (Ebnet et al. 1999; Table1). In contrast,Chlamydomonas opsin in 12% of the transformantsscreened was reduced only by 40% (Fuhrmann et al.2001; Table1). The underexpressing phenotype in Vol-voxwas lost after a few months (Fuhrmann et al. 2001).

To reduce screening efforts and to increase thenumber of transformants that express antisense RNA athigh levels, it appeared reasonable to design a constructthat would generate a message containing the codingregion for a selectable marker and the antisense cDNA.Theble gene that confers resistance against antibiotics ofthe bleomycin family (Stevens et al. 1996) appeared mostsuitable for this purpose, since it was shown to be ex-pressed at fairly high levels in Chlamydomonas (Lum-breras et al. 1998). We generated a construct in whichthe ble gene, driven by the HSP70A-RBCS2 promoter,contained a 278-bp HSP70B antisense cDNA fragmentbetween the ble stop codon and the RBCS2 3 UTR(Fig.3). About 12% of the drug-resistant transformantsfailed to induce the HSP70B gene after heat shock (ourunpublished data). Interestingly, HSP70B-underex-

pressing strains were found only among transformantsthat expressed the ble-antisense construct at low levelsand, therefore, were resistant only to low concentrationsof the antibiotic (our unpublished data). Moreover, insome transformants, the underexpression phenotype waslost over time, but neither was the expression of the ble-antisense construct, nor the drug resistance. Thus, itappears that in these transformants the signal triggeringRNA silencing was lost at a step downstream from theantisense transcript. A similar construct, in which theCOP cDNA instead of HSP70B and the RBCS2 pro-moter alone instead of the HSP70A-RBCS2 tandempromoter were used (Fig.3), affected COP gene

expression in 4% of the transformants; COP proteinlevels were reduced at most by 30% (Fuhrmann et al.2001; Table1).

In summary, antisense constructs have been shown toreduce expression of five different target genes in Volvoxand Chlamydomonas. As little as 195 bp of target se-quences were sufficient. In Chlamydomonas, target geneexpression at the protein level was reduced at most by50%, whereas in Volvox target gene expression wasdiminished by more than 95%. The frequency of un-derexpressing transformants ranged from 0.3 to 50%and loss of underexpression phenotypes was observed insome cases. Co-expression of a selectable marker and

antisense cDNA appeared to improve the frequency ofunderexpressing strains among transformants, but notthe degree of underexpression.

RNA silencing triggered in Chlamydomonasby invertedrepeat constructs

The breakthrough tool for the triggering of RNAsilencing in Chlamydomonas was developed by Fuhr-mann et al. (2001). The authors used a genomic clone of

the COP gene including its own promoter, the first fiveexons, and the first four introns. Right after the fifthexon they cloned the COP cDNA covering the first fiveexons (400 bp) in antisense orientation; a terminatorwas not included (Fuhrmann et al. 2001; Fig.4). Forty-two percent of the transformants screened showed re-duced COP gene expression with reductions in COPprotein levels by up to 98% (Table2). The concept toseparate sense and antisense arms of inverted repeatconstructs by spliceable introns was shown before intobacco and Arabidopsis to trigger RNA silencing withhigh efficiency (Smith et al.2000). The authors suggestedthat intron excision by the spliceosome might improveduplex formation. Alternatively, the assembly of thehairpin RNA into spliceosomes might facilitate its pas-sage from the nucleus to the cytosol.

The genomic-sense/cDNA-antisense strategy ofFuhrmann et al. (2001) was subsequently used todownregulate a number of different genes in Chla-mydomonas. Notably, this strategy remained robustalthough several modifications were introduced. Forexample, the number of exons (and introns) of the target

gene was increased: Pan et al. (2004) used eight exonswhile Huang and Beck (2003) used even ten (Fig.4). Panet al. (2004) reported that in 12% of the transformantsscreened, expression of the target gene (an aurora kinasetermed CALK) at the protein level was reduced by up to95%. Similarly, expression of phototropin (PHOT) was

Fig. 4 Constructs used in Chlamydomonas to trigger RNA silenc-ing by inverted repeats based on genomic sense/cDNA antisense.Symbols are as described in Fig. 3. Constructs are not drawn toscale

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Table2

Chlamydomonasgenesa

ffectedbygenomicsense/cDNAantisenseinvertedrepeatconstructs

Gene

downregulated

Constructtype

(c)cDNA

(g)genomicDN

A

(s)sense

(as)antisense

Regionof

targetgene

affected

Promoter

driving

construct

Terminator

ofconstruct

Recipient

strainused

Selectionmarker

used(S)ame

plasmid

(C)otransformation

%

transformants

showing

do

wnregulation

%

ofWT(R)

mRNA/(P)

proteinleftin

mostaffected

strains

Stability

of

phenotype

References

COP

Hairpin

g(s)/c(as)

ex1-5

COP

None

cw15arg-A

ARG7(C)

42

%

(of48)

25%

(P)

Fuhrmann

etal.

(2001)

CALK

Hairpin

g(s)/c(as)

ex1-8

CALK

CALK

B215

NIT1(C)

12%

(5/42)

570%

(P)

Panetal.

(2004)

PHOT

Hairpin,

loopg(s)/c(as)

1.4kb

(ex1-1

0)

PHOT

None

cc124

aphVIII(S)

10

%

(8/80)