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The unicellular green alga Chlamydomonas reinhardtii is a model organism for various studies in biology. CC-124 is a laboratory strain widely used as a wild type. However, this strain is known to carry agg1 mutation, which causes cells to swim away from the light source (negative phototaxis), in contrast to the cells of other wild-type strains, which swim toward the light source (positive phototaxis). Here we identified the causative gene of agg1 (AGG1) using AFLP-based gene mapping and whole genome next-generation sequencing. This gene encodes a 36-kDa protein containing a Fibronectin type III domain and a CHORD-Sgt1 (CS) domain. The gene product is localized to the cell body and not to flagella or basal body.
The biflagellated unicellular green alga Chlamydomonas reinhardtii is a model organism used in various fields of biology, such as flagellar motility, photosynthesis, photomovement and sexual reproduction. One of its strong advantages is the feasibility of genetic studies. C. reinhardtii has two mating types (mt+ and mt−) and undergoes sexual reproduction under nitrogen-starved conditions, which results in the production of four daughter cells amenable to classical tetrad analysis.
Among several wild-type strains of C. reinhardtii, the most widely used are CC-125 (mt+) and CC-124 (mt−). This pair has descended from Ebersold/Levine 137c strain, from which many mutants have been isolated . However, strictly speaking, CC-125 and CC-124 are mutants: both carry mutations in NIT1 and NIT2 loci and cannot grow on nitrate as the sole nitrogen source , . Moreover, CC-124, but not CC-125, carries a mutation in the AGG1 locus, which causes strong negative phototaxis; cells form aggregates at the bottom of a test tube under room light, in contrast to the positively phototactic CC-125 tending to swim toward the surface of the medium . Hereinafter we refer to CC-124 as agg1.
Since the agg1 mutant has been used as a wild type, the mutation in the AGG1 locus must be unintentionally present in many mutant strains . The sign of phototaxis is regulated by various cellular factors, such as the phase of circadian rhythm, reduction-oxidation poise and internal cAMP level , , . In order to precisely understand the phenotypes of mutants deficient in the phototactic signaling pathway, we need to determine the presence or absence of agg1 in experimental strains. Identification of the AGG1 gene is therefore important for studies using C. reinhardtii and related green algae, particularly in the field of photobiology and behavior. Classical genetic studies based on tetrad analysis have mapped the AGG1 locus to linkage group XIV (currently chromosome 13) . However, the locus has not been sufficiently narrowed down to allow determination of the causative gene.
In this study, we identified the agg1 mutation by AFLP-based gene mapping and next-generation sequencing. The causative gene encodes a novel protein containing a Fibronectin type III domain and a CHORD-Sgt1(CS) domain. This protein was found localized to the cell body and not to the flagella.
Chlamydomonas reinhardtii strains, CC-125 (AGG1; mt+) and CC-124 (agg1; mt−), were obtained from the Chlamydomonas Center (http://www.chlamy.org/). These strains and the transformants agg1::AGG1-3×hemagglutinin (HA) and bld12::SAS6-HA were grown in Tris-acetate-phosphate (TAP) medium with aeration at 25 °C on a 12 h/12 h light/dark cycle , . For immunofluorescence microscopy, cells were grown under constant illumination.
CC-125 was crossed to agg1 (mt−) to obtain an agg1 (mt+) strain. The agg1 (mt+) strain was crossed to a polymorphic strain, S1C5 (mt−, CC-1952; ), for AFLP-based gene mapping. Recombination frequencies between agg1 and genetic markers were determined by detecting polymorphic PCR products in 77 progenies that displayed strong negative phototaxis . The agg1 mutation was mapped to a 1537-kb region between two genetic markers on chromosome 13: STS79-178 (5′-TAGGGACACCCAAGGTAATGAGCA-3′, 5′-ACGCTCAACTGTTCTAGACCCGAG-3′ and 5′-CCGGAAGGCTACGAATGAGATACA-3′) and SSR116–16 (5′-CTCGGGTGAGCTGCAATCAGTAG-3′ and 5′-CCTGTAAGCCCAGACAGGTCAAAC-3′).
Cell walls were removed from cells of agg1 and AGG1 (selected from the progenies of an agg1× CC-125 cross) strains by autolysin treatment . DNA was prepared using a DNeasy Plant Mini kit (QIAGEN) following the manufacturer's instructions. Two micrograms of each DNA sample was fragmented using a Covaris sonicator. ~300 bp DNA fragments were then purified using a Pippin Prep system (Sage Science) and were used to construct sequence libraries using an Illumina TruSeq library prep kit (Illumina) essentially following the manufacturer's instruction except that the number of PCR cycles in the amplification enrichment step was reduced to six to minimize the PCR amplification bias. The libraries were sequenced using Illumina HiSeq 2000 to produce 2×101 bp paired-end reads. In total, 48.2 M (9.7 Gbp) and 71.6 M paired-end reads (14.5 Gbp) were obtained for agg1 and the AGG1 strain, respectively.
Sequence reads were aligned onto the Joint Genome Institute (JGI) version 5.3.1 (Creinhardtii_236.fa.gz) Chlamydomonas genome sequence (https://phytozome.jgi.doe.gov/pz/portal.html#! info? alias=Org_Creinhardtii) using bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). The resulting SAM (Sequence Alignment/Map) files were converted to BAM (Binary Sequence Alignment/Map) and sorted by SAMtools version 0.1.18 (http://samtools.sourceforge.net/). To identify the mutation in the mapped region of agg1 genome, the alignment data were visualized using IGV software (version 2.3.39; https://www.broadinstitute.org/igv/) and compared to each other or to the genome database.
For cloning of AGG1 cDNA, total RNA was prepared and reverse-transcription PCR was performed following the method of , using primers 5′-GCGCCATATGTCCTGGCTCAGCAGCTTT-3′ and 5′-GCGCAGATCTTCAATCCCAGCTGGGCCGG-3′.
For detection of transposon insertion at 5′ untranslated region (UTR) of AGG1, the following primers were used: #1: 5′-CTAACGCAACACGGCAGTTA-3′, #2: 5′-CCTGCATTGAGACTGCGCCC-3′, #3:5′-TTAGGGCCAGTGGTGGTTTA-3′ (see Fig. 1C for details).
AGG1 cDNA with a 6×His tag at the C terminus was cloned into a bacterial expression vector, pMaL-c2X (New England Biolabs). AGG1 was expressed as a fusion protein with maltose-binding protein (MBP). MBP-AGG1-6×His was purified using amylose resin (New England Biolabs) and used to immunize a guinea pig. The obtained antiserum was blot-purified using AGG1 after proteolytic removal of MBP- and 6×His-tags.
The cDNA of AGG1 with a 3×HA tag at the C terminus was cloned into the pGenD expression vector containing the paromomycin resistance gene aphVIII , . The DNA construct was introduced into agg1 cells by electroporation (NEPAGENE) . A transformant that displayed positive phototaxis and expressed AGG1-3×HA was isolated.
Dish phototaxis assay was performed following the method of . Tube phototaxis assay was performed following the method of  with modifications. Briefly, cells were washed with an assay solution (5 mM Hepes (pH 7.4), 0.2 mM EGTA, 1 mM KCl, and 0.3 mM CaCl2) and kept under red light for ~1 h before the assays. Cell suspensions (~7 ml, ~1×107 cells/ml) were put in test tubes (18 mm×180 mm) and illuminated with a white LED (ATTO Flatviewer; ATTO) from the top. Cell distribution patterns in both assays were photographed.
Immunofluorescence microscopy was carried out as described previously . Anti-HA tag antibody (1:200, 11867423001; Roche Applied Science) and anti-acetylated α-tubulin antibody (1:200, ab24610; abcam) were used as primary antibodies. Anti-mouse IgG antibody conjugated with Alexa Fluor 350 (1:200, A11045; Life technologies) and anti-rat IgG antibody conjugated with DyLight 549 (1:200, 612-142-120; ROCKLAND) were used as secondary antibodies. Images were taken with a CCD camera (DP73; Olympus).
Western blot analysis of cell fractions was carried out using anti-HA (11867423001; Roche Applied Science) and anti-ODA-IC2 (D6168; Sigma Aldrich) antibodies as primary antibodies and anti-rat IgG (NA935V; GE Health Care), anti-mouse IgG (NA931V; GE Health Care), and anti-guinea pig IgG (A7289; Sigma-Aldrich) antibodies as secondary antibodies. The cell fractions were prepared following the procedures described previously .
The AGG1 locus was narrowed down to an ~1537-kb region on chromosome 13 by linkage mapping (see Section 2). The causal mutation of agg1 was searched for in the mapped region by a visual comparison of genome sequence of the agg1 mutant with that of an agg1-free progeny from the cross agg1× CC-125 or with that of the strain CC-503 registered in the JGI database using the IGV browser (see Section 2). We found three agg1-specific mutations in the mapped region: in Cre13.g590400, paired-reads of the agg1 genome did not exactly map to the 5′ terminus of the first exon in the reference database, suggesting an insertion; Cre13.g603000t2.1 and Cre13.g604250 have a single nucleotide substitution in an intron (13:4,387,946 bp C to A, and 13:4,655,586 bp G to A, respectively). Among these, the mutation in the Cre13.g590400 is the most likely candidate. Sanger DNA sequencing of this locus revealed that an insertion of a transposon, X56231.1 (also known as TOC1) in the 5′ UTR of Cre13.g590400 ,  (Fig. 1A). It is a non-autonomous version of TOC3, a retroelement encoding a tyrosine recombinase, widespread in the C. reinhardtii genome .
The gene product of Cre13.g590400 is predicted to be a novel 317-amino-acid protein containing a Fibronectin type III domain and a CHORD-Sgt1(CS) domain (Fig. 1B). The Fibronectin type III domain has been found in a variety of proteins and does not suggest specific protein functions. The CS domain is the core of NudC, a protein associated with dynein and involved in nuclear migration; because of this, Cre13.g590400 has been annotated as a NudC-like protein (33% identical to murine NudC) , .
To determine whether Cre13.g590400 is in fact the causal gene of agg1, we examined the correlation between the presence of the transposon at the 5′ UTR of Cre13.g590400 and the phototactic behavior of the cell. We assayed tetrads from the agg1× CC-125 cross for the sign of phototaxis either by a “test-tube assay” (Materials and Methods), or by visual inspection under the microscope. In test-tube assays, negatively phototactic cells tend to aggregate at the test tube bottom in response to the room light (“agg” phenotype) (Fig. 2A). In 80 tetrads (320 progenies) examined, progenies with opposite phototactic signs were always segregated 2:2, and the two progenies displaying negative phototaxis always contained the inserted transposon, as detected by PCR (Fig. 2B).
The segregation indicates that the transposon insertion in the Cre13.g590400 gene of agg1 blocks its expression and cause negative phototaxis. To examine this possibility, we introduced Cre13.g590400 cDNA in an expression vector into the agg1 mutant. As expected, the transformed cells expressing the Cre13.g590400 protein tagged with 3×HA showed positive phototaxis under conditions where the agg1 mutant showed negative phototaxis (Fig. 3A).
To detect the AGG1 protein (the product of the Cre13.g590400 gene, GenBank accession#: XP_001692941) in the cell, we raised antiserum against bacterially expressed protein. Western blot analysis of whole cell extract using the affinity-purified antiserum detected a band with the expected molecular mass of AGG1 (35.8 kDa) in wild type, and a band expected for 3×HA-tagged AGG1 (40.6 kDa) in the transformant, but no bands in the agg1 mutant (Fig. 3B). From these results, we concluded that Cre13.g590400 is the causal gene of the agg1 mutation.
Western blot analysis of fractions from wild type cells indicated that AGG1 localizes to the cell body (Fig. 4A). Consistent with this observation, immunofluorescence images of the agg1::AGG1-3×HA transformant using an anti-HA antibody, which detects only AGG1-3×HA in western blotting, showed that AGG1 is present as small particles in the cell body (Fig. 4B and C). The signal did not appear to be concentrated at the eyespot or basal body. TargetP (http://www.cbs.dtu.dk/services/TargetP/), an analytical tool to predict the subcellular localization of eukaryotic proteins, suggests that AGG1 has a plant-type mitochondrial targeting peptide (mTP score=0.810) . However, we have thus far been unable to detect mitochondrial localization of AGG1. Further studies are necessary to determine its subcellular localization.
In this study, we identified the causal gene of the agg1 mutation, which is carried by CC-124, a widely used wild-type strain of C. reinhardtii. In the agg1 mutant, a transposon, TOC1, is inserted in the 5′ UTR of the Cre13.g590400 gene and blocks the expression of a 317 amino-acid polypeptide (AGG1) that localizes to the cell body. As stated in Introduction, agg1 mutation may well be distributed among many kinds of C. reinhardtii mutants used in various research fields. Identification of the agg1 gene sequence must be useful for detection and elimination of the agg1 mutation from those mutants.
How does agg1 display strong negative phototaxis? Detailed analyses of wild-type and agg1 flagellar movements revealed that, in wild-type cells, the trans-flagellum (the one farthest from the eyespot) beats stronger than the cis-flagellum (the one nearest to the eyespot) after photoreception, whereas in agg1 cells, the cis-flagellum beats stronger than the trans-flagellum after photostimulation , . The beating balance between the cis- and trans-flagella has been shown to be regulated by submicromolar Ca2+ , . However, in vitro motility reactivation of demembranated cell models in Ca2+ buffers showed no significant difference in the Ca2+ sensitivity between wild-type and agg1 flagellar axonemes . Thus, it is unlikely that the loss of AGG1 in the cell body directly affects flagellar function. Recently, cAMP is also shown to affect the beating balance between two flagella in vitro . AGG1 may modulate the signaling pathway in which photoreception causes a change in cellular signals, such as cAMP or Ca2+ concentrations.
The properties of AGG1, including the function of Fibronectin type III and CS-domains, remain to be clarified. The CS-domain is known to be involved in a dynein-regulating protein NudC and Chlamydomonas has a NudC-like protein that regulates axonemal dyneins. However, it is unlikely that AGG1 is involved in dynein regulation since it is localized in the cell body and Chlamydomonas has no cytoplasmic dynein functioning in the cell body. BLAST search suggests that Volvox carteri and Gonium pectorale have AGG1 homologs, Vocar20015120m.g (XP_002949933.1) (62.1% similarity) and GPECTOR_33g581 (KXZ47699.1) (76.8% similarity), which also have Fibronectin type III- and CS-domains. It is interesting to note that these two organisms are colonial algae and may not need to regulate the beating balance of two flagella , . This feature also suggests that AGG1 is not directly involved in the regulation of flagellar beating, although it should be tested by determination of AGG1 localization in these algae or by knock-down/out experiments.
A previous study showed that the lack of AGG2 or AGG3 gene products also results in the “agg” phenotype . AGG2 is a membrane protein that localizes to the proximal flagellar region, and AGG3 is a flavodoxin that localizes to the flagellar matrix. These proteins interact with each other and are suggested to function in a pathway that also involves AGG1 . However, our results clearly showed that the localization of AGG1 differs from that of AGG2 or AGG3. Volvox carteri has multiple AGG2 homologs Vocar.0001s1693.1 (XP_002951946.1), Vocar.0001s1548.1 (XP_002947691.1), Vocar.0017s0113.1 (XP_002953333.1), and Vocar.0017s0112.1 (XP_002953334.1), and an AGG3 homolog Vocar.0028s0169.1 (XP_002947514.1). Gonium pectorale has an AGG2 homolog GPECTOR_15g310 (KXZ50626.1) and an AGG3 homolog GPECTPR_60g718 (KXZ44941.1). Intriguingly, AGG2 is conserved in plants (e.g. Oryza sativa indica group EAY92439.1; Medicago truncatula AFK38344.1) and so is AGG3 (e.g. Citrus clementine XP_006452110.1; Brassica oleracea var. oleracea XP_013625297.1). Because these plants do not have AGG1 homologs, this also indicates that AGG1 functions in a different pathway from that involves AGG2 and AGG3. For determination of the functional relationship between AGG1, AGG2 and AGG3, identification of the structural basis for the punctate localization of AGG1 and analysis of light-induced changes in cytoplasmic factors in agg1~agg3 mutants should be important future challenges.
We thank Dr. Tatsuya Kitazume, Ms. Hisayo Asao (NIBB), and Ms. Mishio Toh (Univ. Tokyo) for Illumina sequencing, Ms. Naomi Miyamoto (Hosei Univ.) for linkage mapping, Dr. Toru Hisabori (Tokyo Tech) for fruitful discussion, and Dr. Ritsu Kamiya (Gakushuin Univ.) for critical reading of this manuscript. This work was supported by JSPS KAKENHI Grant numbers 25291058, 26650093, 15H01206, and 15H01314 to KW, National Institute for Basic Biology Collaborative Research Program 14-733 to KW and Network Joint Research Center for Materials and Devices 2015298 to MH.
Appendix ATransparency document associated with this article can be found in the online version at 10.1016/j.bbrep.2016.07.016.