Development of a Heat-Shock Inducible Gene Expression System in the Red Alga Cyanidioschyzon merolae

The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.


Introduction
The unicellular red algae Cyanidiales are defined as thermoacidophiles because they grow at an extremely low pH (0.05-5) and relatively high temperature (35-56uC) [1]. Among the Cyanidiales, the unicellular red alga, Cyanidioschyzon merolae, contains just one nucleus, mitochondrion and chloroplast per cell [2]. Cell division and organelle division are tightly synchronized by the light/dark cycle [3]. These features make this alga a model organism for studying the mechanisms of chloroplast and mitochondrial division [4]. In addition, the complete determination of the nuclear, mitochondrion and chloroplast genomes [5,6,7,8], as well as the establishment of genetic manipulation methods, such as transient plasmid expression [9] and gene targeting by homologous recombination [10,11,12], have contributed to the growing interest in C. merolae as a model organism. The C. merolae nuclear genome size is 16.5 Mbp with 4,774 protein-coding genes. The genome content is extremely simple, with a low level of redundancy in photosynthetic eukaryotes. In addition, most of the genes lack introns [7,8,13]. Thus, in addition to studies on chloroplast and mitochondrial division, C. merolae has become a promising organism for the study of cell biology and metabolisms in photosynthetic eukaryotes, such as vacuolar inheritance [14,15], the dynamics of the endoplasmic reticulum [16] and Golgi apparatus [17], nitrogen assimilation [18,19], chromosome organization [20], and circadian rhythms [21].
The simple nature of the cellular architecture and genome content of C. merolae has facilitated the characterization of fundamental intracellular phenomena. However, since such phenomena are essential to cellular growth and survival, inactivation or modification of the mechanisms by gene manipulation would likely be lethal. To overcome this critical issue, analyses of primary defects using conditional mutations, an approach which is not effective under permissive environmental conditions but can be effective under certain tightly restricted conditions, is greatly desired.
To this end, we have developed an inducible gene expression system using a heat-shock promoter in C. merolae, which we report in this study. As an example of an application of the system, expression of a dominant negative form of the DRP5B/CmDnm2 protein was induced by heat shock, after which the effect on chloroplast division and cell cycle progression was examined.

Algal culture
Cyanidioschyzon merolae 10D or stable transformants were grown in 50 mL of Allen's medium in a 100 mL test tube under continuous light (100 mE m 22 s 21 ) with aeration (500 mL ambient air min 21 ) [22].

Semi-quantitative RT-PCR
Cells were harvested by centrifugation at 8006g, frozen in liquid nitrogen and stored at 280uC until use. Total RNA was extracted with the RNeasy Mini Kit and RNase-Free DNase set (Qiagen, Venlo, Netherlands). cDNA was synthesized from the total RNA using 6 nucleotide random primers with ThermoScript RT (Life Technologies, Carlsbad, CA). The primers used for semiquantitative RT-PCR are listed in Table S1.

DNA construction for the superfolder GFP (sfGFP) expressing strain
According to the amino acid sequence of original sfGFP [23], the oligonucleotides encoding sfGFP for the expression in C. merolae were artificially synthesized so as to introduce the mutations S30R, Y39N, Q80R, F99S, N105T, Y145F, M153T, V163A, I171V, A206V and L231H (accession no. AB971579) into the nucleotide sequence encoding the enhanced GFP (EGFP; [24]). For further construction, BamHI and NotI sites were fused to the 59-and 39-terminals of the sequence, respectively.
To produce a strain which constitutively expresses sfGFP, a plasmid containing APCC promoter-sfGFP-URA was constructed by eliminating the E2F orf from the plasmid APCC promoter-sfGFP-pmE2F-URA [21]. The regions other than pmE2F were amplified from the original plasmid by the primers sfGFP720taaRbt and btUTR(+1), then circularized using an In-Fusion cloning kit (Takara Bio, Shiga, Japan).
To prepare a URA (the URA5.3 gene; CMK046C) expressing strain as a control, APCC promoter-EGFP was eliminated from the plasmid containing APCC promoter-EGFP-URA (pD184-O250-EGFP-URA Cm-Cm ; [11]). The regions other than APCC promoter-EGFP were amplified from the original plasmid using the primers d184(+25)Rura d184(+25)R and CmUra(2897)F and the fragment was circularized using an In-Fusion cloning kit. The primer sequences for the construction are listed in Table S1. The plasmid pD184-O250-EGFP-URA Cm-Cm [11] was used to produce the strain constitutively expressing EGFP.

Construction of the plasmids for the transient transformation assays
To transiently express the sfGFP driven by the upstream region of C. merolae CMJ101C from a plasmid, we constructed plasmids in which a series of different lengths of the CMJ101C upstream region were fused to the sfGFP orf and the Nos terminator. BamHIand NotI-digested sfGFP was cloned between the BamHI and NotI sites of the pI050P-GFP vector [25] to produce the pI050P-sfGFP plasmid. A series of different length of the upstream region of CMJ101C (21000 to +2, 2750 to +2, 2500 to +2, 2345 to +2, 2250 to +2, 2200 to +2 and 2100 to +2) were amplified by PCR. For the forward primers, 101_21000_Xba, 101_2750_Xba, 101_2500_Xba, 101_2345_Xba, 101_2250_Xba, 101_2200_Xba The expression pattern of CMJ101C mRNA after a 0,24 hr heat shock at 50uC. (C) C. merolae wild-type cells were cultured at 34uC before the heat shock. The expression pattern of CMJ101C mRNA before and 0,210 min after one-hour heat shock at 50uC. mRNA levels were examined by semi-quantitative RT-PCR. TIM13 (mitochondrial intermembrane space complex subunit, CMB148C) was used as a quantitative control. C in (A) represents the mRNA level in the cells cultured at 42uC continuously. doi:10.1371/journal.pone.0111261.g001 and 101_2100_Xba were used. For the single reverse primer, 101_+ 2_BglII was used. The amplified fragments were digested with XbaI and BglII and cloned between the XbaI and BamHI sites of pI050P-sfGFP. For convenience, the resultant plasmids were named P-1000, P-750, P-500, P-345, P-250, P-200, and P-100, respectively.

Construction of the plasmids for the stable transformants
To prepare the stable transformants which express sfGFP by 200, 250, or 345-bp upstream region of CMJ101C orf, upstream region comprising CMJ101C, sfGFP and the nos terminator was amplified with the primers CMD184D,+25-M13F_Inf_1 and M13 R. The amplified products were replaced with APCC promoter-EGFP in pD184-O250-EGFP-Ura Cm-Cm [11] by ligation with a linearized vector that was amplified from the plasmid pD184-O250-EGFP-Ura Cm-Cm by the primers CMD184C_+25R and M13R_CmUra-897,Inf_1 using In-Fusion cloning kit.
To produce the GFP-DRP5B or GFP-DRP5B K135A expressing the stable transformants, the stop codon of sfGFP in pI050P-sfGFP was eliminated. The promoter region of the resultant plasmid pI050P-sfGFP (-stop) was then substituted with a 200-bp upstream region of CMJ101C. The 200-bp upstream region of CMJ101C was amplified with the primers 101_2200_Xba and 101_+2_BglII. The amplified fragment was digested with XbaI and BglII and cloned between the XbaI and BamHI sites of pI050P-sfGFP (-stop). The resultant plasmid was digested with NotI and C. merolae DRP5B orf (CMN262C), which was amplified with the primers GFP_Not_Dynamin_inf_1 and GFP_Not_Dynamin_inf_2, was inserted by the In-Fusion cloning kit. For the cloning of the dominant negative form of DRP5B, Lys-135 was replaced by Ala using the primers Dynamin_K44A_sense and Dynamin_K44A antisense. The primer sequences for the construction are listed in Table S1.

Transformation of C. merolae
Transient transformation was performed using a polyethylene glycol (PEG)-mediated protocol, as described previously [9]. To produce stable transformants, 6 mg of the PCR product obtained by the primers D184(1270)F and D184(+1448)R from the aforementioned plasmids were introduced via the PEG-mediated protocol into C. merolae M4, which has a mutation in the URA gene [10], and the transformants were selected as described previously [19].

Quantitative PCR Analyses
Quantitative-PCR analyses were performed using a StepOne-Plus Real-Time PCR System (Life Technologies) and a 20 ml reaction mixture containing 2 ml template DNA, 0.25 nM primers and 10 ml Power SYBR Green Master Mix (Life Technologies). To examine copy number of the introduced DNA fragment, standard curves were constructed using serially diluted solutions of DNA isolated from CmsfGFP-1 strain cells and the relevant sets of primers. The value of GFP (from 638 to 717 bp in the orf) in each strain was normalized with the data of a region (from 741 to 841 bp in CMD184C orf) located outside of the introduced DNA fragment and then the copy number was displayed relative to an EGFP strain, which contains ,1 copy of EGFP sequence [11]. To examine cell cycle progression of the heat-shocked culture, standard curves were constructed by using serially diluted solutions of cDNA that was prepared from S-200 cells cultured at 42uC under light with the relevant sets of primers. The value of PCNA (from 468 to 524 bp in the orf) and CDC20 (from 419 to 473 bp in the orf) were normalized with the data of DRP3 (from 1981 to 2050 bp in the orf). The primer sequences for the quantitative-PCR analyses are shown in Table S1.
Immunoblot analyses 5610 8 cells were harvested by centrifugation at 8006g for 5 min and stored at 280uC. Cells were suspended in SDS-PAGE sample buffer (50 mM Tris, pH 6.8, 6% 2-mercaptoethanol, 2% sodium dodecyl sulfate, 10% glycerol) and sonicated on ice for 6 cycles (10 s on and 30 s off at 310 W; Bioruptor UCW-310, Cosmobio, Tokyo, Japan). After centrifugation at 15,0006g for 10 min at 4uC, the supernatant fraction was separated by SDS-PAGE and analyzed by immunoblotting with a mouse monoclonal anti-GFP antibody (Living Colors A.v. Monoclonal Antibody (JL-8), Clontech, Mountain View, CA) as the primary antibody at a dilution of 1:1,000 and goat anti-mouse IgG-HRP antibody (Life Technologies) as the secondary antibody at a dilution of 1:2,000.

Fluorescence microscopy
The cells were examined using an epifluorescence microscope (BX51, Olympus, Tokyo, Japan) equipped with a CCD camera (DP70, Olympus). The GFP fluorescence and autofluorescence of chlorophyll were captured using an Olympus WIB cube.

Results and Discussion
Expression pattern of CMJ101C mRNA To develop an inducible gene expression system in C. merolae using a heat-shock promoter, we searched for C. merolae HSP genes that are transcribed only when cells are exposed to elevated temperature (i.e. with no basal level expression at a lower temperature).
In the C. merolae genome database (http://merolae.biol.s. u-tokyo.ac.jp/), 14 genes are predicted to encode heat shock proteins. Among these 14 genes, the transcripts of 13 were found in the previous expressed sequence tag (EST) data that was obtained from the culture at the optimal temperature (42uC), while the transcript of one gene, CMJ101C (A BLASTP search in the NCBI database showed that CMJ101C encodes a member of the Hsp20/alpha crystallin family of proteins) that was not found in the EST data. To examine whether CMJ101C is transcribed only at higher temperatures, C. merolae cells cultured at 34uC for one day were shifted to 34,62uC for 20 min. By RT-PCR analyses, CMJ101C mRNA was detected at only 38uC or higher ( Figure 1A) and the mRNA level exhibited a maximum at 50uC.
To determine the optimum duration of the heat shock for inducing CMJ101C transcription, the cells were shifted from 34uC to 50uC. The mRNA level increased for 10 min after the temperature shift and the level was maintained for 24 hr ( Figure 1B). After the 60-min heat shock, the CMJ101C mRNA level rapidly decreased over 30 min (Figure 1C), while a marginal transcript level was detected for at least 210 min, suggesting that CMJ101C transcription ceased immediately after the end of the heat shock. These results led us to explore the usage of the CMJ101C promoter for an inducible gene expression system in C. merolae.
sfGFP functions as a reporter protein in C. merolae To examine the transcriptional activity of the CMJ101C promoter, we searched for GFP variants that are appropriate for C. merolae culture at higher temperatures (34,50uC). The folding of enhanced GFP (EGFP) is optimized at 37uC [24]. In contrast, superfolder GFP (sfGFP; [23]) exhibits bright fluorescence in extreme thermophiles, even at 70uC [26]. In order to compare the efficiency of EGFP and sfGFP as reporter proteins in C. merolae, we prepared strains that constitutively express EGFP or sfGFP (one copy of the orf was integrated into the genome, Figure 2A) under the C. merolae constitutive APCC promoter from a genomic-neutral locus [11]. By quantitative-PCR analyses, we confirmed that a single GFP sequence was integrated into the genome in the transformants (Figure 2A). Immunoblot analysis with an using the anti-GFP antibody showed that the level of sfGFP was higher than that of EGFP in C. merolae cells cultured at 42uC, a temperature which is optimal for C. merolae growth ( Figure 2B). In addition, fluorescence microscopy revealed that sfGFP-expressing cells exhibited brighter fluorescence than EGFPexpressing cells ( Figure 2C). Because the nucleotide sequences encoding the EGFP and sfGFP that we expressed in C. merolae do not contain any rare codons of C. merolae, the above results suggest that sfGFP is more stable than EGFP in C. merolae when the cells were cultured at 42uC. Thus, we used sfGFP as the reporter protein for further investigation.
Determination of the optimal CMJ101C promoter region for the heat-shock-inducible gene expression system Upstream of the CMJ101C orf, the CMJ100C orf locates in the opposite orientation and the interval between the two orfs is 345 bp ( Figure 3A). It is well known that there are conserved cisregulatory promoter elements called heat shock elements in the upstream region of heat shock genes (reviewed in [27]), but there are no typical heat shock elements (at least of the three nGAAn elements) in the 345-bp region. In order to define the region that is optimal for transcription specifically in response to heat shock, we fused a series of CMJ101C upstream sequences of different length to sfGFP orf and the constructs (plasmids; P-100 to P-1,000) were transiently transformed into C. merolae wild-type cells ( Figure 3A). The transient transformation of C. merolae cells resulted at most in 2,10% of transformants in a population [9]. After transforma- Quantitative PCR analyses showing that a single copy of EGFP or sfGFP was integrated into the nuclear genome of the respective transformants. The CMD184C-mid region, which locates outside of the introduced DNA fragment (a) and GFP sequence (b), was investigated by quantitative PCR. The value of (b) in each strain was normalized against that of (a) to estimate the copy number. The copy number is shown relative to an EGFP strain, which contains ,1 copy of the EGFP sequence [11]. The bars indicate the SD (n = 3). (B) EGFP and sfGFP protein levels were compared by immunoblotting. Two sfGFP strains were isolated independently (sfGFP-1, sfGFP-2). The URA strain was used as a negative control, in which the URA gene (URA5.3, CMK046C) was introduced into the genomic-neutral locus of C. merolae M4 that has a mutation in the URA5.3 gene. Coomassie Brilliant Blue (CBB) staining of the gel is shown as a loading control. (C) Fluorescence of EGFP and sfGFP were observed by fluorescence microscopy. The fluorescence from GFP and autofluorescence of chlorophyll were captured simultaneously. The images were captured using the same exposure time (1 sec). The scale bar = 5 mm. doi:10.1371/journal.pone.0111261.g002 tion, cells were cultured at 36uC for 24 h and then the temperature was shifted to 50uC.
After 1-h heat shock at 50uC, GFP fluorescence-positive cells were detected, especially in the cultures transformed with P-200, P-250 and P-345. However, the fluorescence-positive cells were also observed before the heat shock treatment in the culture transformed with P-250 ( Figures 3B, 3C). Consistent with this observation, the immunoblot analysis showed that GFP was expressed in the cultures transformed with P-200, P-250 and P-345 after 1-h heat shock at 50uC, and that GFP was expressed before the heat shock in the culture transformed with P-250 ( Figure 3D).
The above results obtained by the transient expression of GFP from the plasmids suggest that a 200-bp or 345-bp upstream sequence of CMJ101C orf is suitable for the heat shock inducible gene expression system, but the plasmid copy number of the transformants varies with the method, so it is difficult to exactly compare the transcriptional activity of the upstream sequences before and after the heat shock. Thus, we prepared the stable transformants (S-200, S-250 and S-345) in which a single promoter (the 200, 250 or 345-bp upstream region of CMJ101C orf) and GFP orf fusion was inserted into the same genomicneutral site for further evaluation.
To evaluate the basal level of GFP expression before the induction by heat shock, the S-200, S-250 and S-345 stable transformants were cultured at 35, 37, 40, 42, and 45uC for one day. It was shown by semi-quantitative RT-PCR that GFP mRNA was detected in the S-200 cells at only 42uC and 45uC. In contrast, in the S-250 and S-345 cells, the mRNA was detected at all of the temperatures tested, even though a higher level of mRNA was detected at 42uC and 45uC ( Figure 4A). Consistent with the results of RT-PCR, GFP fluorescence was detected in the S-200 cells at only 42uC and 45uC. In contrast, some of the S-250 and S-345 cells exhibited GFP fluorescence at lower temperatures (S-250 at 35, 37 and 40uC, and S-345 at 37 and 40uC)( Figure 4B). The strongest fluorescence was observed at 45uC in all of the transformants. These results indicate that the 200-bp upstream region of CMJ101C orf is the most suitable for the heat shock inducible system in the sense of there being little leakage of expression at lower temperatures.

Determination of the optimum temperature and duration for heat-shock inducible gene expression in the stable transformants
To determine the optimum temperature at which heat shock induces gene expression in the stable transformants, the S-200 cells were shifted from 34uC to 34-62uC and cultured for one hour. The semi-quantitative RT-PCR showed that the GFP mRNA level peaked at 50-58uC ( Figure 5A). Immunoblotting and fluorescence microscopy showed that the GFP protein and fluorescent level peaked at 50-54uC ( Figure 5B, 5C). Based on these results, we fixed the heat shock temperature at 50uC for further analyses.
To determine the optimum duration of the 50uC heat shock to induce gene expression in the stable transformants, the S-200 cells were shifted from 34uC to 50uC and cultured for 120 min. GFP mRNA was detected in within 5 min of the temperature shift and reached the maximum level at 30 min ( Figure 6A). The GFP protein was detected at 30 min and reached the maximum level at 60 min ( Figure 6B). GFP fluorescence-positive cells were detected at 30 min and the strongest fluorescence was detected at 60 min ( Figure 6C). Thus, for the expression of GFP, at least 30-min heat shock is required and the protein expression level increased for a further 60 min after the temperature shift. Because GFP mRNA was detected for at least 24 hours in cells cultured at 50uC (Figure 6D), the duration of translational induction is likely to be able to be extended for particular purposes.
After the one hour heat shock at 50uC, the GFP mRNA level decreased rapidly ( Figure 7A). In contrast, the GFP protein and fluorescence was detected even one day after the heat shock ( Figures 7B, 7C). This difference between the mRNA and protein level is probably because of the high stability of GFP [28]. In conclusion, for the expression of a target gene by the 200-bp upstream region of CMJ101C orf, a heat shock at 50uC for at least  Effect of DRP5B mutation on chloroplast division and cell cycle progression: An example of the application of the C. merolae heat-shock gene expression system To apply the inducible gene expression system to an investigation of chloroplast division, we expressed a dominant negative form of DRP5B/CmDmm2 which has a mutation in the GTPase domain. Because the C. merolae cell, as in the case of many other algal species, contains only a single chloroplast, the blockage of chloroplast division is probably lethal and thus conditional mutation, such as the inducible expression system employed in this study, is required to characterize the effect of the mutation on chloroplast division.
DRP5B is a member of a eukaryotic dynamin family of selfassembling GTPases and localizes on the cytosolic side of the chloroplast division site, where it is involved in the division process [29,30]. Conventional dynamin, which is involved in endocytosis, consists of five domains: an N-terminal GTPase domain, a middle domain, a pleckstrin homology domain, a GTPase effecter domain and a proline-rich domain (Reviewed in [31], Figure 8A). GTPase activity is essential for membrane fission in endocytosis and the K44A mutation in human dynamin 1 is known to abolish the activity of dynamin binding GTP, and the expression of the K44A mutant protein exhibits dominant-negative effects [32]. The GTP binding domain is well conserved in the DRP5B of algae and plants ( Figure 8A). It has been shown that DRP5B is required for normal chloroplast division based on the impairment of chloroplast division in DRP5B knockout or null mutants in Arabidopsis thaliana [30] and Physcomitrella patens [33]. In addition, DRP5B is recruited to the chloroplast division site before the onset of division site constriction and persists there throughout the division process [29]. However, it is not known how GTP-binding and/or GTP hydrolysis by DRP5B is involved in chloroplast division. To investigate the role of GTP-binding and/or hydrolysis by DRP5B in chloroplast division in C. merolae, we induced the expression of GFP-DRP5B or GFP-DRP5B K135A, the mutation of which corresponds to K44A of the human dynamin1 mutation, by the heat shock system in the stable transformants ( Figure 8A).
In exponentially proliferating culture of both of the transformants, the GFP signal was not detected at 40uC. The cells were transferred to a dark condition to stop cell growth and new entrance of G1-phase cells into the S phase and then were heatshocked at 50uC for 1 h ( Figure 8B). After the heat shock, the GFP signal was detected at the chloroplast division site (Figures 8C, 8D. indicated with single, double and triple arrowheads), although additional fluorescent speckles were observed in the cytosol of the GFP-DRP5B K135A expressing cells ( Figure 8D). Cells were heatshocked again for 1 h (4-5 h in Figure 8B). After 24-h incubation under dark at 42uC, GFP-DRP5B cells had completed chloroplast division and subsequent cytokinesis, and thus GFP fluorescence was not detected. In contrast, GFP fluorescence was still observed in some GFP-DRP5B K135A cells ( Figure 8D). After 1-h heat shock, GFP fluorescence was observed at the chloroplasts division site before and during the earlier and later stages of the division site constriction in the both transgenic lines. In contrast, after 24-h incubation under dark, GFP-DRP5B K135A cells with GFP fluorescence were at the final stage of chloroplast division and displayed aberrant shapes in which two daughter chloroplasts and the cells were still connected by a small DRP5B K135A positive tube-like structure ( Figures 8D, E, indicated by the arrowheads in D). DNA-specific fluorescent dye (DAPI) -staining showed that these aberrant shaped cells contain two daughter nuclei on opposite sides of the tube-like structure ( Figure 8F). These results suggest that the expression of DRP5B K135A inhibited the final scission of the division site, but not the earlier stage of division site constriction in the course of chloroplast division. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage, although the completion of cytokinesis is blocked, probably induced by obstruction brought about by the tubular bridge connecting the two daughter chloroplasts.  In the GTPase domain, four GTP binding motifs (G1, G2, G3 and G4) contribute to the GTP binding. Partial alignment of the DRP5B proteins showed the conservation of the G1 motif. The DRP5B amino acid sequences (Chlamydomonas reinhardtii, GI: 30349146; Chlorella variabilis, GI: 552813628; Ostreococcus tauri, GI: 308803420; Arabidopsis thaliana, GI: 30349146; Physcomitrella patens, GI: 224434564; Cyanidioschyzon merolae, GI: 544214467) were aligned using ClustalW [35,36]. Alanine substitution for the conserved lysine residue within G1 of human dynamin 1 is known to result in a dominant negative effect When GFP-DRP5B and GFP-DRP5B K135A expressing cells were transferred to light condition after 24-h dark period, the transcript levels of both S-phase marker PCNA (CMS101C) and M-phase marker CDC20 (CMA138C) [22] increased for 12 hour (24 h to 36 h in Figure 8G) and then decreased (36 h to 48 h in Figure 8G). These results indicate that both GFP-DRP5B K135A expressing cells entered and finished the next cell cycle even though final stage of chloroplast division and cytokinesis had been blocked.
In conclusion, we developed a heat-shock inducible gene expression system in C. merolae. By using the CMJ101C promoter, the heat shock at 50uC for 1 h resulted in the induction of maximum level of mRNA and protein expression. In addition, the results show that the level of expression is adjustable by changing the duration or temperature of the administered heat shock.
As an example of an application of the system, here we have examined the function of DRP5B in chloroplast division by expressing the dominant negative form. Although it has not been tested in this study, this system is probably also applicable to the knockdown expression of genes of interest by heat shock induction of antisense RNA expression. The C. merolae genome does not encode RNAi machinery components such as Dicer [34]. However, gene expression knockdown by antisense RNA has been achieved by the transient introduction of plasmids in C. merolae [25].
The use of the authentic URA (CMK046C) selection marker guarantees single-copy insertion of constructs into the genome [11,20]. In contrast, a recent study showed that the use of an artificial URA gene results in genomic insertion of multiple copies (5,50 copies) of a construct into genomic-neutral loci [11]. By combining the heat shock inducible expression system and the artificial URA marker, it is likely to be possible to further increase the induction level of mRNA or antisense RNA.