Effect of marker-free transgenic Chlamydomonas on the control of Aedes mosquito population and on plankton.

BACKGROUND: More than half of the world's population suffers from epidemic diseases that are spread by mosquitoes. The primary strategy used to stop the spread of mosquito-borne diseases is vector control. Interference RNA (RNAi) is a powerful tool for controlling insect populations and may be less susceptible to insect resistance than other strategies. However, public concerns have been raised because of the transfer of antibiotic resistance marker genes to environmental microorganisms after integration into the recipient genome, thus allowing the pathogen to acquire resistance. Therefore, in the present study, we modified the 3-hydroxykynurenine transaminase (3hkt) and hormone receptor 3 (hr3) RNAi vectors to remove antibiotic resistance marker genes and retain the expression cassette of the inverse repeat sequence of the 3hkt/hr3 target gene. This recombinant microalgal marker-free RNAi insecticide was subsequently added to the suburban water in a simulated-field trial to test its ability to control mosquito population. METHODS: The expression cassette of the 3hkt/hr3 inverted repeat sequence and a DNA fragment of the argininosuccinate lyase gene without the ampicillin resistance gene were obtained using restriction enzyme digestion and recovery. After the cotransformation of Chlamydomonas, the recombinant algae was then employed to feed Aedes albopictus larvae. Ten and 300 larvae were used in small- and large-scale laboratory Ae.albopictus feeding trials, respectively. Simulated field trials were conducted using Meishe River water that was complemented with recombinant Chlamydomonas. Moreover, the impact of recombinant microalgae on phytoplankton and zooplankton in the released water was explored via high-throughput sequencing. RESULTS: The marker-free RNAi-recombinant Chlamydomonas effectively silenced the 3hkt/hr3 target gene, resulting in the inhibition of Ae. albopictus development and also in the high rate of Ae. albopictus larvae mortality in the laboratory and simulated field trials. In addition, the results confirmed that the effect of recombinant Chlamydomonas on plankton in the released water was similar to that of the nontransgenic Chlamydomonas, which could reduce the abundance and species of plankton. CONCLUSIONS: The marker-free RNAi-recombinant Chlamydomonas are highly lethal to the Ae. albopictus mosquito, and their effect on plankton in released water is similar to that of the nontransgenic algal strains, which reduces the abundance and species of plankton. Thus, marker-free recombinant Chlamydomonas can be used for mosquito biorational control and mosquito-borne disease prevention.

Control of Aedes mosquito populations using recombinant microalgae expressing short hairpin RNAs and their effect on plankton.

New biocontrol strategies are urgently needed to combat vector-borne infectious diseases. This study presents a low-cost method to produce a potential mosquito insecticide that utilizes the microalgae released into suburban water sources to control mosquito populations. Chlorella microalgae are ubiquitous in local waters, which were chosen as the host for genetic transfection. This species facilitated the recombinant algae to adapt to the prevailing environmental conditions with rapid growth and high relative abundance. The procedure involved microalgae RNAi-based insecticides developed using short hairpin RNAs targeting the Aedes aegypti chitin synthase A (chsa) gene in Chlorella. These insecticides effectively silenced the chsa gene, inhibiting Aedes metamorphosis in the laboratory and simulated-field trials. This study explored the impact of recombinant microalgae on the phytoplankton and zooplankton in suburban waters. High-throughput sequencing revealed that rapid reproduction of recombinant Chlorella indirectly caused the disappearance of some phytoplankton and reduced the protozoan species. This study demonstrated that a recombinant microalgae-based insecticide could effectively reduce the population of Aedes mosquitoes in the laboratory and simulated field trials. However, the impact of this technology on the environment and ecology requires further investigation.

Development of an RNAi-based microalgal larvicide for the control of Aedes aegypti.

BACKGROUND: Mosquito-borne diseases affect over half of the human population globally. Multiple studies have shown that chemical insecticides are ineffective because of resistance. Therefore, environmentally safe mosquito population control tools need to be developed. Ribonucleic acid interference (RNAi) is a reverse genetic mechanism recently introduced as a new pest control tool. This technique represents a new class of biorational technology that could combat the increased global incidence of insecticide resistance. The technique has the potential of becoming a critical component of integrated vector control programs. METHODS: A 3-hydroxykynurenine transaminase (3-HKT) RNAi expression plasmid was constructed, generated and transformed into Chlamydomonas and Chlorella algae. The transgenic algae were then used to feed Ae. aegypti mosquito larvae. The feeding experiments were conducted on a small and large scale with 10 and about 300 larvae, respectively. The mortality rate of the larvae was calculated over 30 days. In addition, histological examination of the insect tissues was performed to examine the extent of tissue damage. RESULTS: The integumentary system and midguts of larvae fed with transgenic Chlamydomonas were severely damaged. The mortality rate of the larvae fed with transgenic Chlamydomonas ranged from 60 to 100% in small-scale tests. The survival rate of adult mosquitoes was 0.0% in a large-scale feeding experiment when the larvae were fed with transgenic Chlamydomonas. Moreover, when the larvae were fed with transgenic Chlorella, the mortality rate ranged from 6.7% to 43% compared to that fed wild-type Chlorella. CONCLUSIONS: 3HKT RNAi transgenic algae are in some scales lethal to Ae. aegypti. The findings of this study indicate that technology based on microalgae RNAi may provide a new way to control mosquito populations.

Controlling the development of the dengue vector Aedes aegypti using HR3 RNAi transgenic Chlamydomonas.

The Aedes aegypti mosquito plays an important role in the spread of diseases, including epidemic ones, such as dengue fever, Zika virus disease, yellow fever, and chikungunya disease. To control the population of Ae.aegypti, we transferred an HR3 RNAi fragment into the microalgae Chlamydomonas, which serves as food for Ae.aegypti larvae. Results showed that the HR3 RNAi transgenic algal strains were lethal to Ae.aegypti. The integumentary system of larvae fed with HR3 RNAi transgenic algal strains was severely damaged. Muscles of the larvae were unevenly distributed and disordered, and their midgut showed disintegration of the intestinal cavity. RNA-Seq results demonstrated that on the 4th day of inoculation with the transgenic algae, the abundance of early expressed genes in the 20E signal transduction pathway of larvae fed with the HR3 RNAi transgenic algal strain significantly reduced. These genes include E74, E75, E93, and 20E receptor complex EcR/USP and FTZ-F1 gene regulated by HR3. In later experiments, a scale test of 300 Ae.aegypti eggs per group was carried out for 30 days, and the survival rate of Ae.aegypti fed with the HR3 RNAi transgenic strain was only 1.3%. These results indicate that the HR3 RNAi transgenic strain exerts obvious insecticidal effect.

Genome-Wide Identification of Calcium-Dependent Protein Kinases in Chlamydomonas reinhardtii and Functional Analyses in Nitrogen Deficiency-Induced Oil Accumulation.

Calcium-dependent protein kinases (CDPKs) are recognized as important calcium (Ca2+) sensors in signal transduction and play multiple roles in plant growth and developmental processes, as well as in response to various environmental stresses. However, little information is available about the CDPK family in the green microalga Chlamydomonas reinhardtii. In this study, 15 CrCDPK genes were identified in C. reinhardtii genome, and their functions in nitrogen (N) deficiency-induced oil accumulation were analyzed. Our results showed that all CrCDPK proteins harbored the typical elongation factor (EF)-hand Ca2+-binding and protein kinase domains. Phylogenetic analysis revealed that these CrCDPKs were clustered into one group together with a subclade of several CPKs from Arabidopsis and rice, clearly separating from the remaining AtCPKs and OsCPKs. These genes were located in 10 chromosomes and one scaffold of C. reinhardtii and contained 6-17 exons. RNA sequencing and quantitative reverse transcription (qRT)-PCR assays indicated that most of these CrCDPKs were significantly induced by N deficiency and salt stress. Lanthanum chloride (LaCl3), a plasma membrane Ca2+ channel blocker, limited oil accumulation in C. reinhardtii under N-deficient conditions, suggesting that Ca2+ was involved in N deficiency-induced oil accumulation. Furthermore, RNA interference (RNAi) silencing analyses demonstrated that six CrCDPKs played positive roles and three CrCDPKs played negative roles in N deficiency-induced oil accumulation in C. reinhardtii.

Low-Temperature- and Phosphate Deficiency-Responsive Elements Control DGTT3 Expression in Chlamydomonas reinhardtii.

acyl-CoA:Diacylglycerol acyltransferases (DGAT) catalyse the final step of the triacylglycerol biosynthesis. Two major gene families have been shown to encode DGATs, DGAT1, and DGAT2. Abiotic factors such as low temperatures, nitrogen, or phosphorus deficiency was reported to play important roles in the growth and development in green algae. Whether DGATs are induced by low temperatures or phosphorus deficiency, and the corresponding promoter elements are not reported yet. In this study, we found DGTT3 to have a significant response to low temperatures, phosphorus deficiency, and other stresses, such as high concentrations of NaCl, 20 µM GA, and 20 µM abscisic acid. The promoter element of DGTT3 was then studied by deletion and scanning mutagenesis method. Results revealed that the - 319/- 247 region is essential for low-temperature and phosphate-deficiency-mediated induction of DGTT3 expression. The sequence from - 312 to - 299 of the CAATAGACTGCTGCT was the core sequence of the cold responsive element, which facilitated the promoter response to cold induction. Meanwhile, the sequence from - 319 to - 275 was critical to phosphate-deficiency regulation. Furthermore, the relationship between DNA methylation and transgenic silence in -N condition was analyzed, and results showed that the DNA methylation rate of the transformed insertion region was high. This phenomenon was responsible for the decrease in ARS gene expression in the transgenic algal strain under -N conditions.

CrMAPK3 regulates the expression of iron-deficiency-responsive genes in Chlamydomonas reinhardtii.

BACKGROUND: Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption. Nevertheless, the response, transmission, and regulation of downstream gene expression in algae cells have not to be investigated. Considering that the MAPK pathway is essential for abiotic stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas. RESULTS: Arabidopsis MAPK gene sequences were used as entry data to search for homologous genes in Chlamydomonas reinhardtii genome database to investigate the functions of mitogen-activated protein kinase (MAPK) gene family in C. reinhardtii under iron-free conditions. Results revealed 16 C. reinhardtii MAPK genes labeled CrMAPK2-CrMAPK17 with TXY conserved domains and low homology to MAPK in yeast, Arabidopsis, and humans. The expression levels of these genes were then analyzed through qRT-PCR and exposure to high salt (150 mM NaCl), low nitrogen, or iron-free conditions. The expression levels of these genes were also subjected to adverse stress conditions. The mRNA levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were remarkably upregulated under iron-deficient stress. The increase in CrMAPK3 expression was 43-fold greater than that in the control. An RNA interference vector was constructed and transformed into C. reinhardtii 2A38, an algal strain with an exogenous FOX1:ARS chimeric gene, to silence CrMAPK3. After this gene was silenced, the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1 were decreased. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced. CONCLUSION: CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.

A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination.

Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies have revealed that members of the SidE effector family of the bacterial pathogen Legionella pneumophila attack several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. In this case, ubiquitin is first activated by ADP-ribosylation at Arg42 by a mono-ADP-ribosyltransferase activity; the intermediate is then cleaved by a phosphodiesterase activity also residing within SdeA, concomitant with the attachment of ubiquitin to serine residues of substrate proteins via a phosphoribosyl linker. Here we demonstrate that the effect of SidEs is antagonized by SidJ, an effector encoded by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ reverses ubiquitination of SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. SidJ also displays classical deubiquitinase activity but does not require catalytic cysteine residues. Further, these deubiquitinase activities of SidJ are essential for its role in L. pneumophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a ubiquitin-deconjugating enzyme that functions to impose temporal regulation on the activity of SidE effectors. SidJ may be important in future studies of signaling cascades mediated by this unique ubiquitination, one that also potentially regulates cellular processes in eukaryotic cells.

Iron deficiency response gene Femu2 plays a positive role in protecting Chlamydomonas reinhardtii against salt stress.

BACKGROUND: Iron deficiency related gene, Femu2, encodes protein homologous to a C2H2-type zinc finger protein, which participates in the regulation of FOX1 gene induced by iron (Fe) deficiency in Chlamydomonas reinhardtii. In this study, we investigate the gene function of Femu2 in response to salt stress in C. reinhardtii. METHODS: Femu2-overexpressing and Femu2-silencing transgenic cells were analyzed under salt stress. Several physiological indices were measured, and global changes in gene expression were investigated via RNA-seq. RESULTS: Compared with that of the non-treated control, the transcript levels of Femu2 were dramatically induced by iron deficiency and can also be significantly induced after algal cell exposure to Tris-acetate-phosphate (TAP) medium with 100 and 150mM NaCl. The promoter also responded to NaCl induction. Femu2-overexpressing transgenic algal cells exhibited significantly enhanced tolerance to salt stress. Conversely, Femu2-silencing cells showed higher sensitivity to salt stress than the control. Physiological analyses revealed that the overexpression of Femu2 increased the contents of proline and soluble sugars in transgenic cells under high salinity and that silencing Femu2 resulted in increased malondialdehyde level and decreased superoxide dismutase activity. RNA-seq results showed that a total of 248 genes have opposite expression profiles and that 5508 and 2120 genes were distinctly up-regulated or down-regulated in Femu2-overexpressing and Femu2-silencing transgenic cells under salt stress, respectively. CONCLUSION: Femu2 may play an important positive role in protecting C. reinhardtii against salt stress. GENERAL SIGNIFICANCE: The results of this study indicated that Femu2 may be useful in improving plant salt tolerance.

Genome-wide survey and expression analysis of Chlamydomonas reinhardtii U-box E3 ubiquitin ligases (CrPUBs) reveal a functional lipid metabolism module.

E3 ubiquitin ligases determine the substrate specificity of ubiquitination. Plant U-box (PUB) E3 ligases, with a typical 70-amino acid U-box domain, participate in plant developmental processes and environmental responses. Thus far, 64 PUB proteins have been identified in Arabidopsis and 77 PUB proteins have been identified in Oryza. However, detailed studies on U-box genes in the model microalgae Chlamydomonas reinhardtii are lacking. Here, we present a comprehensive analysis of the genes encoding U-box family proteins in C. reinhardtii. Following BLASTP analysis, 30 full-length U-box genes were identified in the C. reinhardtii genome sequence. Bioinformatics analyses of CrPUB genes were performed to characterize the phylogenetic relationships, chromosomal locations and gene structures of each member. The 30 identified CrPUB proteins are clustered into 3 distinct subfamilies, and the genes for these proteins are unevenly distributed among 14 chromosomes. Furthermore, the quantitative real-time RT-PCR or semi-quantitative RT-PCR analysis of 30 CrPUB mRNA abundances under nitrogen starvation showed that 18 CrPUB genes were induced by N starvation and that 7 genes were repressed in the N-poor environment. We selected five CrPUB genes exhibiting marked changes in expression under N-free conditions for further analysis in RNAi experiments and examined the oil content of these gene-silenced transgenic strains. The silencing of CrPUB5 and CrPUB14, which are typically down-regulated under N starvation, induced 9.8%-45.0% and 14.4%-61.8% lipid accumulation, respectively. In contrast, the silencing of CrPUB11, CrPUB23 and CrPUB28, which are markedly up-regulated under N-free conditions, decreased the lipid content by 5.5%-27.8%, 8.1%-27.3% and 6.6%-27.9%, respectively. These results provide a useful reference for the identification and functional analysis of this gene family and fundamental information for microalgae lipid metabolism research.

A photoperiod-regulating gene CONSTANS is correlated to lipid biosynthesis in Chlamydomonas reinhardtii.

Background. The regulation of lipid biosynthesis is essential in photosynthetic eukaryotic cells. Thus far, no regulatory genes have been reported in the lipid metabolism pathway. Plant CONSTANS (CO) gene regulates blooming by participating in photoperiod and biological clock. Apart from regulating photoperiod, the Chlamydomonas CO gene also regulates starch content. Results. In this study, the results showed that, under HSM-S condition, cells accumulated more lipids at short-day conditions than at long-day conditions. The silencing of the CrCO gene via RNA interference resulted in an increase in lipid content and an increase in triacylglyceride (TAG) level by 24.5%. CrCO RNAi strains accumulated more lipids at short-day conditions than at long-day conditions. The decrease in CrCO expression resulted in the increased expression of TAG biosynthesis-related genes, such as DGAT2, PAP2, and PDAT3, whereas CIS and FBP1 genes showed a decrease in their mRNA when the CrCO expression was suppressed. On the other hand, the overexpression of CrCO resulted in the decrease in lipid content and TAG level. Conclusions. The results of this study revealed a relationship between CrCO gene and lipid metabolism in Chlamydomonas, suggesting that increasing oil by suppressing CrCO expression in microalgae is feasible.

Expression activation and functional analysis of HLA3, a putative inorganic carbon transporter in Chlamydomonas reinhardtii.

The CO2 concentrating mechanism (CCM) is a key component of the carbon assimilation strategy of aquatic microalgae. Induced by limiting CO2 and tightly regulated, the CCM enables these microalgae to respond rapidly to varying environmental CO2 supplies and to perform photosynthetic CO2 assimilation in a cost-effective way. A functional CCM in eukaryotic algae requires Rubisco sequestration, rapid interconversion between CO2 and HCO3(-) catalyzed by carbonic anhydrases (CAs), and active inorganic carbon (Ci) uptake. In the model microalga Chlamydomonas reinhardtii, a membrane protein HLA3 is proposed to be involved in active Ci uptake across the plasma membrane. In this study, we use an artificially designed transcription activator-like effector (dTALE) to activate the expression of HLA3. The successful activation of HLA3 expression demonstrates dTALE as a promising tool for gene-specific activation and investigation of gene function in Chlamydomonas. Activation of HLA3 expression in high CO2 acclimated cells, where HLA3 is not expressed, resulted in increased Ci accumulation and Ci-dependent photosynthetic O2 evolution specifically in very low CO2 concentrations, which confirms that HLA3 is indeed involved in Ci uptake, and suggests it is mainly associated with HCO3(-) transport in very low CO2 concentrations, conditions in which active CO2 uptake is highly limited.

A C2H2 zinc finger protein FEMU2 is required for fox1 expression in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii fox1 gene encodes a ferroxidase that is involved in cellular Fe uptake and highly induced during Fe deficient conditions. In an effort to identify fox1 promoter regulatory elements, an insertional library was generated in a transgenic Chlamydomonas strain (2A38) harboring an arylsulfatase (ARS) reporter gene driven by the fox1 promoter. Mutants with a defective response to low iron conditions were selected for further study. Among these, a strain containing a disrupted femu2 gene was identified. Activation of the fox1 promoter by the femu2 gene product was confirmed by silencing the femu2 gene using RNA interference. In three femu2 RNAi transgenic lines (IR3, IR6, and IR7), ARS reporter gene activities declined by 84.3%, 86.4%, and 88.8%, respectively under Fe deficient conditions. Furthermore, RT-PCR analysis of both the femu2 mutant and the RNAi transgenic lines showed significantly decreased transcript abundance of the endogenous fox1 gene under Fe deficient conditions. Amino acid sequence analysis of the femu2 gene product identified three potential C2H2 zinc finger (ZF) motifs and a nuclear localization study suggests that FEMU2 is localized to the nucleus. In addition, a potential FEMU2 binding site ((G/T)TTGG(G/T)(G/T)T) was identified using PCR-mediated random binding site selection. Taken together, this evidence suggests that FEMU2 is involved in up-regulation of the fox1 gene in Fe deficient cells.

Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii.

The regulation of lipid biosynthesis is important in photosynthetic eukaryotic cells. This regulation is facilitated by the direct synthesis of fatty acids and triacylglycerol (TAG), and by other controls of the main carbon metabolic pathway. In this study, knockdown of the mRNA expression of the Chlamydomonas phosphoenolpyruvate carboxylase isoform 1 (CrPEPC1) gene by RNA interference increased TAG level by 20 % but decreased PEPC activities in the corresponding transgenic algae by 39-50 %. The decrease in CrPEPC1 expression increased the expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, CrPEPC1 over-expression decreased TAG level by 37 % and increased PEPC activities by 157-184 %. These observations suggest that the lipid content of algal cells can be controlled by regulating the CrPEPC1 gene.

Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii.

Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%-17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%-21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.

Effect of the expression and knockdown of citrate synthase gene on carbon flux during triacylglycerol biosynthesis by green algae Chlamydomonas reinhardtii.

BACKGROUND: The regulation of lipid biosynthesis is essential in photosynthetic eukaryotic cells. This regulation occurs during the direct synthesis of fatty acids and triacylglycerols (TAGs), as well as during other controlling processes in the main carbon metabolic pathway. RESULTS: In this study, the mRNA levels of Chlamydomonas citrate synthase (CrCIS) were found to decrease under nitrogen-limited conditions, which suggests suppressed gene expression. Gene silencing by RNA interference (RNAi) was conducted to determine whether CrCIS suppression affected the carbon flux in TAG biosynthesis. Results showed that the TAG level increased by 169.5%, whereas the CrCIS activities in the corresponding transgenic algae decreased by 16.7% to 37.7%. Moreover, the decrease in CrCIS expression led to the increased expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, overexpression of CrCIS gene decreased the TAG level by 45% but increased CrCIS activity by 209% to 266% in transgenic algae. CONCLUSIONS: The regulation of CrCIS gene can indirectly control the lipid content of algal cells. Our findings propose that increasing oil by suppressing CrCIS expression in microalgae is feasible.

A novel negative Fe-deficiency-responsive element and a TGGCA-type-like FeRE control the expression of FTR1 in Chlamydomonas reinhardtii.

We have reported three Fe-deficiency-responsive elements (FEREs), FOX1, ATX1, and FEA1, all of which are positive regulatory elements in response to iron deficiency in Chlamydomonas reinhardtii. Here we describe FTR1, another iron regulated gene and mutational analysis of its promoter. Our results reveal that the FeREs of FTR1 distinguish itself from other iron response elements by containing both negative and positive regulatory regions. In FTR1, the -291/-236 region from the transcriptional start site is necessary and sufficient for Fe-deficiency-inducible expression. This region contains two positive FeREs with a TGGCA-like core sequence: the FtrFeRE1 (ATGCAGGCT) at -287/-279 and the FtrFeRE2 (AAGCGATTGCCAGAGCGC) at -253/-236. Furthermore, we identified a novel FERE, FtrFeRE3 (AGTAACTGTTAAGCC) localized at -319/-292, which negatively influences the expression of FTR1.

An Fe deficiency responsive element with a core sequence of TGGCA regulates the expression of FEA1 in Chlamydomonas reinharditii.

Iron is essential to the unicellular green alga Chlamydomonas, but the molecular mechanism for response to iron deficiency remains largely unknown. In previous studies, we have identified FOX1 and ATX1 FEREs (Fe deficiency-responsive elements) as important regulation components of iron response in this organism. Here we present another iron regulated gene FEA1, which promoter was analysed by using a 5'-and 3'-end deletion and a scanning mutagenesis assay. The results reveal that the co-existence of -273/-188 and -118/-49 regions from transcriptional start site of FEA1 were sufficient and necessary for Fe deficiency-induced expression. Further deletion analysis indicates both -273/-253 and -103/-85 regions are essential for inducible expression. The scanning mutagenesis analysis of these regions identifies two cis-acting elements: the FeaFeRE1 at -273/-259 (CTGCGGTGGCAAAGT) and FeaFeRE2 at -106/-85 (CCGCCGCNNNTGGCACCAGCCT). Sequence comparison of FeaFeRE1 and FeaFeRE2 reveals a core sequence of TGGCA, which had been found in our previously reported Fe-deficiency-inducible gene ATX1. Moreover, we show that the promoter region of several genes, including FRE1, IRT1, ISCA, ZRT1, ZRT5, NRAMP2 and COPT1, also contains this core sequence, suggesting that at least two classes FeRE elements exist in Clamydomonas, one in FEA1 and ATX1 and others the second in FOX1, FEA2, MTP4, NRAMP3 and RBOL1.