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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. Graphical Abstract

The objectives of this study were to determine nutrient removal rates and algal community variation using the isolated microalgal strains Chlorella sp. and Scenedesmus sp. from an urban river water. The concentration of total nitrogen (TN) and total phosphorus (TP) in river water declined after pouring into Chlorella sp. and Scenedesmus sp., it was indicated that the Scenedesmus sp. had respective advantage in removing nitrogen (86% removal rate) and Chlorella sp. in removing phosphorous (95% removal rate). The algae community composition showed extreme sensitivity to change in the joint of the Scenedesmus or Chlorella, respectively, the lower diversity and higher dominance of algae can be observed in Scenedesmus group, there existed an opposite tendency in Chlorella group. The results demonstrated that the high potential of using Chlorella sp. and Scenedesmus sp. for nutrient removal from riverwater.

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

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.

The Lys-63–linked polyubiquitination of target proteins is considered a fundamentally different process from conventional Lys-48–linked polyubiquitination. This process is deemed analogous to other post-translational regulatory processes, such as phosphorylation and sumoylation, which alter target protein activities and are reported to be involved in several cellular processes. In this study, we report that CrUBC2 , a yeast and Arabidopsis MMS2/UEV homolog gene in Chlamydomonas reinhardtii , is able to functionally rescue yeast mms2 mutant, leading to increased DNA damage tolerance. The overexpression of the CrUBC2 improves the algal ability to tolerate DNA damage, while silencing CrUBC2 sensitizes cells to DNA damage, indicating that CrUBC2 is involved in DNA-damage response in Chlamydomonas . Moreover, the overexpression of CrUBC2 increases algal cell lipid, while the silencing of CrUBC2 gene decreases lipid content, implying that a protein substrate of CrUBC13–CrUBC2 polyubiquitination is involved in lipid accumulation.

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.

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.

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. This article is protected by copyright. All rights reserved.

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.

Background Iron deficiency related gene, Femu2 , encodes protein homologous to a C 2 H 2 -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 150 mM 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.

Microalgae usually accumulate triacylglycerols (TAGs) under stress conditions, especially during nitrogen (N) starvation. However, little information is available regarding the underlying mechanism. In the present study, a green algae Micractinium pusillum accumulated significant amounts of TAGs after 6 d of N starvation. Aiming at fundamental elucidation of the molecular mechanisms involved in N starvation-induced TAG accumulation, the global gene expression changes were identified by using suppression subtractive hybridization (SSH). In total, 290 expressed sequence tags (ESTs) showed significant differential expression between the treated and control samples. The expression profiles of 19 ESTs have been confirmed by quantitative real-time PCR (qPCR). Homology analysis showed that the 174 N deficiency-induced ESTs were mainly involved in carbohydrate metabolism, pyruvate and acetyl-CoA synthesis, isoprenoids biosynthesis, and TAG degradation, as well as a severe down-regulation in the expression of 116 genes related to photosynthesis, cell growth, amino acid synthesis and cell cycle regulation. A significant decrease in carbohydrate content was also observed in the N-deficient algal cells. These results suggested that the carbon sources for TAG biosynthesis were largely derived from carbohydrate metabolism rather than from photosynthesis. The results of the qPCR showed that, compared to the control, the transcript levels of genes related to pyruvate and acetyl-CoA synthesis were dramatically increased over 100 times after N starvation, which suggests that pyruvate and acetyl-CoA play important roles in driving carbon flow into TAG biosynthesis.

Many microalgae accumulate oils, especially under stress conditions, thus have attracted more and more attention as a potential feedstock for biodiesel production. However, little information is available for molecular mechanisms that control oil accumulation in microalgae. In this study, we investigated the accumulation of oils during nitrogen (N) deprivation in the model green algae Chlamydomonas reinhardtii . Our results indicated that oil content significantly increased after N deficiency in C. reinhardtii , which was accompanied by a drastic decrease (63–91%) in the transcript level of phosphoenolpyruvate carboxylase 2 (PEPC2), an enzyme that converts phosphoenolpyruvate (PEP) to oxaloacetate (OAA), which conjugates with acetyl-CoA to generate citric acid. Furthermore, down regulation of pepc2 gene by RNAi silencing in C. reinhardtii led to 14–28% increase in oil content. These results revealed a negative correlation between pepc2 mRNA abundance and oil content in C. reinhardtii .

We have reported three Fe-deficiency-responsive elements (FEREs),FOX1, ATX1,andFEA1, all of which are positive regulatory elements in response to iron deficiency inChlamydomonas reinhardtii. Here we describeFTR1, another iron regulated gene and mutational analysis of its promoter. Our results reveal that the FeREs ofFTR1distinguish itself from other iron response elements by containing bothnegativeandpositiveregulatory regions. InFTR1, 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 ofFTR1.

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.

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. PlantCONSTANS (CO)gene regulates blooming by participating in photoperiod and biological clock. Apart from regulating photoperiod, theChlamydomonas COgene 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 theCrCOgene via RNA interference resulted in an increase in lipid content and an increase in triacylglyceride (TAG) level by 24.5%.CrCORNAi strains accumulated more lipids at short-day conditions than at long-day conditions. The decrease inCrCOexpression resulted in the increased expression of TAG biosynthesis-related genes, such asDGAT2, PAP2,andPDAT3, whereasCISandFBP1genes showed a decrease in their mRNA when theCrCOexpression was suppressed. On the other hand, the overexpression ofCrCOresulted in the decrease in lipid content and TAG level.Conclusions.The results of this study revealed a relationship betweenCrCOgene and lipid metabolism inChlamydomonas, suggesting that increasing oil by suppressingCrCOexpression in microalgae is feasible.

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.

Microalgal oils have been considered as a promising feedstock for biodiesel production, which can potentially completely substitute fossil fuels. In the present study, 4500 mutants were obtained from Chlamydomonas reinhardtii CC124 through ultraviolet (UV) induced mutagenesis. After screening these mutants using HSM and HSM-N media, a mutant CC124-M25 with high oil content was selected under an HSM-N medium condition. Compared with the wildtype CC124, the mutant CC124-M25 has a similar growth rate, but the oil content increased by 10%, reaching up to 42%, which has a good prospect for biodiesel production. Optimization studies showed that the nitrogen-free medium with 20 mmol/L potassium element, called N0P20 medium is the most suitable for oil accumulation in the mutant, in which the oil content can reach a maximum of 0.5 g/g. The oil content of the mutant cells grown in the medium contain 2 g/L sodium acetate also can reach a maximum of 0.5 g/g , suggesting that 2 g/L sodium acetate is optimum for oil accumulation. In addition, ISSR markers were used to analyze the difference between the mutant CC124-M25 and the wildtype CC124. In total, 4 out of 100 primers could amplify distinct bands with good polymorphsim. The results showed that the polymorphism information content or CC124 was 78% and that for the mutant CC124-M25 was 50%, suggesting the genetic stability of the mutant obtained by UV mutagenesis. Key words: Chlamydomonas reinhardtii, UV mutagenesis, orthogonal, inter-simple sequence repeat (ISSR)

Figure S1Amino acid sequence alignment of the homologues of CrDGAT2 from C.xDownload (.81 MB ) Figure S1Amino acid sequence alignment of the homologues of CrDGAT2 from C.

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.

We investigated the promoter region of atx1, which encodes a copper chaperone in response to iron deficiency induction. Deletion analysis of the promoter region from the 5' and 3' ends revealed that the -532/-461 and -320/-276 regions were necessary and sufficient for iron deficiency-inducible expression. Further deletion analysis showed that two of the Fe deficiency-responsive elements (FeREs) localized within the -532/-511 and -306/-276 regions, in which AtxFeRE1 at -529/-515 (GTCGCACTGGCATGT) and AtxFeRE2 at -300/-286 (GCAGCGATGGCATTT) had been identified, respectively, with a conserved sequence of GNNGCNNTGGCATNT, differing from all known FeREs found in other organisms.