The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas

Schroda, Michael; Blöcker, Dagmar; Beck, Christoph F.

doi:10.1046/j.1365-313x.2000.00652.x

Cited by 306 publications

(294 citation statements)

References 49 publications

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“…A hairpin RNAi plasmid containing inverted pairs of the third exon of MTD1, with the third intron serving as the middle loop, was driven by the constitutive HSP70A/rbcS2 promoter ( Figure 5A) (Schroda et al 2000;Koblenz et al 2003). This RNAi construct was cotransformed into wild-type mt À cells with pSI103.…”

Section: Resultsmentioning

confidence: 99%

Gametogenesis in the Chlamydomonas reinhardtii minus Mating Type Is Controlled by Two Genes, MID and MTD1

Lin

Goodenough

2007

Genetics

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In the unicellular algae Chlamydomonas reinhardtii, the plus and minus mating types are controlled by a complex locus, MT, where the dominant MID gene in the MT À locus has been shown to be necessary for expression of minus-specific gamete-specific genes in response to nitrogen depletion. We report studies on MID expression patterns during gametogenesis and on a second gene unique to the MT À locus, MTD1. Vegetative cells express basal levels of MID. An early activation of MID transcription after nitrogen removal, and its sequence similarity to plant RWP-RK proteins involved in nitrogen-responsive processes, suggest that Mid conformation/activity may be nitrogen sensitive. A second stage of MID upregulation correlates with the acquisition of mating ability in minus gametes. Knockdown of MTD1 by RNAi in minus strains results in a failure to differentiate into gametes of either mating type after nitrogen deprivation. We propose that intermediate Mid levels are sufficient to activate MTD1 transcription and to repress plus gametespecific genes and that MTD1 expression in turn allows the threshold-level MID expression needed to turn on minus gamete-specific genes. We further propose that an MTD1-equivalent system, utilizing at least one gene product encoded in the MT 1 locus, is operant during plus gametogenesis.C HLAMYDOMONAS reinhardtii is a flagellated unicellular green alga that has two mating types, plus and minus, determined by the mating type (MT ) loci (MT 1 and MT À ). The center of this $1-Mb locus of recombinational suppression carries translocations and inversions and is called the rearranged (R) domain (Ferris and Goodenough 1994). Both housekeeping and sex-limited genes are found in this region (Ferris et al. 2002), similar to mating-type loci and sex chromosomes in other organisms (Graves 2006). Six unique regions (a-f ) are found within the R domain, three (ac) specific to MT 1 and three (d-f ) specific to MT À . Four genes have been identified in these regions: MTA1 (MT locus, region a) in a, FUS1 (fusion) in c, MTD1 (MT locus, region d) in d, and MID (minus dominance) in f (Ferris et al. 2002). The two MT À -specific genes are the focus of this study.In response to nitrogen starvation, haploid vegetative Chlamydomonas cells differentiate into gametes. Gametes of opposite mating type are able to agglutinate and fuse to form zygotes (Harris 1989). Occasionally, heterozygous mt 1 /mt À diploids form after mating, resume vegetative growth, and differentiate as gametes with N-starvation. The fact that these diploids always mate as minus indicates that minus is dominant to plus (Harris 1989), a phenomenon found to be controlled by the MID gene (Galloway and Goodenough 1985). MID encodes a transcription factor in the RWP-RK family that also includes several proteins in higher plants that are suggested to exert their function during nitrogen limitation (Schauser et al. 1999(Schauser et al. , 2005Borisov et al. 2003).Previous studies revealed that MID is necessary and sufficient to convert wild-type ...

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Section: Resultsmentioning

confidence: 99%

Gametogenesis in the Chlamydomonas reinhardtii minus Mating Type Is Controlled by Two Genes, MID and MTD1

Lin

Goodenough

2007

Genetics

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“…This approach also would examine whether lipid yields could be altered by increased abundance of a GPDH in C. reinhardtii. Both of the GPD2 and GPD3 cDNA constructs were transformed into wild-type C. reinhardtii (CC-48 background) under the control of the previously used tandem HSP70A-RBCS2 promoter (Schroda et al, 2000;Bajhaiya et al, 2016b). Two GPD2 overexpression lines (GPD2-OE1 and GPD2-OE2) and three GPD3 overexpression lines (GPD3-OE1, GPD3-OE2, and GPD3-OE3) were examined further, with each line showing a significant increase in GPD2 or GPD3 expression relative to the wild type (Fig.…”

Section: Gpd2 and Gpd3 Overexpression Lines Show Distinct Growth Phenmentioning

confidence: 99%

Two Glycerol-3-Phosphate Dehydrogenases from Chlamydomonas Have Distinct Roles in Lipid Metabolism

et al. 2017

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The metabolism of glycerol-3-phosphate (G3P) is important for environmental stress responses by eukaryotic microalgae. G3P is an essential precursor for glycerolipid synthesis and the accumulation of triacylglycerol (TAG) in response to nutrient starvation. G3P dehydrogenase (GPDH) mediates G3P synthesis, but the roles of specific GPDH isoforms are currently poorly understood. Of the five GPDH enzymes in the model alga Chlamydomonas reinhardtii, GPD2 and GPD3 were shown to be induced by nutrient starvation and/or salt stress. Heterologous expression of GPD2, a putative chloroplastic GPDH, and GPD3, a putative cytosolic GPDH, in a yeast gpd1D mutant demonstrated the functionality of both enzymes. C. reinhardtii knockdown mutants for GPD2 and GPD3 showed no difference in growth but displayed significant reduction in TAG concentration compared with the wild type in response to phosphorus or nitrogen starvation. Overexpression of GPD2 and GPD3 in C. reinhardtii gave distinct phenotypes. GPD2 overexpression lines showed only subtle metabolic phenotypes and no significant alteration in growth. In contrast, GPD3 overexpression lines displayed significantly inhibited growth and chlorophyll concentration, reduced glycerol concentration, and changes to lipid composition compared with the wild type, including increased abundance of phosphatidic acids but reduced abundance of diglycerides, triglycerides, and phosphatidylglycerol lipids. This may indicate a block in the downstream glycerolipid metabolism pathway in GPD3 overexpression lines. Thus, lipid engineering by GPDH modification may depend on the activities of other downstream enzyme steps. These results also suggest that GPD2 and GPD3 GPDH isoforms are important for nutrient starvation-induced TAG accumulation but have distinct metabolic functions.

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“…CrGEX1 is driven by a RBCS-HSP70 chimeric promoter and contains the RBCS terminator from Chlamydomonas (Schroda et al 2000). One microgram of linearized plasmid was introduced into 21gr (mt + ) cells by electroporation (Shimogawara et al 1998).…”

Section: Expressing the Crgex1-flag Transgene In The Chlamydomonas Gementioning

confidence: 99%

Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates

et al. 2013

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Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa.

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The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas

Cited by 306 publications

References 49 publications

Gametogenesis in the Chlamydomonas reinhardtii minus Mating Type Is Controlled by Two Genes, MID and MTD1

Gametogenesis in the Chlamydomonas reinhardtii minus Mating Type Is Controlled by Two Genes, MID and MTD1

Two Glycerol-3-Phosphate Dehydrogenases from Chlamydomonas Have Distinct Roles in Lipid Metabolism

Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates

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