Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum

Abstract

Viruses are considered key players in phytoplankton population control in oceans. However, mechanisms that control viral gene expression in prominent microalgae such as diatoms remain largely unknown. In this study, potential promoter regions isolated from several marine diatom-infecting viruses (DIVs) were linked to the egfp reporter gene and transformed into the Pennales diatom Phaeodactylum tricornutum. We analysed their activity in cells grown under different conditions. Compared to diatom endogenous promoters, novel DIV promoter (ClP1) mediated a significantly higher degree of reporter transcription and translation. Stable expression levels were observed in transformants grown under both light and dark conditions, and high levels of expression were reported in cells in the stationary phase compared to the exponential phase of growth. Conserved motifs in the sequence of DIV promoters were also found. These results allow the identification of novel regulatory regions that drive DIV gene expression and further examinations of the mechanisms that control virus-mediated bloom control in diatoms. Moreover, the identified ClP1 promoter can serve as a novel tool for metabolic engineering of diatoms. This is the first report describing a promoter of DIVs that may be of use in basic and applied diatom research.

Non-model model organisms

“…In addition, CRISPR-Cas9 genome editing has recently become feasible in model diatoms [19, 20], and several diatom viruses have been sequenced [21] which may provide useful resources for building tools as so many animal viruses have done before them. Owing to the relative ease of adapting these modern genetic tools to diatoms, several labs are engaging additional tools with promising results, including proximity proteomics, live cell microscopy, and super-resolution fluorescence microscopy [22].…”

Genetic and metabolic engineering in diatoms

“…Heterologous or synthetic promoters are required to drive transgene expression without a negative effect on endogenous regulatory networks. Examples of such promoters are the Lhcf2 promoter from P. tricornutum, which is used in Fistulifera species [9], and the Lhcf1 promoter from C. fusiformis, which is used in P. tricornutum [21]. In addition, viral promoters have been reported to be functional in P. tricornutum species.…”

“…Thus, the CaMV (cauliflower mosaic virus) promoter, the CMV (cytomegalovirus) promoter and the RSV promoter (Rous sarcoma virus) drive reporter gene expression [22]. Similarly, the ClP1 promoter from diatom-infecting viruses can drive stable expression and does so to levels higher than those observed with endogenous diatom promoters and other viral promoters (CaMV, CMV and RSV) [21]. Overall, the diversity of diatom promoters now available opens up new opportunities to control gene expression at specific times during culture and to fine-tune enzyme levels, thereby facilitating metabolic pathway engineering.…”

“…Both pennate and centric diatoms have been successfully transformed with this technique (table 1). Transformation efficiency is from 10 27 to 10 28 diatom cells mg 21 DNA[8,21,35,36]. Biolistic guns can be used with diverse species, and to introduce multiple transgenes simultaneously…”

Motilidade da associação entre uma diatomácea cêntrica e um ciliado peritríquio e suas possíveis consequências ecológicas

“…Pertencentes à classe Bacillariophyceae, as espécies deste grupo possuem frústula, constituída por sílica e composta por duas valvas (tecas), o que faz com que elas exerçam um importante papel no ciclo biogeoquímico global da sílica, já que utilizam este componente para a biogênese de sua parede celular (Hopkins & Drum, 1966;Kadono et al, 2015). Em termos morfológicos, as diatomáceas são tradicionalmente divididas em penadas e cêntricas, sendo distinguidas por meio por meio das ornamentações das estruturas na parede celular (Hendey, 1964;Lee, 2008).…”