Kroth, Peter G.
A strategy to complement PtAUREO1a in TALEN knockout strains of Phaeodactylum tricornutum
2019-05, Madhuri, Shvaita, Río Bártulos, Carolina, Serif, Manuel, Lepetit, Bernard, Kroth, Peter G.
The recent availability of genome editing tools like TALEN (Transcription activator-like effector nuclease) and CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats) for the diatom Phaeodactylum tricornutum has dramatically increased the options to explore diatom biology via reverse genetics. In order to verify that an observed phenotype indeed is directly related to a specific gene knockout and not due to a secondary effect, complementation of the inactivated gene with the wildtype gene and restoration of the wild type phenotype is an essential tool in molecular biology. So far, no strategy for a complementation method has been published for P. tricornutum. Here we demonstrate, as a proof-of principle, the complementation of P. tricornutum AUREO1a knockout strains previously created by TALEN technology. These strains are deficient in the PtAureo1a gene, which is encoding a blue-light dependent transcription factor. pPTbsr, a modified pPha-T1 vector with an antibiotic resistance cassette against Blasticidin served as a complementation vector. In order to avoid the modification of the complementing gene via the potentially still active TALEN nucleases, we have modified the TALEN binding sites of the complementing PtAureo1a gene using synonymous codons. The altered PtAureo1a gene along with its native promoter and terminator was transformed by particle gun bombardment into PtAUREO1a TALEN knockout strains of P. tricornutum. The integration and the expression of PtAUREO1a was confirmed by PCR and western blotting. Due to random integration within the genome, the expression level of the complemented gene may be variable in different lines. Physiological parameters indicated the successful rescue of the wild type phenotype in several lines that showed a similar PtAUREO1a protein content as wild type cells. Our method provides a rapid and efficient tool to complement knockout lines generated by genome editing approaches in P. tricornutum.
Isolation of Plastid Fractions from the Diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum
2018, Schober, Alexander, Flori, Serena, Finazzi, Giovanni, Kroth, Peter G., Río Bártulos, Carolina
The so-called "complex" plastids from diatoms possessing four envelope membranes are a typical feature of algae that arose from secondary endosymbiosis. Studying isolated plastids from these algae may allow answering a number of fundamental questions regarding diatom photosynthesis and plastid functionality. Due to their complex architecture and their integration into the cellular endoplasmic reticulum (ER) system, their isolation though is still challenging. In this work, we report a reliable isolation technique that is applicable for the two model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. The resulting plastid-enriched fractions are of homogenous quality, almost free from cellular contaminants, and feature structurally intact thylakoids that are capable to perform oxygenic photosynthesis, though in most cases they seem to lack most of the stromal components as well as plastid envelopes.
Intracellular distribution of the reductive and oxidative pentose phosphate pathways in two diatoms
2009, Gruber, Ansgar, Weber, Till, Río Bártulos, Carolina, Vugrinec, Sascha, Kroth, Peter G.
Diatoms contribute a large proportion to the worldwide primary production and are particularly effective in fixing carbon dioxide. Possibly because diatom plastids originate from a secondary endocytobiosis, their cellular structure is more complex and metabolic pathways are rearranged within diatom cells compared to cells containing primary plastids. We annotated genes encoding isozymes of the reductive and oxidative pentose phosphate pathways in the genomes of the centric diatom Thalassiosira pseudonana and the pennate diatom Phaeodactylum tricornutum and bioinformatically inferred their intracellular distribution. Prediction results were confirmed by fusion of selected presequences to Green Fluorescent Protein and expression of these constructs in P. tricornutum. Calvin cycle enzymes for the carbon fixation and reduction of 3-phosphoglycerate are present in single isoforms, while we found multiple isoenzymes involved in the regeneration of ribulose-1,5-bisphosphate. We only identified one cytosolic sedoheptulose-1,7-bisphosphatase in both investigated diatoms. The oxidative pentose phosphate pathway seems to be restricted to the cytosol in diatoms, since we did not find stromal glucose-6-phosphate dehydrogenase and 6-phosphogluconolactone dehydrogenase isoforms. However, the two species apparently possess a plastidic phosphogluconolactonase. A 6-phosphogluconolactone dehydrogenase is apparently plastid associated in P. tricornutum and might be active in the periplastidic compartment, suggesting that this compartment might be involved in metabolic processes in diatoms.