Development of an inducible artificial microRNA system for Chlamydomonas reinhardtii as a tool for investigation of essential gene function
Stefan Schmollinger, Daniela Strenkert, Corinna Gruber, and Michael Schroda
Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany
Conventional RNA silencing strategies employing inverted repeat or antisense constructs suffer from severe drawbacks like off-target effects by unpredictable generation of siRNAs or silencing of the RNAi construct. To overcome problems with these strategies constructs for routine expression of artificial microRNAs (amiRNAs) have been developed for Chlamydomonas reinhardtii in the past years. However, assigning gene function on the basis of a phenotype observed in constitutive knock-out/down mutants may be corrupted by the appearance of strong secondary phenotypes, and moreover, this approach is also not suitable for studying functions of essential genes. For this purpose we equipped a recently developed amiRNA expression vector with the NIT1 promoter, which is repressed by ammonium and activated by nitrate to create an inducible artificial microRNA system. We tested this inducible amiRNA vector with heat shock factor 1 (HSF1) and chloroplast HSP70 (HSP70B) as targets. Transcripts in transformants of both targets were already reduced ~60 min after transfer to nitrate-containing medium. In contrast, HSF1 protein levels started to decline only ~8 h after the shift and were strongly reduced after 24 h, suggesting that HSF1 is a quite stable protein and is diluted out by growth. Transformants developed thermosensitivity only on nitrate and thermosensitivity correlated with strong reduction in HSF1 protein levels hence supporting our earlier conclusion that HSF1 is a key regulator for thermotolerance in Chlamydomonas. Downregulation of the HSP70B gene resulted in a slight reduction of the protein when growth arrested ~24 hours after the switch to nitrate, demonstrating the importance of this chaperone.
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