| During the last decades, the green alga Chlamydomonas reinhardtii has emerged as a powerful model system for chloroplast molecular biology studies. Yet, our knowledge of the function of essential chloroplast genes remains very limited because it is not possible to obtain homoplasmic disruptions of these loci. In order to circumvent this problem, we developed a novel repressible chloroplast gene expression system. The system is based on a genetically engineered version of the nucleus-encoded chloroplast Nac2 protein which is specifically required for stabilization of the endogenous psbD mRNA encoding the D2 reaction center polypeptide of PSII (Nickelsen et al., 1999; Surzycki and Rochaix, 2007). Expression of Chlamydomonas genes can be tightly controlled by the artificial fusion of a B12-responsive promoter and a TPP-riboswitch within the 5' UTR (Croft et al., 2005; Croft et al., 2007). Thus, when the nac2 mutant strain is transformed with a chimeric Nac2 gene driven by the vitamin-reponsive promoter and 5' UTR, conditional repression of D2 synthesis and, in turn, loss of PSII activity, is indeed observed when B12 and TPP are added to the growth medium. Recent studies demonstrated that the Nac2 vitamin-responsive system can also be used to achieve conditional knock-down of essential chloroplast genes, by fusion of the Nac2 target site, the psbD 5' UTR, to the chloroplast coding sequence of interest, in a constitutive photoautrophic strain where the endogenous psbD 5'UTR has been replaced with another chloroplast 5' UTR. I will present the conditional knock-down of three essential plastid ORFs such as the ribosomal protein rps12, the DNA-dependent polymerase gene rpoA and the proteolytic subunit of the ATP-dependent Clp protease clpP1 and give some future directions about their potential use in the field of retrograde signaling research to identify novel components involved in the crosstalk between the chloroplast and the other genetic compartments.
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