Loss of chloroplast ClpP elicits an autophagy-like response in Chlamydomonas

Abstract

Chloroplast genomes contain a single ClpP1 gene encoding one of the catalytic subunits of the evolutionarily conserved ATP-dependent Clp protease. Efforts to inactivate this protease in the chloroplast through targeted disruption of the clpP1 gene have failed, suggesting that it is essential for cell survival in plants. To circumvent this problem, a repressible chloroplast gene expression system was developed in the green unicellular alga Chlamydomonas reinhardtii. This system takes advantage of the nuclear Nac2 gene fused to the MetE promoter and Thi4 riboswitch, which can be repressed by adding vitamin B12 and thiamine to the growth medium. Nac2 encodes a chloroplast protein that interacts specifically with the 5?UTR of the psbD mRNA and is involved in processing/ translation of this transcript. Loss of Nac2 leads to the specific degradation of psbD mRNA. Because the psbD 5?UTR is necessary and sufficient for the Nac2-dependent stability of psbD mRNA, this dependence can be transferred to any chloroplast gene by linking its coding sequence to the psbD 5 'UTR. In this way it was possible to repress the clpP1 gene in a reversible way with vitamins.