Antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functionalin vivofluorescent reporter in the chloroplast ofChlamydomonas reinhardtii

Although over 30 years have passed sinceChlamydomonas reinhardtiichloroplast transformation was first achieved, robust genetic engineering of the chloroplast still remains a challenging task. The glass-bead transformation method has enabled simple and accessible chloroplast transformation of theC. reinhardtiiTN72 strain, allowing generation of marker-free transplastomic strains for low-cost experimentation. However, lack of functionalin vivofluorescent reporters limit research and widespread development of chloroplast engineering. Here, we developed a chloroplast codon-optimised mNeonGreen fluorescent reporter, which can be detectedin vivothrough fluorescence microscopy and fluorometry, by context-aware construct engineering, leading up to a ∼6-fold increase in fluorescence. We found evidence for chloroplast post-transcriptional regulation of gene expression derived from formation of antisense pairing of mRNAs due to transcriptional readthrough of the convergent adjacent gene, which was validated through detection of the double-stranded RNA. In addition, engineering approaches were used to modulate transcriptional readthrough, allowing a better understanding of context effects that are relevant for heterologous expression. Finally, we characterised a suite of regulatory parts for transgene expression in a sense-transcriptional context regarding to the selectable marker, achieving up to ∼2-fold increase in mNeonGreen fluorescence levels regarding to the control, with the use of PrrnS,5’atpAand 3’rbcLendogenous regulatory sequences from the chloroplast ofC. reinhardtii. This work provides new tools for studying basic aspects of the molecular biology in the chloroplast inC. reinhardtii, as well as evidence for fundamental processes of gene regulation that may enable developing rules for more efficient chloroplast engineering.