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

Abstract

Although over 30 years have passed sinceChlamydomonas reinhardtiichloroplast transformation was first achieved, genetic engineering of the chloroplast still remains an arduous 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 functional fluorescent reporters limit research and widespread development of chloroplast engineering. Here, we developed a codon-optimised mNeonGreen fluorescent reporter, which can be detectedin vivothrough fluorometry and microscopy. We found evidence for chloroplast post-transcriptional regulation of gene expression derived from formation of antisense pairing of mRNAs due to readthrough of convergent adjacent genes. In addition, synthetic biology approaches were used to modulate transcriptional readthrough, allowing a better understanding of context effects relevant for heterologous expression. This work provides new tools to study 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.

Advancing chloroplast synthetic biology through high-throughput plastome engineering ofChlamydomonas reinhardtii

“…This observation is in line with a recent report that the 3’UTR at the psbH site is important in controlling read-through activity of neighboring genes, which in turn can modulate transgene expression. 67 Overall, these experiments showed that most constructs behaved similar independent of their genomic position, but also highlighted that validating individual constructs at the psbH site is important for quantifying actual transgene expression strength.…”

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform

“…To facilitate our initial experiments, we engineered a ‘universal test construct’, designed to be a standard tool for troubleshooting chloroplast cell-free extracts. This construct comprised genetic elements of viral origins, specifically the T7 RNA polymerase promoter, gene10 5′UTR, and the Tobacco Mosaic Virus (TMV) 3′UTR, , each chosen for their proven efficacy in driving strong gene expression in chloroplasts. To support broader research efforts, we have made this ‘universal test construct’ accessible to the scientific community through Addgene (ID 216625).…”