Context‐dependent antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functional in vivo fluorescent reporter in the chloroplast of Chlamydomonas reinhardtii
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Arcadia Science)
Abstract
Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast‐optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context‐aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post‐transcriptional regulation, pointing to novel strategies for optimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass‐bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5′atpA, and 3′ rbcL in a sense‐transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play.
Article activity feed
-
-
-
most of the signal presumably occupying the stroma of the chloroplast. A greater accumulation of mNG fluorescence was observed in the center of chloroplast lumen, with the highest green signal presumably located in the zone where pyrenoid is located (Figure 5a, progressions 3-4). Notably, most of the mNG fluorescence signal does not co-localize with chloroplast fluorescence.
It is interesting to see the stratification of chloroplast signal between the mNG fluorescence and chloroplast autofluorescence. Is the bulk of the nucleus within the empty space of the chloroplast autofluorescence? What accounts for the DAPI staining throughout the cell body?
-
Other works have described that exclusion of proteins higher than ∼50kDa from the pyrenoid matrix may occur [Mackinder et al., 2018], this might be happening with mNG due its effective molecular weight observed at ∼70kDa by native-PAGE in this work.
Would it be possible to synthesize this reporter with a lower MW to explore the heterogenous distribution further?
-