Abstract
Communication between chloroplasts and the nucleus is one of the milestones of the evolution of plants on earth. Proteins encoded by ancestral chloroplast-endogenous genes were transferred to the nucleus during the endosymbiotic evolution and originated this communication, which is mainly dependent on specific transit-peptides. However, the identification of nuclear-encoded proteins targeted to the chloroplast lacking these canonical signals suggests the existence of an alternative cellular pathway tuning this metabolic crosstalk. Non-coding RNAS (NcRNAs) are increasingly recognized as regulators of gene expression as they play roles previously believed to correspond to proteins. Avsunviroidae family viroids are the only noncoding functional RNAs that have been reported to traffic inside the chloroplasts. Elucidating mechanisms used by these pathogens to enter this organelle will unearth novel transport pathways in plant cells. Here we show that a viroid-derived NcRNA acting as a 5′UTR-end mediates the functional import of Green Fluorescent Protein (GFP) mRNA into chloroplast. This claim is supported by the observation at confocal microscopy of a selective accumulation of GFP in the chloroplast of the leaves expressing the chimeric vd-5′UTR/GFP and by the detection of the GFP mRNA in chloroplasts isolated from cells expressing this construct. These results support the existence of an alternative signaling mechanism in plants between the host cell and chloroplasts, where an ncRNA functions as a key regulatory molecule to control the accumulation of nuclear-encoded proteins in this organelle. In addition, our findings provide a conceptual framework to develop new biotechnological tools in systems using plant chloroplast as bioreactors. Finally, viroids of the family Avsunviroidae have probably evolved to subvert this signaling mechanism to regulate their differential traffic into the chloroplast of infected cells.
Highlights
Chloroplasts are the hallmark organelle of photosynthetic eukaryotes [1,2]
We report that a viroid-derived noncoding RNAs (ncRNAs) sequence acting as 59UTR mediates the specific import of a functional Green Fluorescent Protein (GFP) mRNA into the chloroplasts of N. benthamiana cells, supporting the existence of a novel signaling mechanism between the host cell and these organelles
To evaluate if an ncRNA could regulate the transport of mRNAs to chloroplasts, we constructed a chimeric DNA containing a modified Eggplant latent viroid (ELVd) [19] cDNA sequence fused as an untranslated region (UTR) to the 59end of the cDNA of the Green Fluorescence Protein (GFP) (Figure 1A)
Summary
Chloroplasts are the hallmark organelle of photosynthetic eukaryotes [1,2]. They are responsible for many metabolic processes, including photosynthesis and biosynthesis of diverse essentials primary and secondary metabolites [3,4,5]. It is widely accepted that this genome-coordinated mechanism is mediated by proteins encoded in the nucleus, synthesized in the cytosol in the corresponding precursor form (containing a targeting signal called transit peptide, TP) and imported into the organelle [7].
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