Noncoding RNA mediated traffic of foreign mRNA into chloroplasts reveals a novel signaling mechanism in plants

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.

Figure 1. Transcriptional fusion of viroid derived 5′UTR end to GFP cDNA.

(A) Physical map of the vd-5′UTR/GFP construct. (B) Serological detection of the vd-5′UTR/GFP. Total proteins were extracted from infiltrated leaves, electrophoresed in 10% SDS-PAGE (upper panel) and blotted for serological detection (lower panel). The vd-5′UTR/GFP and the GFP were clearly detected and show similar relative electrophoretic mobility, indicating that the viroid derived sequence acts as a true untranslated RNA. Three 1∶5 serial dilutions are sown for each construct. MwS: Molecular weight standard.

Figure 2. Details of the fusion ELVd-derived RNA/GFP mRNA.

(A) Partial DNA sequence of the vd-5′UTR-GFP construct (AN - HM136583) used in this study. (B) In silico predicted translation of the vd-5′UTR-GFP transcript.

Figure 3. The GFP arising from vd-5′UTR/GFP transcripts accumulate specifically in chloroplasts.

Confocal microscope observation of the N. benthamiana leaves expressing unmodified GFP (left panels), vd-5′UTR-GFP (central panels) or OE23/GFP (right panels). As observed, the vd-5′UTR/GFP mimics the cellular localization of the OE23/GFP construct that accumulates specifically in chloroplasts.

Figure 4. Vd-5′UTR mediates the traffic of functional GFP-mRNA to chloroplasts.

(A) Confocal microscope observation of chloroplasts isolated from leaves expressing GFP or vd-5′UTR/GFP. (B) RT-PCR analysis of GFP transcripts extracted from chloroplasts isolated from leaves infiltrated with both constructs. Chloroplast ribosomal RNA 28 s was amplified as a control of isolated-chloroplasts integrity.