Expression and subcellular targeting of green fluorescent protein using a zucchini yellow mosaic virus-based vector

Document Type : Research Article

Authors

1 Environmental Research Institute, ACECR

2 Departement Of Plant Pathology, Faculty of Agriculture, Tarbiat Modaress University

3 Department of Plant Virology, Faculty of Agriculture, National Chung Hsing University

Abstract

The present study investigated the feasibility of transient expression and successful subcellular targeting of Green Fluorescent Protein (GFP) using a plant viral expression system. For this purpose, zucchini yellow mosaic virus (ZYMV, Potyvirus cucurbitaflavitesselati) was employed to express GFP in cucurbit plants as the systemic host for ZYMV. The GFP-encoding gene was inserted between the P1 and HC-Pro open reading frames in the ZYMV vector. Additionally, a chloroplast signal peptide and the SEKDEL sequence were incorporated to direct GFP to the chloroplast and endoplasmic reticulum, respectively. The infectivity of the recombinant vectors was evaluated by inoculating Chenopodium quinoa, a local lesion host for ZYMV. Chlorotic local lesions developed on the inoculated leaves of C. quinoa. An extract from a single local lesion was used to inoculate squash plants, which were then sampled at 5, 10, 15, and 20 days post-inoculation. The samples were analyzed using indirect ELISA, RT-PCR, fluorescence microscopy, and confocal imaging. The presence of ZYMV recombinants in the inoculated plants was confirmed by RT-PCR. ZYMV-expressed GFP was detected using ELISA, fluorescence microscopy, and confocal imaging. Data analysis revealed that GFP was successfully expressed in squash leaves, and the SEKDEL sequence significantly enhanced the expression levels of the recombinant protein. Overall, the findings of this study demonstrate that the use of plants as hosts for the expression of foreign proteins remains a rapidly evolving field, and this approach offers a rapid and efficient method for producing high quantities of recombinant proteins in plants.

Keywords

References

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Iranian Journal of Plant Pathology
Volume 60, Issue 1
April 2025
Pages 79-98

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