Functional characterization of the EMCV IRES in plants

Area of Science:

• Molecular Biology
• Plant Biotechnology
• Virology

Background:

• Eukaryotic mRNA translation typically requires a 5' cap structure.
• Animal viruses like Picornaviruses use internal ribosome entry sites (IRES) for cap-independent translation.
• IRES elements have been found in some non-plant eukaryotes but not in endogenous plant genes.

Purpose of the Study:

• To investigate the potential of animal virus-derived IRES elements to mediate cap-independent translation in plants.
• To construct and test a bicistronic reporter gene system in plants using the encephalomyocarditis virus (EMCV) IRES.

Main Methods:

• Construction of a plant bicistronic gene with GFP and luciferase ORFs separated by the EMCV IRES, under the CaMV 35S promoter.
• Northern blot analysis to detect bicistronic transcript expression.
• In vivo imaging and Western blot analysis to confirm protein expression and independent translation.

Main Results:

• Expression of the bicistronic transcript was confirmed in transgenic plants.
• Both Green Fluorescent Protein (GFP) and luciferase activities were detected, indicating functional protein production.
• Western blot analysis confirmed the independent translation of both reporter proteins, mediated by the EMCV IRES.

Conclusions:

• The encephalomyocarditis virus (EMCV) IRES element can function in plant cells to mediate cap-independent translation of a downstream open-reading frame.
• This IRES activity was observed in transgenic seedlings, particularly in leaves.
• This finding opens possibilities for engineering cap-independent translation in plants using viral IRES elements.