SignificanceWe present identification of the luciferase and enzymes of the biosynthesis of a eukaryotic luciferin from fungi. Fungi possess a simple bioluminescent system, with luciferin being only two enzymatic steps from well-known metabolic pathways. The expression of genes from the fungal bioluminescent pathway is not toxic to eukaryotic cells, and the luciferase can be easily co-opted to bioimaging applications. With the fungal system being a genetically encodable bioluminescent system from eukaryotes, it is now possible to create artificially bioluminescent eukaryotes by expression of three genes. The fungal bioluminescent system represents an example of molecular evolution of a complex ecological trait and with molecular details reported in the paper, will allow additional research into ecological significance of fungal bioluminescence.

Autoluminescent plants that express a bacterial bioluminescence gene cluster 1 have not been widely adopted due to requisite expression in plastids and low light output. Alternatively, we have engineered tobacco lines expressing a fungal bioluminescent system 2 , which converts caffeic acid present in all plants into luciferin, and report self-sustained luminescence easily visible to the naked eye. Our findings might underpin development of a suite of imaging tools for plants.

Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of β-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-β-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-β-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 μg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza.