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.

In contrast to fluorescent proteins, light emission from luciferase reporters requires exogenous addition of a luciferin substrate. Bacterial bioluminescence has been the single exception, where an operon of five genes is sufficient to produce light autonomously. Although commonly used in prokaryotic hosts, toxicity of the aldehyde substrate has limited its use in eukaryotes 1 . Here we demonstrate autonomous luminescence in a multicellular eukaryotic organism by incorporating a recently discovered fungal bioluminescent system 2 into tobacco plants. We monitored these light-emitting plants from germination to flowering, observing temporal and spatial patterns of luminescence across time scales from seconds to months. The dynamic patterns of luminescence reflected progression through developmental stages, circadian oscillations, transport, and response to injuries. As with other fluorescent and luminescent reporters, we anticipate that this system will be further engineered for varied purposes, especially where exogenous addition of substrate is undesirable.

Noncoding RNAs play a crucial role in various cellular processes in living organisms, and RNA functions heavily depend on molecule structures composed of stems, loops, and various tertiary motifs. Among those, the most frequent are A-minor interactions, which are often involved in the formation of more complex motifs such as kink-turns and pseudoknots. We present a novel classification of A-minors in terms of RNA secondary structure where each nucleotide of an A-minor is attributed to the stem or loop, and each pair of nucleotides is attributed to their relative position within the secondary structure. By analyzing classes of A-minors in known RNA structures, we found that the largest classes are mostly homogeneous and preferably localize with known A-minor co-motifs, such as tetraloop–tetraloop receptor and coaxial stacking. Detailed analysis of local A-minors within internal loops revealed a novel recurrent RNA tertiary motif, the across-bulged motif. Interestingly, the motif resembles the previously known GAAA/11nt motif but with the local adenines performing the role of the GAAA-tetraloop. By using machine learning, we show that particular classes of local A-minors can be predicted from sequence and secondary structure. The proposed classification is the first step toward automatic annotation of not only A-minors and their co-motifs but various types of RNA tertiary motifs as well.