VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype.

Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work, we developed a technology called VipariNama (ViN) in which vectors based on the tobacco rattle virus are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, ViN accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

Building customizable auto-luminescent luciferase-based reporters in plants.

Bioluminescence is a powerful biological signal that scientists have repurposed as a reporter for gene expression in plants and animals. However, there are downsides associated with the need to provide a substrate to these reporters, including its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP) in planta using a composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP in planta to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for future construction of programmable auto-luminescent plant traits, such as light driven plant-pollinator interactions or light emitting plant-based sensors.

Many animals have evolved the capacity to produce light from chemical reactions. For example, an enzyme known as luciferase in fireflies produces light by acting on a molecule called luciferin. Scientists have identified the enzymes that drive several of these systems and used them to build reporters that can study the activity of genes in the tissues of plants and other lifeforms over space and time. However, these reporters often require chemicals to be added to the tissues to produce light. These chemicals tend to be expensive and may not penetrate evenly into the tissues of interest, limiting the potential applications of the reporters in research studies. Recently, it has been discovered that fungi have a bioluminescence pathway that converts a molecule known as caffeic acid into luciferin. Caffeic acid is a common molecule in plants, therefore, it is possible the fungal bioluminescence pathway could be used to build reporters that produce light without needing the addition of chemicals. Now, Khakhar et al. have inserted the genes that encode the enzymes of the fungal bioluminescence pathway into tobacco plants. The experiments found that this was sufficient to turn caffeic acid into molecules of luciferin which are able to produce light. Inserting the same genes into several other plant species, including tomatoes and dahlias, produced similar results. Further experiments showed that the fungal bioluminescence pathway can be used to build reporters that monitor the activity of plant genes throughout living tissues and over a period of several days as well as examine the response to plant hormones. Alongside studying the activities of genes in plants, Khakhar et al. propose that the toolkit developed in this work could be used to generate plants with luminescence that can be switched on or off as desired. This could have many uses including helping plants attract insects to pollinate flowers and building plant biosensors that emit light in response to environmental signals.