Episome-Based Gene Expression Modulation Platform in the Model Diatom Phaeodactylum tricornutum.

Chemically inducible gene expression systems have been an integral part of the advanced synthetic genetic circuit design and are employed for precise dynamic control over genetically engineered traits. However, the current systems for controlling transgene expression in most algae are limited to endogenous promoters that respond to different environmental factors. We developed a highly efficient, tunable, and reversible episome-based transcriptional control system in the model diatom alga, Phaeodactylum tricornutum. We assessed the time- and dose-response dynamics of each expression system using a reporter protein (eYFP) as a readout. Using our circuit configuration, we found two inducible expression systems with a high dynamic range and confirmed the suitability of an episome expression platform for synthetic biological applications in diatoms. These systems are controlled by the presence of ß-estradiol and digoxin. Addition of either chemical to transgenic strains activates transcription with a dynamic range of up to ∼180-fold and ∼90-fold, respectively. We demonstrated that our episome-based transcriptional control systems are tunable and reversible in a dose- and time-dependent manner. Using droplet digital polymerase chain reaction (PCR), we also confirmed that inducer-dependent transcriptional activation starts within minutes of inducer application without any detectable transcript in the uninduced controls. The system described here expands the molecular and synthetic biology toolkits in algae and will facilitate future gene discovery and metabolic engineering efforts.

Engineering synthetic regulatory circuits in plants.

Plant synthetic biology is a rapidly emerging field that aims to engineer genetic circuits to function in plants with the same reliability and precision as electronic circuits. These circuits can be used to program predictable plant behavior, producing novel traits to improve crop plant productivity, enable biosensors, and serve as platforms to synthesize chemicals and complex biomolecules. Herein we introduce the importance of developing orthogonal plant parts and the need for quantitative part characterization for mathematical modeling of complex circuits. In particular, transfer functions are important when designing electronic-like genetic controls such as toggle switches, positive/negative feedback loops, and Boolean logic gates. We then discuss potential constraints and challenges in synthetic regulatory circuit design and integration when using plants. Finally, we highlight current and potential plant synthetic regulatory circuit applications.

Quantitative characterization of genetic parts and circuits for plant synthetic biology.

Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.

Simple and efficient methods to generate split roots and grafted plants useful for long-distance signaling studies in Medicago truncatula and other small plants.

BACKGROUND: Long distance signaling is a common phenomenon in animal and plant development. In plants, lateral organs such as nodules and lateral roots are developmentally regulated by root-to-shoot and shoot-to-root long distance signaling. Grafting and split root experiments have been used in the past to study the systemic long distance effect of endogenous and environmental factors, however the potential of these techniques has not been fully realized because data replicates are often limited due to cumbersome and difficult approaches and many plant species with soft tissue are difficult to work with. Hence, developing simple and efficient methods for grafting and split root inoculation in these plants is of great importance. RESULTS: We report a split root inoculation system for the small legume M. truncatula as well as robust and reliable techniques of inverted-Y grafting and reciprocal grafting. Although the split root technique has been historically used for a variety of experimental purposes, we made it simple, efficient and reproducible for M. truncatula. Using our split root experiments, we showed the systemic long distance suppression of nodulation on a second wild type root inoculated after a delay, as well as the lack of this suppression in mutants defective in autoregulation. We demonstrated inverted-Y grafting as a method to generate plants having two different root genotypes. We confirmed that our grafting method does not affect the normal growth and development of the inserted root; the composite plants maintained normal root morphology and anatomy. Shoot-to-root reciprocal grafts were efficiently made with a modification of this technique and, like standard grafts, demonstrate that the regulatory signal defective in rdn1 mutants acts in the root. CONCLUSIONS: Our split root inoculation protocol shows marked improvement over existing methods in the number and quality of the roots produced. The dual functions of the inverted-Y grafting approach are demonstrated: it is a useful system to produce a plant having roots of two different genotypes and is also more efficient than published shoot-to-root reciprocal grafting techniques. Both techniques together allow dissection of long distance plant developmental regulation with very simple, efficient and reproducible approaches.

The ROOT DETERMINED NODULATION1 gene regulates nodule number in roots of Medicago truncatula and defines a highly conserved, uncharacterized plant gene family.

The formation of nitrogen-fixing nodules in legumes is tightly controlled by a long-distance signaling system in which nodulating roots signal to shoot tissues to suppress further nodulation. A screen for supernodulating Medicago truncatula mutants defective in this regulatory behavior yielded loss-of-function alleles of a gene designated ROOT DETERMINED NODULATION1 (RDN1). Grafting experiments demonstrated that RDN1 regulatory function occurs in the roots, not the shoots, and is essential for normal nodule number regulation. The RDN1 gene, Medtr5g089520, was identified by genetic mapping, transcript profiling, and phenotypic rescue by expression of the wild-type gene in rdn1 mutants. A mutation in a putative RDN1 ortholog was also identified in the supernodulating nod3 mutant of pea (Pisum sativum). RDN1 is predicted to encode a 357-amino acid protein of unknown function. The RDN1 promoter drives expression in the vascular cylinder, suggesting RDN1 may be involved in initiating, responding to, or transporting vascular signals. RDN1 is a member of a small, uncharacterized, highly conserved gene family unique to green plants, including algae, that we have named the RDN family.