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.

ROOT DETERMINED NODULATION1 Is Required for M. truncatula CLE12, But Not CLE13, Peptide Signaling through the SUNN Receptor Kinase.

The combinatorial interaction of a receptor kinase and a modified CLE peptide is involved in several developmental processes in plants, including autoregulation of nodulation (AON), which allows legumes to limit the number of root nodules formed based on available nitrogen and previous rhizobial colonization. Evidence supports the modification of CLE peptides by enzymes of the hydroxyproline O-arabinosyltransferase (HPAT/RDN) family. Here, we show by grafting and genetic analysis in Medicago truncatula that, in the AON pathway, RDN1, functioning in the root, acts upstream of the receptor kinase SUNN, functioning in the shoot. As expected for a glycosyltransferase, we found that RDN1 and RDN2 proteins are localized to the Golgi, as was shown previously for AtHPAT1. Using composite plants with transgenic hairy roots, we show that RDN1 and RDN2 orthologs from dicots as well as a related RDN gene from rice (Oryza sativa) can rescue the phenotype of rdn1-2 when expressed constitutively, but the less related MtRDN3 cannot. The timing of the induction of MtCLE12 and MtCLE13 peptide genes (negative regulators of AON) in nodulating roots is not altered by the mutation of RDN1 or SUNN, although expression levels are higher. Plants with transgenic roots constitutively expressing MtCLE12 require both RDN1 and SUNN to prevent nodule formation, while plants constitutively expressing MtCLE13 require only SUNN, suggesting that the two CLEs have different requirements for function. Combined with previous work, these data support a model in which RDN1 arabinosylates MtCLE12, and this modification is necessary for the transport and/or reception of the AON signal by the SUNN kinase.

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.

Multiple Autoregulation of Nodulation (AON) Signals Identified through Split Root Analysis of Medicago truncatula sunn and rdn1 Mutants.

Nodulation is energetically costly to the host: legumes balance the nitrogen demand with the energy expense by limiting the number of nodules through long-distance signaling. A split root system was used to investigate systemic autoregulation of nodulation (AON) in Medicago truncatula and the role of the AON genes RDN1 and SUNN in the regulatory circuit. Developing nodule primordia did not trigger AON in plants carrying mutations in RDN1 and SUNN genes, while wild type plants had fully induced AON within three days. However, despite lacking an early suppression response, AON mutants suppressed nodulation when roots were inoculated 10 days or more apart, correlated with the maturation of nitrogen fixing nodules. In addition to correlation between nitrogen fixation and suppression of nodulation, suppression by extreme nutrient stress was also observed in all genotypes and may be a component of the observed response due to the conditions of the assay. These results suggest there is more than one systemic regulatory circuit controlling nodulation in M. truncatula. While both signals are present in wild type plants, the second signal can only be observed in plants lacking the early repression (AON mutants). RDN1 and SUNN are not essential for response to the later signal.

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 M. truncatula SUNN gene is expressed in vascular tissue, similarly to RDN1, consistent with the role of these nodulation regulation genes in long distance signaling.

Encoding a conserved protein of unknown function, the Medicago truncatula RDN1 gene is involved in autoregulation of nodulation through signaling in the root.  In contrast, the SUNN kinase in M. truncatula has been shown by grafting of mutant scions to control nodule number in the root by communication of a signal from the shoot to the root.  GUS staining patterns resulting from expression of the SUNN promoter fused to uidA showed expression of SUNN in most parts of plant including the root, but confined to the vascular tissue, a pattern that overlaps with that published for RDN1.  Real Time qRT-PCR analysis showed levels of both SUNN RNA and RDN1 RNA did not change significantly during early nodulation signaling (0-72 hours after inoculation).  The similarity in expression in cell types strongly suggests vascular signaling for nodule number regulation, while the lack of changes over early nodule development suggest post transcriptional mechanisms such as protein association or phosphorylation transmit the signal.  

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.

The lss supernodulation mutant of Medicago truncatula reduces expression of the SUNN gene.

The number of nodules that form in a legume when interacting with compatible rhizobia is regulated by the plant. We report the identification of a mutant in nodule regulation in Medicago truncatula, like sunn supernodulator (lss), which displays shoot-controlled supernodulation and short roots, similar to sunn mutants. In contrast with the sunn-1 mutant, nodulation in the lss mutant is more extensive and is less sensitive to nitrate and ethylene, resembling the sunn-4 presumed null allele phenotype. Although the lss locus maps to the SUNN region of linkage group 4 and sunn and lss do not complement each other, there is no mutation in the genomic copy of the SUNN gene or in the 15-kb surrounding region in the lss mutant. However, expression of the SUNN gene in the shoots of lss plants is greatly reduced compared with wild-type plants. Analysis of cDNA from plants heterozygous for lss indicates that lss is a cis-acting factor affecting the expression of SUNN, and documented reversion events show it to be unstable, suggesting a possible reversible DNA rearrangement or an epigenetic change in the lss mutant. Assessment of the SUNN promoter revealed low levels of cytosine methylation in the 700-bp region proximal to the predicted transcription start site in both wild-type and lss plants, indicating that promoter hypermethylation is not responsible for the suppression of SUNN expression in lss. Thus, lss represents either a distal novel locus within the mapped region affecting SUNN expression or an uncharacterized epigenetic modification at the SUNN locus.