Cytoplasmic male sterility and abortive seed traits generated through mitochondrial genome editing coupled with allotopic expression of atp1 in tobacco.

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

Targeted DNA excision in Arabidopsis by a re-engineered homing endonuclease.

BACKGROUND: A systematic method for plant genome manipulation is a major aim of plant biotechnology. One approach to achieving this involves producing a double-strand DNA break at a genomic target site followed by the introduction or removal of DNA sequences by cellular DNA repair. Hence, a site-specific endonuclease capable of targeting double-strand breaks to unique locations in the plant genome is needed. RESULTS: We engineered and tested a synthetic homing endonuclease, PB1, derived from the I-CreI endonuclease of Chlamydomonas reinhardtii, which was re-designed to recognize and cleave a newly specified DNA sequence. We demonstrate that an activity-optimized version of the PB1 endonuclease, under the control of a heat-inducible promoter, is capable of targeting DNA breaks to an introduced PB1 recognition site in the genome of Arabidopsis thaliana. We further demonstrate that this engineered endonuclease can very efficiently excise unwanted transgenic DNA, such as an herbicide resistance marker, from the genome when the marker gene is flanked by PB1 recognition sites. Interestingly, under certain conditions the repair of the DNA junctions resulted in a conservative pairing of recognition half sites to remove the intervening DNA and reconstitute a single functional recognition site. CONCLUSION: These results establish parameters needed to use engineered homing endonucleases for the modification of endogenous loci in plant genomes.

Crosstalk between endogenous and synthetic components--synthetic signaling meets endogenous components.

Synthetic biology uses biological components to engineer new functionality in living organisms. We have used the tools of synthetic biology to engineer detector plants that can sense man-made chemicals, such as the explosive trinitrotoluene, and induce a response detectable by eye or instrumentation. A goal of this type of work is to make the designed system orthogonal, that is, able to function independently of systems in the host. In this review, the design and function of two partially synthetic signaling pathways for use in plants is discussed. We describe observed interactions (crosstalk) with endogenous signaling components. This crosstalk can be beneficial, allowing the creation of hybrid synthetic/endogenous signaling pathways, or detrimental, resulting in system noise and/or false positives. Current approaches in the field of synthetic biology applicable to the design of orthogonal signaling systems, including the design of synthetic components, partially synthetic systems that utilize crosstalk to signal through endogenous components, computational redesign of proteins, and the use of heterologous components, are discussed.

Programmable ligand detection system in plants through a synthetic signal transduction pathway.

BACKGROUND: There is an unmet need to monitor human and natural environments for substances that are intentionally or unintentionally introduced. A long-sought goal is to adapt plants to sense and respond to specific substances for use as environmental monitors. Computationally re-designed periplasmic binding proteins (PBPs) provide a means to design highly sensitive and specific ligand sensing capabilities in receptors. Input from these proteins can be linked to gene expression through histidine kinase (HK) mediated signaling. Components of HK signaling systems are evolutionarily conserved between bacteria and plants. We previously reported that in response to cytokinin-mediated HK activation in plants, the bacterial response regulator PhoB translocates to the nucleus and activates transcription. Also, we previously described a plant visual response system, the de-greening circuit, a threshold sensitive reporter system that produces a visual response which is remotely detectable and quantifiable. METHODOLOGY/PRINCIPAL FINDINGS: We describe assembly and function of a complete synthetic signal transduction pathway in plants that links input from computationally re-designed PBPs to a visual response. To sense extracellular ligands, we targeted the computational re-designed PBPs to the apoplast. PBPs bind the ligand and develop affinity for the extracellular domain of a chemotactic protein, Trg. We experimentally developed Trg fusions proteins, which bind the ligand-PBP complex, and activate intracellular PhoR, the HK cognate of PhoB. We then adapted Trg-PhoR fusions for function in plants showing that in the presence of an external ligand PhoB translocates to the nucleus and activates transcription. We linked this input to the de-greening circuit creating a detector plant. CONCLUSIONS/SIGNIFICANCE: Our system is modular and PBPs can theoretically be designed to bind most small molecules. Hence our system, with improvements, may allow plants to serve as a simple and inexpensive means to monitor human surroundings for substances such as pollutants, explosives, or chemical agents.

Engineering key components in a synthetic eukaryotic signal transduction pathway.

Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB-VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB-VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved-signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways.

Construction of a fluorescent biosensor family.

Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand-mediated hinge-bending motion. Construction of cysteine mutations at these locations then permits site-specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three-dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.