Robust soybean leaf agroinfiltration.

KEY MESSAGE: A robust agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and Agrobacterium culture conditions are important for successful agroinfiltration. Agroinfiltration of Nicotiana benthamiana has emerged as a workhorse transient assay for plant biotechnology and synthetic biology to test the performance of gene constructs in dicot leaves. While effective, it is nonetheless often desirable to assay transgene constructs directly in crop species. To that end, we innovated a substantially robust agroinfiltration method for Glycine max (soybean), the most widely grown dicot crop plant in the world. Several factors were found to be relevant to successful soybean leaf agroinfiltration, including genotype, surfactant, developmental stage, and Agrobacterium strain and culture medium. Our optimized protocol involved a multi-step Agrobacterium culturing process with appropriate expression vectors, Silwet L-77 as the surfactant, selection of fully expanded leaves in the VC or V1 stage of growth, and 5 min of vacuum at - 85 kPa followed by a dark incubation period before plants were returned to normal growth conditions. Using this method, young soybean leaves of two lines-V17-0799DT, and TN16-5004-were high expressors for GUS, two co-expressed fluorescent protein genes, and the RUBY reporter product, betalain. This work not only represents a new research tool for soybean biotechnology, but also indicates critical parameters for guiding agroinfiltration optimization for other crop species. We speculate that leaf developmental stage might be the most critical factor for successful agroinfiltration.

Phytosensors: harnessing plants to understand the world around us.

Although plants are sessile, their ubiquitous distribution, ability to harness energy from the sun, and ability to sense above and belowground signals make them ideal candidates for biosensor development. Synthetic biology has allowed scientists to reimagine biosensors as engineered devices that are focused on accomplishing novel tasks. As such, a new wave of plant-based sensors, phytosensors, are being engineered as multi-component sense-and-report devices that can alert human operators to a variety of hazards. While phytosensors are intrinsically tied to agriculture, a new generation of phytosensors has been envisioned to function in the built environment and even in austere environments, such as space. In this review, we will explore the current state of the art with regard to phytosensor engineering.

Development of new binary expression systems for plant synthetic biology.

KEY MESSAGE: A novel plant binary expression system was developed from the compactin biosynthetic pathway 27 of Penicillium citrinum ML-236B. The system achieved >fivefold activation of gene expression in 28 transgenic tobacco. A diverse and well-characterized genetic toolset is fundamental to achieve the overall goals of plant synthetic biology. To properly coordinate expression of a multigene pathway, this toolset should include binary systems that control gene expression at the level of transcription. In plants, few highly functional, orthogonal transcriptional regulators have been identified. Here, we describe the process of developing synthetic plant transcription factors using regulatory elements from the Penicillium citrinum ML-236B (compactin) pathway. This pathway contains several genes including mlcA and mlcC that are transcriptionally regulated in a dose-dependent manner by the activator mlcR. In Nicotiana benthamiana, we first expressed mlcR with several cognate synthetic promoters driving expression of GFP. Synthetic promoters contained operator sequences from the compactin gene cluster. Following identification of the most active synthetic promoter, the DNA-binding domain from mlcR was used to generate chimeric transcription factors containing variable activation domains, including QF from the Neurospora crassa Q-system. Activity was measured at both protein and RNA levels which correlated with an R2 value of 0.94. A synthetic transcription factor with a QF activation domain increased gene expression from its synthetic promoter up to sixfold in N. benthamiana. Two systems were characterized in transgenic tobacco plants. The QF-based plants maintained high expression in tobacco, increasing expression from the cognate synthetic promoter by fivefold. Transgenic plants and non-transgenic plants were morphologically indistinguishable. The framework of this study can easily be adopted for other putative transcription factors to continue improvement of the plant synthetic biology toolbox.

Genome-Editing of FtsZ1 for Alteration of Starch Granule Size in Potato Tubers.

Genome-editing has enabled rapid improvement for staple food crops, such as potato, a key beneficiary of the technology. In potato, starch contained within tubers represents the primary product for use in food and non-food industries. Starch granules are produced in the plastids of tubers with plastid size correlated with the size of starch grana. The division of plastids is controlled by proteins, including the tubulin-like GTPase FtsZ1. The altered expression of FtsZ1 has been shown to disrupt plastid division, leading to the production of "macro-plastid"-containing plants. These macro-chloroplast plants are characterized by cells containing fewer and enlarged plastids. In this work, we utilize CRISPR/Cas9 to generate FtsZ1 edited potato lines to demonstrate that genome-editing can be used to increase the size of starch granules in tubers. Altered plastid morphology was comparable to the overexpression of FtsZ1 in previous work in potato and other crops. Several lines were generated with up to a 1.98-fold increase in starch granule size that was otherwise phenotypically indistinguishable from wild-type plants. Further, starch paste from one of the most promising lines showed a 2.07-fold increase in final viscosity. The advantages of enlarged starch granules and the potential of CRISPR/Cas9-based technologies for food crop improvement are further discussed.

Genetic Engineering of Potato (Solanum tuberosum) Chloroplasts Using the Small Synthetic Plastome "Mini-Synplastome".

In the rapidly expanding field of synthetic biology, chloroplasts represent attractive targets for installation of valuable genetic circuits in plant cells. Conventional methods for engineering the chloroplast genome (plastome) have relied on homologous recombination (HR) vectors for site-specific transgene integration for over 30 years. Recently, episomal-replicating vectors have emerged as valuable alternative tools for genetic engineering of chloroplasts. With regard to this technology, in this chapter we describe a method for engineering potato (Solanum tuberosum) chloroplasts to generate transgenic plants using the small synthetic plastome (mini-synplastome). In this method, the mini-synplastome is designed for Golden Gate cloning for easy assembly of chloroplast transgene operons. Mini-synplastomes have the potential to accelerate plant synthetic biology by enabling complex metabolic engineering in plants with similar flexibility of engineered microorganisms.

Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements.

While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.

High-Throughput Transfection and Analysis of Soybean (Glycine max ) Protoplasts.

With the advent of plant synthetic biology, there is an urgent need to develop plant-based systems that are able to effectively enhance the speed of design-build-test cycles to screen large numbers of synthetic constructs. Thus far, protoplasts have served to fill this need, with cell suspension cultures serving as the primary source tissue to enable high-throughput protoplast experimentation. The possibility to use low-cost food-grade enzymes for cell wall digestion along with polyethylene glycol (PEG)-mediated transfection makes protoplasts particularly suited to automation and high-throughput screening. In other systems for which synthetic biology is well established (model bacteria and yeast), libraries of components, i.e., promoters, 5' untranslated regions, 3' untranslated regions, terminators, and transcription factors, serve as the basis for the design of complex genetic circuits. In order for synthetic biology to make similar strides in plant biology, well-characterized libraries of functional genetic parts for plants are required, necessitating the need for high-throughput protoplast assays.In this chapter, we describe an optimized method for the preparation of soybean (Glycine max ) dark-grown cell suspension cultures, followed by protoplast isolation, automated transfection , and subsequent screening.

Mini-synplastomes for plastid genetic engineering.

In the age of synthetic biology, plastid engineering requires a nimble platform to introduce novel synthetic circuits in plants. While effective for integrating relatively small constructs into the plastome, plastid engineering via homologous recombination of transgenes is over 30 years old. Here we show the design-build-test of a novel synthetic genome structure that does not disturb the native plastome: the 'mini-synplastome'. The mini-synplastome was inspired by dinoflagellate plastome organization, which is comprised of numerous minicircles residing in the plastid instead of a single organellar genome molecule. The first mini-synplastome in plants was developed in vitro to meet the following criteria: (i) episomal replication in plastids; (ii) facile cloning; (iii) predictable transgene expression in plastids; (iv) non-integration of vector sequences into the endogenous plastome; and (v) autonomous persistence in the plant over generations in the absence of exogenous selection pressure. Mini-synplastomes are anticipated to revolutionize chloroplast biotechnology, enable facile marker-free plastid engineering, and provide an unparalleled platform for one-step metabolic engineering in plants.

Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology.

Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana. Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.

MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology.

Plant synthetic biology is a rapidly evolving field with new tools constantly emerging to drive innovation. Of particular interest is the application of synthetic biology to chloroplast biotechnology to generate plants capable of producing new metabolites, vaccines, biofuels, and high-value chemicals. Progress made in the assembly of large DNA molecules, composing multiple transcriptional units, has significantly aided in the ability to rapidly construct novel vectors for genetic engineering. In particular, Golden Gate assembly has provided a facile molecular tool for standardized assembly of synthetic genetic elements into larger DNA constructs. In this work, a complete modular chloroplast cloning system, MoChlo, was developed and validated for fast and flexible chloroplast engineering in plants. A library of 128 standardized chloroplast-specific parts (47 promoters, 38 5' untranslated regions [5'UTRs], nine promoter:5'UTR fusions, 10 3'UTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors) were mined from the literature and modified for use in MoChlo assembly, along with chloroplast-specific destination vectors. The strategy was validated by assembling synthetic operons of various sizes and determining the efficiency of assembly. This method was successfully used to generate chloroplast transformation vectors containing up to seven transcriptional units in a single vector (∼10.6-kb synthetic operon). To enable researchers with limited resources to engage in chloroplast biotechnology, and to accelerate progress in the field, the entire kit, as described, is available through Addgene at minimal cost. Thus, the MoChlo kit represents a valuable tool for fast and flexible design of heterologous metabolic pathways for plastid metabolic engineering.

Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants.

CRISPR/Cas9 has been widely applied to various plant species accelerating the pace of plant genome editing and precision breeding in crops. Unintended effects beyond off-target nucleotide mutations are still somewhat unexplored. We investigated the degree and patterns of epigenetic changes after gene editing. We examined changes in DNA methylation in genome-edited promoters of naturally hypermethylated genes (AT1G72350 and AT1G09970) and hypomethylated genes (AT3G17320 and AT5G28770) from Arabidopsis. Transgenic plants were developed via Agrobacterium-mediated floral dip transformation. Homozygous edited lines were selected from segregated T2 plants by an in vitro digestion assay using ribonucleoprotein complex. Bisulfite sequencing comparisons were made between paired groups of edited and non-edited plants to identify changes in DNA methylation of the targeted loci. We found that directed mutagenesis via CRISPR/Cas9 resulted in no unintended morphological or epigenetic alterations. Phenotypes of wild-type, transgenic empty vector, and transgenic edited plants were similar. Epigenetic profiles revealed that methylation patterns of promoter regions flanking target sequences were identical among wild-type, transgenic empty vector, and transgenic edited plants. There was no effect of mutation type on epigenetic status. We also evaluated off-target mutagenesis effects in the edited plants. Potential off-target sites containing up to 4-bp mismatch of each target were sequenced. No off-target mutations were detected in candidate sites. Our results showed that CRISPR/Cas9 did not leave an epigenetic footprint on either the immediate gene-edited DNA and flanking DNA or introduce off-target mutations.