Engineering Plant Cell Fates and Functions for Agriculture and Industry.

Many plant species are grown to enable access to specific organs or tissues, such as seeds, fruits, or stems. In some cases, a value is associated with a molecule that accumulates in a single type of cell. Domestication and subsequent breeding have often increased the yields of these target products by increasing the size, number, and quality of harvested organs and tissues but also via changes to overall plant growth architecture to suit large-scale cultivation. Many of the mutations that underlie these changes have been identified in key regulators of cellular identity and function. As key determinants of yield, these regulators are key targets for synthetic biology approaches to engineer new forms and functions. However, our understanding of many plant developmental programs and cell-type specific functions is still incomplete. In this Perspective, we discuss how advances in cellular genomics together with synthetic biology tools such as biosensors and DNA-recording devices are advancing our understanding of cell-specific programs and cell fates. We then discuss advances and emerging opportunities for cell-type-specific engineering to optimize plant morphology, responses to the environment, and the production of valuable compounds.

Transcriptome-informed identification and characterization of Planococcus citri cis- and trans-isoprenyl diphosphate synthase genes.

Insect physiology and reproduction depend on several terpenoid compounds, whose biosynthesis is mainly unknown. One enigmatic group of insect monoterpenoids are mealybug sex pheromones, presumably resulting from the irregular coupling activity of unidentified isoprenyl diphosphate synthases (IDSs). Here, we performed a comprehensive search for IDS coding sequences of the pest mealybug Planococcus citri. We queried the available genomic and newly generated short- and long-read P. citri transcriptomic data and identified 18 putative IDS genes, whose phylogenetic analysis indicates several gene family expansion events. In vitro testing confirmed regular short-chain coupling activity with five gene products. With the candidate with highest IDS activity, we also detected low amounts of irregular coupling products, and determined amino acid residues important for chain-length preference and irregular coupling activity. This work therefore provides an important foundation for deciphering terpenoid biosynthesis in mealybugs, including the sex pheromone biosynthesis in P. citri.

An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis.

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report a new assembly of the N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis.

Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana.

Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species-specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low-cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9-based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

A mini-TGA protein modulates gene expression through heterogeneous association with transcription factors.

TGA (TGACG-binding) transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigated the role of StTGA2.1, a potato (Solanum tuberosum) TGA lacking the full bZIP, which we named a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirmed that StTGA2.1 overexpression compensates for SA-deficiency, indicating a distinct mechanism of action compared with model plant species. To understand the underlying mechanisms, we showed that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigated the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirmed that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases (StPRX), which are known to play role in immune response. Finally, via structural modeling and molecular dynamics simulations, we hypothesized that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to distinct functions and that such events should be studied carefully in other protein families.

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana.

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally important compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. Here we use transcriptomic analysis to identify glycosyltransferases that are upregulated in response to biosynthetic intermediates and produce plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produces a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) is critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases does not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

Cas9-Mediated Targeted Mutagenesis in Plants.

Genome engineering technologies enable targeted mutations to be induced at almost any location in plant genomes. In particular, Cas9 nucleases use easily recoded RNA guides to target user-defined sequences and generate double-stranded breaks (DSB) that are then repaired by the cell's endogenous repair mechanisms. Incorrect repair results in mutations at the target. When the targets are in coding sequences, this often results in loss-of-function mutations. In this chapter, we describe a method to rapidly design and assemble RNA-guided Cas9 constructs for plants and test their ability to induce mutations at their intended targets in rapid assays using both Agrobacterium-mediated transient expression and PEG-mediated DNA delivery to protoplasts, the latter of which can be adapted to a wide range of plant species. We describe a PCR-based method for detecting mutagenesis and outline the steps required to segregate the Cas9 transgene from the targeted mutation to enable the production of transgene-free mutated plants. These techniques are amenable to a range of plant species and should accelerate the application of Cas-9-mediated genome engineering for basic plant science as well as crop development.

Insect pest management in the age of synthetic biology.

Arthropod crop pests are responsible for 20% of global annual crop losses, a figure predicted to increase in a changing climate where the ranges of numerous species are projected to expand. At the same time, many insect species are beneficial, acting as pollinators and predators of pest species. For thousands of years, humans have used increasingly sophisticated chemical formulations to control insect pests but, as the scale of agriculture expanded to meet the needs of the global population, concerns about the negative impacts of agricultural practices on biodiversity have grown. While biological solutions, such as biological control agents and pheromones, have previously had relatively minor roles in pest management, biotechnology has opened the door to numerous new approaches for controlling insect pests. In this review, we look at how advances in synthetic biology and biotechnology are providing new options for pest control. We discuss emerging technologies for engineering resistant crops and insect populations and examine advances in biomanufacturing that are enabling the production of new products for pest control.

Biofoundry-assisted expression and characterization of plant proteins.

Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterization has remained a technical bottleneck, often requiring significant effort to optimize expression and purification protocols. To leverage the ability of biofoundries to accelerate design-built-test-learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimization and enables rapid screening of enzyme activity. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using Escherichia coli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification and improved expression/folding. We next optimized automated assembly of miniaturized cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11-amino acid tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional assays can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.

Measurement of Transgene Copy Number in Plants Using Droplet Digital PCR.

Transgenic plants are produced both to investigate gene function and to confer desirable traits into crops. Transgene copy number is known to influence expression levels, and consequently, phenotypes. Similarly, knowledge of transgene zygosity is desirable for making quantitative assessments of phenotype and tracking the inheritance of transgenes in progeny generations. Since the first transgenic plants were produced, several methods for determining copy number have been applied, including Southern blotting, quantitative real-time PCR, and more recently, sequencing methods; however, each method has specific disadvantages, compromising throughput, accuracy, or expense. Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of occurrence of specific sequences to be accurately estimated. Confidence increases with the number of partitions; therefore, the availability of emulsion technologies that enable reactions to be divided into tens of thousands of nanodroplets allows accurate determination of copy number in what has become known as digital droplet PCR (ddPCR). ddPCR offers similar benefits of low costs and scalability as other PCR techniques but with superior accuracy and reliability. Graphic abstract: Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of transgene copy number to be accurately assessed.

A biofoundry workflow for the identification of genetic determinants of microbial growth inhibition.

Biofoundries integrate high-throughput software and hardware platforms with synthetic biology approaches to enable the design, execution and analyses of large-scale experiments. The unique and powerful combination of laboratory infrastructure and expertise in molecular biology and automation programming, provide flexible resources for a wide range of workflows and research areas. Here, we demonstrate the applicability of biofoundries to molecular microbiology, describing the development and application of automated workflows to identify the genetic basis of growth inhibition of the plant pathogen Streptomyces scabies by a Pseudomonas strain isolated from a potato field. Combining transposon mutagenesis with automated high-throughput antagonistic assays, the workflow accelerated the screening of 2880 mutants to correlate growth inhibition with a biosynthetic gene cluster within 2 weeks.

Evaluation of existing guidelines for their adequacy for the molecular characterisation and environmental risk assessment of genetically modified plants obtained through synthetic biology.

Synthetic Biology (SynBio) is an interdisciplinary field at the interface of engineering and biology aiming to develop new biological systems and impart new functions to living cells. EFSA has been asked by the European Commission to evaluate SynBio developments in agri-food with the aim of identifying the adequacy of existing guidelines for risk assessment and determine if updated guidance is needed. The scope of this opinion covers the molecular characterisation and environmental risk assessment of such genetically modified plants obtained through SynBio, meant to be for cultivation or food and feed purposes. The previous work on SynBio by the non-food scientific Committees (2014, 2015) was used and complemented with the output of a horizon scanning exercise, which was commissioned by the EFSA to identify the most realistic and forthcoming SynBio cases of relevance to this remit. The horizon scan did not identify other sectors/advances in addition to the six SynBio categories previously identified by the non-food scientific committees of the European Commission. The exercise did show that plant SynBio products reaching the market in the near future (next decade) are likely to apply SynBio approaches to their development using existing genetic modification and genome editing technologies. In addition, three hypothetical SynBio case studies were selected by the working group of the Panel on Genetically Modified Organisms (GMO), to further support the scoping exercise of this Scientific Opinion. Using the selected cases, the GMO Panel concludes that the requirements of the EU regulatory framework and existing EFSA guidelines are adequate for the risk assessment of SynBio products to be developed in the next 10 years, although specific requirements may not apply to all products. The GMO Panel acknowledges that as SynBio developments evolve, a need may exist to adjust the guidelines to ensure they are adequate and sufficient.

80 questions for UK biological security.

Multiple national and international trends and drivers are radically changing what biological security means for the United Kingdom (UK). New technologies present novel opportunities and challenges, and globalisation has created new pathways and increased the speed, volume and routes by which organisms can spread. The UK Biological Security Strategy (2018) acknowledges the importance of research on biological security in the UK. Given the breadth of potential research, a targeted agenda identifying the questions most critical to effective and coordinated progress in different disciplines of biological security is required. We used expert elicitation to generate 80 policy-relevant research questions considered by participants to have the greatest impact on UK biological security. Drawing on a collaboratively-developed set of 450 questions, proposed by 41 experts from academia, industry and the UK government (consulting 168 additional experts) we subdivided the final 80 questions into six categories: bioengineering; communication and behaviour; disease threats (including pandemics); governance and policy; invasive alien species; and securing biological materials and securing against misuse. Initially, the questions were ranked through a voting process and then reduced and refined to 80 during a one-day workshop with 35 participants from a variety of disciplines. Consistently emerging themes included: the nature of current and potential biological security threats, the efficacy of existing management actions, and the most appropriate future options. The resulting questions offer a research agenda for biological security in the UK that can assist the targeting of research resources and inform the implementation of the UK Biological Security Strategy. These questions include research that could aid with the mitigation of Covid-19, and preparation for the next pandemic. We hope that our structured and rigorous approach to creating a biological security research agenda will be replicated in other countries and regions. The world, not just the UK, is in need of a thoughtful approach to directing biological security research to tackle the emerging issues.

The wheat Sr22, Sr33, Sr35 and Sr45 genes confer resistance against stem rust in barley.

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley production in Africa and Europe. In contrast to wheat with 60 designated stem rust (Sr) resistance genes, barley's genetic variation for stem rust resistance is very narrow with only ten resistance genes genetically identified. Of these, only one complex locus consisting of three genes is effective against TTKSK, a widely virulent Pgt race of the Ug99 tribe which emerged in Uganda in 1999 and has since spread to much of East Africa and parts of the Middle East. The objective of this study was to assess the functionality, in barley, of cloned wheat Sr genes effective against race TTKSK. Sr22, Sr33, Sr35 and Sr45 were transformed into barley cv. Golden Promise using Agrobacterium-mediated transformation. All four genes were found to confer effective stem rust resistance. The barley transgenics remained susceptible to the barley leaf rust pathogen Puccinia hordei, indicating that the resistance conferred by these wheat Sr genes was specific for Pgt. Furthermore, these transgenic plants did not display significant adverse agronomic effects in the absence of disease. Cloned Sr genes from wheat are therefore a potential source of resistance against wheat stem rust in barley.

Phytobricks: Manual and Automated Assembly of Constructs for Engineering Plants.

Phytobricks are standardized DNA parts for plants that can be assembled hierarchically into transcriptional units and, subsequently, into multigene constructs. Phytobricks each contain the sequences of one or more functional elements that comprise eukaryotic transcription units, with sequence features that enable them to be used interchangeably in one-step cloning reactions to facilitate combinatorial assembly. The simplicity and efficiency of this one-step reaction has enabled Phytobrick assembly to be miniaturized and automated on liquid handing platforms. In this method, we describe how to design and construct new Phytobricks as well as how to assemble them in both manual and nanoscale automated one-step reactions. Finally, we describe a high-throughput method for sequence verification of assembled plasmids.

Rational design of minimal synthetic promoters for plants.

Promoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors. Previously, a paucity of data for cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilize plant transcriptional machinery for gene expression. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling the computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices.

Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis.

The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.

Bioengineering horizon scan 2020.

Horizon scanning is intended to identify the opportunities and threats associated with technological, regulatory and social change. In 2017 some of the present authors conducted a horizon scan for bioengineering (Wintle et al., 2017). Here we report the results of a new horizon scan that is based on inputs from a larger and more international group of 38 participants. The final list of 20 issues includes topics spanning from the political (the regulation of genomic data, increased philanthropic funding and malicious uses of neurochemicals) to the environmental (crops for changing climates and agricultural gene drives). The early identification of such issues is relevant to researchers, policy-makers and the wider public.