Biology subjects

Orzaez, D.

Publications and source records attributed to Orzaez, D..

At least 19 recordsLinked to original sources

bioRxiv2025

Bioluminescent sentinel plants enable autonomous diagnostics of viral infections

Plants engineered with synthetic genetic programs can transform how we monitor and manage the extension of crop pests and diseases. Here, we establish a bioluminescent platform in Nicotiana benthamiana for autonomous viral sensing based on the fungal bioluminescence pathway (FBP). We first demonstrate that recombinant viruses can deliver missing pathway components, enabling spatially resolved tracking of infection dynamics. Leveraging this starting point, we developed a dual-output sentinel circuit that uses a protease-responsive Bioluminescence Resonance Energy Transfer (BRET) module to report infection through a virus-triggered spectral shift in luminescence. In the absence of infection, plants emit a stable yellow glow indicating system integrity. Upon infection with potyviruses, cleavage of the BRET fusion by the virus-encoded NIa-Pro protease activates a distinct colour change detectable with low-cost imaging. This modular design is compatible with other pathogens carrying specific proteases and supports future multiplexing strategies. Our results highlight the potential of synthetic sentinel gene circuits as autonomous biosensors for precision crop protection.

synthetic biology↗

bioRxiv2024

CuBe: a geminivirus-based copper-regulated expression system suitable for post-harvest activation.

The growing demand for sustainable platforms for biomolecule manufacturing has fueled the development of plant-based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large-scale production due to high operational costs and batch-to-batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)-derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulfate (CuSO4), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harboring the gene of interest (GOI) flanked by cis-acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper-inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast-derived CUP2-Gal4 copper-responsive transcriptional activator for Rep/RepA expression, and (iv) a copper-inducible Flp recombinase to minimize basal Rep/RepA expression. Copper sulfate application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti-SARS-CoV-2 antibody upon copper treatment. Notably, the system is also functional with post-harvest copper application, a strategy with potential advantages for large-scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost-effective and scalable production of recombinant proteins in plants.

synthetic biology↗

bioRxiv2023

Assessment of tobacco and N. benthamiana as biofactories of irregular monoterpenes for sustainable crop protection.

Irregular monoterpenes are important precursors of different compounds employed in pest control such as insecticides and insect sex pheromones. Metabolically engineered plants are appealing as biofactories of such compounds, but specially as potential live biodispensers of related bioactive volatiles, which could be continuously emitted to the environment from different plant tissues. Here we assess the use of cultivated tobacco and Nicotiana benthamiana as biofactories for the irregular monoterpenes chrysanthemol and lavandulol. We evaluate the impact of high levels of constitutive metabolite production on the plant physiology and biomass, and their biosynthetic dynamics for different plant tissues and developmental stages. As an example of an active pheromone compound, we super-transformed the best lavandulol-producing tobacco line with an acetyl transferase gene to obtain a tobacco lavandulyl acetate biodispenser emitting up to 0.63 mg of lavandulyl acetate per plant every day. We estimate that with these volatile emission levels, between 200 and 500 plants per hectare would be sufficient to ensure a daily emission of pheromones comparable to commercial lures. This is an important step towards plant-based sustainable solutions for pest control, and it lays the ground for further developing biofactories for other irregular monoterpenoid pheromones, whose biosynthetic genes are yet unknown.

molecular biology↗

bioRxiv2023

Identification and characterisation of Planococcus citri cis- and trans-isoprenyl diphosphate synthase genes, supported by short- and long-read transcriptome data

Many insect species rely on diverse terpenoids for their development and interorganismal interactions. However, little is known about terpenoid biosynthesis in insects. The monoterpenoid sex pheromones of mealybugs and scale insects (Coccoidea) are particularly enigmatic, with several species producing unique structures presumed to result from the irregular coupling activity of unidentified isopentenyl diphosphate synthases (IDSs). Enzymes capable of similar transformations have previously only been described from a few plant, bacterial and archaeal species. To investigate if insect irregular monoterpenes can be biosynthesised by similar enzymes, we performed a comprehensive search for IDS coding sequences in the genome of Planococcus citri, a widespread agricultural pest. We complemented the available P. citri genome data with newly generated short- and long-read transcriptome data. The identified candidate genes had homology to both short- and long-chain IDSs and some appeared to be paralogous, indicating gene duplications and consequent IDS gene family expansion in P. citri. We tested the activity of eleven candidate gene products, confirming in vitro regular activity for five enzymes, one of which (transIDS5) also produced the irregular prenyl diphosphates, maconelliyl and lavandulyl diphosphate. Targeted mutagenesis of selected aspartates and a lysine in the active site of transIDS5 uncovered their importance for chain-length preference and irregular coupling. This work provides an important foundation for deciphering terpenoid biosynthesis in mealybugs, as well as a potential source of enzymes for the biotechnological production of sustainable insect pest management products.

biochemistry↗

bioRxiv2023

A quantitative autonomous bioluminescence reporter system with a wide dynamic range for Plant Synthetic Biology

Engineered autonomous bioluminescence (EAB) offers many potential applications in Plant Synthetic Biology, notably as in vivo reporter system. Current EAB reporter configurations are limited for quantitative applications due to low dynamic range. We reconfigured the Neonothopanus nambi fungal bioluminescence (NeoLuc) pathway to serve as a high-throughput and inexpensive reporter for quantitative analysis of gene expression. We showed that by configuring the first committed step in the pathway (HispS) as the transcriptional entry point instead of the fungal luciferase, the dynamic range of the output increased dramatically, equaling that of the FLuc/RLuc reporter, and outperforming it in high throughput capacity. Furthermore, the inclusion of an enhanced GFP as normalizer allowed transient ratiometric measurements in N. benthamiana. Fast and rich datasets generated by the NeoLuc/eGFP system enabled us to undertake the optimization of new challenging synthetic gene circuits, including a complex agrochemical/optogenetic dual input switch for tight control of engineered metabolic pathways.

synthetic biology↗

bioRxiv2022

A multi-omic Nicotiana benthamiana resource for fundamental research and biotechnology

Nicotiana benthamiana is an invaluable model plant and biotechnology platform. To further improve its usefulness and versatility, we have produced high quality chromosome level genome assemblies and multi-omic datasets for both the ubiquitously used LAB strain and a distantly related wild accession, QLD, as well as mapping their single nucleotide polymorphisms with two additional laboratory and four additional wild accessions. LAB and QLD have many genetic, functional, and metabolic differences. These coupled with their high inter-fertility and equally efficient transient and stable transformation and genome editing provide a powerful resource partnership. Their [~]3Gb allotetraploid genomes show advanced diploidisation with major chromosome loss and rearrangement, extensive homoeologous gene loss, and widespread segmental allopolyploidy. Recent bursts of Copia mobility, not seen in other Nicotiana genomes, have probably aided N. benthamianas adaptation to a spectrum of Australian ecologies.

plant biology↗

bioRxiv2022

Dually biofortified cisgenic tomatoes with increased flavonoids and branched-chain amino acids content

Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched-chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant-derived foods contain only small amounts of BCAAs and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically-engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide-resistant version of an acetolactate synthase (mSlALS) gene expressed broadly, and a MYB12-like transcription factor (SlMYB12) expressed in a fruit-specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21-fold compared to wild type levels), Valine (9-fold), Isoleucine (3-fold), and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64-fold) and quercetin (45-fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption.

bioengineering↗

bioRxiv2022

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.

synthetic biology↗

bioRxiv2022

GB_SynP: a modular dCas9-regulated synthetic promoter collection for fine-tuned recombinant gene expression in plants.

Programable transcriptional factors based on the CRISPR architecture are becoming commonly used in plants for endogenous gene regulation. In plants, a potent CRISPR tool for gene induction is the so-called dCasEV2.1 activation system, which has shown remarkable genome-wide specificity combined with a strong activation capacity. To explore the ability of dCasEV2.1 to act as a transactivator for orthogonal synthetic promoters, a collection of DNA parts was created (GB_SynP) for combinatorial synthetic promoter building. The collection includes (i) minimal promoter parts with the TATA box and 5UTR regions, (ii) proximal parts containing single or multiple copies of the target sequence for the gRNA, thus functioning as regulatory cis boxes, and (iii) sequence-randomized distal parts that ensure the adequate length of the resulting promoter. A total of 35 promoters were assembled using the GB_SynP collection, showing in all cases minimal background and predictable activation levels depending on the proximal parts used. GB_SynP was also employed in a combinatorial expression analysis of an auto-luminescence pathway in Nicotiana benthamiana, showing the value of this tool in extracting important biological information such as the determination of the limiting steps in an enzymatic pathway.

synthetic biology↗

bioRxiv2022

Potato virus X -delivered CRISPR activation programs lead to strong endogenous gene induction and transient metabolic reprogramming in Nicotiana benthamiana.

Programmable transcriptional regulators based on CRISPR architecture are promising tools for the control of plant gene expression. In plants, CRISPR gene activation (CRISPRa) has been shown effective in modulating development processes, such as the flowering time, or customising biochemical composition. The most widely used method for delivering the CRISPR components into the plant is Agrobacterium tumefaciens-mediated genetic transformation, either transient or stable. However, due to their versatility and their ability to move, virus-derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular the gRNA, which represents the variable component in CRISPR strategies. In this work we describe a Potato virus X (PVX)-derived vector that, upon agroinfection in N. benthamiana, serves as a vehicle for gRNAs delivery, producing a highly specific Virus-Induced Gene Activation (VIGA). The system works in combination with a Nicotiana benthamiana transgenic line carrying the remaining complementary CRISPRa components, specifically the dCasEV2.1 cassette, which has previously been shown to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non-invasive spraying method, we show here that gRNAs-mediated activation programs move locally and systemically generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this PVX-based virus-induced gene reprogramming (VIGR) strategy results in program-specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid-enriched metabolite profiles.

synthetic biology↗

bioRxiv2021

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.

CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations in place, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold for the endogenous N. benthamiana DFR gene, with negligible expression in the absence of the trigger. The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

synthetic biology↗

bioRxiv2021

Custom-made design of metabolite composition in N. benthamiana leaves using CRISPR activators.

Transcriptional regulators based on CRISPR architecture expand our ability of reprogramming endogenous gene expression in plants. One of their potential applications is the customization of plant metabolome through the activation of selected enzymes in a given metabolic pathway. Using the previously described multiplexable CRISPR activator dCasEV2.1, we assayed the selective enrichment in Nicotiana benthamiana leaves of four different flavonoids, namely naringenin, eriodictyol, kaempferol and quercetin. After careful selection of target genes and guide RNAs combinations, we created successful activation programs for each of the four metabolites, each program activating between three and seven genes, and with individual gene activation levels ranging from 4- to 1500-fold. Metabolic analysis of the flavonoid profiles of each multigene activation program showed a sharp and selective enrichment of the intended metabolites and their glycosylated derivatives. Remarkably, principal component analysis of untargeted metabolic profiles clearly separated samples according to their activation treatment, and hierarchical clustering separated the samples in five groups, corresponding to the expected four highly enriched metabolite groups, plus an un-activated control. These results demonstrate that dCasEV2.1 is a powerful tool for re-routing metabolic fluxes towards the accumulation of metabolites of interest, opening the door for custom-made design of metabolic contents in plants.

synthetic biology↗

bioRxiv2021

CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors

The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR) components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-associated (Cas) nuclease. Here we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors, derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.

plant biology↗

bioRxiv2021

Production of Volatile Moth Sex Pheromones in Transgenic Nicotiana benthamiana Plants

Plant-based bio-production of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here we describe the engineering of transgenic plants producing (Z)-11-hexadecen-1-ol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them in Nicotiana benthamiana plants. The constructs comprised the Amyelois transitella Atr{Delta}11 desaturase gene, the Helicoverpa armigera farnesyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, whose severity correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered harbouring an intact pheromone pathway and producing moderate levels of Z11-16OAc (11.8 {micro}g g-1 FW), next to high levels of Z11-16OH (111.4 {micro}g g-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g-1 FW per day for Z11-16OH and 9.44 ng g-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone bio-dispenser strategies and establish a roadmap for biotechnological improvements.

synthetic biology↗

bioRxiv2021

Strong and tunable anti-CRISPR/Cas9 activity of AcrIIA4 in plants

This study describes the strong anti-CRISPR activity of the bacterial AcrIIA4 protein in Nicotiana benthamiana, a model plant used as molecular farming platform. The results demonstrate that AcrIIA4 abolishes site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. We also show that AcrIIA4 represses CRISPR/dCas9-based transcriptional activation (CRISPRa) of both reporter and endogenous genes in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas9 activity of AcrIIA4 reported here opens new possibilities for customized control of gene editing and gene expression in plants.

synthetic biology↗

bioRxiv2020

Pilot production of SARS-CoV-2 related proteins in plants: a proof of concept for rapid repurposing of indoors farms into biomanufacturing facilities

The current CoVid-19 crisis is revealing the strengths and the weaknesses of the worlds capacity to respond to a global health crisis. A critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. On the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions. In this work we explore how, in a context of rapid exchange of scientific information, plant biofactories can serve as a rapid and easily adaptable solution for local manufacturing of bioreagents, more specifically recombinant antibodies. For this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against SARS-CoV-2 in Nicotiana benthamiana. For the design of the antibodies we took advantage, among other data sources, of the DNA sequence information made rapidly available by other groups in preprint publications. mAbs were all engineered as single-chain fragments fused to a human gamma Fc and transiently expressed using a viral vector. In parallel, we also produced the recombinant SARS-CoV-2 N protein and its Receptor Binding Domain (RBD) in planta and used them to test the binding specificity of the recombinant mAbs. Finally, for two of the antibodies we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. Our results indicate that gram amounts of anti-SARS-CoV-2 antibodies could be easily produced in little more than 6 weeks in repurposed greenhouses with little infrastructure requirements using N. benthamiana as production platform. Similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid therapeutic responses.

synthetic biology↗

bioRxiv2020

Edition of complex gene families in tobacco with GoldenBraid 4.0, a multipurpose web-based platform for plant genome engineering

CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy in polyploid crops. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, multiplex constructs comprising tandemly arrayed gRNAs are difficult to assemble, this hampering more widespread use. Here we present a comprehensive upgrade of the popular cloning platform GoldenBraid (GB), in which, on top of its classical multigene cloning software, we integrate new assembly tools for two-dimensions gRNA multiplexing with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the gRNA unspaced approaches, respectively. As functional validation, we show, among others, the assembly of up to 17 tandemly-arrayed gRNAs constructs against a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in tobacco. With these constructs we generated a collection of Cas9-free SPL mutants harboring up to 9 biallelic mutations in a single generation. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR activators and repressors using single and multiplexing gRNAs is also validated. With the incorporation of the new CRISPR tools and parts collection, GB4.0 turns an unprecedentedly comprehensive open platform for plant genetic engineering.

molecular biology↗

bioRxiv2020

Efficient Cas9 multiplex editing using unspaced gRNA arrays engineering in a Potato virus X vector

SummarySystems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as the Cas nucleases or the guide RNA (gRNA). While the Cas nucleases are constant elements in editing approaches, gRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of gRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing (VIGE). In this work, we engineered Potato virus X (PVX) to build a vector able to easily express one or more gRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expressed Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced gRNAs achieving highly efficient multiplex editing in a few days in adult plant tissues. We also demonstrate that genome modifications are inherited in plants regenerated from infected tissues. In sum, the new PVX VIGE vector allows easy, fast and efficient expression of gRNAs arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in important crops of the family Solanaceae.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗