Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning

Genetic engineering of complex metabolic pathways and multiple traits often requires the introduction of multiple genes. The construction of plasmids carrying multiple DNA fragments plays a vital role in these processes. In this study, the Gibson assembly and Gateway cloning combined Pyramiding Stacking of Multigenes (PSM) system was developed to assemble multiple transgenes into a single T-DNA. Combining the advantages of Gibson assembly and Gateway cloning, the PSM system uses an inverted pyramid stacking route and allows fast, flexible and efficient stacking of multiple genes into a binary vector. The PSM system contains two modular designed entry vectors (each containing two different attL sites and two selectable markers) and one Gateway-compatible destination vector (containing four attR sites and two negative selection markers). The target genes are primarily assembled into the entry vectors via two parallel rounds of Gibson assembly reactions. Then, the cargos in the entry constructs are integrated into the destination vector via a single tube Gateway LR reaction. To demonstrate PSM’s capabilities, four and nine gene expression cassettes were respectively assembled into the destination vector to generate two binary expression vectors. The transgenic analysis of these constructs in Arabidopsis demonstrated the reliability of the constructs generated by PSM. Due to its flexibility, simplicity and versatility, PSM has great potential for genetic engineering, synthetic biology and the improvement of multiple traits

RMDAP: A Versatile, Ready-To-Use Toolbox for Multigene Genetic Transformation

Background: The use of transgenes to improve complex traits in crops has challenged current genetic transformation technology for multigene transfer. Therefore, a multigene transformation strategy for use in plant molecular biology and plant genetic breeding is thus needed. Methodology/Principal Findings: Here we describe a versatile, ready-to-use multigene genetic transformation method, named the Recombination-assisted Multifunctional DNA Assembly Platform (RMDAP), which combines many of the useful features of existing plant transformation systems. This platform incorporates three widely-used recombination systems, namely, Gateway technology, in vivo Cre/loxP and recombineering into a highly efficient and reliable approach for gene assembly. RMDAP proposes a strategy for gene stacking and contains a wide range of flexible, modular vectors offering a series of functionally validated genetic elements to manipulate transgene overexpression or gene silencing involved in a metabolic pathway. In particular, the ability to construct a multigene marker-free vector is another attractive feature. The built-in flexibility of original vectors has greatly increased the expansibility and applicability of the system. A proof-ofprinciple experiment was confirmed by successfully transferring several heterologous genes into the plant genome. Conclusions/Significance: This platform is a ready-to-use toolbox for full exploitation of the potential for coordinate regulation of metabolic pathways and molecular breeding, and will eventually achieve the aim of what we call ‘‘one-sto

One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE

Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes

GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology

[EN] Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.This work was supported by the Spanish Ministry of Economy and Competitiveness (grant no. BIO2010-15384), by a Research Personnel in Training fellowship to A.S.-P., and by a Junta de Ampliacion de Estudios fellowship to M.V.-V.Sarrion-Perdigones, A.; Vázquez Vilar, M.; Palací Bataller, J.; Castelijns, B.; Forment Millet, JJ.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.... (2013). GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiology. 162(3):1618-1631. https://doi.org/10.1104/pp.113.217661S16181631162

ROLE FOR HYDROGEN PEROXIDE DURING ABIOTIC AND BIOTIC STRESS SIGNALING IN PLANTS

Plants must adapt to negative environmental conditions that limit their growth, development and yield. Common for such stress conditions is that they induce the accumulation of harmful reactive oxygen species (ROS). ROS, such as hydrogen peroxide (H2O2), are now also considered to be important signal molecules that regulate the defense response of plants to stress. In this thesis, different strategies were pursued to identify genes that are involved in the stress response of plants

The generation of biofortified and weed-resistant cereal plants through genetic engineering

El desenvolupament de cultius biofortificats amb un major contingut de minerals mitjançant l'enginyeria genètica pot resultar útil per combatre la malnutrició a curt o mitjà termini. En aquest projecte ens vam proposar desenvolupar plantes d'arròs transgèniques que acumulessin més calci (Ca) i seleni (Se) al gra. Les línies transgèniques van ser caracteritzades a nivell molecular, i es va avaluar com afectava l'expressió dels transgens a l'acumulació de minerals i al metabolisme del Ca i del Se en les plantes transgèniques. L’Striga perjudica greument la producció de cereals a l'Àfrica. L'ús de l'enginyeria genètica pot permetre el desenvolupament de noves varietats de cereals que produeixin menys quantitat d'estrigolactones, fent-les així resistents a l'Striga. En aquest estudi vam desenvolupar línies de blat de moro transgèniques que expressaven una construcció genètica de RNAi, per tal de disminuir l'expressió de dos dels gens involucrats en la biosíntesi d'estrigolactones, el Zmd27 i el Zmccd8.El desarrollo de cultivos biofortificados con un mayor contenido de minerales mediante la ingeniería genética puede resultar útil para combatir la malnutrición a corto o medio plazo. En este proyecto nos propusimos desarrollar plantas de arroz transgénicas que acumulasen más calcio (Ca) y selenio (Se) en el grano. Las líneas transgénicas resultantes fueron caracterizadas a nivel molecular, y se evaluó cómo afectaba la expressión de los transgenes en la acumulación de minerales, así como en el metabolismo del Ca y del Se en las plantas transgénicas. El género Striga perjudica gravemente la producción de cereales en África. El uso de la ingeniería genética puede permitir el desarrollo de nuevas variedades de cereales que produzcan menos cantidad de estrigolactonas, haciéndolas así resistentes a la Striga. En éste estudio desarrollamos líneas de maíz transgénicas que expresaban niveles más bajos de dos de los genes involucrados en la biosíntesis de estrigolactonas, el Zmd27 y el Zmccd8.The generation of biofortified staple crops with enhanced mineral content through genetic engineering is a promising approach to counter malnutrition in the short- and middle-term. We aimed to create a population of transgenic rice plants with increased capacity for calcium (Ca) and selenium (Se) accumulation in the grain. Transgenic rice lines were characterized at molecular level, and the impact of transgene expression on mineral accumulation and endogenous Ca and Se metabolism was preliminary evaluated. Cereal production in Africa is severely hampered by Striga infection. Genetic engineering can be used to develop Striga resistant cereal varieties through reducing strigolactone production. We generated transgenic maize plants expressing RNAi constructs targeting two genes involved in the strigolactone biosynthetic pathway, Zmd27 and Zmccd8. Expression of target genes was effectively down-regulated and distinctive phenotypes were observed in both transgenic lines. Strigolactone levels were drastically reduced in Zmccd8 line and offers great potential for Striga control

Optimierung der Photosynthese von C3-Pflanzen : C 4 -ähnlicher Zyklus und chloroplastidärer Bypass

Most agronomic traits are polygenic in nature and rely on complex metabolic and regulatory pathways. Genetic modifications of these traits or introduction of new pathways require the transfer of multiple genes into the plant genome. Most of the existing methods like retransformation, cotransformation and crossing suffer from certain deficiencies like high expenditure of time or the integration of multiple copies (Dafny-Yelin und Tzfira, 2007; Naqvi et al., 2010). The transfer of the genes as a single multigene construct offers considerable advantages. Up to now there are only few efficient methods for assembly and subsequent transformation of multigene constructs into plants. In the context of this work a Gateway-based method could be established, which enables fusion of multiple fragments. The system consists of a Gateway-compatible destination vector and two special entry vectors with attR cassettes, which are flanked by incompatible attL sequences. By alternate use of the two entry vectors multiple transgenes can be recombined into the destination vector. Multigene constructs with 7 gene expression cassettes could be transferred successfully into tobacco by agrobacterium-mediated transformation. By biolistic transformation a multigene construct with 5 gene expression cassettes could be transferred into rice. Due to the bifunctionality of RUBISCO, the key enzyme of photosynthesis, besides carboxylation also oxygenation of ribulose-1,5-bisphosphat is catalyzed in the chloroplasts of higher plants (Bowes et al., 1971). Glycolate formed by this process is toxic and unusable for the plant (Zelitch et al., 2008) and have to be metabolized in the photorespiration. The conversion of glycolate not only consumes ATP and reduction equivalents, it also leads to loss of 25% carbon fixed in this metabolite. For C3 plants photorespiration means a significant reduction of photosynthesis efficiency. On the contrary C4 plants can considerably reduce the oxygenase activity of RUBISCO due to their CO2 concentration mechanism. By use of the MultiRound Gateway technology tobacco plants could be generated, which expressed the heterologous enzymes for a single cell C4 cycle following the example of Hydrilla verticillata. Besides a NADP-ME type C4 cylce, a NAD-ME type and a PCK type C4 cycle were constructed. However, there was no evidence for a CO2 concentration inside the chloroplasts. The heterologous expression of both malic enzymes (NADP-HvMe and NAD-EcMe) strongly affects plant growth. The higher the ME activities were, the slower was the plant growth. All types had in common a higher nitrogen content, which resulted in a lower C/N ratio. Also by use of the MultiRound Gateway technology rice plants with a chloroplastic photorespiratory bypass could be generated. The bypass relies on the catabolic glycolate pathway from E. coli and converts the glycolate inside the chloroplasts. This is intended to increase the CO2 concentration in the vicinity of RUBISCO and thereby suppress photorespiration. In Arabidopsis this pathway led to promising results (Kebeish et al., 2007). The expression of all genes required for this pathway (TSR, GCL, glcD, glcE, glcF) could be verified on the RNA level. To sum up, it can be said that the MultiRound Gateway technology is well suited for the assembly of multigene constructs and the constructed destination vector can be transferred into plants by agrobacterium-mediated transformation as well as biolistic transformation. The generated plants can serve as starting basis for further attempts to establish a single cell C4 cycle. A better balanced relation of enzymatic activities may avoid the negative effects on plant growth caused by high ME activities

Engineering the multigene pathways for CO 2 concentration mechanism and bypassing the photorespiration in C 3 plants

The present study shows an improvement in photosynthesis in C3 plants by two different approaches. The first approach is aimed to increasing the CO2 concentration in the proximity of Rubisco. The second approach is aimed at bypassing the ‘wasteful’ reaction of photorespiration in C3 plants. In both approaches, multiple transgene metabolic pathways are created and successfully introduced in C3 plants. With the help of multiple transgene metabolic pathways the final products such as CO2 is observed to successfully reach Rubisco. The multiple transgenes are introduced into the tobacco (Nicotiana tabacum) plant through Agrobacterium transformation. The MultiRound Gateway technology is used to successfully deliver the combination of two multiple transgene pathways in to the Agrobacterium. The first approach is intended to create a single cell C4 pathway in a C3 plant, similar like in Hydrilla verticillata C4 plants, Novel pathway (HC4l) mimic Hydrilla in which carbon channels from cytoplasm to chloroplast ultimately resulting in an increase of CO2 under the vicinity of Rubisco. In this approach, to create the first multigene transgenic pathway, seven different heterologaus enzymes are used. They are: phosphoenolpyruvate carboxylase (PEPC) derived from Hydrilla verticilliata, NAD malate dehydrogenase (MDH) derived from Maize, NAD malic enzyme (ME) derived from E. coli, lactate dehydrogenase (LDHA) derived from mouse liver, lactate dehydrgenase (LDHB) derived from rat heart, phosphoenolpyruvate synthase (PEPS) from E. coli, and malate lactate antiporter (Mle) derived from Bacillus subtilitis. The MultiRound Gateway technology is successfully used to transfer these seven transgenes in to plant with the help of two entry vector and one destination vector approach. The RT-PCR is used to quantify the expression level and the efficiency of the expression of transgenes in the study plant. The findings from the RT-PCR indicate successful expression of transgenes in the test plants. The enzymes of respective transgenic are analyzed with enzymatic assays and it also reveals the presence of active protein in the plant studied. In the second approach, another combination of transgenic pathway is introduced into C3 plants to bypass the wasteful reaction and flux of photorespiration. The transgenic pathway used in this approach included genes such as malate synthase (MS) from E. coli, NAD malic enzyme (ME) from E. coli, phosphoenolpyruvate synthase (PPS), enolase from E. coli and phosphoglycerate mutase (PGM) from E. coli. The aim of this transgenic pathway is to reduce the photorespiration to a greater degree than previously observed in plants with GTDEF pathway. The plants used for this approach already possessed the GTDEF pathway. Similar to the first approach, the MultiRound Gateway technology is used to transfer all transgenes by using two entry and one destination vector. The analysis by enzyme assays shows the active presence of the test proteins the plant studied. The influence of introduction of five genes pathway in a plant is measured through physiological, biochemical and by photosynthetic parameters. Metabolism of glycolate pathway derived from E. coli and was established in the chloroplast of Arabidopsis thaliana plants (Kebeish et al., 2007). This pathway (GTDEF) showed lower compensation point, higher glucose, fructose and end product of the photosynthesis as compared to the wild type plants. The present study reveals that the introduction of the novel pathway consisting five genes (MMPEM pathway) into the plants existing GTDEF pathway results in further decrease in the compensation point (Г*), a noticeable increase in glucose, fructose and other end product of photosynthesis. The study also reveals that the plants with combination of GTDEF pathway and five gene pathway has higher fresh as well as dry weight as compared to plants with only GTDEF pathway. The research shows that with the successful introduction of multigene complex metabolic pathways in C3 plants, the higher rate of photosynthesis is possible