Transformed Plant Cells Research Articles

Identification of a di-glucose conjugate of 4-hydroxybenzoic acid in bamboo cells expressing bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase.

Rational metabolic-flow switching is an effective strategy that we proposed for producing exogenous high-value natural products using transformed plant cells. In an earlier proof-of-concept study, we generated bamboo (Phyllostachys nigra; Pn) cells expressing the 4-hydroxycinnamoyl-CoA hydratase/lyase gene of Pseudomonas putida KT2440 (PpHCHL). The encoded enzyme catalyzes the formation of 4-hydroxybenzaldehyde and vanillin from p-coumaroyl-CoA and feruloyl-CoA, respectively. The PpHCHL-transformed Pn cells accumulated mono-glucose conjugates (glucoside and glucose ester) of 4-hydroxybenzoic acid and vanillic acid, indicating that the products (aldehydes) of the PpHCHL-catalyzed reaction were oxidized by endogenous enzyme(s) in Pn cells. In this study, we re-examined the extracts of PpHCHL-transformed Pn cells to screen for additional 4-hydroxybenzoic acid derivatives. An unidentified compound was detected exclusively in the PpHCHL-transformed Pn cells. This compound was purified via column chromatography and then identified as a di-glucose conjugate of 4-hydroxybenzoic acid (i.e., β-D-glucopyranosyl 4-O-β-D-glucopyranosylbenzoate), implying that some of the mono-glucose conjugates of 4-hydroxybenzoic acid were converted to the di-glucose conjugate by endogenous enzyme(s) in Pn cells. The maximum production titer of this di-glucose conjugate in the suspension-cultured cells was 0.38 g l-1, which was the second highest titer among the four glucose conjugates produced by the PpHCHL-transformed Pn cells. The study findings further support the utility of PpHCHL-transformed Pn cells for the bioproduction of 4-hydroxybenzoic acid and its derivatives.

Microbiome and plant cell transformation trigger insect gall induction in cassava.

Several specialised insects can manipulate normal plant development to induce a highly organised structure known as a gall, which represents one of the most complex interactions between insects and plants. Thus far, the mechanism for insect-induced plant galls has remained elusive. To study the induction mechanism of insect galls, we selected the gall induced by Iatrophobia brasiliensis (Diptera: Cecidomyiidae) in cassava (Euphorbiaceae: Manihot esculenta Crantz) as our model. PCR-based molecular markers and deep metagenomic sequencing data were employed to analyse the gall microbiome and to test the hypothesis that gall cells are genetically transformed by insect vectored bacteria. A shotgun sequencing discrimination approach was implemented to selectively discriminate between foreign DNA and the reference host plant genome. Several known candidate insertion sequences were identified, the most significant being DNA sequences found in bacterial genes related to the transcription regulatory factor CadR, cadmium-transporting ATPase encoded by the cadA gene, nitrate transport permease protein (nrtB gene), and arsenical pump ATPase (arsA gene). In addition, a DNA fragment associated with ubiquitin-like gene E2 was identified as a potential accessory genetic element involved in gall induction mechanism. Furthermore, our results suggest that the increased quality and rapid development of gall tissue are mostly driven by microbiome enrichment and the acquisition of critical endophytes. An initial gall-like structure was experimentally obtained in M. esculenta cultured tissues through inoculation assays using a Rhodococcus bacterial strain that originated from the inducing insect, which we related to the gall induction process. We provide evidence that the modification of the endophytic microbiome and the genetic transformation of plant cells in M. esculenta are two essential requirements for insect-induced gall formation. Based on these findings and having observed the same potential DNA marker in galls from other plant species (ubiquitin-like gene E2), we speculate that bacterially mediated genetic transformation of plant cells may represent a more widespread gall induction mechanism found in nature.

An efficient and broadly applicable method for transient transformation of plants using vertically aligned carbon nanofiber arrays.

Transient transformation in plants is a useful process for evaluating gene function. However, there is a scarcity of minimally perturbing methods for gene delivery that can be used on multiple organs, plant species, and non-excised tissues. We pioneered and demonstrated the use of vertically aligned carbon nanofiber (VACNF) arrays to efficiently perform transient transformation of different tissues with DNA constructs in multiple plant species. The VACNFs permeabilize plant tissue transiently to allow molecules into cells without causing a detectable stress response. We successfully delivered DNA into leaves, roots and fruit of five plant species (Arabidopsis, poplar, lettuce, Nicotiana benthamiana, and tomato) and confirmed accumulation of the encoded fluorescent proteins by confocal microscopy. Using this system, it is possible to transiently transform plant cells with both small and large plasmids. The method is successful for species recalcitrant to Agrobacterium-mediated transformation. VACNFs provide simple, reliable means of DNA delivery into a variety of plant organs and species.

The power and versatility of genome editing tools in crop improvement

The power and versatility of genome editing tools in crop improvement

A Review of Nanotechnology as a Novel Method of Gene Transfer in Plants

Purpose: Nanotechnology has evolved as an effective tool in numerous fields including agriculture, medicine and engineering. Recently it’s potential as an alternative genetic transformation method has been identified. However, a comprehensive understanding over nanoparticles and their behavior in living cells is important to realize the full potential of this technology in biotechnological applications. Therefore, we review the application potential of widely employed nanoparticles in plant transformation here.Literature/Background: Development of new crop varieties with desirable traits via biotechnological applications is a solution for challenges associated with climate change and higher population growth. In such aspects, transformation of plant cells which is known as the process of changing one’s genome by integration exogenous DNA, is an absolute necessity and results far better and improved stable characteristics in original. Rigid and multi layered cell wall impedes penetration of exterior biomolecules and hence causes the transformation process complicated. Even though, numerous conventional methods have been established for plant transformation, lower transformation efficiency, tissue damage and random integration of transgenes warrants the need for novel approaches. In this context, novel techniques have been explored and as a result nanoparticles have been found effective in transformation of protoplasts as well as intact plant cells. Nanoparticles internalized either via endocytosis or direct penetration release transgenes from nanoparticle-DNA complexes and result in transient or stable expression. Nanoparticles ensure higher transformation efficiency, no transgenic silencing and protection of biogenic molecules from degradation by intracellular nucleases.

Gene Downregulation in Potato Roots Using Agrobacterium rhizogenes-Mediated Transformation.

Agrobacterium rhizogenes has the ability to transform plant cells by transferring the T-DNA from the Ri plasmid to the plant cell genome. These infected plant cells divide and organize the formation of adventitious roots, called hairy roots. When the A. rhizogenes is additionally transformed with a binary vector, the cells infected can indeed be transformed with this second T-DNA producing transgenic hairy roots. In this chapter, we present the protocol to produce transgenic hairy roots from in vitro potato (Solanum tuberosum) plants injected with transformed A. rhizogenes, generating plants with a wild-type shoot and a transgenic root system. Specifically, we detail the procedure to obtain in vitro-cultured hairy roots with a downregulated gene of interest, by using a Gateway-based binary vector able to produce a RNA hairpin triggering the RNA interference mechanism (hpRNAi). We also present the protocol to analyze the downregulation of the target gene in hairy roots by means of reverse-transcription reaction followed by real-time PCR (qPCR).

Biotransformation of papaverine and in silico docking studies of the metabolites on human phosphodiesterase 10a

The metabolism of papaverine, the opium benzylisoquinoline alkaloid, with Aspergillus niger NRRL 322, Beauveria bassiana NRRL 22864, Cunninghamella echinulate ATCC 18968 and Cunninghamella echinulate ATCC 1382 has resulted in O-demethylation, O-methylglucosylation and N-oxidation products. Two new metabolites (4″-O-methyl-β-D-glucopyranosyl) 4′-demethyl papaverine and (4″-O-methyl-β-D-glucopyranosyl) 6-demethyl papaverine, (Metabolites 5 and 6) together with 4′-O-demethylated papaverine (Metabolite 1), 3′-O-demethylated papaverine (Metabolite 2), 6-O-demethylated papaverine (Metabolite 3) and papaverine N-oxide (Metabolite 4) were isolated. The structure elucidation of the metabolites was based primarily on 1D, 2D-NMR analyses and HRMS. These metabolism results were consistent with the previous plant cell transformation studies on papaverine and isopapaverine and the microbial metabolism of papaveraldine. In silico docking studies of the metabolites using crystals of human phosphodiesterase 10a (hPDE10a) revealed that compounds 4, 1, 6, 3, and 5 possess better docking scores and binding poses with favorable interactions than the native ligand papaverine.

Development and validation of a UHPLC-ESI-QTOF mass spectrometry method to analyze opines, plant biomarkers of crown gall or hairy root diseases

Opines are low-molecular-weight metabolites specifically biosynthesized by agrobacteria-transformed plant cells when plants are struck by crown gall and hairy root diseases, which cause uncontrolled tissue overgrowth. Transferred DNA is sustainably incorporated into the genomes of the transformed plant cells, so that opines constitute a persistent biomarker of plant infection by pathogenic agrobacteria and can be targeted for crown gall/hairy root disease diagnosis. We developed a general, rapid, specific and sensitive analytical method for overall opine detection using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-MS-QTOF), with easy preparation of samples. Based on MS, MS/MS and chromatography data, the detection selectivity of a wide range of standard opines was validated in pure solution and in different plant extracts. The method was successfully used to detect different structural types of opines, including opines for which standard compounds are unavailable, in tumors or hairy roots induced by pathogenic strains. As the method can detect a wide range of opines in a single run, it represents a powerful tool for plant gall analysis and crown gall/hairy root disease diagnosis. Using an appropriate dilution of plant extract and a matrix-based calibration curve, the quantification ability of the method was validated for three opines belonging to different families (nopaline, octopine, mannopine), which were accurately quantified in plant tissue extracts.

Enabling Transgenic Plant Cell–Derived Biomedicines with Nanotechnology

Transgenic plants are promising factories for manufacturing pharmaceutical small molecules or proteins safe for human consumption. Eukaryotic plant cells can synthesize proteins with precise posttranslational modifications, but microbes cannot. Conventional transformation methods (e.g., electroporation, Agrobacterium‐mediated transformation, and biolistic particle delivery) often suffer from inefficient transformation, damage to plant tissues and cells, or applicability to a narrow range of plant species. Notably, the cell wall poses a physical barrier that obstructs effective delivery of nucleic acids to plant cells. Nanoparticles (NPs) are emerging carriers of nucleic acids to plants because they are sufficiently small to diffuse through the cell wall, enter plant cells without the aid of external forces, and inflict limited damage to the plant cells in a broad variety of plants. Herein, three areas of the phytonanotechnology field that merit comprehensive investigations are outlined, namely, the design considerations of NPs for gene delivery to plant cells, homing of NPs to specific organelles (e.g., nucleus and chloroplast), and transformation of plant suspension cells. This perspective concludes with recent insights into scale‐up production of therapeutics by using bioreactors. NPs are poised to catalyze the transformation of plant cells for producing therapeutics in bioreactors at reduced costs, high purity, and improved scale.

DNA-free genome editing

<p indent=0mm>CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been extensively used for gene editing of many plant species, including model plants such as <italic>Arabidopsis</italic> and rice, and major crops such as maize and wheat, as well as woody plants like poplar, bamboo, apple and citrus, etc. This technique has not only significantly promoted basic research, but has also facilitated the agronomic traits improvement of economically important crops or trees. Typically, the CRISPR/Cas system required its integration and expression of exogenous DNA in the genome of host plants, which lead to the cleavage of site-specific DNA double-strand breaks, and introduced the genome modifications during the DNA repair process. However, the integration of CRISPR/Cas elements brings several concerns for commercialization and basic research. First, the inserted CRISPR/Cas cassette is constitutively expressed in plant cells, and may increase the chance of off-target changes, as well as make it difficult to evaluate the stability and penetrance of the initially observed phenotypes. Second, plants with a foreign gene integrated into its genome were regarded as classic genetically modified organisms, which may potentially lead to the escape of the gene into the environment, leading to legislation concerns and strict government policy regulation about such organisms. Therefore, one of the main challenges in using the CRISPR/Cas system in commercial agriculture is to improve the plant’s agronomic traits by genome editing, meanwhile obtaining transgene-free mutant plants with stable transmissible properties. DNA-free genome editing technology in plants is new and began in 2015, and since then great advances have been made on producing transgenic-free genome-edited plants. The exogenous CRISPR/Cas cassette and other transgenes could be eliminated by multiple approaches after the target gene has been edited. For example, the CRISPR/Cas expression cassette can be removed through genetic segregation for plants that are reproduced sexually, and the end products are CRISPR-edited but transgene-free. Transgene-free gene-edited plants may also be generated by non-transgenic approaches, such as transient expression of mRNA encoding Cas nuclease and guide RNA without DNA integration, preassembled ribonucleoprotein and sgRNA complex transfection, fluorescence marker-assisted transgene cassette selection and elimination, virus-mediated and nano-material mediated genome editing technologies. By using these methods, people can obtain the genome-edited end plant product with increased agronomic traits, while no additional foreign DNA fragments have been introduced into the plant genome. Until now, at least 14 transgene-free and genome-edited plant species have been generated using this technology. In this review, we have highlighted these achievements, including methods, applications and the possibilities of their commercialization in plant breeding. We also discuss the current problems in using this technology, such as very low efficiency, limited gene modification types and labor-consuming aspects. With the development of new plant cell transformation methods and the applications of new typical CRISPR/Cas systems, the potential strategies for improving this revolutionary technology in the future are also discussed.

Food/Feed and Environmental Risk Assessment of Insect Resistant Genetically Modified Maize 1507 for Cultivation, Import, Processing, Food and Feed Uses under Directive 2001/18/EC and Regulation (EC) No 1829/2003 (C/ES/01/01, C/NL/00/10, EFSA/GMO/NL/2004/02)

Food/Feed and Environmental Risk Assessment of Insect Resistant Genetically Modified Maize 1507 for Cultivation, Import, Processing, Food and Feed Uses under Directive 2001/18/EC and Regulation (EC) No 1829/2003 (C/ES/01/01, C/NL/00/10, EFSA/GMO/NL/2004/02)

Bioproduction of glucose conjugates of 4-hydroxybenzoic and vanillic acids using bamboo cells transformed to express bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase

Rational metabolic-flow switching, which we proposed recently, is an effective strategy to produce an exogenous high-value natural product using transformed plant cells; the proof of this concept was demonstrated using bamboo (Phyllostachys nigra; Pn) cells as a model system. Pn cells were transformed to express 4-hydroxycinnamoyl-CoA hydratase/lyase of Pseudomonas putida KT2440 (PpHCHL), which catalyzes the formation of 4-hydroxybenzaldehyde and vanillin from p-coumaroyl-CoA and feruloyl-CoA, respectively. The PpHCHL-transformed cells accumulated glucose conjugates of 4-hydroxybenzoic acid and vanillic acid, indicating that the PpHCHL products (aldehydes) were further metabolized by inherent enzymes in the Pn cells. The production titers of 4-hydroxybenzoic acid glucose ester, vanillic acid glucose ester, and 4-hydroxybenzoic acid glucoside reached 1.7, 0.17, and 0.14g/L at the maximum, respectively. These results proved the versatility of Pn cells for producing vanillin-related compounds based on rational metabolic-flow switching.

Final Health and Environmental Risk Assessment of Genetically Modified Maize 59122

Final Health and Environmental Risk Assessment of Genetically Modified Maize 59122

Differential expression of calcium-dependent protein kinase genes (CDPK1–14) in Rubia cordifolia callus cultures transformed with the rolB and rolC genes

Integration of Agrobacterium rhizogenes T-DNA region leads to multiple changes in the physiology and biochemistry of the transformed plant cells. We have previously shown that one of these possible effects may be related to the changes in the expression profile of calcium-dependent protein kinase (CDPK) genes. Since the key determinants of T-DNA are rol genes, in this work, we studied the individual action of the rolB and rolC genes on the expression of CDPK genes in transgenic cells of Rubia cordifolia calli. Rol genes caused a general increase in total R. cordifolia CDPK transcripts abundance. Expression of RcCDPK2, RcCDPK7, RcCDPK8, RcCDPK9 and RcCDPK11 was 1.3- to 3.5-fold higher in both rolB- and rolC-transformed cells than in non-transformed cells, whereas expression of RcCDPK10 was 1.9-fold lower. Expression of RcCDPK1, RcCDPK13 and RcCDPK14 was up-regulated by the rolB gene, and the transcriptional level of RcCDPK4 and RcCDPK5 was higher in the rolC-expressing cells. Our results indicate that complex but often divergent effects of rolB and rolC on transformed plant cells may be attributed to changes caused by these genes on particular CDPK isoforms.

Cyclic di-GMP inactivates T6SS and T4SS activity in Agrobacterium tumefaciens.

SummaryThe Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di‐GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c‐di‐GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c‐di‐GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c‐di‐GMP being a key second messenger that silences energy‐costing systems during early colonization phase and biofilm formation, while low c‐di‐GMP levels unleash T6SS and T4SS to advance plant colonization.

The Agrobacterium VirD5 protein hyperactivates the mitotic Aurora kinase in host cells

Summary Aided by translocated virulence proteins, Agrobacterium tumefaciens transforms plant cells with oncogenic T‐DNA. In the host cells the virulence protein VirD5 moves to the nucleus, where it becomes localized at the kinetochores, and disturbs faithful chromosome segregation, but the molecular mechanism underlying this remains unknown.To gain more insight, we screened amongst the kinetochore proteins for VirD5 interactors using bimolecular fluorescence complementation assays, and tested chromosome segregation in yeast cells.We found that VirD5 interacts with the conserved mitotic Aurora kinase Ipl1 in yeast and likewise with plant Aurora kinases. In vitro VirD5 was found to stimulate the activity of Ipl1. Phosphorylation of substrates by Ipl1 in vivo is known to result in the detachment between kinetochore and spindle microtubule. This is necessary for error correction, but increased Ipl1/Aurora kinase activity is known to cause spindle instability, explaining enhanced chromosome mis‐segregation seen in the presence of VirD5. That activation of the Ipl1/Aurora kinase at least partially underlies the toxicity of VirD5 became apparent by artificial boosting the activity of the specific counteracting phosphatase Glc7 in vivo, which relieved the toxicity.These findings reveal a novel mechanism by which a pathogenic bacterium manipulates host cells.

Transient Gene Expression as a Tool to Monitor and Manipulate the Levels of Acidic Phospholipids in Plant Cells.

Anionic phospholipids represent only minor fraction of cell membranes lipids but they are critically important for many membrane-related processes, including membrane identity, charge, shape, the generation of second messengers, and the recruitment of peripheral proteins. The main anionic phospholipids of the plasma membrane are phosphoinositides phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), phosphatidylserine (PS), and phosphatidic acid (PA). Recent insights in the understanding of the nature of protein-phospholipid interactions enabled the design of genetically encoded fluorescent molecular probes that can interact with various phospholipids in a specific manner allowing their imaging in live cells. Here, we describe the use of transiently transformed plant cells to study phospholipid-dependent membrane recruitment.

A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

SummaryThe genetic transformation of plant cells is critically dependent on the availability of efficient selectable marker gene. Sulfonamides are herbicides that, by inhibiting the folic acid biosynthetic pathway, suppress the growth of untransformed cells. Sulfonamide resistance genes that were previously developed as selectable markers for plant transformation were based on the assumption that, in plants, the folic acid biosynthetic pathway resides in the chloroplast compartment. Consequently, the Sul resistance protein, a herbicide‐insensitive dihydropteroate synthase, was targeted to the chloroplast. Although these vectors produce transgenic plants, the transformation efficiencies are low compared to other markers. Here, we show that this inefficiency is due to the erroneous assumption that the folic acid pathway is located in chloroplasts. When the RbcS transit peptide was replaced by a transit peptide for protein import into mitochondria, the compartment where folic acid biosynthesis takes place in yeast, much higher resistance to sulfonamide and much higher transformation efficiencies are obtained, suggesting that current sul vectors are likely to function due to low‐level mistargeting of the resistance protein to mitochondria. We constructed a series of optimized transformation vectors and demonstrate that they produce transgenic events at very high frequency in both the seed plant tobacco and the green alga Chlamydomonas reinhardtii. Co‐transformation experiments in tobacco revealed that sul is even superior to nptII, the currently most efficient selectable marker gene, and thus provides an attractive marker for the high‐throughput genetic transformation of plants and algae.

The rolB plant oncogene affects multiple signaling protein modules related to hormone signaling and plant defense

The rolB plant oncogene of Agrobacterium rhizogenes perturbs many biochemical processes in transformed plant cells, thereby causing their neoplastic reprogramming. The oncogene renders the cells more tolerant to environmental stresses and herbicides and inhibits ROS elevation and programmed cell death. In the present work, we performed a proteomic analysis of Arabidopsis thaliana rolB-expressing callus line AtB-2, which represents a line with moderate expression of the oncogene. Our results show that under these conditions rolB greatly perturbs the expression of some chaperone-type proteins such as heat-shock proteins and cyclophilins. Heat-shock proteins of the DnaK subfamily were overexpressed in rolB-transformed calli, whereas the abundance of cyclophilins, members of the closely related single-domain cyclophilin family was decreased. Real-time PCR analysis of corresponding genes confirmed the reliability of proteomics data because gene expression correlated well with the expression of proteins. Bioinformatics analysis indicates that rolB can potentially affect several levels of signaling protein modules, including effector-triggered immunity (via the RPM1-RPS2 signaling module), the miRNA processing machinery, auxin and cytokinin signaling, the calcium signaling system and secondary metabolism.

Agrobacterium-Mediated Transformation Of Plant Cells

Agrobacterium-Mediated Transformation Of Plant Cells