Agrobacterium Tumefaciens Cells Research Articles

DNA methylation by CcrM contributes to genome maintenance in the Agrobacterium tumefaciens plant pathogen.

The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric chromosome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and represses gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. Further analyses show that GcrA promotes the expression of the RepCCh2 initiator, most likely through the repression of a RepECh2 anti-sense RNA. Altogether, we propose that replication at ori1 leads to a transient hemi-methylation and activation of the gcrA promoter, allowing repCCh2 activation by GcrA and contributing to initiation at ori2. This study then uncovers a novel and original connection between CcrM-dependent DNA methylation, a conserved epigenetic regulator and genome maintenance in an Alphaproteobacterial pathogen.

Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546.

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24-36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes' integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum.

Agroinoculation of Geminiviral Infectious Clones into Plants.

Most geminiviruses are not transmitted by mechanical inoculation. Therefore, pathogenicity and plant-pathogen interaction studies rely on agroinoculation using infectious clones, which involves cloning the geminiviral genome in a binary vector (see previous chapter for details). A suspension containing the infectious clone inserted into Agrobacterium tumefaciens cells is then inoculated into plants, i.e., agroinoculated. Below is a simple protocol for agroinoculation of an infectious geminivirus clone into plants.

Efficient Agrobacterium tumefaciens-mediated genetic transformation of Aloe vera

Genetic transformation of plants has emerged as a core research tool for the functional characterization of genes and cultivar improvement in the field of plant biology. Effectually, high transformation efficiency is a prerequisite for accomplishing the stated targets. Aloe vera, a tropical cactus plant in distinction to the family Liliaceae, has seized a long history of secular acceptance as a therapeutic agent and is reasonably the most popular herbal remedy these days. However, the genetic transformation efficiency of Aloe vera is relatively low and seldom reported formerly. This study aims to optimize Agrobacterium-mediated transformation in Aloe vera by refining several parameters like explant selection, the extent of explant injury during infection, Agrobacterium concentration, co-cultivation pH, and duration and desiccation of plant tissue to improve the infection efficiency. The results showed that nearly 83% transient transformation efficiency was attained by suspending the Agrobacterium tumefaciens (AGL1 strain) cells at a concentration of OD600: 0.4 in co-cultivation media at pH-5.6 to infect the stem of aloe by desiccation, followed by 3 days of co-cultivation. Desiccation during infection had proved to enhance T-DNA delivery, whereas a higher extent of explant injury was found to curtail the infection efficiency. The molecular analysis of the putative transformants was carried out by following GUS histochemical assay and PCR. Thereupon, Southern blotting was used to substantiate the authentication of positive transgenic plants and analyze the copy number of the hptII gene in the Aloe vera genome. A transformation efficiency of over 2% was obtained, which is higher than the previous report. This study bestows an improved Agrobacterium-mediated transformation protocol based on desiccation in Aloe vera, which might help in facilitating various functional genomic studies and gene regulation in the aloe plant, eventually allowing the genetic modification of aloe species in an effective manner. This research deliberates a meliorated Agrobacterium-mediated transformation methodology for Aloe vera, which may facilitate various gene functional investigations and bioengineering of aloe species in an effective salutary way.

Tracking of plasmodesmata localization with the use of fluorescently labelled VAP27 protein

Plasmodesmata play a crucial role in plant development processes, biotic and abiotic stress responses. The thick space inside of a plasmodesma between the plasma membrane and desmotubule is tethered by spoke-like elements. VAP27 is a transmembrane protein localized in plasmodesmata that acts as their tether. The aim of the study was to investigate the integration of fluorescently labelled VAP27 protein into plasma membrane in Nicotiana benthamiana plasmodesmata which would enable the tracking of N. benthamiana plasmodesmata localization in vivo. The research was done by infiltration of N. benthamiana with the Agrobacterium tumefaciens cells. A. tumefaciens were transformed with the GreenGate assembled plasmid containing the gene coding for Nterminally labelled VAP27 with EGFP. The EGFP fluorescence was detected by confocal imaging of N. benthamiana leaves. Prior to confocal imaging, the leaves were infiltrated with aniline blue, which acted as plasmodesmata marker. The findings revealed the colocalization of positions of VAP27-EGFP expression and plasmodesmata. The conclusion was made that the integration of VAP27-EGFP into the plasma membrane in plasmodesmata was successful.

Genome Analysis of Phytophthora nicotianae JM01 Provides Insights into Its Pathogenicity Mechanisms.

Phytophthora nicotianae is a widely distributed plant pathogen that can cause serious disease and cause significant economic losses to various crops, including tomatoes, tobacco, onions, and strawberries. To understand its pathogenic mechanisms and explore strategies for controlling diseases caused by this pathogen, we sequenced and analyzed the whole genome of Ph. nicotianae JM01. The Ph. nicotianae JM01 genome was assembled using a combination of approaches including shotgun sequencing, single-molecule sequencing, and the Hi-C technique. The assembled Ph. nicotianae JM01 genome is about 95.32 Mb, with contig and scaffold N50 54.23 kb and 113.15 kb, respectively. The average GC content of the whole-genome is about 49.02%, encoding 23,275 genes. In addition, we identified 19.15% of interspersed elements and 0.95% of tandem elements in the whole genome. A genome-wide phylogenetic tree indicated that Phytophthora diverged from Pythium approximately 156.32 Ma. Meanwhile, we found that 252 and 285 gene families showed expansion and contraction in Phytophthora when compared to gene families in Pythium. To determine the pathogenic mechanisms Ph. nicotianae JM01, we analyzed a suite of proteins involved in plant–pathogen interactions. The results revealed that gene duplication contributed to the expansion of Cell Wall Degrading Enzymes (CWDEs) such as glycoside hydrolases, and effectors such as Arg-Xaa-Leu-Arg (RXLR) effectors. In addition, transient expression was performed on Nicotiana benthamiana by infiltrating with Agrobacterium tumefaciens cells containing a cysteine-rich (SCR) protein. The results indicated that SCR can cause symptoms of hypersensitive response. Moreover, we also conducted comparative genome analysis among four Ph. nicotianae genomes. The completion of the Ph. nicotianae JM01 genome can not only help us understand its genomic characteristics, but also help us discover genes involved in infection and then help us understand its pathogenic mechanisms.

Candidate Effectors from Botryosphaeria dothidea Suppress Plant Immunity and Contribute to Virulence.

Fungal effectors play important roles in host–pathogen interactions. Botryosphaeria dothidea is an ascomycetous fungus that is responsible for the diseases of hundreds of woody plant species, including apple ring rot, which seriously affects apples worldwide. However, little is known about the effectors of B. dothidea. In this study, we analyzed the B. dothidea genome and predicted 320 candidate effector genes, 124 of which were successfully amplified and cloned. We investigated the effects of these genes on plant cell death in Nicotiana benthamiana while using a transient expression system. Twenty-four hours after initial inoculation with Agrobacterium tumefaciens cells carrying candidate effectors, the infiltrated leaves were challenged with A. tumefaciens cells carrying the BAX gene. In total, 116 candidate effectors completely inhibited, while one partially inhibited, the programmed cell death (PCD) of N. benthamiana induced by BAX, whereas seven candidate effectors had no effect. We then further tested seven candidate effectors able to suppress BAX-triggered PCD (BT-PCD) and found that they all completely inhibited PCD triggered by the elicitors INF1, MKK1, and NPK1. This result suggests that these effectors were activated in order to suppress pathogen-associated molecular pattern-triggered immunity. The signal peptides of these candidate effectors exhibited secretory activity in yeast (pSUC2 vector). Moreover, the respective deletion of Bdo_11198 and Bdo_12090 significantly reduced the virulence of B. dothidea. These results suggest that these effectors play important roles in the interaction of B. dothidea with its hosts.

Quorum quenching acylase impacts the viability and morphological change of Agrobacterium tumefaciens cells

Acylase is known as a quorum quenching enzyme that degrades N-acyl-homoserine lactones (AHLs), a key signaling molecule in a quorum sensing (QS) mechanism. Acylase I cleaves the acyl-chain in the chemical structures of AHLs, thereby exerting an anti-biofilm effect by the inhibition of bacterial cell-cell communication and resultant secretion of extracellular polymeric substances (EPS). However, the physical and physiological impacts of acylase on bacterial cells remain to be systematically elucidated. This study, therefore, investigated the effect of active and inactive acylase addition on the growth, viability, and cell morphologies of Agrobacterium tumefaciens. For comparison, active and inactive lysozymes were taken as positive controls. The results showed that active acylase inhibited A.tumefaciens cell growth at concentrations ranging from 0.1 to 1000μgmL-1, and so did active lysozyme. Fluorescent detection by Live/Dead staining underpinned that cell viability of A.tumefaciens decreased at concentrations higher than 0.1μgmL-1 for both acylase and lysozyme, although lysozyme inflicted higher degree of cellular damage. Moreover, atomic force microscopy unraveled a noticeable distortion of A.tumefaciens cells by both acylase and lysozyme. Together, the results showed that acylase not only blocked AHLs-based QS mechanisms but also compromised cell viability and altered surface morphology of A.tumefaciens cells, as observed by the addition of hydrolase.

Identification of Critical Surface Parameters Driving Lectin-Mediated Capture of Bacteria from Solution.

Lectin-functional interfaces are useful for isolation of bacteria from solution because they are low-cost and allow nondestructive, reversible capture. This study provides a systematic investigation of physical and chemical surface parameters that influence bacteria capture over lectin-functionalized polymer interfaces and then applies these findings to construct surfaces with significantly enhanced bacteria capture. The designer block copolymer poly(glycidyl methacrylate)- block-poly(vinyldimethyl azlactone) was used as a lectin attachment layer, and lectin coupling into the polymer film through azlactone-lectin coupling reactions was first characterized. Here, experimental parameters including polymer areal chain density, lectin molecular weight, and lectin coupling buffer were systematically varied to identify parameters driving highest azlactone conversions and corresponding lectin surface densities. To introduce physical nanostructures into the attachment layer, nanopillar arrays (NPAs) of varied heights (300 and 2100 nm) were then used to provide an underlying surface template for the functional polymer layer. Capture of Escherichia coli on lectin-polymer surfaces coated over both flat and NPA surfaces was then investigated. For flat polymer interfaces, bacteria were detected on the surface after incubation at a solution concentration of 103 cfu/mL, and a corresponding detection limit of 1.7 × 103 cfu/mL was quantified. This detection limit was 1 order of magnitude lower than control lectin surfaces functionalized with standard, carbodiimide coupling chemistry. NPA surfaces containing 300 nm tall pillars further improved the detection limit to 2.1 × 102 cfu/mL, but also reduced the viability of captured cells. Finally, to investigate the impact of cell surface parameters on capture, we used Agrobacterium tumefaciens cells genetically modified to allow manipulation of exopolysaccharide adhesin production levels. Statistical analysis of surface capture levels revealed that lectin surface density was the primary factor driving capture, as opposed to exopolysaccharide adhesin expression. These findings emphasize the critical importance of the synthetic interface and the development of surfaces that combine high lectin densities with tailored physical features to drive high levels of capture. These insights will aid in design of biofunctional interfaces with physicochemical surface properties favorable for capture and isolation of bacteria cells from solutions.

Expression of Hemagglutinin-Neuraminidase (HN) and Fusion (F) Epitopes of Newcastle Disease Virus (NDV) in Chlamydomonas reinhardtii

Owing to their unique characteristics which combines the properties of both prokaryotes and eukaryotes, microalgae have emerged as an ideal platform for heterologous production of recombinant proteins including subunit vaccines. In an attempt to develop recombinant vaccine against Newcastle Disease, an agrobacterium-mediated genetic transformation was carried out to express a chimeric gene construct including Hemagglutinin-Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) in Chlamydomonas reinhardtii. Four tandem repeat of HN epitope with 96bp length followed by three tandem repeat of F epitope of NDV with 153bp length were used. Microalgal cells (Chlamydomonas reinhardtii) were co-cultivated with Agrobacterium tumefaciens cells harboring foreign gene construct and then transferred to selection medium. Single colonies representing putative transformation events were screened in selection medium enriched with kanamycin. PCR assay confirmed integration of F-HN sequence in microalgal nuclei. RT-PCR assay showed that the F-HN sequence was expressed in transformed colonies. Finally, translation of the foreign gene was confirmed by protein dot blotting, western blot and Elisa assay. The results of this experiment may contain both research and practical implications.

An improved cucumber mosaic virus-based vector for efficient decoying of plant microRNAs.

We previously devised a cucumber mosaic virus (CMV)-based vector system carrying microRNA target mimic sequences for analysis of microRNA function in Arabidopsis thaliana. We describe an improved version in which target mimic cloning is achieved by annealing two partly-overlapping complementary DNA oligonucleotides for insertion into an infectious clone of CMV RNA3 (LS strain) fused to the cauliflower mosaic virus-derived 35S promoter. LS-CMV variants carrying mimic sequences were generated by co-infiltrating plants with Agrobacterium tumefaciens cells harboring engineered RNA3 with cells carrying RNA1 and RNA2 infectious clones. The utility of using agroinfection to deliver LS-CMV-derived microRNA target mimic sequences was demonstrated using a miR165/166 target mimic and three solanaceous hosts: Nicotiana benthamiana, tobacco (N. tabacum), and tomato (Solanum lycopersicum). In all three hosts the miR165/166 target mimic induced marked changes in developmental phenotype. Inhibition of miRNA accumulation and increased target mRNA (HD-ZIP III) accumulation was demonstrated in tomato. Thus, a CMV-derived target mimic delivered via agroinfection is a simple, cheap and powerful means of launching virus-based miRNA mimics and is likely to be useful for high-throughput investigation of miRNA function in a wide range of plants.

Plant cell death caused by fungal, bacterial, and viral elicitors: protective effect of mitochondria-targeted quinones.

Chitosan (partially deacetylated chitin), a component of fungal cell walls, caused epidermal cell (EC) death in the leaves of pea (Pisum sativum L.) and tobacco Nicotiana tabacum or Nicotiana benthamiana detected by destruction of cell nuclei. The mitochondria-targeted quinone SkQ1 prevented the destruction of EC nuclei induced by chitosan. Chitosan increased and SkQ1 suppressed the activity of protein kinases in N. benthamiana and P. sativum and eliminated the effect of chitosan. Chitosan induced the generation of reactive oxygen species (ROS) in the guard cells (GC) of pea plants. Treatment with chitosan or H2O2 did not cause destruction of GC nuclei; however, it resulted in disruption of the permeability barrier of the plasma membrane detected by propidium iodide fluorescence. Treatment with bacterial lipopolysaccharide but not peptidoglycan caused destruction of pea EC nuclei, which was prevented by SkQ1. Leaves of tobacco plants containing the N gene responsible for resistance to tobacco mosaic virus (TMV) were infiltrated with Agrobacterium tumefaciens cells. These cells contained a genetic construct with the gene of the helicase domain of TMV replicase (p50); its protein product p50 is a target for the N-gene product. As a result, the hypersensitive response (HR) was initiated. The HR manifested itself in the death of leaves and was suppressed by SkQ3. Treatment of tobacco epidermal peels with the A. tumefaciens cells for the p50 gene expression stimulated the destruction of EC nuclei, which was inhibited by SkQ1 or SkQ3. The p50-lacking A. tumefaciens cells did not induce the destruction of EC nuclei. The protective effect of mitochondria-targeted antioxidants SkQ1 and SkQ3 demonstrates the involvement of mitochondria and their ROS in programmed cell death caused by pathogen elicitors.

Involvement of Rad52 in T‐DNA circle formation during Agrobacterium tumefaciens‐mediated transformation of Saccharomyces cerevisiae

Agrobacterium tumefaciens cells carrying a tumour inducing plasmid (Ti-plasmid) can transfer a defined region of transfer DNA (T-DNA) to plant cells as well as to yeast. This process of Agrobacterium-mediated transformation (AMT) eventually results in the incorporation of the T-DNA in the genomic DNA of the recipient cells. All available evidence indicates that T-strand transfer closely resembles conjugal DNA transfer as found between Gram-negative bacteria. However, where conjugal plasmid DNA transfer starts via relaxase-mediated processing of a single origin of transfer (oriT), the T-DNA is flanked by two imperfect direct border repeats which are both substrates for the Ti-plasmid encoded relaxase VirD2. Yeast was used as a model system to investigate the requirements of the recipient cell for the formation of T-DNA circles after AMT. It was found that, despite the absence of self-homology on the T-DNA, the homologous repair proteins Rad52 and Rad51 are involved in T-DNA circle formation. A model is presented involving the formation of T-DNA concatemers derived from T-strands by a process of strand-transfer catalysed by VirD2. These concatemers are then resolved into T-DNA circles by homologous recombination in the recipient cell.

A Technique for Agrobacterium-Mediated Transformation via Germinating Seeds of Soybean

The plasmid (with resistant the herbicide glyphosate gene EPSPS and glufosinate resistance screening gene Bar) was transferred into soybean [Glycine max (L.) Merr.] by agrobacterium-mediated transformation, and the Bar gene as the selectable marker coupled with glufosinate as a selective agent is described. This method used for transformation requires without tissue culture steps. Soybean seeds were germinated for 1 day, plumule, cotyledonary node, and adjacent regions were wounded with the tip. The wounded half-seeds were inoculated with agrobacterium tumefaciens cells harboring a recombinant DNA that contained the EPSPS and Bar genes. Polymerase chain reaction and two herbicides screened proved that the EPSPS gene has been transferred into soybean successfully; the transformation rate of soybean about was 0.37%.

Design and make up 35S::DREB1A molecular construct to produce environmental stress tolerant plants

Environmental stresses affect plant growth and cause losses worth hundreds of million dollars of agricultural industry each year. Many genes induced in response to environmental stresses. The DREB1A gene is a stress-inducible transcription factor that its overexpression increased tolerance to environmental stress in transgenic plants. DNA was extracted from Arabidopsis thaliana var. Col.0 plants and DREB1A gene amplified by particular primers. After purification, PCR products were cloned into pGEMT-EASY vector and transferred into E. coli strain DH5α competent cells. Blue/white colonies were analyzed and present of the DREB1A gene revealed by restriction analysis and sequencing test. A 668-bp XbaI/BamHI digested fragment of DREB1A gene from pGEMT::DREB1A construct cloned in pBI121 plasmid. The recombinant plasmids were transferred into Agrobacterium tumefaciens cells strain of LBA4404 and transformed cells were cultured on selective medium that supplied with kanamycin and rifampycin. As a result of gel electrophoresis of Agrobacterium colonies-PCR product, band existing on expected size show that the DREB1A gene was cloned in to pBI121 binary vector. At time, Agrobacterium cells containing of pBI121::DREB1A construct are using for product of environmental stress tolerant plants.

Agroinoculation of the cloned infectious cDNAs of Lettuce chlorosis virus results in systemic plant infection and production of whitefly transmissible virions

Lettuce chlorosis virus (LCV) is a single stranded, positive strand RNA virus that is solely transmitted by specific whitefly vectors (Bemisia tabaci biotypes A and B) but not by mechanical leaf-rub inoculation. The roles of viral encoded proteins involved in the infection cycle of LCV have not yet been characterized due to the lack of reverse genetic tools. We present here a report of the successful development of an Agrobacterium-mediated inoculation system for the cloned cDNA constructs of LCV. The cDNAs of both LCV RNAs 1 and 2 were engineered into binary vectors in which the expression of LCV RNAs was regulated under a Cauliflower mosaic virus (CaMV) 35S promoter. In addition, by engineering the sequence elements of the Hepatitis delta virus ribozyme and the nopaline synthase 3′ untranslated region immediately downstream of the last nucleotide of LCV RNAs 1 and 2 in the binary vector constructs, the in planta produced LCV transcripts were expected to bear authentic 3′ termini. Both constructs were transformed into Agrobacterium tumefaciens cells and infiltrated in Nicotiana benthamiana plants. Three to four weeks post-agroinoculation, the N. benthamiana plants developed typical interveinal chlorosis and LCV infection was detected in the systemic leaves by reverse transcription-PCR. Virions purified from the LCV-infected N. benthamiana plants were flexuous rod-shaped and were transmissible by both B. tabaci biotypes A and B following membrane feeding. These results support the conclusion that Agrobacterium-mediated inoculation of LCV binary vectors in N. benthamiana plants results in LCV infection and the production of biologically active, whitefly transmissible virions. This system represents an important tool for use with reverse genetics designed for the study of LCV gene functions.

Regeneration of transgenic plants expressing the GFP gene from rape cotyledonary and leaf explants: Effects of the genotype and ABA

Cotyledons of five-day-old seedlings and leaves of 6-week-old plants of two rape cultivars (Brassica napus L., cvs. Westar and Podmoskovnyi) were co-cultured with the culture of Agrobacterium tumefaciens cells comprising the genetic construct with the marker gfp gene, on Murashige and Skoog nutrient medium supplemented with benzyladenine, NAA, and ABA in various combinations. A capacity for regeneration on both types of explants was rather high, but leaf explants produced weakly differentiated shoots and most of them were vitrificated. On cotyledonary explants of transformed rape plants of both cultivars expressing the gfp gene, regeneration frequency was 70%. On leaf explants, it was much lower (47% in cv. Westar and 28% in cv. Podmoskovnyi). The gfp gene was expressed on all stages of shoot development. On primary, starting differentiation calli, we observed the strongest fluorescence of GFP in meristematic and vascular tissues. On leaf blades, GFP fluorescence was much brighter in old than young leaves; often it was observed only in the cell groups; it. PCR analysis of seed generation of transformants showed that some plants did not follow the Mendelian inheritance of a monogenic trait (transgene) in self-pollinated plants. This phenomenon could be explained as a result of meiotic recombination or production of genotypic chimeric organisms at regeneration.

Simple and Robust <em>in vivo</em> and <em>in vitro</em> Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Transgenic Lines of Oryza Sativa (Basmati 2000) Expressing Xa21 Demonstrate Enhanced Resistance to Bacterial Blight

An efficient transformation system for rice was established by co-cultivating calli, derived from 21-day-old scutellum, with Agrobacterium tumefaciens cells (OD600 = 0.04), maintained on filter paper, moistened with 4 mL of a medium, and supplemented with 400 μM acetosyringone, for a period of 2 days. Presence of the transgene was confirmed by polymerase chain reaction, and stable integration and copy number of the transgene were determined by Southern blot analysis. Among seven plants analyzed, six possessed single T-DNA integration events, while one plant was found to have two integrated copies of the T-DNA. A total of 45 T0 plants were grown in the greenhouse to obtain the T1 generation. T1 plants evaluated for presence of the transgene and for response to inoculation with the bacterial leaf blight pathogen, Xanthomonas oryzae pv. Oryzae, exhibited Mendelian segregation (3:1) for the transgene as well as enhanced resistance to bacterial blight

Transformation of potato via Agrobacterium coated microparticle bombardment

The transformation of potato (Solanum tuberosum L. cv. Desiree) was extended by the Agrobacterium-mediated biolistic method. Using this approach transgenic shoots could be obtained at a similar frequency to that achieved through conventional biolistics. Leaves from shoot cultures were bombarded with gold particles coated in Agrobacterium tumefaciens cells harboring a binary plasmid encoding three genes of interest in the T-DNA. Nine shoots were obtained from 20 shots, with selection of transgenic shoots on a series of media containing progressively increasing concentrations of hygromycin from 5 to 20 mg dm−3.