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1.  A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated genetic transformation 
F1000Research  2013;2:33.
Agrobacterium is a phytopathogenic bacterium that induces crown gall disease in many plant species by transferring and integrating a segment of its own DNA (T-DNA) into its host genome. Whereas Agrobacterium usually does not trigger an extensive defense response in its host plants, it induces the expression of several defense-related genes and activates plant stress reactions. In the complex interplay between Agrobacterium and its host plant, Agrobacterium has evolved to take advantage of these plant defense pathways for its own purpose of advancement of the infection process. For example, Agrobacterium utilizes the host stress response transcriptional regulator VIP1 to facilitate nuclear import and proteasomal uncoating of its T-DNA during genetic transformation of the host cell. In Arabidopsis, the VIP1 gene expression is repressed by WRKY17, a negative regulator of basal resistance to Pseudomonas. Thus, we examined whether WRKY17 is also involved in plant susceptibility to genetic transformation by Agrobacterium. Using reverse genetics, we showed that a wrky17 mutant displays higher expression of the VIP1 gene in roots, but not in shoots. In a root infection assay, the wrky17 mutant plants were hyper-susceptible to Agrobacterium compared to wild type plants. WRKY17, therefore, may act as a positive regulator of Arabidopsis resistance to Agrobacterium. This notion is important for understanding the complex regulation of Agrobacterium-mediated genetic transformation; thus, although this paper reports a relatively small set of data that we do not plan to pursue further in our lab, we believe it might be useful for the broad community of plant pathologists and plant biotechnologists.
PMCID: PMC3799546  PMID: 24358874
2.  Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter 
Molecular microbiology  1999;32(6):1239-1253.
The Agrobacterium tumefaciens VirB4 ATPase functions with other VirB proteins to export T-DNA to susceptible plant cells and other DNA substrates to a variety of prokaryotic and eukaryotic cells. Previous studies have demonstrated that VirB4 mutants with defects in the Walker A nucleotide-binding motif are non-functional and exert a dominant negative phenotype when synthesized in wild-type cells. This study characterized the oligomeric structure of VirB4 and examined the effects of Walker A sequence mutations on complex formation and transporter activity. VirB4 directed dimer formation when fused to the amino-terminal portion of cI repressor protein, as shown by immunity of Escherichia coli cells to λ phage infection. VirB4 also dimerized in Agrobacterium tumefaciens, as demonstrated by the recovery of a detergent-resistant complex of native protein and a functional, histidine-tagged derivative by precipitation with anti-His6 antibodies and by Co2+ affinity chromatography. Walker A sequence mutants directed repressor dimerization in E. coli and interacted with His-VirB4 in A. tumefaciens, indicating that ATP binding is not required for self-association. A dimerization domain was localized to a proposed N-terminal membrane-spanning region of VirB4, as shown by the dominance of an allele coding for the N-terminal 312 residues and phage immunity of host cells expressing cI repressor fusions to alleles for the first 237 or 312 residues. A recent study reported that the synthesis of a subset of VirB proteins, including VirB4, in agrobacterial recipients has a pronounced stimulatory effect on the virB-dependent conjugal transfer of plasmid RSF1010 by agrobacterial donors. VirB4′312 suppressed the stimulatory effect of VirB proteins for DNA uptake when synthesized in recipient cells. In striking contrast, Walker A sequence mutants contributed to the stimulatory effect of VirB proteins to the same extent as native VirB4. These findings indicate that the oligomeric structure of VirB4, but not its capacity to bind ATP, is important for the assembly of VirB proteins as a DNA uptake system. The results of these studies support a model in which VirB4 dimers or homomultimers contribute structural information for the assembly of a transenvelope channel competent for bidirectional DNA transfer, whereas an ATP-dependent activity is required for configuring this channel as a dedicated export machine.
PMCID: PMC3918219  PMID: 10383764
3.  Agrobacterium aiming for the host chromatin 
Agrobacterium genetically transforms its hosts by transferring a segment of DNA (T-DNA) into the host cell and integrating it into the host genome. Integration requires a close interaction between T-DNA, which is packaged into a nucleoprotein complex (T-complex) by bacterial virulence (Vir) proteins, and the host chromatin. This interaction is facilitated by the host protein VIP 1, which binds both to the major protein component of the T-complex, VirE2, and to the core histones. Recently, VIP1 has been demonstrated to mediate the interaction between plant nucleosomes and VirE2-DNA complexes (i.e., synthetic T-complex-like structures) in vitro. Here, we discuss major implications of these observations—such as the possible role of core histone modifications, proteasomal uncoating of the T-complex mediated by the bacterial F-box protein VirF, and the need for changes in chromatin structure to render it accessible to the T-DNA integration—for the process of chromatin targeting of foreign DNA and its integration into the eukaryotic genome.
PMCID: PMC2649301  PMID: 19513263
histones; nucleosomes; VirE2-interacting protein 1 (VIP1); VirE2; chromatin targeting; T-complex
4.  Agrobacterium induces expression of a host F-box protein required for tumorigenicity 
Cell host & microbe  2010;7(3):197-209.
In plant-pathogen interactions, the host defends against the invading pathogen and the pathogen aims to suppress or subvert this defense. Whereas the defense suppression strategy is relatively well understood for many pathogens, the mechanisms by which pathogens can actively utilize the defense machinery of the host remain obscure. We report that Agrobacterium, a microorganism that elicits neoplastic growths on many plant species, induces expression of a plant defense-related F-box protein, VBF, which it incorporates into its own pathway for genetic transformation. Our data suggest that VBF may function to uncoat the bacterial transferred DNA from its associated virulence VirE2 and host VIP1 proteins via the SCFVBF pathway. Suppression of VBF elevates the intracellular content of VIP1, but renders the plant largely resistant to Agrobacterium, indicating that, in the infection pathway, VBF is functionally epistatic to VIP1. When expressed in Agrobacterium and exported into the plant cell, VBF facilitates tumor formation.
PMCID: PMC3427693  PMID: 20227663
5.  A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane. 
Journal of Bacteriology  1989;171(3):1616-1622.
The VirA protein is one of two proteins required for transcriptional activation of Agrobacterium tumefaciens virulence genes in response to phenolic compounds released by plants during infection. We describe two experimental approaches which indicate that this protein has a transmembrane topology. First, spheroplasts of Escherichia coli or wild-type A. tumefaciens expressing the VirA protein were treated with proteinase K to digest periplasmic proteins, and the remaining proteins were immunologically stained on Western blots (immunoblots) by using anti-VirA antibody. Second, transposon TnphoA was used to generate translational fusions between virA and phoA, the latter of which is the structural gene for alkaline phosphatase. Both techniques indicated that VirA spans the cytoplasmic membrane, with approximately 275 amino acids near the amino terminus being localized in the periplasmic space and the rest of the protein being localized in the cytoplasm. We also show that overexpression of VirA in E. coli is deleterious to cell growth and that this phenomenon depends on the synthesis of either the second hydrophobic core or some nearby portion of the VirA protein.
PMCID: PMC209789  PMID: 2921246
6.  Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. 
Journal of Bacteriology  1995;177(17):4890-4899.
The transfer of DNA from Agrobacterium tumefaciens into a plant cell requires the activities of several virulence (vir) genes that reside on the tumor-inducing (Ti) plasmid. The putative transferred intermediate is a single-stranded DNA (T strand), covalently attached to the VirD2 protein and coated with the single-stranded DNA-binding protein, VirE2. The movement of this intermediate out of Agrobacterium cells and into plant cells requires the expression of the virB operon, which encodes 11 proteins that localize to the membrane system. Our earlier studies showed that the IncQ broad-host-range plasmid RSF1010, which can be transferred from Agrobacterium cells to plant cells, inhibits the transfer of T-DNA from pTiA6 in a fashion that is reversed by overexpression of virB9, virB10, and virB11. Here, we examined the specificity of this inhibition by following the transfer of other T-DNA molecules. By using extracellular complementation assays, the effects of RSF1010 on movement of either VirE2 or an uncoated T strand from A. tumefaciens were also monitored. The RSF1010 derivative plasmid pJW323 drastically inhibited the capacity of strains to serve as VirE2 donors but only partially inhibited T-strand transfer from virE2 mutants. Further, we show that all the virB genes tested are required for the movement of VirE2 and the uncoated T strand as assayed by extracellular complementation. Our results are consistent with a model in which the RSF1010 plasmid, or intermediates from it, compete with the T strand and VirE2 for a common transport site.
PMCID: PMC177262  PMID: 7665465
7.  Generation of Composite Plants in Medicago truncatula used for Nodulation Assays 
Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.
PMCID: PMC3197321  PMID: 21490571
8.  Effects of Arbuscular Mycorrhization in Sterile and Non-sterile Soils 
Mycorrhiza, a mutualistic association between fungi and higher plants, has been documented extensively, but much less is known about the development of arbuscular mycorrhizal (AM) fungi and their effects on the growth of peanuts (Arachis hypogea L.). Therefore, the mycorrhizal status of Glomus spp. was investigated in the following diverse substrate soil conditions: non-autoclaved soil, autoclaved soil and autoclaved soil plus soil microbiota. The results indicated that both the arbuscular mycorrhizae, Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe, and Glomus fasciculatum (Thaxter) Gerd. & Trappe emend. Walker & Koske were infective to peanut, but displayed a differential impact on peanut growth depending on the microbial biomass content of the substrate soils. G. mosseae proved to be the most effective at improving peanut growth.
PMCID: PMC3819066  PMID: 24575190
Arachis hypogea; Soil Microbiota; Mycorrhizal Colonisation; AM Fungi
9.  Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3 
Nucleic Acids Research  2003;31(3):860-868.
Agrobacterium tumefaciens causes crown gall disease on a variety of plants. During the infection process Agrobacterium transfers a nucleoprotein complex, the VirD2 T-complex, and at least two Vir proteins, VirE2 and VirF, into the plant cell via the VirB/VirD4 type IV secretion system. Recently, we found that T-DNA could also be transferred from Agrobacterium to Saccharomyces cerevisiae. Here, we describe a novel method to also detect trans-kingdom Vir protein transfer from Agrobacterium to yeast, using the Cre/lox system. Protein fusions between Cre and VirE2 or VirF were expressed in Agrobacterium. Transfer of the Cre-Vir fusion proteins from Agrobacterium to yeast was monitored by a selectable excision event resulting from site-specific recombination mediated by Cre on a lox-flanked transgene in yeast. The VirE2 and VirF proteins were transported to yeast via the virB-encoded transfer system in the presence of coupling factor VirD4, analogous to translocation into plant cells. The yeast system therefore provides a suitable and fast model system to study basic aspects of trans-kingdom protein transport from Agrobacterium into host cells. Using this method we showed that VirE2 and VirF protein transfer was inhibited by the presence of the Osa protein. Besides, we found evidence for a novel third effector protein, VirE3, which has a similar C-terminal signature to VirE2 and VirF.
PMCID: PMC149200  PMID: 12560481
10.  Cultivation of Chlamydia trachomatis in cycloheximide-treated mccoy cells. 
Journal of Clinical Microbiology  1977;6(4):328-331.
An isolation technique for Chlamydia trachomatis using McCoy cells is described. In contrast to earlier techniques employing such cells, no pretreatment of the cells was used. The glutarimide antibiotic cycloheximide was added to the culture medium used for incubating the cells after infection. Cycloheximide was used at concentrations that depressed, but did not completely inhibit, the metabolism of the eucaryotic host cells. In studies on different immunotypes of C. trachomatis cultured in the yolk sac of embryonated hen eggs, the cycloheximide technique was compared with a method using pretreatment of cells with 5-iodo-2-deoxyuridine. The cycloheximide method gave greater numbers of inclusion-forming units per cover slip for all the immunotypes of trachoma-inclusion conjunctivitis agents tested, i.e., A through I. In a study of 194 cervical and urethral specimens from women, cycloheximide treatment of McCoy cells was found to be more efficient than 5-iodo-2-deoxyuridine treatment for the isolation of C. trachomatis.
PMCID: PMC274768  PMID: 562356
11.  Lactimidomycin, Iso-migrastatin and Related Glutarimide-containing 12-membered Macrolides are Extremely Potent Inhibitors of Cell Migration 
Migrastatin (1), iso-migrastatin (5) and lactimidomycin (7) are all glutarimide-containing polyketides known for their unique structures and cytotoxic activities against human cancer cell lines. Migrastatin, a strong inhibitor of tumor cell migration, has been an important lead in the development of antimetastatic agents. Yet studies of the related 12-membered macrolides iso-migrastatin, lactimidomycin and related analogs have been hampered by their limited availability. We report here the production, isolation, structural characterization and biological activities of iso-migrastatin, lactimidomycin, and 23 related congeners. Our studies showed that, as a family, the glutarimide-containing 12-membered macrolides are extremely potent cell migration inhibitors with some members displaying activity on par or superior to that of migrastatin as exemplified by compounds 5, 7, and 9–12. On the basis of these findings, the structures and activity of this family of compounds as cell migration inhibitors are discussed.
PMCID: PMC2651819  PMID: 19132897
12.  Drug-Induced (Thalidomide) Malformations 
Canadian Medical Association Journal  1962;87(24):1259-1262.
Phocomelia (flipper-like limbs) has long been recognized as a rare malformation. Numerous cases of phocomelia and other congenital malformations have recently been reported in the United Kingdom, Australia, Europe and Canada in which the common factor appears to have been the administration of the hypnotic compound thalidomide during early pregnancy. Two additional cases of infants born with phocomelia, amelia and alimentary abnormalities are presented. In both of these cases the administration of thalidomide was initiated early during pregnancy (five to eight weeks after the last normal menstrual period) and maintained for several weeks. Thalidomide (alpha-phthalimido glutarimide) is related chemically to other glutarimides currently in clinical use. The possibility that these compounds and/or their metabolites may induce teratogenic effects warrants consideration. Emphasis is added to the view that caution should be exercised when prescribing new drugs.
PMCID: PMC1920822  PMID: 20327332
13.  Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells 
MicrobiologyOpen  2013;3(1):104-117.
Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20–25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli.
PMCID: PMC3937733  PMID: 24376037
Agrobacterium tumefaciens; BiFC; protein translocation; Saccharomyces cerevisiae; split GFP; type 4 secretion system; VirE2.
14.  Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein. 
Journal of Bacteriology  1992;174(21):7033-7039.
VirA and VirG activate the Agrobacterium tumefaciens vir regulon in response to phenolic compounds, monosaccharides, and acidity released from plant wound sites. VirA contains an amino-terminal periplasmic domain and three cytoplasmic domains: a linker, a protein kinase, and a phosphoryl receiver. We constructed internal deletions of virA that truncate one or more domains and tested the ability of the resulting proteins to mediate environmentally responsive vir gene activation in vivo. The periplasmic domain is required for sensing of monosaccharides (in agreement with earlier results), while the linker domain is required for sensing of phenolic compounds and acidity. The phosphoryl receiver domain of VirA plays an inhibitory role in signal transduction that may be modulated by phosphorylation. The carboxy terminus of the protein was also dispensable for tumorigenesis, while the periplasmic domain was required.
PMCID: PMC207384  PMID: 1400253
15.  Model for inactivation and disposal of infectious human immunodeficiency virus and radioactive waste in a BL3 facility. 
A method is described for autoclaving low levels of solid infectious, radioactive waste. The method permits steam penetration to inactivate biologic waste, while any volatile radioactive compounds generated during the autoclave process are absorbed. Inactivation of radiolabeled infectious waste has been problematic because the usual sterilization techniques result in unacceptable radiation handling practices. If autoclaved under the usual conditions, there exists a high probability of volatilization or release of radioisotopes from the waste. This results in the radioactive contamination of the autoclave and the laboratory area where steam is released from the autoclave. Our results provide a practical method to inactivate and dispose of infectious radioactive waste. For our research, Bacillus pumilus spore strips and vaccinia virus were used as more heat-resistant surrogates of the human immunodeficiency virus (HIV). These surrogates were used because HIV is difficult to grow under most conditions and is less heat tolerant than the surrogates. In addition, B. pumilus has defined cell death values, whereas such values have not been established for HIV. Both B. pumilus and vaccinia virus are less hazardous to work with. The autoclave method is time efficient and can be performed by laboratory personnel with minimal handling of the waste. Furthermore, waste site handlers are able to visually inspect the solid waste containers and ascertain that inactivation procedures have been implemented.
PMCID: PMC183304  PMID: 2310182
16.  VirE2: A Unique ssDNA-Compacting Molecular Machine  
PLoS Biology  2008;6(2):e44.
The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.
Author Summary
The importation of genetic material into cells is a common and fundamental mechanism occurring in bacterial conjugation, DNA uptake, and Agrobacterium plant infection and is, for instance, responsible for antibiotic resistance spread. Previous studies suggested that this process relied only on the activity of complex molecular machines pumping the single-stranded DNA (ssDNA) into the recipient cell. Here, we show that proteins provided by the pathogenic organism and translocated prior to the arrival of ssDNA into the recipient cell also play a fundamental role. These proteins not only bind to ssDNA to protect it but also rearrange ssDNA into a compact helix, thus generating a contractile force that pulls the DNA into the host. Interestingly, the production of mechanical energy occurs solely through the free-energy gain during the binding of VirE2 to ssDNA without the need for an external source of energy, such as nucleotide hydrolysis.
Combining single-molecule techniques and biological assays, the authors show that VirE2 proteins could mediate the active translocation ofAgrobacterium's genetic material into plant cells.
PMCID: PMC2253637  PMID: 18303950
17.  Small heat-shock protein HspL is induced by VirB protein(s) and promotes VirB/D4-mediated DNA transfer in Agrobacterium tumefaciens 
Microbiology  2009;155(Pt 10):3270-3280.
Agrobacterium tumefaciens is a Gram-negative plant-pathogenic bacterium that causes crown gall disease by transferring and integrating its transferred DNA (T-DNA) into the host genome. We characterized the chromosomally encoded alpha-crystallin-type small heat-shock protein (α-Hsp) HspL, which was induced by the virulence (vir) gene inducer acetosyringone (AS). The transcription of hspL but not three other α-Hsp genes (hspC, hspAT1, hspAT2) was upregulated by AS. Further expression analysis in various vir mutants suggested that AS-induced hspL transcription is not directly activated by the VirG response regulator but rather depends on the expression of VirG-activated virB genes encoding components of the type IV secretion system (T4SS). Among the 11 virB genes encoded by the virB operon, HspL protein levels were reduced in strains with deletions of virB6, virB8 or virB11. VirB protein accumulation but not virB transcription levels were reduced in an hspL deletion mutant early after AS induction, implying that HspL may affect the stability of individual VirB proteins or of the T4S complex directly or indirectly. Tumorigenesis efficiency and the VirB/D4-mediated conjugal transfer of an IncQ plasmid RSF1010 derivative between A. tumefaciens strains were reduced in the absence of HspL. In conclusion, increased HspL abundance is triggered in response to certain VirB protein(s) and plays a role in optimal VirB protein accumulation, VirB/D4-mediated DNA transfer and tumorigenesis.
PMCID: PMC2885654  PMID: 19556291
18.  Epigenetic control of Agrobacterium T-DNA integration 
Biochimica et biophysica acta  2011;1809(8):388-394.
To genetically transform plants, Agrobacterium transfers its T-DNA into the host cell and integrates it into the plant genome, resulting in neoplastic growths. Over the past two decades, a great deal has been learned about the molecular mechanism by which Agrobacterium produces T-DNA and transports it into the host nucleus. However, T-DNA integration, which is the limiting, hence, the most critical step of the transformation process, largely remains an enigma. Increasing evidence suggests that Agrobacterium utilizes the host DNA repair machinery to facilitate T-DNA integration. Meanwhile, it is well known that chromatin modifications, including the phosphorylation of histone H2AX, play an important role in DNA repair. Thus, by implication, such epigenetic codes in chromatin may also have a considerable impact on T-DNA integration, although the direct evidence to demonstrate this hypothesis is still lacking. In this review, we summarize the recent advances in our understanding of Agrobacterium T-DNA integration and discuss the potential link between this process and the epigenetic information in the host chromatin.
PMCID: PMC3108046  PMID: 21296691
Agrobacterium; T-DNA integration; DSB repair; chromatin modifications; histone codes
19.  Constitutive Activation of Two-Component Response Regulators: Characterization of VirG Activation in Agrobacterium tumefaciens 
Journal of Bacteriology  2006;188(14):5204-5211.
Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.
PMCID: PMC1539974  PMID: 16816192
20.  Engineering a thermo-stable superoxide dismutase functional at sub-zero to >50°C, which also tolerates autoclaving 
Scientific Reports  2012;2:387.
Superoxide dismutase (SOD) is a critical enzyme associated with controlling oxygen toxicity arising out of oxidative stress in any living system. A hyper-thermostable SOD isolated from a polyextremophile higher plant Potentilla atrosanguinea Lodd. var. argyrophylla (Wall. ex Lehm.) was engineered by mutation of a single amino acid that enhanced the thermostability of the enzyme to twofold. The engineered enzyme was functional from sub-zero temperature to >50°C, tolerated autoclaving (heating at 121°C, at a pressure of 1.1 kg per square cm for 20 min) and was resistant to proteolysis. The present work is the first example to enhance the thermostability of a hyper-thermostable protein and has potential to application to other proteins for enhancing thermostability.
PMCID: PMC3339387  PMID: 22548128
21.  Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool 
Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this “natural genetic engineer” for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.
PMCID: PMC150518  PMID: 12626681
Journal of Bacteriology  1962;83(3):668-672.
Johnson, R. C. (Fort Detrick, Frederick, Md.) and N. D. Gary. Nutrition of Leptospira pomona. I. A chemically defined substitute for rabbit serum ultrafiltrate. J. Bacteriol. 83:668–672. 1962.—Poor growth of Leptospira pomona, strain Wickard, in a medium containing exhaustively dialyzed rabbit serum was corrected by addition of rabbit serum ultrafiltrate. A dialyzed serum medium was used to investigate the nutritional qualities of various compounds when tested as substitutes for the ultrafiltrate fraction. l-Asparagine was the only amino acid that markedly stimulated growth. l-Glutamine was active only when autoclaved (converted to the ammonium salt of pyrrolidone carboxylic acid), and NH4Cl and urea satisfactorily replaced l-asparagine. Results indicated that amino acids functioned primarily as a nitrogen source in this medium.
Thiamine was the only vitamin required for growth. The lag phase was usually decreased by 1 day with NaHCO3.
A medium composed of 0.02 m phospate buffer (pH 7.4), 15% dialyzed rabbit serum, thiamine (5 μg/ml), and NH4Cl (10−3m) supported growth equivalent to that obtained with undialyzed rabbit serum medium. L. pomona was transferred six times, in this medium and seven other serotypes were transferred three times, without any decrease in amount of growth. Growth of L. pomona was initiated with 20 organisms per ml.
PMCID: PMC279326  PMID: 14452094
23.  Tissue culture independent transformation of the forage crop sunnhemp (Crotalaria juncea L.): an easy method towards generation of transgenics 
A transformation system which is free of in vitro plant regeneration following Agrobacterium infection is established for the forage legume, Sunnhemp (Crotalaria juncea L.) where in the entire embryo axis of the germinating seed was used as the target tissue for transformation. After standardization of transformation conditions, the cotyledonary node of the embryo axis was infected with Agrobacterium host LBA 4404 harboring the recombinant vector pCAMBIA 2301. The bivalent 1D gene of the two major foot and mouth disease virus (FMDV) serotypes ‘O’ and ‘A22’ and the neomycin phosphotransferase (nptII) gene were used as the markers for optimization of the protocol. The embryo axes were pricked randomly on the cotyledonary node and co-cultivated with Agrobacterium. The germlings were then allowed to grow under standard growth room conditions in to mature fertile plants. 60 T0 plants were established from 3 separate experiments. Three hundred seeds from the 60 T0 plants were sown to raise the T1 generation of which 180 were analyzed for integration of bivalent FMDV gene 1D “O” and “A22” and the nptII gene. Eighteen out of these 180 plants amplified both the marker genes. Two independent transgenic lines 24 and 37, showed elevated levels of expression of 12 μg and 8 μg (per gm of fresh leaf) of the bivalent ID antigen “O” and “A22” . The results showed that the transformation efficiency was 3 %. To the best of our knowledge, this is the first successful attempt of Agrobacterium tumefaciens mediated transformation of Sunnhemp. The protocol can generate whole plant transformants with relative ease and should be compatible to all genotypes of Sunnhemp.
PMCID: PMC3550535  PMID: 23573040
Sunnhemp; Agrobacterium tumefaciens; FMDV -1D gene; nptII gene markers; in planta transformation
24.  In planta transformation of pigeon pea: a method to overcome recalcitrancy of the crop to regeneration in vitro 
Development of transgenics in pigeon pea remains dogged by poor plant regeneration in vitro from transformed tissues and low frequency transformation protocols. This article presents a non-tissue culture-based method of generating transgenic pigeon pea (Cajanus cajan (L.) Millisp.) plants using Agrobacterium-Ti plasmid-mediated transformation system. The protocol involves raising of whole plant transformants (T0 plants) directly from Agrobacterium-infected young seedlings. The plumular and intercotyledonary meristems of the seedling axes are targeted for transformation. The transformation conditions optimized were, pricking of the apical and intercotyledonary region of the seedling axes of two-day old germinating seedlings with a sewing needle, infection with Agrobacterium (LBA4404/pKIWI105 carrying uid A and npt II genes) in Winans’ AB medium that was added with wounded tobacco leaf extract, co-cultivation in the same medium for 1h and transfer of seedlings to soilrite for further growth and hardening and subsequent transfer of seedlings to soil in pots in the greenhouse. Out of the 22–25 primary transformants that survived infection-hardening treatments from each of the three experiments, 15 plants on the average established on the soil under greenhouse conditions, showed slow growth initially, nevertheless grew as normal plants, and flowered and set seed eventually. Of the several seeds harvested from all the T0 plants, six hundred were sown to obtain progeny (T1) plants and 350 of these were randomly analysed to determine their transgenic nature. PCR was performed for both gus (uid A) and npt II genes. Forty eight of the 350 T1 plants amplified both transgenes. Southern blot analysis substantiated the integration and transmission of these genes. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and is applicable across different genotypes/cultivars of the crop and offers immense potential as a supplemental or an alternative protocol for generating transgenic plants of difficult-to-regenerate pigeon pea. Further, the protocol offers the option of doing away with a selection step in the procedure and so facilitates transformation, which is free of marker genes.
PMCID: PMC3550634  PMID: 23572898
Cajanus cajan; Transformation; Tissue culture-independent plant regeneration
25.  The Conjugal Intermediate of Plasmid RSF1010 Inhibits Agrobacterium tumefaciens Virulence and VirB-Dependent Export of VirE2 
Journal of Bacteriology  1998;180(15):3933-3939.
Agrobacterium tumefaciens causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from A. tumefaciens to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 between Agrobacterium strains or from Agrobacterium to plants also requires the virB-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of Agrobacterium inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with virE mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.
PMCID: PMC107378  PMID: 9683491

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