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PubMed2006

Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation.

Higher eukaryotes sense microbes through the perception of pathogen-associated molecular patterns (PAMPs). Arabidopsis plants detect a variety of PAMPs including conserved domains of bacterial flagellin and of bacterial EF-Tu. Here, we show that flagellin and EF-Tu activate a common set of signaling events and defense responses but without clear synergistic effects. Treatment with either PAMP results in increased binding sites for both PAMPs. We used this finding in a targeted reverse-genetic approach to identify a receptor kinase essential for EF-Tu perception, which we called EFR. Nicotiana benthamiana, a plant unable to perceive EF-Tu, acquires EF-Tu binding sites and responsiveness upon transient expression of EFR. Arabidopsis efr mutants show enhanced susceptibility to the bacterium Agrobacterium tumefaciens, as revealed by a higher efficiency of T-DNA transformation. These results demonstrate that EFR is the EF-Tu receptor and that plant defense responses induced by PAMPs such as EF-Tu reduce transformation by Agrobacterium.

Agrobacterium tumefaciens↗
PubMed2005

Agrobacterium-mediated high efficiency transformation of tall fescue (Festuca arundinacea).

Tall fescue (Festuca arundinacea) is the predominant cool-season pasture grass in the USA. Embryogenic calluses were induced from seeds/caryopsis of elite tall fescue cultivars Jesup and Kentucky-31, and were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. Agrobacterium strains LBA4404 and EHA105 harboring pCAMBIA vectors or the super-binary vector pTOK233 were used to infect the embryogenic callus pieces. The number of hygromycin resistant calluses obtained per dish of infected callus pieces was in the range of 2.0-5.8, and the number of transgenic plants recovered per dish of infected callus pieces was in the range of 0.4-1.7. When transformation efficiency was calculated based on the number of transgenic plants recovered and the number of original intact calluses used, the transformation frequency was in the range of 1.9-8.7%. The use of the easily available pCAMBIA vectors could produce equivalent results as the superbinary vector pTOK233. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. Expression of the transgenes was confirmed by northern hybridization analysis, GUS staining, and detection of GFP signals. Fertile transgenic plants were obtained after vernalization in the greenhouse. Progeny analysis revealed Mendelian inheritance of the transgenes.

Agrobacterium tumefaciens↗
PubMed2005

Agrobacterium-mediated transformation of Sclerotinia sclerotiorum.

Ascospores from the phytopathogenic fungus Sclerotinia sclerotiorum were transformed to hygromycin B resistance by co-cultivation with Agrobacterium tumefaciens. Transformed spores germinated and grew on PDA supplemented with 100 ug/ml hygromycin B. The presence of mitotically stable hph gene integration at random sites in the genome was confirmed by PCR and Southern blot analysis. A transformation frequency of 8 x 10(-5) was achieved in five separate experiments. This study is the first report of success co-cultivating A. tumefaciens with S. sclerotiorum. This report of a reproducible Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in S. sclerotiorum and provide a simple and reliable method for genetic manipulation.

Agrobacterium tumefaciens↗
PubMed1998

vir-Gene-inducing activities of hydroxycinnamic acid amides in Agrobacterium tumefaciens.

Expression of Agrobacterium tumefaciens virulence genes and transformation of dicots by this organism are dependent upon host plant phenolic compounds. Several alkylsyringamides have recently been shown to be powerful inducers of these vir-genes. These synthetic amides, and especially ethylsyringamide, are much stronger inducers than syringic acid. In this work, four alkylamides derived from ferulic or sinapic acids were synthesized by a dicyclohexylcarbodiimide method and tested for their potential to induce vir-gene expression on A. tumefaciens strains harbouring virB::lacZ or virE::lacZ fusion plasmids. Their effectiveness was compared to that of ethylsyringamide and tyraminylferulamide, a naturally occurring amide in plants. Whatever the amine moiety of the amide (ethylamine, propylamine, tyramine or beta-alanine ethyl ester) conjugation of the acid functional group clearly diminished the toxicity to the bacteria of the respective acid at high concentration and thereby increased the vir-inducing potential. However, none of the inducers tested exhibited higher activity than acetosyringone, the reference compound for vir-gene induction, with the exception of ethylsyringamide at concentrations above 1mM. When tested on Agrobacterium tumefaciens strain A348(pSM243cd), ethylferulamide and ethylsinapamide were more efficient than the corresponding phenolic acids but only above 100 microM.

Acetophenones↗
PubMed1998

Purification of two lectins from a nopalin Agrobacterium tumefaciens strain.

Lectins were evidenced on the surface of one Agrobacterium tumefaciens wild strain (82,139) by agglutination test and neoglycoprotein labelling. Bacteria were incubated in the presence of various fluorescein-labelled neoglycoproteins and the binding was assessed by a fluorimetric method. Among the fluorescein-labelled neoglycoproteins tested, the one bearing alpha-D-galactosyl residues was the most efficient. The labelling was optimal at pH 5.0 and naught at pH above 7. The binding was specifically inhibited by homologous fluorescein-free neoglycoproteins. A galactoside-specific lectin was purified to homogeneity by affinity chromatography on agarose-A4 substituted with alpha-D-galactopyranosyl residues. Upon polyacrylamide gel electrophoresis, a single band (M(r) 58,000) was detected. This alpha-D-galactoside-specific lectin agglutinated preferentially human B red blood cells at pH 5.0. Another lectin specific for alpha-L-rhamnoside (M(r) 40,000) not retained on the immobilised galactose was purified by affinity chromatography on alpha-L-rhamnosyl substituted agarose-A4. This L-rhamnoside-specific lectin preferentially agglutinated horse erythrocytes. On the basis of their M(r) and on their sugar specificity, these two lectins are novel lectins with regard to the known sugar-binding proteins present in the Rhizobiaceae family: Agrobacterium, Rhizobium or Bradyrhizobium strains.

Agglutination↗
PubMed2003

The VirE2 protein of Agrobacterium tumefaciens: the Yin and Yang of T-DNA transfer.

Agrobacterium tumefaciens has evolved a unique mechanism to solve the problem of transferring DNA across five bilayers; the inner and outer membranes of the bacterium, the plasma membrane of the plant cell and the double membrane formed by the nuclear envelope. The two first and two last seem to be mediated by, respectively, the type IV secretion system in Agrobacterium and the nuclear pore complex in the plant cell, but the mechanism by which the transferred DNA (T-DNA) crosses the plant membrane still remains a mystery. New biophysical experiments suggest that, in addition to its established role as a single-stranded DNA (ssDNA)-binding protein, the VirE2 protein forms a channel in the plant membrane allowing the passage of the T-DNA into the cell. Such a role would be reminiscent of translocator molecules secreted by the type III secretion system of pathogenic bacteria and inserting into the host eukaryotic plasma membrane. The implications for the structure of the protein, its regulation and role in vivo are discussed.

Agrobacterium tumefaciens↗
PubMed2002

An Agrobacterium gene involved in tumorigenesis encodes an outer membrane protein exposed on the bacterial cell surface.

A gene designated as aopB was identified which was involved in tumorigenesis of Agrobacterium tumefaciens. aopB is located on the circular chromosome as a single copy. This gene shares high homology with ropB, a Rhizobium leguminosarum gene encoding an outer membrane protein. A transposon mutant CGI1 containing a gfp-tagged transposon insertion at aopB caused attenuated tumors on plants when inoculated at a low cell concentration (5x10(7) cells/ml). The mutation did not affect the bacterial growth on different media. A broad host range plasmid containing the wild type aopB could restore the tumor formation ability of CGI1 to the wild type level. When both aopB-gfp and aopB-phoA fusions were used to study the aopB gene expression, we found that the aopB gene was inducible by acidic pH but not by plant phenolic compound acetosyringone. aopB encodes a putative protein of 218 amino acids with a predicted molecular weight of 22.8 kDa. TnphoA transposon mutagenesis of aopB, subcellular fractionation and whole cell ELISA experiments indicated that AopB is an outer membrane protein exposed on the bacterial cell surface. It appeared that AopB was exclusively present in the outer membrane and not in other fractions. The vir gene induction assays showed that the aopB gene was not required for the expression of the Ti plasmid encoded vir genes that are essential for tumorigenesis. The C-terminal half of AopB is slightly homologous to some of the bacterial porin proteins and some of plant dehydrins. The role of AopB in Agrobacterium-plant interaction is discussed.

Agrobacterium tumefaciens↗
PubMed1998

Characterization of an unusual sensor gene (virA) of Agrobacterium.

Previous studies have shown that the virulence(vir) genes of Agrobacterium tumefaciens strain KU12 are induced by a unique set of phenolic compounds that are non-functional in most strains of Agrobacterium. Further, strain KU12 is not induced by phenolic compounds that induce the vir genes in other strains. Previous studies have shown that these differences in inducing activity result from differences in the sensor protein for these signal molecules, the VirA protein. To gain some understanding of the basis for these differences in sensing ability, we sequenced the entire virA locus of pTiKU12, including its promoter region and compared this sequence with five different published virA sequences that respond in different ways to inducing compounds. The virA gene of KU12 is composed of an open single reading frame coding for 851 aa. At the aa level, the VirA protein of pTiKU12 is 45, 45, 49, 49 and 64% identical to the VirA proteins from pTiA6, pTi15955, pRiA4, pTiC58 and pTiAg162, respectively. The transcription start sites of pTiKU12 and pTiA6 virA genes differ significantly when mapped by primer extension. Unlike all other vir genes, except the virA gene of pTiAg162, pTiKU12 virA is constitutively expressed, and its synthesis is not induced by phenolic compounds. The lack of induction is accounted for by the fact that the promoter region does not have the conserved VirG-binding dodecadeoxynucleotide sequence (vir-box) that was previously identified in all promoter regions of inducible vir genes.

Agrobacterium tumefaciens↗
PubMed1998

A chemotaxis cluster from Agrobacterium tumefaciens.

We report the DNA sequence of a 9.6-kb region of the Agrobacterium tumefaciens chromosome containing a putative 8-kb chemotaxis operon. The putative operon begins with orf1, whose predicted protein product shows strong sequence identity to methyl-accepting chemotaxis proteins (MCPs), followed by orf2, cheY1, cheA, cheR, cheB, cheY2, orf9, orf10. All of the identified homologues show a high degree of sequence conservation with their counterparts in the che operons from Sinorhizobium meliloti and Rhodobacter sphaeroides, and are arranged in a similar order. Mutations in orf1 and cheA result in impaired chemotaxis, whereas deletion of orf10, appears to have no effect on chemotaxis or motility. Although the putative operon does not contain a cheW homologue, heterologous probing and PCR using consensus primers indicates that cheW maps elsewhere in the Agrobacterium genome.

Agrobacterium tumefaciens↗
PubMed2000

Construction of an efficient expression system for Agrobacterium tumefaciens based on the coliphage T5 promoter.

A versatile expression vector utilizing a promoter of coliphage T5, P(N25) (Gentz and Bujard, 1985. J. Bacteriol. 164, 70-77) and a derivative of the IncW broad-host-range plasmid pJB20 (Beaupré et al., 1997. J. Bacteriol. 179, 78-89) has been developed. This vector successfully expresses virulence proteins of Agrobacterium tumefaciens encoded by virG and a mutant allele of virA, virA (delta1-284, G665D) in Escherichia coli as well as in A. tumefaciens. The signal transduction proteins VirA (delta1-284, G665D) and VirG are fully functional when expressed in Agrobacterium, and the P(N25) driven expression overrides the complex transcriptional regulation present with the native promoters. This expression system will enable a more detailed analysis of the activation events in signal transduction in A. tumefaciens, and we expect it to be useful in other prokaryotes.

Acetophenones↗
PubMed2000

The T-pilus of Agrobacterium tumefaciens.

T-pilus biogenesis uses a conserved transmembrane nucleoprotein- and protein-transport apparatus for the transport of cyclic T-pilin subunits to the Agrobacterium cell surface. T-pilin subunits are processed from full-length VirB2 pro-pilin into a cyclized peptide, a rapid reaction that is Agrobacterium specific and can occur in the absence of Ti-plasmid genes.

Agrobacterium tumefaciens↗
PubMed2002

GATEWAY vectors for Agrobacterium-mediated plant transformation.

Agrobacterium tumefaciens is the preferred method for transformation of a wide range of plant species. Commonly, the genes to be transferred are cloned between the left and right T-DNA borders of so-called binary T-DNA vectors that can replicate both in E. coli and Agrobacterium. Because these vectors are generally large, cloning can be time-consuming and laborious. Recently, the GATEWAY conversion technology has provided a fast and reliable alternative to the cloning of sequences into large acceptor plasmids.

Agrobacterium tumefaciens↗
PubMed2003

Agrobacterium tumefaciens as an agent of disease.

Twenty-six years ago it was found that the common soil bacterium Agrobacterium tumefaciens is capable of extraordinary feats of interkingdom genetic transfer. Since this discovery, A. tumefaciens has served as a model system for the study of type IV bacterial secretory systems, horizontal gene transfer and bacterial-plant signal exchange. It has also been modified for controlled genetic transformation of plants, a core technology of plant molecular biology. These areas have often overshadowed its role as a serious, widespread phytopathogen - the primary driver of the first 80 years of Agrobacterium research. Now, the diverse areas of A. tumefaciens research are again converging because new discoveries in transformation biology and the use of A. tumefaciens vectors are allowing the development of novel, effective biotechnology-based strategies for the control of crown gall disease.

Agrobacterium tumefaciens↗
PubMed2005

Agrobacterium phytochrome as an enzyme for the production of ZZE bilins.

Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2.

Agrobacterium tumefaciens↗
PubMed2000

pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation.

Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids. Its size and copy number in Escherichia coli offers increased efficiencies in routine in vitro recombination procedures. pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain. pSoup provides replication functions in trans for pGreen. The removal of RepA and Mob functions has enabled the size of pGreen to be kept to a minimum. Versions of pGreen have been used to transform several plant species with the same efficiencies as other binary Ti vectors. Information on the pGreen plasmid system is supplemented by an Internet site (http://www.pgreen.ac.uk) through which comprehensive information, protocols, order forms and lists of different pGreen marker gene permutations can be found.

Agrobacterium tumefaciens↗
PubMed2001

Expression of virB2 protein-containing structures on Agrobacterium in mating cultures.

Exocellular structures containing VirB2 proteins were, for the first time, localized on the surface of Agrobacterium by transmission electron microscopy. Using colloidal gold (CG)-labeled VirB2-specific antibodies, it was shown that VirB2 proteins enter into the composition of short surface pili, which emerge at the poles of acetosyringone (AS)-inducedAgrobacterium cells. However, cells of the Ti plasmidless A. tumefaciens strain UBAPF-2 and cells not induced with AS were incapable of pilus synthesis. In suspension, mating Agrobacterium cells were connected together by short thick bridges. It was found that these bridges did not include as part of their structure CG-labeled VirB1 and VirB2 proteins. We did not find the tetracycline-resistant transconjugants after mating of A. tumefaciens donor cells harboring binary systems with plasmid-free A. tumefaciens GM-I9023 in vir-induced and vir-uninduced conditions. However, the same strains can transfer pSUP106 plasmid via a vir-dependent way. We found that activated vir genes slightly stimulate pTd33 plasmid transfer via a tra-dependent pathway to plasmid-free strain UBAPF-2. It seems, that vir-induced T-DNA/plasmid DNA transfer machinery is not essential for the conjugation process between agrobacterial cells but may participate in this process.

Agrobacterium tumefaciens↗
PubMed2002

High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system.

For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar (confers resistance to bialaphos) and the beta-glucuronidase (GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where bar and GUS are contained on single T-DNA vectors in two separate Agrobacterium strains; (3) and a single T-DNA binary vector containing both bar and GUS as control treatment. Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment. A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the cis control vector treatment. For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R0 plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment. A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R0 events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R1 generation. Of these R0 events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R1 generation. A total of 64.4% of the R0 2 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R1 progeny. A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R0 plants displaying both transgenic phenotypes makes this method less efficient. Molecular analyses were then used to confirm that the observed segregation of R1 phenotypes were highly correlated to genetic segregation of the bar and GUS genes. A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.

Agrobacterium tumefaciens↗
PubMed2003

Catalytic analysis of a recombinant D-hydantoinase from Agrobacterium tumefaciens.

The D-hydantoinase gene of a wild strain of Agrobacterium tumefaciens BQL9 had 99.78% nucleotide sequence identity with other available Agrobacterium genes. The resulting amino acid sequence showed two important substitutions affecting two alpha-helixes in the secondary structure of the protein. The union of Mn2+ to the protein was essential for activating the enzyme and was independent of the temperature. D-Hydantoinase only was inactivated in the presence of 70 mM EDTA and at over 40 degrees C. The enzyme showed both hydantoinase and pyrimidinase activities, but only with the D-enantiomers of the substrates. Activity was greater for substrates with apolar groups in the number 5 carbon atom of the hydantoin. The native structure of the N-terminal end of this D-hydantoinase proved to be indispensable to its enzymatic activity.

Agrobacterium tumefaciens↗