Agrobacterial Strains Research Articles

Development of Agrobacterium-mediated transient expression system in Caragana intermedia and characterization of CiDREB1C in stress response

BackgroundThe Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants. Despite this transient expression system has been successfully applied in a number of plant species, it is poorly developed in Caragana intermedia.ResultsIn this study, we established an Agrobacterium-mediated transient expression system in C. intermedia leaves and optimized the effect of different Agrobacterial strains, several surfactants and the concentration of Silwet L-77, which would affect transient expression efficiency. Among the 5 Agrobacterial strains examined, GV3101 produced the highest GUS expression level. Besides, higher level of transient expression was observed in plants infiltrated with Silwet L-77 than with Triton X-100 or Tween-20. Silwet L-77 at a concentration of 0.001% greatly improved the level of GUS transient expression. Real-time PCR showed that expression of CiDREB1C was highly up-regulated in transiently expressed plants and reached the highest level at the 2nd day after infiltration. Based on this optimized transient transformation method, we characterized CiDREB1C function in response to drought, salt and ABA treatment. The results showed that transiently expressed CiDREB1C in C. intermedia leaves could enhance the survival rate and chlorophyll content, and reduce the lodging rate compared with the control seedlings under drought, salt and ABA treatments. Furthermore, the rate of leaf shedding of CiDREB1C transient expression seedlings was lower than that of the control under ABA treatment.ConclusionsThe optimized transient expression condition in C. intermedia leaves were infiltrated with Agrobacterial strains GV3101 plus Silwet L-77 at a concentration of 0.001% added into the infiltration medium. Transiently expressed CiDREB1C enhanced drought, salt and ABA stress tolerance, indicated that it was a suitable and effective tool to determine gene function involved in abiotic stress response in C. intermedia.

Prediction and identification of novel sRNAs involved in Agrobacterium strains by integrated genome-wide and transcriptome-based methods.

Small RNAs (sRNAs) are a class of gene regulators in bacteria, playing a central role in their response to environmental changes. Bioinformatic prediction facilitates the identification of sRNAs expressed under different conditions. We propose a novel method of prediction of sRNAs from the genome of Agrobacterium based on a positional weight matrix of conditional sigma factors. sRNAs predicted from the genome are integrated with the virulence-specific transcriptome data to identify putative sRNAs that are overexpressed during Agrobacterial virulence induction. A total of 384 sRNAs are predicted from transcriptome data analysis of Agrobacterium fabrum and 100-500 sRNAs from the genome of different Agrobacterial strains. In order to refine our study, a final set of 10 novel sRNAs with best features across different replicons targeting virulence genes were experimentally identified using semi-quantitative polymerase chain reaction. Since Ti plasmid plays a major role in virulence, out of 10 sRNAs across the replicons, 4 novel sRNAs differentially expressed under virulence induced and non-induced conditions are predicted to be present in the Ti plasmid T-DNA region flanking virulence-related genes like agrocinopine synthase, indole 3-lactate synthase, mannopine synthase and tryptophan monooxygenase. Further validation of the function of these sRNAs in conferring virulence would be relevant to explore their role in Agrobacterium-mediated plant transformation.

Jacob’s ladder Polemonium caeruleum L. and black salsify Sсorzonera hispanica L. in vitro culture

Aim. Micropropagation of Jacob’s ladder Polemonium caeruleum L. and black salsify Scorzonera hispanica L., obtaining root culture and regenerated plants. Methods. In vitro plant cultivation, medium composition modification for micropropagation, inoculation of explants with agrobacterial strains. Results. In vitro cultures of Jacob’s ladder and black salsify have been obtained, the optimal medium composition has been determined for the effective plants multiplication, rooting and growth, root cultures and regenerated plants of studied species have been obtained. Conclusions. Obtained technology of in vitro culture establishment of P. caeruleum and S. hispanica can be used for plants microclonal propagation so as root culture and regenerated plants acquiring due to the agrobacterial transformation – for further studies of secondary metabolism of these plants.
 Keywords: P. caeruleum L., S. hispanica L., micropropagation, phytohormones, root culture.

Agrobacterium rhizogenes-mediated transformation of a dioecious plant model Silene latifolia

Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S. latifolia tissues leading to transient and stable expression of the GUS reporter. The use of Agrobacterium rhizogenes strains resulted in the highest transformation efficiency (up to 4.7% of stable transformants) in hairy root cultures. Phenotypic and genotypic analyses of the T1 generation suggested that the majority of transformation events contain a small number of independent T-DNA insertions and the transgenes are transmitted to the progeny in a Mendelian pattern of inheritance. In short, we report an efficient and reproducible protocol for leaf disc transformation and subsequent plant regeneration in S. latifolia, based on the unique combination of infection with A. rhizogenes and plant regeneration from hairy root cultures using synthetic cytokinins. A protocol for the transient transformation of S.latifolia protoplasts was also developed and applied to demonstrate the possibility of targeted mutagenesis of the sex linked gene SlAP3 by TALENs and CRISPR/Cas9.

Quorum-Dependent Mannopine-Inducible Conjugative Transfer of an Agrobacterium Opine-Catabolic Plasmid

The Ti plasmid in Agrobacterium tumefaciens strain 15955 carries two alleles of traR that regulate conjugative transfer. The first is a functional allele, called traR, that is transcriptionally induced by the opine octopine. The second, trlR, is a nonfunctional, dominant-negative mutant located in an operon that is inducible by the opine mannopine (MOP). Based on these findings, we predicted that there exist wild-type agrobacterial strains harboring plasmids in which MOP induces a functional traR and, hence, conjugation. We analyzed 11 MOP-utilizing field isolates and found five where MOP induced transfer of the MOP-catabolic element and increased production of the acyl-homoserine lactone (acyl-HSL) quormone. The transmissible elements in these five strains represent a set of highly related plasmids. Sequence analysis of one such plasmid, pAoF64/95, revealed that the 176-kb element is not a Ti plasmid but carries genes for catabolism of MOP, mannopinic acid (MOA), agropinic acid (AGA), and the agrocinopines. The plasmid additionally carries all of the genes required for conjugative transfer, including the regulatory genes traR, traI, and traM. The traR gene, however, is not located in the MOP catabolism region. The gene, instead, is monocistronic and located within the tra-trb-rep gene cluster. A traR mutant failed to transfer the plasmid and produced little to no quormone even when grown with MOP, indicating that TraRpAoF64/95 is the activator of the tra regulon. A traM mutant was constitutive for transfer and acyl-HSL production, indicating that the anti-activator function of TraM is conserved.

A novel 5-enolpyruvylshikimate-3-phosphate synthase from Rahnella aquatilis with significantly reduced glyphosate sensitivity.

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroAR.aquatilis, was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroAE.coli), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroAR.aquatilis were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroAE.coli. To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.

Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The beta-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85-90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.

Biological Control of Grape Crown Gall by Rahnella aquatilis HX2.

Crown gall induced by Agrobacterium vitis is a worldwide plant disease in grape-growing regions. Rahnella aquatilis HX2, a new isolate from vineyard soil in Beijing, showed a significant inhibition effect on the development of crown galls in grapevines. In field trials, immersion of the basal ends of grape cuttings with HX2 cell suspension inhibited or completely prevented crown gall formation caused by A. vitis K308 in the roots of the plants from the cuttings. The 3-year average disease incidence in grape plants treated with HX2 was 30.8% compared to 93.5% in plants without HX2. The culture supernatant of HX2 exhibited a stronger inhibition effect on disease development than did the cell suspension. HX2 could be found in the grape rhizosphere, grown under field conditions, for up to 90 days after inoculation. There was no significant difference in the mean population sizes of root microflora between plants treated and not treated with HX2. The inhibition effect of HX2 on crown gall in sunflower, caused by different agrobacterial strains, varied between 30.7 and 100%, depending on strains. Our results showed that Rahnella aquatilis HX2 may be used as a biological control agent for crown gall disease of grapes.

Genetically Transformed Plant Roots as a Model for Studying Specific Metabolism and Symbiotic Contacts of the Root System

Genetic transformation of plants mediated by Ri plasmid of Agrobacterium rhizogenes occupies a special place in plant cell engineering, since this technique based on a natural phenomenon allows cultivation of separately growing roots on hormone-free media. Application of wild-type unmodified agrobacterial strains allows us to obtain root cultures capable of long-term growth in vitro due to an increased sensitivity of the cells to auxins while other biochemical properties remain unaltered. A collection of pRi T-DNA transformed roots of certain dicotyledons was constructed; some strains of it are used to study synthesis of secondary metabolites in root cells. The in vitro cultivated roots could synthesize root-specific metabolites, which allows their large-scale application for biotechnological production of ecologically pure crude drugs. Cocultivation of pRi T-DNA transformed roots with arbuscular mycorrhizal fungi makes possible vital study of all stages of obligate symbiont development and interaction with plant roots. Mixed axenic culture of AM fungi and pRi T-DNA transformed plants can be used to construct a collection of the most valuable endomycorrhizal fungal species and to produce considerable quantities of homogeneous fungal inoculums.

Phylogeny of Rhizobium galegae with respect to other rhizobia and agrobacteria.

PCR-RFLP with nine restriction enzymes was applied to the 16S and 23S rRNA genes of 42 rhizobial and agrobacterial strains to determine the phylogenetic position of Rhizobium galegae and increase the understanding of the evolution of ribosomal operons. The strains were selected based on previous phylogenetic studies. PCR primers were designed so that they amplified a 2.3 kb fragment of the 23S rRNA gene (excluding the B8 loop). Universal primers rD1 and fD1 were used to amplify the full-length 16S rRNA. The RFLP analysis resulted in 27 and 32 different restriction patterns for 16S and 23S, respectively. The RFLP patterns were transformed to genetic distances and dendrograms were constructed from the data using the unweighted pair group method with averages. The shapes of the dendrograms derived from the analysis of the 16S and 23S rRNA genes correlated well, with only a few strains having different positions. The 23S tree generally had deeper branching than the 16S tree, allowing better discrimination between species and strains. The combined data from the two analyses described 36 genotypes. The eight R. galegae strains formed a homogeneous cluster in all dendrograms. The RFLP analysis was confirmed by partial sequence analysis of the 16S rRNA gene (the first 800 bp), which correlated well with full-length 16S rRNA sequence analysis. The 16S data placed R. galegae near the genus Agrobacterium with Agrobacterium vitis as its nearest neighbour, whereas in the 23S and the combined dendrograms it showed closer affinity to the genus Rhizobium.

Variability of the 5'-end of the large subunit rDNA and presence of a new short class of rRNA in Rhizobiaceae.

A highly variable region of DNA was found between positions 115 and 388 (Escherichia coli numeration) of the large subunit (ls) rRNA genes of 55 rhizobial and agrobacterial strains. In each case this heterogeneity was accompanied by the presence of a new rRNA species approximately 130 bp long. This novel rRNA species corresponded to the 5'-end of the ls rRNA genes. An additional rRNA processing site was located in the central region of the remaining ls rRNA of many of the Rhizobium leguminosarum and Rh. etli strains, and in all of the agrobacteria studied, excepting the type strain of Agrobacterium vitis NCPPB 3554 and Agrobacterium sp. strain ChAg4.

Enhanced oxidative-stress defense in transgenic potato expressing tomato Cu,Zn superoxide dismutases

The two cDNAs coding for the cytosolic (cyt) and the chloroplast-located (chl) Cu,Zn superoxide dismutases (SODs) of tomato (Perl-Treves et al. 1988) were cloned into respective binary vectors and mobilized into Agrobacterium strains. Potato tuber discs were infected with either of the two agrobacterial strains and cultured on selective medium containing kanaymcin. The integration of either of the cyt or the chl SOD transgenes was verified by Southern-blot hybridization. The enzymatic activity of the additional tomato chl Cu,Zn SOD could be distinguished from endogenous SOD activity since the latter isozyme migrated faster on SOD-activity gels. Several transgenic potato lines harboring either the cyt or the chl SOD genes of tomato showed elevated tolerance to the superoxide-generating herbicide paraquat (methyl viologen). After exposure of shoots to paraquat, tolerance was recorded either by scoring symptoms visually or by measurements of photosynthesis using the photoacoustic method. Root cultures from transgenic lines that harbored the additional cyt Cu,Zn SOD gene of tomato were tolerant to methyl viologen up to 10(-5) M; a lower tolerance was recorded in roots of transgenic lines that expressed the additional chl Cu,Zn SOD of tomato.

The tropical legume Sesbania rostrata: Tissue culture, plant regeneration and infection with Agrobacterium tumefaciens and Rhizogenes strains

Conditions were examined for callus induction and in vitro morphogenesis of Sesbania rostrata. A protocol for organogenesis from different S. rostrata explants (cotyledons, hypocotyls, immature embryos) was established and used to regenerate plants. The cytokinin BAP was found to be essential for shoot formation at concentrations of 0.2–1.0 mg/l. SH medium, free of hormones or supplemented with 0.1 mg/l naphthaleneacetic acid (NAA), was found to stimulate root development of the regenerated plantlets. The susceptibility of S. rostrata to Agrobacterium mediated infection/transformation was tested using different wild type A. tumefaciens (C58 and B6S3) and A. rhizogenes (15834) strains. An extensive systemic infection of S. rostrata by the agrobacterial strains was observed, presumably occurring via spread of the bacteria in the vascular bundles.