Evolutionary classification of tumor- and root-inducing plasmids based on T-DNAs and virulence regions.

Tumor-inducing (Ti) and root-inducing (Ri) plasmids of Agrobacterium that display a large diversity are involved in crown gall and hairy root plant diseases. Their phylogenetic relationships were inferred from an exhaustive set of Ti and Ri plasmids (including 36 new complete Ti plasmids) by focusing on T-DNA and virulence regions. The opine synthase gene content of T-DNAs revealed 13 opine types corresponding to former classifications based on opines detected in diseased plants, while the T-DNA gene content more finely separate opine types in 18 T-DNA organizations. This classification was supported by the phylogeny of T-DNA oncogenes of Ti plasmids. The five gene organizations found in Ti/Ri vir regions was supported by the phylogeny of common vir genes. The vir organization was found to be likely an ancestral plasmid trait separating "classic" Ti plasmids (with one or two T-DNAs) and "Ri and vine-Ti" plasmids. A scenario generally supported by the repABC phylogeny. T-DNAs likely evolved later with the acquisition of opine characteristics as last steps in the Ti/Ri plasmid evolution. This novel evolutionary classification of Ti/Ri plasmids was found to be relevant for accurate crown gall and hairy root epidemiology.

Pathogenicity, Phylogenetic relationship and NGS based identification and assembly of tumorigenic Agrobacterium radiabacter plasmidic and chromosomic reads isolated from Prunus duclcis.

Although many Agrobacterium radiobacter strains have already been identified, only a few genomes of strains belonging to genomovar G4 have been sequenced so far. In this study, we report the first virulent genome sequence of Agrobacterium radiobacter strain tun 183, which is highly virulent to almond specie. The genome size was estimated to be 5.53 Mb, with 57.9%GC content. In total, 6486 genes encoding proteins and 61 genes encoding RNAs were identified in this genome. Comparisons with the available sequenced genomes of genomovar G4 as well as with other A. sp. were conducted, revealing a hexapartite genome containing circular and linear chromosomes in addition to two accessory plasmids and a tumor inducing plasmid (pTi) in strain tun 183. The phylogenetic analysis of recA gene clearly showed the clustering of tun 183 strain within genomovar G4, supporting the monophyly within this genomovar.

A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples.

A new dual plasmid calibrator for the quantification of the construct specific GM canola Oxy-235 with duplex real-time PCR.

To overcome the difficulties of obtaining the Certified Reference Material (CRM) and according to the key documents of the European Union Reference Laboratory (EU-RL), a new standard reference molecule containing the construct specific of the canola event Oxy-235 (3'-junction Nitrilase/Tnos) and the canola endogenous reference gene (acety-CoA-carboxylase) was constructed and used for duplex real-time quantitative analysis. The limits of detection (LOD) were less than 5 Haploid Genome Copy (HGC) and the limits of quantification (LOQ) were about 10 HGC. Furthermore, mixed GM and non-GM canola samples were analysed with duplex QRT-PCR to evaluate the performance criteria as required for validation procedures in the EU-RL, namely, the precision and the accuracy. The accuracy expressed as bias ranged from 2% to 10% and the precision (repeatability and reproducibility) expressed as the RSDr and RSDR was from 2.2 to 5.12 and 2.15 to 5.46 respectively. All these indicated that the developed construct specific method and the reference molecule are suitable for the identification and the quantification of the canola event Oxy-235.