Complete genomic sequence and phylogenomics analysis of Agrobacterium strain AB2/73: a new Rhizobium species with a unique mega-Ti plasmid

Abstract

Background The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors. Results We obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7012 genes (6948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in ridéopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin. Conclusions The comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium.

Genomics of the “tumorigenes” clade of the family Rhizobiaceae and description of Rhizobium rhododendri sp. nov.

“…AB2/73 which was isolated from Lippia canescens in the USA (Anderson & Moore, 1979). Recently, Hooykaas and Hooykaas (2021) suggested that this strain belongs to a novel Rhizobium species, which is closely related to Rhizobium dioscoreae according to our results. The second putative species includes Ti plasmid‐carrying strains 17‐2069‐2b and 17‐2069‐2c isolated from blackberry (Weisberg et al, 2022).…”

“…For all analyses reported in this study, annotations produced by Prokka (Galaxy Version 1.13) (Seemann, 2014) were used. Annotation of particular sequences of interest and metabolic pathway prediction were performed using eggNOG-mapper (version emapper-2.1.9) (Cantalapiedra et al, 2021) based on eggNOG orthology data (Huerta-Cepas et al, 2018), as well as with BlastKOALA (last accessed on November 2022) (Kanehisa et al, 2016). For eggNOGmapper, sequence searches were performed using DIAMOND version 2.0.11 (Buchfink et al, 2021).…”

“…However, another member of this genus, Rhizobium tumorigenes , was recently isolated from cane gall tumors on thornless blackberries (Kuzmanović et al, 2018). In addition, genomic analyses now suggest that the tumorigenic strain AB2/73 (Anderson & Moore, 1979), initially identified as a biovar 2 strain ( R. rhizogenes ) (Unger et al, 1985), actually belongs to a novel, so far undescribed Rhizobium species (Hooykaas & Hooykaas, 2021).…”

Development specifies, diversifies and empowers root immunity

“…R. rhizogenes changes plant organ morphology (Veena & Taylor, 2007 ; Bourras et al , 2015 ), known as hairy root disease, by integrating a part of a root‐inducing plasmid containing so‐called root oncogenic loci genes into the host cell genome. The expression of root oncogenic loci genes leads to the formation of new roots as a disease phenotype (Veena & Taylor, 2007 ; Hooykaas & Hooykaas, 2021 ), which involves induced expression of KNOTTED1‐LIKE HOMEOBOX ( KNOX ) TFs and cell cycle regulator genes in the host plant (Stieger et al , 2004 ). KNOX TFs are known to participate in meristem maintenance and organ patterning (Hake et al , 2004 ).…”

Identification and combinatorial engineering of indole-3-acetic acid synthetic pathways in Paenibacillus polymyxa

“…The IAM pathway of IAA synthesis is the best-characterized pathway in bacteria which mainly exists in Pseudomonas savastanoi and A. tumefaciens [ 41 – 43 ]. The main genes driving the IAM pathway are iaam and iaah , which encode tryptophan monooxygenase and indole-3-acetamide hydrolase, respectively [ 44 , 45 ]. Tryptophan monooxygenase catalyzes the conversion of L-tryptophan to indole-3-acetamide (IAM), and indole-3-acetamide hydrolase further catalyzes the hydrolysis of IAM to IAA [ 46 ].…”