Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsThe 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.
Highlights
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
General features of the genome of Agrobacterium (Rhizobium) strain AB2/73 We obtained the full genomic sequence of AB2/73 by Unicycler hybrid assembly of short-reads from Illumina sequencing and long-reads from MinION Nanopore sequencing
Clusters of Orthologous Groups (COG) functional categories were assigned to predicted coding sequences, as can be seen on the circular maps in Figs. 1 and 2 and Comparative genomics and phylogenetic relatedness The strain AB2/73 was originally classified as a biotype 2 Agrobacterium, nowadays Rhizobium rhizogenes [19]
Summary
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. Hooykaas and Hooykaas BMC Microbiol (2021) 21:295 bacteria, A. rhizogenes for hairy root-inducing bacteria and A. radiobacter for avirulent strains. This classification appeared to be based on plasmid-determined properties, with Ti plasmids responsible for the induction of crown gall and Ri plasmids responsible for induction of hairy root. To biovar 2 bacteria the species name Rhizobium rhizogenes was given and to biovar 3 bacteria the name Allorhizobium vitis [5]. These very different bacteria may contain virtually identical Ti plasmids. Four new tumorigenic strains isolated from blackberry galls were found to belong to the genus Rhizobium, but differ from R. rhizogenes and were given the species name Rhizobium tumorigenes [6]
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