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Evolutionary Relatedness and Classification of Tumor-Inducing and Opine-Catabolic Plasmids in Three Rhizobium rhizogenes Strains Isolated from the Same Crown Gall Tumor.

ABSTRACT: Plasmids play a crucial role in the ecology of agrobacteria. In this study, we sequenced tumor-inducing (Ti) and opine-catabolic (OC) plasmids in three Rhizobium rhizogenes (Agrobacterium biovar 2) strains isolated from the same crown gall tumor on "Colt" cherry rootstock and conducted comparative genomic analyses. Tumorigenic strains C5.7 and C6.5 carry nopaline-type Ti plasmids pTiC5.7/pTiC6.5, whereas the nonpathogenic strain Colt5.8 carries the nopaline-type OC plasmid pOC-Colt5.8. Overall, comparative genomic analysis indicated that pTiC5.7/pTiC6.5 and related Ti plasmids described before (pTiC58 and pTi-SAKURA) originate from a common ancestor, although they have diverged during evolution. On the other hand, plasmid pOC-Colt5.8 was most closely related to the well-known OC plasmid pAtK84b; however, analysis suggested that they had different evolutionary histories and seem to share a more distant common ancestor. Although the reconstruction of the evolutionary history of Ti and OC plasmids is still speculative, we hypothesized that nopaline-type Ti plasmid might originate from the nopaline-type OC plasmid. Our results suggested that OC plasmids are widespread and closely associated with crown gall tumors. Finally, we proposed a thorough scheme for classification of Ti and OC plasmids that is based on separate comparative analysis of each functional element of the plasmid studied.

SUBMITTER: Kuzmanovic N

PROVIDER: S-EPMC6546132 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Deoxyfructosyl glutamine (DFG, referred to elsewhere as dfg) is a naturally occurring Amadori compound found in rotting fruits and vegetables. DFG also is an opine and is found in tumors induced by chrysopine-type strains of Agrobacterium tumefaciens. Such strains catabolize this opine via a pathway coded for by their plasmids. NT1, a derivative of the nopaline-type A. tumefaciens strain C58 lacking pTiC58, can utilize DFG as the sole carbon source. Genes for utilization of DFG were mapped to the 543-kb accessory plasmid pAtC58. Two cosmid clones of pAtC58 allowed UIA5, a plasmid-free derivative of C58, harboring pSa-C that expresses MocC (mannopine [MOP] oxidoreductase that oxidizes MOP to DFG), to grow by using MOP as the sole carbon source. Genetic analysis of subclones indicated that the genes for utilization of DFG are located in a 6.2-kb BglII (Bg2) region adjacent to repABC-type genes probably responsible for the replication of pAtC58. This region contains five open reading frames organized into at least two transcriptional soc (santhopine catabolism) groups: socR and socABCD. Nucleotide sequence analysis and analyses of transposon-insertion mutations in the region showed that SocR negatively regulates the expression of socR itself and socABCD. SocA and SocB are responsible for transport of DFG and MOP. SocA is a homolog of known periplasmic amino acid binding proteins. The N-terminal half of SocB is a homolog of the transmembrane transporter proteins for several amino acids, and the C-terminal half is a homolog of the transporter-associated ATP-binding proteins. SocC and SocD could be responsible for the enzymatic degradation of DFG, being homologs of sugar oxidoreductases and an amadoriase from Corynebacterium sp., respectively. The protein products of socABCD are not related at the amino acid sequence level to those of the moc and mot genes of Ti plasmids responsible for utilization of DFG and MOP, indicating that these two sets of genes and their catabolic pathways have evolved convergently from independent origins.

Project description:BACKGROUND: Crown gall (CG) (Agrobacterium tumefaciens) and the root lesion nematodes (RLNs) (Pratylenchus vulnus) are major challenges faced by the California walnut industry, reducing productivity and increasing the cost of establishing and maintaining orchards. Current nematode control strategies include nematicides, crop rotation, and tolerant cultivars, but these methods have limits. Developing genetic resistance through novel approaches like RNA interference (RNAi) can address these problems. RNAi-mediated silencing of CG disease in walnut (Juglans regia L.) has been achieved previously. We sought to place both CG and nematode resistance into a single walnut rootstock genotype using co-transformation to stack the resistance genes. A. tumefaciens, carrying self-complimentary iaaM and ipt transgenes, and Agrobacterium rhizogenes, carrying a self-complimentary Pv010 gene from P. vulnus, were used as co-transformation vectors. RolABC genes were introduced by the resident T-DNA in the A. rhizogenes Ri-plasmid used as a vector for plant transformation. Pv010 and Pv194 (transgenic control) genes were also transferred separately using A. tumefaciens. To test for resistance, transformed walnut roots were challenged with P. vulnus and microshoots were challenged with a virulent strain of A. tumefaciens. RESULTS: Combining the two bacterial strains at a 1:1 rather than 1:3 ratio increased the co-transformation efficiency. Although complete immunity to nematode infection was not observed, transgenic lines yielded up to 79% fewer nematodes per root following in vitro co-culture than untransformed controls. Transgenic line 33-3-1 exhibited complete crown gall control and 32% fewer nematodes. The transgenic plants had thicker, longer roots than untransformed controls possibly due to insertion of rolABC genes. When the Pv010 gene was present in roots with or without rolABC genes there was partial or complete control of RLNs. Transformation using only one vector showed 100% control in some lines. CONCLUSIONS: CG and nematode resistance gene stacking controlled CG and RLNs simultaneously in walnuts. Silencing genes encoding iaaM, ipt, and Pv010 decrease CG formation and RLNs populations in walnut. Beneficial plant genotype and phenotype changes are caused by co-transformation using A. tumefaciens and A. rhizogenes strains. Viable resistance against root lesion nematodes in walnut plants may be accomplished in the future using this gene stacking technology.

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Evolutionary Relatedness and Classification of Tumor-Inducing and Opine-Catabolic Plasmids in Three Rhizobium rhizogenes Strains Isolated from the Same Crown Gall Tumor.

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