Research Article

Unexpected conservation and global transmission of agrobacterial virulence plasmids

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Science  05 Jun 2020:
Vol. 368, Issue 6495, eaba5256
DOI: 10.1126/science.aba5256

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Agrobacteria virulence writ large

Plasmids are widespread among bacteria and are important because they spread virulence and antibiotic resistance traits, among others. They are horizontally transferred between strains and species, so it is difficult to work out their evolution and epidemiology. Agrobacteria, a diverse grouping of species that infect plants, inject oncogenic Ti and Ri plasmids, which cause crown galls and hairy root diseases, respectively. The upside is that these plasmids have become valuable biotechnological tools. Weisberg et al. combed through an 80-year-old collection of Agrobacterium strains but found a surprisingly low diversity of plasmids. It is puzzling how limited the number of plasmid lineages is despite reported high levels of plasmid recombination, but what is clear is how plant production systems have influenced plasmid spread into various genomic backbones.

Science, this issue p. eaba5256

Structured Abstract


Plasmids are autonomously replicating, nonessential DNA molecules that accelerate the evolution of many important bacterial-driven processes. For example, plasmids spread antibiotic resistance genes, which are a pressing problem for human and animal health. Plasmids can also encode complex traits that allow bacteria to interact intimately with eukaryotes. Acquisition of an oncogenic tumor-inducing (Ti) or root-inducing (Ri) plasmid by saprophytic soil agrobacteria changes them into pathogens capable of genetically transforming and causing disease in a broad range of plant species.

Plasmids are also biotechnology tools that can advance our understanding of life. They can be used to generate organisms with unusual traits and innovative applications. The potential for using oncogenic plasmids to accelerate research was recognized early in their discovery. Along with strains of agrobacteria, disarmed plasmids are mainstays as tools in plant biology and plant genetic engineering.


Inferring evolutionary relationships is foundational for classifying plasmids, accurately assessing the influence of plasmids on disease outbreaks, developing appropriate strategies for mitigating disease, and expediting efforts to leverage plasmid diversity for biotechnology. However, such research is complicated because plasmids consist of diverse structural variants and are extraordinarily dynamic, modular molecules that can be reshuffled and broadly transmitted horizontally.

We focused on oncogenic plasmids of agrobacteria because of their important roles in causing disease and as biotechnology tools. Two genomic datasets were developed. One consisted of diverse, broadly sampled historical strains and was intended to serve as the basis for an evolutionary framework. The other consisted of contemporaneous strains hierarchically sampled from managed plant production sites, for the purpose of calibrating epidemiology methods. The datasets were combined to identify epidemiological patterns.


We combined analyses of chromosomal ancestry and plasmids to uncover their contributions and accurately model the global spread of disease. Phylogenetic, genomic, and time tree analyses of thousands of strains from the Rhizobiales order yielded a robust phylogenetic history of agrobacteria. We developed a strategy that uses phylogenetic and network approaches as well as different scales of genetic information to infer the evolution of diverse oncogenic plasmids. By combining results, we uncovered global epidemiological patterns supporting movement of pathogens clonally and plasmids horizontally in space and time.

This study has three major findings: (i) Lineages of agrobacteria emerged independently and at different times from within a genus-level group that also circumscribes multiple lineages of rhizobia. (ii) Agrobacterial Ti and Ri plasmids are descended from only six and three lineages (types), respectively. Few evolutionary events are sufficient to explain the relationships observed among types. Each type is subject to different pressures and shows different degrees of within-group variation, but their evolution is nonetheless guided by similar principles. The extent of modularity is high, and genes and functional modules are frequently reshuffled via recombination within conserved loci. Yet plasmid diversification is nonetheless constrained by the spatial structure of loci that interact genetically. (iii) Transmission of oncogenic plasmids, especially within agricultural settings, promotes the massive spread of disease.


Our strategy for inferring the evolution and transmission of virulence plasmids has potential applications in plant and human or animal health and food safety, as well as for understanding the ecology and evolution of other plasmid-mediated processes such as mutualistic symbioses. In addition, this strategy can be applied to study other mobile and modular elements, such as integrative conjugative elements and pathogenicity or symbiosis islands. We have shown that plasmids once viewed as too diverse to be classified have distinct lineages, and that accurate modeling of the spread of disease can be accomplished by robustly defining their evolutionary relationships.

Combined genomic analyses of chromosomal and plasmid identities to model disease spread.

Genomic data from hundreds of strains of agrobacteria were parsed and analyzed to infer the evolutionary histories of chromosomes and oncogenic Ti and Ri plasmids. The data were overlaid to uncover the roles of bacteria and plasmids in the global spread of disease.


The accelerated evolution and spread of pathogens are threats to host species. Agrobacteria require an oncogenic Ti or Ri plasmid to transfer genes into plants and cause disease. We developed a strategy to characterize virulence plasmids and applied it to analyze hundreds of strains collected between 1927 and 2017, on six continents and from more than 50 host species. In consideration of prior evidence for prolific recombination, it was surprising that oncogenic plasmids are descended from a few conserved lineages. Characterization of a hierarchy of features that promote or constrain plasticity allowed inference of the evolutionary history across the plasmid lineages. We uncovered epidemiological patterns that highlight the importance of plasmid transmission in pathogen diversification as well as in long-term persistence and the global spread of disease.

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