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.

The type VI secretion system (T6SS) is used by gram-negative bacteria to translocate effectors that can antagonize other bacterial cells. Models predict the variation in collections of effector and cognate immunity genes determine competitiveness and can affect the dynamics of populations and communities of bacteria. However, the outcomes of competition cannot be entirely explained by compatibility of effector-immunity (EI) pairs. Here, we characterized the diversity of T6SS loci of plant-pathogenic Agrobacterium tumefaciens and showed that factors other than EI pairs can impact interbacterial competition. All examined strains encode T6SS active in secretion and antagonism against Escherichia coli. The spectra of EI pairs as well as compositions of gene neighborhoods are diverse. Almost 30 in-planta competitions were tested between different genotypes of A. tumefaciens. Fifteen competitions between members of different species-level groups resulted in T6SS-dependent suppression in in-planta growth of prey genotypes. In contrast, ten competitions between members within species-level groups resulted in no significant effect on the growth of prey genotypes. One strain was an exceptional case and, despite encoding a functional T6SS and toxic effector protein, could not compromise the growth of the four tested prey genotypes. The data suggest T6SS-associated EI pairs can influence the competitiveness of strains of A. tumefaciens, but genetic features have a significant role on the efficacy of interbacterial antagonism.

Pto is a member of a multigene family and encodes a serine/threonine kinase that mediates gene-for-gene resistance to strains of Pseudomonas syringae pv. tomato expressing avrPto. The inferred amino acid sequence of the Pto homologs from both resistant (LpimPth2 to LpimPth4) and susceptible (LescFen, LescPth2 to LescPth5) haplotypes suggested that most could encode functional serine/threonine kinases. In addition, the activation segments of the homologs are similar in sequence to that of Pto, and some have residues previously identified as required for binding of AvrPto by Pto in the yeast two-hybrid system. The Pto homologs were therefore characterized for transcription, for the ability of their products to interact with AvrPto in the yeast two-hybrid system, for their autophosphorylation activity, and for their potential to elicit cell death in the presence of and absence of a ligand, as well as their dependence on Prf. LpimPth5, LpimPth4, and LescPth4 were not transcribed at levels detectable by reverse transcription-polymerase chain reaction. The interaction with AvrPto was unique to Pto in the yeast two-hybrid system. LescPth2 autophosphorylated in vitro as a fusion protein. LpimPth2, LpimPth3, LpimPth4, LescPth3, and LescPth4 did not autophosphorylate in vitro. Transient expression of wild-type Fen and wild-type LpimPth3, as well as LescFen, LescPth3, and LescPth5 with perturbations in their P+1 loop caused cell death in Nicotiana benthamiana. LpimPth3 and LescPth3 with amino acid substitutions in the P+1 loop also elicited cell death in tomato; this was dependent on the presence of wild-type Prf. Consequently, some homologs could potentially encode functional resistance proteins. LescPth5 induced cell death specifically in response to expression of AvrPto in tobacco in a Prf-dependent manner; this is consistent with a homolog from a 'susceptible' haplotype encoding a minor recognition determinant.