Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus,
some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model
predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central
to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a
hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of
Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was
identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to
phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from
phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion
protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating
cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was
specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is
synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a
mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four
horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse
clade of Rhodococcus
