The phytopathogenic actinomycete Rhodococcus fascians D188 relies mainly on the linear plasmid-encoded fas operon for its virulence. The bacteria secrete six cytokinin bases that synergistically redirect the developmental program of the plant to stimulate proliferation of young shoot tissue, thus establishing a leafy gall as a niche. A yeast-based cytokinin bioassay combined with cytokinin profiling of bacterial mutants revealed that the fas operon is essential for the enhanced production of isopentenyladenine, trans-zeatin, cis-zeatin, and the 2-methylthio derivatives of the zeatins. Cytokinin metabolite data and the demonstration of the enzymatic activities of FasD (isopentenyltransferase), FasE (cytokinin oxidase/dehydrogenase), and FasF (phosphoribohydrolase) led us to propose a pathway for the production of the cytokinin spectrum. Further evaluation of the pathogenicity of different fas mutants and of fas gene expression and cytokinin signal transduction upon infection implied that the secretion of the cytokinin mix is a highly dynamic process, with the consecutive production of a tom initiation wave followed by a maintenance flow.Rhodococcus fascians is a phytopathogenic actinomycete with a very broad host range that causes important commercial losses in the ornamentals industry because it triggers severe malformations of shoots, referred to as leafy galls (Depuydt et al. 2008b). In strain D188, the virulence determinants are encoded by a large conjugative linear plasmid, pFiD188 , and the pathology is induced by the secretion of a mix of six synergistically acting cytokinins: isopentenyladenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and their 2-methylthio (2MeS) derivatives (Pertry et al. 2009). In Arabidopsis thaliana, these cytokinins are perceived by the receptors AHK3 and CRE1/AHK4 (Pertry et al. 2009), activating a signaling cascade that stimulates cell proliferation and meristematic gene expression and, ultimately, results in the establishment of a specific niche (Depuydt et al. 2008a.Comparison of the cytokinin profiles of two near-isogenic strains, D188 and its plasmid-free derivative, D188-5, has shown that a basal level of the six cytokinins is produced by a chromosomally encoded pathway. However, the much higher levels of iP, cZ, tZ, 2MeScZ, and 2MeStZ secreted by strain D188 strongly suggest an additional linear plasmid-encoded de novo biosynthetic pathway (Pertry et al. 2009). By sequence analysis of pFiD188, the fas operon was identified, consisting of six genes putatively involved in cytokinin biosynthesis and essential for virulence (Fig. 1). FasA is similar to P450-type cytochrome monooxygenases. The N-terminal region of FasB corresponds to 4Fe-3S-type ferredoxins of Actinomycetes, whereas its carboxy-terminus is homologous to the α subunit of pyruvate dehydrogenase. FasC is similar to the β subunit of the latter enzyme. Both FasB and FasC have a binding site for the cofactor thiamine pyrophosphate (Crespi et al. 1994). FasD is an isopentenyltransferase (Ipt) protein that mediates th...
