Decades ago, the importance of cytokinins (CKs) during Rhodococcus fascians pathology had been acknowledged, and an isopentenyltransferase gene had been characterized in the fas operon of the linear virulence plasmid, but hitherto, no specific CK(s) could be associated with virulence. We show that the CK receptors AHK3 and AHK4 of Arabidopsis thaliana are essential for symptom development, and that the CK perception machinery is induced upon infection, underlining its central role in the symptomatology. Three classical CKs [isopentenyladenine, trans-zeatin, and cis-zeatin (cZ)] and their 2-methylthio (2MeS)-derivatives were identified by CK profiling of both the pathogenic R. fascians strain D188 and its nonpathogenic derivative D188 -5. However, the much higher CK levels in strain D188 suggest that the linear plasmid is responsible for the virulenceassociated production. All R. fascians CKs were recognized by AHK3 and AHK4, and, although they individually provoked typical CK responses in several bioassays, the mixture of bacterial CKs exhibited clear synergistic effects. The cis-and 2MeS-derivatives were poor substrates of the apoplastic CK oxidase/dehydrogenase enzymes and the latter were not cytotoxic at high concentrations. Consequently, the accumulating 2MeScZ (and cZ) in infected Arabidopsis tissue contribute to the continuous stimulation of tissue proliferation. Based on these results, we postulate that the R. fascians pathology is based on the local and persistent secretion of an array of CKs.phytopathogen ͉ actinomycete ͉ phytohormone T he fine-tuned balance of plant regulators has a key role in the growth and development of plants. Many plant-associated bacteria can influence their hosts by either modulating the phytohormone production or producing the phytohormones themselves. The main advantages for the bacteria are increased nutrient release, suppression of defense, and/or disease establishment (1, 2). Hyperplasia-inducing bacteria, such as Pantoea agglomerans and Pseudomonas savastanoi, secrete high amounts of cytokinins (CKs) and auxins to facilitate or initiate gall development (3, 4), and Agrobacterium tumefaciens genetically transforms plant cells to convert them into CK and auxin (and opine) factories (5).In contrast to the undifferentiated galls induced by the bacteria mentioned above, the Actinomycete Rhodococcus fascians that shares persistence strategies with the closely related human pathogen Mycobacterium tuberculosis (6) provokes the formation of differentiated leafy galls, consisting of numerous shoot primordia whose further outgrowth is inhibited (7). The shooty symptoms can be partially mimicked by exogenous addition of CKs (8, 9), and analyses of culture supernatants of different nonisogenic virulent and avirulent R. fascians strains grown under rich culture conditions identified 11 different CKs: methylaminopurine, 2-methylthioisopentenyladenine (2MeSiP), iP, cis-zeatin (cZ), trans-zeatin (tZ), dihydrozeatin (DZ), 2MeScZ, and their respective ribosides (10-14). Except for iP, the p...

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...

Three virulence loci (fas, ftt, and hyp) of Rhodococcus fascians D188 have been identified on a 200-kb conjugative linear plasmid (pFiD188). The fas locus was delimited to a 6.5-kb DNA fragment by insertion mutagenesis, single homologous disruptive recombination, and in trans complementation of different avirulent insertion mutants. The locus is arranged as a large operon containing six open reading frames whose expression is specifically induced during the interaction with host plants. One predicted protein is homologous to P-450 cytochromes from actinomycetes. The putative ferredoxin component is of a novel type containing additional domains homologous to transketolases from chemoautotrophic, photosynthetic, and methylotrophic microorganisms. Genetic analysis revealed thatfas encodes, in addition to the previously identified ipt, at least two new genes that are involved in fasciation development, one of which is only required on older tobacco plants.The interaction between Rhodococcus fascians and host plants leads to the loss of apical dominance and the development of multiple malformed shoots at the site of infection (fasciation). This proliferation is governed by multiple bacterial loci located both on a linear Fi plasmid and on the chromosome (reference 8 and unpublished results). From the three identified loci on pFiD188, only inactivation of one (fas) leads to a complete loss of phytopathogenicity. In this locus, an open reading frame (ORF) coding for an isopentenyltransferase (ipt) has been identified by sequence homology to other cytokinin biosynthesis genes and by biochemical approaches (8). Expression of ipt in R. fascians is regulated by an inducing factor which can only be detected in significant amounts in extracts from tumor tissues (induced by R. fascians) (8).In this paper, we present genetic data that allowed the definition of the fas locus on a 6.5-kb DNA fragment whose complete nucleotide sequence was determined. Six ORFs directed in the same orientation as the previously defined ipt were identified. ORFi and the amino-terminal part of ORF2 have extensive homologies, to P-450 cytochromes and ferredoxins from actinomycetes, whereas the carboxy-terminal portion of ORF2 has conserved regions homologous to the aminoterminal part of transketolases from chemoautotrophic, photosynthetic, or methylotrophic microorganisms.The isolation of new fas mutants and complementation analysis revealed that fas encodes new genes involved in fasciation of the host plant.MATERIALS AND METHODS Bacterial strains and plasmids. Growth conditions and antibiotic concentrations for R. fascians strains have been described previously (11). The Escherichia coli strains used for

Rhodococcus fascians can interact with many plant species and induce the formation of either leafy galls or fasciations. To provoke symptoms, R. fascians strain D188 requires pathogenicity genes that are located on a linear plasmid, pFiD188. The fas genes are essential for virulence and constitute an operon that encodes, among other functions, a cytokinin synthase gene. Expression of the fas genes is induced by extracts of infected plant tissue only. We have isolated an AraC-type regulatory gene, fasR, located on pFiD188, which is indispensable for pathogenesis and for fas gene expression. The combined results of our experiments show that in vitro expression of the fas genes in a defined medium is strictly regulated and that several environmental factors (pH, carbon and nitrogen sources, phosphate and oxygen content, and cell density) and regulatory proteins are involved. We further show that expression of the fas genes is controlled at both the transcriptional and the translational levels. The complex expression pattern probably reflects the necessity of integrating a multitude of signals and underlines the importance of the fas operon in the pathogenicity of R. fascians.The gram-positive bacterium Rhodococcus fascians (58) infects diverse plant species. Infection of dicotyledonous plants can result in the local proliferation of meristematic tissue, leading to galls that are covered with leaflets, known as leafy galls (17, 61). On monocotyledonous plants, such as lilies, R. fascians provokes severe malformations of the bulbs and the formation of long side shoots (37, 60), resulting in abnormal plants that are unfit for commercial use (2, 18). Infection of tobacco seedlings with R. fascians strongly inhibits growth, accompanied by arrested root development, thickening and stunting of the hypocotyl, and inhibition of leaf formation (10).In 1966, the production of cytokinins was inferred as a major virulence determinant of R. fascians (31, 57). In our laboratory, in R. fascians strain D188, genes involved in pathogenicity were shown to be located on a large, conjugative, linear, fasciationinducing plasmid (pFiD188) (10). Random mutagenesis of pFiD188 led to the identification of three virulence loci, of which the best characterized is the essential fas locus. This locus consists of an operon of six genes, of which the most important are a cytochrome P450 homologue gene (ORF1) and an isopentenyl transferase (ipt) gene (ORF4) homologous to ipt genes of other phytopathogens (10, 11). The ipt genes are typically involved in the biosynthesis of isopentenyl AMP (i 6 AMP), a general precursor of several cytokinins (29). However, the chemical structure of the compound resulting from the action of the fas gene products remains to be determined. Two other pFiD188-located virulence loci, hyp and att, are necessary for balanced virulence because mutations in these regions result in hypervirulence and attenuated virulence, respectively (10).Expression of the fas genes is induced by extracts of infected plant tissues and not of...