Rhodococcus fascians Impacts Plant Development Through the Dynamic Fas-Mediated Production of a Cytokinin Mix

Abstract

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...

Bacterial and Plant Signal Integration via D3-Type Cyclins Enhances Symptom Development in the Arabidopsis-Rhodococcus fasciansInteraction

“…Therefore, R. fascians apparently succeeds in obtaining the maximum effect with minimum effort. The bacteria secrete a relatively low amount of cytokinins, but by producing a changing mixture of cytokinins, they prevent a complete breakdown by the host (Pertry et al, 2009(Pertry et al, , 2010. Then, cytokinin-activated plant Put production acts as a secondary and amplifying messenger of the cytokinin signal.…”

“…Therefore, we evaluated whether Put affected bacterial growth by following the turbidity of cultures and by counting colony-forming units present in infected Arabidopsis plants, both grown in the presence or absence of 1 mM Put. We also monitored the expression of the fasA gene involved in cytokinin production in cultures of the GUS reporter strain D188-pSPIPfasAgus (Pertry et al, 2010) incubated under optimal conditions for fas gene expression (Temmerman et al, 2000) and in bacterial cells present in plant tissues, again in the presence or absence of 1 mM Put. No significant differences were measured for either of these parameters (Supplemental Table S1), implying that the improved symptom establishment on Put is caused by an enhancement of the plant's signal transduction process implicated in symptom induction.…”

“…Cytokinins are the main virulence factors of R. fascians, and they are encoded by the fas operon located on the linear plasmid (Pertry et al, 2010), but the bacterium also secretes indole-3-acetic acid (IAA). Although auxin production is mediated by the Figure 3.…”

“…The ahk3ahk4 mutant has been proposed to be unresponsive to R. fascians, possibly because AHK2, the only functional cytokinin receptor in this mutant, is not sufficiently sensitive to the bacterial cytokinins and/or because the downstream signaling that it controls is not involved in symptom development (Pertry et al, 2009(Pertry et al, , 2010. At 7 and 14 dpi, the levels of free Spd and Spm, which were slightly reduced upon infection with strain D188, were comparable in the receptor mutant and wild-type plants (Supplemental Fig.…”

“…The bacteria disturb the plant's hormone balance by producing morphogenic signals, such as auxins and cytokinins, that provoke the outgrowth of existing and the development of new shoot meristems de O Manes et al, 2001;Vandeputte et al, 2005;Pertry et al, 2009), ultimately leading to the establishment of a leafy gall. In strain D188, virulence is conferred by a linear plasmid, pFiD188, that harbors the fasciation (fas) genes essential for cytokinin biosynthesis and symptom development (Crespi et al, 1992;Stange et al, 1996;Pertry et al, 2010). The bacteria secrete a mixture of synergistically acting cytokinins, some of which accumulate in infected plant tissues (Pertry et al, 2009).…”