Wim Temmerman

Identification of Rhodococcus fascians cytokinins and their modus operandi to reshape the plant

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 virulence-associated 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.

Rhodococcus fascians impacts plant development through the dynamic Fas-mediated production of a cytokinin mix

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.

A nucleotide substitution in one of the beta-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-yl-carbamate.

We characterized a Trichoderma viride strain that is resistant to the antimitotic drug methyl benzimidazole-2-yl-carbamate (MBC). This species has two β-tubulin genes (tub1 and tub2) and by reverse genetics we showed that a mutation in the tub2 gene confers MBC resistance in this strain. Comparison of the tub2 sequence of the mutant strain with that of the wild type revealed that a single amino acid substitution of tyrosine for histidine at position 6 is responsible for the MBC tolerance. Furthermore, we showed that this gene can be used as a homologous dominant selectable marker in T. viride transformation. Both tubulin genes were completely sequenced. They differ by 48 residues and the degree of identity between their deduced amino acid sequences is 86.3%.

Leafy Gall Formation Is Controlled by fasR, an AraC-Type Regulatory Gene in Rhodococcus fascians

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 fas locus of the phytopathogen Rhodococcus fascians affects mitosis of tobacco BY-2 cells.

The effect of Rhodococcus fascians, the causal agent of leafy gall disease, on the mitotic behavior of synchronized tobacco Bright Yellow‐2 (BY‐2) cells was investigated. Incubation of aphidicolin‐synchronized BY‐2 cells with R. fascians cells specifically resulted in a broader mitotic index peak, an effect that was linked to an intact and expressed fas virulence locus. The obtained results pointed towards an effect of R. fascians on the prophase of mitosis. The relevance of these results to the virulence of the bacterium is discussed.