The Cytokinin Complex Associated With Rhodococcus fascians: Which Compounds Are Critical for Virulence?

Abstract

Virulent strains of <i>Rhodococcus fascians</i> cause a range of disease symptoms, many of which can be mimicked by application of cytokinin. Both virulent and avirulent strains produce a complex of cytokinins, most of which can be derived from tRNA degradation. To test the three current hypotheses regarding the involvement of cytokinins as virulence determinants, we used PCR to detect specific genes, previously associated with a linear virulence plasmid, including two methyl transferase genes (<i>mt1</i> and <i>mt2</i>) and <i>fas4</i> (dimethyl transferase), of multiple strains of <i>R. fascians</i>. We inoculated <i>Pisum sativum</i> (pea) seeds with virulent and avirulent strains of <i>R. fascians</i>, monitored the plants over time and compared these to mock-inoculated controls. We used RT-qPCR to monitor the expression of <i>mt1, mt2</i>, and <i>fas4</i> in inoculated tissues and LC-MS/MS to obtain a comprehensive picture of the cytokinin complement of inoculated cotyledons, roots and shoots over time. The presence and expression of <i>mt1</i> and <i>mt2</i> was associated with those strains of <i>R. fascians</i> classed as virulent, and not those classed as avirulent. Expression of <i>mt1, mt2</i>, and <i>fas4</i> peaked at 9 days post-inoculation (dpi) in cotyledons and at 15 dpi in shoots and roots developed from seeds inoculated with virulent strain 602. Pea plants inoculated with virulent and avirulent strains of <i>R. fascians</i> both contained cytokinins likely to have been derived from tRNA turnover including the 2-methylthio cytokinins and <i>cis</i>-zeatin-derivatives. Along with the isopentenyladenine-type cytokinins, the levels of these compounds did not correlate with virulence. Only the novel 1- and 2-methylated isopentenyladenine cytokinins were uniquely associated with infection by the virulent strains and are, therefore, the likely causative factors of the disease symptoms.