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

Abstract

Virulent strains of Rhodococcus fascians 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 ( mt1 and mt2 ) and fas4 (dimethyl transferase), of multiple strains of R. fascians . We inoculated Pisum sativum (pea) seeds with virulent and avirulent strains of R. fascians , monitored the plants over time and compared these to mock-inoculated controls. We used RT-qPCR to monitor the expression of mt1, mt2 , and fas4 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 mt1 and mt2 was associated with those strains of R. fascians classed as virulent, and not those classed as avirulent. Expression of mt1, mt2 , and fas4 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 R. fascians both contained cytokinins likely to have been derived from tRNA turnover including the 2-methylthio cytokinins and cis -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.

Role of Cytokinins for Interactions of Plants With Microbial Pathogens and Pest Insects

“…The Gram positive bacterium Rhodococcus fascians causes leafy galls symptoms in plants which resemble to the symptoms caused by exogeneous applied CK to plants. Moreover, CK pathways downstream of AHK3 and AHK4 are similarly important for leafy gall formation (Jameson et al, 2019). However, CK-induced susceptibility is interconnected to an early, AHK3 and AHK4-dependent, transcriptional reprogramming that renders host cells more receptive to A. tumefaciens (Sardesai et al, 2013).…”

Functional Genomics Insights Into the Pathogenicity, Habitat Fitness, and Mechanisms Modifying Plant Development of Rhodococcus sp. PBTS1 and PBTS2

“…The host range of these bacteria encompasses a broad spectrum of mostly herbaceous dicotyledonous plants, although some woody plants and few monocotyledons are sensitive as well (Putnam and Miller, 2007;Depuydt et al, 2008b). Key to symptom development is the bacterial production of an array of cytokinins via gene products of the fasciation (fas) operon encoded on a linear virulence plasmid in most pathogenic isolates (Crespi et al, 1992;Pertry et al, 2009Pertry et al, , 2010Francis et al, 2012;Creason et al, 2014;Radhika et al, 2015;Jameson et al, 2019) and the concomitant modification of the hormone landscape of the infected plant (Depuydt et al, 2008a(Depuydt et al, , 2009aStes et al, 2011aStes et al, , 2012Stes et al, , 2015Jameson et al, 2019). Additionally, the pathogenic model strain D188 possesses a chromosomal locus vic (virulence in chromosome) implicated in symptom persistence that encodes a malate synthase believed to be involved in the catabolism of leafy gall-specific nutrients (Vereecke et al, 2002).…”

Virulent Rhodococcus fascians Produce Unique Methylated Cytokinins

“…We recently showed that both mt1 and mt2, and fasD, are expressed in peas inoculated with a virulent strain but not in tissues inoculated with an avirulent strain or in controls. 1-MeiP and 2-MeiP were detected in pea tissues inoculated with the virulent strain [15].…”

“…However, these data derive from a comparison between plants inoculated by a virulent strain and a mock-inoculated control, and lack a comparative analysis with avirulent-inoculated plants. We have shown that both virulent and avirulent strains of R. fascians can produce the cytokinins implicated in the 'Trick-with-the-Cytokinin-Mix' hypothesis in planta [13][14][15]. Moreover, both a virulent and an avirulent strain caused increased expression of amino acid (AAP) and sugar (SWEET and SUT) transporters and cell wall invertases (CWINV) [13,14].…”

“…Both avirulent (epiphytic) as well as virulent strains of R. fascians have been shown to extrude multiple different cytokinins into culture, most of which can, however, be derived from tRNA breakdown e.g., [9][10][11]. Likewise, multiple cytokinins can be extracted from plants inoculated with both virulent and avirulent strains e.g., [12][13][14][15]. Critically, the levels of individual cytokinins extracted from tissues inoculated with virulent strains have never been sufficiently elevated relative to mock-, or avirulent-inoculated plants to be convincing as the cause of the shooty galls.…”