Analysis of Genome Sequences from Plant Pathogenic Rhodococcus Reveals Genetic Novelties in Virulence Loci

Abstract

Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus.

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

“…Determination of the fresh weight of the plants revealed that D188 decreased the biomass of the plants to 10% of that of the non-infected controls, whereas plants infected with either PBTS1 or PBTS2 reached a fresh weight that was approximately 20% of that of the uninfected controls (Figure 2E). In line with these findings, when seedlings of N. benthamiana, which has also been used as a host for leafy gall inducers (Creason et al, 2014), were planted in substrate co-inoculated with PBTS1 and PBTS2, their growth was severely stunted, flowering was delayed, and the number of flowers was reduced (Figure 2F).…”

“…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).…”

“…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). Despite the absolute requirement of the linear plasmid for virulence in most leafy gall inducers, an important contribution of co-option of chromosomal and plasmid-encoded functions seems to guarantee a successful interaction with plants (Creason et al, 2014;Francis et al, 2016). As a result of metagenomic studies in search of biostimulants, it has now become evident that diverse Rhodococcus species are established members of the plant microbiome (Francis and Vereecke, 2019).…”

“…In agreement with the similarities between the symptoms associated with the leafy gall and PBTS syndromes, in both PBTS1 and PBTS2, the presence was demonstrated by PCR of the fasD gene, encoding an isopentenyl transferase that mediates the rate-limiting step of cytokinin biosynthesis (Crespi et al, 1992), the fasA gene coding for a P450 monooxygenase involved in zeatin production (Pertry et al, 2010; Genbank accession MF278335 for PBTS2 fasA), and vic (Stamler et al, 2015b). Although the presence of fasD is 100% correlated with virulence in leafy gall inducers (e.g., Crespi et al, 1992;Stange et al, 1996;Nikolaeva et al, 2009;Creason et al, 2014), its role in the PBTS strains remains to be determined. Moreover, the synergistic activity of both species in symptom development and the absence of genuine leafy galls in the PBTS syndrome suggest that the strategies used by these bacteria differ from those of leafy gall inducers.…”

Infection byRhodococcus fasciansmaintains cotyledons as a sink tissue for the pathogen

“…Subsequent investigations will focus on expression of RfMT1 and RfMT2 and levels of methylated cytokinins [newly identified from Rhodococcus-infected plant tissues by Radhika et al (2015)], as well as the methylthio-and cis-forms of the cytokinins. We will then be able to compare, in pea, the impact of the methylated cytokinins (Radhika et al, 2015), with the 'trickwith-the-mix' hypothesis developed based on experiments with R. fascians and arabidopsis (Pertry et al, 2009(Pertry et al, , 2010, with the suggestion by Creason et al (2014) that only one cytokinin type (the iP-type) is necessary for disease symptoms to be manifested.…”