Phytopathogenic Rhodococcus Have Diverse Plasmids With Few Conserved Virulence Functions

Abstract

Rhodococcus is a genus of Gram-positive bacteria with species that can cause growth deformations to a large number of plant species. This ability to cause disease is hypothesized to be dependent on a cluster of three gene loci on an almost 200 kb-sized linear plasmid. To reevaluate the roles of some of the genes in pathogenicity, we constructed and characterized deletion mutants of fasR and four fas genes. Findings confirmed that fasR, which encodes a putative transcriptional regulator, is necessary for pathogenesis. However, three of the fas genes, implicated in the metabolism of plant growth promoting cytokinins, are dispensable for the ability of the pathogen to cause disease. We also used long-read sequencing technology to generate high quality genome sequences for two phytopathogenic strains in which virulence genes are diverged in sequence and/or hypothesized to have recombined into the chromosome. Surprisingly, findings showed that the two strains carry extremely diverse virulence plasmids. Ortholog clustering identified only 12 genes present on all three virulence plasmids. Rhodococcus requires a small number of horizontally acquired traits to be pathogenic and the transmission of the corresponding genes, via recombination and conjugation, has the potential to rapidly diversify plasmids and bacterial populations.

Keywords: Gram-positive; Rhodococcus; cytokinins; evolution; virulence plasmids.

FIGURE 1

Most fas deletion mutants are pathogenic. (A) Wild type and deletion mutants of Rhodococcus strain D188 were inoculated onto seedlings or meristems of N. benthamiana. Red circles circumscribe leafy galls. This is a composite figure in which photographs were taken of plants treated in different experiments. Images of representative plants are shown. In all experiments, positive and negative control treatments were included. (B) At 7 dpi, roots were imaged and measured. Differences were compared relative to the corresponding D188 control group (* is not significant with a p-value threshold ≤0.05). Similar results were reproduced in at least two additional repeats of the experiments.

FIGURE 2

The fasR gene is necessary for virulence of D188 and A21d2. (A) Scaled schematic of the fasR coding sequences of D188 (blue) and A21d2 (red). The first three in-frame ATG codons of fasR_D188 are shown. The location of the single base insertion introduced to cause a frameshift (fs) in fasR_D188 is also indicated. A possible 5′ extension to fasR_A21d2 is depicted by a pink-colored box. An alignment of the first ∼100 amino acids of the FasR variants of D188 and A21d2 is shown below the schematic. The first three methionine residues of FasR of D188 are boxed in red. (B) Wild type and mutant strains of D188 and A21d2 were inoculated onto seedlings or meristems of N. benthamiana. Red circles circumscribe leafy galls. Images of representative plants are shown. This is a composite figure in which photographs were taken of plants treated in different experiments. Images of representative plants are shown. In all experiments, positive and negative control treatments were included. (C) At 7 dpi, roots were imaged and measured. Differences were compared relative to the corresponding D188 or A21d2 control groups (* is not significant with a p-value threshold ≤0.05). Similar results were reproduced in at least two additional repeats of the experiments.

FIGURE 3

Pathogenic strains A21d2 and A25f have novel virulence plasmids. (A) Graphs of hybrid assembled genome sequences of A21d2 and A25f. Sizes of the replicons are shown. For the pA25f, a predicted structure is presented. The relative depth of coverage of plasmid contigs is shown in parentheses. (B) Venn diagram of gene homolog similarity. The sequences of all genes on the plasmids of A21d2, A25f, and D188 were translated and clustered on the basis of homology.

FIGURE 4

Phylogeny of virulence genes of plant pathogenic Rhodococcus. Maximum likelihood phylogenetic trees of translated sequences of (A) attB, (B) fasR, and (C) fasD. The trees were midpoint rooted and a subset of clades of Rhodococcus virulence proteins are shown. Tips with identical sequences are indicated. Large clades were collapsed and labeled. Bootstrap values greater than 50 are shown.