Phage selection drives resistance-virulence trade-offs in Ralstonia solanacearum plant-pathogenic bacterium irrespective of the growth temperature

Abstract

While temperature has been shown to affect the survival and growth of bacteria and their phage parasites, it is unclear if trade-offs between phage resistance and other bacterial traits depend on the temperature. Here, we experimentally compared the evolution of phage resistance-virulence trade-offs and underlying molecular mechanisms in phytopathogenic Ralstonia solanacearum bacterium at 25 °C and 35 °C temperature environments. We found that while phages reduced R. solanacearum densities relatively more at 25 °C, no difference in the final level of phage resistance was observed between temperature treatments. Instead, small colony variants (SCVs) with increased growth rate and mutations in the quorum-sensing (QS) signaling receptor gene, phcS, evolved in both temperature treatments. Interestingly, SCVs were also phage-resistant and reached higher frequencies in the presence of phages. Evolving phage resistance was costly, resulting in reduced carrying capacity, biofilm formation, and virulence in planta, possibly due to loss of QS-mediated expression of key virulence genes. We also observed mucoid phage-resistant colonies that showed loss of virulence and reduced twitching motility likely due to parallel mutations in prepilin peptidase gene, pilD. Moreover, phage-resistant SCVs from 35 °C-phage treatment had parallel mutations in type II secretion system (T2SS) genes (gspE and gspF). Adsorption assays confirmed the role of pilD as a phage receptor, while no loss of adsorption was found with phcS or T2SS mutants, indicative of other downstream phage resistance mechanisms. Additional transcriptomic analysis revealed upregulation of CBASS and type I restriction-modification phage defense systems in response to phage exposure, which coincided with reduced expression of motility and virulence-associated genes, including pilD and type II and III secretion systems. Together, these results suggest that while phage resistance-virulence trade-offs are not affected by the growth temperature, they could be mediated through both pre- and postinfection phage resistance mechanisms.

Keywords: experimental evolution; phage defense systems; phage resistance; small colony variant (SCV); trade-offs; virulence.

Figure 1.

Schematic representation of the experimental design (A), where Ralstonia solanacearum RS-N was evolved in the absence and presence of phage NNP42 at 25 or 35 °C for 12 days with 2-day serial transfers. Bacterial and phage densities were recorded at every transfer (2 days) and final time point samples were used to quantify changes in R. solanacearum phage resistance, growth and virulence, and to identify parallel mutations between treatment replicates. Population density dynamics of R. solanacearum (B) and NNP42 phage (C) during the selection experiment. N = 10 for (B) and (C) and bars show the standard error of the mean (± 1 SEM). In (D), stacked histograms show the frequencies of mucoid (pink, “M”) and small colony variants (purple, “SCV”) in each sampled treatment replicate at the Day 12 (N = 4). In (B) and (D), the “+P” and “–P” denote for phage presence and absence, respectively. (E) Example morphologies for SCV (top) and mucoid (bottom) colony morphologies on TTC agar plates.

Figure 2.

Evolution of phage resistance and correlated changes with growth and virulence in R. solanacearum at the end of the selection experiment (day 12). (A–F) show evolved clones’ traits relative to ancestral strain (dashed lines) regarding phage resistance (A), maximum growth rate (B), maximum population density (C), twitching motility (D), biofilm formation (E), and virulence in planta (F), respectively. In (A–F), small letters above boxplots show significances between treatments (P < .05), blue and red panel headings denote for prior temperature treatments and M (pink color) and SCV (purple color) denote for mucoid and non-mucoid colonies, respectively. No mucoid colonies were found at 35 °C temperature treatment in the presence of phage. N = 4 for all treatments and boxplots show the interquartile range (25%–75% of the data), the median as lines and replicate means as dots (based on eight clones isolated per replicate for A–C and four mucoid clones and four SCVs isolated per treatment for D–F).

Figure. 3

Mutations identified across different loci of clones evolved at 25 °C (blue circles) and 35 °C (red circles) in the absence (A and C) and presence of phages (B and D). (A) and (B) show mucoid and (C) and (D) SCV colony types, respectively. In all panels, the size and color of smaller dots on the circles (whole genome composed of chromosome and megaplasmid) denote for the number of parallel mutations in given loci, and black and gray text color indicate whether mutations were in the chromosome or megaplasmid, respectively. IGR denotes for mutations observed on intergenic regions (small white dots). Mutations that appeared in all clones and ancestral strain are not shown in the figure.

Figure 4.

R. solanacearum transcriptomic analysis in the absence and presence of phage. (A) Differential expression of genes in the presence of phage with a false discovery rate < 0.05 and |log₂FC| > 1. (B) Differential expression of genes in the presence of phage that were found to be mutated in phage treatments at the end of the selection experiment (significantly expressed genes are shown with stars with a false discovery rate < 0.05 and |log₂FC| > 1). (C) Differential expression of genes linked to phage defense systems, motility, and virulence in the presence of phage (only genes with padj < .05 and |log₂FC| > 2 are shown). In all panels, red and yellow colors denote upregulated and downregulated genes, respectively, while gray color in (A) denotes for nonsignificance.