DOI: 10.5061/dryad.tdz08kq5d
title: Phage selection drives resistance-virulence trade-offs in Ralstonia solanacearum plant pathogenic bacterium irrespective of the growth temperature

description: 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 experimental growth conditions
 selected for small colony variants (SCVs) with increased growth rate and
 mutations in the quorum-sensing (QS) signalling receptor gene, phcS.
 Interestingly, SCVs were also phage-resistant and reached higher
 frequencies in the presence of phages in both temperature environments.
 Evolving phage resistance was costly in terms of reduced carrying
 capacity, biofilm formation and reduced 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 genes encoding type II secretion system (T2SS) genes
 (gspE and gspF), indicating that defects in pseudopilus made bacterium
 resistant to the phage. Additional transcriptomic analysis revealed
 upregulation of CBASS and type Ⅰ restriction-modification phage defence
 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
 phage resistance-virulence trade-offs are not affected by the growth
 temperature but can be mediated through both pre- and post-infection phage
 resistance mechanisms.
description: # Phage selection drives resistance-virulence trade-offs in Ralstonia
 solanacearum plant pathogenic bacterium irrespective of the growth
 temperature
 [https://doi.org/10.5061/dryad.tdz08kq5d](https://doi.org/10.5061/dryad.tdz08kq5d) This is an experiment focused on the coevolution of Ralstonia solanacearum and phages. We gathered information concerning the growth of R. solanacearum and the presence of phages at various stages of the experiment. Subsequent to isolating the evolved strains, we conducted assessments of their fundamental characteristics, encompassing growth parameters, motility, biofilm formation capability, and pathogenicity. Furthermore, we performed genomic analysis on these evolved strains, identified pivotal genes through SNP analysis, and corroborated the functionality of these genes via experimental assays. ## Description of the data and file structure Datasets included: 1. transfer OD • every two days’ OD600 value of each replicate in R. solanacearum -phage selection experiment * in the row of “phage”, “1” represents in the presence of phage, and “0” represents in the absence of phage 2. phage density * every four days’ phage density of each replicate of treatments with the presence of phage in R. solanacearum -phage selection experiment 3. scv frequency * the densities and frequencies of SCV and mucoid ones in each replicate of the end of R. solanacearum -phage selection experiment. 4 out of 10 replicates of selection experiment were randomly chosen here. * in the row of “phage”, “1” represents in the presence of phage, and “0” represents in the absence of phage 4. resistance to phage * the resistance to ancestral phage of all isolates (N=128) from all treatments, and together with the phage resistance of ancestral RS-N * the resistance to ancestral phage of each tested clone were replicated three times, and only average resistance to phage were shown here * the “treatment” row refers to the evolution trajectory of isolates, in detail, “35\_1” represents evolved with phage at 35°C, “35\_0” represents evolved without phage at 35°C, “25\_1” represents evolved with phage at 25°C, and “25\_0” represents evolved without phage at 25°C 5. maximum growth rate and 6. carrying capacity have the same dataset structure with dataset 4) 7. twitching, 8. biofilm and 9. disease incidence were the twitching ability, biofilm formation ability and ability of causing tomato wilt disease of a subset of all isolates (N=28) 10. snp\_indel * variant calling of a subset isolates from all treatments comparing with RS-N using Snippy * Missing values are denoted by “n/a” 11. gene blast in uniprot * blast variant gene sequence in UniProt (https://www.uniprot.org) to ensure the gene name and related protein 12. transcriptome * changes in RS-N gene expression in response to phage infection 13. anc RS growth at 30°C * RS-N density dynamics in the presence of phage and absence of phage at 30°C for 24 hours 14. adsorption assay * adsorption rate of NNP42 phage to RS-N (anc) and phcS, gspE, gspF and pilD mutants isolated at the end of the selection experiment 15. one-step growth cuve * One-step growth curve of NNP42 phage infecting RS-N. 16. kegg enrichment * KEGG enrichment analysis for differentially expressed genes in the presence of phage ## Sharing/Access information ## Code/Software The R code is employed for various tasks, including generating line plots, boxplots, and more. Additionally, it encompasses code for computing bacterial growth parameters like maximum growth rate and maximum density. Furthermore, it incorporates scripts for performing SNP analysis and creating circular genome plots.