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

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.