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 degrees C and 35 degrees C temperature environments. We found that while phages reduced R. solanacearum densities relatively more at 25 degrees 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 degrees 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. Ralstonia solanacearum is a notorious plant-pathogenic bacterium, capable of infecting several important crop plants. In the absence of effective chemical control strategies, phage therapy has been proposed as an alternative strategy for biocontrol of R. solanacearum. Here we tested whether phage efficacy is limited by the growth temperature, which could potentially affect the rate of phage resistance. We find that phage resistance evolves regardless of the temperature, leading to trade-offs with pathogen virulence. Sequencing and validation experiments with mutants revealed that phage uses type IV pilus as a receptor, while defects in type II secretion system (T2SS) and quorum-sensing signaling genes provided phage resistance irrespective of phage adsorption. Activation of restriction-modification and abortive infection (CBASS) phage defense systems upon short-term phage exposure was also observed, which was coupled with downregulation of several virulence-associated genes. Together, these results suggest that phages could be used to select less virulent R. solanacearum strains, expanding their biocontrol potential beyond pathogen density control.