A high-throughput virulence screening method for the Ralstonia solanacearum species complex

Authors: Schachterle, Jeffrey; Huang, Qi

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/10/2021
Publication Date: 6/15/2021
Citation: Schachterle, J.K., Huang, Q. 2021. A high-throughput virulence screening method for the Ralstonia solanacearum species complex. Journal of Microbiological Methods. https://doi.org/10.1016/j.mimet.2021.106270.
DOI: https://doi.org/10.1016/j.mimet.2021.106270

Interpretive Summary: Ralstonia solanacearum is a plant pathogenic bacterium that causes wilting diseases of many plants, including the economically important brown rot of potato. Because of the serious economic impact of this disease, the strain of this bacterial species that causes the disease is quarantined from entering the United States, Canada, and Europe. ARS scientists developed a novel high-throughput, accurate, and economical method to determine what contributes to the bacterium’s ability to cause disease at cool temperatures. Using this method, they tested thousands of mutants of the bacterium that they had created in the lab, and found a protein produced by the bacterium that is associated with its ability to cause plant infection under cool temperatures. This research may contribute to the development of effective controls to safeguard United States agriculture from this pathogen.

Technical Abstract: Ralstonia solanacearum is the causative agent for wilting diseases of many plants, including the economically important brown rot of potato. Of great concern are strains of R. solanacearum capable of causing disease in potato at cool temperatures, which are select agents in the United States. To gain a better understanding of cool-virulence mechanisms, we first developed a high-throughput virulence screen using seedlings of Nicotiana glutinosa that is suitable for evaluation of disease symptoms caused by R. solanacearum under both cool (20oC) and warm (28oC) temperature conditions. We then generated a library of transposon mutants in the cool-virulent strain R. solanacearum strain UW551 and screened 2,500 mutants using our seedling screen for significantly reduced virulence at 20oC. We obtained three isolates of a single mutant that exhibited reduced virulence at 20oC but full virulence at 28oC, as compared to the wild-type strain. Sequencing of this mutant revealed that the transposon was inserted in an intergenic region between a type III secretion system effector gene ripS1 and a major facilitator superfamily protein gene. Gene expression analysis showed that expression of ripS1 was altered by the transposon insertion, but not the major facilitator superfamily protein gene. The effector, RipS1, has unknown function and is part of a family of effectors belonging to the largest known type III effectors. The functional connection between RipS1 and cool-virulence of R. solanacearum UW551 suggests that RipS1 (and/or its upstream promoter element) may serve as a potential target for development of cool-virulence-specific diagnostic tools to differentiate the highly regulated cool-virulent strains from non-cool-virulent strains of R. solanacearum. Both our screen method and results provide valuable tools for future work in understanding cool-virulence of R. solanacearum and development of proper control strategies to combat this important plant pathogen.