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4'-Phosphopantetheinyl transferases (PPTases) play important roles in the posttranslational modifications of bacterial carrier proteins, which are involved in various metabolic pathways. Here, we found that RsacpS and RspcpS encoded a functional AcpS-type and Sfp-type PPTase, respectively, in Ralstonia solanacearum GMI1000, and both are capable of modifying R. solanacearum AcpP1, AcpP2, AcpP3, and AcpP5 proteins. RspcpS is located on the megaplasmid, which does not affect strain growth and fatty acid synthesis but significantly contributes to the virulence of R. solanacearum and preferentially participates in secondary metabolism. We found that deletion of RspcpS did not affect the abilities of cellulose degradation, biofilm formation, and resistance to NaCl, sodium dodecyl sulfate, and H 2 O 2 and attenuated R. solanacearum pathogenicity only in the assay of soil-drenching infection but not stem injection of tomato. It is hypothesized that RsPcpS plays a role in cell viability in complex environments and in the process during which the strain recognizes and approaches plants. These results suggest that both RsAcpS and RsPcpS may be potential targets for controlling diseases caused by R. solanacearum ., Competing Interests: The author(s) declare no conflict of interest.