Natural variation in the hrpL promoter renders the phytopathogen Pseudomonas syringae pv. actinidiae nonpathogenic

The genetic basis underlying loss-of-virulence mutations that arise among natural phytopathogen populations is not well documented. In this study, we examined the virulence of 377 isolates of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) that were isolated from 76 kiwifruit orchards suffering from bacterial canker disease. Eighty-four nonpathogenic isolates were identified in 40 orchards. A nonpathogenic isolate G166 was found to be defective in hrpL transcription and the downstream type III secretion system (T3SS)-dependent phenotypes. Comparative genomics and complementary expression assay revealed that a single-base "G" insertion in the hrpL promoter blocks gene transcription by reducing promoter activity. The electrophoretic mobility shift assay showed that the genetic variation impairs σ⁵⁴ /promoter binding during gene transcription under hrp-inducing conditions, resulting in lower expression of hrpL. A PCR-restriction fragment length polymorphism assay was performed to trace the evolutionary history of this mutation, which revealed the independent onset of genetic variations in natural Psa3 populations. We also found that nonpathogenic variants outperformed virulent Psa3 bacteria for both epiphytic and apoplast colonization of kiwifruit leaves in mixed inoculations. Our study highlights a novel mechanism for loss of virulence in Psa3 and provides insight into bacterial adaptive evolution under natural settings.