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Abstract
Burkholderia onion pathogens are a routine threat to onion production in the United States causing diseases such as slippery skin and sour skin. Onion in response to pathogen attack produces toxic organosulfur defense compounds called thiosulfinates which induce thiol stress and inactivates essential bacterial enzymes. The thiosulfinate tolerance gene (TTG) cluster has been shown to confer resistance to these compounds in Pantoea ananatis, another bacterial onion pathogen. However, its presence and function in Burkholderia onion pathogens remain unexplored. Using comparative genomics and allicin zone of inhibition assays, we identified the widespread distribution of TTG cluster in onion and non-onion isolated Burkholderia onion pathogens and demonstrated their role in allicin tolerance in vitro. Phenotypic assays revealed that in Burkholderia gladioli pv. alliicola (Bga), the TTG cluster performs variable virulence role depending on the tissue type. Given the limited understanding of the genetic basis of virulence in Bga, we further characterized the putative genetic determinants of virulence in strain 20GA0385. Candidate virulence factors/regulators were identified using in silico analysis and deletion mutants were generated using allelic exchange. Infection assays demonstrated that, like TTG cluster, the phytotoxin toxoflavin and the type II secretion system perform onion tissue specific virulence role in Bga. We also observed decoupling of Bga symptoms production and corresponding in planta population particularly in the scale tissue. The tissue specific virulence role of the TTG cluster is surprising, given the higher thiosulfinate content in onion bulbs compared to leaves. Using an altR promoter reporter construct, we found that its de-repression is transient during early infection but disappears with the onset of Bga induced scale necrosis. However, in the presence of a cell death-inducing factor, de-repression persists, and TTG mutant recovery from scale tissue is significantly reduced compared to the WT strain. The ability of Bga WT and P. ananatis WT strains to partially rescue the TTG mutant phenotype in the ZOI assay suggests that Bga may secrete factors that inactivate or suppress thiosulfinates. Future studies should focus on identifying the primary necrosis-inducing factor and potential Bga secreted factors conferring thiosulfinate resistance.