Transposon sequencing reveals Burkholderia gene fitness in a spaceflight-relevant plant-pathogen interaction

The spaceflight environment imparts unique selective pressures on the plants and microbes of plant growth chambers on the International Space Station (ISS), which generally manifests through genetic signatures associated with a heightened response to stress. Terrestrially, a baseline understanding of the gene fitness response for any plant growth promoting microbe when in a tripartite relationship with host and pathogen is currently unknown and is important to characterize before closed-environment spaceflight implementation. To that end, this study evaluated the behavior of an ISS plant habitat isolate of Burkholderia contaminans as tomato seeds transition to seedlings and assessed gene fitness during challenge with Fusarium oxysporum f. sp. lycopersici (FOL), the causal agent of Fusarium wilt. To probe mechanisms underlying the bacterial-fungal interaction between B. contaminans and FOL in the tomato root zone, a genome-wide transposon mutant library was developed of the B. contaminans isolate. Using a seed film delivery method vetted for spaceflight, a Tn-Seq library of B. contaminans was applied to Solanum lycopersicum cv. Red Robin seeds. Transposon sequencing (Tn-Seq) analysis revealed that the type II secretion system (T2SS) was critical for root zone establishment, whereas a Nudix hydrolase was specifically important for responding to FOL infection, and provided further confirmation that antifungal and siderophore-producing gene clusters were not. Overall design: In each plant growth vessel (magenta jar) two seed films were placed in one corner of each for the following scenarios: a) “NoSeed”-a control where only the B. contaminans Tn-Seq library (108 CFU/mL) is applied, b) “Root” or “Shoot”-a treatment where B. contaminans Tn-Seq library (108 CFU/mL) plus seed is applied and c) “FOLRoot” or “FOLShoot”-a treatment of B. contaminans Tn-Seq library (108 CFU/mL) plus seed for additional FOL challenge. The final 108 CFU/mL count was obtained by adding 10 µL droplets of the bacterial cultures at 1010 CFU/mL to the seed film. The B. contaminans Tn-Seq library was revived by slightly thawing the freezer stock and pipetting 100 mL of Tn-Seq library into 3 mL of TSB containing 50 mg/mL kanamycin and incubated at 35°C with shaking at 200 rpm for 16 hours. Three separate Tn-Seq starter cultures were prepared to serve as timepoint 0 (T0) and to inoculate the seed films as the three biological replicates (TnA, TnB, and TnC) of the experiment. Cells which were not used for the experiment were retained as T0 for Tn-Seq sequencing. Magenta vessels were placed into a controlled-environment chamber set to 50% RH and ambient CO2. At seven days after initiation, vessels were thinned to one plant per vessel and 10 µL of 103 CFU/mL FOL culture was applied to the corner opposite to the established plant in the fungal control and experimental treatment vessels. Plants were grown for 28 days total before they were photographed and harvested. At harvest, shoot and root tissues were separated and placed into 50 mL Falcon tubes with 10 mL of 50 mg/mL kanamycin in TSB media and allowed to grow for 16 hours at 35°C with shaking at 200 rpm to select for the transposed B. contaminans Tn-Seq library. Plant material was removed from the Falcon tube, the bacterial culture was pelleted by centrifugation at 3000 rpm for 5 min, and media decanted. Three cell pellets were obtained for scenarios a, b roots, b shoots, c roots, c shoots, and timepoint 0 for a total of 18 cell pellets for DNA extraction. A total of 19 Tn-Seq libraries were prepared including the input library that made the freezer stock.