All strains used in this study are listed in Table 1, and primers used for the strain construction are in Table 2. Unless otherwise specified, strains were routinely grown in LB medium (Miller, 1972). Minimal A medium was prepared as in Ludwig-Müller (2015). A kanamycin marked ipdC mutant was constructed using Datsenko and Wanner mutagenesis (Datsenko and Wanner, 2000) with primers CEC207 and CEC209. The deletion was confirmed with primers BA505 and CEC135. An unmarked mutant was constructed by removing the FRT-kanR-FRT cassette with plasmid pCP20 as in Datsenko and Wanner (2000). The deletion was confirmed with primers CEC134 and CEC135. RIVET reporters were constructed as in Merighi et al. (2005) by placing tnpR immediately downstream of the ipdC stop codon or by placing tnpR immediately downstream of the promoter region in a stream where ipdC has been removed between the start and stop codons. Constructs were confirmed with primers CEC134 and BA184 or CEC210 and BA184. Activation of the RIVET reporter is scored as the loss of the tetracycline resistance marker in the recovered cells (this occurs because activation of the promoter of interest leads to the expression of the TnpR recombinase and the resulting excision of the tetracycline-resistance marker encoded within “res” sites that are substrates for TnpR; Merighi et al., 2005). A plasmid carrying constitutively expressed gfp reporter driven by the Salmonella dppA promoter (Noel et al., 2010) was introduced into strains as needed via electroporation. To ascertain that the genetic manipulations did not result in a growth deficiency, strains were grown in LB broth shake cultures at 22°C and 37°C for 10–24 h and OD600 measurements were taken periodically, at 30 min or 2 h intervals.

Salmonella enterica sv Typhimurium (S. Typhimurium thereafter) cultures were grown overnight from glycerol stocks at 37°C. 1 mL was washed 3x in PBS and was diluted 1,000x in 5 mL Minimal A medium with or without 1 mM tryptophan. Cultures were incubated at 22°C for 72 h. Supernatant was harvested from 1 mL of culture by centrifugation for 15 min at 7,000 × g. The supernatant was transferred to a 12 mm × 75 mm glass tube and 2 mL of fresh Salkowski reagent R1 (Glickmann and Dessaux, 1995) was added and vortexed to mix. The tubes were incubated for 30 min at 30°C in the dark, aliquots were then withdrawn and A540 was measured using the Shimadzu biospec-mini.

Hundred millliter of Salmonella culture filtrates were extracted twice with 150 and 100 mL of ethyl acetate. The extracts were rotary evaporated over a water bath at 40°C. The resulting yellow oily residue was brought up in 600 μl of methanol. The extracts and standards (IAA, tryptophol and IPyA) were subject to reverse phase (C₁₈) liquid chromatography using Waters Atlantis dC18-5 μM column using isocratic elution (72 water 7.2% acetic acid 20.8% methanol), as before (Brandl and Lindow, 1996; Brandl et al., 1996). Eluting substances were detected with a UV/VIS detector set to 230 and 280 nm (Brandl and Lindow, 1996; Brandl et al., 1996). Low Resolution Electrospray Ionization LCMS was carried in a Thermo Scientific LTQ LC-MS instrument using a Grace Vydac 218TP C18 (5 μ, 7.5 cm, 2.1 mm ID) column. LCMS was run using a gradient of 10% acetonitrile (with 0.1% formic acid) and 90% water (with 0.1% formic acid) to 100% acetonitrile (with 0.1% formic acid) from 0 to 15 min and then 100% acetonitrile with formic acid was continued till 21 min.

Seeds of M. truncatula A17 (wild type) and transgenic line containing the GH3::GUS reporter (van Noorden et al., 2007) were surface sterilized in ethanol and then in a diluted chlorox bleach solution as before (Mathesius et al., 2003) and stratified overnight at 4°C. Imbibed seeds were transferred into individual growth pouches and were left to germinate at room temperature overnight. Once radicles reached ~1 cm, six seedlings (per treatment) were inoculated with 100 μl of the suspension containing ~5,000–10,000 CFU of either CEC1000 (S. Typhimurium 14028 marked with a kanamycin cassette in a neutral site) or CEC1002. Plants were watered as needed with the N-free Hoagland solution. To detect activation of the GH3::GUS reporter, seedlings were withdrawn after 48 hrs and 6 days past infection, and stained with X-gluc (10 mg/ml) in 0.2 mM Phosphate buffer (pH 7.2) overnight. They were then washed 3 times with cold phosphate buffer to remove excess substrate and the seedlings were kept in 50% ethanol/phosphate buffer at 4C. They were imaged under a dissecting microscope Olympus MVX10 fit with a MicroFIRE camera (Optronics).

For the reporter assays, washed RIVET reporter suspensions (containing ~10,000–100,000 CFU) were inoculated onto M. truncatula seedlings as described in Supplemental Information. At indicated time intervals, seedlings were excised from pouches, blended with a glass mortar and pestle and dilution plated onto XLD with kanamycin. Bacteria were patched from these onto LB with tetracycline.

To detect changes in lateral root formation, seeds of M. truncatula A17 were surface sterilized, imbibed and then transferred into growth pouches. Seedlings were inoculated with 100 μl of the washed bacterial suspension containing ~5,000–10,000 CFU of either CEC1000 (S. Typhimurium 14028 marked with a kanamycin cassette in a neutral site) or CEC1002 (ipdC::kan).

BALB/c mice were inoculated by oral gavage, which results in a systemic infection. Prior to the infections, S. enterica sv. Typhimurium 14028 and CEC1002 were streaked on LB agar and incubated at 37°C overnight, colonies were harvested and re-suspended in 1 ml of sterile PBS. Each suspension was adjusted to a final dilution of ~10⁸ CFUs/ml. Groups (n = 3) of female BALB/c mice received three doses of Salmonella in 0.2 ml of PBS. Inoculum doses were confirmed by serial titration, and were found to be 10⁶, 10⁴, and 10² CFU per injection dose for the wild type, and 2.2 × 10⁶, 2.2 × 10⁴, and 2.2 × 10² CFU per injection for the ipdC mutant. Animals were observed daily for 7 days after infection and distressed animals presenting signs of morbidity were euthanized, their organs were not sampled. The experiment was terminated at 7 days, and all surviving animals were sacrificed. Liver, spleen, large intestine, and Peyer's patches were harvested. Tissues were weighed, and homogenized in sterile PBS in TissueLyser II (Qiagen). Bacterial homogenates were serially diluted in PBS and plated onto Xylose-Lysine Deoxycholate (XLD) agar (Beckton, Dickinson and Company) plates, followed by incubation at 37°C overnight for bacterial CFU counts. All animal care and procedures were in accordance with institutional policies for animal health and well-being and approved by the University of Florida Institutional Animal Care and Use Committee (IACUC).

Synteny analysis was carried out using default parameters in the SyntTax web server (Oberto, 2013). Sequences were imported from GenBank. UV peak integration was conducted with proprietary Waters software, and analysis of mass spectral data was carried out using Thermo Scientific proprietary software. Means and standard error of the mean were calculated in Microsoft Excel v. 14.0.0.

The ability of Salmonella to produce IAA was first tested in culture in Minimal A medium using a colorimetric assay based on the Salkowski reagent. While there was no auxin production by either the wild type or the isogenic ipdC mutant grown in unammended Minimal A medium, production of IAA was detected in the medium supplemented with 1 mM L-tryptophan, the precursor for the IPyA pathway (Figure 2A). The intensity of absorbance at 540 nm after addition of the Salkowski reagent, which correlates with the presence of indole compounds, including IAA, was significantly lower in the culture filtrate extract of the ipdC mutant than that of the wild type strain (Figure 2A). When culture filtrate extracts were separated using HPLC, UV peaks corresponding to IAA and tryptophol were observed for the wild type, but were strongly reduced for the ipdC mutant (Figure 2B). Tryptophol is a product of a reduction of indole-3-acetaldehyde, the product of the reaction catalyzed by ipdC. The production of IAA by the wild type S. enterica sv. Typhimurium 14028 was confirmed by LC-MS/MS. Production of IAA was significantly reduced (but not completely eliminated) in the isogenic strain lacking ipdC (Figure 2C). In laboratory cultures, expression of the ipdC RIVET (recombinase-based in vivo expression technology) reporter was low (less than 10%) after 24 h of incubation, however, it increased by 72 h (Figure 2D). Consistent with the production of IAA in laboratory media, a recombinase in vivo expression technology (RIVET) reporter in ipdC was expressed in a laboratory medium at 45–55% (Figure 2D). Expression of the reporter in the ipdC background was considerably lower than in the wild type, suggesting a feedback regulation. However, supplementation of the medium with 50 μM IAA did not significantly affect expression of the ipdC RIVET reporter in the wild type or in the ipdC mutant. Despite clear differences in the amount of IAA produced in a minimal medium with and without tryptophan, the ipdC RIVET reporter was expressed at similar levels in the minimal medium with and without tryptophan (Figure 2D), suggesting that the availability of the substrate is critical for the function of the IpdC enzyme (as indirectly evidenced by the accumulation of IAA in culture) but not for transcription of the encoding gene.

To determine whether Salmonella produces auxin on plant surfaces and whether this bacterially-produced plant hormone has a function during colonization of plant hosts, ipdC expression was assessed in the M. truncatula rhizosphere. The ipdC RIVET reporter was resolved at ~5–8% during the first 3 days of root colonization, reaching 22 and 37% at seven and 10 days post-inoculation, respectively (Figure 3A). When compared with the expression of the same reporter in Minimal A medium (Figure 2D), it does not appear that ipdC is expressed stronger in the rhizosphere, nevertheless, it is clear that ipdC is expressed in S. Typhimurium on roots during colonization. Next, we tested the consequences of the ipdC mutation using a GH3::GUS reporter in M. truncatula. Genes belonging to the GH3 (Gretchen Hagen 3) family are among the regulators that control the dynamic process of endogenous auxin homeostasis (Yang et al., 2015). This GH3::GUS reporter is normally expressed in the stele of the root, while expression in the cortical cells results from exposure to exogenous IAA, including that produced by bacteria such as by the symbiotic Sinorhizobium meliloti (Mathesius et al., 1998, 2000; van Noorden et al., 2007). As shown in Figure 3B, the GH3::GUS reporter was broadly induced in the cortical tissue of M. trucatula roots after inoculation with the S. Typhimurium wild type strain, but only at discrete locations in the cortex after that with the ipdC mutant. Taken together, the activation of the plant GH3::GUS reporter by Salmonella and the expression of the Salmonella ipdC gene in the rhizosphere suggest that this human pathogen synthesizes auxin via IpdC during plant colonization and in quantities that are detectable by the plant cells and sufficient to cause changes in plant gene expression.

To determine how Salmonella may benefit from the production of IAA, we first tested whether perception of the bacterially-produced IAA led to observable phenotypic changes in the inoculated plants. Secondary root initiation is a phenotype known to be regulated by IAA (Spaepen et al., 2014; Bensmihen, 2015). Therefore, we evaluated formation of secondary roots in the seedlings inoculated with the wild type Salmonella or the isogenic ipdC mutant. As shown in Figure 3C, few secondary roots had emerged in 2-week old M. truncatula seedlings that were not inoculated with Salmonella and treated with the nutrient solution only (control). Seedlings inoculated with the S. Typhimurium wild type strain showed 5–12 secondary roots, while seedlings inoculated with the ipdC mutant had 2–7 secondary roots per plant. When the wild type and the ipdC mutant were tagged with a plasmid constitutively expressing GFP, the ipdC mutant was found to be evenly distributed throughout the root surface and on root hairs, while the wild type tended to form large aggregates at the sites of the lateral root emergence (Figure 4). However, we did not observe differences in the total number of bacteria recovered from the seedlings (data not shown). In laboratory LB shake cultures, the wild type and the ipdC mutant grew with the same kinetics and to the same final optical densities at 22 and 37°C.

We hypothesized that because IAA is a plant hormone, the function of ipdC may be confined to the interaction of Salmonella with its plant hosts. To test this hypothesis, the comparative ability of the S. Typhimurium wild type and ipdC mutant to colonize mice following an oral gavage was determined. The hypothesis that the mutation in idpC impacts virulence in mice was proven null (Figure 5). While the ipdC mutant did not differ from the wild type in its ability to establish within the intestine, it was deficient in the colonization of the liver and the spleen (Figure 5). However, due to the small number of animals infected per dose, robust statistical analyses of these data were not possible.

The reviewer AHCVB declared a shared affiliation, with no collaboration, with several of the authors (CC, MdM, and MT) to the handling Editor. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.