Salicylate

The β-Galactosidase assay was carried out in triplicate; overnight cultures were diluted with N medium and grown for 4 h, and the activity was determined as described previously (55 ). Mg²⁺ was added to 0.01 mM (namely, low Mg²⁺), and 10 mM (high Mg²⁺). Complementation was carried out by using complementing plasmid pemrR-FLAG, and heterologous expression of emrR from this plasmid was induced by adding 0.2 mM IPTG or indicated specifically. When needed, salicylate (Sigma-Aldrich) or dopamine (Alfa Aesar, Thermo Fisher Scientific) was added to the required concentrations. Data correspond to three independent assays conducted in duplicate, and all values were means ± standard deviations.

A laboratory colony of H. zea, generously provided by Dr. May R. Berenbaum (Department of Entomology, University of Illinois at Urbana-Champaign), was maintained in an insectary kept at 28 °C with a photoperiod of 16 h light: 8 h dark on a semi-synthetic control diet containing wheat germ [25 ]. Induction treatments were performed as described by Li et al. [9 (link)]. The analytical grade plant allelochemicals, xanthotoxin, chlorogenic acid, indole-3-carbinol, flavone, rutin, gossypol, 2-tridecanone, quercetin, coumarin and plant signal molecules jasmonate and salicylate, used in this study, were obtained from Sigma (Sigma-Aldrich, St. Louis, MO, USA) (Figure 1). In brief, 30 newly molted 5th instar larvae were allowed to feed on control diets or control diets containing 0.1% plant xenobiotics for 48 h. Three independent biological replicates of the control diet or each plant xenobiotic treated diet were prepared for subsequent RNA extraction. Midguts and fat bodies were then dissected out, flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent RNA extraction.

A strain called IE01 carrying two fluorescent reporters was engineered to measure the activity of the WT mar core network. It is based on E. coli K-12 MG1655. The strain contains a chromosomal copy of the gene coding for a yellow fluorescent protein (YFP) under the control of the marRAB promoter, and the gene coding for a cyan fluorescent protein (CFP) expressed with a constitutive promoter as a control.^{50 (link)} Four more strains called TC01, DB01, IE02, and TC02 were constructed by deleting fundamental genes of the system, with the application of the knockout protocol on the strain IE01.^{51 (link)} TC01 is a ∆marA strain, DB01 a ∆marB strain, IE02 a ∆rob strain, and TC02 a ∆marA∆rob strain.
Medium LB was always used for overnight cultures. Minimal medium M9 (1x M9 salts, 2 mM MgSO₄, 0.1 mM CaCl₂, 0.4% glucose, 0.05% casamino acids, 0.05% vitamin B1) was used to grow cells during characterization experiments. To induce the marRAB promoter, different concentrations of salicylate and cAMP (Sigma Aldrich) were used. Note that glucose inhibits the production of internal cAMP.

Routine chemicals used for protein expression and purifications were purchased from Sigma-Aldrich, Fisher Scientific, VWR, Research Products International, or MP Biomedicals. NMR isotopes were purchased from Sigma-Aldrich [glucose and D₂O for four samples used for nuclear Overhauser effect spectroscopy (NOESY) experiments] or Cambridge Isotope Laboratories (all other samples). Salicylate, adenosine triphosphate (ATP), and dithiothreitol (DTT) were purchased from Sigma-Aldrich, GoldBio, and VWR, respectively. Unless otherwise noted, Escherichia coli strains for expression were purchased from Novagen. E. coli ΔEntD cell lines used in the expression of T1 were courtesy of C. Chalut and C. Guilhot (CNRS, Toulouse, France). Cloning and mutagenesis enzymes and primers were purchased from New England BioLabs and Integrated DNA Technologies, respectively. The adenylation domain YbtE and thioesterase SrfAD were prepared as described in (23 (link)), with vectors provided by the C. T. Walsh laboratory (previously at Harvard Medical School). The precursors to prepare stereospecific ¹H-¹³C-Me samples were a gift from H. Arthanari (Dana-Farber Cancer Institute).

PPs were prepared as previously reported [6 (link)]. Among PPs, the structures of PP1S and PP2S tested in this study are presented in Supplementary Figure S4. Isoniazid, rifampicin, streptomycin, ethambutol, pyrazinamide, ETH, and salicylate were purchased from Sigma-Aldrich (USA).