15n ammonium chloride

At 5 wk, 150 µl of ³³P‐orthophosphate (1.5 MBq; [³³P]‐phosphoric acid; Hartmann Analytic, Braunschweig, Germany) and ¹⁵N ammonium chloride (1.5 mg ml⁻¹; Sigma‐Aldrich) were supplied in an aqueous solution to one of the two mesh‐walled cores in each pot via the capillary tube. In half of the pots, the labelled cores were rotated every other day to sever the hyphal connection between core and plant roots (n = 6, ‘rotated’ cores). The nonlabelled cores in these pots were kept static to preserve the hyphal link. In the remaining half of the pots, the labelled cores were kept static, to preserve hyphal connections (n = 6, ‘static’ cores), and the nonlabelled cores were rotated.

¹³C₆‐D‐glucose and ¹⁵N‐ammonium chloride were purchased from Sigma‐Aldrich, Germany and Euriso‐Top, France, respectively. For over‐expression of recombinant proteins in E. coli, the following vector systems were used: pET11d (Novagen®) was used for all eIF4GII constructs, active sLb^pro and sLb^proC51A, pET3d (Novagen®, formally known as pET8c) for HRV2 2A^pro, HRV2 2A^proC106S, and CVB4 2A^proC110A VP1₈ and _pproExHTA (Invitrogen®) for eIF4E.

To produce uniformly ¹³C, ¹⁵N-labelled pili in vivo, cells were grown in M9 minimal media supplemented with ¹³C–glucose (4 g/L) (Sigma) and ¹⁵N–ammonium chloride (0.5 g/L) (Sigma). The 6 × 1.5 L culture was grown at 37 °C to an optical density (OD₆₀₀) of 0.8, induced with 0.1% arabinose and further incubated overnight. All other aspects of pilus expression and subsequent purification were performed as described above for the production of short pili from the Fim-Ara plasmid. For the in vitro production of uniformly ¹³C, ¹⁵N-labelled pili, FimA was produced as described^{46 (link)}, but with isotope-supplemented M9 media (as above). The purification of FimA and its subsequent assembly into pilus rods was carried out as described in ref. ^{46 (link)}. For the production of ¹³C, ¹⁵N-labelled FimD-cat pili, FimA was produced as described above using isotope-supplemented M9 media. Labelled FimA was then refolded by rapid dilution in the presence of FimC to form FimA–FimC complexes and FimD-catalysed assembly was performed as described earlier for the preparation of unlabelled FimD-cat pili. All labelled pili were pelleted by ultracentrifugation at 200,000 × g, for 2 h at 4 °C with an MLA-80 rotor (Beckman Coulter) followed by washing with 50 mM Tris-HCl [pH 8.0], 1 mM EDTA. These steps were repeated four times before finally resuspending in 500 µL of the same buffer.

Gelatin from porcine skin (300 bloom) was purchased from Electron Microscopy Sciences (Hatfield, PA, United States). 4-hydroxy-3-cinnamaldehyde (coniferyl aldehyde; CA), deuterated alanine, ammonium chloride, and 98 atom percent ¹⁵N ammonium chloride were purchased from Sigma Aldrich (St. Louis, MO, United States). Potassium acetate was purchased from Fisher Scientific (Hampton, NH). The gold sputter target was purchased from Ted Pella, Inc. (Redding, CA, United States). B73, Mo17, MxB, and BxM maize seeds were obtained courtesy of Dr. Marna Yandeau-Nelson at Iowa State University.