Spectinomycin

For the fungal transformation, we used an AtMT protocol that was previously described [21] (link) with slight modifications. The hygromycin-resistant transformants were selected on the PDA medium containing 100 µg/ml of hygromycin B (Wako Chemicals, Osaka, Japan), 50 µg/ml of cefotaxim (Wako Chemicals, Osaka, Japan), and 50 µg/ml of spectinomycin (Wako Chemicals, Osaka, Japan). The bialaphos-resistant transformants were selected on an SD medium containing 10 µg/mL of bialaphos (Meiji Seika Kaisha, Ltd., Tokyo, Japan), 100 µg/ml of cefotaxim, and 100 µg/ml of spectinomycin. The sulfonylurea-resistant transformants were selected on an SD medium containing 4 µg/ml of chlorimuron-ethyl (Chem Service West Chester, PA, USA.), 100 µg/ml of cefotaxim, and 100 µg/ml of spectinomycin.

WT plants used in this study were Ler and Col-0. Alleles of rfc3 were described previously (rfc3-2) [34 (link)] or obtained from the Arabidopsis Biological Resource Center (rfc3-3 [Salk_015990] and rfc3-4 [Sail_557_D02]). pWOX5::GFP [79 (link)], pPLT3::CFP [44 (link),80 (link)], QC25::GUS and QC184::GUS [81 (link)], p35S::RecATP-CFP [35 (link)], and prps17-1 [45 (link)] were reported previously. The reporter lines or mutants were introgressed into the Ler background by at least four successive genetic crosses. Plants were cultured under long-day conditions (16 h light, 8 h dark) at 22 °C. The light intensity was 50 or 20 μmol m⁻² s⁻¹ when rock wool or solid medium was used, respectively. Half-strength Murashige and Skoog (MS) medium [82 (link)] supplemented with 3% or 2% (w/v) sucrose and solidified with 0.5% (w/v) gellan gum was used to observe root phenotypes. Spectinomycin (Wako Pure Chemical, Osaka, Japan) was added to MS medium at final concentrations up to 6 mg/L as a plastid ribosome inhibitor. Plants were grown on rock wool covered with powdered peat moss to observe shoot phenotypes or to generate transgenic plants. N. benthamiana was grown on peat containing nutrients (Sakatanotane Co., Yokohama, Japan) and Arabidopsis was grown with 0.5 g/L Hyponex (Hyponex Japan, Osaka, Japan) supplied daily as fertilizer.

S. pneumoniae strains were cultured at 37°C in Todd-Hewitt broth (BD Biosciences, Franklin Lakes, NJ, United States) supplemented with 0.2% yeast extract (THY; BD Biosciences). Spectinomycin (Wako Pure Chemical Industries, Osaka, Japan) was added to the medium to a concentration of 120 μg/mL for mutant selection and maintenance. The S. pneumoniae TIGR4 isogenic bgaA (ΔbgaA) mutant strain was generated as previously described (Mori et al., 2012 (link); Yamaguchi et al., 2019b (link)). Briefly, the upstream region of bgaA, an aad9 cassette, and the downstream region of bgaA were combined by PCR using the primers summarized in Supplementary Table 1. The PCR product was transformed with synthesized CSP2 to construct the mutant strains by double-crossover recombination (Bricker and Camilli, 1999 (link)). The mutation was confirmed by site-specific PCR with isolated genomic DNA from the mutant strains. For growth measurement, overnight cultures of each strain were back-diluted 3:100 into fresh THY and grown at 37°C. Growth was monitored by measuring the OD₆₀₀ values every 30 min. The starting point was set at an OD₆₀₀ value of approximately 0.1. The experiment was repeated three times and the data is provided as Supplementary Data 1.