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10 protocols using Agar

For ER stress recovery assay, 5-day-old seedlings were transferred to half-strength LS liquid buffer containing either 0.5 μg/mL Tm (Sigma-Aldrich, St. Louis, MO, USA) or DMSO-only as mock, and treated for 6 h. After the drug treaTment, seedlings were transferred to 100 × 100 square Petri dishes with grid (Fisher Scientific, Hampton, NH, USA) containing half-strength LS medium (Caisson Labs, Ontario, Canada) supplemented with 1% sucrose (Sigma-Aldrich, St. Louis, MO, USA), 1.2% Agar (Acumedia, Lansing, MI, USA). Each square plate was split into two portions, of which each side accommodated either Tm-treated seedlings or DMSO-treated seedlings, which served as a biological replicate (n = 6 per replicate). Then, the seedlings grew vertically under the normal growth condition. For adaptive ER stress assay, 5-day-old seedlings were transferred to half-strength LS liquid buffer containing either 0.5 μg/mL Tm (Sigma-Aldrich, St. Louis, MO, USA) or DMSO as mock, and treated for 12 and 24 h. For chronic ER stress assay, seeds were plated on half-strength LS medium (Caisson Labs, Ontario, Canada) supplemented with 1% sucrose (Sigma-Aldrich, St. Louis, MO, USA), 1.2% Agar (Acumedia, Lansing, MI, USA), and 15, 25, 50 ng/mL Tm (Sigma-Aldrich, St. Louis, MO, USA) or DMSO only as mock.
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2

Evaluating Alkaline Protease Production in P. aeruginosa

P. aeruginosa strains were grown in LB broth (Neogen, Lansing, MI, USA) at 37 °C for 24 h in the presence (1/2, 1/4, and 1/8 MIC) or absence of bio-AgNPs. Then, 10 µL of supernatant from treated and untreated P. aeruginosa PAO1 and PA14 was added to a milk agar plate (pH 10.0) containing 1.0% w/v milk powder (Acumedia, Lansing, MI, USA), 0.1% w/v peptone (Acumedia, Lansing, MI, USA), 0.5% w/v NaCl (Synth, Diadema, SP, Brazil), and 2.0% w/v agar (Acumedia, Lansing, MI, USA) and was incubated at 37 °C for 24 h. Alkaline protease production was indicated by the formation of a clear halo around colonies. Halo diameters were measured and compared with those of the control [26 (link)].
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P. aeruginosa isolates were seeded on LB agar (Neogen, USA) and incubated at 37°C for 24 h. Then, one colony of each isolate was inoculated, in the presence and absence (control) of ½ MIC bio-AgNPs, on the surface of swimming agar plates containing 1.0% tryptone (Acumedia, USA), 0.5% sodium chloride (Casa Americana, Brazil), and 0.3% agar (Acumedia, USA), previously equilibrated to room temperature. Plates were incubated without inversion for 24 h at 30°C (Inoue et al., 2008 (link)).
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Surface-sterilized seeds were plated on half-strength Linsmaier Skoog (LS) medium (Caisson Labs, Ontario, Canada) supplemented with 1% sucrose (Sigma-Aldrich, St. Louis, MO, USA), and 1.2% Agar (Acumedia, Lansing, MI, USA). Appropriate antibiotics was also added for screening transgenic lines. After stratification in the dark at 4 °C for 2 days, seeds were transferred to a controlled growth chamber with 80 μmol m−2 s−1 under 16 h light:8 h dark with 22 °C.
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Surface-sterilized seeds were directly plated on half-strength Linsmaier and Skoog (LS) medium containing 1.5% w/v sucrose and 1.2% Agar (Acumedia, http://foodsafety.neogen.com/pdf/Acumedia_PI/7558_PI.pdf) at 4°C for 2 days in the dark and then grown at 21°C under a 16-h light/8-h dark cycle for 12 days before photographed. For chronic ER stress assays, 50 ng ml−1 Tm (Sigma-Aldrich T7765, http://www.sigmaaldrich.com/) or an equivalent volume of DMSO (Tm solvent) was added to the medium above.
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A. thaliana ecotype Columbia-0 (Col-0) was used as the wild-type control. The following mutants and transgenic lines, which are in Col-0 background, were used in this study: bzip28–2 (SALK_132285), bzip60–2 (SAIL_283_B03), bzip28–2 bzip60–1 (SALK_132285 SALK_050203), gbf2–1 (SALK_206654), gbf2–2 (SALK_205706), gbf2–3 (SALK_087916), nf-yc2 (SALK_111422), wrky8 (SALK_050194), lbd3 (SAIL_659_D08), anac092 (SALK_090154), anac036 (SAIL_600_D02), hb28 (SALK_096579), erf11 (SALK_085781), nac2 (SALK_037700), rap2.6 (SALK_051006), anac062 (WiscDsLoxHs100_07A), gbf1 (SALK_027691), gbf3 (SALK_056627), GBF2ox (CS2104585) and pGBF2:GBF2-Ypet (CS71581). All T-DNA single and high-order mutants used in this study were confirmed to be homozygous before the analysis. Primers used for genotyping are presented in Table S3. Surface-sterilized seeds were plated on half-strength Linsmaier Skoog (LS) medium (Caisson Labs, Ontario, Canada) supplemented with 1% sucrose (Sigma-Aldrich, St. Louis, MO, USA), and 1.2% Agar (Acumedia, Lansing, MI, USA). Appropriate antibiotics was also added for the screening of transgenic lines. After stratification in the dark at 4 °C for 2 days, plates were transferred to a controlled growth chamber with 80 μmol m−2 s−1 under 16 h light:8 h dark with 22 °C.
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Bio-AgNPs were synthesized by reduction of silver nitrate catalyzed by a cell-free enzyme preparation from fungus Fusarium oxysporum (strain 551). The fungal inoculum was obtained from the Laboratory of Molecular Genetics, University of São Paulo, Piracicaba, São Paulo State, Brazil. F. oxysporum was grown for 7 days at 28 °C on 0.5% w/v yeast extract (Neogen, USA), 2% w/v malt extract (Neogen, USA), 2% w/v agar (Acumedia, Lansing, MI, USA), and distilled water. Then, the fungal biomass (0.1 g/mL) was mixed with sterile distilled water and incubated at 28 °C for 72 h under stirring (150 rpm). Next, the cell-free filtrate was mixed with 0.01 M silver nitrate (AgNO3, Sigma–Aldrich, Steinheim am Albuch, Germany ) and incubated for 15 days at 28 °C in the dark. Finally, Bio-AgNPs were washed with distilled water, centrifuged at 27,000× g and 4 °C for 30 min, and incubated in an ultrasonic bath for 30 min. Washing steps were repeated three times [17 (link)].
These bio-AgNPs have been synthesized, quantified, and characterized by our research group and these results have been previously published [18 (link)]. According to Scandorieiro et al. [18 (link)], the nanoparticle used in this study exhibited a plasmonic absorption band in the visible region of the spectrum (near 420 nm), spherical shape, and an average size and zeta potential of 73.1 ± 0.5 nm and −24.2 ± 2.1 mV, respectively.
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Surface-sterilized seeds were plated on half-strength Linsmaier Skoog (LS) medium (Caisson Labs) supplemented with 1% sucrose (Sigma-Aldrich) and 1.2% agar (Acumedia). An appropriate antibiotic was also added for screening of complementation transgenic lines. After stratification in the dark at 4 °C for 2 days, plates were transferred to a controlled growth chamber with 80 μmol m−2 s−1 under 16 h light:8 h dark at 22 °C.
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Quercetin (≥95 %), tannic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), (+) - 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid - Trolox (≥97 %), type I agarose and anhydrous potassium persulfate (K2S2O8), dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (Aldrich Brasil Ltda., Brazil). l-ascorbic acid (≥99.0 %), ethanol PA (99.5 %), and anhydrous sodium carbonate Na2CO3 (≥99.5) were obtained from Synth (LABSYNTH, Brazil), while ultrapure monohydrate gallic acid and aluminum chloride (III) (≥99.5 %) from Vetec (VETEC-Sigma-Aldrich, Brazil). The Folin-Ciocalteu reagent and bovine serum albumin–BSA were acquired from Merck (Merck, USA). Glacial acetic acid PA and sodium hydroxide were obtained from Dinâmica (Dinâmica, Brazil. Nutrient broth, malt extract, yeast extract, potato extract, and agar were purchased from Acumedia (Acumendia, USA). DMSOd6 and TMSP-d4 (2,2,3,3-d4-3- sodium trimethylsilylpropionate) were purchased from Cambridge Isotope Laboratories, Inc. (USA). The reagents used in antimicrobial susceptibility tests were ciclopirox olamine [(LOPROX®), 10 mg g−1, Sanofi-Aventis, Brazil)], DMSO [(≥99.0), Sigma-Aldrich (Aldrich Brasil Ltda., Brazil], DMEM Mix F12, penicillin G, gentamicin, amphotericin B, glucose, l-glutamine, and fetal from Gibco (Thermo Fisher Scientific, Brazil).
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P. aeruginosa strains were cultured in the presence (1/2, 1/4, and 1/8 MIC) or absence of bio-AgNPs in LB broth (Neogen, Lansing, MI, USA) at 37 °C for 24 h. Then, 10 µL of inoculum was placed at the center of a cetyltrimethylammonium bromide (CTAB) agar plate containing 0.09% w/v monobasic potassium phosphate (Synth, Diadema, SP, Brazil), 0.11% w/v bibasic sodium phosphate (Synth, Diadema, SP, Brazil), 0.25% w/v sodium nitrate (Synth, Diadema, SP, Brazil), 0.01% w/v calcium chloride (Synth, Diadema, SP, Brazil), 0.04% w/v magnesium sulfate (Synth, Diadema, SP, Brazil), 0.2% w/v CTAB (Sigma–Aldrich, Steinheim am Albuch, Germany), 0.005% w/v methylene blue (Synth, Diadema, SP, Brazil), 0.5% w/v glucose (Synth, Diadema, SP, Brazil), and 2.0% w/v agar (Acumedia, Lansing, MI, USA). Plates were incubated at 37 °C for 48 h [25 (link)]. Rhamnolipid production was determined by measuring the halo of blue precipitate surrounding colonies.
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