Mediation of pathogen resistance by exudation of antimicrobials from roots- Bais et al.

Supplementary Online Material:

Supplementary Methods:

Plant material and growth conditions of Arabidopsis thaliana in vitro and in sterilized planting mix

Wild-type Arabidopsis thaliana ecotype Columbia (Col-0) seeds (Lehle Seeds, Round Rock, TX) were surface sterilized using sodium hypochlorite (0.3% v/v), and germinated on solidified Murashige and Skoog basal medium (MS)1 in a growth chamber at 25 oC, with a photoperiod of 16 hours light and eight hours dark. For liquid (in vitro) root infection assays, 25-day-old seedlings were transferred to 6 ml 12-well culture plates (Fisher Co) each holding 2 ml of liquid MS basal media containing 3% sucrose, incubated on an orbital platform shaker at 90 rpm, and illuminated under cool white fluorescent light (45 mmol m-2 s-1) with a photoperiod of 16 hours light / eight hours dark at 25 ± 2 oC for two to three days prior to infection. For the planting mix inoculation assay, Arabidopsis plants were germinated on sterilized planting mix (Jiffy Co. Riverside, CA). Prior to bacterial inoculation, 25-day-old plants growing in sterilized planting mix were transferred to Magenta boxes (Magenta Corp. Chicago, IL) and were incubated in a growth chamber at 30 oC with 12 hours of light and watered daily to provide ~100% humidity.

Bacterial strains and culture conditions

P. syringae pv. tomato DC3000 (Pst DC3000)2, P. syringae pv. phaseolicola NPS3121 (Psp NPS3121)3, P. syringae pv. glycinea 29-2 race 4 (Psg A29-2)2, P. syringae pv. phaseolicola race 6 (Psp rc6)4, P. syringae pv. syringae B728a (Pss B728a)5, and P. syringae pv. maculicola strains ES4326 (Psm ES4326)6, M1 (Psm M1)6, and M4 (Psm M4)7 have been previously described. Pst DC3000 hrpL- and Pst DC3000 hrpH- (hrcC-) (Rifr Cmr)8 mutants were obtained from Dr. S.-Y. He, Michigan State University (personal communication). P. syringae DC3000 cor [Pst DC3000 cor- (DB5A6)]9 and coronatine were obtained from Dr. Carol L. Bender, Oklahoma State University. A coronatine-non producing mutant (Pst DC3000 cor-) constructed in the Pst DC3000 strain with Tn5 insertions in cfa-710 gene, involved in biosynthesis of coronatine, was also obtained from Dr. Carol L. Bender, Oklahoma State University. P. syringae cultures were maintained and grown on Luria-Bertani (LB) medium with appropriate antibiotic selection at 28 oC. In some experiments bacterial cells were grown in MS medium as described above. Sinorhizobium meliloti 1021 strain was obtained from Dr. A.M. Hirsch (University of California) and was cultured in LB medium.

In vitro root pathogenicity assay

Twenty-five-day-old Arabidopsis seedlings growing in MS media in 12-well tissue culture plates (see above) were inoculated with P. syringae strains that had been grown to OD600 0.02 - 0.04 in LB medium at 28 oC to give an initial dose of ~2.5 x 107 CFU/ml. The inoculated plants were incubated at 30 oC in a controlled environmental incubator shaker at 30 rpm with a 16/8 h day/night cycle. Disease symptoms were assessed on a daily basis and photographed on day 7 after inoculation. Twenty plants per treatment were used for analysis of disease symptoms and mortality rates. In parallel experiments, Arabidopsis plants were infected with mixed cultures of Pst DC3000 + Psp NPS3121 or Pst DC3000 + Psg A29-2 in the ratio of ~2.5 x 107: ~ 2.5 x 107 (cfu/ml). Disease symptoms were recorded and photographed on day 7 as described above.

Pathogenicity assay and bacterial counts

Pots of sterilized planting mix, each containing a single four-week-old Arabidopsis plant, were flooded with 10 ml of a P. syringae or S. meliloti culture grown to OD600 = 0.2 - 0.4 in LB medium at 28 oC to give an inoculum of ~1 - 5 x 108 CFU/g of planting mix. Alternatively, plants were flooded with 10 ml of a 1:1 mixture of Pst DC3000 + Psp NPS3121 or Pst DC3000 + Psg A29-2. Plants were incubated in a growth chamber at 30 oC and 80% relative humidity with a 12/12 hour light/dark cycle and assessed every day for disease symptoms. Twenty plants per treatment were used for analysis of mortality rates. Root bacterial number was determined at 7 days after inoculation. For bacterial counts on root surfaces, 500 mg fresh weight root tissue from inoculated plants in sterilized planting mix were washed with distilled water, homogenized in 1ml of 2 % NaCl with a tissue grinder, filtered, diluted in 2 % NaCl, and plated on LB agar plates with an appropriate antibiotic selection medium to determine bacterial cell counts as previously described11. Rifampicin (20 µg ml-1) (Sigma Chemical Co) was used to distinguish Pst DC3000 from Psp NPS3121 in mixed inoculation experiments. Streptomycin (50 µg ml-1) (Sigma Chemical Co) was used to distinguish Psg A29-2 from Pst DC3000 in mixed inoculation experiments. Each data point represents five replicates. All bacterial growth assays were repeated, and only results that were observed consistently are shown.

Activated carbon supplementation

Activated charcoal (30 mg/g planting mix) (Merck, Co., NJ) was added to sterilized planting mix by mixing it with water (1.5 g in 5 ml of water for 50 g planting mix) and pipetting the suspension around four-week-old Arabidopsis rosettes. Plants were subsequently infected with P. syringae strains as described above. As observed in in vitro experiments, plants infected with Pst DC3000 and activated charcoal treated plants infected with the non-pathogenic P. syringae strains, first showed disease symptoms of leaf chlorosis at 4 days after inoculation. Arabidopsis plants not supplemented with activated charcoal and those inoculated with the non-pathogenic strains showed no disease symptoms and very low mortality rates (~5 %) even after two weeks. Planting mix was autoclaved before seeding in all the root inoculation assays. Disease symptoms were assessed on a daily basis and photographed on day 7 after inoculation. Twenty plants per treatment were used for analysis of disease symptoms and mortality rates. Five plants per treatment were used for analysis of root-bacterial counts.

Microscopy

Confocal scanning laser (CSL) microscopy, performed four days post-inoculation to observe root colonization of P. syringae pathovar and non-pathovar infected roots as previously described3. Fluorescence microscopy for bacterial viability was performed using Molecular-Probes BacLight Bacterial Viability Kit (Eugene, OR, USA) as described previously11. Arabidopsis roots treated with COR were also micro-graphed as per the methodology described in the literature11.

Media extraction and HPLC analysis

To examine rhizosecreted secondary metabolites from A. thaliana roots, liquid media samples from in vitro-grown Arabidopsis plants with well-differentiated roots were lyophilized (Virtis, Genesis, 25LL), extracted and analyzed by HPLC as previously described12. Briefly, root exudates (2 ml) from pathogen-treated and untreated plants were collected from 40 wells to make a final volume of 80 ml for lyophillization. The same procedure was conducted for each treatment. Controls included HPLC analysis of MS basal liquid media, methanol extracts in the absence/presence of untreated control plants, exudates of untreated control plants, and HPLC analysis of individual bacterial suspensions. Compounds detected in media from P. syringae-infected plants were not detected in any of the control experiments without plants. Peak volumes of ten commercially purchased compounds (Sigma Chemical Co.) identified previously in Arabidopsis root exudates12 were used to determine the corresponding compounds in root exudates and to calculate the concentrations of these ten compounds in root exudates on the seventh day following inoculation. The gradient used in this study was slightly modified from the previous work12 by supplementing 0.3% of acetic acid diluted in water. Quantification of each marker compound in the root exudates were performed by comparing the retention time of standards and calculating the area under the curve/peak.

Growth inhibition assays

P. syringae strains were tested for inhibition of growth by root exudates, by the ten commercially purchased compounds described above, and by a mixture of these ten compounds (Table 1). The ten individual compounds were tested at concentrations detected in the root exudates from non-infected plants (~58.6-189.1 mM). Initial stock solutions (1 mg mL-1) of each compound were prepared in methanol. Bacteriostatic assays were performed in 96-well, sterile, flat bottom microtiter plates. P. syringae cultures were grown overnight in LB medium at 28 oC, diluted to OD600 = 0.02 in LB, and in each well, 5 µl of the bacterial suspension was added to a total volume of 100 µl of LB in each well. Positive control wells contained 5 µL of bacteria alone with the highest volume of methanol used. Broth micro-dilution antimicrobial susceptibility testing was carried out in accordance with published NCCLS methods13. The plates were covered with sterile lids and placed in polystyrene boxes lined with moistened filter paper to maintain high humidity, and incubated at 28 oC. The absorbance of each well was determined at 600 nm with an Opsys MR microtiter plate reader (Dynex Technologies, USA). Total exudates from untreated plants and Pst DC3000, Psp NPS3121 and Psg A29-2 were lyophilized, concentrated, re-dissolved in MS media at 25% v/v and were subsequently administered for the antimicrobial assays as described above. Each assay was performed five times with three replicates.


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