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 mol 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
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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
o
C. 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
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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
1 ml 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
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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. syringaeinfected 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.
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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 noninfected plants (~58.6-189.1 M). 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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