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Mediation of pathogen resistance by exudation of antimicrobials from roots- Bais et al.
Supplementary Online Material:
Legends to Supplementary Figures S1-S8:
Figure S1. Pathogenicity of various P. syringae strains in vitro. Under in vitro conditions plants treated
with Pst DC3000 or Pst DC3000 cor- first showed disease symptoms (necrotic roots and leaf chlorosis) at
3 day post-inoculation. Plant inoculated with the seven non-pathogenic P. syringae strains also showed
rotting symptoms at the end of two weeks post-inoculation under in vitro conditions. Disease symptoms
were assessed on a daily basis and photographed on day 7 after inoculation. In vitro experiments with
Arabidopsis plants treated with Pst DC3000 showed high mortality rates (~80%). Experiments were
repeated twice with each experiment containing three replicates of twenty plants each. Infection was
performed as described in Supplementary Methods and symptoms or plant mortality were recorded 7 days
post-inoculation. Arrows in the panel are indicative of disease symptoms.
Figure S2. (A) Pathogenicity of various P. syringae strains in a sterilized planting mix-based assay in the
presence or absence of activated charcoal. Arrows are indicative of disease symptoms. Disease symptoms
with Pst DC3000 and activated charcoal-treated plants subsequently infected with pathogens and nonpathogens appeared at day 4 post-inoculation. Arabidopsis plants not supplemented with activated
charcoal and infected with the non-pathogenic strains showed no disease symptoms and low mortality
rates (~5 % in 7 days) at the end of two weeks. Planting mix experiments with Arabidopsis plants treated
with Pst DC3000, Pst DC3000 cor- and the non-pathogenic strains under activated charcoal
supplementation showed high mortality rates (~65-80%). (B) To confirm the adsorbing capacity of
activated charcoal on root exuded antimicrobials, A. thaliana plants were treated with activated charcoal
(30 mg g-1 soil) for two days and then the roots were subsequently washed with water to get rid of excess
root-bound charcoal. Washed plants were subsequently transferred to greenhouse soil pots to allow the
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normal exudation of compounds by plant roots. Plants were infected five days later with Psp NPS3121 or
Psg A29-2. Disease symptoms were assessed on a daily basis and photographed on day 7 after
inoculation. Experiments were repeated twice with each experiment consisting of 3 replicates of twenty
plants each.
Figure S3. Quantification of plant mortality and root colonization in the sterilized planting mix-based
pathogenicity assay. (A) To measure pathogenicity, plant mortality was monitored after inoculation with
various pathogenic and non-pathogenic P. syringae strains in the presence and absence of activated
charcoal. Data are the average of two independent experiments. Bars show one standard error. Two-way
ANOVA for plant mortality: Ftreatment= 12.30, df =1,31, P<0.005. (B) Root colonization of P. syringae
strains shown by colony formation units (cfu) in the presence and absence of activated charcoal. In
parallel experiments, Arabidopsis plants were infected with mixed cultures of Pst DC3000 + Psg A29-2
in the ratio of ~2.5 x 107: ~ 2.5 x 107 (cfu/ml). In the mixed inoculation treatment of Pst DC3000 plus Psg
A29-2, the group bars in panel B depict CFUs for Pst DC3000. The CFUs for Psg A29-2 under nonactivated charcoal and activated charcoal treatment were ~7.8 x 105 / g roots and ~6.3 x 105/ g roots
respectively. Data are the average of two independent experiments with each experiment consisting of
three replicates of five plants each. Initial root-associated bacterial counts at the time of inoculation were
on the order of ~102 CFU per gram of root tissue. Bars show one standard error. Two-way ANOVA for
total bacterial counts: Ftreatment= 11.45, df =1,28, P<0.005. Inoculation was performed as described in
Methods, and plant mortality and bacterial growth were scored 7 days post-inoculation.
Figure S4. Confocal scanning laser (CSL) microscopy of Arabidopsis roots infected with the P. syringae
pathovars (A) Psg A29-2, (B) Pst DC3000, or (C) Psp NPS3121 in the presence and absence of activated
charcoal. (Brackets in panels indicate the root zone). A live-dead BacLight bacterial viability kit (See
Supplemental Methods) was used to detect live bacteria. Bacteria on the root surface were stained with
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propidium iodide to visualize the polysaccharides present in the bacterial cell wall (green fluorescence).
Panel B shows intact biofilm formation by Pst DC3000 in the presence and absence of activated charcoal
as represented by green fluorescence. The bars in all three panels indicate 50 µm. Micrographs of roots
were captured 7 days post-inoculation of P. syringae pathovars.
Figure S5. Kinetics of ten marker secondary metabolites in the root exudates of A. thaliana infected with
different P. syringae pathovars obtained on the 0th, 3rd, 5th, and 7th days post-inoculation. Different panels
indicate the quantification of each metabolite on respective days under various infection regimes. Data are
the average of ten independent experiments. Lines show one standard error. Two-way ANOVA for each
metabolite concentration: Ftreatment= 12.41, df =1,58, P<0.001.
Figure S6. Chromatographic kinetics of root exudation obtained on the 3rd, 5th, 7th days post-inoculation
of Arabidopsis plants infected with different P. syringae pathovars. (A) Untreated control, (B) Psp
NPS3121, (C) Pst DC3000, (D) Pst DC3000 hrpL-, (E) Pst DC3000 cor-, (F) Pst DC3000 hrcCtreatment. The peaks numbered 1-10 correspond to the 10 compounds listed in Table 1 and supplemental
Table S1
Figure S7. Chromatographic kinetics of root exudation obtained on the 3rd, 5th, 7th days post-inoculation
of Arabidopsis plants infected with different P. syringae pathovars, (A) Untreated control, (B) Psg A29-2,
(C) Psg A29-2 + Pst DC3000, (D) Psp NPS3121 + Pst DC3000, (E) Psp NPS3121. The peaks numbered
1-10 correspond to the 10 compounds listed in Table 1 and supplemental Table S1. (F) Concentrations of
ten marker secondary metabolites in the root exudates of A. thaliana infected with different P. syringae
pathovars on day 7. Numbers on the x axis refer to the compounds detected in the root exudates: (1)
Butanoic acid, (2) trans-Cinnamic acid, (3) o-Coumaric acid, (4) p-Coumaric acid, (5) 3- Indolepropanoic
acid, (6) Ferulic acid, (7) Vanillic acid, (8) Methyl p-Hydroxybenzoate, (9) p-Hydroxybenzamide, (10)
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Syringic acid. Data are the average of ten independent experiments. Bars show one standard error. Twoway ANOVA for metabolite concentration: Ftreatment= 12.41, df =1,58, P<0.001.
Figure S8. Bacteriostatic activities of root exudates and antimicrobial compounds. Exudates were from
untreated plants, from plants infected with Psp NPS3121, from plants infected with Psg A29-2, and from
plants infected with Pst DC3000. Bacteriostatic activity of a cocktail of the ten characterized secondary
metabolites was also tested. The compounds are (1) Butanoic acid (189.1 M), (2) trans-Cinnamic acid
(139.8 M), (3) o-Coumaric acid (146.9 M), (4) p-Coumaric acid (152.1 M), (5) 3- Indolepropanoic
acid (156.8 M), (6) Ferulic acid (89.12 M), (7) Vanillic acid (91.8 M), (8) Methyl p-Hydroxybenzoate
(145.2 M), (9) p-Hydroxybenzamide (58.6 M), (10) Syringic acid (65.2 M). Net bacterial growth was
calculated by subtracting the OD600 at the beginning from the OD600 after 24 hours of incubation. Percent
inhibition (%I) was calculated using net bacterial growth based on OD600 readings with the following
formula: (untreated-treated/untreated) x (100). Values are mean ± S. D., (n=5). Data are the average of
five independent experiments. Bars show one standard error. Two-way ANOVA for percentage
inhibition: Ftreatment= 13.23, df =1,41, P<0.005. Total exudates from untreated plants and Pst DC3000, Psp
NPS3121 and Psg A29-2 were concentrated, re-dissolved in MS media at 25% v/v and were subsequently
administered for the antimicrobial assays as described above. The concentration referred to in all the
treatments represents the approximate biological concentration in the untreated Arabidopsis root exudates.
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