of 6
Supplemental
Fig
ures
1
2
Fig.
S1
.
Fungi from identified bacterial
-
fungal pairings are sensiti
ve to PCA.
.
A)
Lecythophora
and
3
Aspergillus
species grown on PDA, PDA supplemented with 300 μM PCA, and finally in the presence of
4
PCA along with co
-
isolated bacterial partners, a
Paraburkholderia
species and a
Luteibacter
species,
5
respectively. Plates were grown for 48 hrs at 30
º
C.
B)
Phenazine production is required to inhibit growth
6
of the
Aspergillus
species
.
Pseudomonas chlororaphis
and
Pseudomonas fluorescens
inhibit fungal
7
growth, but the
::phzD
and
::phzB
strains,
respectively, fail to inhibit fungal growth after growing on
8
PDA for 48 hrs at 30 ºC.
9
Fig.
S
2
.
Bacterial aggregates accumulate along the ridge of co
-
colonies treated with PCA.
A band of
10
bacteria can be seen in both treated and control colonies between the inner and outer zones. In the PCA
11
treated colony, this band is thicker with more well formed aggregates, and is representative of the region
12
larger aggregates are visible to t
he eye during co
-
colony growth on PCA.
Samples were cleared using the
13
MiPACT protocol, and i
mages were captured on a Nikon
Ti2 Eclipse.
Red represents the signal produced
14
by eubacterial HCR probes
coupled to Alexa fluor
647 nm
.
.
15
Fig.
S
3
.
P. edwinii
does not degrade PCA in liquid culture
.
P. edwinii
was grown in potato dextrose
16
broth
shaking at 30
º
C spiked with and without 300 μM PCA, and with PCA and its fungal partner. PCA
17
concentration was measured every 24 hours for three days. A control lacking
P. edwinii
was included to
18
confirm PCA did not degrade over this time period. Quantification occurred
by measuring absorbance at
19
365 nm. Error bars represent standard deviation of four biological replicates.
20
21
Fig.
S
4
.
Morphology of
WT and
Δ
hrcA
P. edwinii
and properties of the
Aspergillu
s isolate
with and
22
without PCA challenge
.
A)
Comparison of the
Aspergillus
isolate grown alone (left
, top
), with WT
P.
23
edwinii
(center), and with the
Δ
hrcA
mutant (
bottom
). Note large aggregates
in the Δ
hrcA
mutant despite
24
the absence of PCA
B)
WT (
left
) the the Δ
hrcA
mutant (
right
) grown in the
absence (
top
) and presence
25
(center) of PCA, and next to its partner fungus when challenged with PCA (
bottom
). The WT strain
26
shows a thickening of the colony in response to PCA, as well as an increase sheen. A similar thickening
27
and sheen is visible for the
Δ
hrcA
mutant in all conditions.
C)
The
Aspergillus
isolate produces an
28
oxidizing environment when growing on potato dextrose agar in the absence of PCA, but generates a
29
more reducing environment during challenge with 300 μM PCA. Error bars represent the s
tandard
30
deviation of 3 measurements at each depth. All colonies were grown for 48 hours at 30 ºC.
31
32
33
Supplemental Tables
34
Su
ppleme
nt
ary Table 1.
List of
transposon
mutants
found in P. edwinii
that
alter its ability to protect its
35
p
artner fungus
, including
phenotype
(left)
,
closest
homologue of P.
edwinii gene each transposon hit was
36
found in
based
on
NCBI
BLAST
using
arbitrary
PCR on the mutant
strains
, and any
apparent homologue
37
the gene encodes.
38
39
Supplementary Table 2.
List of strains and plasmids used in this study.
40
41
42
S
u
p
p
l
e
m
e
n
t
a
l
References
43
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Mellado, J., Martínez
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Aguilar, L., Paredes
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Valdez, G., and Estrada
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carboxamide
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Chiang, S.L., and Rubin, E.J. (2002). Construction of a mariner
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77
Figure S1
Fungus (
-
) PCA
Fungus (+) PCA
Co
-
colony (+) PCA
Aspergillus sp.
Lecythophora
sp.
+
Paraburkholderia
sp.
+
Luteibacter
sp.
2 mm
P.
chlororaphis
P.
chlororaphis
::
phzB
P. fluorescens
P. fluorescens
::
phzD
Aspergillus sp.
A
B
Figure S2
Eubacteria HCR
DAPI
Merge
(
-
) PCA
(+) PCA
Figure S3
Figure S4
2 mm
Aspergillus
Alone
Aspergillus
+
Δ
hrcA
Aspergillus
+ WT
Δ
hrcA
WT
(+) PCA
(+) PCA +
Aspergillus
sp
Fungus
(μm)
A
B
C