Supporting Information Figs S1 & S2, Methods S1 and legends to Videos S1–S10
Robust anti-oxidant defences in the rice blast fungus Magnaporthe oryzae confer tolerance to
the host oxidative burst.
Marketa Samalova, Andreas J. Meyer, Sarah J. Gurr and Mark D. Fricker
(a)
-56
-38
-21
-3
15
32
50
67
85
102 120 137 155
(f)
173 190 208 225s
-89
bc
mc
ac
gt
ap
(b)
(g)
-61
-33
-5
23
51
79
107 135
163
F/F0
0
F/F0
1
0.2 0.4 0.6 0.8
1
Grx1-roGFP2
ratio
(e)
1
bc
0.9
0.8
ac
0.7
all
0.6
mc
0.5
60
120
time (s)
180
240
Grx1-roGFP2 ratio
1.1
1.2
1.1
1
0.9
0.8
0.7
mc
0.6
0.5
0.4
-60
red.
0.4 0.6
ox.
0.8
1
1.2
ratio
(j)
(i)
1.1
0
60
120
time (s)
180
240
Grx1-roGFP2 ratio
(d)
0
0
(h)
F/F0
ratio
0.4
-60
0.2 0.4 0.6 0.8
Fluorescence (F/F0)
Grx1-roGFP2
ratio
(c)
Fluorescence (F/F0)
191 219 247 274 302 330 358s
F/F0
F/F0
bc
mc
ac
gt
1
0.9
0.8
all
0.7
0.6
mc
0.5
-120 -60 0
60 120 180 240 300 360
time (s)
red.
0.4 0.6
1.2
1.1
1
0.9
0.8
0.7 bc
0.6
mc
0.5 ac
0.4
-120 -60 0
ox.
0.8
1
1.2
60 120 180 240 300 360
time (s)
Fig. S1 Functional isolation of septal compartments in developing spores in M. oryzae,
measured using fluorescent recovery after photobleaching (FRAP) of Grx1-roGFP2.
Functional connectivity between different cells in developing germlings was examined using
fluorescence recovery after photobleaching (FRAP) of the Grx1-roGFP2 signal in the mid cell.
Examples are shown for growing germ tubes (a–e), and during germ tube swelling (f–j). Following
a high-intensity laser pulse when the 405 and 488 nm lasers were increased to full power and over a
region-of-interest covering the mid cell to bleach the Grx1-roGFP2 fluorescence, the 488nm signal
dropped to around half of the initial value (b, g), expressed as a proportion of the initial
fluorescence on a pixel-by-pixel basis (F/F0). During germination and germ tube emergence little or
no recovery was observed, with only slight convergence of signals in the apical cell and middle cell
over the first 30s post-bleach, whilst the total for all three cells (black line) remained constant (d).
At later stages of development, the Grx1-roGFP2 signal was able to progressively equilibrate
between two or more of the cells in the germling (g, i). In this case signals from all three cells
converged on the average value for the germling post-bleach (i). This would be consistent with
exchange through opening of the septal pores. The Grx1-roGFP2 ratio in the bleached middle cell
showed transient oxidation following the bleach, that returned to resting levels over c. 60 s (e, j),
whilst the ratio from the other cells remained constant. Note, the absolute ratio values reflect an
average across the whole cell and include the contribution of both cytoplasm and vacuole.
Fluorescent images are every 6th image from a multichannel (x,y,t) 3-D series collected at 2.3 s (a–
c) or 3.6 s (f–h) intervals. Scale bars = 5 µm.
Fig. S2 Occlusion of septal pores in developing spores in M. oryzae observed using
transmission electron microscopy.
Septal pores were observed between spore cells using transmission electron microscopy, along with
a cluster of associated electron dense Woronin bodies. In some cases the septal pore was open (a, c),
but in several instances, the pore was partially (d), or fully occluded by an electron dense plug (b,
e). Scale bars = 1 µm, a, b and 100 nm (c–e).
Methods S1 Transmission electron microscopy of conidia from M. oryzae
Conidia were incubated for 1–3 h at 22ºC in the light with gentle shaking, collected by
centrifugation (4000 g, 5 min) and the pellet fixed in 2.5% (v/v) glutaraldehyde in 0.1 M sodium
phosphate buffer, pH 7.2 overnight. Conidia were post-fixed in osmium tetroxide (1% w/v in 0.1 M
buffer), contrasted with uranyl acetate (2% w/v in dH2O), dehydrated through increasing
concentrations of ethanol (70–100%) and embedded in Spurr’s resin (Agar Scientific, UK). Ultrathin sections (50–80 nm) were viewed with a JEM-1010 TEM (Jeol USA Inc., Peabody, MA,
USA).
Legends to Videos S1–S10
Video S1 Measurement of the redox potential of the glutathione pool in M. oryzae germlings using
recombinant Grx1-roGFP2.
Video S2 Measurement of ROS using H2DCFDA in developing spores and during appressorium formation
in M. oryzae.
Video S3 Simultaneous measurement of mitochondrial membrane potential, ROS and Grx1-roGFP2 ratio in
a swelling germ tube of M. oryzae.
Video S4 Simultaneous measurement of mitochondrial membrane potential, ROS and Grx1-roGFP2 ratio in
a developing appressorium of M. oryzae.
Video S5 Formation of glutathione-bimane and subsequent vacuolar sequestration in a developing germling
of M.oryzae.
Video S6 Animated 3-D projections of vacuolar segmentation in spores of M. oryzae during the early stages
of germination.
Video S7 Animated 3-D projections of Grx1-roGFP2 fluorescence expressed in M. oryzae and the
corresponding ratio images, 16 h or 24 h after inoculation on a susceptible rice variety, CO39.
Video S8 Animated 3-D projections of Grx1-roGFP2 fluorescence expressed in M. oryzae, and the
corresponding ratio images, 48 h after inoculation on a susceptible rice variety, CO39.
Video S9 Animated 3-D projections of Grx1-roGFP2 fluorescence expressed in M. oryzae, and the
corresponding ratio images, 16 h or 24 h after inoculation on a resistant rice variety, IR68.
Video S10 Animated 3-D projections of Grx1-roGFP2 fluorescence expressed in M.oryzae, and the
corresponding ratio images, 48 h after inoculation on a resistant rice variety, IR68