figure s1. germination rate after repeated passage of the msb seeds from each generation of the msb...
DESCRIPTION
x 10 4 cfu/g Brown rice Polished rice 95%90% Figure S3. The number of viable bacterial in MucoRice-CTB brown rice and polished rice Viable bacteria in powdered brown rice or polished rice (polishing rate, 95% or 90%) were calculated. The polishing percentage indicates the rice weight ratio after compared with before polishing. This experiment was performed twice, and the mean values are shown.TRANSCRIPT
Figure S1. Germination rate after repeated passage of the MSB
Seeds from each generation of the MSB were sowed. All five passages of the MSB passed the specification for germination rate (90% or greater).
40
60
80
100G
erm
inat
ion
rate
[%]
20
0
Passages from MSB
P1 P2 P3 P4 P5
Figure S2. Gradual decrease in the moisture content of harvested MucoRice-CTB grains
Harvested rice attached to ears was dried in the ventilated incubator for 7 days; the moisture content in the powdered brown rice was measured on days 0, 1, 3, and 7 by using the loss-on-drying method. The data from the five lots of cultivation were averaged and are shown. The error bars represent the standard deviation calculated from the combined data from the five lots.
20
30
40M
oist
ure
cont
ent o
fric
e gr
ain[
%]
10
0
Period of drying [days]
0 1 3 7
1.0
2.0
3.0
4.0
x 104 cfu/g
Brown rice
Polished rice
95% 90%
Figure S3. The number of viable bacterial in MucoRice-CTB brown rice and polished rice
Viable bacteria in powdered brown rice or polished rice (polishing rate, 95% or 90%) were calculated. The polishing percentage indicates the rice weight ratio after compared with before polishing. This experiment was performed twice, and the mean values are shown.
WT MucoRice-CTB (MSB)
A
Bars 500 µm
Figure S4. Location of CTB in MucoRice-CTB MSB seed
Immunohistochemistry showed that CTB accumulated in the endosperm cells under the aleurone layer of the MucoRice-CTB MSB seeds but was not detected in a WT-rice seeds. (A) and (B) Stained with anti-CTB antibody. (C) Stained with anti-rabbit IgG (negative control).
B C
Figure S5. Densitometric chart (A) on SDS-PAGE (B) of MucoRice-CTB drug substance
(A) SDS-PAGE gel capture image used to generate the densitogram. (B) A densitogram for 8 samples including protein marker, house standard lot 1, MucoRice-CTB drug substance (lot 2) and recombinant CTB. The densitogram was generated from each lane’s top to bottom along the red lines in (A) and correspondingly charted from left to right in (B). Lane description; 1-2 and 11-12: blank, 3: Protein marker, 4-6: MucoRice-CTB (house standard: lot 1), 7-9: MucoRice-CTB (lot 2) and 10: SR-CTB (N4Q), recombinant CTB.
B
1 2 3 4 5 6 7 8 9 10 11 12laneA
Lane 3Lane 7
Lane 4Lane 8
Lane 5Lane 9
Lane 6Lane 10
A B
Figure S6. CT challenge test in mice immunized with MucoRice-CTB
After mice were orally immunized a total of 5 times at 2-week intervals with MucoRice-CTB lot 2 or non-transgenic Oryza sativa L cv. Nipponbare (WT) rice powder suspended in PBS, CTB-specific serum IgG (A), serum IgA (B), and fecal IgA (C) responses were significantly induced in mice immunized with MucoRice-CTB lot 2 but not in those that received WT rice. In contrast to the mice that received WT rice, mice immunized with MucoRice-CTB lot 2 showed no signs of diarrhea and had a low intestinal water content. **, P < 0.01, MucoRice-CTB versus WT rice.
C D
**17
15
13
11
9
7Rec
ipro
cal L
og2 T
iter
WT rice MucoRice-CTB
CTB-specific Serum IgG
WT rice MucoRice-CTB
**CTB-specific Serum IgA
10
8
7
6
5
4
Rec
ipro
cal L
og2 T
iter
9
**9
8
76
5
3
Rec
ipro
cal L
og2 T
iter
WT rice MucoRice-CTB
CTB-specific Fecal IgA
4
**CT-induced Diarrhea
WT rice MucoRice-CTB
1600
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800
0C
T-in
duce
d In
test
inal
Flu
id V
olum
e (µ
l)
400