Supporting Information Figs S1–S6
Fig. S1 Peptides identified in frass of FAW larvae fed two maize genotypes were aligned to the
RIP2 amino acid sequence. The underlined regions represent identified peptides found in frass.
Peptides underlined in red and green represent those found in frass from caterpillars that fed on
Tx601 and Mp704, respectively. The region between arrows is removed during the proteolytic
processing of RIP1 protein (Walsh et al., 1991) and we are predicting that similar processing
occurs in RIP2. The gray shadowed boxes represent conserved amino acid sequences in both
RIP1 and RIP2.
Walsh TA, Morgan AE, Hey TD. 1991. Characterization and molecular cloning of a proenzyme form of a
ribosome-inactivating protein from maize. Novel mechanism of proenzyme activation by
proteolytic removal of a 2.8-kilodalton internal peptide segment. J Biol Chem 266(34): 2342223427.
35 -
1
14
2
3
4
5
6
7
8
9
10
11
15
35 25 Fig. S2 Immunoblot analysis of RIP2 in several maize inbreds and teosinte (Zea mays
parviglumis and Zea mays mexicana).Plants were fed by S. frugiperda larvae (three larvae per
plant) for 24 h. Leaf tissues at the feeding sites were collected. Equal amounts of proteins (50 µg)
were loaded in each well. The proteins were separated by SDS-PAGE and analyzed by
immunoblot using anti-RIP antibody. The polypeptide corresponds to 30 kD proenzyme form of
RIP2. Lanes 1 to 15 are the following. 1: Mp704, 2: Mp496; 3: CML131; 4: OH43; 5: Mo17; 6:
Ab24E; 7: CML139; 8: Mp708; 9: KI3; 10: CML67; 11: Tx601; 12: B73; 13: W64A; 14: Zea
mays parviglumis; 15: Zea mays mexicana.
12
13
Control
Caterpillar fed
1’ 2’ 3’ 4’ 5’ 1 2 3 4 5
Fig. S3 Systemic expression of RIP2 protein in maize leaves after larval feeding. Maize V8 stage
plants were fed by FAW larvae (two or three larvae per plant) for 24 h. The larvae were placed
on eleventh leaf in a small bag that limited their movement. Leaf samples from the feeding site
(0 cm; lane 1) and distal to the site (3 and 5 cm; lane 2 and 3) were collected. Samples were
taken from other leaves (unexpanded ninth and tenth leaf; lane 4 and 5) of the same plant.
Undamaged plants were as control groups. Lane 1’ to 5’ represent the similar tissue sections
from undamaged plants. Each treatment represents four to five biological samples. Equal
amounts of protein (50 µg) were loaded in each well and samples were analyzed on immunoblots.
Stages
V2
V4
V6
V8
V10
C F
C F
C F
C F
C F
Fig. S4 Developmental expression of RIP2 protein in maize leaves after larval feeding. Maize
plants at various vegetative stages (V2, V4, V6, V8, and V10) were fed with larvae (five larvae
per plant) for 24 h (larval feeding=F). Undamaged plants were as control groups (control=C). In
each stage, 2 to 6 plants were harvested for control and feeding treatments. Equal amounts of
protein (50 µg) were loaded in each well and samples were analyzed on immunoblots.
(a)
0.03%
MEJA
C
W
J
(b)
0.10%
WJ
Ponceau S
Stain
C
W
J
WJ
C
Ponceau S
Stain
Fig. S5 Effect of phytohormones (MeJA and ABA) on RIP2 protein expression in maize leaves.
(a) Methyl jasmonate (MeJA): Maize plants were damaged by paper punch (wounded only=W).
Wounded plants were sprayed with 0.01% to 0.1% MeJA (wounded+MeJA=WJ). Undamaged
plants were also treated with 0.01% to 0.1% MEJA (MEJA=J). Undamaged plants without
treatment were control groups (control=C). Leaf samples were collected after 24 h. (b) Abscisic
acid (ABA): Maize plants were damaged by paper punch (W). Wounded plants were sprayed
with 300µM ABA (wounded+ABA=WA). Undamaged plants were also treated with 300µM ABA
(ABA=A). Undamaged plants without treatment were control groups (C). Leaf samples were
collected after 24 h. Each treatment represents two biological samples. Equal amounts of
protein (50 µg) were loaded in each well. A Ponceau S Stain membrane showing the 55 kD
Rubisco large subunit were the loading controls.
A
Fig. S6 The identified peptide sequences from two polypeptides of rRIP2 treated with papaincoated beads (Fig 7, lane 2) were aligned to the RIP2 amino acid sequence. The underlined
regions represent identified peptides found in these two polypeptides. Peptides underlined in
green and red represent those found in the upper and lower bands in the Fig 7, lane 2,
respectively. The region between arrows is removed during the proteolytic processing of RIP1
protein (Walsh et al., 1991) and we are predicting that similar processing occurs in RIP2.
Walsh TA, Morgan AE, Hey TD. 1991. Characterization and molecular cloning of a proenzyme form of a
ribosome-inactivating protein from maize. Novel mechanism of proenzyme activation by
proteolytic removal of a 2.8-kilodalton internal peptide segment. J Biol Chem 266(34): 2342223427.