Supplemental Data. Vieira et al. (2014). Plant Cell 10.1105/tpc.114.126425
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Supplemental Figure 1. Free-GFP protein expression during gall development. In vivo localization of GFP (35S:GFP) in galls induced by M. incognita. Free GFP localizes both in nuclei and cytoplasm of giant cells. Asterisk, giant cell; n, nematode. Bars = 20 µm.
GFP Merge
3DAI
14DAI
21DAI
*
**
* *
Supplemental Figure 1. Free-GFP protein expression during gall development.
In vivo localization of GFP (35S:GFP) in galls induced by M. incognita. Free GFP localizes both in nuclei and cytoplasm of giant cells. Asterisk, giant
cell; n, nematode. Bars = 20 µm.
*
n
n
* *
n***
*
n n
**
**
*
***
**
n n
***
**
*
Supplemental Data. Vieira et al. (2014). Plant Cell 10.1105/tpc.114.126425
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Supplemental Figure 2. GFP-KRP6 localization in Arabidopsis during cell division. Time lapse during mitotic cell division in stable KRP6OE Arabidopsis root seedlings. The time is indicated at the right side of each confocal image. Intermittent white lines indicate the separation plane of two new daughter cells formed after cell division. Scale bar = 5 µm.
Supplemental Figure 2. GFP-KRP6 localization in Arabidopsis during cell division.
Time lapse during mitotic cell division in Arabidopsis root cells. The time is indicated at the right side of each confocal image. Intermittent white
lines indicate the separation plane of two new daughter cells formed after cell division. Scale bar = 5 µm.
0’ 1.3’ 4’ 6.5’ 8’
Supplemental Data. Vieira et al. (2014). Plant Cell 10.1105/tpc.114.126425
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Supplemental Figure 3. Effect of ectopic expression of KRP6 on cell cycle progression in an Arabidopsis cell culture, independently obtained from the transgenic culture used in Figure 7. (A) BrdU labeling index (dashed lines) and M/A index (solid lines) after aphidicolin synchronization of PSB-D (blue lines) and 35S:GFP-KRP6 (green lines) cell cultures. (B) Total protein extracts of PSB-D and transgenic Arabidopsis cultures overexpressing GFP-KRP6 after release from the aphidicolin block, analyzed 2-hourly by immunoblot analysis for CDKA;1 and CDKB1;1.
Supplemental Figure 3. Effect of ectopic expression of KRP6 on cell cycle progression in an Arabidopsis cell culture, independently
obtained from the transgenic culture used in Figure 7.
(A) BrdU labeling index and M/A index after aphidicolin synchronization of PSB-D and 35S:GFP-KRP6 cell cultures.
(B) Total protein extracts of PSB-D and transgenic Arabidopsis cultures overexpressing GFP-KRP6 after release from the aphidicolin
block, analyzed 2-hourly by immunoblot analysis with antisera against CDKA;1 and CDKB1;1.
!
A
B
Supplemental Data. Vieira et al. (2014). Plant Cell 10.1105/tpc.114.126425
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Supplemental Figure 4. GFP-KRP6 overexpression causes leaf serration in seedlings. (A) GFP-KRP6 seedlings showing serrated phenotype on the first leaves (white arrows). (B) Wild-type seedling. Arabidopsis thaliana Col-0 transformants expressing 35S:GFP-KRP6 were selected on Kanamycin, transferred to ½ Ms medium and photographed 4 days later. (C) The transcript levels of the KRP6OE line 1 selected for the RKN inoculation tests were examined by RT-PCR-based DNA gel blot analysis, using 7-day-old whole seedlings. (D) Example of three-month-old soil grown KRP6OE plants (T3) obtained from independent transformation events, showing the typical serrated leaf phenotype in comparison to untransformed control plant (Columbia-0) of similar age.
Supplemental Figure 4. GFP-KRP6 overexpression causes leaf serration in seedlings.
(A) GFP-KRP6 seedlings showing serrated phenotype on the first leaves (white arrows). (B) Wild-type seedling. Arabidopsis thaliana Col-0
transformants expressing 35S:GFP-KRP6 were selected on Kanamycin, transferred to ½ Ms medium and photographed 4 days later.
(C) The transcript levels of the KRP6OE line 1 selected for the RKN inoculation tests were examined by RT-PCR-based DNA gel blot
analysis, using 7-day-old whole seedlings.
(D) Example of three-month-old soil grown KRP6OE plants (T3) obtained from independent transformation events, showing the typical
serrated leaf phenotype in comparison to untransformed control plant (Columbia-0) of similar age.!
!
D
KRP6OE
Col-0
Line 1 Line 2
Supplemental Data. Vieira et al. (2014). Plant Cell 10.1105/tpc.114.126425
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Supplemental Figure 5. Micronuclei formed within 10% percentage KRP6OE giant cells. (A) Average number of nuclei per giant cell section at 7 and 14 DAI. These values are representative of the number of nuclei per giant cell section. The nuclei were counted in the largest giant cells (two to three) in each gall, using 15 galls for each line. Error bars represent standard deviation and asterisks indicate values that are significantly different from wild-type at the same time point (Student’s t-test, P<0.05). (B-D) Micronuclei within giant cells at 7DAI (C) and 14DAI (D-D’) compared to wild-type (B). Bright-field images of longitudinal gall sections stained with toluidine blue. Asterisk, giant cell; n, nematode. Scale bars = 50 µm.
Supplemental Figure 5. Micronuclei formed within 10% percentage KRP6OE giant cells.
(A) Average number of nuclei per giant cell section at 7 and 14 DAI. These values are representative of the number of nuclei per giant
cell section. The nuclei were counted in the largest giant cells (two to three) in each gall, using 15 galls for each line. Error bars
represent standard deviation and asterisks indicate values that are significantly different from wild-type at the same time point
(Student’s t-test, P<0.05).
(B-D) Micronuclei within giant cells at 7DAI (C) and 14DAI (D-D’) compared to wild-type (B). Bright-field images of longitudinal
gall sections stained with toluidine blue. Asterisk, giant cell; n, nematode. Scale bars = 50 µm.
!
!
Supplemental Data. Vieira et al. (2014). Plant Cell 10.1105/tpc.114.126425
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Supplemental Figure 6. Electron micrographs of giant cells of galls in Arabidopsis wild-type plants compared to KRP6OE line. (A-B) Galls in wild-type roots. (A) A giant cell surrounded by neighbouring cells at 14 DAI, and (B) a detailed section of a giant cell and the nucleus distribution at 21 DAI. (C-D) Galls in roots of KRP6OE line. (C) A giant cell surrounded by multiple neighbouring cells with various shapes and sizes at 14 DAI, and (D) a detailed section showing an increase in the number of nuclei in a single giant cell at 21 DAI. NC, neighboring cell; Asterisks, giant cell.
Supplemental Figure 6. Electron micrographs of giant cells of galls in Arabidopsis wild-type plants compared to KRP6OE line.
(A-B) Galls in wild-type roots. (A) A giant cell surrounded by neighbouring cells at 14 DAI, and (B) a detailed section of a giant
cell and the nucleus distribution at 21 DAI.
(C-D) Galls in roots of KRP6OE line. (C) A giant cell surrounded by multiple neighbouring cells with various shapes and sizes at
14 DAI, and (D) a detailed section showing an increase in the number of nuclei in a single giant cell at 21 DAI.
NC, neighboring cell; Asterisks, giant cell.
Supplemental Data. Vieira et al. (2014). Plant Cell 10.1105/tpc.114.126425
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Supplemental Figure 7. Nuclear flow cytometry analyses of roots in wild-type and KRP6OE line. Representative flow cytometry graphics of nuclei isolated from 4-week-old roots (approximately 2000 nuclei were measured per sample). Three independent experiments were performed using roots of six plants of each line and each experiment.
Col.
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Supplemental Figure 7. Nuclear flow cytometry analyses of roots in wild-type and KRP6OE line. Representative flow cytometry
graphics of nuclei isolated from 4-week-old roots (approximately 2000 nuclei were measured per sample). Three independent
experiments were performed using roots of six plants of each line and each experiment.
KRP6OECol-0
Supplemental Data. Vieira et al. (2014). Plant Cell 10.1105/tpc.114.126425
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krp6
ASAIL_54B_B03
krp6
SALK_142997
KRP6
-LP+
KRP6
-R
LB1+
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LB1+
KRP6
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LBa1
+ K
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-RP
KR
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KR
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P
LBa1
+ K
RP6
-RP
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850 pb 1 Kb
1.65 Kb
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KRP6
EF1A
Col-0 krp6
KRP6
EF1A
SALK_142997
Sail_548_B03 SALK_142997
0!
5000!
10000!
15000!
20000!
25000!
0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10" 11" 12" 13" 14" 15" 16" 17" 18" 19" 20"
Giant cell surface (µm2) F
7DAI 14DAI 21DAI
*
*
*
Col-0
krp6 KO
D
0"
5"
10"
15"
20"
25"
30"
35"
40"
Col+0" ick4/krp6"
Galls"
Egg"masses"
Gal
ls a
nd e
gg m
asse
s / p
late
E!
Gal
ls a
nd e
gg m
asse
s / p
late!
0"
5"
10"
15"
20"
25"
30"
35"
40"
Col+0" ick4/krp6"
Galls"
Egg"masses"
E
Gal
ls a
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asse
s / p
late
0"
5"
10"
15"
20"
25"
30"
35"
40"
Col+0" ick4/krp6"
Galls"
Egg"masses"
0"
5"
10"
15"
20"
25"
30"
35"
40"
Col+0" ick4/krp6"
Galls"
Egg"masses"
0"
5"
10"
15"
20"
25"
30"
35"
40"
Col+0" ick4/krp6"
Galls"
Egg"masses"
0"
5"
10"
15"
20"
25"
30"
35"
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Col+0" ick4/krp6"
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Egg"masses"
Sail_54B_B03 SALK_142997
n
n
14DAI 21DAI7DAIG H I
Supplemental Data. Vieira et al. (2014). Plant Cell 10.1105/tpc.114.126425
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Supplemental Figure 8. Characterization of krp6 loss-of-function lines. (A) Graphic representation of KRP6, with relative position of T-DNA insertion for the SAIL_54B_B03 and SALK_142997 lines. (B) PCR analysis of single T-DNA insertion into the KRP6 gene for the SAIL_54B_B03 and SALK_142997 lines. (C) RT-PCR performed with locus-specific primers revealed the absence of full-length transcripts of krp6 knockout lines. (D-E) Nematode infection tests of krp6 loss-of-function lines; (D) SAIL_54B_B03 and (E) SALK_142997 lines compared to wild-type. Data shown represents means ± SD from two independent biological experiments, using 60 nematode-infected seedlings of each line. Pair wise comparisons were made by Student’s t-test (P≤0.05), revealing no statistically differences within the lines of each test respectively. (F) Measurements (µm2) of giant cells surface in the wild-type and the krp6 line (SAIL_54B_B03) at 7, 14 and 21 DAI. Asterisks show statistical differences of each time point after nematode infection (P<0.05). (G-I) Histological analysis of the krp6 line (SALK_142997) on M. incognita-induced galls in Arabidopsis roots. Bright-field images of longitudinal sections of galls stained with toluidine blue at 7 (G), 14 (H) and 21 (I) DAI, showing the presence of cell wall stubs (red arrows). Asterisk, giant cell; n, nematode. Bars = 50 µm.
Supplemental Data. Vieira et al. (2014). Plant Cell 10.1105/tpc.114.126425
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Supplemental Table 1. List of primers. KRP6 promoter primers Forward: 5’-ATAGAAAAGTTGTGTGTTATTTGTTAGTGGATTTTTGATTTTGG-3’ Reverse: 5’-TGTACAAACTTGCGATTTTTTCTCTCTTGGATTTTTGTGTG-3’ RT-PCR primers KRP6 Forward: 5’-TCGAAACCGAAACCGAAA-3’ Reverse: 5’-TCTTCGTTGCCGATGATG-3’ OXA1 Forward: 5’-TACCTGATCTGCCTCCACCT-3’ Reverse: 5’-AACAGGACTCAGCGATGTTG-3 Elongation Factor 1α (EF1A) Forward: 5’- TGAGCACGCTCTTCTTGCTTTCA-3’ Reverse: 5’- GGTGGTGGCATCCATCTTGTTACA-3 krp6 mutant identification SAIL_548_B03 Forward: 5’-ATTCATCACCGGACTCTCATG-3’ Reverse: 5’-TGGTATTTGCTTTTTGATGGC-3’ LB1: 5’-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3’ SALK_142997 Forward: 5’-AATTTATTGTTCCGACCACCAC-3’ Reverse: 5’-ACACCAAACGACGAACTGTTC-3’ LBa1: 5’-TGGTTCACGTAGTGGGCCATCG-3’
