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348 349 16
Dang et al. p. 17 350 Figure legends
351 352 Fig. 1 Quantitative RT-PCR analysis of five MoSET1-dependent transcription factors
353 at hyphal, conidial and infectious stages in P. oryzae. Actin was used to normalize
354 mRNA expression level. Data show fold change (relative to mRNA quantity in hyphae)
355 ± standard error (n = 3). Asterisks are given to indicate significant difference at p < 0.05
356 (*) and p < 0.01 (**) (two-tailed t-test).
357 358 Fig. 2 Quantitative RT-PCR analysis of MGG_00472 and MGG_07386 mRNA in
359 candidates of their knock-down mutants. Actin was used to normalize mRNA
360 expression level. Data show fold change (relative to mRNA quantity in the wild-type
361 strain) ± standard error (n = 3). Different characters indicate significant differences by
362 Tukey's HSD (p < 0.05).
363 364 Fig. 3 Phenotypic characterization of knock-out and -down mutants of MoSET1-
365 dependent TFs in P. oryzae. a Diameters of fungal colonies were measured at 9 days
366 after inoculation on rich agar medium. b Conidiation was evaluated by counting the
367 number of conidia under a light microscopy as described in details in Materials and
368 methods. c-d The rates of conidial germination (c) and appressorium formation (d) were
369 measured by observing conidial suspension on hydrophobic surface under a light
370 microscope after 5 h (conidial germination) and 24 h (appressorium formation)
371 incubation at 25̊C.
372 Black bars indicate the wild-type strain Br48 (WT) and grey bars represent knock-out
373 and -down mutants of MoSET1-dependent TFs and their gene complemented strains
374 (c∆mgg_04699 and c∆mgg_06898). Data show fold change (relative to the wild-type
375 strain) ± standard error (n = 3). Different characters in the graphs indicate significant
376 differences by Tukey's HSD (p < 0.05) ND, not determined.
377 378 Fig. 4 Inoculation test of knock-out and -down mutants of MoSET1-dependent TFs in
379 P. oryzae. a Infection assay was performed on the wheat cultivar Norin 4 at 23̊C. Four
380 to five days after inoculation, symptoms on the inoculated plants were evaluated. Letters
17
Dang et al. p. 18 381 under pictures of infected leaves indicate disease index values by a grading method
382 (Hyon et al., 2012). This experiment was repeated at least three times, and
383 representative samples are presented. b The rates of infection hyphae formation in
384 infected leaves. The black bar indicates the wild-type strain Br48 (WT) and grey bars
385 represent knock-out and –down mutants of MoSET1-dependent TFs and a gene
386 complemented strain (c∆mgg_04699). Error bars represent standard errors of the mean
387 (n = 10). Different characters in the graph indicate significant differences by Tukey's
388 HSD (p < 0.05).
389 390 Fig. 5 Schematic diagram of putative MoSET1 regulatory network during infection-
391 related morphogenesis in P. oryzae.
18
Dang et al. p.19
3.E+01
25
Fold change
(relative to Hyphae)
8.E+00
23
**
**
4.E+00
22
2.E+00
21
Hyphae
Spore
infection (5hpi)
infection (12hpi)
**
2.E+01
24
**
**
1.E+00
5.E‐01
2-1
3.E‐01
2-2
**
2-3
1.E‐01
**
2-4
6.E‐02
MGG_00472
MGG_04699
MGG_06898
MGG_07386
**
MGG_07450
3.E‐02
-5
Fig 1. Quantitative RT‐PCR analysis of five MoSET1‐dependent transcription factors at hyphal, conidial and infectious stages in P. oryzae. Actin was used to normalize mRNA expression level. Data show fold change (relative to mRNA quantity in hyphae) ±standard error (n = 3). Asterisks are given to indicate significant difference at p< 0.05 (*) and p< 0.01 (**) (two‐tailed t‐test). Dang et al. p.20 1.2
Fold change
(relative to WT)
MGG_00472
MGG_07386
0.8
0.6
0.4
0.2
Fig 2. Quantitative RT‐PCR analysis of MGG_00472 and MGG_07386 mRNA in candidates of their knock‐down mutants. Actin was used to normalize mRNA expression level. Data show fold change (relative to mRNA quantity in the wild‐type strain) ±standard error (n = 3). a‐c, Different characters indicate significant differences by Tukey's HSD (P < 0.05).
Dang et al. p.21 a, vegetative growth
ab
bc
ab
ab
bc
0.75
0.5
0.25
Fold change (relative to WT)
b, conidiation
ab
0.75
0.5
0.25
c, germination
0.75
0.5
0.25
d, appressorium formation
0.75
0.5
0.25
N.D.
N.D.
N.D.
N.D.
Fig 3. Phenotypic characterization of knock‐out and ‐down mutants of MoSET1‐
dependent TFs in P. oryzae. a, Diameters of fungal colonies were measured at 9 days after inoculation on rich agar medium. b, Conidiation was evaluated by counting the number of conidia under a light microscopy as described in details in Materials and method. c‐d, The rates of conidial germination (c) and appressorium formation (d) were measured by observing conidial suspension on hydrophobic surface under a light microscope after 5 h (conidial germination) and 24 h (appressorium formation) incubation at 25̊C. Black bars indicate the wild‐type strain Br48 (WT) and grey bars represent knock‐
out and –down mutants of MoSET1‐dependent TFs and their gene complemented strains (c∆mgg_04699 and c∆mgg_06898). Data show fold change (relative to the wild‐type strain) ±standard error (n = 3). a‐d, Different characters in the graphs indicate significant differences by Tukey's HSD (P < 0.05). ND, not determined.
Dang et al. p.22 a
5G
2-3BG
5G
3-4BG
5B
5G
ab
(%)
100
Rates of infection
hyphae formation
80
60
40
20
Fig. 4. Inoculation test of knock‐out and ‐down mutants of MoSET1‐
dependent TFs in P. oryzae. a, Infection assay was performed on the wheat cultivar Norin 4 at 23̊C. Four to five days after inoculation, symptoms on the inoculated plants were evaluated. Letters under pictures of infected leaves indicate disease index values by a grading method (Hyon et al., 2012). This experiment was repeated at least three times, and representative samples are presented. b, The rates of infection hyphae formation in infected leaves. The black bar indicates the wild‐type strain Br48 (WT) and grey bars represent knock‐out and –
down mutants of MoSET1‐dependent TFs and a gene complemented strain (c∆mgg_04699). Error bars represent standard errors of the mean (n = 10). a‐d, Different characters in the graph indicate significant differences by Tukey's HSD (P < 0.05). Dang et al. p.23 MoSET1
TFs
vegetative growth
sporulation
appressorium formation
Fig. 5. Schematic diagram of putative MoSET1 regulatory network during infection‐related morphogenesis in P. oryzae.
virulence
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