Table 1.
Expression of WRKY genes in syncytia and control root segments.
Figure 1.
GUS expression of WRKY6, WRKY11 and WRKY17 in syncytia.
GUS staining of promoter::GUS lines for WRKY6, WRKY11 and WRKY17 was performed for 3, 5, 7, 12, and 15 dpi syncytia and uninfected roots. N = nematode, S = syncytia and bar = 500 µm in case of WRKY6 and WRKY11 and bar = 100 µm in case of WRKY17.
Figure 2.
Nematode resistance test for WRKY6 overexpression line and mutant.
The resistance of overexpression line of WRKY6 along with its knock out mutant compared to wild type plants after infection with H. schachtii. A: Number of male and female nemaodes per cm of root length calculated at 15 dpi setting the wild type as 100%. Different letters indicate significant differences (P<0.05; ANOVA and LSD). The statistical significance was determined by three independent replicates. Values are means ± SE, n = 15. The bar shows standard error for the mean. B: Size of female syncytia and female nematodes at 14 dpi. Ten syncytia were selected randomly from three independent replicates (total = 30) and the size of syncytia and associated female nematodes was determined. Data were analysed for significance difference using ANOVA (P<0.05) and LSD. Values are means ± SE.
Figure 3.
Nematode resistance test for WRKY11 and WRKY17 mutants.
The resistance of single and double mutants of WRKY11 and WRKY17 as compared to wild type plants after infection with H. schachtii. A: Number of male and female nemaodes per cm of root length calculated at 15 dpi setting the wild type as 100%. Different letters indicate significant differences (P<0.05; ANOVA and LSD). The statistical significance was determined by three independent replicates. Values are means ± SE, n = 15. The bar shows standard error for the mean. B: Size of female syncytia and female nematodes at 14 dpi. Ten syncytia were selected randomly from three independent replicates (total = 30) and the size of syncytia and associated female nematodes was determined. Data were analysed for significance difference using ANOVA (P<0.05) and LSD. Values are means ± SE.
Figure 4.
Expression of WRKY33 in response to nematode infection.
Expression of WRKY33 in wild type plants was determined by qRT-PCR in 5 and 15 dpi syncytia and uninfected root segments (containing elongation zones without root tips from 15-d-old seedling). The data included three independent biological and three technical replicates. The WRKY33 expression values are relative to un-infected control roots and were normalized using 18S as a housekeeping gene. Values are means ± SE, n = 3. The bar shows standard error for the mean.
Figure 5.
Analysis of pWRKY33::GUS expression in syncytia.
GUS staining of a pWRKY33::GUS line was performed for 1, 2, 5, 7, 10 and 15 dpi syncytia and uninfected roots. N = nematode, S = syncytia and bar = 100 µm.
Figure 6.
Nematode resistance test for WRKY33 overexpression lines.
The resistance of overexpression lines of WRKY33 with constitutive 35S promoter (A&D) and syncytia specific promoters Pdf2.1 (B&E) and Miox5 (C&F) was compared to wild type plants after infection with H. schachtii. A–C: Number of male and female nemaodes per cm of root length calculated at 15 dpi setting the wild type as 100%. Asterisks indicate significant differences (*, P<0.05 and **, P<0.01; ANOVA and LSD). The statistical significance was determined by three independent replicates. Values are means ± SE, n = 15. The bar shows standard error for the mean. D–F: Size of female syncytia and female nematodes at 14 dpi. Ten syncytia were selected randomly from three independent replicates (total = 30) and the size of syncytia and associated female nematodes was determined. Data were analysed for significance difference using ANOVA (P<0.05) and LSD. Values are means ± SE.
Figure 7.
Nematode resistance test for wrky33 knock out mutant.
The resistance of wrky33 knock out mutant (wrky33-1) was compared to wild type plants after infection with H. schachtii. A: Number of male and female nemaodes per cm of root length calculated at 15 dpi setting the wild type as 100%. Asterisks indicate significant differences (P<0.01; T-test). The statistical significance was determined by three independent replicates. Values are means ± SE, n = 15. The bar shows standard error for the mean.
Figure 8.
Nematode resistance test for MKK4DD overexpression lines.
The resistance of MKK4DD lines was compared to wild type plants after infection with H. schachtii. A: Number of male and female nemaodes per cm of root length calculated at 15 dpi setting the wild type as 100%. Different letters indicate significant differences (P<0.05; ANOVA and LSD). The statistical significance was determined by three independent replicates. Values are means ± SE, n = 15. The bar shows standard error for the mean. B: Size of female syncytia and female nematodes at 14 dpi. Ten syncytia were selected randomly from three independent replicates (total = 30) and the size of syncytia and associated female nematodes was determined. Data were analysed for significance difference using ANOVA (P<0.05) and LSD. Values are means ± SE.
Figure 9.
The pad3 mutant is more susceptible.
The resistance of pad3 knock out mutant compared to wild type plants after infection with H. schachtii. A: Number of male and female nemaodes per cm of root length calculated at 15 dpi setting the wild type as 100%. Asterisks indicate significant differences (P<0.05; T-test). The statistical significance was determined by three independent replicates. Values are means ± SE, n = 15. The bar shows standard error for the mean. B: Size of female syncytia and female nematodes at 14 dpi. Ten syncytia were selected randomly from three independent replicates (total = 30) and the size of syncytia and associated female nematodes was determined. Data were analysed for significance difference using T-test. Values are means ± SE.
Figure 10.
The WRKY33 pathway leading to camalexin as related to H. schachtii infection.
The phosphorylation cascade involving WRKY33 leading to camalexin. P, phosphorylation. ↓ indicates that the expression of the gene is downregulated in syncytia induced by H. schachtii as compared to control root segments while = indicates no change in expression according to data from Szakasits et al. (2009). + and – indicate that increase or decrease leads to enhanced resistance or susceptibility, respectively.