Figures (7)  Tables (0)
    • Figure 1. 

      Response of apple rootstock 'M9T337' to F. solani infection and expression levels of MdWRKY-TFs after F. solani infection detected by qRT-PCR. (a) Images of 'M9T337' taken on the 3, 6, 9 and 12 dpi after infection by F. solani. Scale bar = 5 cm. (b)–(i) qRT-PCR was used to detect the expression level of MdWRKY2, MdWRKY4, MdWRKY10, MdWRKY20, MdWRKY25, MdWRKY33, MdWRKY44, MdWRKY58. For (b)–(i), values were means ± s.d. of three independent biological replicates. Bars not labeled with same letters in each panel indicate values are significantly different at p < 0.05, based on one-way ANOVA and Duncan's tests.

    • Figure 2. 

      Phylogenetic analysis and subcellular localization analysis of MdWRKY20. (a) Phylogenetic relationship and subgroup classification of MdWRKY20, AtWRKY, and other Group I WRKYs proteins. The WRKY20 sequence of apple and other species was derived from NCBI database (www.ncbi.nlm.nih.gov), and the WRKY protein sequence of Arabidopsis was derived from TAIR database (www.arabidopsis.org). The phylogenetic tree was created using MEGA software (version 5.1) by the adjacency method. MdWRKY20 is highlighted with black circles in the figure. (b) 35S::MdWRKY20-GFP vectors were transformed into the leaves of Nicotiana benthamiana. The nucleus were labeled with red fluorescent protein H2B. Three biological repeats were performed. Scale bar = 50 μm.

    • Figure 3. 

      Functional characteristics of 'Orin' callus overexpressing MdWRKY20. (a) MdWRKY20 CDs was inserted into PRI101-AN vector with CaMV 35S promoter and green fluorescent protein (GFP) sequence. (b) Phenotype of wild type (WT) and MdWRKY20-OE callus 4 d after the infection of F. solani. (c) RT-PCR based validation of MdWRKY20-OE in 'Orin' callus. (d) The diameter of the plaque extension of different callus after infection with F. solani. (c), (d), Values are means ± s.d. of three independent biological replicates. Bars not labeled with the same letters in each panel indicate values are significantly different at p < 0.05, based on one-way ANOVA and Duncan's tests.

    • Figure 4. 

      qRT–PCR analysis of MdPR1, MdPR2, MdPR3, and MdPR4 expressions in MdWRKY20-OE and wild type apple callus with F. solani infection. Values are means ± s.d. of three independent biological replicates. Bars not labeled with the same letters in each panel indicate values are significantly different at p < 0.05, based on one-way ANOVA and Duncan's tests.

    • Figure 5. 

      MdWRKY20 binds to the MdPR1 promoter. (a) The optimal 3-AT concentration was determined by cloning proMdPR1 into the pHIS2 vector. (b) MdWRKY20 interacted with MdPR1 promoter fragments as per the Y1H assay. (c) The W-box I and W-box II elements were used in the EMSA. (d) EMSA analysis revealed that MdWRKY20 binds to the W-box II of the MdPR1 promoter (e) Luciferase reporter (LUC) assays showed the MdWRKY20-mediated activation of proMdPR1.

    • Figure 6. 

      Germination percentage and phenotypic analysis of Arabidopsis under control and F. solani treatment. The germination rates of Arabidopsis seeds treated with (a) 0 mg/L , (b) 100 mg/L, and (c) 200 mg/L fermentation extract of F. solani. The phenotype of Arabidopsis was recorded by taking photos on the (d)−(f) 7 dpi, and (g) 21 dpi treated with (d) 0 mg/L, (e) 100 mg/L, and (f) 200 mg/L fermentation extract of F. solani. For (d)–(f) bars = 2 cm. (h) Root length of Arabidopsis after 7 d of treatment with control and F. solani. (i) Fresh weight of Arabidopsis after 7 d of treatment with control and F. solani. * 0.01 ≤ p < 0.05, ** 0.001 < p ≤ 0.01.

    • Figure 7. 

      Effects of F. solani treatment on several parameters of Arabidopsis leaves. The influence of F. solani treatment on (a), (b) active oxygen levels, (c)−(e) the activity of antioxidant enzymes, and (f) the content of malondialdehyde in Arabidopsis leaves. The (g) NBT staining, and (h) DAB staining of Arabidopsis leaves. For (a)–(f), values were means ± s.d. of three independent biological replicates. Bars not labeled with the same letters in each panel indicate values are significantly different at p < 0.05, based on one-way ANOVA and Duncan's tests. For (g) and (h), the deeper the color, the more content of O2 or H2O2 in plant tissues. Bars = 1 cm.