Figure 1.
Characterization and sequence analysis of GhWRKY40.
(A) Alignment of the amino acid sequences of GhWRKY40 and the representative related proteins AtWRKY18 (NP_567882), BnWRKY18 (ACN89257), AtWRKY40 (NP_178199) and BnWRKY40 (ACQ76806). Amino acids with 100% identity are shaded in black. The approximately 60-amino acid WRKY domain and the C and H residues in the zinc-finger motif (C-X4–5-C-X22–23-H-X1-H) are marked by a two-headed arrow and dot, respectively. The highly conserved amino acid sequence WRKYGQK in the WRKY domain is boxed. The putative nuclear localization signals are marked by lines. (B) Phylogenetic relationship between GhWRKY40 and other plant WRKY proteins. A neighbor-joining phylogenetic tree was created using MEGA 4.1 software. GhWRKY40 is boxed. Each gene name is followed by its protein ID. The abbreviations of the gene names are indicated as follows: Gh, Gossypium hirsutum; At, Arabidopsis thaliana; Os, Oryza sativa; Bn, Brassica napus; Cs, Cucumis sativus and Gm, Glycine max.
Figure 2.
Subcellular localization of the GhWRKY40 protein and transcriptional activation of the GhWRKY40 gene.
(A) Schematic diagram of the 35S-GhWRKY40::GFP fusion protein construct and the 35S-GFP construct. (B) Transient expression of the 35S-GFP and 35S-GhWRKY40::GFP constructs in onion epidermal cells. Green fluorescence corresponding to the expressed proteins was observed with a fluorescence microscope 24 h after particle bombardment. The nuclei of the onion cells were visualized by DAPI staining. (C) Transactivation of the GhWRKY40 gene in yeast. The vector pGBKT7 was used as a control. The transformed yeast culture was streaked onto SD/-Trp or SD/-Trp-His-Ade medium, and the α-galactosidase activity was determined. Three independent experiments were performed.
Figure 3.
Expression of the GhWRKY40 gene in response to stress.
Seven-day-old cotton seedlings in hydroponic culture were treated with R. solanacearum (A), wounding (B), 100 µM H2O2 (C), 100 µM MeJA (D), 2 mM SA (E) or 5 mM ET released from ethephon (F). Total RNA was isolated at the indicated times after the treatment and subjected to qPCR analysis. The ubiquitin gene was employed as an internal control. This experiment was repeated at least twice. The values indicated by the different letters are significantly different at P<0.01, as determined using Duncan's multiple range tests.
Table 1.
Putative cis-elements in the GhWRKY40 promoter.
Figure 4.
Analysis of ROS accumulation after wounding in WT and OE plants.
(A–B) Wound-induced H2O2 and O2− accumulation, as detected via DAB staining and NBT staining, respectively. (C) The phenotype of leaf disks from WT and OE plants that were incubated in different concentrations of MV (0, 200, 400 or 600 mM). (D) Relative chlorophyll content in the leaf disks from (C). Disks floated in water were used as a control. The presented data are the means ± standard error of three independent experiments. The different letters above the columns indicate significant differences (P<0.01) according to Duncan's multiple range test, which was performed using SAS version 9.1 software.
Figure 5.
qPCR analysis of stress-related gene expression in WT and OE plants under normal conditions (A) and after wounding (B).
The data are presented as the mean ± standard error of three independent experiments. The values indicated by the different letters are significantly different at P<0.01, as determined using Duncan's multiple range tests.
Figure 6.
GhWRKY40 overexpression enhances susceptibility to R. Solanacearum in transgenic plants.
(A) Phenotype of WT and OE lines after 5 days of incubation with R. solanacearum. (B–C) Relative transcript levels of defense-related genes in non-infected and infected WT and OE plants were analyzed by qPCR. The data are presented as the mean ± standard error of three independent experiments. The values indicated by the different letters are significantly different at P<0.01, as determined using Duncan's multiple range tests.
Figure 7.
Interaction between GhWRKY40 and GhMPK20/GhMPK6a in yeast and onion epidermal cells.
(A) Transformants grown on DDO, QDO or QDO/X/A. (B) In vitro BiFC analysis of the GhWRKY40 -interacting protein GhMPK20 in co-transformed into onion epidermal cells. The yellow fluorescence indicates the interaction between GhWRKY40 and GhMPK20. The fluorescent signals were observed using a confocal microscope. Scale bar = 20 µm.