Fig 1.
Dendrogram showing relationships of wheat TaWRKY49 and TaWRKY62 with other plant WRKY proteins.
The GenBank accession numbers of the WRKY proteins used for constructing the phylogenetic tree are given in S2 Table. Ta, Triticum aestivum; At, Arabidopsis thaliana; Hv, Hordeum vulgare, Os, Oryza sativa. The phylogenetic tree was constructed with MEGA4.0 using a bootstrap test of phylogeny with a minimum evolution test and a parameter of 1000 replications.
Fig 2.
Expression levels of TaWRKY49 and TaWRKY62 exposed to high temperature after inoculation with Puccinia striiformis f. sp. tritici.
Expression levels of (A) TaWRKY49 and (B) TaWRKY62 subjected to low temperature (LT) (constant 15°C) and high temperature (HT) [15°C for 192 h after Pst inoculation, then 20°C for 24 h, and back to 15°C after inoculation with Puccinia striiformis f. sp. tritici (Pst). LT Mock, low temperature treatment without inoculation of Pst; HT Mock, high temperature treatment without inoculation of Pst. The arrow indicates the beginning of the HT treatment. Relative gene expression levels were related to the level observed at 0 hpi. Three biological replicates were performed independently for each treatment. Error bars indicate standard error.
Fig 3.
Phenotypes of TaWRKY49- and TaWRKY62-silenced leaves when inoculated with Pst subjected to low and high temperature.
The phenotypes of (A, B and C) TaWRKY49 or (D, E and F) TaWRKY62-silenced wheat leaves after Puccinia striiformis f. sp. tritici (Pst) inoculation at high temperature (HT) [15°C for the first 192 h post inoculation (hpi), then 20°C for 24 h, and back to 15°C] and low temperature (LT) (constant 15°C). (A, D) Mild chlorotic mosaic symptoms of BSMV at 9 days post-inoculation (dpi) (Mock: plants treated with FES buffer). Disease symptoms on the fourth leaves that were pre-inoculated with BSMV-derived RNAs, challenged with Pst race CYR32, and then subjected to (B, E) LT and (C, F) HT treatments. Disease symptoms were photographed on 14 dpi. Mock1 and Mock2: wheat plants were pre-inoculated with FES buffer, then inoculated with CYR32 and subjected to the LT and HT treatments, respectively. (G) Expression level of TaWRKY49 in the fourth leaves of the plants that were pre-inoculated with BSMV: 00 or BSMV: TaWRKY49-as on the second leaves, followed by inoculation of Pst subjected to the HT or LT treatment. (H) Expression level of TaWRKY62 in the fourth leaves of the plants that were pre-inoculated with BSMV: 00 or BSMV: TaWRKY62-as on the second leaves, followed by inoculation of Pst subjected to the HT or LT treatment. 0 hptt: 192 hours post-inoculation (hpi) of Pst from which HT was applied. Three biological replicates were performed independently for each treatment. Error bars indicate standard error.
Fig 4.
Histological observation of Puccinia striiformis f. sp. tritici (Pst) development in TaWRKY49-silenced leaves of Xiaoyan 6 at the low temperature (15°C) treatment.
Photographs were obtained from BSMV:00-infected (top panels) and BSMV:TaWRKY49-as-infected (bottom panels) leaves inoculated with Pst race CYR32 under an epifluorescence (left panels) or light microscope (right panels) at 120 h post-inoculation (hpi). SV, substomatal vesicle; IH, initial hyphae; HMC, haustorial mother cell; SH, secondary hyphae. Scale bars = 100 μm.
Table 1.
Histological observations of Puccinia striiformis f. sp. tritici (Pst) development and host responses in TaWRKY49- and TaWRKY62-silenced wheat leaves under the low temperature (LT, 15°C) treatment.
Fig 5.
Histological observation of TaWRKY49- and TaWRKY62-silenced wheat leaves subjected to high temperatures after inoculation with Pst.
(A) Puccinia striiformis f. sp. tritici (Pst) development in TaWRKY49-silenced leaves under the low temperature (LT) (constant 15°C) and high temperature (HT) [15°C for the first 192 h post-inoculation (hpi), then 20°C for 24 h, and back to 15°C] treatments: 1 = colony at LT, 2 = colony at HT, 3 = uredinia at LT, and 4 = uredinia at HT. SV = substomatal vesicle, IH = initial hyphae, HMC = haustorial mother cell, SH = secondary hyphae, NC = necrotic cell and U = uredinia. Scale bars = 100 μm. Photographs 1 and 2 were taken at 24 h post temperature treatment (hptt), while those of 3 and 4 were taken at 48 hptt. (B) Length of fungal colonies, (D) uredinium length and (F) number of necrotic cells from TaWRKY49-silenced leaves and from TaWRKY62-silenced leaves (C, E, G respectively) subjected to the LT and HT treatments after Pst inoculation. 0 hptt: 192 hours post inoculation (hpi), from which HT was applied. Error bars indicate standard error.
Fig 6.
Detection of reactive oxygen species in gene-silenced leaves exposed to high temperature after Pst inoculation.
(A) Histochemical localization of H2O2 (top panels) and O2− (bottom panels) at the infection sites in BSMV:TaWRKY49-as-inoculated leaves subjected to the high temperature (HT) [15°C for the first 192 post-inoculation (hpi), then 20°C for 24 h, and back to 15°C] after inoculation with Puccinia striiformis f. sp. tritici (Pst). Photographs were obtained using light microscopy or epifluorescence after the HT treatment. * = guard cells of stoma harboring substomatal vesicle or mesophyll cells in contact with hyphae showing reddish-brown (H2O2 accumulation) or blue (O2− accumulation) staining, SV = substomatal vesicle, IH = infection hyphae, U = uredinia. Bars = 100 μm. Percentages of infection sites exhibiting accumulation of (B) H2O2 and (C) O2− in the TaWRKY49-silenced leaves when exposed to the HT and low temperature (LT) (constant 15°C) after inoculation with Pst. Relative expression of catalase (CAT) and peroxidase (POD) (D, F) TaWRKY49-silenced and (E, G) TaWRKY62-silenced leaves exposed to LT and HT after Pst inoculation. 0 hptt: 192 hours post inoculation (hpi) from which HT was applied. Three biological replicates were performed independently for each treatment. Error bars indicate standard error.
Fig 7.
Relative expression analyses of defense-related genes in TaWRKY49- or TaWRKY62-silenced Xiaoyan 6 leaves of subjected to high temperature after inoculation with Puccinia striiformis f. sp. tritici (Pst).
A-F shows the expression level of pathogenesis-related (TaPR1.1), pathogen-induced ethylene response factor 1 (TaPIE1) and allene oxide synthase (TaAOS) genes in TaWRKY49-silenced or TaWRKY62-silenced leaves when exposed to the high temperature (HT) [15°C for the first 192 h post-inoculation (hpi), then 20°C for 24 h, and back to 15°C] and low temperature (LT) (constant 15°C) after inoculation with Pst. 0 hptt: 192 hours post inoculation (hpi) from which HT was applied. Three biological replicates were performed independently for each treatment. Error bars represent standard error.
Table 2.
Expression patterns of TaWRKY49 and TaWRKY62 under different abiotic stresses and in different wheat tissues.
Fig 8.
Model showing WRKY-mediated regulation of plant defense responses and signalings in high-temperature induced resistance to Pst.
The solid line with an arrow denotes enhancement; solid line without an arrow denotes suppression; solid line with an arrow and a symbol “?” denotes unknown; and the dotted lines denote no effect. Pst, Puccinia striiformis f. sp. tritici; JA, jasmonic acid; H2O2, hydrogen; SA, salicylic acid; ET, ethylene; and ROS, reactive oxygen species.