Wheat transcription factor TaWRKY70 is positively involved in high-temperature seedling plant resistance to Puccinia striiformis f. sp. tritici

Abstract

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating disease of wheat (Triticum aestivum) worldwide. Wheat high-temperature seedling plant (HTSP) resistance to Pst is non-race-specific and durable. WRKY transcription factors have been proven to play important roles in plant defence responses to attacks by several pathogens. However, there is no direct evidence as to whether WRKY transcription factors play a role in HTSP resistance to Pst. We isolated a WRKY gene, named TaWRKY70, from wheat cultivar Xiaoyan 6. The expression level of TaWRKY70 was increased significantly when exposed to high temperatures (HTs) during the initial symptom expression stage of Pst infection. The expression of this gene increased in plants treated with ethylene (ET), salicylic acid (SA) and cold (4°C) stresses, but decreased in plants treated with methyl jasmonate (MeJA) and heat (40°C) stresses. Silencing of TaWRKY70 led to greater susceptibility to Pst (in terms of the increase in length of uredinial pustules and the decrease in the number of necrotic cells) compared with non-silenced plants when exposed to HT during the initial symptom expression stage of Pst infection, coinciding with expression changes of the ET- and SA-responsive genes TaPIE1 and TaPR1.1. In contrast, the expression level of the jasmonic acid (JA)-responsive gene TaAOS was not affected by TaWRKY70. These results indicate that TaWRKY70 is positively involved in HTSP resistance, during which SA and ET signalling are probably activated.

Keywords: Puccinia striiformis f. sp. tritici; WRKY70 transcription factor; high-temperature seedling plant resistance; virus-induced gene silencing.

Figure 1

Dendrogram of TaWRKY70 with other WRKYs from various plant species. The GenBank accession numbers of the WRKY proteins used for the construction of the phylogenetic tree are shown in Table S2 (see Supporting Information). At, Arabidopsis thaliana; Hv, Hordeum vulgare; Os, Oryza sativa; Ta, Triticum aestivum; Zm, Zea mays.

Figure 2

Expression profiles of TaWRKY70 in different wheat tissues. The values are means of three independent biological replicates. Error bars indicate standard errors. **Significant difference (Student's t‐test, P < 0.01) from the mean TaWRKY70 relative expression level in the leaf.

Figure 3

Expression patterns of TaWRKY70 under different stress conditions. (a) Expression levels of TaWRKY70 in response to low temperature (LT) (constant 15°C) and high temperature (HT) [15°C initially, then 20°C from 192 h post‐inoculation (hpi) for 24 h, and back to 15°C again] treatments after inoculation with Puccinia striiformis f. sp. tritici (Pst). LT mock, low temperature without inoculation of Pst; HT mock, high temperature without inoculation of Pst. The expression levels were determined by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis and were relative to the level observed at 0 hpi. The values are the means of three independent biological replicates. The error bars indicate standard errors. The arrow indicates the beginning of HT treatment. (b) Expression levels of TaWRKY70 in response to hormone treatment. ABA, abscisic acid; ET, ethylene; H₂O₂, hydrogen peroxide; MeJA, methyl jasmonate; SA, salicylic acid; hpt, hours post treatment. (c) Expression levels of TaWRKY70 in response to cold and heat stresses. The values are the means of three independent biological replicates. The error bars indicate standard errors. Analysis of variance (ANOVA) was conducted by Duncan's multiple range test (P = 0.05); the means of the TaWRKY70 expression levels do not differ significantly if they contain at least one common lowercase letter between time points for each treatment.

Figure 4

Functional analyses of TaWRKY70 during high‐temperature seedling plant (HTSP) resistance to Puccinia striiformis f. sp. tritici (Pst) using a Barley stripe mosaic virus (BSMV)‐induced gene silencing (VIGS) system. (a) Mild chlorotic mosaic symptoms of BSMV at 9 days post‐inoculation (dpi) (mock, plants treated with FES buffer). Disease symptoms on the fourth leaves pre‐inoculated with BSMV‐derived RNAs, challenged with Pst race CYR32 and then subjected to low‐temperature (LT, 15 ºC) (b) and high‐temperature (HT, 20 ºC) (c) treatments. 0 h post‐temperature treatment (hptt), 192 h post‐inoculation (hpi) from which HT was applied; disease symptoms were photographed at 14 dpi. Mock1 and Mock2, wheat plants were pre‐inoculated with FES, and then inoculated with CYR32 and subjected to LT and HT treatments, respectively. (d) Efficiency of silencing of TaWRKY70 by VIGS under LT treatment after Pst inoculation. **Student's t‐test (P < 0.01) indicates significant differences in the mean TaWRKY70 expression level between the BSMV:WRKY70‐as‐inoculated plants and BSMV:00‐inoculated plants. (e) Induction of TaWRKY70 in the fourth leaves pre‐inoculated with BSMV:00 or BSMV:TaWRKY70‐as under HT or LT treatments. The error bars represent standard errors. Analysis of variance (ANOVA) was conducted and followed by Duncan's multiple range test (P = 0.01); the means of the TaWRKY70 expression level do not differ significantly if they contain at least one common lowercase letter.

Figure 5

Histological observation of Puccinia striiformis f. sp. tritici (Pst) in the leaves of TaWRKY70‐knockdown wheat plants under the low‐temperature (LT, 15 ºC) treatment. Photographs were obtained from Barley stripe mosaic virus (BSMV):00‐infected (top panels) and BSMV:TaWRKY70‐as‐infected (bottom panels) leaves inoculated with Pst race CYR32 under an epifluorescence (left panels) or light (right panels) microscope at 120 h post‐inoculation (hpi). HMC, haustorial mother cell; IH, initial hyphae; SH, secondary hyphae; SV, substomatal vesicle. Bars, 100 μm.

Figure 6

Differences in Puccinia striiformis f. sp. tritici (Pst) development and the wheat response between TaWRKY70‐silenced and TaWRKY70‐induced plants during the initial symptom expression stage of stripe rust development. (a) Fungal colonies under low‐temperature (LT, 15 ºC) (top panels) and high‐temperature (HT, 20 ºC) (centre panels) treatments, as well as uredinia (bottom panels), under epifluorescence (left panels) or light (right panels) microscopy. HMC, haustorial mother cell; IH, initial hyphae; NC, necrotic cell; SH, secondary hyphae; SV, substomatal vesicle; U, uredinia. Bars, 100 μm. Colony length (b), uredinium length (c) and numbers of necrotic cells (d). 00, plants inoculated with BSMV:00 vectors were used as a control; TaWRKY70‐as, plants inoculated with BSMV:TaWRKY70‐as vectors for the knockdown of TaWRKY70. The values are the means of three independent biological replicates. The bars show standard errors. Analysis of variance (ANOVA) was conducted by Duncan's multiple range test (P = 0.01); the same lowercase letter indicates that the level does not differ significantly.

Figure 7

Transcriptional changes in the defence‐related genes TaPR1.1 (a), TaPIE1 (b) and TaAOS (c) in TaWRKY70‐induced [under high‐temperature (HT, 20 ºC) treatment] wheat leaves during the initial symptom expression stage of Puccinia striiformis f. sp. tritici development. The expression levels of these genes were determined by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The presented transcript levels are relative to the reference gene 26S (ATP‐dependent 26S proteasome regulatory subunit). The bars show standard errors. Analysis of variance (ANOVA) was conducted by Duncan's multiple range test (P = 0.01) to analyse the gene expression differences among different vector‐inoculated plants for each time point; the means of the gene expression levels do not differ significantly if they are indicated by the same lowercase letter.

Figure 8

Model of the regulation of TaWRKY70 in jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) signalling during the high‐temperature (HT)‐induced wheat defence responses to Puccinia striiformis f. sp. tritici (Pst).