The Wall-Associated Receptor-Like Kinase TaWAK7D Is Required for Defense Responses to Rhizoctonia cerealis in Wheat

Abstract

Sharp eyespot, caused by necrotrophic fungus Rhizoctonia cerealis, is a serious fungal disease in wheat (Triticum aestivum). Certain wall-associated receptor kinases (WAK) mediate resistance to diseases caused by biotrophic/hemibiotrophic pathogens in several plant species. Yet, none of wheat WAK genes with positive effect on the innate immune responses to R. cerealis has been reported. In this study, we identified a WAK gene TaWAK7D, located on chromosome 7D, and showed its positive regulatory role in the defense response to R. cerealis infection in wheat. RNA-seq and qRT-PCR analyses showed that TaWAK7D transcript abundance was elevated in wheat after R. cerealis inoculation and the induction in the stem was the highest among the tested organs. Additionally, TaWAK7D transcript levels were significantly elevated by pectin and chitin treatments. The knock-down of TaWAK7D transcript impaired resistance to R. cerealis and repressed the expression of five pathogenesis-related genes in wheat. The green fluorescent protein signal distribution assays indicated that TaWAK7D localized on the plasma membrane in wheat protoplasts. Thus, TaWAK7D, which is induced by R. cerealis, pectin and chitin stimuli, positively participates in defense responses to R. cerealis through modulating the expression of several pathogenesis-related genes in wheat.

Keywords: Rhizoctonia cerealis; defense response; wall-associated receptor kinase; wheat (Triticum aestivum).

Figure 1

Transcript profiles of TaWAK7D responding to Rhizoctonia cerealis in wheat. (A) The transcriptional level of TaWAK7D was obviously increasing in the resistant lines (RIL-R) than in the susceptible lines (RIL-S) at 4 and 10 dpi with R. cerealis. The expression level of TaWAK7D in RIL-S plants at mock was set to 1. (B) Transcript profiles of TaWAK7D in R. cerealis-resistant wheat line CI12633 at none, 1, 2, 4, 7 and 10 dpi with R. cerealis Rc207. TaWAK7D transcript level of none was set to 1. (C) Expression patterns of TaWAK7D in four wheat cultivars with different resistance degrees at 4 dpi with R. cerealis. The expression level of TaWAK7D in Yangmai 9 was set to 1. (D) Expression pattern of TaWAK7D in spikes, leaves, sheathes and stems of CI12633 at 4 dpi with R. cerealis or mock. The transcriptional level of TaWAK7D in spikes with mock treatment was set to 1. TaActin was used as the internal control. Significant differences were determined based on three technical repeats (Student’s t-test: * p < 0.05; ** p < 0.01). Bars indicate the standard error of the mean.

Figure 2

Sequence and phylogenetic analyses of TaWAK7D. (A) Gene structure of TaWAK7D. The white box indicates UTR, black boxes represent exons and black lines indicate introns. (B) Schematic diagram of the TaWAK7D protein. The colored regions indicate different domains. (C) A phylogenetic tree of TaWAK7D and 15 other WAK members from monocots and dicots. The bootstrapped phylogenetic tree was constructed using the neighbor-joining method (MEGA 6.0). The red blot indicates the position of TaWAK7D.

Figure 3

Subcellular localization of TaWAK7D in wheat protoplasts cells. The control GFP and fused TaWAK7D–GFP are transiently expressed in mesophyll protoplasts cells. The red colour was auto-fluorescence from wheat chloroplast. Scale bars = 20 μm (wheat protoplasts).

Figure 4

Schemata of recombinant BSMV:TaWAK7D construct and si-Fi software off-target prediction. (A) Schemata of recombinant BSMV:TaWAK7D construct. The orientation of the TaWAK7D insert is indicated by dark box. (B) SIFI software off-target prediction results.

Figure 5

Silencing of TaWAK7D impairs resistance of the wheat cv. CI12633 to R. cerealis. (A) Typical symptom of BSMV in the fourth leaves of wheat plants infected by BSMV:GFP or BSMV:TaWAK7D at 15 dpi with BSMV and RT-PCR analysis of the transcription of the BSMV CP gene. (B) qRT-PCR analysis of the relative transcript level of TaWAK7D in BSMV:GFP or BSMV:TaWAK7D wheat plants. The transcript level of TaWAK7D in BSMV:GFP-infected wheat CI12633 plants was set to 1. Significant differences were determined based on three technical repeats (t-test: * p<0.01) (C) Sharp eyespot symptoms of BSMV:TaWAK7D- and BSMV:GFP-inoculated CI12633 plants at 20 dpi with R. cerealis. Bar represents 1 cm. (D) Disease lesion size in TaWAK7D-silencing and BSMV GFP control CI12633 plants at 40 dpi with R. cerealis. Significant differences were determined based on 12 independent biological replications. Bars indicate standard error of the mean. (E) Sharp eyespot symptoms of the BSMV:GFP and BSMV:TaWAK7D-inoculated CI12633 plants at 40 dpi with R. cerealis. IT indicates sharp eyespot infection type of each wheat plant. (F) Mean infection types of CI12633 plants infected by BSMV:GFP or BSMV:TaWAK7D in three batches. Significant differences were determined based on 11–15 independent biological replications (Student’s t-test: * p < 0.05). Bars indicate standard error of the mean.

Figure 6

Trypan blue staining and RcActin transcript level in BSMV:TaWAK7D- and BSMV:GFP-infected wheat CI12633 plants. Trypan blue staining for the detection of the R. cerealis hyphae on the base leaf sheath of the BSMV:TaWAK7D (A) and BSMV:GFP (B) inoculated CI12633 plants at 4 dpi with R. cerealis Rc207. Bar = 20 μm. (C) qRT-PCR analysis of RcActin gene in stems of BSMV:TaWAK7D- and BSMV:GFP-infected wheat CI12633 plants at 10 dpi with R. cerealis. RcActin transcription represents the relative biomass of R. cerealis. Significant differences were determined based on three technical repeats (Student’s t-test: ** p < 0.01). The expression level of RcActin in BSMV:GFP-infected wheat CI12633 plants was set to 1.

Figure 7

Transcript profiles of TaWAK7D and pathogenesis-related genes in BSMV:GFP- and BSMV:TaWAK7D-infected wheat plants after infection by pathogens. Relative transcript abundances of β-1,3-Glucanase, PR1, PR17 Chitinase3 and Chitinase4 in BSMV:TaWAK7D-infected CI12633 plants were quantified relative to those in BSMV:GFP-infected control plants after R. cerealis inoculation for 10 days. Statistically significant differences between BSMV:TaWAK7D-infected and BSMV:GFP-infected wheat plants were determined based on three biological replications (Student’s t-test: ** p< 0.01). Bars indicate the standard error of the mean. TaActin was used as an internal control.

Figure 8

Transcript profiles of TaWAK7D responding to exogenous pectin and chitin treatments. (A) Transcript profiles of TaWAK7D in wheat cv. CI12633 leaves treated by 100 μg/mL exogenous pectin. (B) Transcript profiles of TaWAK7D in leaves of wheat cv. CI12633 after exogenous application of 100 μg/mL chitin. The transcript level of TaWAK7D in mock-treated wheat plants is set to 1. Statistically significant differences are analyzed based on three biological replications (Student’s t-test: ** p < 0.01). Error bars indicate the SE.