A Novel Wall-Associated Kinase TaWAK-5D600 Positively Participates in Defense against Sharp Eyespot and Fusarium Crown Rot in Wheat

Abstract

Sharp eyespot and Fusarium crown rot, mainly caused by soil-borne fungi Rhizoctonia cerealis and Fusarium pseudograminearum, are destructive diseases of major cereal crops including wheat (Triticum aestivum). However, the mechanisms underlying wheat-resistant responses to the two pathogens are largely elusive. In this study, we performed a genome-wide analysis of wall-associated kinase (WAK) family in wheat. As a result, a total of 140 TaWAK (not TaWAKL) candidate genes were identified from the wheat genome, each of which contains an N-terminal signal peptide, a galacturonan binding domain, an EGF-like domain, a calcium binding EGF domain (EGF-Ca), a transmembrane domain, and an intracellular Serine/Threonine protein kinase domain. By analyzing the RNA-sequencing data of wheat inoculated with R. cerealis and F. pseudograminearum, we found that transcript abundance of TaWAK-5D600 (TraesCS5D02G268600) on chromosome 5D was significantly upregulated, and that its upregulated transcript levels in response to both pathogens were higher compared with other TaWAK genes. Importantly, knock-down of TaWAK-5D600 transcript impaired wheat resistance against the fungal pathogens R. cerealis and F. pseudograminearum, and significantly repressed expression of defense-related genes in wheat, TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase4. Thus, this study proposes TaWAK-5D600 as a promising gene for improving wheat broad resistance to sharp eyespot and Fusarium crown rot (FCR) in wheat.

Keywords: Fusarium pseudograminearum; Rhizoctonia cerealis; sharp eyespot; wall-associated receptor-like kinase; wheat (Triticum aestivum).

Figure 1

Chromosomal distribution of the typical TaWAK genes in wheat. The 140 TaWAK genes were unevenly distributed on 20 wheat chromosomes. The bar indicates the length of chromosome in megabases (MB).

Figure 2

The transcript profiles of 140 TaWAKs in the wheat RNA-sequencing (RNA-seq) data. (A) The transcript levels of 140 TaWAKs upon R. cerealis infection in the resistant recombinant inbred lines (RILs) derived from the cross ‘Shanhongmai’ × ‘Wenmai 6′. (B) The transcript profiles of 140 TaWAKs upon F. pseudograminearum infection. The RNA-seq data upon F. pseudograminearum infection were checked from the online RNA-seq data (http://www.wheat-expression.com/ accessed on 25 August 2022) [32].

Figure 3

TaWAK-5D600 is involved in wheat responses to both sharp eyespot and Fusarium crown rot. (A) The transcript levels and fold change of the significantly upregulated 27 TaWAK genes in the RILs-R response to R. cerealis infection. (B) The transcript levels and fold change of the 27 R. cerealis induced TaWAK genes upon F. pseudograminearum infection in the online RNA-seq data (http://www.wheat-expression.com/ accessed on 25 August 2022) [32]. (C) Transcript levels of TaWAK-5D600 in sharp eyespot-resistant wheat line CI12633 at non-treatment and 4, 7, and 10 dpi with R. cerealis Rc207. (D) The transcript patterns of TaWAK-5D600 in FCR-mildly-resistant wheat line CI12633 at non-treatment and 1 and 2 dpi with F. pseudograminearum WHF220. TaWAK-5D600 transcript level at non-treatment is set to 1. TaActin gene was used as the internal control (t-test: * p < 0.05; ** p < 0.01).

Figure 4

Phylogenetic tree, conserved-domain, and gene-structure analyses of TaWAK-5D600. (A) A phylogenetic tree of TaWAK-5D600 and other 18 WAK proteins from different plants. The position of TaWAK-5D600 was indicated by a red blot. (B) Gene structure of TaWAK-5D600; black boxes represent exons and black lines indicate introns. (C) Schematic diagram of the TaWAK-5D600 protein. The conserved protein domains of TaWAK-5D600 were represented by different colored boxes. (D) Subcellular localization of TaWAK-5D600 in wheat protoplasts cells (bars = 20 μm).

Figure 5

Silencing of TaWAK-5D600-compromised wheat resistance both to sharp eyespot and Fusarium crown rot. (A) Typical BSMV symptoms on wheat leaves at 15 dpi infected with BSMV and transcripts of BSMV coat protein (CP) gene detecting by RT-PCR assays. (B) The silencing efficiency of TaWAK-5D600 detecting by RT-qPCR assay. The transcript level of TaWAK-5D600 in BSMV:GFP (control) wheat seedlings was set to 1. (C) Sharp eyespot symptoms on TaWAK-5D600-silenced and BSMV:GFP (control) wheat plants at 30 dpi with R. cerealis. (D) Disease indexes (DIs) of TaWAK-5D600-silenced and BSMV:GFP (control) wheat plants at 30 dpi with R. cerealis in two independent batches (t-test: ** p < 0.01). (E) Fusarium crown rot symptoms on TaWAK-5D600-silenced and control wheat plants at 30 dpi with F. pseudograminearum. (F) Disease index (DI) of TaWAK-5D600-silenced and control wheat plants at 30 dpi with F. pseudograminearum WHF220 in two independent batches (t-test: ** p < 0.01). Bars indicate SEs of the mean.

Figure 6

Transcript profiles of TaWAK-5D600 and defense-related genes in BSMV:GFP (control) and BSMV:TaWAK-5D600-infected wheat seedlings. Relative transcript abundances of TaWAK-5D600 and the tested genes TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase4 in BSMV: TaWAK5D600-infected CI12633 seedlings were quantified relative to those in BSMV:GFP (control) seedlings. Statistically significant differences were calculated based on three replications via a Student’s t-test (** p < 0.01). TaActin was used as an internal control.