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

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.


Introduction
As one of the most important staple cereal crops, bread wheat (Triticum aestivum) provides about a fifth of the total calories consumed around the world [1]. However, wheat grain production is threatened by various soil-borne viruses and fungal pathogens, such as the fungi Rhizoctonia cerealis and Fusarium pseudograminearum [2][3][4][5]. These two pathogens often cause stem necrosis and plant wilt of wheat, leading to severe production reduction and grain losses. Planting and application of resistant wheat cultivars are the most effective methods to control these soil-borne pathogens [6][7][8]. To improve wheat resistance against fungal diseases, it is vital to identify resistance or key immune-regulated genes in wheat.
Wall-associated kinase (WAK) proteins belong to a unique plant subfamily of receptorlike kinases (RLKs) and are likely linked to the cell wall by the galacturonan-binding domain [9,10]. Generally, the typical WAK proteins can be distinguished by several sequence characteristics, including an N-terminal signal peptide, an extracellular cell wall galacturonan-binding domain (GUB), an epidermal growth factor domain (EGF), a calciumbinding EGF domain (EGF-Ca), a transmembrane region (TM), and an intracellular conserved Serine/Threonine protein kinase (STK) domain [9,10]. The WAK-like (WAKL) 2 of 15 proteins usually contain the extracellular cell wall galacturonan-binding domain (GUB) and intracellular conserved Serine/Threonine protein kinase (STK) domain, while other domains vary among different WAKL proteins [9]. Several studies have demonstrated that some WAK or WAKL proteins play an important role in plant innate immunity to pathogens. The extracellular domains of WAKs or WAKL proteins are involved in cytoplasm-cell wall communication, while the intracellular kinase domain is related to the activation of cytoplasmic signaling cascades in plant defense responses [11]. When plants are invaded by pathogens, WAK or WAKL proteins can perceive short oligogalacturonic acid fragments (OGs) released by plant cell wall wounding, and trigger defense signaling in plants. In Arabidopsis, the AtWAK1 is significantly induced by OGs, initiating immunity against the necrotrophic fungus Botrytis cinerea [12]. AtWAKL22 and AtWAKL10 participate in resistance to Fusarium wilt disease [13] and defense against Pseudomonas syringae, respectively [14]. In maize (Zea mays), ZmWAK-RLK1 (encoded by Htn1) and ZmWAK qHSR1 confer quantitative resistance to the northern corn leaf blight and maize smut diseases, respectively [15,16]. In rice (Oryza sativa), OsWAK1, OsWAK14, OsWAK91, and OsWAK92 contribute to resistance against the blast fungus Magnaporthe oryzae [17,18], and the WAK gene Xa4 could confer durable resistance against Xanthomonas oryzae pv.oryzae [19]. In wheat (Triticum aestivum), Stb6, encoding a WAKL protein, confers resistance against Zymoseptoria tritici [20], and TaWAK6 participates in wheat adult plant resistance to leaf rust (Puccinia triticina) [21]. Our laboratory showed that two wheat WAK genes, TaWAK2A-800 and TaWAK-6D, and a TaWAKL gene TaWAK-7D (lacking the signal peptide in protein) participate in defense responses to Fusarium graminearum, R. cerealis, and F. pseudograminearum [22][23][24]. In Gossypium hirsutum, the wall-associated kinase GhWAK7A positively regulates cotton response to infections of both soil-borne fungal pathogens Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum [25].
Based on a high-quality version of a plant genome assembled, genome-wide identification of the WAK gene family has been reported in several plant species, such as Arabidopsis [9], rice [26], tomato [27], cotton [28], barley [29], and wheat [30]. For instance, in the Arabidopsis genome, there are five WAK and twenty-one WAKL genes, and the extracellular domain of WAKL proteins only show 18% to 22% identities to the WAKs [9]. In rice genome, 125 genes were identified as belonging to the WAK subfamily [26]. In tomato, 11 typical SlWAK and 18 SlWAKL genes were identified from the tomato genome, and these SlWAK or SlWAKL genes were distributed in nine of twelve chromosomes [27]. In cotton, 29 GhWAK genes were identified, and all cotton GhWAK proteins can be divided into five clades [28]. In barley genome, 91 HvWAK genes were identified [29]. Recently, 320 genes belonging to the WAK subfamily containing WAK_GUB domain, TM domain, and protein kinase domain were identified from wheat genome using the Pfam and SMART websites with three domains. They were divided into three phylogenetic groups [30]. However, the WAK and WAKL genes in wheat were not distinguished according to the typical characteristics of WAK kinase.
In this study, we genome-widely identified the typical WAK (not WAKL) genes in the wheat genome. According to the conserved characteristics of Arabidopsis WAK1-5, we identified a total of 140 TaWAK candidate genes in the wheat genome. These TaWAK genes were located on the 20 chromosomes of wheat genome, except chromosome 4B. By analyzing the RNA-sequencing (RNA-seq) data of wheat against R. cerealis and F. pseudograminearum, we found that a TaWAK gene TaWAK-5D600 (TraesCS5D02G268600) located on chromosomes 5D was significantly upregulated, and that its upregulated transcript levels in response to both pathogens were higher compared with other TaWAK genes. Knockdown of TaWAK-5D600 impaired wheat resistance against the fungal pathogens R. cerealis and F. pseudograminearum in wheat and repressed expression of defense-related genes in wheat, TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase4. This study proposes TaWAK-5D600 as a promising gene for enhancing broad resistance to sharp eyespot and Fusarium crown rot (FCR) in wheat. WAK proteins were characterized by an N-terminal signal peptide, a galacturonan  binding domain (GUB), an EGF-like domain, a calcium binding EGF domain (EGF-Ca), a transmembrane domain, and an intracellular C-terminal STK domain [9,25]. In this study, we used the five Arabidopsis AtWAK proteins as queries, and first performed a local BLAST search (e-value < 1 × 10 −10 ) against the wheat genome database [31]. Secondly, the conserved domains of these candidate proteins were manually predicted through the SMART website (http://smart.embl-heidelberg.de/ accessed on 24 August 2022). Based on the conserved characteristics of Arabidopsis WAK1-5, proteins lacking typical domains of WAKs were screened out from the candidate proteins. As a result, a total of 140 TaWAK candidate genes, encoding typical WAK proteins possessing an N-terminal signal peptide, a GUB domain, an EGF-like domain, an EGF-Ca domain, a transmembrane domain, and an intracellular conserved STK domain, were identified from the wheat genome [1]. These TaWAK genes were located on 20 wheat chromosomes, but not on chromosome 4B (Figure 1). The majority of TaWAK genes are tandemly arranged on each chromosome, and the quantities of TaWAK genes from the most to the least are chromosomes 6 (45 TaWAK genes), chromosomes 5 (33 TaWAK genes), chromosomes 2 (25 TaWAK genes), chromosomes 1 (20 TaWAK genes), chromosomes 7 (nine TaWAK genes), chromosomes 3 (five TaWAK genes), and chromosomes 4 (three TaWAK genes). The length of these TaWAK proteins ranged from 663 to 791 (amino acid, aa), with an average of 719.84 aa. The molecular weight values of these TaWAKs proteins ranged from 71.19 kDa to 87.05 kDa, with an average of 79.40 KDa, and isoelectric point (pI) values ranged from 4.89 to 8.66, with an average of 6.57 (Table S1). genes. Knock-down of TaWAK-5D600 impaired wheat resistance against the fung pathogens R. cerealis and F. pseudograminearum in wheat and repressed expression of de fense-related genes in wheat, TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase This study proposes TaWAK-5D600 as a promising gene for enhancing broad resistanc to sharp eyespot and Fusarium crown rot (FCR) in wheat.

Identification of Typical WAK Family Genes in Wheat
WAK proteins were characterized by an N-terminal signal peptide, a galacturona binding domain (GUB), an EGF-like domain, a calcium binding EGF domain (EGF-Ca), transmembrane domain, and an intracellular C-terminal STK domain [9,25]. In th study, we used the five Arabidopsis AtWAK proteins as queries, and first performed local BLAST search (e-value < 1 × 10 -10 ) against the wheat genome database [31]. Secondl the conserved domains of these candidate proteins were manually predicted through th SMART website (http://smart.embl-heidelberg.de/ accessed on 24 August 2022). Based o the conserved characteristics of Arabidopsis WAK1-5, proteins lacking typical domains o WAKs were screened out from the candidate proteins. As a result, a total of 140 TaWA candidate genes, encoding typical WAK proteins possessing an N-terminal signal pep tide, a GUB domain, an EGF-like domain, an EGF-Ca domain, a transmembrane domain and an intracellular conserved STK domain, were identified from the wheat genome [1 These TaWAK genes were located on 20 wheat chromosomes, but not on chromosome 4 ( Figure 1). The majority of TaWAK genes are tandemly arranged on each chromosom and the quantities of TaWAK genes from the most to the least are chromosomes 6 (4 TaWAK genes), chromosomes 5 (33 TaWAK genes), chromosomes 2 (25 TaWAK genes chromosomes 1 (20 TaWAK genes), chromosomes 7 (nine TaWAK genes), chromosomes (five TaWAK genes), and chromosomes 4 (three TaWAK genes). The length of these Ta WAK proteins ranged from 663 to 791 (amino acid, aa), with an average of 719.84 aa. Th molecular weight values of these TaWAKs proteins ranged from 71.19 kDa to 87.05 kD with an average of 79.40 KDa, and isoelectric point (pI) values ranged from 4.89 to 8.6 with an average of 6.57 (Table S1).

TaWAK-5D600 Are Involved in Wheat Responses against R. cerealis and F. pseudograminearum
To investigate the potential roles of TaWAKs in defense responses against infection of R. cerealis, we first analyzed the RNA-seq data of wheat inoculated with R. cerealis. Moreover, to explore which TaWAK genes were involved in wheat resistance to F. pseudograminearum, we also checked the transcript levels of whole 140 WAK genes in the online RNA-seq data (http://www.wheat-expression.com/ accessed on 25 August 2022) [32]. The results indicated that, after inoculating with R. cerealis or F. pseudograminearum, the expression of TaWAK genes presented a similar tendency in wheat, and compared with the mock treatment, 36 of the 140 TaWAK genes were upregulated after inoculating with both of the two pathogens (Figure 2A,B). This suggested that some TaWAK genes involved in defense responses might be conserved between R. cerealis and F. pseudograminearum.
To further analyze the transcript profiles of these upregulated TaWAK genes, we calculated the transcript changing fold of these TaWAK genes upon the R. cerealis. As shown in Figure 3A, among the whole 140 TaWAKs in wheat, 27 TaWAK genes were upregulated higher than 1.5-fold at 4 d post-inoculation (dpi) with R. cerealis compared to the mock treatment. Additionally, we also calculated the transcript change fold of these 27 TaWAK genes upon the F. pseudograminearum. In total, 11 of the 27 TaWAK genes were also upregulated higher than 1.5-fold at 36 h inoculated with F. pseudograminearum compared to the mock treatment ( Figure 3B). In particular, two homologous genes on chromosomes 5A and 5D, TraesCS5A02G261200 (named TaWAK-5A200) and TraesCS5D02G268600 (named TaWAK-5D600), showed higher transcript fold, and upregulated levels responding to both pathogens compared with other TaWAK genes. As shown in Figure 3A, the transcript levels of TaWAK-5A200 and TaWAK-5D600 were elevated to 4.17-fold and 4.33-fold at 4 dpi with R. cerealis compared with mock treatment and increased to 5.06-fold and 17.91-fold at 10 dpi with R. cerealis compared with mock treatment, respectively. Additionally, at 36 h inoculated with F. pseudograminearum, the transcript levels of TaWAK-5A200 and TaWAK-5D600 were elevated to 1.91-fold and 3.73-fold compared with mock treatment ( Figure 3B). These data suggest that the two homoeologous TaWAK genes, especially TaWAK-5D600, might be involved in wheat resistant responses to both sharp eyespot and FCR. Here, we focused on the functional investigation of TaWAK-5D600.
Subsequently, RT-qPCR assays were used to verify the transcript patterns of TaWAK-5D600 in wheat stems/sheaths infected in time-course by R. cerealis or F. pseudograminearum. As shown in Figure 3C, TaWAK-5D600 transcript was significantly upregulated after R. cerealis infection and peaked at 10 dpi (~8.56-fold over non-treatment). Furthermore, TaWAK-5D600 transcript was significantly upregulated toward F. pseudograminearum. As shown in Figure 3D, TaWAK-5D600 transcript abundance increased to 1.68-and 3.54-fold at 1 dpi and 2 dpi with F. pseudograminearum compared with non-treatment. Taken together, the transcript patterns of TaWAK-5D600 indicate that the gene was involved in wheat defense responses to both R. cerealis and F. pseudograminearum infections.

TaWAK-5D600 Is Required for Wheat Defense against R. cerealis and F. pseudograminearum
To identify the defense function of TaWAK-5D600 against R. cerealis and F. pseudograminearum in wheat, the TaWAK-5D600 was silenced by a Barley stripe mosaic virus (BSMV) induced silencing (VIGS) assay. As shown in Figure 5A, at 15 dpi infected by BSMV, typical stripe virus infection symptoms appeared on the wheat leaves and the TaWAK-5D600 sequence cloned from the resistant wheat cultivar CI12633 contains an open reading frame with 2232 bp length sequence ( Figure 4B). The predicted TaWAK-5D600 protein consists of 743 amino acid (aa) residues with a predicted Mw of 82.02 kDa and theoretical pI of 6.15. As shown in Figure 4C, the TaWAK-5D600 protein contains a signal peptide (no. 1-28 aa), a cell wall extracellular GUB domain (no. 35-142 aa), an EGF domain (no. 248-296 aa), an EGF-Ca domain (no. 297-336 aa), a transmembrane region (no. 348-370 aa), and an intracellular STK domain (no. 420-689 aa).
To investigate the sub-localization of TaWAK-5D600 in wheat cells, the p35S:GFP-TaWAK-5D600 fusion vector was constructed, and then the resulting p35S:GFP-TaWAK-5D600 vector or control p35S:GFP vector DNAs were separately introduced into wheat mesophyll protoplasts and transiently expressed ( Figure 4D). The confocal microscopic observation results showed that GFP-TaWAK-5D600 protein mainly distributed at the plasma membrane, while the control p35S:GFP protein diffused both in the nucleus and cytoplasm.
The results indicate that TaWAK-5D600 mainly localizes at the plasma membrane.

TaWAK-5D600 Is Required for Wheat Defense against R. cerealis and F. pseudograminearum
To identify the defense function of TaWAK-5D600 against R. cerealis and F. pseudograminearum in wheat, the TaWAK-5D600 was silenced by a Barley stripe mosaic virus (BSMV) induced silencing (VIGS) assay. As shown in Figure 5A, at 15 dpi infected by BSMV, typical stripe virus infection symptoms appeared on the wheat leaves and the transcript of BSMV coat protein (CP) gene was detected, which indicates the BSMV had successfully infected the test wheat plants. In addition, RT-qPCR assays results showed the transcript abundance of TaWAK-5D600 was significantly lower in the BSMV: TaWAK-5D600 plants than in BSMV:GFP (control) CI12633 plants, indicating the TaWAK-5D600 was successfully silenced in the VIGS wheat plants ( Figure 5B).
Subsequently, these TaWAK-5D600-silenced or BSMV:GFP (control) wheat seedlings were inoculated with R. cerealis strain Rc207 or F. pseudograminearum WHF220 as previously described [8,34]. At 30 dpi with R. cerealis Rc207, the TaWAK-5D600-silenced wheat seedlings displayed more serious disease symptoms of sharp eyespot, including bigger necrotic areas and higher infection types (ITs) ( Figure 5C). As shown in Figures 5D and S1, the disease severity quantified results showed that the average ITs of TaWAK-5D600-silenced wheat seedlings were 2.65 and 2.50, and the corresponding disease indexes (DIs) were 53 and 50, while the average ITs of BSMV: GFP (control) CI12633 seedlings were 1.40-1.45, and their disease indexes were 28 and 29 in two VIGS batches. These results indicate that TaWAK-5D600-silenced wheat plants were more susceptible than the control wheat plants, and the TaWAK-5D600 was required for wheat resistance against R. cerealis.
In addition, after inoculation with F. pseudograminearum WHF220, the TaWAK-5D600silenced wheat plants also exhibited greater disease severity of Fusarium crown rot symptoms than the BSMV:GFP-infected CI12633 plants ( Figure 5E). Furthermore, the disease severity quantified results showed that the average disease indexes were 38.8 and 37.6 (corresponding ITs of control wheat plants were only 2.9 and 2.8), while the average disease indexes of TaWAK-5D600-silenced plants were 67.5 and 61.2 (corresponding ITs were 5.0 and 4.5), respectively ( Figure 5F, Figure S2). These results suggest that TaWAK-5D600 was also required for wheat resistance to F. pseudograminearum.

TaWAK-5D600 Positively Regulated Expression of Several Defense-Related Genes
The RT-qPCR assays were used to examine transcription profiles of wheat defenserelated genes in TaWAK-5D600-silenced and BSMV:GFP (control) wheat seedlings. As shown in Figure 6, the transcript levels of TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase4 were significantly lower in TaWAK-5D600-silenced CI12633 wheat seedlings compared with the BSMV:GFP (control) wheat seedlings, suggesting that TaWAK-5D600 regulates positively the expression of several defense-associated genes in wheat seedlings.
transcript of BSMV coat protein (CP) gene was detected, which indicates the BSMV had successfully infected the test wheat plants. In addition, RT-qPCR assays results showed the transcript abundance of TaWAK-5D600 was significantly lower in the BSMV: Ta-WAK-5D600 plants than in BSMV:GFP (control) CI12633 plants, indicating the Ta-WAK-5D600 was successfully silenced in the VIGS wheat plants ( Figure 5B). fense-related genes in TaWAK-5D600-silenced and BSMV:GFP (control) wheat seedlings. As shown in Figure 6, the transcript levels of TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase4 were significantly lower in TaWAK-5D600-silenced CI12633 wheat seedlings compared with the BSMV:GFP (control) wheat seedlings, suggesting that Ta-WAK-5D600 regulates positively the expression of several defense-associated genes in wheat seedlings. 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.

Discussion
Genome-wide sequences analysis of the TaWAK genes could provide valuable information for identifying new resistant genes against fungal pathogens in wheat. Xia et al., using the Arabidopsis AtWAK/AtWAKL proteins from Arabidopsis as queries, filtered all candidate genes using three conserved domains of AtWAK/AtWAKL (WAK_GUB domain, TM domain, and protein kinase domain), eventually identifying 320 TaWAK genes in wheat genome [30]. However, in their study, the WAK and WAKL genes are not strictly distinguished according to the typical characteristics of WAK kinase. In this study, we mainly focused on the typical WAK (not WAKL) genes in the wheat genome. Using Arabidopsis WAK1-5 as queries, according to the conserved characteristics of WAK proteins (a WAK_GUB domain, an EGF-like domain, an EGF-Ca domain, a transmembrane domain (TM), and an intracellular cytosolic STK domain) [9,25], a total of 140 TaWAK candidate genes were identified from the wheat genome. This work will be helpful to better understand the roles of TaWAK gene members in wheat.
To further investigate the potential functions of TaWAK genes in defense responses against the two pathogens, we checked the transcript levels of whole 140 TaWAK genes in wheat RNA-seq data upon R. cerealis infection, and the online RNA-seq data upon F. pseudograminearum infection (http://www.wheat-expression.com/ accessed on 25 August 2022) [32]. The results indicated that with R. cerealis or F. pseudograminearum infection, the expression of TaWAK genes presented a similar tendency in wheat. It suggested that some TaWAK genes involved in defense responses were conserved between R. cerealis and F. pseudograminearum, and 11 TaWAK genes were significantly upregulated higher than 1.5-fold compared to the mock treatment both upon the R. cerealis and F. pseudograminearum infections. Among them, the two homologous genes (TraesCS5A02G261200, TraesCS5D02G268600), especially the TaWAK on chromosome 5D, showed higher transcript fold change, and higher transcript expression levels response to both pathogens compared with other TaWAK genes. Furthermore, the RT-qPCR assays also showed that the TaWAK-5D600 gene was involved in wheat defense responses both against R. cerealis and F. pseudograminearum, and it was therefore selected for further experimental analysis by the barley yellow dwarf virus (BSMV)-mediated gene silencing (VIGS) assays. Silencing of TaWAK-5D600 impaired wheat resistance to both of the two pathogens in wheat. Thus, our data revealed that TaWAK-5D600 was a promising gene for enhancing wheat broad resistance to both sharp eyespot and FCR. Besides resistance in planted wheat cultivars, the epidemics and severity of both sharp eyespot and FCR diseases are also influenced by temperature and humidity in diverse agro-ecological conditions. In this study, we mainly focused on the transcript changing fold of TaWAK genes upon the infection of the R. cerealis and F. pseudograminearum and validated the resistance function of TaWAK-5D600 to both sharp eyespot and FCR by VIGS assays. Additionally, by aligning the sequences of TaWAK-5D600 and allele genes in natural populations, we found that there are many variation sites in the genome sequence of TaWAK-5D600 among natural populations ( Figure S3, http://wheat.cau.edu.cn/WheatUnion accessed on 28 February 2023) [35], suggesting that the resistance function of TaWAK-5D600 might be differentiated among various wheat varieties. In the future, the gene may be applied in breeding resistant wheat varieties by molecular marker-assistant selection and geneediting methods.
The phylogenetic analysis results showed that the TaWAK-5D600 were clustered on the same branch with T. aestivum sharp eyespot and FCR-resistant protein TaWAK-6D [24] and sharp eyespot-resistant protein TaWAK-7D [23]. Meanwhile, pairwise sequence alignment showed that the TaWAK-5D600 protein only shared 40.65% and 49.68% proteins sequence identities with TaWAK-7D and TaWAK-6D, suggesting that TaWAK-5D600 is a novel WAK protein involved in wheat disease resistance responses. The current subcellularlocalization analysis showed that GFP-TaWAK-5D600 protein mainly distributed at the plasma membrane in wheat mesophyll protoplasts. This was consistent with the sublocalization of some WAKs proteins reported [23][24][25]36,37]. Many plasma membranelocalized RLK proteins were shown to be responsible for triggering a series of intracellular innate immune responses during pathogens infection. For instance, in cotton, silencing of GhWAK7A repressed the expression level of GhMPK3 and GhWRKY30 relative to control plants [25]. In rice, OsWAK25 overexpression increased the expression level of PR10 and PBZ1 [37]. The maize smut-resistant gene ZmqHSR1 could also elevate the transcript level of ZmPR-1 and ZmPR5 during pathogen infection [16]. In previous studies, a series of defense-related genes, such as TaSERK1, TaMPK3, TaPR1, TaChitinase 3, and TaChitinase 4, have been proven to be related to innate immunity of wheat to R. cerealis [38,39]. In this study, silencing of TaWAK-5D600 not only repressed the transcript levels of immunityrelated genes (TaSERK1, TaMPK3, TaPR1, TaChitinase 3, and TaChitinase 4) in wheat, but also compromised wheat resistance to both sharp eyespot and FCR infection. These data suggest that TaWAK-5D600 positively participates in wheat innate immunity against the two fungal pathogens.

Plants and Fungal Materials
A sharp eyespot-and Fusarium crown rot-medium-resistant wheat cultivar, CI12633, was used in the BSMV-mediated silencing assays. All wheat seedlings were cultured for 14 h under 23 • C light conditions and 10 h under 15 • C dark conditions in greenhouse. The sharp eyespot pathogen (Rhizoctonia cerealis strain Rc207) and Fusarium crown rot pathogen (Fusarium pseudograminearum strain WHF220) were isolated and donated by Prof. Jinfeng Yu and Dr. Li Zhang (Shandong Agricultural University, China). The sharp eyespot-resistant wheat RILs used in RNA-sequencing were kindly donated by Prof. Jizeng Jia (Chinese Academy of Agricultural Sciences).

Identification of WAK Gene Family Members in Wheat
The TaWAK genes were identified according to previously published methods [9,25]. At first, the protein sequences of AtWAK1-5 were used as queries to perform local BLAST searches (e-value < 1 × 10 −10 ) against the wheat genome database by utilizing the software Tbtools [1,31]. Subsequently, the conserved domains of the remaining candidate protein sequences were manually checked using the online SMART website (http://smart.emblheidelberg.de/ accessed on 24 August 2022), and proteins with typical characteristics of TaWAK proteins were screened out. Eventually, proteins with an N-terminal signal peptide, an extracellular cell wall galacturonan-binding domain (GUB), an epidermal growth factor domain (EGF), a calcium-binding EGF domain (EGF-Ca), a transmembrane region (TM), and an intracellular conserved Serine/Threonine protein kinase (STK) domain were regarded as TaWAK family members in wheat.

Phylogenetic, Gene Structure, and Conserved Motifs Analysis
The neighbor-joining method (MEGA 6.0) was used to construct the phylogenetic tree through the neighbor-joining method with 1000 bootstrap replicates. The basic characteristics of protein sequences were predicted through the online software ExPASy7 (https://www.expasy.org/ accessed on 27 August 2022).

RNA-Seq Data Analysis
At mock treatment and 4 and 10 dpi with R. cerealis infection, the leaf sheaths of the resistant recombinant inbred lines (RILs-R) wheat seedlings derived from the cross 'Shanhongmai' × 'Wenmai 6 were used for RNA extraction and RNA sequencing [22]. To explore which TaWAK genes were involved in wheat resistance to R. cerealis, we checked the transcript levels of whole 140 TaWAK genes in the RNA-seq data of R. cerealis infection.

VIGS and Assessment for Wheat Resistance to R. cerealis and F. pseudograminearum
The Barley stripe mosaic virus (BSMV)-mediated silencing (VIGS) assays were used to assess the resistant function of TaWAK-5D600 against R. cerealis or F. pseudograminearum. A 192 bp fragment of TaWAK-5D600 in the anti-sense orientation was sub-cloned into the RNA γ of BSMV, to form a BSMV: TaWAK-5D600 recombinant γ construct ( Figure S4). Subsequently, the BSMV: TaWAK-5D600 and BSMV: GFP virus construct were transcribed into RNAs in vitro and used to infect CI12633 wheat seedlings at the three-leaf stage. After 15 days infected with BSMV, the fourth leaves of the BSMV-infected wheat seedlings were sampled to evaluate the silencing efficiency of TaWAK-5D600. Eventually, the TaWAK-5D600-silenced and the BSMV: GFP-infected CI12633 wheat seedlings were further used in resistance assessment to sharp eyespot or FCR as previously described [8,34].

Conclusions
We identified a novel cell wall-associated kinase gene, TaWAK-5D600, in wheat defense responses to both R. cerealis and F. pseudograminearum infection. TaWAK-5D600 is required for wheat defense responses against the two pathogens and TaWAK-5D600 positively regulates the expression of several immunity-related genes, including TaSERK1, TaMPK3, TaPR1, TaChitinase 3, and TaChitinase 4. Thus, TaWAK-5D600 positively participates in defense against R. cerealis and F. pseudograminearum infections through modulating the transcript of several defense-related genes in wheat. TaWAK-5D600 is a promising gene for enhancing wheat broad resistance to both sharp eyespot and FCR.