Skip to main content
NCBI home page
Molecular Plant Pathology logoLink to Molecular Plant Pathology
. 2018 Oct 16;19(12):2561–2574. doi: 10.1111/mpp.12729

A malectin‐like/leucine‐rich repeat receptor protein kinase gene, RLK‐V, regulates powdery mildew resistance in wheat

Ping Hu 1,2, Jiaqian Liu 1, Jiefei Xu 1, Chuanyu Zhou 1, Shuqi Cao 1, Weihao Zhou 1, Zhenpu Huang 1, Sufan Yuan 1, Xiue Wang 1, Jin Xiao 1, Ruiqi Zhang 1, Haiyan Wang 1, Shouzhong Zhang 1, Liping Xing 1,✉, Aizhong Cao 1,✉
PMCID: PMC6637979  PMID: 30030900

Summary

Pattern recognition receptors (PRRs) can trigger plant immunity through the recognition of pathogen‐associated molecular patterns. In this study, we report that a malectin‐like/leucine‐rich repeat receptor protein kinase gene, RLK‐V, from Haynaldia villosa putatively acts as a PRR to positively regulate powdery mildew resistance caused by Blumeria graminis f. sp. tritici (Bgt) in wheat. RLK‐V has two alternatively spliced transcripts corresponding to an intact RLK‐V1.1 and a truncated RLK‐V1.2 caused by intron retention. Expression analysis showed that both transcripts could be up‐regulated by Bgt in resistant materials, whereas the functional RLK‐V1.1 was expressed only after Bgt inoculation. Promoter activity assays indicated that RLK‐V could respond to Bgt even in susceptible wheat. Silencing of RLK‐V in Pm21‐carrying resistant materials resulted in compromised resistance to Bgt. In addition, over‐expression of RLK‐V1.1 in Pm21‐lacking susceptible Yangmai158 and SM‐1 by single‐cell transient expression and stable transformation in Yangmai158 could improve powdery mildew resistance. We propose that RLK‐V regulates basal resistance to powdery mildew, which is also required for broad‐spectrum resistance mediated by the Pm21 gene. Over‐expression of RLK‐V1.1 could trigger cell death in Nicotiana benthamiana, and RLK‐V1.1 transgenic wheat accumulated more reactive oxygen species and displayed a stronger hypersensitive response than did the recipient, which led to enhanced Bgt resistance. However, constitutive activation of RLK‐V1.1 resulted in the abnormal growth of transgenic plants.

Keywords: disease resistance, malectin‐like/leucine‐rich repeat receptor protein kinase, powdery mildew, RLK‐V, wheat.

Introduction

Plants have evolved a sophisticated defence mechanism to protect themselves from pathogen attack. Passive defence is mediated by physical and chemical barriers on the plant surface, and active immune defence includes pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI). Pattern recognition receptors (PRRs) play important roles in PTI by recognizing PAMPs, such as chitin in fungi, or damage‐associated molecular patterns (DAMPs), such as oligogalacturonides released by the attacked host (Boller and Felix, 2009; Macho and Zipfel, 2014). The structure and composition of PAMPs are often conserved for the entire class of a particular pathogen, and so the PTI induced by PAMPs is usually involved in basal immunity and broad‐spectrum resistance (Hurni et al., 2015). The broad‐spectrum resistance mediated by resistance (R) genes can also be activated by the recognition of PAMP‐like conserved factors for a particular pathogen (Zhu et al., 2017). Occasionally, there is crosstalk between the broad‐spectrum resistance pathway mediated by R genes and the basal resistance pathway mediated by PRRs (Kou and Wang, 2010).

PRRs are surface localized and have been shown to be either receptor‐like kinases (RLKs) or receptor‐like proteins (RLPs). RLKs typically contain an N‐terminal signal peptide, and extracellular ligand‐binding, transmembrane and cytoplasmic kinase domains, whereas RLPs lack the intracellular signalling domain (Fritz‐Laylin et al., 2005). According to the specific sequence motifs in the extracellular ligand‐binding domain, such as leucine‐rich repeats (LRRs), lectin‐like domain, lysine (Lys) motifs, cysteine‐rich domain, epidermal growth factor (EGF)‐like domain and malectin‐like domain (MLD), RLKs are grouped into several subfamilies (Wu and Zhou, 2013). Basal defence and some broad‐spectrum resistance are induced by PTI; for example, flagellin and elongation factor‐Tu (EF‐Tu) recognition by PRRs always leads to the rapid production of reactive oxygen species (ROS), the activation of mitogen‐activated protein kinases and calcium‐dependent protein kinases, and callose deposition (Boller and Felix, 2009).

Alternative splicing has been found to occur frequently in the primary transcripts of intron‐containing genes of eukaryotic organisms (Duan et al., 2016; Marquez et al., 2012), and can increase protein diversity, with different structures, functions or subcellular localization, and affect the stability of mRNA (Liu et al., 2018; Staiger and Brown, 2013). Alternative splicing is widely involved in development, particularly in plant defence responses. In rice, the LAMMER kinase gene, OsDR11, conferring resistance against Xanthomonas oryzae pv. oryzae is alternatively spliced, and the two forms of OsDR11 play opposite roles in the resistance pathway, with OsDR11L as a negative regulator and OsDR11S as a positive regulator. In addition, the transcription and kinase activity of OsDR11L is suppressed by OsDR11S (Duan et al., 2016). The tobacco N gene can generate two splice variants: before Tobacco mosaic virus (TMV) infection, the truncated transcript is prevalent, but, after TMV infection, the full‐length transcript becomes more prevalent. Both forms are required for full resistance to TMV. A similar phenomenon has also been observed in the Arabidopsis bacterial resistance gene RPS4 (Dinesh‐Kumar and Baker, 2000; Zhang and Gassmann, 2003, 2007 ). Medicago truncatula RCT1 gene‐mediated resistance to Colletotrichum trifolii also requires the regular and alternative transcripts (Tang et al., 2013). Much attention has been paid to the alternative splicing of R genes, but there is little known about the alternative splicing of RLKs. One recent study has reported that a Nicotiana tabacum S‐domain RLK gene has two different splice forms, with the full‐length transcript induced by lipopolysaccharide treatment, but the shorter transcript generated without treatment (Sanabria and Dubery, 2016).

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive diseases of wheat. The exploration and utilization of powdery mildew resistance genes in wheat breeding have been a focus for some time. Pm21, a broad‐spectrum powdery mildew resistance gene located on chromosome 6VS of the wild species Haynaldia villosa (2n = 14, VV), has received much attention and has been widely used in production. In a previous study, a cryptic alien chromatin introgression line, NAU427, harbouring the Pm21 locus, was developed and used to clone Pm21 successfully (Xing et al., 2018). A malectin‐like/LRR receptor protein kinase gene, RLK‐V, was also found to be located in the introgressed chromatin of NAU427. Here, we report that RLK‐V, with two transcript isoforms, putatively acts as a PRR to positively regulate powdery mildew resistance in wheat, in both the basal resistance stage and in a Pm21‐mediated broad‐spectrum resistance process.

Results

The malectin‐like/LRR RLK gene, RLK‐V, is alternatively spliced

NAU427 was identified as a cryptic introgression line carrying the Pm21 locus from the wild species H. villosa, and the genomic sequence of the introgression chromatin in NAU427 was determined by anchoring the molecular markers to the scaffolds via Chicago long‐range linkage by Dovetail Genomics (Santa Cruz, CA, USA) (Xing et al., 2018). In the introgressed segment, a malectin‐like/LRR RLK gene, RLK‐V, was identified. In this study, a pair of gene‐specific primers designed according to the genomic sequence of RLK‐V was used for physical mapping by polymerase chain reaction (PCR) (Table S1, see Supporting Information). A polymorphism occurred specifically in the resistant Pm21‐containing materials, including H. villosa, Triticum durum–H. villosa amphiploid (AABBVV), wheat–H. villosa translocation line T6VS·6AL and NAU427 (Fig. 1a), confirming that RLK‐V was indeed located in the H. villosa chromatin introgressed segment of NAU427, as predicted by sequencing. After BLAST searching the RNA‐Seq database of H. villosa, a cDNA sequence of RLK‐V with a predicted open reading frame (ORF) was obtained, and specific primers (Table S1) were then designed to clone the full‐length coding sequence.

Figure 1.

Figure 1

Open in a new tab

Chromosomal location, alternative splicing and sequence alignment of RLK‐V. (a) Chromosomal location of RLK‐V by polymerase chain reaction (PCR) using various cytogenetic stocks: 1, Chinese Spring (AABBDD); 2, Haynaldia villosa (VV); 3, Triticum durum–H. villosa amphiploid (AABBVV); 4, Nannong9918 (wheat–H. villosa translocation line T6VS·6AL); 5, NAU427 (resistant cryptic alien introgression with Pm21). The arrow indicates the 373‐bp band specific for RLK‐V. (b) The response pattern of RLK‐V1.1 and RLK‐V1.2 to Blumeria graminis f. sp. tritici (Bgt) in leaves of Shi4185R analysed by reverse transcription‐polymerase chain reaction (RT‐PCR). (c) The alternative splicing of RLK‐V. The sequence from 1323 to 1361 bp in the red frame indicates the alternative splice site, and the underlined ‘TAG’ indicates the introduced termination codon leading to the pre‐termination of RLK‐V1.2. (d) Analysis of the genomic structures of RLK‐V1.1 and RLK‐V1.2. ATG, translation start codon; TAG, translation stop codon. The numbers indicate the nucleotide length of each substructure. (e) Alignment of RLK‐V with its orthologues on the short arm of chromosome 6A, 6B and 6D of wheat. Sequences of RLK‐6A, RLK‐6B and RLK‐6D were downloaded from UniProtKB and the corresponding accession numbers were W5GEX4, A0A1D6B0L1 and A0A1D6BEW2, respectively. The red frame indicates the conserved GDPC motif, and the blue frames indicate the conserved VAVK, HRD and DFG motifs. LRR, leucine‐rich repeat; MLD, malectin‐like domain; TM, transmembrane domain. [Colour figure can be viewed at wileyonlinelibrary.com]

Surprisingly, two alternative transcripts, denoted RLK‐V1.1 and RLK‐V1.2, were obtained from H. villosa cDNA. The ORF of RLK‐V1.1 comprises a 2691‐bp nucleotide sequence encoding a predicted protein of 896 amino acids. The ORF of RLK‐V1.2 was 1326 bp in length, with an additional 39‐bp sequence that was retained from the fifth intron and a terminator codon was introduced at the nucleotide positions 1324–1326 bp; therefore, the predicted protein was truncated to 441 amino acids (Fig. 1c). The genomic sequence of RLK‐V1.1 consisted of 12 introns and 13 exons, and its alternative splicing in the fifth intron introduced a termination codon leading to the premature termination of RLK‐V1.2 (Fig. 1d). The two alternatively spliced transcripts were also detected in the wheat–H. villosa translocation T6VS·6AL cv. Nannong9918. RLK‐V1.1 protein was predicted to have an extracellular signal peptide domain (1–18 amino acids), an MLD (28–358 amino acids) and a complete LRR domain (412–483 amino acids) at the N‐terminus, and a transmembrane domain (TM; 517–539 amino acids) and an intracellular serine/threonine kinase domain (576–845 amino acids) at the C‐terminus. RLK‐V1.2 only contained the intact signal peptide domain and the MLD at the N‐terminus, but a truncated LRR domain (412–441 amino acids) at the C‐terminus (Fig. 1e).

A number of plant RLK family members were downloaded from the National Center for Biotechnology Information (NCBI) and UniProtKB databases and were used to construct a phylogenetic tree. As expected, five LysM‐RLKs, four lectin‐RLKs, four LRR‐RLKs, five CRK‐RLKs and seven WAK‐RLKs could be clustered into five subgroups, respectively. However, the four MLD‐RLKs displayed higher diversity and could not be clustered into a single branch. RLK‐V1.1, IOS1 and FRK1 were clustered into one separate branch, but HvLEMK1 was clustered into the LRR‐RLK subgroup because of its long LRR domain (Fig. S1, see Supporting Information).

RLK‐V is up‐regulated by Bgt, as revealed by gene expression analysis in Pm21‐containing resistant materials

The expression level of RLK‐V was analysed in four materials containing both the Pm21 and RLK‐V genes, including H. villosa with the whole VV genome, Nannong9918 with the whole chromosome arm 6VS, and Shi4185R and Yangmai158R with the cryptic introgressed segment of 6VS as in NAU427. In H. villosa, the expression level of RLK‐V was highest in the leaves, but very low in the stems, roots and spikes (Fig. 2a). The result also indicated that RLK‐V was up‐regulated by Bgt inoculation in H. villosa (Fig. 2b), Nannong9918 (Fig. 2c), Shi4185R (Fig. 2d) and Yangmai158R (Fig. 2e). Alternative splicing of genes in plants sometimes correlates with the sensing of external changes; therefore, the response of the two transcripts of RLK‐V to Bgt infection was studied in Shi4185R. Truncated RLK‐V1.2 was expressed constitutively and up‐regulated by Bgt, but RLK‐V1.1 expression was only induced by Bgt (Fig. 1b).

Figure 2.

Figure 2

Open in a new tab

Expression analysis of both transcripts of RLK‐V by quantitative real‐time reverse transcription polymerase chain reaction (qRT‐PCR). (a) Expression pattern of RLK‐V in different tissues of Haynaldia villosa. (b–e) The response patterns of RLK‐V to Blumeria graminis f. sp. tritici (Bgt) in the leaves of H. villosa (b), Nannong9918 (c), Shi4185R (d) and Yangmai158R (e) inoculated with Bgt. The values are the means of three technical replicates of one biological experiment. Error bars indicate the standard error. All three biological replicates showed similar results. Different letters indicate significant differences assessed using Duncan’s honestly significant difference test (P < 0.05).

Although RLK‐V is not Pm21 itself, the up‐regulated expression pattern of RLK‐V in all of the tested materials containing both the Pm21 and RLK‐V genes, and the alternative splicing correlating with Bgt inoculation, indicated that RLK‐V is closely involved in powdery mildew resistance.

The promoter of RLK‐V is Bgt responsive, as revealed by a promoter activity assay in susceptible materials

It would be interesting to study whether RLK‐V could also respond to Bgt in susceptible materials without Pm21. The lack of RLK‐V in common wheat means that it is impossible to analyse the gene expression level by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) using the primers specific to RLK‐V. Therefore, the activity of the native promoter of RLK‐V was tested in susceptible Yangmai158 and SM‐1 by a transient expression assay. The native promoter of the RLK‐V gene was cloned from the upstream region of the ORF with 2444 bp. An expression vector, pWMB002:RLK‐Vpro:GUS, was constructed by fusing the promoter of the RLK‐V gene to the β‐glucuronidase (GUS) gene which was served as a reporter. The native promoter showed stronger Bgt‐induced activity than did the constitutive maize ubiquitin promoter in both materials (Fig. 3a–i). Thus, RLK‐V could respond to Bgt not only in Pm21‐containing resistant wheat, but also in susceptible wheat.

Figure 3.

Figure 3

Open in a new tab

Activity assay of the RLK‐V native promoter. Leaves expressing the β‐glucuronidase (GUS) gene driven by the maize ubiquitin promoter (Ubi) without Blumeria graminis f. sp. tritici (Bgt) inoculation (a, e) and with Bgt inoculation (b, f). Leaves expressing the GUS gene driven by the native promoter of RLK‐V without Bgt inoculation (c, g) and with Bgt inoculation (d, h). (i) The numbers of Ubi‐driven GUS‐expressing cells in leaves without Bgt inoculation (Ubi) and with Bgt inoculation (Ubi+Bgt), and the numbers of P‐RLK‐V‐driven GUS‐expressing cells in leaves without Bgt inoculation (P‐RLK‐V) and with Bgt inoculation (P‐RLK‐V+Bgt). The values are the means of three biological replicates. **Significant differences with one‐way analysis of variance (ANOVA) least‐significant difference (LSD) analysis (P < 0.01). Scale bar, 100 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

A series of cis‐elements related to pathogen response and stress tolerance regulation were predicted using the promoter database PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al., 2002). Several pathogen‐responsive related motifs were found, such as ‘GACTTT’ motifs, which are the core sequence of the ‘WT‐box’, and ‘TTGACC’ motifs, which make up the ‘W‐box’ (Table S2, see Supporting Information).

Silencing of RLK‐V compromises powdery mildew resistance in Pm21‐containing materials

RLK‐V was up‐regulated and alternatively spliced by Bgt inoculation in Pm21‐containing materials; therefore, the Barley stripe mosaic virus‐based virus‐induced gene silencing (BSMV‐VIGS) system was used to characterize the function of the RLK‐V gene in Bgt‐resistant Nannong9918, Shi4185R and Yangmai158R. The nucleotide coding sequence of the kinase domain specific to the RLK‐V gene was designed to induce target silencing. The fourth fully expanded leaves of BSMV:RLK‐V‐infected individuals were detached, followed by inoculation with fresh Bgt conidia and RNA extraction; leaves of the same age from BSMV:γ‐infected individuals were collected as controls. The expression levels of the endogenous RLK‐V gene were significantly decreased, by 5–10‐fold, in the fourth leaves, as assessed by qRT‐PCR (Fig. 4a1–a3). Microscopic observation of Bgt stained with Coomassie blue showed obvious hyphal and conidiophore development at 60 h after infection (hai) (Figs 4b1,b2, S2a1,a2, see Supporting Information), at 4 days after infection (dai) (Figs 4c1,c2, S2b1,b2) and at 7 dai (Figs 4d1,d2, S2c1,c2) in BSMV:RLK‐V‐infected leaves in comparison with fungal development in the controls in Nannong9918. Similar results were obtained in Shi4185R (Fig. 4e1,e2) and Yangmai158R (Fig. 4f1,f2) with BSMV‐VIGS; the Bgt hypha developed normally in BSMV:RLK‐V‐infected leaves. Therefore, silencing of the RLK‐V gene could compromise the resistance mediated by Pm21.

Figure 4.

Figure 4

Open in a new tab

Functional analysis of RLK‐V by Barley stripe mosaic virus‐based virus‐induced gene silencing (BSMV‐VIGS) in three Pm21‐carrying resistance materials. (a1–a3) Expression of RLK‐V at 14 days in BSMV:RLK‐V‐infected leaves was compared with that in BSMV:γ‐infected controls of Nannong9918 (a1), Shi4185R (a2) and Yangmai158R (a3). CK represents plants inoculated with BSMV:γ, and 1 and 2 represent plants inoculated with BSMV:RLK‐V. The values are the means of three technical replicates of one biological experiment. Error bars indicate the standard error. **Significant differences assessed using one‐way analysis of variance (ANOVA) least‐significant difference (LSD) analysis (P < 0.01). Development of Blumeria graminis f. sp. tritici (Bgt) in BSMV:RLK‐V‐infected leaves using BSMV:γ‐infected leaves as a control. Bgt development at 60 h after infection (hai) (b1, b2), 4 d after infection (dai) (c1, c2) and 7 dai (d1, d2) in Nannong9918, and at 7 dai in Shi4185R (e1, e2) and Yangmai158R (f1, f2). Arrows indicate spore (Sp), appressorium (App), secondary hyphae (SH) and conidiophore (CH). Scale bar, 50 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Transient over‐expression of RLK‐V1.1, but not RLK‐V1.2, improves Bgt resistance in susceptible Yangmai158 and SM‐1

The single‐cell transient over‐expression assay has been successfully used to elucidate gene function involved in the resistance against Bgt infection in barley and wheat (Cao et al., 2011; Chen et al., 2016; Shirasu et al., 1999;Yahiaoui et al., 2006). GUS‐stained epidermal cells attacked by Bgt spores were selected to observe haustorium formation and to calculate the haustorium index (HI). In leaves of the susceptible variety Yangmai158, transient over‐expression of RLK‐V1.1 significantly decreased the HI of Bgt‐interacting epidermal cells from 51.1% in GUS gene‐transformed leaves to 34.1% in GUS+RLK‐V1.1‐co‐transformed leaves; however, the over‐expression of RLK‐V1.2 did not change the HI (Fig. 5a). In the leaves of the susceptible mutant SM‐1, transient over‐expression of RLK‐V1.1 significantly decreased the HI of Bgt‐interacting epidermal cells from 68.8% in GUS gene‐transformed leaves to 41.7% in GUS+RLK‐V1.1‐co‐transformed leaves, which was not significantly different from the HI of GUS‐transformed leaves of Nannong9918 (Fig. 5b). Although RLK‐V1.1 could repress HI formation significantly, we also observed that hyphae developed quickly in haustorium‐formed RLK‐V1.1‐transformed cells in SM‐1, whereas there was no hyphal development at all in Nannong9918. The results indicate that RLK‐V1.1 positively regulates powdery mildew resistance by the inhibition of haustorium formation of Bgt; however, the splice variant RLK‐V1.2 does not improve resistance.

Figure 5.

Figure 5

Open in a new tab

Powdery mildew resistance analysis of RLK‐V by single‐cell transient over‐expression and stable transformation (a) Haustorium index (HI) in GUS+RLK‐V1.1 and GUS+RLK‐V1.2 co‐transformed cells in comparison with that in GUS‐transformed cells of susceptible Yangmai158. (b) HI in GUS+RLK‐V1.1 co‐transformed cells in comparison with that in GUS‐transformed cells in susceptible SM‐1 and resistant Nannong9918. Different letters indicate significant differences assessed using Duncan’s honestly significant difference test (P < 0.05). (c, d) Powdery mildew resistance evaluation of RLK‐V1.1‐ and RLK‐V1.2‐over‐expressing transgenic plants in the T0 generation. Yangmai158, the negative plant RLK‐V1.1‐T0‐165 and RLK‐V1.2‐T0‐10 were used as susceptible controls, and Nannong9918 was used as the resistant control. Photographs were taken at 7 days after Blumeria graminis f. sp. tritici (Bgt) inoculation. The red arrow indicates cell death. (e) The derived T1 generation plants over‐expressing RLK‐V1.1 were also evaluated for Bgt resistance, and the negative RLK‐V1.1‐T1‐165 was used as a susceptible control. Photographs were taken at 7 days after Bgt inoculation. The red arrow indicates cell death. [Colour figure can be viewed at wileyonlinelibrary.com]

Stable over‐expression of RLK‐V1.1 enhances the powdery mildew resistance of the susceptible recipient Yangmai158

Over‐expression vectors of pBI220:RLK‐V1.1 and pBI220:RLK‐V1.2 were co‐transformed with pAHC20 (carrying a bar gene as a selection marker) by particle bombardment into the callus induced from immature embryos of Yangmai158, separately. PCR analysis identified eight and four independent T0 generation transgenic plants from the 375 and 145 regenerated plants, respectively (Fig. S3a,b, see Supporting Information). Powdery mildew resistance evaluation at the seedling stage indicated that Yangmai158, as the susceptible recipient control, showed high susceptibility with a grade 4 scale infection type after inoculation with Bgt. Nannong9918, as the resistant control, showed high resistance to Bgt with a grade 0 scale infection type; all eight RLK‐V1.1‐positive transgenic plants showed enhanced resistance to Bgt with grade 0–2 scale infection type (Fig. 5c); however, the four RLK‐V1.2‐positive transgenic plants still showed high susceptibility, with grade 4 scale infection type, similar to Yangmai158 (Fig. 5d). The four selected T1 generation positive transgenic lines, RLK‐V1.1‐T1‐149, RLK‐V1.1‐T1‐203, RLK‐V1.1‐T1‐206 and RLK‐V1.1‐T1‐228, which were progenitors derived from the corresponding T0 generation plants, were further identified as positive transgenic lines by PCR (Fig. S3c). The qRT‐PCR results of the above four lines indicated that RLK‐V1.1 expression levels were significantly increased compared with that in the non‐transformed Yangmai158 and in one negative transgenic control plant (Fig. S3d). The four lines also exhibited strong cell death after Bgt inoculation and increased resistance to Bgt (Fig. 5c,e). These results indicate that RLK‐V1.1 contributes to the enhanced resistance of transgenic plants to wheat powdery mildew, and induced cell death may play an important role.

Resistance mediated by RLK‐V1.1 is associated with H2O2 accumulation and cell death

H2O2 accumulation is reported to be involved in cell death during plant–pathogen interactions. To study whether H2O2 accumulation correlates with RLK‐V1.1‐mediated resistance, H2O2 accumulation at infection sites and the development of Bgt of the positive transgenic line RLK‐V1.1‐T1‐228 were compared with those of the negative transgenic line RLK‐V1.1‐T1‐165 and Yangmai158 at 24, 48 and 72 hai. Based on histological observations, the infected cells were classified into three types: type a, no H2O2 production; type b, H2O2 accumulation only at the interaction sites; type c, H2O2 accumulation in whole cells (Fig. 6a). Statistical analysis at 72 hai showed that a higher percentage of ‘type b’ and ‘type c’, but a lower percentage of ‘type a’, infected cells were observed in the positive transgenic line RLK‐V1.1‐T1‐228 than in the negative transgenic line RLK‐V1.1‐T1‐165 and Yangmai158 (Fig. 6c). In addition, the number of Bgt colonies and the hyphal branches of each colony in RLK‐V1.1‐T1‐228 were lower than those in Yangmai158 and RLK‐V1.1‐T1‐165 (Fig. 6b). This finding indicates that H2O2 may mediate a critical resistance pathway and induce cell death, which may help to suppress the growth of Bgt fungus in RLK‐V1.1‐over‐expressing plants.

Figure 6.

Figure 6

Open in a new tab

Reactive oxygen species (ROS) accumulation and the development of Blumeria graminis f. sp. tritici (Bgt) in transgenic plants and controls. (a) Three types of Bgt‐infected cell. Type a, no H2O2 production; type b, H2O2 accumulation only at the interaction sites; type c, H2O2 accumulation in the whole cell. H2O2 accumulation and Bgt development in wheat leaves of Yangmai158 (b1–b3), the positive transgenic line RLK‐V1.1‐T1‐228 (b4–b6) and the negative control RLK‐V‐T1‐165 (b7–b9) at 24, 48 and 72 h after infection (hai). The arrows indicate H2O2 accumulation. Scale bar, 100 μm. (c) Comparison of the numbers of the three types of infected cell at 72 hai. Different letters indicate significant differences assessed using Duncan’s honestly significant difference test (P < 0.05). [Colour figure can be viewed at wileyonlinelibrary.com]

Transient over‐expression of RLK‐V1.1 can trigger strong cell death in Nicotiana benthamiana

The intact RLK‐V1.1 and truncated RLK‐V1.2 (Fig. 7a) were fused to a C‐terminal green fluorescent protein (GFP) tag under the control of the 35S promoter. To study whether RLK‐V could trigger cell death, the fused gene was transiently over‐expressed in N. benthamiana leaves using Agrobacterium‐mediated infiltration. At approximately 40 h after Agrobacterium‐infiltration, obvious cell death was induced by the intact RLK‐V1.1, but not by truncated RLK‐V1.2 (Fig. 7b). This result indicates that the intact RLK‐V1.1 protein is partially auto‐active in N. benthamiana.

Figure 7.

Figure 7

Open in a new tab

Over‐expression of RLK‐V1.1 triggers cell death in Nicotiana benthamiana. (a) Schematic diagrams of the RLK‐V1.1 and RLK‐V1.2 domain structures. Individual domains of RLK‐V are represented by different coloured boxes. LRR, leucine‐rich repeat; MLD, malectin‐like domain; SP, signal peptide TM, transmembrane domain. (b) RLK‐V‐GFP fusion proteins were transiently expressed by Agrobacterium infiltration in N. benthamiana, and cell death triggered by each protein was visualized at 40 h after infection (hai) before and after staining with trypan blue. Red circles indicate cell death triggered by RLK‐V1.1 in the infiltrated area, and white circles indicate no cell death. GFP, green fluorescent protein. [Colour figure can be viewed at wileyonlinelibrary.com]

Discussion

RLK‐V1.1 positively regulates Bgt resistance in the basal resistance pathway and in the Pm21‐mediated broad‐spectrum resistance pathway

Several MLD‐RLKs have been identified to be involved in plant immune responses, especially in PTI. For example, IOS1 not only forms a complex with FLS2 and EFR to mediate BAK1‐dependent PTI, but also forms a complex with CERK1 to mediate BAK1‐independent PTI (Yeh et al., 2016). FRK1 is regulated by AtWRKY6 to mediate flg22‐triggered PTI (Asai et al., 2002). HvLEMK1 contributes to non‐host resistance to Bgt in barley and quantitative host resistance to Bgt in wheat (Rajaraman et al., 2016).

RLK‐V silencing by BSMV‐VIGS in Nannong9918, Shi4185R and Yangmai158R significantly compromised resistance, leading to the accelerated growth of Bgt; however, resistance was not completely lost in the gene‐silenced leaves. Therefore, it implied that the RLK‐V1.1 gene is involved in the Pm21‐mediated resistance pathway. In addition, RLK‐V was also up‐regulated greatly by chitin treatment (Fig. S4, see Supporting Information). More importantly, the RLK‐V1.1 transgenic lines showed quantitative resistance to wheat powdery mildew. Therefore, we suspect that RLK‐V1.1 is involved in the basal resistance in wheat. We hypothesize that RLK‐V is involved in PAMP recognition, leading to PTI activation at the early stage of Bgt infection, and the activated immunity response overlaps with and is required by the Pm21‐mediated broad‐spectrum resistance pathway.

Resistance mediated by RLK‐V1.1 is tightly correlated with ROS accumulation and cell death

Cell death was thought to be one of the most important strategies for plants to resist the invasion of biotrophic pathogens (Gill et al., 2015), and ROS has been found to play an important role in plant–pathogen interactions by acting as a cell death inducer, signal transduction factor or direct antimicrobial molecule (Thordal‐Christensen et al., 1997). Cell death has also been found to contribute to resistance pathways mediated by RLKs. Over‐expression of Arabidopsis CRK28 increased disease resistance to Pseudomonas syringae and induced spontaneous cell death in both Arabidopsis and N. benthamiana, but few transformants were obtained from Col‐0 transformed with 35S:CRK28; furthermore, the surviving T1 transgenics exhibited abnormal development and low fertility (Yadeta et al., 2017). Similar phenomena have also been observed in transgenic plants of CRK4, CRK5, CRK13, CRK19 and CRK20 (Acharya et al., 2007; Chen et al., 2003, 2003). Over‐expression of pathogen‐inducible Arabidopsis LecRK‐IX.1 and LecRK‐IX.2 resulted in elevated ROS and enhanced systemic acquired resistance to P. syringae pv. tomato DC3000; at the same time, it induced spontaneous cell death in both Arabidopsis and N. benthamiana (Luo et al., 2017; Wang et al., 2015). LecRK‐V, a lectin‐like receptor kinase protein, can enhance wheat resistance to powdery mildew by inducing strong cell death and ROS accumulation (Wang et al., 2018). In our study, over‐expression of RLK‐V1.1 in N. benthamiana showed a spontaneous cell death phenotype. In addition, ROS accumulation and cell death were also observed in RLK‐V1.1 transgenic plants. Therefore, the effect of RLK‐V1.1 on powdery mildew resistance is perhaps tightly correlated with its ability to enhance ROS accumulation and trigger cell death.

Sequence comparison of RLK‐V with the identified functional MLD‐RLKs and its orthologous genes in wheat

The protein sequences of RLK‐V1.1, IOS1, FRK1 and HvLEMK1 were compared, and the similarity between RLK‐V1.1 and the other three RLKs was lower than 40%. The protein structure and phylogenetic analysis indicated that RLK‐V1.1 was evolutionarily similar to IOS1 and FRK1, but distant from HvLEMK1 (Fig. S1). In the extracellular part of RLK‐V1.1, the MLD is upstream of a short LRR domain, and a conserved glycine‐aspartic acid‐proline‐cysteine (GDPC) sequence is located in the interspace, as in IOS1 and FRK1. However, in HvLEMK1, the MLD is downstream of a long LRR domain, but the GDPC motif is absent. In the intracellular part of RLK‐V1.1, the RD‐kinase domain possesses highly conserved valine‐alanine‐valine‐lysine (VAVK), histidine‐arginine‐aspartic acid (HRD) and aspartic acid‐phenylalanine‐glycine (DFG) motifs, as in IOS1 and FRK1, which are necessary for catalytic activity and autophosphorylation of the activation loop (Johnson et al., 1996). However, in HvLEMK1, the VAVK motifs is absent.

RLK‐V1.1 was compared with its orthologous genes from common wheat. RLK‐V1.1 shared the highest homology with an orthologous gene from chromosome 6B, whereas the sequence from chromosome 6D was most divergent (Fig. 1e). Nine amino acid polymorphisms are specific to RLK‐V1.1, which are located in the MLD, LRR, transmembrane and kinase regions (Fig. 1e). It will be studied whether these polymorphisms have effects on immunity function. RLK‐V was strongly up‐regulated by Bgt inoculation, but the orthologue of RLK‐V from common wheat cannot be up‐regulated or is only slightly up‐regulated. Thus, the cis‐acting elements were also compared by multiple sequence alignment. It was found that the ‘GACTTT’ motif in the promoter of RLK‐V was absent in its orthologues (Fig. S5, see Supporting Information). The ‘GACTTT’ motif, identified as the core sequence of the WT‐box, was found in six MAMP‐responsive sequences and was reported to interact with WRKY70 (Machens et al., 2014). In a future study, the core cis‐acting element responsive to Bgt will be determined by promoter activity assays.

Alternative splicing of RLK‐V balances development and disease resistance

Alternative splicing is a basal mechanism of plants to address environmental stresses (Liu et al., 2018), and many cases of alternative splicing of resistance genes during pathogen invasion have been reported. The Arabidopsis bacterial resistance gene RPS4 has a different alternatively spliced transcript when challenged with pathogens. When truncated RPS4 proteins are transiently expressed in N. benthamiana, they can induce cell death without recognition of AvrRps4, and the different spliced proteins possess different protein stabilities (Zhang and Gassmann, 2007). The powdery mildew resistance genes MLA6 and MLA13 are alternatively spliced during barley–pathogen interactions (Halterman et al., 2003). In this study, the truncated transcript RLK‐V1.2 was expressed without Bgt inoculation and increased after Bgt inoculation, but the alternatively spliced transcript RLK‐V1.1 occurred only after Bgt invasion. Functional RLK‐V1.1 could induce cell death in N. benthamiana by transient assay, and over‐expression of RLK‐V1.1 in wheat could also induce cell death after Bgt inoculation. However, truncated RLK‐V1.2 could not induce cell death in N. benthamiana or enhance resistance in wheat. A hypersensitive response is advantageous for disease resistance; however, an excessive hypersensitive response in plants usually leads to abnormal growth. It was observed that an abnormal phenotype and low fertility were accompanied by enhanced resistance in CaMV35S‐driven RLK‐V1.1 transgenic plants (Fig. S6, see Supporting Information), but not in RLK‐V1.2‐over‐expressing transgenic plants. Therefore, the alternative splicing of RLK‐V may act as a regulator to balance plant development and disease resistance.

Experimental Procedures

Plant material

Haynaldia villosa (2n = 14, VV), with a powdery mildew resistance gene Pm21 on the short arm of chromosome 6V, was introduced from Cambridge Botanical Garden, Cambridge, UK. Triticum durum–H. villosa amphiploid (AABBVV), the wheat–H. villosa translocation T6VS·6AL cv. Nannong9918 and the Pm21‐containing resistant cryptic alien introgression line NAU427 (Xing et al., 2018) were developed by the Cytogenetic Institute of Nanjing Agricultural University (CINAU), Nanjing China. Powdery mildew‐susceptible common wheat Chinese Spring was collected and maintained by CINAU. Susceptible wheat cv. Yangmai158 was collected from Yangzhou Academy of Agricultural Sciences and susceptible wheat cv. Shi4185 was collected from the Shijiazhuang Academy of Agricultural Sciences, and preserved by CINAU. Powdery mildew‐resistant Shi4185R and Yangmai158R are Pm21‐containing lines developed by backcross using NAU427 as the Pm21‐donor parent and Shi4185 and Yangmai158 as the recurrent parent, respectively. SM‐1 was an ethyl methane sulphonate (EMS)‐induced susceptible mutant of Nannong9918. Haynaldia villosa, Nannong9918, Shi4185R and Yangmai158R were used for gene expression analysis. Yangmai158, SM‐1 and Nannong9918 were used for transient single‐cell over‐expression, and Yangmai158 was used as the recipient for genetic transformation. Nannong9918, Shi4185R and Yangmai158R were used for VIGS. Nicotiana benthamiana plants were used for Agrobacterium‐mediated transient expression.

Bgt race preparation and evaluation of powdery mildew resistance

Mixed races of Bgt were collected in the field of Jiangpu experimental station (Nanjing, China) and preserved on seedlings of the highly susceptible variety Sumai3 in a glasshouse at 22 °C/14 h light and 18 °C/10 h dark day/night conditions (with 70% humidity). For RNA extraction, seedling leaves were harvested after inoculation with Bgt at different time points. For resistance evaluation, the leaves of the gene‐silenced plants and transgenic plants were placed on 20 mg/L benzimidazole agar medium and inoculated with fresh mixed Bgt under 22 °C/16 h light and 18 °C/8 h dark day/night conditions (relative humidity of 80%). The resistance level was recorded at 7 days after Bgt inoculation. The infection types (ITs) were scored as grades 0–4: 0, no visible symptoms; 0; necrotic flecks; 1, highly resistant; 2, moderately resistant; 3, moderately susceptible; 4, highly susceptible (Liu et al., 1999). The ITs of the transgenic plants were deduced from three independent experiments.

PCR amplification for mapping of RLK‐V on 6VS

Chinese spring, H. villosa, T. durum–H. villosa amphiploidy, Nannong9918 and NAU427 were used as cytogenetic stocks for the chromosomal location of RLK‐V. The primer pair RLK‐V‐CL (Table S1), which was designed according to the genomic sequence of RLK‐V, were synthesized by Invitrogen Life Technologies (Shanghai, China) and used for RLK‐V chromosomal location. Each PCR contained a 10‐μL volume including 2 × PCR Mix (Vazyme, Nanjing, China), 2 μm of each primer and 100 ng of DNA template. PCR was performed with incubation at 94 °C for 3 min, followed by 33 cycles of 94 °C for 30 s, 56 °C for 45 s and 72 °C for 1 min, and then incubation for 10 min at 72 °C. The PCR products were separated in 8% non‐denaturing polyacrylamide gels (Acrylamide : N, N'‐methylene‐bis‐acylamide= 39 : 1) and the bands were visualized by silver staining.

Cloning of RLK‐V and sequence analysis

The genomic sequence of RLK‐V was downloaded from the long‐range chromosome assembly database of flow‐sorted 6VS·6AL from Nannong9918 (Xing et al., 2018). The genomic sequence of RLK‐V was used to BLAST the database of H. villosa RNA‐Seq, and an assembly sequence with an ORF was obtained. Then, the primer pair RLK‐V‐CDS (Table S1) was designed to clone the coding sequence from the cDNA of H. villosa inoculated with Bgt. The PCR cycle was performed at 95 °C for 5 min, followed by 33 cycles of 95 °C for 15 s, 56 °C for 15 s and 72 °C for 3 min, and then incubation for 10 min at 72 °C. The Phanta Max Super‐Fidelity DNA polymerase was used for amplification (Vazyme , Nanjing, China). The PCR product was cloned into pTOPO‐Blunt Vector (Aidlab, Beijing, China) for sequencing at GENEWIZ (Suzhou, China). The putative domain of the cloned genes was analysed using SMART (https://smart.embl-heidelberg.de/). The phylogenetic tree was obtained by MEGA6 software through the neighbour‐joining algorithm, and bootstrapping was performed 1000 times to obtain support values for each branch.

Expression analysis of RLK‐V by qRT‐PCR and RT‐PCR

Total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The first‐strand cDNA was synthesized using 1 μg of total RNA by HiScript Q RT SuperMix for qRT‐PCR (Vazyme, Nanjing, China). Primer pair RLK‐V‐RT (Table S1) was used for expression analysis of RLK‐V by qRT‐PCR. The primer pair AS‐RLK‐V (Table S1) flanking the fifth intron, which could amplify 261 bp from RLK‐V1.1 and 300 bp from RLK‐V1.2, was used for alternative splicing analysis by RT‐PCR. qRT‐PCR was performed with AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). The program used was as follows: 5 min at 95 °C, followed by 40 cycles at 95 °C for 10 s and 60 °C for 20 s. The comparative 2–ΔΔCT method was used to quantify relative gene expression. Statistical analyses were conducted using the IBM SPSS Statistics 19 system (Amok, NY, USA) by Duncan’s honestly significant difference test (P < 0.05). SigmaPlot 12.5 supplied by Systat Software (San Jose, CA, USA) was used to develop the figures.

Cloning and activity assay of the native promoter of RLK‐V

The predicted promoter of RLK‐V, about 2444 bp upstream of the transcription start site, was amplified using the primer pair RLK‐V‐Promoter (Table S1) from the genomic DNA of H. villosa. The cis‐acting element was analysed with the promoter database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al., 2002). The promoter amplified by the primer pair RLK‐V‐Promoter‐GUS (Table S1) was inserted into pWMB002 upstream of the GUS gene to replace the maize ubiquitin promoter. The recombinant pWMB002:RLK‐Vpro:GUS was transiently transformed into wheat leaves by particle bombardment, and the promoter activity was compared between non‐inoculated leaves and Bgt‐inoculated leaves at 40 hai with GUS staining of the transformed leaf epidermal cells.

BSMV‐VIGS

BSMV‐VIGS was performed as described by Wang et al. (2010) with some modifications. The 225‐bp fragment located on the 3ʹ end of RLK‐V was amplified using the primer pair VIGS‐RLK‐V (Table S1) and inserted into the γ‐strain of BSMV to produce the vector BSMV:RLK‐V. In order to evaluate the function of RLK‐V in different genetic backgrounds, three resistance materials, Nannong9918, Shi4185R and Yanmgai158R, carrying both RLK‐V and Pm21 genes, were used for gene silencing assay. The second fully expanded leaves were infected with in vitro‐transcribed virus BSMV:RLK‐V, using BSMV:TaPDS‐ and BSMV:γ‐infected leaves as controls. The fourth fully expanded leaves with clear virus infection symptoms were cut for disease resistance evaluation and for RNA extraction. At 60 hai, 4 dai and 7 dai, leaves were bleached with destaining solution (ethanol : acetic acid, 3 : 1) and stained with Coomassie blue (6 mg/mL) to observe fungal development under an Olympus BX‐60 microscope (Tokyo,Japan). The silencing efficiency of the target gene was evaluated by qRT‐PCR using the primer pair VIGS‐RLK‐V‐Q (Table S1) on an LC 480II (Roche, Basel, Switzerland).

Single‐cell transient over‐expression assay

The primer pairs RLK‐V‐pBI220–F/RLK‐V1.1‐pBI220–R and RLK‐V‐pBI‐220–F/RLK‐V1.2‐pBI220–R (Table S1) were used to amplify the ORF fragments of RLK‐V1.1 and RLK‐V1.2, respectively. The amplified fragments were placed under the control of the 2×CaMV35S promoter to construct the recombinant vectors pBI220:RLK‐V1.1 and pBI220:RLK‐V1.2. The single‐cell transient over‐expression assay was performed according to Shirasu et al. (1999). Seedlings of Yangmai158, SM‐1 and Nannong9918 were grown under conditions of 16 h of light (18 °C)/8 h of dark (16 °C) with a relative humidity of 80%. At the two‐leaf stage, primary leaves were cut and placed on 20 mg/L benzimidazole agar medium. The pWMB002 vector containing the GUS reporter was mixed with the recombinant vector pBI220:RLK‐V1.1 or pBI220:RLK‐V1.2 for coating of the particles (molar ratio of 1 : 1 in 1 μg of total DNA). The particles coated with pWMB002+PBI220:RLK‐V1.1 or pWMB002+PBI220:RLK‐V1.2 were delivered into wheat epidermal cells through particle bombardment (PDS‐1000/He, Bio‐Rad , Hercules, CA, USA) using particles coated with pWMB002 as control. The bombarded leaves were incubated in the dark at 18 °C for 6 h and then inoculated with a high density of Bgt mixed spores. The leaves were stained for GUS at 42–48 hai to count the haustorium‐forming cells in Bgt‐interacting and GUS‐expressing epidermal cells. HI, the percentage of haustorium‐forming cells in the Bgt‐interacting and GUS‐expressing cells of the total Bgt‐interacting and GUS‐expressing cells, is presented as the mean of three independent replicates. In each replicate, at least 150 Bgt‐interacting and GUS‐expressing cells were observed.

Wheat transformation

Calluses were cultured from immature embryos of susceptible wheat variety Yangmai158, and the pBI220:RLK‐V1.1 and pBI220:RLK‐V1.2 vectors were co‐transformed with the plasmid pAHC20 into the callus of Yangmai158 by particle bombardment. The genomic DNA of the T0 regenerated plants and the derived T1 generations was obtained to identify positive plants with the RLK‐V‐GM‐35S‐F and RLK‐V‐GM‐R primer pair (Table S1). The forward primer was located on the sequence of the 35S promoter and the reverse primer was located on RLK‐V. The PCR cycle was as follows: 94 °C for 3 min, followed by 33 cycles of 94 °C for 45 s, 57 °C for 45 s and 72 °C for 1 min, and then incubation for 10 min at 72 °C. The powdery mildew resistance of transgenic plants was evaluated at the seedling stage using Nannong9918 as the resistant control and Yangmai158 as the susceptible control.

Detection of H2O2 with 3,3′‐diaminobenzidine (DAB) staining

In order to detect the accumulation of H2O2, the third leaves cut from Nannong9918, Yangmai158 and T1 generation transgenic plants at 24, 48 and 72 h after Bgt inoculation were incubated in DAB solution (1 mg/mL, pH 3.8) for 8 h and then bleached in destaining solution (ethanol : acetic acid, 3 : 1). The leaves were observed under an Olympus BX‐60 microscope.

Agrobacterium‐mediated transient expression for cell death observation

The full length of RLK‐V1.1 was amplified from the coding sequence (CDS) of RLK‐V1.1 using the primer pairs RLK‐V1.1‐L1F/RLK‐V1.1‐L2688R (Table S1); similarly, the fragment of RLK‐V1.2 was amplified from the CDS of RLK‐V1.2 using the primer pairs RLK‐V1.1‐L1F/RLK‐V1.2‐L1323R (Table S1). The pCambia1305.1‐GFP vector was digested by SpeI and BamHI, and then the digested vector was ligated with the RLK‐V fragments to produce the recombinant fusion vector pCambia1305.1:RLK‐V‐GFP. The Agrobacterium tumefaciens strain GV3101 carrying the corresponding vectors was infiltrated into N. benthamiana leaves as described by Van Ooijen et al. (2008). Agrobacterium suspensions at an optical density at 600 nm (OD600) of 0.5 were infiltrated into the leaves of 4–5‐week‐old N. benthamiana for cell death observation.

Trypan blue staining was performed to detect dead cells in the plants (Thordal‐Christensen et al., 1997). To visualize cell death on N. benthamiana leaves, the infiltrated leaves were stained with trypan blue solution (4 mg/mL) for at least 6 h at 37 °C, and then bleached in destaining solution (ethanol : acetic acid, 3 : 1) overnight.

Supporting information

Fig. S1  Phylogenetic analysis of RLK‐V1.1 and 28 RLKs. The phylogenetic tree was constructed using MEGA 6.0 software by the neighbor‐joining algorithms. Boots strapping was performed 1000 times to obtain support values for each branch. The 29 RLKs were clustered into six groups, including MLD‐LRR‐RLK, LysM‐RLK, Lectin‐RLK, LRR‐RLK, CRK‐RLK and WAK‐RLK. The phylogenetic tree indicated that RLK‐V1.1 is close to IOS1 and FRK1 but distant from HvLEMK1.

The accession number of the selected RLK proteins in Genebank are as follows: AtFRK1 (NP_179509.1), AtIOS1 (NP_175591), NtCERK1 (XP_016459329), AtCERK1 (NP_566689), HvLysM‐RLK (CAJ14969), ZmLysM‐RLK (ACG29351), OsCERK1 (XP_015611968), HvLecRK (CAH17379), TaLecRK (ACL36476), AtLecRK (NP_191529), OsLecRK (BAD07507), HvLEMK1 (KR610392.1), TaLRR‐RLK1 (AHG54199), TaLRR‐RLK2 (AHG54200), HvBAK1 (ABN05373), AtBAK1 (NP_567920), AtCRK13 (NP_001078435), OsCRK25 (XP_015646489), OsCRK10 (XP_015646832), OsCRK15 (XP_015645524), TaCRK1 (AGY79316), TaWAK2 (AAY34781), TaWAK4 (AAY34782)，TaWAK (ALJ11037), AtWAK1 (NP_564137.1), AtWAK3 (Q9LMN8.2), OsWAK91 (XP_015611274.1), OsWAK92 (XP_015611270.1).

Fig. S2  Development of Bgt in the BSMV:RLK‐V‐infected leaves using the BSMV:γ‐infected leaves as a control. Bgt development at 60 hai (a1–a2), 4 dai (b1–b2), and 7 dai (c1–c2) in Nannong9918 were observed under low magnification. Arrows indicate spore (Sp) and secondary hyphae (SH). (Scale bar = 50 μm)

Fig. S3  Molecular identification of RLK‐V transgenic plants and gene expression analysis in the T0 and T1 generations. (a, b) Molecular identification of RLK‐V1.1 T0 generation positive transgenic plants, using RLK‐V1.1‐T0‐165 as the negative control (a); and RLK‐V1.2 T0 generation positive transgenic plants, using RLK‐V1.2‐T0‐10 as the negative control (b). (c) Molecular identification of RLK‐V1.1 T1 generation positive transgenic plants using RLK‐V1.1‐T1‐165 as the negative control. (d) RLK‐V1.1 expression in the leaves of RLK‐V1.1‐T1‐149, RLK‐V1.1‐T1‐203, RLK‐V1.1‐T1‐206, and RLK‐V1.1‐T1‐228 lines. The qRT‐PCR values were normalized to Ta‐Tubulin and presented as fold changes relative to Yangmai158. RLK‐V1.1‐T1‐165 and the recipient Yangmai158 were used as the negative controls. ** indicates significantly differences assessed using one‐way ANOVA LSD analysis (P < 0.01).

Fig. S4  Response of RLK‐V to chitin in the leaves of H. villosa. The values are the means of three technical replicates of one biological experiment. Error bars indicate the standard error. All two biological replicates showed the similar results. Different letters indicate significantly differences assessed using Duncan’s honest significant difference test (P < 0.05).

Fig. S5  Comparison of the promoter sequences of the orthologous genes from different genomes. The promoter sequences of P‐RLK‐6AS and P‐RLK‐6BS from T. aestivum, P‐RLK‐A from T. urartu, and P‐RLK‐B from Ae. speltoides were downloaded from IWGSC and multiple compared using DNAMAN6.0. The sequence underlined in red indicates the Insert/Deletion sites specific to the RLK‐V promoter. The red frame indicates the core motif of the WT‐box. The yellow frame indicates the initiation codon ‘ATG’.

Fig. S6  Abnormal development of T1 generation transgenic plants of RLK‐V1.1 at the seedling and adult stage. The positive individuals segregated from the T1 generation showed severely abnormal growth and relatively low fertility compared with the segregated negative plants. (a) The T1 generation line RLK‐V1.1‐208 and the negative control RLK‐V1.1‐165. The red arrow indicated the positive individuals. (b) The T1 generation line RLK‐V1.1‐203, 1 and 2 indicate the negative individuals and 8 indicates the positive individual. (c) The T1 generation line RLK‐V1.1‐228, 1 and 2 indicate the negative individuals and 6, 7, and 8 indicate the positive individuals. (Scale bar = 5 cm)

Click here for additional data file. (296.6KB, tif)

 

Click here for additional data file. (2.5MB, tif)

 

Click here for additional data file. (894.4KB, tif)

 

Click here for additional data file. (97KB, tif)

 

Click here for additional data file. (8.6MB, tif)

 

Click here for additional data file. (1.6MB, tif)

Table S1  Primers list

Click here for additional data file. (18.4KB, docx)

Table S2  Cis‐acting elements involved in stress‐responses and pathogen‐responses predicted in the promoter of RLK‐V

Click here for additional data file. (14.3KB, docx)

Acknowledgements

This work was supported by the Important National Science and Technology Specific Projects of Transgenic Research (Grant No. 2018ZX0800905B), the National Natural Science Foundation of China (Grant Nos. 31771779, 31671685 and 31471489), the Fundamental Research Funds for the National Central Universities (Grant Nos. KYZ201601 and KYYJ201602), the Natural Science Foundation of Jiangsu Province (Grant No. SBK2017020263), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (Grant No. KYCX17_0555) and the National Training Program of Innovation and Entrepreneurship for Undergraduates (Grant No. 201710307017).

†The accession number of RLK‐V in GenBank is MG256391.

Contributor Information

Liping Xing, Email: xingliping@njau.edu.cn.

Aizhong Cao, Email: caoaz@njau.edu.cn.

References

  1. Acharya, B.R. , Raina, S. , Maqbool, S.B. , Jagadeeswaran, G. , Mosher, S.L. , Appel, H.M. , Schultz, J.C. , Klessig, D.F. and Raina, R. (2007) Overexpression of CRK13, an Arabidopsis cysteine‐rich receptor‐like kinase, results in enhanced resistance to Pseudomonas syringae . Plant J. 50, 488–499. [DOI] [PubMed] [Google Scholar]
  2. Asai, T. , Tena, G. , Plotnikova, J. , Willmann, M.R. , Chiu, W.L. , Gomez‐Gomez, L. , Boller, T. , Ausubel, F.M. and Sheen, J. (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature, 415, 977–983. [DOI] [PubMed] [Google Scholar]
  3. Boller, T. and Felix, G. (2009) A renaissance of elicitors: perception of microbe‐associated molecular patterns and danger signals by pattern‐recognition receptors. Annu. Rev. Plant Biol. 60, 379–406. [DOI] [PubMed] [Google Scholar]
  4. Cao, A.Z. , Xing, L.P. , Wang, X.Y. , Yang, X.M. , Wang, W. , Sun, Y.L. , Qian, C. , Ni, J.L. , Chen, Y.P. , Liu, D.J. , Wang, X.E. and Chen, P.D. (2011) Serine/threonine kinase gene Stpk‐V, a key member of powdery mildew resistance gene Pm21, confers powdery mildew resistance in wheat. Proc. Natl. Acad. Sci. USA, 108, 7727–7732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen, K.G. , Du, L.Q. and Chen, Z.X. (2003) Sensitization of defense responses and activation of programmed cell death by a pathogen‐induced receptor‐like protein kinase in Arabidopsis . Plant Mol. Biol. 53, 61–74. [DOI] [PubMed] [Google Scholar]
  6. Chen, K.G. , Fan, B.F. , Du, L.Q. and Chen, Z.X. (2004) Activation of hypersensitive cell death by pathogen‐induced receptor‐like protein kinases from Arabidopsis . Plant Mol. Biol. 56, 271–283. [DOI] [PubMed] [Google Scholar]
  7. Chen, T.T. , Xiao, J. , Xu, J. , Wan, W.T. , Qin, B. , Cao, A.Z. , Chen, W. , Xing, L.P. , Du, C. , Gao, X.Q. , Zhang, S.Z. , Zhang, R.Q. , Shen, W.B. , Wang, H.Y. and Wang, X.E. (2016) Two members of TaRLK family confer powdery mildew resistance in common wheat. BMC Plant Biol. 16(1), 1–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dinesh‐Kumar, S.P. and Baker, B.J. (2000) Alternatively spliced N resistance gene transcripts: their possible role in tobacco mosaic virus resistance. Proc. Natl. Acad. Sci. USA, 97, 1908–1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duan, L. , Xiao, W. , Xia, F. , Liu, H. , Xiao, J. , Li, X. and Wang, S. (2016) Two different transcripts of a LAMMER kinase gene play opposite roles in disease resistance. Plant Physiol. 172, 1959–1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fritz‐Laylin, L.K. , Krishnamurthy, N. , Tor, M. , Sjolander, K.V. and Jones, J.D.G. (2005) Phylogenomic analysis of the receptor‐like proteins of rice and Arabidopsis . Plant Physiol. 138, 611–623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gill, U.S. , Lee, S. and Mysore, K.S. (2015) Host versus nonhost resistance: distinct wars with similar arsenals. Phytopathology, 105, 580–587. [DOI] [PubMed] [Google Scholar]
  12. Halterman, D.A. , Wei, F. and Wise, R.P. (2003) Powdery mildew‐induced Mla mRNAs are alternatively spliced and contain multiple upstream open reading frames. Plant Physiol. 131, 558–567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hurni, S. , Scheuermann, D. , Krattinger, S.G. , Kessel, B. , Wicker, T. , Herren, G. , Fitze, M.N. , Breen, J. , Presterl, T. , Ouzunova, M. and Keller, B. (2015) The maize disease resistance gene Htn1 against northern corn leaf blight encodes a wall‐associated receptor‐like kinase. Proc. Natl. Acad. Sci. USA, 112, 8780–8785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnson, L.N. , Noble, M.E.M. and Owen, D.J. (1996) Active and inactive protein kinases: structural basis for regulation. Cell, 85, 149–158. [DOI] [PubMed] [Google Scholar]
  15. Kou, Y.J. and Wang, S.P. (2010) Broad‐spectrum and durability: understanding of quantitative disease resistance. Curr. Opin. Plant Biol. 13, 181–185. [DOI] [PubMed] [Google Scholar]
  16. Lescot, M. , Dehais, P. , Thijs, G. , Marchal, K. , Moreau, Y. , Van de Peer, Y. , Rouze, P. and Rombauts, S. (2002) PlantCARE, a database of plant cis‐acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 30, 325–327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu, Z.S. , Qin, J.X. , Tian, X.J. , Xu, S.B. , Wang, Y. , Li, H.X. , Wang, X.M. , Peng, H.R. , Yao, Y.Y. , Hu, Z.R. , Ni, Z.G. , Xin, M.M. and Sun, Q.X. (2018) Global profiling of alternative splicing landscape responsive to drought, heat and their combination in wheat (Triticum aestivum L.). Plant Biotechnol. J. 16, 714–726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liu, Z.Y. , Sun, Q.X. , Ni, Z.F. and Yang, T.M. (1999) Development of SCAR markers linked to the Pm21 gene conferring resistance to powdery mildew in common wheat. Plant Breed. 118, 215–219. [Google Scholar]
  19. Luo, X.M. , Xu, N. , Huang, J.K. , Gao, F. , Zou, H.S. , Boudsocq, M. , Coaker, G. and Liu, J. (2017) A lectin receptor‐like kinase mediates pattern‐triggered salicylic acid signaling. Plant Physiol. 174, 2501–2514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Machens, F. , Becker, M. , Umrath, F. and Hehl, R. (2014) Identification of a novel type of WRKY transcription factor binding site in elicitor‐responsive cis‐sequences from Arabidopsis thaliana . Plant Mol. Biol. 84, 371–385. [DOI] [PubMed] [Google Scholar]
  21. Macho, A.P. and Zipfel, C. (2014) Plant PRRs and the activation of innate immune signaling. Mol. Cell, 54, 263–272. [DOI] [PubMed] [Google Scholar]
  22. Marquez, Y. , Brown, J.W.S. , Simpson, C. , Barta, A. and Kalyna, M. (2012) Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis . Genome Res. 22, 1184–1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rajaraman, J. , Douchkov, D. , Hensel, G. , Stefanato, F.L. , Gordon, A. , Ereful, N. , Caldararu, O.F. , Petrescu, A.J. , Kumlehn, J. , Boyd, L.A. and Schweizer, P. (2016) An LRR/Malectin receptor‐like kinase mediates resistance to non‐adapted and adapted powdery mildew fungi in barley and wheat. Front. Plant Sci. 71836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanabria, N.M. and Dubery, I.A. (2016) Alternative splicing of the receptor‐like kinase Nt‐Sd‐RLK in tobacco cells responding to lipopolysaccharides: suggestive of a role in pathogen surveillance and perception? FEBS Lett. 590, 3628–3638. [DOI] [PubMed] [Google Scholar]
  25. Shirasu, K. , Nielsen, K. , Piffanelli, P. , Oliver, R. and Schulze‐Lefert, P. (1999) Cell‐autonomous complementation of mlo resistance using a biolistic transient expression system. Plant J. 17, 293–299. [Google Scholar]
  26. Staiger, D. and Brown, J.W.S. (2013) Alternative splicing at the intersection of biological timing, development, and stress responses. Plant Cell, 25, 3640–3656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tang, F. , Yang, S.M. , Gao, M.Q. and Zhu, H.Y. (2013) Alternative splicing is required for RCT1‐mediated disease resistance in Medicago truncatula . Plant Mol. Biol. 82, 367–374. [DOI] [PubMed] [Google Scholar]
  28. Thordal‐Christensen, H. , Zhang, Z.G. , Wei, Y.D. and Collinge, D.B. (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley–powdery mildew interaction. Plant J. 11, 1187–1194. [Google Scholar]
  29. Van Ooijen, G. , Mayr, G. , Kasiem, M.M.A. , Albrecht, M. , Cornelissen, B.J.C. and Takken, F.L.W. (2008) Structure‐function analysis of the NB‐ARC domain of plant disease resistance proteins. J. Exp. Bot. 59, 1383–1397. [DOI] [PubMed] [Google Scholar]
  30. Wang, X.Y. , Cao, A.Z. , Yu, C.M. , Wang, D.W. , Wang, X.E. and Chen, P.D. (2010) Establishment of an effective virus induced gene silencing system with BSMV in Haynaldia villosa . Mol. Biol. Rep. 37, 967–972. [DOI] [PubMed] [Google Scholar]
  31. Wang, Y. , Cordewener, J.H.G. , America, A.H.P. , Shan, W.X. , Bouwmeester, K. and Govers, F. (2015) Arabidopsis lectin receptor kinases LecRK‐IX.1 and LecRK‐IX.2 are functional analogs in regulating Phytophthora resistance and plant cell death. Mol. Plant–Microbe Interact. 28, 1032–1048. [DOI] [PubMed] [Google Scholar]
  32. Wang, Z.K. , Cheng, J.Y. , Fan, A.Q. , Zhao, J. , Yu, Z.Y. , Li, Y.B. , Zhang, H. , Xiao, J. , Muhammad, F. , Wang, H.Y. , Cao, A.Z. , Xing, L.P. and Wang, X.E. (2018) LecRK‐V, an L‐type lectin receptor kinase in Haynaldia villosa, plays positive role in resistance to wheat powdery mildew. Plant Biotechnol. J. 16, 50–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wu, Y. and Zhou, J.M. (2013) Receptor‐like kinases in plant innate immunity. J. Integr. Plant Biol. 55, 1271–1286. [DOI] [PubMed] [Google Scholar]
  34. Xing, L.P. , Hu, P. , Liu, J.Q. , Witek, K. , Zhou, S. , Xu, J.F. , Zhou, W.H. , Gao, L. , Huang, Z.P. , Zhang, R.Q. , Wang, X.E. , Chen, P.D. , Wang, H.Y. , Jones, J.D.G. , Karafiátová, M. , Vrána, J. , Bartoš, J. , Doležel, J. , Tian, Y.C. , Wu, Y.F. and Cao, A.Z. (2018) Pm21 from Haynaldia villosa encodes a CC‐NBS‐LRR that confers powdery mildew resistance in wheat. Mol. Plant. 11, 874–878. [DOI] [PubMed] [Google Scholar]
  35. Yadeta, K.A. , Elmore, J.M. , Creer, A.Y. , Feng, B.M. , Franco, J.Y. , Rufian, J.S. , He, P. , Phinney, B. and Coaker, G. (2017) A cysteine‐rich protein kinase associates with a membrane immune complex and the cysteine residues are required for cell death. Plant Physiol. 173, 771–787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yahiaoui, N. , Brunner, S. and Keller, B. (2006) Rapid generation of new powdery mildew resistance genes after wheat domestication. Plant J. 47, 85–98. [DOI] [PubMed] [Google Scholar]
  37. Yeh, Y.H. , Panzeri, D. , Kadota, Y. , Huang, Y.C. , Huang, P.Y. , Tao, C.N. , Roux, M. , Chien, H.C. , Chin, T.C. , Chu, P.W. , Zipfel, C. and Zimmerli, L. (2016) The Arabidopsis Malectin‐Like/LRR‐RLK IOS1 is critical for BAK1‐dependent and BAK1‐independent pattern‐triggered immunity. Plant Cell, 28, 1701–1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang, X.C. and Gassmann, W. (2003) RPS4‐mediated disease resistance requires the combined presence of RPS4 transcripts with full‐length and truncated open reading frames. Plant Cell, 15, 2333–2342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhang, X.C. and Gassmann, W. (2007) Alternative splicing and mRNA levels of the disease resistance gene RPS4 are induced during defense responses. Plant Physiol. 145, 1577–1587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhu, M. , Jiang, L. , Bai, B.H. , Zhao, W.Y. , Chen, X.J. , Li, J. , Liu, Y. , Chen, Z.Q. , Wang, B.T. , Wang, C.L. , Wu, Q. , Shen, Q.H. , Dinesh‐Kumar, S.P. and Tao, X.R. (2017) The intracellular immune receptor Sw‐5b confers broad‐spectrum resistance to Tospoviruses through recognition of a conserved 21‐amino acid viral effector epitope. Plant Cell, 29, 2214–2232. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1  Phylogenetic analysis of RLK‐V1.1 and 28 RLKs. The phylogenetic tree was constructed using MEGA 6.0 software by the neighbor‐joining algorithms. Boots strapping was performed 1000 times to obtain support values for each branch. The 29 RLKs were clustered into six groups, including MLD‐LRR‐RLK, LysM‐RLK, Lectin‐RLK, LRR‐RLK, CRK‐RLK and WAK‐RLK. The phylogenetic tree indicated that RLK‐V1.1 is close to IOS1 and FRK1 but distant from HvLEMK1.

The accession number of the selected RLK proteins in Genebank are as follows: AtFRK1 (NP_179509.1), AtIOS1 (NP_175591), NtCERK1 (XP_016459329), AtCERK1 (NP_566689), HvLysM‐RLK (CAJ14969), ZmLysM‐RLK (ACG29351), OsCERK1 (XP_015611968), HvLecRK (CAH17379), TaLecRK (ACL36476), AtLecRK (NP_191529), OsLecRK (BAD07507), HvLEMK1 (KR610392.1), TaLRR‐RLK1 (AHG54199), TaLRR‐RLK2 (AHG54200), HvBAK1 (ABN05373), AtBAK1 (NP_567920), AtCRK13 (NP_001078435), OsCRK25 (XP_015646489), OsCRK10 (XP_015646832), OsCRK15 (XP_015645524), TaCRK1 (AGY79316), TaWAK2 (AAY34781), TaWAK4 (AAY34782)，TaWAK (ALJ11037), AtWAK1 (NP_564137.1), AtWAK3 (Q9LMN8.2), OsWAK91 (XP_015611274.1), OsWAK92 (XP_015611270.1).

Fig. S2  Development of Bgt in the BSMV:RLK‐V‐infected leaves using the BSMV:γ‐infected leaves as a control. Bgt development at 60 hai (a1–a2), 4 dai (b1–b2), and 7 dai (c1–c2) in Nannong9918 were observed under low magnification. Arrows indicate spore (Sp) and secondary hyphae (SH). (Scale bar = 50 μm)

Fig. S3  Molecular identification of RLK‐V transgenic plants and gene expression analysis in the T0 and T1 generations. (a, b) Molecular identification of RLK‐V1.1 T0 generation positive transgenic plants, using RLK‐V1.1‐T0‐165 as the negative control (a); and RLK‐V1.2 T0 generation positive transgenic plants, using RLK‐V1.2‐T0‐10 as the negative control (b). (c) Molecular identification of RLK‐V1.1 T1 generation positive transgenic plants using RLK‐V1.1‐T1‐165 as the negative control. (d) RLK‐V1.1 expression in the leaves of RLK‐V1.1‐T1‐149, RLK‐V1.1‐T1‐203, RLK‐V1.1‐T1‐206, and RLK‐V1.1‐T1‐228 lines. The qRT‐PCR values were normalized to Ta‐Tubulin and presented as fold changes relative to Yangmai158. RLK‐V1.1‐T1‐165 and the recipient Yangmai158 were used as the negative controls. ** indicates significantly differences assessed using one‐way ANOVA LSD analysis (P < 0.01).

Fig. S4  Response of RLK‐V to chitin in the leaves of H. villosa. The values are the means of three technical replicates of one biological experiment. Error bars indicate the standard error. All two biological replicates showed the similar results. Different letters indicate significantly differences assessed using Duncan’s honest significant difference test (P < 0.05).

Fig. S5  Comparison of the promoter sequences of the orthologous genes from different genomes. The promoter sequences of P‐RLK‐6AS and P‐RLK‐6BS from T. aestivum, P‐RLK‐A from T. urartu, and P‐RLK‐B from Ae. speltoides were downloaded from IWGSC and multiple compared using DNAMAN6.0. The sequence underlined in red indicates the Insert/Deletion sites specific to the RLK‐V promoter. The red frame indicates the core motif of the WT‐box. The yellow frame indicates the initiation codon ‘ATG’.

Fig. S6  Abnormal development of T1 generation transgenic plants of RLK‐V1.1 at the seedling and adult stage. The positive individuals segregated from the T1 generation showed severely abnormal growth and relatively low fertility compared with the segregated negative plants. (a) The T1 generation line RLK‐V1.1‐208 and the negative control RLK‐V1.1‐165. The red arrow indicated the positive individuals. (b) The T1 generation line RLK‐V1.1‐203, 1 and 2 indicate the negative individuals and 8 indicates the positive individual. (c) The T1 generation line RLK‐V1.1‐228, 1 and 2 indicate the negative individuals and 6, 7, and 8 indicate the positive individuals. (Scale bar = 5 cm)

Click here for additional data file. (296.6KB, tif)

 

Click here for additional data file. (2.5MB, tif)

 

Click here for additional data file. (894.4KB, tif)

 

Click here for additional data file. (97KB, tif)

 

Click here for additional data file. (8.6MB, tif)

 

Click here for additional data file. (1.6MB, tif)

Table S1  Primers list

Click here for additional data file. (18.4KB, docx)

Table S2  Cis‐acting elements involved in stress‐responses and pathogen‐responses predicted in the promoter of RLK‐V

Click here for additional data file. (14.3KB, docx)

Articles from Molecular Plant Pathology are provided here courtesy of Wiley

RESOURCES