TaELP2 Interacts With TaDCL1 and Negatively Regulates Wheat Resistance Against Stripe Rust

ABSTRACT

Wheat ( Triticum aestivum ) is an important staple food crop worldwide, playing a fundamental role in global food security. Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a highly destructive disease affecting this crop. The Elongator complex is known to be a key regulator of plant responses to stress; however, little is known about the function of its subunit 2, encoded by the TaELP2 gene, in the wheat–Pst interaction. In this study, we investigated the role of TaELP2 in the susceptibility of wheat to Pst. We found that the transient silencing of TaELP2 using a virus‐induced gene silencing (VIGS) system markedly increased wheat resistance to the pathogen, whereas its overexpression exerted the opposite effect. Subsequently, yeast two‐hybrid (Y2H), luciferase complementation imaging (LCI), pull‐down and co‐immunoprecipitation (Co‐IP) assays demonstrated that TaELP2 directly interacts with DICER‐LIKE (DCL1) protein. Notably, the knockdown of TaDCL1 expression greatly enhanced wheat resistance to stripe rust. Furthermore, Y2H and LCI assays indicated that TaELP2 also interacts with TaELP3. RNA‐sequencing analysis revealed that TaELP2 overexpression downregulates NB‐ARC genes and compromises disease resistance in wheat. Based on these findings, we propose that TaELP2 acts as a negative regulator of wheat stripe rust resistance, probably by interacting with TaDCL1 to modulate the transcription of key disease resistance genes.

TaELP2 negatively regulates wheat stripe rust resistance by inducing alternative splicing of RNA silencing genes and promoting the degradation of disease‐resistance genes via its interaction with TaDCL1.

1. Introduction

Both plants and animals have evolved complex immune systems to defend against microbial pathogens. Unlike animals, plants lack mobile cells specialised for defence. Instead, they rely on individual cells to recognise pathogens and activate immune responses (Jones and Dangl 2006). Plants employ two primary recognition mechanisms, one involving the recognition of pathogen‐ or microbe‐associated molecular patterns (PAMPs/MAMPs) through cell surface pattern‐recognition receptors, and the other the sensing of pathogen effectors via intracellular resistance‐gene products, resulting in PAMP‐triggered immunity (PTI) and effector‐triggered immunity (ETI), respectively (Chisholm et al. 2006; Jones and Dangl 2006). The two immune responses are similar and involve ion fluxes, the phosphorylation of target proteins, the generation of reactive oxygen species (ROS), callose deposition and large‐scale transcriptional reprogramming (Yan et al. 2025). This extensive transcriptional reprogramming is particularly crucial for plant cells to rapidly and efficiently respond to pathogen invasion (DeFraia and Mou 2011), with numerous transcriptional regulators, including the Elongator complex, having been shown to modulate this process (DeFraia et al. 2010).

Elongator was initially identified in yeast as an elongating RNA polymerase II‐associated protein complex (Otero et al. 1999) and was subsequently found to be highly conserved in eukaryotes (Hawkes et al. 2002; Nelissen et al. 2010). In yeast, Elongator is composed of six subunits (ELP1–6) (it should be noted that the acronym ‘ELP’ has also been previously used to describe ‘EDM2‐like proteins’ in Arabidopsis (Eulgem et al. 2007)), which function as a single unit (Krogan and Greenblatt 2001). ELP1 and ELP2 are WD40 proteins that act as a scaffold for complex assembly. ELP1 also contains a functional nuclear localisation sequence that is crucial for the transcriptional regulatory function of Elongator (Fichtner et al. 2003). ELP3 is the catalytic subunit, which harbours a C‐terminal GNAT‐type histone acetyltransferase (HAT) domain and an N‐terminal iron–sulphur radical S‐adenosylmethionine (SAM) domain (Chinenov 2002). The ELP4, ELP5 and ELP6 subunits, which form an accessory subcomplex, each adopt a RecA‐ATPase‐like fold and assemble into a hexameric ring‐shaped structure that is important for recognising histone H3 (Lin et al. 2012). The loss of any Elongator subunit is thought to compromise its integrity, rendering the complex inactive (An et al. 2017; Versées et al. 2010). Notably, however, it has also been reported that ELP2 is dispensable for the integrity of the holo‐Elongator complex, as the complex lacking ELP2 retains the ability to acetylate histones in vitro (Wang et al. 2013). Elongator has been shown to perform distinct cellular functions in diverse organisms, including histone modification, transcriptional silencing, the maintenance of genome stability, DNA methylation and/or demethylation and microRNA biogenesis (Ding and Mou 2015; Fang et al. 2015; Winkler et al. 2002; Woloszynska et al. 2016).

In plants, Elongator participates in both development and responses to abiotic and biotic stresses. Arabidopsis ELP2 and ELP3 mutants exhibit delayed and/or reduced induction of defence genes, including the salicylic acid (SA) biosynthetic pathway marker gene PATHOGENESIS‐RELATED GENE1 (PR1) and the jasmonic acid (JA)/ethylene (ET) defence pathway marker gene PLANT DEFENSIN1.2 (PDF1.2), and display enhanced susceptibility to the hemibiotrophic bacterial pathogen Pseudomonas syringae and the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola (DeFraia et al. 2010, 2013; Wang et al. 2015). In line with this, ELP2 was shown to modulate the dynamic transcriptome changes induced by the avirulent bacterial pathogen P. syringae pv. tomato DC3000/avrRpt2, likely through the regulation of DNA methylation and histone acetylation of genes involved in plant immunity (Wang et al. 2013). Similarly, the overexpression of AtELP3 and AtELP4 was reported to enhance resistance to anthracnose crown rot, powdery mildew and tomato bacterial speck in Fragaria vesca and tomato plants (Pereira et al. 2018; Silva et al. 2017). In addition, TaELP4 positively regulates the innate immune responses of wheat and Arabidopsis thaliana to Rhizoctonia cerealis and B. cinerea by increasing the levels of histone acetylation in defence‐associated genes, thereby upregulating their transcription (Wang et al. 2020). Despite its reported regulatory roles in plant development and immunity, whether Elongator participates in the wheat–Puccinia striiformis f. sp. tritici (Pst; stripe rust) interaction remains unknown.

Plants have evolved complex mechanisms as a means of responding to environmental stimuli, including RNA silencing, which is involved in the regulation of gene expression at the post‐transcriptional or post‐translational level in response to diverse biotic and abiotic stresses (Pumplin and Voinnet 2013). Small RNAs (sRNAs)—including small interfering RNAs (siRNAs), microRNAs (miRNAs) and piwi‐interacting RNAs (piRNAs)—are generated from double‐stranded RNA (dsRNA) precursors by Dicer or DICER‐LIKE (DCL) proteins, and subsequently associate with ARGONAUTE (AGO) family proteins to perform their functions (Choudhary et al. 2019). A. thaliana contains three major classes of sRNAs: miRNAs, heterochromatic small interfering RNAs (hc‐siRNAs) and trans‐acting siRNAs (ta‐siRNAs), which are processed by DCL1, DCL3 and DCL4, respectively (Baulcombe 2004; Bologna and Voinnet 2014). miRNA biogenesis involves the transcription of primary miRNAs (pri‐miRNAs) by RNA polymerase II (RNAPII) and their subsequent processing by Dicer or DCL proteins. DCL1 and pri‐miRNA transcripts are both associated with chromatin at miRNA loci, and the association of DCL1 with chromatin is dependent on the presence of Elongator. In Arabidopsis, Elongator plays an essential role as a bridge coupling the transcription and processing of pri‐miRNAs, thereby facilitating efficient miRNA biogenesis (Fang et al. 2015).

In this study, we investigated the role of wheat Elongator in the regulation of host immune responses to Pst. We focused on the wheat Elongator subunit 2‐encoding gene, designated as TaELP2, which we identified as being strongly responsive to Pst infection based on transcriptomic analysis. Virus‐induced gene silencing (VIGS) and overexpression experiments revealed that TaELP2 functions as a negative regulator of resistance to Pst infection. Additionally, through yeast two‐hybrid (Y2H), luciferase complementation imaging (LCI), pull‐down and co‐immunoprecipitation (Co‐IP) assays, we found that TaELP2 directly interacts with TaDCL1. The silencing of TaDCL1 through VIGS significantly enhanced resistance to Pst infection. RNA‐sequencing analysis revealed that TaELP2 overexpression downregulates NB‐ARC genes and other disease resistance genes. Consistently, after the silencing of the disease resistance genes by VIGS, the disease resistance significantly weakened. Taken together, these results indicate that TaELP2 acts as a negative regulator by reducing the transcriptional abundance of resistance‐related genes.

2. Results

2.1. TaELP2 Plays a Significant Role in Wheat–Pst Interaction and is Localised to the Nucleus and Cytoplasm

Transcriptome analysis of the wheat–Pst interaction revealed that TaELP2 was highly responsive to fungal infection. Reverse transcription‐quantitative PCR (RT‐qPCR) was used to analyse the expression levels of TaELP2 in wheat inoculated with the avirulent Pst race CYR23 (incompatible interaction) and the virulent Pst race CYR31 (compatible interaction) at different time points. The expression of TaELP2 was significantly higher in the compatible interaction than in the incompatible interaction, with a maximum expression level that was 5‐fold higher than that of the control (Figure 1a). We identified three TaELP2 homologues, which were located in chromosomes 1A, 1B and 1D, respectively. Comparative sequence analysis revealed a high degree of conservation among the homologues, displaying 97.69% nucleotide identity (Figure S1) and 97.87% amino acid similarity (Figure S2). Structurally, TaELP2 was found to contain seven WD40 repeat domains with typical features, including an N‐terminal GH dipeptide and a C‐terminal WD dipeptide. To analyse the evolutionary relationships between wheat ELP2 and ELP2 homologues across species, we performed a protein sequence homology search using the NCBI database and constructed a phylogenetic tree with MEGA7 software based on the neighbour‐joining method. The results showed that monocot and dicot ELP2 proteins clustered into distinct branches, with TaELP2 showing the closest evolutionary relationship to barley ELP2 (Figure 1b). To clarify the cellular localisation of TaELP2 in wheat, we transiently expressed a TaELP2‐GFP fusion protein or a GFP empty vector control in wheat protoplasts. We found that TaELP2 was expressed in both the nucleus and cytoplasm, while the fluorescence signal of the GFP control was uniformly distributed throughout the entire cell (Figure 1c).

FIGURE 1.

The molecular characteristics of TaELP2. (a) The transcript levels of TaELP2 in wheat leaves were induced during wheat–Puccinia striiformis f. sp. tritici (Pst) interaction. The leaves of wheat cv. Suwon 11 were inoculated with Pst CYR23 and CYR31 and examined at 0, 12, 24, 48, 72, 96 and 120 h post‐inoculation (hpi). Untreated leaves served as control. (b) Schematic diagram of TaELP2 protein structure. Phylogenetic analysis of TaELP2 and its homologues from dicotyledons and monocotyledons. (c) Subcellular localisation of TaELP2 in wheat protoplasts. Scale bars, 20 μm. Data are presented as mean ± SD from three biological replicates. Statistical significance was determined by Student's t test (*p < 0.05, **p < 0.01).

2.2. The Knockdown of TaELP2 Enhances the Resistance of Wheat to Pst P

To investigate the role of TaELP2 during the infection of wheat by Pst, we used the BSMV‐VIGS system to knock down its expression in two wheat cultivars, Suwon 11 (SY11) and Fielder. For gene silencing, we selected two specific fragments (TaELP2‐1as and TaELP2‐2as) that have no significant similarity to other wheat genes, including WD40 family members (Figure S1). Two weeks after the inoculation of the second leaves of wheat plants with the BSMV strain, chlorotic mosaic symptoms appeared on the fourth leaves of wheat plants. BSMV:TaPDS‐inoculated wheat exhibited pronounced photobleaching, thereby confirming VIGS efficiency (Figure 2a). The BSMV‐treated plants were then inoculated with Pst CYR31, with the disease phenotype being observed after 14 days. Compared with control plants, the number of uredinia on the leaves of TaELP2‐silenced plants was decreased, while necrotic cell death was significantly increased. This phenotype was consistent in both cultivars (Figure 2b). Expression analysis revealed a significant reduction in TaELP2 homologue transcript levels in both silenced cultivars (Figure 2c–e). The fungal biomass in TaELP2‐silenced plants was significantly reduced in both cultivars (Figure 2f). Following Pst inoculation, the expression of the wheat genes TaPR1 and TaPR2 was significantly up‐regulated in TaELP2‐silenced plants compared to the controls (Figure 2g,h).

FIGURE 2.

Silencing of TaELP2 enhanced resistance to Puccinia striiformis f. sp. tritici (Pst). (a) Photobleaching was clearly observed on wheat leaves inoculated with BSMV:TaPDS. Mild chlorotic mosaic symptoms were observed on wheat leaves inoculated with BSMV:TaPDS, BSMV:γ and BSMV:TaELP2 after 12 days. Wheat cultivar Suwon 11 (SY11) carries Yr26; cultivar Fielder carries Yr20. (b) Disease phenotypes of the fourth leaves were pre‐inoculated with BSMV constructs and then inoculated with urediniospores of Pst CYR31 for 14 days. (c–e) Relative transcript levels of TaELP2 in leaves inoculated with the recombinant BSMV by reverse transcription‐quantitative PCR. (f) The biomass ratio (Pst:wheat) of total DNA extracted from different BMSV‐processed wheat leaves at 14 days post‐inoculation (dpi). Ratio of total fungal DNA to total wheat DNA was assessed using PstEF and TaEF‐1α as internal reference genes, respectively. (g, h) Relative transcript levels of TaPR1 and TaPR2 in BSMV‐treated wheat leaves infected by Pst CYR31 at 48 h post‐inoculation (hpi). (i, j) The infection unit areas in BSMV‐inoculated wheat leaves infected by Pst CYR31 at 24, 48 and 120 hpi. (k) The hyphal length in BSMV‐inoculated wheat leaves infected with Pst CYR31 at 48 hpi. Data are presented as mean ± SD from three biological replicates. Statistical significance was determined by Student's t test (**p < 0.01).

To analyse Pst development in TaELP2‐silenced SY11 wheat, WGA staining was performed on inoculated leaves at 24, 48 and 120 h post‐inoculation (hpi) to enable hyphal observation and quantification (Figure S3). Compared with the controls, the silencing of TaELP2 led to significant reductions in both the area colonised by Pst (Figure 2i,j) and the extent of hyphal elongation (Figure 2k). These results demonstrated that the silencing of TaELP2 enhances wheat resistance against Pst.

2.3. The Overexpression of TaELP2 Increases Wheat Susceptibility to Pst

To further investigate the contribution of TaELP2 to Pst resistance in wheat, we generated three independent Fielder lines overexpressing TaELP2 (TaELP2‐OE‐L9, TaELP2‐OE‐L15, TaELP2‐OE‐L27). Transgenic wheat plants of the T₂ generation were subsequently inoculated with CYR23. The results showed that the overexpression of TaELP2 weakened wheat resistance to Pst, as evidenced by a significant increase in uredinia numbers relative to the controls. TaELP2 overexpression in the three lines was confirmed at both the mRNA and protein levels, and no overexpressed TaELP2 was detected in the controls (Figure 3a). RT‐qPCR revealed that the expression of TaELP2 in the transgenic plants was 50–250‐fold that of control plants (Figure 3b). Similarly, fungal biomass was significantly higher in TaELP2‐OE wheat than in WT controls (Figure 3c). To assess the disease‐resistance phenotypes of TaELP2‐OE wheat seedlings, fungal development and host responses were evaluated. Analysis of the extent of ROS accumulation in TaELP2‐OE plants after inoculation with Pst CYR31 indicated that H₂O₂ accumulation was significantly decreased at the infection sites in these plants compared with that in wild‐type (WT) plants, as determined by 3,3′‐diaminobezidine (DAB) staining (Figure 3d,e). In addition, the infection areas and hyphal lengths were markedly increased in the TaELP2‐OE plants (Figure 3f–h). These results indicated that TaELP2 negatively regulates wheat resistance to Pst.

FIGURE 3.

Overexpression of TaELP2 reduces wheat resistance against Puccinia striiformis f. sp. tritici (Pst) and reduces H₂O₂ accumulation in wheat. (a) Disease phenotypes of wild‐type (WT) and TaELP2 overexpression (OE) lines leaves after Pst CYR23 infection for 14 days. Positive transgenic wheat lines were detected by PCR and the TaELP2 protein levels were detected by western blot. (b) Relative transcript levels of TaELP2 in WT and TaELP2‐OE lines at 48 h post‐inoculation (hpi). (c) The fungal biomass ratio of WT and TaELP2‐OE lines leaves infected by Pst CYR23 at 14 days post‐inoculation (dpi). (d, e) Average areas of H₂O₂ accumulation in wheat leaves stained with 3,3′‐diaminobenzidine (DAB). The amount of H₂O₂ produced was quantified as the size of the DAB staining area. (f) The hyphal length in wheat leaves stained with WGA. (g) Histological observations of the WT and TaELP2‐OE lines leaves infected with Pst. Wheat leaves were stained with DAB and wheat germ agglutinin (WGA). The accumulation sites of H₂O₂ were observed under a light microscope and the fungal structures were observed under a fluorescence microscope. SV, substomatal vesicle. Scale bars, 50 μm. (h) The infection unit areas in wheat leaves stained with WGA. Data are presented as mean ± SD from three biological replicates. Statistical significance was determined by Student's t test (*p < 0.05, **p < 0.01).

2.4. TaELP2 Interacts With TaDCL1 and TaELP3

It has been shown that the Elongator complex interacts with the PAZ domain of DCL1 in A. thaliana (Fang et al. 2015). To further explore the mechanisms underlying the negative regulatory effect of TaELP2 on resistance to Pst, we sought to determine whether TaELP2 directly interacts with TaDCL1. First, we identified the wheat homologue DCL1 based on AtDCL1 sequence homology and conducted interaction validation. TaDCL1 contains several domains, including PAZ, DEAD, DRBM and ribonuclease (Figure S4a). Y2H assay showed that a yeast strain transformed with TaDCL1‐PAZ and TaELP2 could grow on SD/−Trp/−Leu/−His/−Ade solid medium containing X‐α‐Gal, forming blue colonies, whereas TaDCL1‐N, TaDCL1‐C and the control strain could not (Figure 4a). This result was further confirmed using a luciferase (LUC) complementation imaging (LCI) assay. Strong luminescence signals were detected in Nicotiana benthamiana leaf areas co‐expressing TaELP2‐Cluc and TaDCL1^PAZ‐Nluc, while no luminescence signal was observed in the controls (Figure 4b). In addition, the interaction between TaDCL1 and TaELP2 was assessed in vitro using a pull‐down assay. TaELP2‐His was detected in the presence of TaDCL1‐GST but not in the negative control containing GST (glutathione S‐transferase) alone (Figure 4c). Finally, a Co‐IP assay was used to verify whether TaELP2 interacts with TaDCL1 in vivo. TaDCL1‐GFP and TaELP2‐mCherry were co‐expressed in N. benthamiana leaves, with GFP and TaELP2‐mCherry co‐expression serving as a control. Western blot analysis was performed using anti‐GFP and anti‐mCherry antibodies. The results showed that after enrichment using GFP‐Trap agarose beads, TaELP2‐mCherry was only detected when it was co‐expressed with TaDCL1‐GFP but not with GFP alone (control) (Figure 4d). The above results indicated that TaELP2 interacts with TaDCL1.

FIGURE 4.

TaELP2 interacts with TaDCL1 in vitro and in vivo. (a) TaELP2 interacts with TaDCL1 in yeast two‐hybrid assays. SD−‐LW: SD−Trp/Leu; SD‐−LWHA: SD−Trp/Leu/His/Ade. Positive control: Interaction of BD‐P53 and AD‐SV40. Negative control: Interaction of BD‐TaELP2 and pGADT7. (b) Luciferase complementation imaging assays determined the interaction of TaELP2 and TaDCL1 in Nicotiana benthamiana leaves. (c) TaELP2 interacts with TaDCL1 in vitro. His pull‐down assay was used to detect the interaction between TaELP2‐His and TaDCL1‐GST. The pull‐down inputs and samples were detected by western blot using anti‐GST or anti‐His antibodies, respectively. (d) TaELP2 interacts with TaDCL1 in vivo. Co‐immunoprecipitation was performed on extracts of N. benthamiana leaves expressing both TaELP2‐mCherry and TaDCL1PAZ‐GFP. Proteins before (Input) and after incubation (IP) were detected with anti‐GFP and anti‐mCherry antibodies.

Elongator is a protein complex originally identified in the yeast Saccharomyces cerevisiae . The core subcomplex comprises ELP1, ELP2 and ELP3, with ELP4–6 forming the accessory subcomplex (Krogan and Greenblatt 2001; Otero et al. 1999). The molecular architecture of the fully assembled Elongator complex consists of a symmetric dimer of ELP1, ELP2 and ELP3, bound to the heterohexameric ring of ELP4–6 (Dauden et al. 2017). ELP1 and ELP2 are primarily responsible for maintaining the structural integrity of the complex. To analyse the interaction between TaELP2 and the other subunits of the Elongator complex, we performed Y2H and LCI assays. The results demonstrated that TaELP2 interacts with TaELP3, but not with ELP1, ELP4, ELP5 or ELP6 (Figure 5).

FIGURE 5.

TaELP2 interaction between and other subunits in the ELP complex. (a) Interaction of TaELP2 and TaELP3 detected by yeast two‐hybrid assay. SD−LW: SD−Trp/Leu; SD−LWH: SD−Trp/Leu/His. Positive control: Interaction of BD‐P53 and AD‐SV40. Negative control: Interaction of BD‐TaELP2 and pGADT7. (b) Luciferase complementation imaging assays in Nicotiana benthamiana leaves showing TaELP2 and TaELP3 interaction.

2.5. The Silencing of TaDCL1 Increases the Wheat Defence Response to Pst

To investigate the potential role of TaDCL1 in the wheat–Pst interaction, we first assessed its expression level in both compatible and incompatible interaction systems using RT‐qPCR. Significant increases in TaDCL1 expression were observed following infection with the virulent CYR31 strain, reaching 10‐, 6‐ and 7‐fold at 24, 48 and 120 hpi, respectively. These results showed that TaDCL1 expression is induced during Pst infection and is significantly higher in the compatible interaction system than in the incompatible interaction system (Figure S4b). The BSMV‐mediated knockdown of TaDCL1 resulted in fewer uredinia being produced on leaves inoculated with CYR31 than in control leaves at 14 days post‐inoculation (dpi). However, TaDCL1‐silenced wheat leaves inoculated with CYR23 exhibited cell death but no sporulation, consistent with the control phenotype (Figure 6a). Furthermore, compared with control plants, TaDCL1 transcript levels were reduced by 70%–80% in TaDCL1‐knockdown plants (Figure 6b), while the Pst biomass was decreased by 60%–70% (Figure 6c). The expression of the plant defence‐related genes TaPR1 and TaPR2 was determined to further assess host resistance under TaDCL1‐knockdown conditions. Compared with control leaves expressing BSMV, TaPR1 expression was significantly increased in TaDCL1‐knockdown plants at both 48 and 120 hpi (Figure 6d). The expression of TaPR2 remained unchanged after 48 hpi, but exhibited a significant 10‐fold upregulation after 120 h (Figure 6e). Moreover, notable ROS accumulation was detected in TaDCL1‐knockdown plants compared with that in control plants, as revealed by DAB staining (Figure 6f). The silencing of TaDCL1 inhibited the progression of Pst infection, resulting in a significant reduction in the size of the infection area (Figure 6g). Combined, the above results indicated that TaDCL1 silencing enhances the wheat defence response to Pst infection.

FIGURE 6.

Functional characterisation of TaDCL1 by virus‐induced gene silencing. (a) Disease phenotypes of wheat leaves pre‐inoculated with BSMV constructs and then infected by Puccinia striiformis f. sp. tritici (Pst) CYR23 or CYR31 at 14 days post‐inoculation (dpi). (b) Relative transcript levels of TaDCL1 in leaves inoculated with the recombinant BSMV at 0, 24, 48 and 120 h post‐inoculation (hpi) by reverse transcription‐quantitative PCR after inoculation with Pst CYR31. The data were normalised to the TaEF gene. (c) The fungal biomass ratio of the control and TaDCL1‐silenced wheat leaves infected by Pst CYR31 at 14 days post‐inoculation (dpi). (d, e) Relative transcript levels of TaPR1 and TaPR2 in the control and TaDCL1‐silenced wheat leaves infected by Pst CYR31 at 48 and 120 hpi. (f) The infection unit areas in the control and TaDCL1‐silenced wheat leaves infected by Pst CYR31 at 120 hpi. (g) Average area of H₂O₂ accumulation in the control and TaDCL1‐silenced wheat leaves stained with 3,3′‐diaminobenzidine (DAB) after inoculation with Pst CYR31. Data are presented as mean ± SD from three biological replicates. Statistical significance was determined by Student's t test (*p < 0.05, **p < 0.01).

2.6. Analysis of Transcriptome Sequencing Data for TaELP2 ‐Overexpressing Wheat Leaves

To further investigate the role of TaELP2 in wheat resistance to Pst, we next undertook an RNA‐seq analysis of leaves from WT and TaELP2‐OE plants. Compared to WT, TaELP2‐OE plants exhibited 1733 differentially expressed genes (DEGs; fold change [FC] > |2|, p < 0.01), of which 1089 were upregulated and 644 downregulated (Figure 7a). KEGG pathway enrichment analysis revealed that the downregulated DEGs were primarily enriched in ‘protein processing in endoplasmic reticulum’, ‘plant–pathogen interaction’ and ‘starch and sucrose metabolism’ (Figure 7b). RT‐qPCR validation of four randomly selected DEGs showed expression patterns consistent with the RNA‐seq data, confirming the reliability of our transcriptome analysis (Figure S5a).

FIGURE 7.

TaELP2 overexpression reduces the expression level of NB‐ARC genes. (a) Volcano plot of upregulated and downregulated DEGs in TaELP2‐OE lines relative to wild type (WT). (b) KEGG pathway enrichment analysis of downregulated genes in the TaELP2‐OE lines. (c) Hierarchical clustering of some NB‐ARC genes in the transgenic overexpression line (TaELP2‐OE) relative to WT plants. The indicated scale is the log₂ value of the normalised level of gene expression. (d) The expression levels of the four NB‐ARC genes in the TaELP2‐OE line and the WT were verified by reverse transcription‐quantitative PCR. (e) Disease phenotype observed after silencing four NB‐ARC genes by BSMV‐virus‐induced gene silencing and then inoculating with Puccinia striiformis f. sp. tritici (Pst) CYR23. (f) Relative expression of NB‐ARC was decreased in NB‐ARC‐knockdown plants inoculated with Pst CYR23. (g) Relative Pst biomass ratio measured using total DNA content from NB‐ARC‐silenced wheat leaves inoculated with Pst CYR23 at 14 days post‐inoculation. Data are presented as mean ± SD from three biological replicates. Statistical significance was determined by Student's t test (*p < 0.05, **p < 0.01).

Transcriptome analysis further revealed that the expression of NB‐ARC resistance genes was downregulated in TaELP2‐OE plants compared to that in WT plants (Figure 7c). To confirm the reliability of the RNA‐seq data, the expression of four NB‐ARC resistance genes was examined by RT‐qPCR, and their expression profiles were noted to be consistent with the sequencing results (Figure 7d). To assess their functional relevance, these four resistance genes were first analysed under Pst induction (Figure S5b). Subsequently, silencing was performed in wheat using VIGS, after which the plants were inoculated with Pst CYR23. The results showed that the number of urediniospores produced on plants with silenced resistance genes was significantly higher than that on control plants (Figure 7e). In line with this, fungal biomass was significantly higher in silenced plants than in the controls at 12 dpi (Figure 7f). The suppression of all four target genes was confirmed by RT‐qPCR (Figure 7g). Together, these data indicated that the silencing of these NB‐ARC genes, which are required for basal resistance, renders wheat susceptible to Pst. Collectively, TaELP2 functions as a negative regulator of this defence pathway by repressing the expression of these NB‐ARC genes, thereby dampening the host immune response and enhancing disease susceptibility.

3. Discussion

Elongator plays an important role in plant development and responses to both biotic and abiotic stresses (Ding and Mou 2015). In Arabidopsis, AtELP2 acts as an accelerator of immune responses and is required for the rapid induction of defence genes, including PR1, PR2 and PR5, as well as the establishment of PTI and ETI (DeFraia et al. 2010). AtELP2 also regulates the expression of a group of major defence genes, such as NPR1, EDS1, PAD4 and EDS5, which are required for the regulation of dynamic, pathogen infection‐induced changes in DNA methylation levels of two key defence genes, NPR1 and PAD4 (Wang et al. 2013). Moreover, using a histone acetylation assay, Wang et al. (2013) showed that histone H3K9/14ac levels in the coding regions of several defence genes, including NPR1, PAD4, EDS1, PR2, PR5, WRK33, ORA59 and PDF1.2, are significantly reduced in Atelp2 mutants, suggesting that AtELP2 regulates histone acetylation levels in these genes. Consistent with this, mutants of the Arabidopsis Elongator catalytic subunit AtELP3/ELO3 lacking conserved residues in either the HAT or radical SAM domains fail to complement the gns2/Atelp3 mutation, indicating that AtELP3 positively regulates plant immunity through these two domains in Arabidopsis (DeFraia et al. 2013). Also in Arabidopsis, Elongator is required for the full activation of the JA/ET defence pathway‐related marker gene PDF1.2 and positively contributes to resistance to the necrotrophic fungal pathogens B. cinerea and A. brassicicola (Wang et al. 2015). Collectively, these findings show that ELP genes play a positive regulatory role in plant responses to pathogen infection. In this study, transcriptome analysis revealed that the expression of the Elongator subunit TaELP2 was induced in response to Pst infection. The silencing of the TaELP2 gene led to an increase in the expression of the PR1 and PR2 genes, and significantly inhibited the growth of Pst (Figure 2). These results indicated that TaELP2 may act as a negative regulator of the wheat–Pst interaction.

In plant cells, ROS plays a key role in the hypersensitive response (HR) and systemic immunity (Myers et al. 2024; Yu et al. 2024). The overexpression of TaELP2 in wheat led to a significant decrease in ROS accumulation, alongside an increase in Pst spore production in the leaf and more pronounced symptoms of infection (Figure 3). ROS can both directly damage pathogens, inducing programmed cell death in plant cells to limit their spread, and act as a signalling molecule in synergy with other defence substances (Mittler et al. 2022; Su et al. 2024). In Arabidopsis, the disruption of the Elongator complex results in increased resistance to oxidative stress, indicating that Elongator functions as a negative modulator of this process (Zhou et al. 2009). Together, these data suggest that TaELP2 exerts a negative regulatory effect on ROS accumulation during Pst infection.

Transcriptional reprogramming is an integral part of plant immunity. Studies have found that the Elongator complex in eukaryotes is involved in both the transcription and processing of miRNA (Fang et al. 2015). The absence of Elongator in A. thaliana leads to a decrease in pre‐miRNA transcription. Meanwhile, Elongator directly interacts with the pri‐miRNA processing complex, promoting pri‐miRNA transcription and processing. Additionally, subunits of Elongator were observed to interact with components of the Arabidopsis dicing complex, together forming discrete nuclear bodies (D‐bodies). The disruption of the Elongator complex led to a defect in DCL1 localisation to D‐bodies or D‐body formation (Fang et al. 2015). The U11/U12‐65K protein, a U12‐type spliceosome component, interacts with ELP2 (Woloszynska et al. 2016). TaELP2 overexpression led to the alternative splicing of genes enriched in pathways such as ‘RNA interference’, ‘siRNA generation’ and ‘post‐transcriptional gene silencing’. Among the five major AS types, A3SS was most frequent, followed by SE, A5SS, RI and MXE (Figure S6). In our study, we found that TaELP2 can directly interact with the PAZ domain of TaDCL1 through Y2H, LCI, pull‐down and Co‐IP assays (Figure 4). DCLs are endonucleases belonging to the RNase III family. In Arabidopsis, the primary function of DCL1 is the synthesis of miRNAs, although it also participates in the synthesis of some endogenous siRNAs (Katiyar‐Agarwal and Jin 2010). Dicer proteins are typically composed of DExD/helicase, Dicer dimerisation, PAZ, RNase III and dsRBD domains, with the PAZ domain being responsible for the recognition and binding of double‐stranded RNA. The distance between the RNase III domain and the PAZ domain of DCL proteins is the main determinant of the size of the miRNA generated. Our findings suggest that the interaction between TaELP2 and TaDCL1. The mechanism may involve the recognition of RNA sequences of downstream target genes through the PAZ domain, leading to the generation of complementary miRNA sequences and, consequently, the silencing of target genes through the silencing complex.

Plants have evolved a variety of mechanisms for resisting invasion by pathogenic bacteria. Increasing evidence supports that miRNAs are involved in both PTI and ETI (Asadi and Millar 2024; Yang et al. 2021). miRNAs regulate a wide range of defence signals and pathways, including the expression of NBS‐LRR genes, hormone signalling and ROS production. In this study, KEGG enrichment analysis showed that the DEGs downregulated in TaELP2‐OE plants were enriched in pathways related to plant hormone signalling, MAPK signalling and plant–pathogen interactions (Figure 7b). In barley and wheat, miR9863a and miR9863b target NBS‐LRR genes, and their overexpression was reported to weaken Mla1‐mediated disease resistance (Liu et al. 2014). The production of ROS and the expression of genes in the SA signalling pathway are inhibited in the Arabidopsis miR156 overexpression mutant, resulting in enhanced susceptibility to P. syringae (Yin et al. 2019). In rice, siR109944 is involved in the modulation of plant immunity, suppressing resistance to sheath blight via its influence on auxin homeostasis. Furthermore, its heterologous overexpression in A. thaliana weakened the plant's resistance to infection by B. cinerea, Sclerotinia sclerotium and Verticillium dahliae (Qiao, Zhao, et al. 2020; Qiao, Zheng, et al. 2020). In the present study, we noted that the overexpression of TaELP2 significantly downregulated the expression of NB‐ARC family genes. Subsequently, the silencing of these genes through VIGS led to a decrease in the resistance of wheat to Pst. Given the demonstrated interaction between TaELP2 and TaDCL1, a key protein in miRNA biogenesis, we hypothesise that this interaction may lead to the production of miRNAs targeting resistance‐related and ROS‐related genes, thereby reducing their expression and weakening the wheat immune response. Concurrently, as Elongator is known to associate with RNA polymerase II to modulate transcription rates, transcriptional repression also remains a plausible mechanism. In future studies, small RNA (sRNA) sequencing or degradome sequencing can be employed to clarify whether the sRNAs modulated by TaELP2 directly target NB‐ARC genes. Therefore, whether the observed downregulation of NB‐ARC genes is primarily due to transcriptional or post‐transcriptional regulation requires further investigation.

In conclusion, in this study, we established TaELP2 as a susceptibility factor during wheat infection by Pst. The silencing of TaELP2 through VIGS enhanced the disease resistance of wheat to Pst via the upregulation of the expression of PR1 and PR2 genes, whereas its overexpression exerted the opposite effect. In addition, using a variety of protein interaction assays, we found that TaELP2 directly interacts with TaDCL1 and TaELP3. Therefore, this study confirms that TaELP2 acts as a negative regulator of stripe rust resistance in wheat; however, the precise mechanistic contribution of transcriptional versus post‐transcriptional control to TaELP2‐mediated regulation of NB‐ARC gene expression remains to be fully elucidated, which will constitute a key focus of subsequent research.

4. Experimental Procedures

4.1. Plant Materials, Strains and Growth Conditions

The wheat cultivar Suwon 11 (SY11) was used for expression pattern analysis and VIGS experiments following inoculation with Pst, the Fielder cultivar was used for VIGS experiments and generating transgenic materials, and Mingxian 169 was employed for Pst propagation. N. benthamiana was used for subcellular localisation analysis of the TaELP2 protein, as well as for Co‐IP and LCI assays. All wheat cultivars were cultivated in an incubator at 16°C under a 16‐h/8‐h light/dark cycle. N. benthamiana plants were grown at 23°C under a 16‐h light/8‐h dark photoperiod. Suwon 11 (carrying Yr26) and Fielder (carrying Yr20) are both susceptible to Pst isolate CYR23 and compatible with isolate CYR31. The physiologically compatible Pst race CYR31 and the incompatible race CYR23 were identified and provided by the Plant Immunity Research Team of Northwest A&F University. The bacterial strains used in this study (Escherichia coli DH5α and Agrobacterium tumefaciens GV3101) were cultured in Luria Bertani (LB) medium containing the corresponding antibiotics at 37°C and 28°C, respectively. The yeast AH109 strain was used for the Y2H assays and was cultured in YPDA medium at 30°C.

4.2. Total RNA Extraction and RT‐qPCR

Total RNA was extracted from wheat using the protocol described by Liu et al. (2015). The full‐length TaELP2 coding sequence was retrieved from the NCBI database. Gene‐specific primers were designed with Primer Premier 5.0 software. Cloning and sequence analyses were performed according to Huai et al. (2019). The expression levels of the TaELP2 gene in wheat leaves infected with Pst at different times were analysed using RT‐qPCR, with wheat elongation factor 1 (TaEF‐1α) serving as the endogenous reference. RT‐qPCR was performed on a Bio‐Rad CFX system, and data were collected and analysed using Bio‐Rad CFX Maestro software. Each experimental sample had three biological replicates. Relative gene expression levels were calculated using the 2^−ΔΔCt method.

4.3. Virus‐Induced Gene Silencing

BSMV‐mediated gene silencing was conducted as described by Huai et al. (2019). Two silencing fragments with lengths of 150 and 300 bp were derived from the conserved region of the TaELP2 gene in wheat and inserted into the BSMV‐VIGS‐γ vector, yielding the recombinant vector. Similarly, BSMV‐VIGS‐γ‐based recombinant vectors were constructed for wheat TaPDS and GFP. The γ‐TaELP2, γ‐TaPDS and γ‐GFP recombinant vectors, and the α and β virus vectors, with verified sequences, were linearised by restriction enzyme digestion and transcribed in vitro. The generated α and β transcripts were mixed with those of γ‐TaPDS (positive control), γ‐GFP (negative control) or γ‐TaELP2 at a ratio of 1:1:1. Each mixture was mechanically inoculated (rubbed) onto the second leaves of wheat seedlings, which were cultured first in the dark at 25°C–28°C for 24 h, and then under normal conditions for 14 days. The fourth leaves of wheat plants were inoculated with Pst CYR23 or CYR31. Leaf samples were collected at 0, 24, 48 and 120 hpi to evaluate silencing efficiency and for histological examination. Disease phenotype was assessed at 12–14 days after inoculation. The experiment was repeated three times.

4.4. Histological Observation

Wheat leaves inoculated with Pst for 24, 48 and 120 h were collected and decolourised using a fixative solution (glacial acetic acid:ethanol, 1:1 v/v). Once all the leaves had decolourised, they were treated with saturated chloral hydrate solution for 24 h, boiled in water, and stained with wheat germ agglutinin (WGA) Alexa‐488 solution for Pst visualisation. The size, hyphal length, numbers of branches, haustoria and haustorial mother cells of Pst were quantified using a BX‐53 microscope equipped with CELLSENS Entry software (Olympus).

4.5. Y2H Assays

To confirm the interaction between TaELP2 and TaDCL1, the C‐terminal, N‐terminal and PAZ domains of TaDCL1 were inserted into the pGADT7 vector (AD). TaELP2‐BD was co‐transformed with the AD empty plasmid, TaDCL1‐C‐AD, TaDCL1‐N‐AD or TaDCL1‐PAZ‐AD, with TaELP2‐BD/AD empty plasmid co‐transformation serving as a negative control. The transformants were spread on SD/−Trp/−Leu and SD/−Trp/−Leu/−His media and cultured in an incubator at 30°C for 2–3 days. The colonies of the experimental group grown on SD/−Trp/−Leu/−His medium were resuspended in double‐distilled water and then transferred to SD/−Trp/−Leu/−His/−Ade medium containing X‐α‐Gal.

4.6. LCI Assays

The coding sequences of the TaELP2 and GUS genes were inserted into the pCAMBIA 1300‐Cluc vector, while TaDCL1^PAZ was inserted into the pCAMBIA 1300‐Nluc vector. The recombinant vectors were separately transformed into A. tumefaciens GV3101. To verify the interaction, a bacterial solution containing TaDCL1^PAZ‐Nluc was mixed in equal proportions with a bacterial solution containing either TaELP2‐Cluc or GUS‐Cluc, and injected on both sides of the abaxial surface of N. benthamiana leaves. The co‐injection of TaDCL1^PAZ‐Nluc and GUS‐Cluc served as the control. The treated plants were cultured at 25°C for approximately 40 h. After removing the leaves, a luciferase substrate was applied to the injection sites, and the leaves were left in the dark for 5 min. The resulting luminescence was then detected using a plant View100 system.

4.7. Pull‐Down Assay

The pull‐down assay was conducted as described by Wang et al. (2022). The coding sequences of TaELP2 and TaDCL1^PAZ were ligated into pET28a and pGEX4T‐1, respectively. These constructs were transformed into E. coli BL21 competent cells and cultured overnight at 16°C in LB liquid medium and then ultrasonicated. Protein interactions were validated using a His Protein Interaction Pull‐Down Kit (Thermo). Horseradish peroxidase (HRP)‐conjugated anti‐His‐tag and anti‐GST‐tag mouse monoclonal antibodies (Beyotime Biotech) were used for western blot analysis.

4.8. Co‐IP Assay

The Co‐IP assay was conducted as described by Wang et al. (2022). The coding sequence of TaELP2 was cloned into the expression vector pBin‐GFP (GFP tag), and the sequence of TaDCL1^PAZ was fused to mCherry. The resulting recombinant plasmids were separately transformed into A. tumefaciens GV3101. The bacterial solution containing GFP‐TaELP2 was mixed in equal proportions with the bacterial solution containing mCherry‐TaDCL1^PAZ at an OD₆₀₀ of 0.6 and injected into N. benthamiana leaves. After approximately 48 h of transient expression in N. benthamiana leaves, total protein was extracted as described by Wang et al. (2022) and incubated with anti‐GFP magnetic beads (Thermo Fisher) at 4°C. The magnetic beads were then collected by centrifugation at 2500 g at 4°C for 5 min and washed four times with phosphate‐buffered saline. Subsequently, the samples were mixed with a 5× protein loading buffer and boiled for 10 min. The proteins in the immunoprecipitates were separated by 10% SDS‐PAGE and transferred to a nitrocellulose membrane. For immunoblot analysis, the nitrocellulose membrane was incubated first with primary antibodies against GFP and mCherry (Beyotime), and then with goat anti‐mouse immunoglobulin G (H + L) secondary antibody (Beyotime).

4.9. Statistical Analysis

All analyses in this study were performed using GraphPad Prism 8. Statistical significance was determined by Student's t‐test, with *p < 0.05, **p < 0.01, and ns indicating statistically significant. In all diagrams, error bars depict the distribution of values, representing the standard deviation (SD) of the mean.

Author Contributions

X.Z., S.G. and M.L. designed the research. M.L. and Y.C. performed the experiments; M.L. analysed the date and made graph. Y.C. collected the experimental procedures. M.L., Y.C. and X.Z. wrote this manuscript. M.L., S.G. and X.Z. revised this manuscript.

Disclosure

Accession numbers: The gene sequences from this study are available in Ensembl Plants under the following accession numbers: TaELP2 (TraesCS1A02G104700, TraesCS1B02G116100, TraesCS1D02G096900); TaDCL1 (TraesCS4D02G342300); TaELP1 (TraesCS2A02G203700); TaELP3 (TraesCS2B02G361800); TaELP4 (TraesCS7A02G522900); TaELP5 (TraesCS4B02G259300); TaELP6 (TraesCS4D02G199700).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Alignment of the cDNA sequences of the three TaELP2 homoealleles. The full‐length cDNA sequences of TaELP‐1A, TaELP2‐1B and TaELP2‐1D show 97.69% identity.

Figure S2: Alignment of the protein sequences of the three homoealleles of TaELP2. Amino acid sequence alignment of TaELP2‐1A, TaELP2‐1B and TaELP2‐1D shows 97.87% identity.

Figure S3: Knocking down TaELP2 reduces development of Pst race CYR31. Pst growth in BSMV:γ, BSMV:TaELP2‐1as and BSMV:TaELP2‐2as infected leaves with subsequent inoculation of Pst CYR31 was observed at 24, 48 hpi and 120 hpi by staining with wheat germ agglutinin conjugated to Alexa‐488 (WGA) under epifluorescence microscopy. BSMV:γ was used as the control. H, Haustoria; SV, Substomatal vesicle; IH, Infection hypha; HMC, Haustorial mother cell.

Figure S4: TaDCL1 is significantly induced by Pst infection. (a) Schematic diagram of TaDCL1 and its domains. (b) The transcript levels of TaDCL1 in wheat leaves were induced during wheat‐Pst interaction. The leaves of wheat Suwon 11 were inoculated with CYR23 and CYR31 at 0, 12, 24, 48, 72, 96 and 120 hpi. Untreated leaves served as control. Data are presented as mean ± SD from three biological replicates. Statistical significance was determined by Student's t‐test (**p < 0.01).

Figure S5: Analysis of TaELP2 overexpression transcriptome data. (a) The expression levels of four down‐regulated DEGs of the ‘plant‐pathogen interaction’ pathways from RNA‐seq data were selected for RT‐qPCR. (b) The transcript levels of the four NB‐ARC genes in wheat leaves were induced during wheat‐Pst interaction. Data are presented as mean ± SD from three biological replicates. Statistical significance was determined by Student's t‐test (**p < 0.01).

Figure S6: AS analysis of TaELP2‐OE transcriptome data. (a) AS‐GO enrichment analysis of differentially expressed genes between TaELP2‐OE line. (b) Distribution of alternative splicing event types in TaELP2‐OE.

Li, M. , Chen Y., Guo S., et al. 2026. “ TaELP2 Interacts With TaDCL1 and Negatively Regulates Wheat Resistance Against Stripe Rust.” Molecular Plant Pathology 27, no. 2: e70225. 10.1111/mpp.70225.

Data Availability Statement

The data that supports the findings of this study are available in the Supporting Information of this article. Accession numbers: The gene sequences from this study are available in Ensembl Plants under the following accession numbers: TaELP2 (TraesCS1A02G104700, TraesCS1B02G116100, TraesCS1D02G096900); TaDCL1 (TraesCS4D02G342300); TaELP1 (TraesCS2A02G203700); TaELP3 (TraesCS2B02G361800); TaELP4 (TraesCS7A02G522900); TaELP5 (TraesCS4B02G259300); TaELP6 (TraesCS4D02G199700).