Abstract
Introduction:
Introduction: Rye (Secale cereale L.) is a cereal crop with superior overall stress tolerance, particularly prominent resistance to stripe rust, cold, and drought. It thus serves as an important donor of resistance genes for other cereal species. Ethylene-insensitive 3/ethylene-insensitive 3-like (EIN3/EIL) proteins are core transcription factors that transduce ethylene signals into downstream transcriptional cascades. However, their systematic characterization and defense-related functions in rye remain unexplored.
Methods:
Here, we performed genome-wide identification of ScEIL genes from the rye reference genome, designated them based on chromosomal distribution, and analyzed their gene structure, collinearity, and cis-elements. We also examined the expression profiles of ScEIL genes under biotic and abiotic stresses, determined the subcellular localization of ScEIL5, and verified its function via VIGS-mediated silencing.
Results:
Seven ScEIL genes (ScEIL1–ScEIL7) were identified. Gene structure analysis revealed a highly conserved N-terminal DNA-binding domain and divergent C-terminal regions. Collinearity analysis showed significant evolutionary divergence of the EIN3/EIL family between monocotyledonous and dicotyledonous plants. Cis-element prediction and expression profiling indicated that ScEIL genes are involved in rye responses to various stress conditions and exhibit significant tissue specificity, with the lowest expression detected in seeds. ScEIL5 is localized to the nucleus and showed approximately 4.8-fold higher expression at 48 h post-inoculation with Puccinia striiformis f. sp. tritici compared with 0 h. VIGS-mediated silencing of ScEIL5 enhanced stripe rust resistance and reduced fungal biomass.
Discussion:
These results suggest that ScEIL5 acts as a negative regulator of stripe rust resistance in rye. This study not only provides valuable insights for further research on the EIN3/EIL gene family, but also offers important clues regarding the role of ScEIL genes in rye’s response to stripe rust stress.
1 Introduction
Rye (Secale cereale L.) is an outcrossing member of the Poaceae family characterized by high dietary fibre, elevated protein content, abundant minerals, and bioactive phenolic compounds. Predominantly cultivated in northern and eastern Europe, it is utilized for grain, forage, and green-manure purposes (Rodehutscord et al., 2016; Torrinha et al., 2019; Ikram et al., 2023). Rye exhibits exceptional tolerance to low temperature, drought, and stripe rust, a characteristic that has drawn extensive attention from researchers to the molecular mechanisms underlying its biotic/abiotic stress tolerance. The species also possesses a large and highly repetitive genome that has only recently been fully sequenced and annotated (Rabanus-Wallace et al., 2021; Bauer et al., 2017).
Ethylene (C2H4) is a gaseous phytohormone that regulates virtually every phase of the plant life cycle, from seed germination and root hair development to fruit ripening and organ senescence (Ju and Chang, 2015; Guo and Ecker, 2004). The canonical ethylene signalling pathway begins with ethylene perception by a family of endoplasmic reticulum-localized receptors (ETR1, ERS1, EIN4, ETR2, and ERS2) that, in the absence of the hormone, activate the Raf-like kinase CTR1 (Kieber et al., 1993). CTR1 phosphorylates and thereby destabilises the positive regulator EIN2, preventing the nuclear accumulation of the EIN3/EIL transcription factors (Alonso et al., 1999; Ju et al., 2012). Upon ethylene binding, receptor activity is repressed, CTR1 is inactivated and EIN2 is dephosphorylated and proteolytically cleaved to release a C-terminal fragment that migrates to the nucleus and stabilises EIN3/EIL proteins (Wen et al., 2012; Qiao et al., 2012). Stabilised EIN3/EIL then bind directly to the promoter of primary ethylene-response genes such as ERF1, initiating transcriptional cascades that reprogram cellular metabolism and development (Solano et al., 1998).
EIN3 was first identified in Arabidopsis thaliana via a screen for ethylene-insensitive mutants that failed to exhibit the triple-response phenotype in etiolated seedlings (Chao et al., 1997). The locus encodes a 628-amino-acid nuclear protein with a highly conserved N-terminal DNA-binding domain (DBD) composed of five α-helices and seven β-sheets that specifically recognises the primary ethylene-response element (PERE; 5’-TGTCTC-3’) and the adjacent GC-rich motif (Solano et al., 1998; Yamasaki et al., 2005). In addition to the DBD, EIN3 contains a proline-rich transcriptional activation domain and a C-terminal region that mediates dimerisation and interaction with the F-box proteins EBF1 and EBF2, which target EIN3 for 26S proteasome-mediated degradation in the absence of ethylene (Wen et al., 2012; Hao et al., 1998; Ohme-Takagi and Shinshi, 1995; Guo and Ecker, 2003; Potuschak et al., 2003). Outside Arabidopsis, EIN3/EIL homologues have been identified in both monocots and dicots, including rice (Mao et al., 2006), maize (Gallie and Young, 2004), wheat (Duan et al., 2013), tomato (Tieman et al., 2001), petunia (Shibuya et al., 2004), and carnation (Iordachescu and Verlinden, 2005).
The functional versatility of EIN3/EIL proteins stems from their ability to integrate ethylene signalling with other hormone pathways, including gibberellin (An et al., 2012), jasmonic acid (Zhu et al., 2011) and salicylic acid (Chen et al., 2009). In Arabidopsis, EIN3 directly represses SID2 expression, thereby attenuating SA biosynthesis and modulating the trade-off between growth and defence (Chen et al., 2009). Conversely, EIN3 and EIL1 synergistically activate HLS1 transcription in cooperation with GA signalling to promote apical hook formation in etiolated seedlings (An et al., 2012). During leaf senescence, EIN3 induces ORE1 expression by suppressing miR164, accelerating chlorophyll degradation and nutrient remobilisation (Li et al., 2013). Similar pleiotropic roles have been reported in crops: overexpression of OsEIL1 in rice enhances ethylene sensitivity and promotes internode elongation in deep-water conditions (Hattori et al., 2009), whereas silencing of LeEILs in tomato extends flower longevity by delaying petal senescence (Iordachescu and Verlinden, 2005). Beyond developmental control, EIN3/EIL proteins play pivotal roles in biotic/abiotic stress adaptation (Chen et al., 2005; Kazan, 2015; Tao et al., 2015). In wheat, TaEIL1 acts as a negative regulator of stripe rust resistance by suppressing pathogenesis-related (PR) gene expression (Duan et al., 2013), whereas in cotton, GhEIL3 enhances drought tolerance by activating ROS-scavenging enzymes and ABA-responsive genes (Zhang et al., 2023). Under salt stress, Arabidopsis EIN3 directly binds to the promoter of ESE1 (ETHYLENE AND SALT-INDUCIBLE ERF1) to activate the expression of peroxidases and superoxide dismutases, thereby mitigating oxidative damage (Zhang et al., 2011). In rice, OsEIL1 positively regulates the expression of OsAPX2 and OsSOD1 under drought and high-salinity conditions (Cao et al., 2006). MdEILs in apples regulate fruit ripening mechanisms and respond to cold treatment (Feng et al., 2025). Recently, Liu et al. (2019) showed that mulberry MnEIL3 confers both salt and drought tolerance by modulating ethylene biosynthetic genes and ABA signalling. Collectively, these studies have demonstrated the functional diversity of EIN3/EIL proteins.
Given rye exhibits distinctive stress tolerance, further elucidation of its EIN3/EIL gene family may facilitate identification of novel molecular mechanisms underlying disease resistance. In this study, we employed bioinformatics to identify and characterize rye EIN3/EIL gene family members. Their responses to biotic and abiotic stresses were quantified by qRT-PCR, and tissue-specific expression patterns were analyzed using publicly available transcriptome datasets. ScEIL5 was significantly upregulated upon stripe rust infection, and VIGS-mediated silencing confirmed its function as a negative regulator of disease resistance. These findings provide valuable insights into the EIN3/EIL gene family and its biological function in rye.
2 Materials and methods
2.1 Genome-wide identification of the EIN3/EIL gene family and biochemical characterization of ScEIL proteins in rye
To identify EIN3/EIL family members in rye, Arabidopsis EIN3/EIL protein sequences were retrieved from TAIR (https://www.arabidopsis.org) and used as BLAST queries against the rye reference genome (Ensembl Plants; https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_902687465.1/). Candidate loci were refined by HMMER3.0 searches (Pfam PF04873) and validated for the presence of the EIN3/EIL conserved domain using the Conserved Domain Database (CDD; https://www.ncbi.nlm.nih.gov/Structure/cdd/) (Marchler-Bauer et al., 2017).
Physicochemical properties of ScEIL proteins were predicted with ExPASy ProtParam (https://web.expasy.org/protparam/) (Artimo et al., 2012), and subcellular localization was inferred with Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/).
2.2 Chromosomal localization and gene duplication events
The chromosomal start and end location information of ScEIL genes was extracted from gene annotation files, and their physical locations were mapped using TBtools software. Intraspecific synteny of the rye EIN3/EIL family was analyzed via the One-Step MCScanX function in TBtools, and the results were visualised in the software. Furthermore, One Step MCScanX-Super Fast was utilised to visualise interspecific collinearity analysis of the EIN3/EIL family in rye and other species (Chen et al., 2023).
2.3 Construction of the phylogenetic tree and analysis of the gene structure
Phylogenetic trees were constructed with MEGA 7.0 using the Neighbor-Joining method (1,000 bootstrap replicates) (Kumar et al., 2016). Conserved motifs were identified with MEME (https://meme-suite.org; max = 12) and visualised together with exon-intron structures via TBtools. Multiple sequence alignment was performed with ClustalX (Thompson et al., 1997) and edited in GeneDoc (Larkin et al., 2007). Tertiary structures were modeled with SWISS-MODEL (https://swissmodel.expasy.org/) using the best Protein Data Bank (PDB) template (Waterhouse et al., 2018).
2.4 Cis- acting elements and functional prediction
Using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database of EIN3/EIL genes promoter sequences (translation initiation site upstream 2 kbp) of cis regulatory elements (Lescot et al., 2002). The regulatory functions of the predicted cis-acting elements were subjected to a classification and statistical analysis.
2.5 Subcellular localization
The CDS sequence of the ScEIL5 gene was amplified by specific primers, and the product was ligated to the pCAMBIA1302-GFP vector to obtain 35S:ScEIL5-GFP. HY5 (nuclear localization protein) was cloned and ligated into pCAMBIA1302-mCherry to obtain 35S:HY5-RFP (Wang et al., 2021). The fusion carrier was introduced into 4-week-old tobacco leaves for instantaneous expression. After infiltration for 48 h, it was observed using a confocal laser scanning microscope (Olympus, Japan). The detailed acquisition parameters were set as follows: excitation wavelength for GFP was 488 nm, emission wavelength was 500–550 nm; excitation wavelength for RFP was 552 nm, emission wavelength was 570–620 nm.
2.6 Plant materials and treatments
Seeds of ‘Gannong NO. 2’ rye provided by the Grassland Science College of Gansu Agricultural University was used as experimental material.
Seeds were surface-sterilized in 1% sodium hypochlorite solution for 5 minutes and rinsed three times with sterile distilled water. Sterilized seeds were germinated on moist filter paper at 25 °C for 3 days. For abiotic stress treatments, germinated seedlings were transferred to Hoagland nutrient solution (pH 5.8) and hydroponically cultured under controlled conditions (16 h light/8 h dark cycle; light intensity: 250 μmol/m2/s; temperature: 25 ± 2 °C; relative humidity: 60% ± 5%) until the three-leaf stage. Uniformly growing seedlings were selected for subsequent stress treatments. Drought stress: Seedlings were transferred to a nutrient solution supplemented with 15% polyethylene glycol (PEG-6000) to simulate drought stress conditions. Low temperature stress: Seedlings were transferred to an incubator maintained at 4 °C, with all other environmental conditions kept unchanged. Leaves were collected at 0 h, 6 h, 12 h, and 24 h post-treatment for each stress. For biotic stress treatment, germinated seeds were sown in 15 cm-diameter flower pots (10 seedlings per pot) and grown under the aforementioned controlled conditions until the three-leaf stage. The biotic stress, Puccinia striiformis f. sp. tritici (Pst), was applied in a uniformly to dewaxed leaves (dewaxed by gentle manual friction with fingers) at 9-12 °C. Inoculated plants were maintained in a high-humidity environment to facilitate pathogen infection. Leaves were harvested at 0, 24, 48 and 72 hours post-inoculation (hpi). Each treatment included three biological replicates. All samples were immediately frozen in liquid nitrogen and stored at −80 °C.
2.7 Gene expression analysis
Tissue-specific expression of ScEIL genes in seeds, roots, stems, and leaves was analyzed based on RNA-seq data retrieved from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (accession no. PRJNA680499). Fragments per kilobase of transcript per million mapped reads (FPKM) was used to quantitatively calculate gene expression levels (Trapnell et al., 2009).
Total RNA was extracted using the Plant RNA Extraction Kit (TIANGEN, China) according to the manufacturer instructions. The reverse transcription of the RNA samples was conducted using the SweScript All-in-One RT SuperMix for qPCR (One-Step gDNA Remover) kit, following the manufacturer protocol. Specific primers were designed based on the gene sequence using the Primer 5.0 software. The primers are listed in Supplementary Table 1. The expression of ScEIL genes was quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on a LightCycler® 96 system. Three technical replicates were run per biological sample. The reaction system included 10 μL of SYBR Premix Ex Taq (2×), 0.4 μL of each primer (10 μM), 2 μL of template (approximately 100 ng/μL), and 7.2 μL of ddH2O, resulting in a total volume of 20 μL. The qRT-PCR program was as follows: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 15 s, annealing/extension of the primer at 60 °C for 30 s, with fluorescence signal collection. A total of 35 cycles were performed. Actin (GenBank Accession: FJ032189.1) was used as the internal parameter to calculate the relative expressions via the Ct (2−ΔΔCt) method (Livak and Schmittgen, 2001; Ren et al., 2023).
2.8 Virus-induced gene silencing
The function of ScEIL5 in rye was analyzed using a virus-induced gene silencing (VIGS) approach. Plasmid construction for gene silencing was performed as previously described by Holzberg et al. (2002). The specific primers were designed to amplify gene-specific fragments of ScEIL5 (Supplementary Table 1), incorporating PacI and NotI restriction sites. The amplified fragments replaced the phytoene desaturase (PDS) fragment in the γ-PDS positive control vector, resulting in the construction of two vectors, Barley stripe mosaic virus (BSMV):γ-ScEIL5-1as and BSMV:γ-ScEIL5-2as. BSMV:α and BSMV:γ vectors were digested with MluI, while BSMV:β was digested with SpeI. Subsequently, both BSMV:γ-PDS, BSMV:γ-ScEIL5-1as and BSMV:γ-ScEIL5-2as were digested with BssHII. The digested fragments were recovered and purified. Using these linearized products as templates, in vitro transcription was performed with the T7 RiboMAX kit (Promega, USA). A mixture of 0.5 uL BSMV:α, BSMV:β, and BSMV:γ (or BSMV:γ-PDS, BSMV:γ-ScEIL5-1as, and BSMV:γ-ScEIL5-2as) was combined with 8.5 uL of FES buffer and friction inoculation onto the second leaf at the three-leaf stage. The inoculation method followed (Panwar and Kanyuka, 2022). FES buffer was used as a negative control, and BSMV:γ-PDS served as a positive control. Plants were incubated at 25 °C for 12 days until photobleaching symptoms appeared on BSMV: PDS-inoculated leaves. Once photobleaching was observed, the fourth leaf was inoculated with Pst CYR34. The inoculated leaves were collected at 0, 24, 72, and 120 hpi for silencing efficiency analysis. Additional samples were harvested at 24, 72 and 120 hpi for microscopic examination.
2.9 Histological analysis
Using the method described by Redkar et al. (2018), Pst-inoculated leaves were first subjected to decolorization treatment in ethanol:acetic acid (1:1, v/v). Subsequently, cleared tissues were washed with 50 mM Tris-HCl (pH 7.4) and incubated with wheat germ agglutinin Alexa-488 (product number W11261, Thermo Fisher Scientific). Finally, samples were photographed under an Olympus BX53 microscope. Infection areas, hyphal lengths, and haustorial mother cell numbers were quantified using CellSens Entry software.
For H2O2 detection, leaves were immersed in a 3,3’-diaminobenzidine (DAB) solution (1 mg/mL, pH 3.8) and incubated under light for 6 h. After destaining in ethanol:acetic acid (1:1, v/v), samples were photographed under an Olympus BX53 microscope. DAB-stained areas were quantified with CellSens Entry.
2.10 Statistical analysis
All data are presented as the mean ± standard deviation (SD) from at least three independent biological replicates. Statistical analyses were performed using IBM SPSS 20.0 Statistics software. Normality and homogeneity of variances were assessed using Shapiro–Wilk and Levene’s tests, respectively. Since the assumptions were not met, the nonparametric Kruskal–Wallis H test was performed. Post hoc multiple comparisons were conducted using Dunn’s test with Bonferroni correction to identify pairwise differences. Statistical significance was set at p < 0.05, and significant differences are indicated by different lowercase letters.
3 Results
3.1 Genome-wide identification, chromosome localization and gene structure analysis of ScEILs in rye
To identify EIN3/EIL genes in rye, we used A. thaliana (AtEIL, n=6) and wheat (TaEIL, n=21) protein sequences as queries to search the rye genome. The obtained sequences were then extracted using the HMM. Subsequently, candidate gene sequences were filtered through the Conserved Domain Database (CDD) to eliminate redundant sequences. Ultimately, seven ScEIL genes were identified. According to the gene annotation information file (GFF3), these seven ScEIL genes were located on six chromosomes (Figure 1A). Based on their chromosomal order, these genes were named ScEIL1 to ScEIL7. ScEIL1 and ScEIL2 were located on the same chromosome (chr2R), while the remaining five ScEIL genes were distributed on chr3R, chr4R, chr5R, chr6R, and chr7R, respectively.
Figure 1
Twelve conserved motifs were identified based on MEME online analysis. Motifs 1–5 were located at the N-terminus (Figure 1B) and are present in all seven ScEIL proteins. This indicated that the EIN3/EIL protein sequences of rye were highly conserved at the N-terminus, a feature that is also present in EIN3/EIL proteins of other species. Notably, ScEIL proteins that are closely related on the phylogenetic tree have similar structural compositions, suggesting that they may have functional redundancy. Some of these motifs were found to be unique to members of group A (motif 11) or group C (motifs 6 and 12), suggesting that these motifs may have unique functions. In addition, protein domain analysis showed that groups A and C contained the EIN3-specific domain, which is a unique domain defining the EIN3 subfamily, whereas group B contained the EIN3 superfamily domain, which represents a conserved domain shared by the entire EIN3/EIL family (Figure 1C). To further explore the gene structure of ScEILs, the exon-intron organization of these genes was analyzed. As shown in Figure 1D, the number and length of introns varied substantially among ScEIL genes. Group B contained the highest number of introns. Specifically, ScEIL4 harbored two introns and ScEIL5 contained three introns.
3.2 Phylogenetic analysis and physicochemical properties of the ScEIL proteins
To investigate the evolutionary relationships of EIN3/EIL proteins, a phylogenetic tree was constructed using 43 amino acid sequences from Triticum aestivum (TaEILs, n=21), Zea mays (ZmEILs, n=9), A. thaliana (AtEILs, n=6), and S. cereale (ScEILs, n=7). The phylogenetic tree clearly clustered the EIN3/EIL proteins into three distinct clades (group A, B, and C), with each clade comprising members from both monocot and dicot species (Figure 2). The close clustering of ScEILs with TaEILs illustrated the tight kinship between rye and wheat and indicated strong functional conservation, consistent with previous classifications in Arabidopsis and wheat.
Figure 2
Analysis of the physicochemical properties revealed that ScEIL proteins ranged in length from 446 amino acids (ScEIL2 and ScEIL6) to 639 amino acids (ScEIL7) (Table 1). Molecular weights ranged from 49.29 kDa (ScEIL2) to 70.89 kDa (ScEIL7). Isoelectric points ranged from 5.12 (ScEIL7) to 5.89 (ScEIL5), indicating that these proteins are acidic. Hydropathy indices ranged from -0.547 (ScEIL1) to -0.721 (ScEIL7), indicating that these proteins are hydrophilic. In addition, ScEIL4 was identified as a stable protein (instability index < 40), while the other proteins were unstable (instability index > 40). Protein stability here refers to predicted structural stability and resistance to degradation. These values represent in silico predictions of protein structural stability rather than experimentally determined functional stability. Subcellular localization prediction analysis indicated that all members of the ScEIL protein family were localized in the nucleus.
Table 1
| Name | ID | Length (aa) | WM (kDa) | pI | Gravity | Instability index | Subcellular location |
|---|---|---|---|---|---|---|---|
| ScEIL1 | SECCE2Rv1G0071970.1 | 623 | 67.68 | 5.68 | -0.547 | 62.46 | Nucleus. |
| ScEIL2 | SECCE2Rv1G0105520.1 | 446 | 49.29 | 5.52 | -0.654 | 52.63 | Nucleus. |
| ScEIL3 | SECCE3Rv1G0202840.1 | 454 | 50.62 | 5.3 | -0.715 | 62.09 | Nucleus. |
| ScEIL4 | SECCE4Rv1G0244500.1 | 614 | 68.09 | 5.52 | -0.649 | 37.44 | Nucleus. |
| ScEIL5 | SECCE5Rv1G0334180.1 | 611 | 68.25 | 5.89 | -0.685 | 45.76 | Nucleus. |
| ScEIL6 | SECCE6Rv1G0414700.1 | 446 | 49.3 | 5.52 | -0.654 | 57.69 | Nucleus. |
| ScEIL7 | SECCE7Rv1G0482440.1 | 639 | 70.89 | 5.12 | -0.721 | 51.63 | Nucleus. |
The physicochemical properties of the ScEIL proteins.
WM, Molecular weight; pI, Isoelectric point; Gravity: Grand average of hydropathy.
3.3 Gene duplication and collinearity analysis of ScEILs
To investigate the expansion mechanism of the ScEIL gene family in rye, we analyzed gene duplication events. We identified two segmental duplication events: one between ScEIL4 and ScEIL5 (on chr4R and chr5R), and another between ScEIL1 and ScEIL7 (on chr2R and chr7R) (Figure 3A). These duplicated sequence pairs, belonging to groups B and C, further indicate that fragment duplication may be a major driving force for the expansion of the ScEIL gene family in rye, providing a genetic basis for functional divergence and evolution of this gene family.
Figure 3
To investigate the evolutionary conservation and divergence of ScEIL genes in rye, we conducted a collinearity analysis of EIN3/EIL genes in rye, wheat, maize, and Arabidopsis (Figure 3B). The results revealed multiple homologous gene pairs between rye and both wheat and maize, indicating that EIN3/EIL genes in these species exhibit a high degree of evolutionary conservation. This reflects their close phylogenetic relationship within the grass family. During the evolution of the EIN3/EIL gene family, some ancestral gene characteristics may have been retained. No homologous gene pairs were identified between rye and Arabidopsis EIN3/EIL genes, reflecting that due to the long evolutionary distance between these species, the conservation of EIN3/EIL genes has been significantly reduced.
3.4 Sequence and structural characterization of the DNA-binding domain in ScEIL transcription factors
Multiple sequence alignment of seven ScEIL and three AtEIL proteins revealed seven conserved regions (Figure 4A). Five basal regions (BRI-BRV), one acidic region (AR), and one proline-rich domain (PR) were identified. The DBD region exhibited high conservation, particularly in the α1, α3, and α4 helical regions. Key amino acid residues (such as Arg and Lys) were completely conserved, which may be crucial for DNA-binding activity. Additionally, the C-terminal PR region of ScEIL proteins showed significant variation, which may be related to specific regulation of their transcriptional activation functions.
Figure 4
Homology modeling showed that the DNA-binding domain (DBD) of ScEIL proteins consists of five α-helices (Figure 4B). These helical structures form a characteristic three-dimensional fold that is expected to mediate specific binding to target DNA sequences.
3.5 Cis-acting element analysis
Analysis of cis-acting elements in ScEIL gene promoters revealed the presence of elements involved in responses to biotic/abiotic stress, growth and development, and phytohormones (Figure 5). Among these, elements associated with plant growth and development were the most abundant (n = 252), followed by those related to biotic/abiotic stress (n = 117), while phytohormone-responsive elements were the least abundant (n = 63).
Figure 5
Among the 18 biotic/abiotic stress-related elements, G-box and ARE-involved in light responsiveness and antioxidant defense—were identified. The LTR element, associated with low-temperature stress responses, was also present. MBS, related to drought induction, and TC-rich repeats, involved in defense and pathogen stress responses, were additionally detected.
Among the 11 growth and development-related elements, the core promoter element TATA-box, involved in transcription initiation complex assembly, was included. CAAT-box, which enhances transcriptional efficiency; GCN4_motif, involved in endosperm development and amino acid metabolism; and HD-Zip 1, related to light responsiveness and organ development, were also identified.
Among the eight phytohormone-responsive elements, ABRE, responsive to abscisic acid (ABA), was present. AuxRR-core, responsive to auxin; TGA-element, involved in salicylic acid signaling; GA_motif, responsive to gibberellin; and P-box, regulating seed germination and stem elongation, were also identified.
3.6 Gene expression levels under biotic/abiotic stress
To elucidate the potential roles of ScEIL genes in rye under abiotic stress, we analyzed their expression patterns under drought and low-temperature stress using qRT-PCR. Under low-temperature stress, ScEIL genes exhibited diverse expression profiles in leaves. For instance, ScEIL1, ScEIL2, ScEIL5, and ScEIL7 were significantly upregulated at 6 h, with expression levels of ScEIL1 and ScEIL2 increasing 14.7- and 14.5-fold compared to 0 h, respectively (Figure 6A). This strong induction suggests that these genes may play key roles in regulating rye response to low temperature stress. In contrast, ScEIL3 and ScEIL7 showed continuous upregulation but were significantly downregulated at 24 h.
Figure 6
Under drought stress, ScEIL genes displayed distinct expression dynamics. ScEIL3, ScEIL5, and ScEIL6 were initially upregulated but declined at 24 h (Figure 6B). ScEIL2 showed a gradual downregulation throughout the treatment period. In contrast, ScEIL4 expression steadily increased over 24 h, reaching 24.8-fold induction at 24 h. These differential expression patterns suggest that ScEIL genes may contribute specifically and distinctly to drought stress responses.
To investigate the role of ScEIL genes under biotic stress, we analyzed their expression following inoculation with Puccinia striiformis f. sp. tritici (Pst) CYR34. The results revealed a general trend of initial downregulation, followed by upregulation and then a second downregulation (Figure 6C). Notably, ScEIL5 expression was significantly induced at 48 h, increasing 4.8-fold compared to 0 h. These findings suggest that ScEIL5 plays a crucial role in rye defense response to stripe rust.
3.7 The tissue-specific expression of the EIN3/EIL gene family in rye and subcellular localization analysis of ScEIL5
We analyzed the tissue-specific expression patterns of ScEIL genes by interrogating a public rye transcriptome database, examining data from seeds, roots, stems, and leaves (Figure 7A). The results showed that ScEIL genes exhibited significant tissue-specific expression. ScEIL1 was predominantly expressed in roots and leaves. ScEIL7 was predominantly expressed in leaves. ScEIL2 showed high expression in stems. ScEIL5 and ScEIL6 were also highly expressed in stems and leaves. Expression of ScEIL genes in seeds was generally low, with only ScEIL1 and ScEIL5 showing detectable levels. This tissue-specific expression pattern suggests that ScEIL genes may play differentiated regulatory roles in the growth, development, or physiological processes of different rye tissues.
Figure 7
Previous experiments have shown that ScEIL5 may play an important role in resistance to Pst infection. Therefore, we investigated the subcellular localization of ScEIL5. A ScEIL5-GFP fusion expression vector was constructed, with an empty GFP vector used as a control. Subcellular localization was observed following transient transformation of tobacco leaves. The results showed that the green fluorescence signal of the ScEIL5-GFP fusion protein was mainly concentrated in the nucleus and completely overlapped with the red fluorescence signal of the nuclear marker (Figure 7B). This indicated that ScEIL5 was a nuclear-localized protein, consistent with its function as a transcription factor that exerts gene regulatory activity in the nucleus.
3.8 Inhibiting ScEIL5 can enhance the resistance of rye to Pst
To investigate the role of ScEIL5 in rye resistance to stripe rust, we employed virus-induced gene silencing (VIGS) to knock down ScEIL5 expression. Two independent VIGS constructs (BSMV: ScEIL5-1as and BSMV: ScEIL5-2as) targeting distinct regions of ScEIL5 were designed and analyzed separately in this study. Viral infection was confirmed by photobleaching in BSMV: PDS-inoculated plants at 12 days post-inoculation (dpi) (BSMV: Barley stripe mosaic virus; PDS: Phytoene desaturase) (Figure 8A). Subsequently, all BSMV-inoculated plants were challenged with Pst CYR34. Silencing efficiency was assessed by qRT-PCR, which revealed that both independent VIGS constructs (BSMV:ScEIL5-1as and BSMV:ScEIL5-2as) significantly reduced ScEIL5 transcript levels compared with the BSMV:γ empty vector control at 0, 24, 72, and 120 hours post-inoculation (hpi) with Pst (Figure 8B). The silencing efficiency ranged from 55%–77% for BSMV:ScEIL5-1as and 58%–77% for BSMV:ScEIL5-2as, respectively (Figure 8B). Both constructs exhibited high and consistent silencing efficiency across all time points, further confirming the specificity of gene silencing. At 16 dpi with Pst, phenotypic analysis showed a significant reduction in spore production on ScEIL5-silenced leaves compared with the control (Figure 8A). Furthermore, both infection area and hyphal length were significantly smaller in silenced leaves than in the control at 72 and 120 hpi (Figures 8C–E). Histochemical staining with DAB detected significantly greater H2O2 accumulation in ScEIL5-silenced plants than in the control at 24 and 72 hpi (Figures 8F, G). Consistent with these findings, fungal biomass was also significantly lower in ScEIL5-silenced plants than in the control (Figure 8H). Collectively, our results demonstrate that silencing ScEIL5 enhances resistance to stripe rust in rye, suggesting that ScEIL5 functions as a negative regulator of plant defense.
Figure 8
4 Discussion
The EIN3/EIL gene family has been extensively studied in numerous monocotyledonous plants, including maize (Jyoti et al., 2021), wheat (He et al., 2020), and rice (Aluko et al., 2024), as well as in various dicotyledonous species such as Medicago sativa (Su et al., 2024) and poplar (Liu et al., 2022). Despite the publication of a complete rye genome sequence (Bauer et al., 2017), the EIN3/EIL family in rye remains poorly characterized. In this study, we identified seven members of this transcription factor (TF) family from the rye genome. Phylogenetic analysis grouped these ScEIL proteins into three clades (A, B, and C), with group A containing the most members—a pattern similar to the clustering of TaEILs in wheat (He et al., 2020). Gene structure analysis revealed conserved motifs in the N-terminal region but divergent motifs in the C-terminal region, suggesting that the less conserved C-terminus may determine functional specificity. The EIN3/EIL proteins are nuclear-localised DNA-binding proteins and may be involved in a complex network of primary and secondary metabolic pathways in plants (Yamasaki et al., 2005). The conserved domains BRIII, BRIV, and PR contain amino acid residues essential for DNA binding and signal transduction. Collinearity analysis revealed a more conserved evolutionary relationship between rye EIN3/EIL genes and monocotyledonous wheat and maize, but no collinearity with dicotyledonous A. thaliana. This suggests that the EIN3/EIL family exhibits greater evolutionary divergence between monocotyledonous and dicotyledonous plants.
The cis-acting elements of the gene promoter region are the basic functional elements of the complex regulatory network of gene response (Xu et al., 2024). Cis-acting elements are divided into three categories: biotic/abiotic stress, plant growth and development, and plant hormone response. The composition and number of cis-acting elements vary among genes, enabling them to perform distinct biological functions. Cis-acting element prediction revealed that the promoter region of ScEIL genes were rich in cis-acting elements involved in various reactions, including 18 elements involved in biotic/abiotic stress, 11 elements involved in plant growth and development, and 8 elements involved in phytohormone response. This suggests that ScEILs may be involved in multiple stress responses. Among the components involved in biotic/abiotic stress, the number of G-box elements was the highest, consistent with the results of wheat. The G-box is a pervasive regulatory DNA element in plants that has been demonstrated to induce drought tolerance by forming a complex with the Basic leucine zipper (bZIP) protein (Li et al., 2022). Among the elements related to plant growth and development, the TATA-box was the most common element in the promoter region of eukaryotic genes. The TATA-box is not only involved in initiating transcription machinery assembly but also indirectly involved in plant adaptation to environmental conditions such as light (Savinkova et al., 2023). Among the elements involved in phytohormone response, the largest number was ABRE, which is consistent with findings in wheat and maize. Overexpression of ABscisic acid response element-binding protein 1 (AREB1) in A. thaliana significantly upregulates the gene carrying ABRE motif and enhances drought tolerance in vegetative tissues (Barbosa et al., 2013).
The EIN3/EIL gene family is widely conserved across higher plants. Research has demonstrated that ethylene is capable of responding to various biotic/abiotic stresses, including drought and low temperature, and of enhancing plant tolerance by regulating gene expression. In Medicago sativa, MsEIL1, MsEIL4, and MsEIL5 are involved in responses to drought, cold, and other stresses (Su et al., 2024). In broomcorn millet (Panicum miliaceum L.), PmEIL3 and PmEIL8 were significantly up-regulated in response to low-temperature stress (Yang et al., 2024). Overexpression of MnEIL3 in Arabidopsis increased salt tolerance and drought tolerance (Liu et al., 2019). All these results suggest that the EIN3/EIL gene family plays an important role in abiotic stress. Taking advantage of this natural adaptation, we systematically dissected the expression characteristics of the ScEILs under low-temperature and drought stresses. The results indicate that low temperatures rapidly upregulate the expression of ScEIL1 and ScEIL2, suggesting that these two genes may act as early responsive factors in rye perception of low temperature signals. Within 24 h of drought treatment, the expression of ScEIL4 showed a unimodal increase, with a significantly higher amplitude than that of other members, suggesting that it plays a “dominant switch” role in the drought response module. Tissue-specific expression analysis further indicated that the expression of ScEIL4 was relatively low under normal conditions but was sharply induced under drought stress, which conformed to the typical characteristics of a “stress-specific” regulator.
As a close wild relative of wheat, rye is a valuable genetic reservoir for disease resistance and stress tolerance. The successful introgression of the dominant stripe rust resistance gene Yr9 from rye into wheat underscores its historical importance as a core donor for wheat improvement (Rabanus-Wallace et al., 2021). EIN3/EIL proteins are key transcription factors in the ethylene signaling pathway and have been implicated in pathogen defense across various plant species. In this study, we found that the expression of ScEIL5 in rye was significantly induced at 48 hpi with stripe rust. Analysis of cis-acting elements revealed that the ScEIL5 promoter is enriched with TC-rich repeats, which are associated with defense and stress responsiveness, suggesting a potential role for ScEIL5 in biotic stress responses. Functional validation using VIGS confirmed this hypothesis. Silencing of ScEIL5 led to a significant reduction in fungal growth, as evidenced by lower spore counts, fungal biomass, hyphal length, and infection area compared to the BSMV:γ control. Furthermore, DAB staining revealed a substantially larger area of H2O2 accumulation in ScEIL5-silenced leaves, indicating a strengthened oxidative burst. Collectively, these results demonstrate that ScEIL5 negatively regulates rye’s resistance to stripe rust. This conclusion aligns with previous reports on certain EIN3/EIL family members, such as the negative regulator TaEIL1 in wheat and the significant roles of group B EIN3/EIL proteins in disease resistance (He et al., 2020). Our study not only identifies ScEIL5 as a biotic stress-responsive gene in rye but also provides new insights into the functional diversity of EIN3/EIL genes in stripe rust resistance. Therefore, the continued exploration of resistance genes in rye, coupled with advanced gene editing technologies, represents a promising strategy for developing wheat and related cereals with durable resistance to stripe rust.
5 Conclusion
In this study, we conducted genome-wide identification and characterization of the EIN3/EIL gene family in rye (Secale cereale L.) and identified seven ScEIL genes. Phylogenetic analysis revealed significant evolutionary divergence between monocotyledonous and dicotyledonous EIN3/EIL branches. Gene-structure analysis further indicated high conservation of the N-terminal region. Expression profiling showed that each ScEIL member responds to at least one environmental stress, highlighting their extensive involvement in biotic and abiotic stress-defence networks. VIGS-mediated silencing of ScEIL5 reduced fungal biomass, enhanced stripe-rust resistance, and elevated H2O2 accumulation, demonstrating that ScEIL5 negatively regulates stripe-rust resistance in rye.
Statements
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.
Author contributions
YW: Writing – original draft, Data curation, Investigation. FT: Data curation, Visualization, Conceptualization, Writing – review & editing, Supervision. WD: Funding acquisition, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was supported by National Natural Science Foundation of China (32260339); National Natural Science Foundation of China (32060595); Major Project of Agricultural Biological Breeding (2023ZD0402605-2); Major projects of Gansu Province (21ZD4NA012); Industry Supporting Program of Gansu Province (2022CYZC-49); Tibet major project (XZ202101ZD003N) and key R & D projects in Gansu Province (20YF8NA129).
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2026.1791751/full#supplementary-material
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Summary
Keywords
EIN3/EIL, Secale cereale, stress response, stripe rust, virus-induced gene silencing (VIGS)
Citation
Wang Y, Tao F and Du W (2026) Genome-wide analysis of the EIN3/EIL family in rye and functional identification of ScEIL5 in stripe rust resistance. Front. Plant Sci. 17:1791751. doi: 10.3389/fpls.2026.1791751
Received
20 January 2026
Revised
27 February 2026
Accepted
03 March 2026
Published
19 March 2026
Volume
17 - 2026
Edited by
Humberto Prieto, Agricultural Research Institute, Chile
Reviewed by
Andree Álvarez, Universidad de O’Higgins, Chile
Christian Hertig, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany
Updates
Copyright
© 2026 Wang, Tao and Du.
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*Correspondence: Wenhua Du, duwh@gsau.edu.cn
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.