Genome-Wide Characterization Highlights Key Roles for Bread Wheat MLO Genes in Powdery Mildew and Abiotic Stresses

Abstract

Powdery mildew (PM) is one of the most devastating and widespread foliar diseases globally. Despite the critical need for developing a durable PM resistance, the number of cloned genes remains limited, along with a shortage of Mildew Locus O (MLO) resistance-conferring genes in wheat breeding programs. Here, utilizing the latest wheat reference genome data, we comprehensively identified and characterized 47 MLO genes through a genome-wide search approach. These genes are randomly distributed among 21 wheat chromosomes, harbor seven transmembrane domains, and are predicted to be primarily localized in the plasma membrane. Comparative phylogenetic analysis with model plants classified wheat MLOs into four clades (I–IV) harboring 6, 28, 6, and 7 genes, respectively. The phylogenetic grouping was strongly supported by gene structures and motif distribution among members of different clades. Evolution analysis revealed that the MLO gene arsenal expanded through segmental duplications, and purifying selection is potentially conserving their stress-associated functions. In-silico expression analysis highlighted at least 10 genes with overlapping expression patterns among different growth and development stages and under abiotic and biotic stress conditions. The quantitative real-time polymerase chain reaction (qRT-PCR) validated the differential expression patterns of these 10 overlapping genes in PM-resistant and susceptible wheat genotypes after challenging these with a PM pathogen strain at different time intervals. The identified wheat MLO genes, especially the 10 overlapping genes, highlight untapped genetic diversity for engineering a durable and broad-spectrum tolerance/resistance against abiotic and biotic stresses, especially the PM resistance. Collectively, this study provides a compendium of wheat MLO genes, which could be functionally characterized to confirm their roles in PM resistance and further exploited in wheat breeding programs for the development of climate-resilient cultivars for sustainable wheat production.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12284-025-00832-1.

Introduction

Climate change is triggering profound effects on the incidence and severity of plant diseases (Burdon and Zhan 2020; Öztürk et al. 2025). Several fungal diseases including leaf rust, stripe rust, stem rust, smuts, septoria leaf blotch, fusarium head blight (FHB), and powdery mildew (PM) significantly reduce the wheat yield (Hussain et al. 2022). PM in wheat is caused by Blumeria graminis f. sp. tritici (Bgt), one of the most devastating and widespread foliar diseases. It can cause up to 62% losses to wheat yield (Singh et al. 2016) and results in approximately 5–10% annual yield losses (Savary et al. 2019). Development of PM resistant cultivars is the most sustainable strategy for its control in wheat. To date, over 100 PM resistance genes/alleles have been documented in wheat and its wild relatives, with 20 genes already cloned (Zhu et al. 2025). However, resistance against pathogens breaks down when new pathogen races with mutated effectors interact with host plants repressing the host immune receptors and shielding the pathogen growth and colonization (Sánchez-Vallet et al. 2018). The ongoing development of new PM resistant cultivars is crucial, but higher variability of pathogen strains necessitate continuous identification of resistance sources to ensure the long-term effectiveness of research efforts. Despite a critical need for engineering a durable PM resistance, the number of cloned resistance genes remains limited, and there is a shortage of “easy-to-use” resistance genes in wheat breeding programs.

The Mildew Locus O (MLO) gene family is specific to plants, with a negative regulatory role in disease resistance (Acevedo-Garcia et al. 2014). These ancient genes are distributed across the plant kingdom and have diversified within land plants (Kusch et al. 2016). These genes encode proteins with a conserved core structure of seven transmembrane domains and a variable C-terminus. They also contain a calmodulin-binding domain which is involved in calcium signaling, suggesting these might act as membrane-bound receptors. MLO genes play crucial roles in several defence responses, especially against PM (Kusch et al. 2016).

Loss-of‐function mutations in an MLO gene conferred broad‐spectrum resistance against PM in barley. This PM resistance was durable and lasted for more than 30 years (Jørgensen 1992). These loss-of-function mutations yielded pleiotropic effects such as necrotic leaf spotting and reduced grain yield, however, these were overcome by breeding efforts. In wheat, simultaneous knockout of TaMLO-A1, TaMLO-B1, and TaMLO-D1 genes (renamed as TaMLO3/6-A2, TaMLO3/6-B2, and TaMLO3/6-D2 in this study) using transcription activator–like effector nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system conferred heritable broad-spectrum PM resistance (Wang et al. 2014). Similarly, simultaneous knockout of these genes through Targeting Induced Local Lesions IN Genomes (TILLING) enhanced PM resistance without pleiotropic effects (Acevedo‐Garcia et al. 2017). Recently, CRISPR/Cas9 was used to introduce a 304-kilobase pair deletion in TaMLO-B1 (renamed as TaMLO3/6-B2 in this study) that conferred robust PM resistance without pleiotropic effects. The ectopic activation of tonoplast monosaccharide transporter 3 (TaTMT3B) negated the negative effects of MLO disruption (Li et al. 2022). Additionally, simultaneous mutations in above-mentioned genes provided highly effective PM resistance without pleiotropic effects (Ingvardsen et al. 2023).

This stable nature of mlo-based resistance has resulted in a great interest in these genes. Therefore, genome-wide identification of MLOs has been performed across diverse model and crop plant species including cereals, legumes, fruits, and vegetables (Devoto et al. 2003; Liu and Zhu 2008; Kusch et al. 2016; Rispail and Rubiales 2016; Shi et al. 2020; Tapia et al. 2021; Gong et al. 2025). However, to date, information on genome-wide identification of wheat MLO genes (TaMLOs) is scarce, posing a bottleneck in their detailed functional characterization and further exploitation in wheat breeding programs. The availability of a fully annotated and gold-standard reference genome of bread wheat (Appels et al. 2018; Zhu et al. 2021) provides a strong foundation for comprehensive identification and characterization of this important gene family.

In this study, we performed a genome-wide screening to identify MLO genes in wheat and investigated their evolutionary diversity through phylogenetic, duplication, gene structure, and motif analyses. Furthermore, we performed in-silico expression analysis of MLOs under different growth and development stages, biotic and abiotic stresses, and also validated their expressions under Bgt inoculation through qRT-PCR. Collectively, this study provides a compendium of TaMLOs for functional genomics and subsequent development of PM resistant and climate-resilient wheat cultivars.

Materials and Methods

Sequence Retrieval and Genome-Wide Identification

The latest bread wheat genome annotation data (RefSeq v2.1) (Zhu et al. 2021) were retrieved from the International Wheat Genome Sequencing Consortium (IWGSC) website (https://www.wheatgenome.org/resources/annotations). Previously reported 15 A. thaliana (Devoto et al. 2003) and 12 rice MLO (Liu and Zhu 2008) proteins were retrieved from the Ensemble Plants (https://plants.ensembl.org/index.html). Genome-wide identification of MLOs in wheat genome was performed through the Basic Local Alignment Search Tool (BLAST) function of TBtools (Chen et al. 2020) using A. thaliana and rice MLO peptides as query sequences. The presence of a complete MLO domain in wheat proteins was confirmed through the NCBI Conserved Domain Database (CDD) search tool v3.19 (Wang et al. 2023) and Pfam (http://pfam.xfam.org/). The proteins with incomplete MLO domains were discarded before proceeding with downstream analyses.

Moreover, Expasy server’s ProtParam tool (https://web.expasy.org/protparam/) was employed for estimation of molecular weight and isoelectric point of the MLOs. The subcellular localization was predicted with Bologna Unified Subcellular Component Annotator (BUSCA) server (Savojardo et al. 2018). The cell organelle with highest probability was considered as the potential localization of the protein.

Chromosomal Mapping of TaMLOs

The identified genes were mapped on the 21 wheat chromosomes by using the shinyCircos tool (Yu et al. 2018) while extracting their genomic locations from the GFF3 file.

Phylogenetic Analysis

The wheat MLO peptides were multiple sequence aligned through the MAFFT v7.0 (Katoh et al. 2019), with default settings. The resultant alignment was used to construct a maximum likelihood phylogenetic tree through MEGA v11.0 software (Tamura et al. 2021) using the Jones-Taylor-Thornton (JTT) model for amino acid substitution with 1,000 bootstraps. The resultant tree was visualized with iTol v7 (Letunic and Bork 2024).

For comparative phylogenetic tree, peptides of 47 TaMLOs, 15 AtMLOs, and 12 OsMLOs, were multiple sequence aligned using MAFFT v7.0 (Katoh et al. 2019) with the E-INS-i algorithm. A maximum likelihood tree was inferred with IQ_Tree (Minh et al. 2020) by choosing the JTT + F + ASC + R5 best-fit substitution model with 1,000 bootstraps. The final tree was visualized with iTOL v7 (Letunic and Bork 2024).

The TaMLOs renamed based on phylogenetic relationships with rice and/or Arabidopsis homologs by following the updated guidelines for gene nomenclature in wheat (Boden et al. 2023). For example, five TaMLOs that clustered with OsMLO9 were renamed as TaMLO9, followed by sub-genome specific letters (A, B, D) and corresponding homeologs in the three sub-genomes (A1, B1, D1, A2, D2). Therefore, TaMLO9-A1, TaMLO9-B1 and TaMLO9-D1 indicate 3 homologs of OsMOL9 in wheat located on A, B and D sub-genomes, respectively. These three genes share homeologous status among each other. Whereas TaMLO9-A2 and TaMLO9-D2 indicate two homologs of OsMLO9 in wheat located on A and D sub-genomes, sharing homeologous status between themselves, with lack of B sub-genome homeolog. Thus, the five TaMLO9 genes are homolog to OsMLO9 and are paralogous to each other. The comprehensive details about gene nomenclature are provide in the original study (Boden et al. 2023).

Gene Structure and Motif Analysis

The genomic and coding sequences (CDS) of TaMLOs were used in the Gene Structure Display Server (GSDS) tool (Hu et al. 2015) to visualize the exons, introns, and down/upstream regions of the genes. The conserved motif analysis was performed with MEME 5.5.8 tool (Bailey et al. 2015) with default settings, except for the number of motifs and motif length set as 16 and 50, respectively. The motif data was visualized using TBtools (Chen et al. 2020). All motif sequences were searched in the Simple Modular Architecture Research Tool(SMART) (Letunic et al. 2021) to find the known domains/repeats/motifs. Subsequently, MLO proteins were searched in the Deep Transmembrane Helices Hidden Markov Models (TMHMM)-1.0 server (https://services.healthtech.dtu.dk/services/DeepTMHMM-1.0/) to predict the number of trans-membranes. The Expasy Protparam tool (https://web.expasy.org/protparam/) was used to calculate the leucine percentage in MLO proteins.

Gene Duplication

The CDS of all TaMLOs were multiple sequence aligned and Sequence Demarcation Tool v1.2 (Muhire et al. 2014) was employed to calculate the sequence identities in all possible pairwise combinations. Gene pairs with at least 90% identities (E value < 1e^− 10) were classified as duplicates. The duplicated gene pairs that were located on the same chromosome were considered as tandem duplicates, whereas those which were located on different chromosomes were defined as segmental duplicates. The synonymous (Ks), non-synonymous (Ka) substitutions, and codon selection (Ka/Ks) for the duplicated gene pairs were calculated with TBtools software. An approximate divergence time for the duplicated genes was calculated by using the formula T = Ks/2r × 10^− 6, assuming a substitution rate (r) of 6.5 × 10^− 9 substitutions/synonymous site/year (El Baidouri et al. 2017).

MicroRNA Analysis

For microRNA (miRNAs) identification, the primary transcripts of identified TaMLOs were searched in the psRNATarget server (Dai et al. 2018) with default settings to predict miRNAs potentially targeting the TaMLOs.

In-Silico Expression Analysis

The transcriptome-based expression data of TaMLOs during different growth and development stages, and under different abiotic (cold, drought, heat, and phosphate starvation) and biotic (FHB, PM, and stripe rust) stresses were retrieved from the expVIP Wheat Expression Browser (https://www.wheat-expression.com) as Log₂ Transcripts Per Million (TPM) (Borrill et al. 2016). The expression heatmaps were generated with TBtools.

Quantitative PCR Analysis

Plant Materials, Growth Conditions, and Stress Treatments

A PM-resistant wheat line, N9134, that shows high resistance to all Blumeria graminis f. sp. tritici (Bgt) races in China (Zhang et al. 2014), along with a susceptible wheat cultivar, Fielder, were selected for quantitative real-time polymerase chain reaction (qRT-PCR)-based expression analysis. Seeds of both genotypes were obtained from Peking University Institute of Advanced Agricultural Sciences, China. Healthy seeds of both genotypes were disinfected (0.1% HgCl₂ for 10 min), rinsed with autoclaved distilled water, surface dried on filter paper, sown in an autoclaved soil + peatmoss mixture, and kept under controlled conditions (22 ± 1 °C, 60 ± 5% relative humidity,16 h/8 h light and dark cycle). Two-weeks-old seedlings with consistent growth were inoculated with Bgt conidia, while no inoculation was done on control seedlings. The control and inoculated leaves were harvested at 0, 24, 48, and 72 h post inoculation (hpi). Leaf samples from 3 independent seedlings were collected, flash frozen in liquid nitrogen and kept at − 80 °C until RNA extraction.

RNA Extraction and qRT-PCR Analysis

The total RNA was extracted using the SPARKeasy RNA extraction kit (SparkJade, China) by following manufacturer instructions. After confirming RNA quality through agarose gel electrophoresis, reverse transcription was performed using the SPARKscript II One Step RT-PCR kit. Gene-specific primers were designed through the NCBI Primer-BLAST tool, and qRT-PCR was performed using ChamQ Blue Uninversal SYBR qPCR Master Mix (Vazyme, China) on a QuantStudio™ 5 system (Thermo Fisher Scientific). The 20 µL reaction system included 10 µL SYBR mix, 0.4 µL of each primer pair, 5 µL diluted cDNA, and 4.2 µL RNase-free water. For internal control, TaActin (TraesCS5D02G132200) was used. The qPCR profile was as follows: 95 °C for 1 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The results were analyzed using the 2^− ΔΔCt method (Livak and Schmittgen 2001). The analysis was carried out with three biological replications. A list of primer pairs for 10 TaMLOs and internal control is provided in Table S1.

Results and Discussion

Genome-Wide Identification and Properties of TaMLOs

Taking advantage of the latest wheat genome sequencing data (IWGSC RefSeq v2.1), we initially identified 61 TaMLOs through a stringent BLAST search using the model plants MLO proteins. After discarding redundant genes and those containing incomplete MLO domains (pseudogenes), a total of 47 non-redundant TaMLOs were considered for detailed downstream analyses (Table 1; Fig. 1). The identified TaMLOs were renamed based on phylogenetic relationships with rice and/or Arabidopsis homologs following the updated guidelines for gene nomenclature in wheat (Boden et al. 2023). This repertoire of 47 TaMLOs is the second largest number in flowering plants, following 68 MLOs in octaploid strawberry (Tapia et al. 2021), and could be explained by hexaploid nature of bread wheat. Previously, 15 and 12 MLOs had been reported in Arabidopsis and rice, respectively. Since, these model species are diploid in nature, it can be expected that the hexaploid wheat genome might contain ~ 45 TaMLOs, which is consistent with the findings of this study. The occurrence of additional TaMLOs than expectations could be explained by the large genome size and segmental duplications.

Table 1.

Gene IDs, newly assigned names and genomic properties of identified 47 MLO genes in Triticum aestivum L.

S#	Gene ID	Given name	Chr.	Clade	Gene start	Gene end	Gene size (bp)	Strand
1	TraesCS1A03G0539600	TaMLO1-A1	1A	III	364,505,363	364,509,878	4515	–
2	TraesCS1B03G0628500	TaMLO1-B1	1B	III	398,073,082	398,077,619	4537	–
3	TraesCS1D03G0519600	TaMLO1-D1	1D	III	293,427,154	293,431,832	4678	–
4	TraesCS3A03G0944700	TaMLO2-A1	3A	II	648,479,170	648,482,130	2960	–
5	TraesCS3B03G1076900	TaMLO2-B1	3B	II	689,913,560	689,916,559	2999	+
6	TraesCS3D03G0877800	TaMLO2-D1	3D	II	513,351,507	513,355,165	3658	–
7	TraesCS5A03G1158600	TaMLO3/6-A1	5A	IV	664,743,006	664,756,436	13,430	+
8	TraesCS5A03G1159400	TaMLO3/6-A2	5A	IV	664,797,864	664,801,136	3272	–
9	TraesCS5A03G1150400	TaMLO3/6-A3	5A	IV	661,126,976	661,129,967	2991	–
10	TraesCS4B03G0835400	TaMLO3/6-B1	4B	IV	611,711,110	611,728,680	17,570	+
11	TraesCS4B03G0836900	TaMLO3/6-B2	4B	IV	612,012,622	612,014,655	2033	–
12	TraesCS4D03G0743900	TaMLO3/6-D1	4D	IV	483,173,859	483,176,970	3111	+
13	TraesCS4D03G0744500	TaMLO3/6-D2	4D	IV	483,205,801	483,208,560	2759	–
14	TraesCS2A03G1299000	TaMLO4-A1	2A	I	768,633,437	768,638,338	4901	–
15	TraesCS2B03G1489500	TaMLO4-B1	2B	I	788,988,904	788,994,031	5127	–
16	TraesCS2D03G1262400	TaMLO4-D1	2D	I	638,659,281	638,665,452	6171	–
17	TraesCS2A03G0775400	TaMLO5-A1	2A	II	539,693,546	539,696,928	3382	–
18	TraesCS2B03G0856800	TaMLO5-B1	2B	II	479,483,631	479,486,919	3288	–
19	TraesCS2D03G0715400	TaMLO5-D1	2D	II	400,923,755	400,926,943	3188	–
20	TraesCS7A03G0928000	TaMLO7-A1	7A	II	561,794,230	561,801,544	7314	–
21	TraesCS7B03G0775700	TaMLO7-B1	7B	II	524,484,720	524,491,473	6753	–
22	TraesCS7D03G0891400	TaMLO7-D1	7D	II	493,179,947	493,187,259	7312	–
23	TraesCS6A03G0387200	TaMLO8-A1	6A	II	154,203,217	154,207,538	4321	+
24	TraesCS6B03G0494800	TaMLO8-B1	6B	II	232,292,016	232,296,365	4349	–
25	TraesCS6D03G0340700	TaMLO8-D1	6D	II	148,637,851	148,642,268	4417	–
26	TraesCS1A03G0668200	TaMLO9-A1	1A	II	457,568,166	457,572,624	4458	–
27	TraesCS3A03G0013800	TaMLO9-A2	3A	II	6,965,742	6,973,314	7572	–
28	TraesCS1B03G0759600	TaMLO9-B1	1B	II	484,447,774	484,452,526	4752	–
29	TraesCS1D03G0630400	TaMLO9-D1	1D	II	357,744,682	357,749,428	4746	–
30	TraesCS3D03G0010400	TaMLO9-D2	3D	II	1,805,922	1,810,130	4208	–
31	TraesCS2A03G0163000	TaMLO10-A1	2A	II	42,897,580	42,900,950	3370	–
32	TraesCS2A03G0163100	TaMLO10-A2	2A	II	42,905,631	42,908,828	3197	–
33	TraesCS6A03G0805500	TaMLO10-A3	6A	II	545,643,614	545,648,358	4744	–
34	TraesCS2B03G0227100	TaMLO10-B1	2B	II	65,216,529	65,219,835	3306	–
35	TraesCS2B03G0227200	TaMLO10-B2	2B	II	65,315,357	65,318,657	3300	–
36	TraesCS6B03G0955000	TaMLO10-B3	6B	II	601,764,897	601,769,827	4930	–
37	TraesCS2D03G0160400	TaMLO10-D1	2D	II	37,249,636	37,253,058	3422	–
38	TraesCS2D03G0160500	TaMLO10-D2	2D	II	37,272,260	37,275,524	3264	–
39	TraesCS6D03G0681600	TaMLO10-D3	6D	II	418,198,229	418,205,340	7111	–
40	TraesCS7D03G0041500	TaMLO10-D4	7D	II	8,641,641	8,683,479	41,838	–
41	TraesCS7D03G0062000	TaMLO10-D5	7D	II	14,007,248	14,014,362	7114	+
42	TraesCS4A03G0580300	TaMLO11-A1	4A	I	518,674,297	518,679,167	4870	+
43	TraesCS4B03G0222000	TaMLO11-B1	4B	I	104,220,694	104,226,387	5693	+
44	TraesCS4D03G0182500	TaMLO11-D1	4D	I	69,109,133	69,114,152	5019	+
45	TraesCS5A03G0963900	TaMLO12-A1	5A	III	599,592,378	599,596,257	3879	+
46	TraesCS5B03G1014800	TaMLO12-B1	5B	III	590,155,469	590,160,172	4703	+
47	TraesCS5D03G0918700	TaMLO12-D1	5D	III	481,711,884	481,717,428	5544	+

Fig. 1.

Chromosomal distribution of TaMLOs on 21 bread wheat chromosomes. A, B, and D sub-genomes are represented by three different colors. Links among genes represent duplicated gene pairs. Red color represents segmental duplications and blue color represents tandem duplications

The isoelectric points of identified MLO proteins varied from 7.36 to 9.87, while the molecular weights were in the range of 46.98 to 68.50 kDa. The peptide lengths ranged from 409 to 612 amino acids, and the average length was 514 amino acids. Majority of the TaMLOs had three homeologs in A, B, and D sub-genomes with comparable number of amino acids and molecular weights (Table S2). This range of 400 to 600 amino acids in MLO proteins seem to be conserved across plant lineage, as similar range has been previously reported in 28 plant species (Shi et al. 2020). The subcellular localization of TaMLO proteins was predicted to be the plasma membrane (Table S2). This prediction is in agreement with previous reports in different plant species (Kusch et al. 2016; Shi et al. 2020). These results indicate that MLO proteins might function in signal transduction and cell-to-cell communication.

Distribution of TaMLOs in Wheat Genome

Several TaMLOs which were reported previously could not be mapped onto wheat chromosomes due to non-availability of a reliable reference genome. Since the availability of a gold-standard reference genome, nearly all wheat genes could be assigned to the specific 21 chromosomes (Appels et al. 2018; Zhu et al. 2021). Taking advantage of this gold-standard reference genome, all 47 TaMLOs were identified at the whole genome level and assigned to 21 chromosomes. These genes were randomly distributed among chromosomes with nearly equal contributions of A (~ 34%), B (~ 30%) and D (~ 36%) sub-genomes (Table 1; Fig. 1). This random distribution of plant gene families might originate from long terminal repeat retrotransposons through evolution as reported in rice (Ahmed et al. 2021). Likewise, nearly equal contribution of wheat sub-genomes has been reported in the genome-wide analysis of wheat MADS-box genes (Raza et al. 2022).

Four chromosomes 2A, 2B, 2D, and 5A harbored a maximum of four TaMLOs each, while three chromosomes 4B, 4D and 7D harbored three genes each. Similarly, six chromosomes 1A, 1B, 1D, 6A, 6B, and 6D contained two genes each. Notably, unbalanced homeologs carrying TaMLOs were also observed in this study. For example, three paralogous genes (TaMLO3/6-A1, TaMLO3/6-A2 and TaMLO3/6-A3) were only found on chromosome 5A, with their B and D counterparts missing on the 5B and 5D chromosomes. Likewise, two paralogous genes (TaMLO10-A4 and TaMLO10-A5) were only found on chromosome 7D, with lack of corresponding 7A and 7B localized genes (Fig. 1). These uneven distribution patterns indicate prevalence of unbalanced homeologs, potentially due to divergent evolution and mis-annotations in the reference genome.

Phylogenetic Analysis of TaMLOs

A maximum likelihood tree was constructed to determine evolutionary relationships among TaMLO proteins, which classified these proteins into four distinct clades (Fig. 2a). The clade numbers were assigned following previous classification in A. thaliana (Devoto et al. 2003). Clade II represents the largest clade with 28 members (59.57% TaMLOs), while clades I, III, and IV contained 6, 6, and 7 members, respectively. Clade I contained TaMLO4-A1, TaMLO4-B1, TaMLO4-D1, TaMLO11-A1, TaMLO11-B1, and TaMLO11-D1. Clade III contained TaMLO1-A1, TaMLO1-B1, TaMLO1-D1, TaMLO12-A1, TaMLO12-B1, and TaMLO12-D1, while clade IV contained TaMLO3/6-A1, TaMLO3/6-A2, TaMLO3/6-A3, TaMLO3/6-B1, TaMLO3/6-B2, TaMLO3/6-D1, and TaMLO3/6-D2. Additionally, 28 TaMLOs in clade II were further divided into three subclades, i.e., IIA, IIB, and IIC (Fig. 2a). In many monocots, > 50% of MLOs were classified into clade II while clade IV either has a low number of MLOs or is completely absent in some dicot species (Kusch et al. 2016). Previously known MLOs in wheat (Konishi et al. 2010) and other monocots (B. distachyon, rice, maize, oat, barley, and foxtail millet) were also classified into four clades (Liu and Zhu 2008; Kusch et al. 2016; Nguyen et al. 2016). However, this highly conserved pattern of four clades is exclusive to monocots only. A basal angiosperm, Amborella, has six MLO clades, and is believed to have diverged from the other angiosperms more than 160 million years ago (MYA), before the divergence of monocots and dicots (120 MYA). Therefore, ancestors of monocots might also had six clades, but lost clades V and VI during evolution (Kusch et al. 2016).

Fig. 2.

Comparison of (a) the maximum likelihood phylogenetic tree of TaMLO proteins, with (b) the gene structures of full-length genomic sequences, where red boxes represent the exons, and lines connecting the exons represent introns, while blue boxes represent the upstream and downstream untranslated regions, and (c) the conserved motifs distributed across all TaMLO proteins

The distribution of MLOs into different clades is speculated to be associated with their susceptibility or resistance. Many eudicot MLOs found in clade V are reported to be involved in PM susceptibility. For example, genes governing PM susceptibility in A. thaliana (AtMLO2, AtMLO6, and AtMLO12), pea (Er1/PsMLO1), tomato (SlMLO1), grapevine (VvMLO3 and VvMLO4), cucumber (CsaMLO1, CsaMLO8, and CsaMLO11), and lentil (LcMLO1 and LcMLO3) belong to clade V (Bai et al. 2008; Acevedo-Garcia et al. 2014; Kusch et al. 2016; Polanco et al. 2018). However, in monocots, clade IV is reported to harbor PM susceptibility related MLOs. For example, HvMLO in barley and TaMLO-A1, TaMLO-B1, and TaMLO-D1 (renamed as TaMLO3/6-A2, TaMLO3/6-B2, and TaMLO3/6-D2 in this study) in wheat belong to clade IV (Acevedo‐Garcia et al. 2014). TILLING induced missense mutations in these genes conferred enhanced, yet incomplete, Bgt resistance without negative pleiotropic effects. Therefore, clade IV TaMLOs (TaMLO3/6-A1, TaMLO3/6-A2, TaMLO3/6-A3, TaMLO3/6-B1, TaMLO3/6-B2, TaMLO3/6-D1, and TaMLO3/6-D2) might confer PM susceptibility. These genes are potential targets for antisense silencing and CRISPR/Cas9 mediated gene editing/knockout to confirm and subsequently confer PM resistance in wheat.

Gene Structure and Conserved Motif Analysis

Comparison of Gene Structure with Phylogenetic Classification

The exons and introns are of great importance for plant biodiversity and gene functions. The gene structure analysis revealed that TaMLOs have 9 to 15 exons. A total of 21 MLOs (45%) harbor 13 exons, while 12 genes have 14 exons. Similarly, 15 and 11 exons are present in 5 genes each (Fig. 2b). This exon range is consistent with other plant species e.g., there are 12 to 15 exons in B. distachyon MLOs (Ablazov and Tombuloglu 2016), 13 to 15 exons in lentil MLOs (Polanco et al. 2018), and 12 to 17 exons in chickpea, mung bean, pea, pigeon pea, and common bean MLOs (Rispail and Rubiales 2016). All members of clades I, III, and IV have similar intron/exon distributions, whereas clade II members exhibited three different intron/exon patterns, which is consistent with their classification into sub-clades. Taken together, the observed diversity in gene structures is strongly associated with phylogenetic classification.

Characteristics of Conserved Motifs in TaMLOs

A total of 16 conserved motifs were identified in 47 TaMLO proteins with a width range of 11 (motif 12) to 50 (motif 5) amino acids (Table S3). The nature, number, and patterns of motifs in different phylogenetic clades exhibited conservation as well as diversification; e.g., ten motifs in clade I, 11 to 14 motifs in subgroups of clade II, 15 motifs in clade III, and 12 motifs in clade IV. Eight motifs were conserved among almost all TaMLO proteins e.g., motifs 1 to 4 are present in all 47 MLOs, motifs 6 and 7 are present in 46 MLOs, while motifs 5 and 8 are present in 45 and 42 proteins, respectively. These eight conserved motifs highlight basic structural and functional resemblance among TaMLOs. For further annotation, we searched for all 16 motifs in the SMART database, which revealed that motifs 1 to 8 are an integral part of MLO domain (Table S3, Fig. 2c).

The presence of motifs 1, 2, 7 and 8 in 89–100% TaMLO proteins suggest that these are hydrophobic amino acids, which anchor the MLO proteins in the cell membrane. To confirm this, MLO proteins were searched in Deep TMHMM server and nearly 98% TaMLOs were found to have a seven-transmembrane structure (Fig. 3; Table S2), which is a conserved feature across different plant species (Devoto et al. 2003; Chen et al. 2009). Furthermore, MLO proteins are composed of leucine-rich repeats (9.9 to 13.1%) (Ablazov and Tombuloglu 2016), consistent with findings of this study (8.5 to 12.9%) (Table S2) thus predicting their role in pathogen response.

Fig. 3.

The graphical representation depicting the posterior probabilities for transmembrane, outside (exterior), and inside (cytoplasm) regions. TaMLO1-A1 was used for transmembrane regions prediction through Deep TMHMM server

Comparative Phylogenetic Analysis among Arabidopsis, Rice and Wheat MLOs

To infer evolutionary relationships and TaMLOs renaming, a comparative phylogenetic tree was constructed using MLO proteins from these three species. All proteins were classified into four clades (Fig. 4), with clade numbering adopted from A. thaliana (Devoto et al. 2003). The clades I, II, III, and IV harbored 6, 28, 6, and 7 TaMLO proteins, respectively, consistent with phylogenetic analysis of only wheat proteins (Fig. 2a). Clade I harbored two rice and three Arabidopsis MLOs while clade II harbored 6 OsMLOs, and three AtMLOs. Clade III consisted of two rice, and five Arabidopsis MLOs, while clade IV contained two rice and four Arabidopsis MLOs (Fig. 4). The distribution of MLOs into different clades might be associated with their functions e.g., clade I members, AtMLO4 and AtMLO11, control the root thigmomorphogenesis or root curling responding to tactile stimulus (Chen et al. 2009). Additionally, clade III genes, AtMLO7 and OsMLO12, code for pollen tube perception of the egg (Kessler et al. 2010), and pollen hydration (Yi et al. 2014), respectively. Furthermore, clade IV members are involved in PM susceptibility (see Sect. Phylogenetic Analysis of TaMLOs). Similarly, members of the clade II (OsMLO2), and clade III (AtMLO7) are involved in PM susceptibility (Kessler et al. 2010; Acevedo-Garcia et al. 2014). Therefore, in addition to seven clade IV TaMLOs, some clade III TaMLOs (TaMLO12-A1, TaMLO12-B1, TaMLO12-D1), could also be involved in PM susceptibility In conclusion, comparative phylogenetic analysis provided valuable insights into the evolution and functions of TaMLOs.

Fig. 4.

A comparative phylogenetic analysis of Arabidopsis, rice and wheat MLOs. The maximum likelihood tree was inferred after multiple sequence alignment with MAFFT using IQtree and 1,000 bootstrap values. The numbers at the nodes indicate percent bootstrap values. The Arabidopsis, rice and wheat genes are represented by blue, red and black colors, respectively

Gene Duplication and Evolution Analysis of TaMLOs

Gene duplications are essential for the expansion and evolution of plant gene families. A total of 50 duplicated gene pairs were identified, which correspond to 42 TaMLOs. A total of 94% duplications were segmental, i.e., duplicated genes occurred on different chromosomes. Whereas only 6% of gene pairs were tandem duplicated, i.e., located on the same chromosome (Table S4; Fig. 1). Thus, segmental duplications are more prevalent in wheat MLOs, which is consistent with segmental duplications of MADS-Box genes in wheat (Raza et al. 2022) and APETALA-2 genes in rice (Ahmed et al. 2021).

Furthermore, evolution analysis was performed by calculating the synonymous (Ks) and non-synonymous (Ka) substitution rates per site per year for all duplicate gene pairs. The results revealed that TaMLOs duplicated pairs diverged from each other between 3.60 and 19.83 million years ago (MYA) (Table S4). The Ka/Ks ratio measure the selection pressure on amino-acid substitutions during the evolution. The Ka/Ks values ranged between 0.052 and 0.373, indicating strong purifying selection operating on codons of duplicated genes to conserve their PM response functions. A Ka/Ks value < 1 implies that synonymous substitutions are more prevalent as compared to non-synonymous ones (purifying/negative selection), whereas Ka/Ks value > 1 indicates positive/Darwinian selection. Strong purifying selection was also reported for MLOs in several plant species including B. distachyon, rice, maize, A. thaliana, cucumber, tomato, melon, grapevine, Amborella, and black cottonwood (Liu and Zhu 2008; Shi et al. 2020; Gong et al. 2025). It could be speculated that many plants species have undergone purifying selections to remove deleterious alleles and conserve biotic/abiotic stress associated functions during evolution.

MicroRNAs Targeting the TaMLOs

We identified a total of 121 miRNAs through the psRNATargent server, targeting 41 TaMLOs including eight miRNAs targeting the TaMLO3/6-B2. Likewise, seven miRNAs each targeted the TaMLO3/6-A1 and TaMLO4-D1, while six miRNAs targeted the TaMLO1-B1. Six miRNAs including tae-miR5384-3p, tae-miR9679-5p, tae-miR9657a-3p, tae-miR9781, tae-miR167c-5p, and tae-miR167a are the most prevalent that target the TaMLOs. The miRNAs, their sequences, and target regions are provided in Table S5. The miRNAs are 19–24 nucleotides RNAs that bind with mRNA of a gene by complementary pairing, and either cleave the target or repress its translation to reduce its expression (Budak et al. 2015). Several plant miRNAs are involved in natural stress response by targeting the stress-related genes at transcription and post-transcription levels. Therefore, identified miRNAs can be overexpressed to cleave or silence TaMLOs for conferring PM resistance.

In-Silico Expression Analysis of TaMLOs

At Different Growth and Development Stages

MLO genes expression has been extensively investigated in crop plants during growth and development. In this study, we analyzed TaMLOs expression in several tissues at different time points to get insights into their functional roles. A total of 28 MLOs (~ 60%) were expressed in at least two different tissues during the whole growth and development periods (Fig. 5a). Among these, at least 9 genes (TaMLO9-A1, TaMLO10-A1, TaMLO3/6-A2, TaMLO8-A1, TaMLO3/6-B2, TaMLO8-B1, TaMLO3/6-D2, TaMLO8-D1, and TaMLO10-D4) showed ubiquitous expression in nearly all investigated tissues. Interestingly, all these genes exhibited highly significant expression patterns in the flag leaf sheath and blade during the reproductive phase (30% spike to grain ripening stages). However, several other genes exhibited tissue-specific and growth-stage-specific expression perturbations (Fig. 5a). Collectively, these expression patterns indicate diverse functions of TaMLOs in wheat growth and development. The MLO proteins are well documented to be involved in growth/development e.g., pollen tube reception (Kessler et al. 2010), root thigmomorphogenesis (Chen et al. 2009; Bindzinski et al. 2014), reproductive development (Acevedo-Garcia et al. 2014) in Arabidopsis, and pollen hydration in rice (Yi et al. 2014). Similarly, rice MLOs are also involved in formation of stigma, and root tips, and photoperiod sensitivity (Nguyen et al. 2016), thus highlighting their roles in plant growth/development in addition to immunity.

Fig. 5.

In-silico expression patterns of TaMLOs (a) during different stages of growth and development like seedling, tillering, flag leaf, anthesis, and grain filling, (b) under different abiotic stresses like cold, drought, heat, and phosphate starvation, and (c) under different biotic stresses like powdery mildew, stripe rust, and fusarium head blight. The RNA-seq expression data were retrieved from the expVIP Wheat Expression Browser, and heat maps were generated with TBtools

Under Different Abiotic and Biotic Stresses

We also studied the transcriptional changes in wheat MLOs under four abiotic and three biotic stresses. The abiotic stresses included cold, drought, heat, and phosphate starvation, whereas biotic stresses included fusarium head blight (FHB), powdery mildew (PM) and stripe rust (SR). Almost 65% of genes were expressed after at least one abiotic and/or biotic stress application. Interestingly, all genes that showed ubiquitous expression during different growth and development stages were also highly expressed under abiotic and biotic stresses. Under abiotic stresses, seven genes after two weeks of cold stress (TaMLO1-A1, TaMLO3/6-A2, TaMLO9-B1, TaMLO1-D1, TaMLO9-D1, TaMLO10-D1, and TaMLO10-D4), six genes after 1–12 h of drought stress (TaMLO10-A2, TaMLO10-B1, TaMLO10-B2, TaMLO9-D1, TaMLO10-D1, and TaMLO10-D2), three genes after 1–6 h of heat stress (TaMLO10-B3, TaMLO10-D1, and TaMLO10-D3), and three genes under phosphate starvation at the vegetative stage (TaMLO3/6-A2, TaMLO10-B2, and TaMLO10-D1) exhibited transcriptional perturbations as compared to their respective control (CK) treatments (Fig. 5b). These findings are consistent with previous studies as several Brachypodium (BdMLO2, 6, 7, 8, 9, and 10) and rice (OsMLO1, 3, 4, 5, 7, 8, 9, 10 and 12) genes showed differential expression under drought, cold, and heat stress (Ablazov and Tombuloglu 2016; Nguyen et al. 2016). Thus, there is an eminent role for TaMLOs under abiotic stresses, which can be used to develop climate-smart plants.

Under biotic stress, at least four genes (TaMLO10-A2, TaMLO3/6-A2, TaMLO7-A1, and TaMLO10-D5) exhibited PM-specific transcriptional perturbation when compared with respective control. Whereas, only TaMLO9-D1 showed stripe rust-specific, and five genes (TaMLO10-A2, TaMLO4-A1, TaMLO10-B1, TaMLO10-B2, TaMLO10-D1, and TaMLO10-D2) exhibited FHB-specific expression when compared with respective control. Overall, these transcriptional perturbations under different abiotic and biotic stresses indicate that TaMLOs regulate multifarious processes to successfully complete bread wheat life cycle under continuously changing environmental conditions (Fig. 5c). MLO family genes are unique to plants, governing resistance against PM infections through a negative regulatory pathway. Additionally, they have roles in resistance/susceptibility against wheat stripe rust (Zhang et al. 2014), and rice blast fungus (Nguyen et al. 2016). However, apart from conferring plant immunity, previous studies have also reported their diverse roles in growth and development and stress responses (Bindzinski et al. 2014; Tapia et al. 2021), just like multifarious roles of MADS-box family genes in bread wheat (Raza et al. 2022). For example, a systematic study of rice MLOs indicated their roles in morphological development, photoperiod sensitivity, abiotic stress tolerance and resistance against different biotic stresses(Nguyen et al. 2016). In this study, a substantial number of TaMLOs also showed ubiquitous expression patterns during growth and development, abiotic and biotic stresses. These results support previous findings about functional roles of MLOs and reveal that these plant specific genes contribute paramount roles during successful completion of wheat plant life cycle and survival under unfavorable conditions. Detailed functional characterization of significantl1y perturbed TaMLOs using CRISPR-Cas9 could yield strong candidates for development of climate-resilient and super-immune wheat cultivars.

Quantitative RT-PCR Analysis of TaMLOs

Since our in-silico analysis exhibited higher and overlapping expressions of 10 genes during growth & development, and under abiotic and biotic stresses, therefore, we selected these genes for qRT-PCR validation under PM at four different time points as shown in Figure S1. In general, expressions of these genes started to elevate 24 h post infection (hpi) and reached peak deregulations at 72 hpi. Notably, 7 of these genes belonged to clade II, whereas 3 genes belonged to clade IV, and all shared homeologous relationships among distinct subclades. Homeologs of TaMLO8, TaMLO10, and TaMLO3/6 exhibited significant upregulation in the Bgt-resistant wheat line (N9134) as compared with the susceptible cultivar (Fielder). However, TaMLO9-A1 exhibited upregulation, whereas TaMLO9-D1 showed downregulation in Bgt resistant line (Fig. 6).

Fig. 6.

qRT-PCR based expression validation of TaMLOs. The relative expressions were quantified after 0, 24, 48 and 72 h of post inoculation (hpi) with Bgt. Student’s t-test was used to compare relative expression (P < 0.05) between PM resistant (N9134) and susceptible (Fielder) wheat genotypes

Powdery mildew is one of the most devastating diseases of wheat grown in temperate regions. Several studies have reported transcriptome and real-time gene expression analyses of wheat genes following Bgt inoculation. A large-scale transcriptome analysis of Bgt resistant line revealed distinct expression patterns responding to powdery mildew and stripe rust in wheat (Zhang et al. 2014). Similarly, a gene co-expression network analysis provided novel insights into the dynamic response of wheat to powdery mildew resistance (Hu et al. 2020). In this study, the qRT-PCR-based expressions were consistent with in-silico expression patterns. Four genes (TaMLO8-A1, TaMLO8-D1, TaMLO10-A1, and TaMLO10-D1) exhibited significant upregulation in the resistant line after 24, 48, and 72 hpi with the Bgt as compared with the susceptible cultivar. Likewise, five genes (TaMLO9-A1, TaMLO3/6-A2, TaMLO3/6-B2, TaMLO8-B1, TaMLO3/6-D2, and TaMLO8-D1) exhibited significantly higher expression in the resistant line after 2–3 days hpi, indicating positive regulation of PM resistance. Whereas single gene (TaMLO9-D1) showed downregulation in the resistant wheat line, suggesting a negative regulation of PM resistance (Fig. 6).

These genes are expected to be involved in PM susceptibility, and are candidate to be knocked out to confer PM resistance. Indeed, the simultaneous knock out of TaMLO-A1 TaMLO-B1, and TaMLO-D1 (renamed as TaMLO3/6-A2, TaMLO3/6-B2, and TaMLO3/6-D2, respectively) by TILLING, TALEN, and CRISPR/Cas9 conferred durable PM resistance in wheat without pleiotropic phenotypes (Han et al. 2014; Acevedo-Garcia et al. 2017; Li et al. 2022; Ingvardsen et al. 2023). Interestingly, none of the clade II genes have been characterized yet, but as 7 MLOs belonging to clade II showed differential expression under PM, they are probably involved in PM susceptibility too. As discussed in comparative phylogeny section, clade I and III MLOs are also involved in PM susceptibility, therefore we speculate that MLOs belonging to all four clades in monocots are potentially involved in PM susceptibility.

In future, it would be interesting to clone and functionally characterize these genes for confirming their PM resistance responses and engineering a durable resistance in future wheat cultivars.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

BH: Experiment design, analysis, writing original draft. QR: Analysis, experimentation, writing original draft. HR: Analysis, writing original draft. RMA: Conceptualization, experiment design, supervision. MFA: Analysis. HB: Supervision, editing of draft. ZA: Supervision, editing of draft. All authors reviewed the manuscript.

Funding

The current work was not supported by any grant.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics Approval and Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.