Next Article in Journal
Dose-Dependent Dual Effects of Gradient Ionizing Radiation on Neurocognition
Previous Article in Journal
Spexin as a Novel Biomarker for Predicting Disease Severity and Mortality in Acute Pancreatitis
Previous Article in Special Issue
Reinforced Defenses: R-Genes, PTI, and ETI in Modern Wheat Breeding for Blast Resistance
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 27
Issue 4
10.3390/ijms27041846
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications

by
Jovana Eskildsen
1,
Menghui Dong
1,
Tobias Hanak
1,
Claus Krogh Madsen
1,
Inger Holme
1,
Tamás Plaszkó
1,
Mette Vestergård
1,
Mogens Nicolaisen
1,
Hans Thordal-Christensen
2 and
Henrik Brinch-Pedersen
1,*
1
Department of Agroecology, Aarhus University, 4200 Slagelse, Denmark
2
Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg, Denmark
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2026, 27(4), 1846; https://doi.org/10.3390/ijms27041846
Submission received: 10 December 2025 / Revised: 6 February 2026 / Accepted: 10 February 2026 / Published: 14 February 2026
(This article belongs to the Special Issue Advanced Research of Plant-Pathogen Interaction)
Browse Figures
Review Reports Versions Notes

Abstract

Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is mildew resistance locus o (mlo)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of mlo mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel mlo mutant lines and evaluated their resistance to causal fungus Blumeria hordei. We further examined if mlo knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant mlo lines, including the first mutants with amino acid alterations in the protein’s extracellular region. Mutant lines showed significantly reduced B. hordei colony formation (0.5–5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating mlo-based resistance. Moreover, the study revealed functionally important residues in the protein’s extracellular region. Finally, we present the first evidence of limited mlo-associated effects on root microbiome diversity and relative abundance of microbial taxa.

1. Introduction

Powdery mildew is a common fungal disease in temperate climates, reducing crop yields and grain quality [1,2,3]. The fungus responsible for this disease in barley is the ascomycete obligate biotrophic pathogen Blumeria hordei (Bh), which is one of the most frequent barley pathogens [4,5]. Fortunately for barley farmers, an effective resistance to powdery mildew was discovered in the 1940s. It is attributed to recessive loss-of-function mutations in the Mlo (Mildew Resistance Locus O) and has been widely incorporated into barley breeding programs [6]. This resistance, which is widely prevalent in monocot and dicot plant species, is particularly attractive because it is durable and effective against most fungal isolates [2]. In general, immunity to powdery mildew is manifested at the stage of initial fungal penetration, by local cell-wall appositions known as papillae, and as programmed cell death response, referred to as the hypersensitive reaction (HR), of one or a few plant cells at the attack site. The collective action of these immunities determines the level of resistance of the plant [7]. mlo-based resistance is primarily characterized by unusually large papillae, which mediate early stop of the fungal attack [6,8].
More than 40 distinct mlo mutant alleles have been identified in barley, arising either naturally or through chemical and radiation-induced mutagenesis [2,9]. However, since mlo-based resistance represents a monogenic trait in barley, targeted gene-specific mutations can now be precisely recreated using New Genomic Techniques (NGTs) such as CRISPR/Cas, which has already been successfully applied in barley [10]. Unlike traditional mutagenesis, which introduces numerous background mutations, gene-specific approaches enable the direct assessment of mlo-mediated resistance and its effects such as those on the plant microsphere with unprecedented precision.
The Mlo gene codes for an integral membrane protein with seven transmembrane domains, with yet poorly understood molecular function [11,12,13]. Different protein parts and amino acid residues have been described as highly conserved, or variable, and more or less critical for the powdery mildew resistance [9,11,12]. Overall, cytoplasmatic loops 2 and 3, transmembrane proline, and four extracellular cysteine residues appear to be particularly sensitive to mutations [9,12]. Additionally, the majority of molecularly characterized barley mlo mutants have mutations in the cytoplasmatic parts of the protein, suggested to be the most crucial for powdery mildew resistance [9].
While mutations in the Mlo gene provide notable benefits in terms of disease resistance, they also come with specific adverse pleiotropic effects. Barley mlo mutants exhibit spontaneous mesophyll cell death, loss of chlorophyll, and accelerated leaf senescence, which leads to visible chlorotic and necrotic spots [14,15]. Moreover, mlo mutants show increased susceptibility to certain necrotrophic or hemibiotrophic pathogens, such as Magnaporthe grisea [16], Ramularia collo-cygni [17] and Bipolaris sorokiniana [18].
Furthermore, the pleiotropic effects of mlo mutations extend to below-ground plant parts, although these effects are significantly less explored. Studies have shown that mlo mutations can influence the colonization of root-associated microbes, particularly arbuscular mycorrhizal fungi (AMF) and endophytes, in inoculation experiments [19,20,21]. However, these experiments typically involve single microbial strains and are conducted in sterile conditions or germ-free substrates, lacking the complexity of natural soil microbiota, microbial interactions, and environmental variability. Such insights into microbiome stability are important, as the contribution of the microbiome to plant health, productivity, and stress resilience has become more evident in recent years [22,23,24,25]. Consequently, it is important to better understand if the mlo mutation affects the microbiome community as a whole within the natural soil environments.
This study aimed to develop clean, mlo-specific barley mutants using CRISPR/Cas9 technology and to validate resistance at both the macroscopic and microscopic levels. In addition, we investigated the impact of the mlo knockout mutation on root-associated microbial communities under natural, field-like growth conditions. Finally, we assessed whether the mlo mutation influences the relative abundance of specific bacterial and fungal taxa in the rhizosphere.

2. Results

2.1. Generating New CRISPR/Cas9-Induced mlo Mutants

We used A. tumefaciens with the final transformation construct (Figure 1) to transform cv. ‘Maythorpe’ immature embryos. The protospacer designed showed no obvious off-targets based on analysis using the CasOFFinder tool 2.4 (Supplementary Materials Figure S1). In total, 38 regenerated plants were obtained. They were PCR-screened for the presence of the hygromycin resistance gene, and 30 tested positive (Supplementary Materials). Mutations in the mlo gene were confirmed in all 30 plants by PCR amplification and sequencing of the target region within exon 2. Various indel mutations were detected, resulting in homozygous, heterozygous, biallelic, and chimeric genotypes.
Six T0 plants representing different genotypes were selected to generate the T1 generation. Their seeds were sown, and T1 offspring were genotyped by PCR amplification and sequencing of the targeted mlo region. Based on the results, five distinct homozygous mutant lines were selected for subsequent resistance screening (Figure 1). The lines will further be labeled as mlo-3-3, mlo-6-6, mlo-24-24, mlo-5-5, and mlo-14-14, based on the mutation they are carrying.
For the chosen five genotypes, protein sequences were predicted (Table 1; Supplementary Materials Figure S2). Three lines had in-frame mutations (mlo-3-3, mlo-6-6, and mlo-24-24) that deleted one (Tyr113del), two (Asp112_Tyr113del), and eight (Pro107_Cys114del) amino acid sequences from the protein, respectively. The rest of the protein was intact. Based on the instability index (II), the protein stability was unaffected (38.94, 39.28, and 38.88, respectively, compared to the wild type 38.88). The other two lines (mlo-5-5 and mlo-14-14) carried frameshift mutations, leading to a premature stop codon at the beginning of exon 5. The amino acid sequences were disrupted downstream of Asp112 and Tyr110, and encoded polypeptides were 173 and 170 aa long, respectively.
Three-dimensional (3D) modeling using the AlphaFold3 algorithm confirmed that for the mutant lines with early stop codon only, a short peptide is formed (Supplementary Materials), while the other mutations still could result in a protein comparable to the wild type, with minor changes to the intracellular fraction (Supplementary Materials). The short peptide model was expected by the impact of the frameshift mutation in the two lines mlo-5-5 and mlo-14-14 (Table 1). The three in-frame mutations had little effect on the protein model, only influencing the intracellular loop part of the 3D model.

2.2. CRISPR-Induced mlo Mutants Provided Powdery Mildew Resistance

To assess resistance in the T2 generation, five selected mutant lines and wild-type cv. ‘Maythorpe’ were inoculated with Bh using the settling tower method. Estimated spore density was four to six spores per square millimeter. Macroscopic resistance was visually scored 7 dpi.
None of the five mutant lines exhibited fungal growth, indicating complete resistance, while wild-type cv. ‘Maythorpe’ showed extensive powdery mildew symptoms (Figure 2). No difference in resistance was observed between mutant lines.
Microscopic analysis was conducted to assess resistance further. One hundred fungal penetration attempts were evaluated per replica, and averages were compared between mutant lines and the wild-type cv. ‘Maythorpe.’ The wild type exhibited a significantly higher number of successful penetration attempts compared to all mutant lines (p < 0.001), with an average of 62.5% (Figure 3). Among the mutants, the frameshift mutation lines mlo-5-5 and mlo-14-14 displayed the lowest frequencies of penetrating appressoria, with means of 22.5% and 18.5%, respectively. However, this did not differ significantly from in-frame mutant line mlo-3-3, which exhibited 24% successful penetration events. Lines mlo-6-6 and mlo-24-24 exhibited the highest penetration level among the mutants, with mean penetration counts of 32.5% and 28.75%, respectively. However, mlo-24-24 did not differ significantly from mlo-5-5 and mlo-3-3.
The mean number of HRs in the wild type occurred in 9% of the attacked epidermal cells, which was significantly lower compared to all mutant lines (p < 0.001). Among mutants, mlo-6-6, mlo-24-24, and mlo-5-5 exhibited the highest average numbers of HRs, with 29.5%, 27%, and 22%, respectively. Nonetheless, the level of HR in mlo-5-5 was not significantly different from those observed in lines mlo-3-3 and mlo-14-14, which displayed 19 and 16 HRs, respectively (Figure 3).
Finally, the number of fungal colonies formed was significantly greater in the wild-type ‘Maythorpe’ compared to all mutant lines (p < 0.001). Wild type had mean of 53.5 colonies counted. On the other hand, the most successful colony establishments among mutants were observed in in-frame mlo-3-3 and mlo-6-6, with 5 and 3 colonies, respectively. These values were not significantly different from those of mlo-14-14 and mlo-24-24, which exhibited 2.5 and 1.75 colonies, respectively. The most pronounced resistance was seen in mlo-5-5, where only 0.5 colonies were formed (Figure 3).
In summary, these microscopy data shows that the obtained mlo-based resistance in all our CRISPR-/Cas9 lines is due to a combination of reduced penetration and HR.

2.3. Mlo Knockout Did Not Alter Overall Microbial Diversity and Only Affected a Few Specific Taxa

The knockout mutant mlo-14-14 and wild-type cv ‘Maythorpe’ were used in the field-like experiment to assess whether knocking out mlo influences the root-associated microbial community. In particular, we evaluated alpha and beta diversity metrics for bacterial and fungal communities at two time points (T1 and T2). No significant differences in alpha diversity, described by richness or evenness, were observed between the two genotypes in bacterial or fungal communities at any of the sampling times (Figure 4a,b). Moreover, beta diversity, assessed via principal coordinate analysis (PCoA) based on Bray–Curtis dissimilarity, revealed no distinct separation between the mutant and wild type at any sampling time regarding bacterial or fungal community composition (Figure 4c,d). PERMANOVA and ANOSIM further confirmed no significant differences (p > 0.05, R ≈ 0) in overall microbial community composition between the mutant and wild type at any time.
To study if the relative abundances of specific ASVs/OTUs were different between the mutant line and wild type, we performed DESeq2 analysis. We identified a few bacterial ASVs and fungal OTUs that were significantly enriched (Padj < 0.05, Log2Fold change > 4) in one of the two lines at different sampling time points. At time point 1, two bacterial ASVs (i.e., ASV1356 and ASV1266), identified as Allorhizobium-Neorhizobium-Rhizobium group, and one fungal OTU (i.e., OTU_117), classified as Rhizopus, were enriched in the wild type. These were absent in the rhizosphere of the mutant line (Figure 5).
At time point 2, four bacterial ASVs (Allorhizobium-Neorhizobium-Rhizobium group ASV1264, Ornithinibacillus ASV365, Bacillus ASV 321, and Flavobacterium ASV725) exhibited significantly higher abundance in the rhizosphere of the mutant line. In contrast, one bacterial ASV, ASV818, classified as Promicromonospora, was enriched in the wild type. For fungi, three OTUs (i.e., Mortierellaceae OTU_449, Auriculariales OTU_22, and Pyrenochaetopsis OTU_405) were significantly more abundant in the rhizosphere of the wild type.

3. Discussion

3.1. Novel mlo Alleles with Mutations in Extracellular Loop 1 Confer Full Resistance to Blumeria hordei

Loss of function mutations in the mlo gene are known to provide durable, broad resistance to powdery mildew in barley [6,26]. Since we aimed to knock out the gene and recreate the loss of function phenotype, we designed the protospacer to target a sequence early in the gene, in exon 2, corresponding to the extracellular loop 1 part of the Mlo protein [11,27]. To date, no known barley mlo mutant carries a mutation in this position [9,27]. Our transformation pipeline was successful, and we proceeded with experiments using two lines with loss-of-function, frameshift mutations and three with in-frame mutations. The resistance scoring on macroscopic and microscopic levels confirmed the loss of function in all cases.
To analyze in-frame mutants, diving deeper into protein structure and mutation position is important. As mentioned, we generated mutations in the extracellular loop 1, a region described as highly variable and generally considered nonessential for the protein function [11,12]. In particular, mlo-3-3, mlo-6-6, and mlo-24-24 had one (Tyr113del), two (Asp112_Tyr113del), and eight (Pro107_Cys114del) amino acid residues deleted. As for the extracellular loop 1, amino acid residues 107 to 113 have been reported to be highly variable in the Mlo gene family [11,12]. Although extracellular loops have been described as highly variable and less critical for the function in powdery mildew resistance, four Cysteine (Cys) residues in those loops were described as invariable [11,12]. It is proposed that they have a role in disulfide bridge formation, which is vital for protein conformational stability, and even Mlo protein dimer/oligomerization, which is important for the protein function [11,12]. Among four invariable Cys residues, one was on the aa position 114, precisely where we targeted. Elliott, Müller [12] delivered the Mlo Cys114A variant through biolistic transformation into the mlo resistant genotype and showed no complementation and resistance preservation, emphasizing the significance of this residue for protein function. On the other hand, the wild-type, susceptible phenotype was successfully restored when the same complementation approach was applied to variable residues 103 and 105 [12].
Going back to our mutants, the invariable Cys114 residue was deleted in line mlo-24-24. Considering its vital role in powdery mildew resistance, we anticipated loss of function in that line. However, all our in-frame mutants showed complete resistance, raising the question about the role of highly variable residues deleted in lines mlo-3-3 and mlo-6-6. They have one or two aa residue(s) right upstream of the Cys114 deleted. Although those are known to be variable [12], we hypothesize that their deletion could affect the Cys position and conformation and impair its ability to form essential disulfide bridges or even oligomers. This would consequently lead to loss of function and a resistant phenotype, as observed. A study by Petersen, Jonson [28] reported on the importance and preference of Cys involved in disulfide bridges towards particular aa as neighbors, suggesting that this could be the mechanism. When comparing the 3D modeling results of our in-frame mutants with the wild-type protein, we can see that changes only occur in the intracellular loop (Supplementary Data). These changes could have an effect on the intracellular loop stability or tethering, leading to impaired disulfide bridges in our in-frame mutation. This is backed by other research, showing that cysteine replacement impairs protein function [29]. Therefore, deeper research on protein structure and biochemistry is necessary to understand the potential influence of mentioned aa residues on protein conformation, folding, disulfide bridge formation, and oligomerization.
As mentioned, all our mutants showed strong resistance to powdery mildew, with penetration success rates varying from 5% to 0.5%. There was no systematic difference between in-frame and frameshift mutant lines in the number of unsuccessful penetration attempts, HRs, or number of colonies. The in-frame mlo-3-3 and mlo-6-6 lines exhibited the highest number of colonies (5 and 3 per 100 attempts, respectively). However, the result was not significantly different from that observed for mlo-24-24 and mlo-14-14.
It is noteworthy that our mutants also show no typical symptoms of other barley mlo mutants. We could not detect any necrotic area under the Bh challenged sites at 7 dpi (Figure 2). While other mlo mutants, like mlo-5 or mlo-28, show these typical necrotic regions, our mutants are in barley the first mutants showing strong resistance without this side-effect [15]. There are reports of one allele from an Ethiopian landrace, showing a tempered resistance to powdery mildew without necrotic lesions, indicating the importance of location for the mutation [30]. There are similar reports from wheat, where usually the same phenotypic effect is observed [31], but novel CRISPR/Cas9-derived alleles show similar effects like our barley mutants [32].
We reported new CRISPR-induced mlo alleles that confer strong resistance to powdery mildew and the first-ever resistant mlo mutants in the extracellular part of the protein. Our study also highlights the potential of using CRISPR/Cas technology in breeding programs for the rapid and straightforward incorporation of valuable traits, such as disease resistance. Furthermore, we provided new insights into protein parts and amino acid residues essential for powdery mildew resistance, paving the way for further research on the topic.

3.2. mlo Knockout Preserves Overall Microbial Diversity but Influences a Few Rhizosphere Taxa

Several studies have examined the effects of the mlo mutation on below-ground plant–microbe interactions, specifically regarding its role in endophyte and mycorrhizal colonization [19,20,21]. However, no study has investigated how the mlo loss of function impacts the entire microbial community in the roots and rhizosphere. To address this knowledge gap, we used amplicon sequencing for analyzing microbial diversity, as well as specific taxa enrichment in mlo-14-14 and wild-type plants under field-like conditions.
We compared alpha and beta diversity between mlo-14-14 and wild type at two time points. Our results suggest no significant influence of the mlo loss of function mutation on either alpha or beta diversity at any time point. It is possible that the Mlo function is localized primarily in above-ground parts, while roots may have other genes playing a more dominant role in immune responses and plant–microbe interactions. Although Jacott, Charpentier [19] reported Mlo expression pattern changes during the interaction with beneficial root microbes, Le Fevre, O’Boyle [33] discovered that the role of Mlo in Phytophthora palmivora colonization was limited to leaves and did not have any detectable effect in roots, suggesting that its influence is exclusive for foliar tissue. Additionally, the root microbiome is known to be shaped by multiple factors, such as soil type, plant development stage, plant genotype, and secondary metabolites [34,35,36,37,38,39]. Hence, it is plausible that a loss of function mutation in this gene may be insufficient to override the mentioned dominant factors.
Recently, the plant microbiome has emerged as a key factor in sustainable agriculture. Microbiome management could soon be integrated into breeding programs, allowing breeders to select genotypes that recruit beneficial microbial communities [40]. With that in mind, understanding the factors that shape plant microbiomes is not only of fundamental scientific interest but also of practical relevance for the future of sustainable agriculture. Concerns about the influence of the mlo mutation, widely used in breeding, on microbial communities have already been raised in the barley scientific circles [41,42]. Our results provide initial indications that root microbiome diversity may not be substantially affected by the mlo knockout mutation.
However, we did detect alterations in relative abundances of a few specific taxa in the mlo mutant at different time points. At TP1, two ASVs within the Allorhizobium-Neorhizobium-Rhizobium group were more abundant in the wild type. At TP2, another ASV assigned to the Allorhizobium-Neorhizobium-Rhizobium group was significantly more abundant in the mlo mutant, suggesting that Mlo has a time-dependent role in the colonization.
Additionally, fungal OTU_117, classified as Rhizopus, was also enriched in the wild type at TP1. The Rhizopus genus is known to be pathogenic, with some members causing root rot disease in Plukenetia volubilis [43] or seedling blight in rice [44]. The Rhizopus enrichment in the wild type may indicate that the mlo knockout also provides resistance to this fungal genus. Studies on the effect of mlo mutation on Rhizopus infections are needed to better understand their interaction.
Additionally, other bacteria were enriched in the mutant at TP2. Those included agriculturally valuable genera Bacillus and Flavobacterium, known to promote growth [45,46,47], increased tolerance to abiotic stress [48,49,50], and disease resistance [51,52,53], and less explored but also growth-promoting Ornithinibacillus [54]. On the other hand, the only genus whose colonization was inhibited by mlo mutation was Promicromonospora, which was reported as growth promoter in some cases [55]. Overall, the mlo mutation appeared to facilitate the recruitment and colonization of roots by a few specific bacterial taxa, but there are no indications that the overall composition or functioning of the bacterial community is significantly modulated by the mutation.
Moreover, specific fungal taxa were enriched in the wild type at TP2. Mortierellaceae are well-described in the plant microbiome context, with reported growth-promoting [56,57] and disease-resistance properties [58]. The genus Pyrenochaetopsis is less documented but also reported to be beneficial, enhancing resistance to Fusarium oxysporum in tomatoes [59] and improving phosphorus uptake [60]. The last enriched fungal taxon was Auriculariales. This taxon is mainly described as a significant wood decomposer [61], especially in forests, but a study reported its presence in rhizosphere soil of switchgrass [62]. No information about its possible function in the context of root microbiome is available. Further research is needed to confirm and understand the nature of associations between Mlo and the mentioned fungal taxa.
Apart from the probable effect of the mlo mutation, the observed differences in specific taxa could be driven by powdery mildew infection that occurred naturally through airborne spores at TP2. The relationship between powdery mildew disease and the barley root microbiome has already been reported [63], and it is known that plant immunity, which is triggered by the Bh infection, also affects microbiome composition [64]. Although no effect was detectable on the overall microbial community, the disease could contribute to the observed changes in the relative abundance of the mentioned taxa. We propose multi-site replication studies to further confirm the observed results.

4. Materials and Methods

4.1. Plant Material and Agrobacterium-Mediated Transformation

Powdery mildew-susceptible barley (Hordeum vulgare ssp. vulgare) cv. ‘Maythorpe’ was used as donor material for the Agrobacterium-mediated transformation and genome editing of Mlo. Cv. ‘Maythorpe’ was sown into the substrate Hawita professional pH 5.2 (sphagnum) (HAWITA Gruppe GmbH, Vechta, Germany), and plants were grown in a growth chamber at 20 °C during the day and 16 °C at night, with a day/night cycle of 16 h/8 h. Water mixed with fertilizers (Azelis brun pioneer basis and NPK Makro 13-2-23+Mg+TE) (Tangaard, Bjæverskov, Danmark) was provided twice a day. The isolation and transformation of immature embryos was performed 12–14 days after pollination, following the protocol described in Holme, Dionisio [65]. After the transformation and regeneration process, plants were grown in greenhouse conditions until full maturity, and the seeds of chosen individuals were harvested.

4.2. Construct Design

To knock out Mlo, we targeted the second exon, and a protospacer sequence was created with the CRISPR tool available on benchling.com. The genome sequence of barley cv. ‘Golden Promise’ was used as a reference. Before designing the protospacer, Mlo was sequenced in cv. ‘Maythorpe’ to confirm that the sequence is the same as the reference. Although the CRISPR tool on benchling.com considers off-targets when designing protospacers, to double-check, we also used the online tool Cas-OFFinder, available at http://www.rgenome.net/cas-offinder/ (accessed on 1 May 2022).
We used a binary vector system based on the destination vector pANIC6A [66]. The entry vector pJG85 was used to clone the protospacer using the In-Fusion® Snap Assembly Master Mix (Takara). Vector pJG80 was used as an entry vector for wheat codon-optimized Cas9, and both entry vectors were cloned into the pANIC6A destination vector, according to Holme, Wendt [67]. After the assembly, the final transformation vector was transformed into the A. tumefaciens strain AGL-0 using the freeze-thaw method [68]. Glycerol stocks were made and kept at −80 °C until usage. The protospacer sequence, target site, and final transformation vector map are available in Figure 1.

4.3. Genotyping Generations T0 and T1

Two to three weeks after regenerating T0 plantlets, emerged leaf samples (two pieces of around 3 cm each) were collected to extract gDNA and genotype each plantlet. gDNA was extracted following the protocol from Pallotta, Graham [69]. First, we checked if the plant was transformed with PCR amplifying the hygromycin resistance gene using the Herculase II DNA polymerase (Agilent Technologies, Santa Clara, CA, USA) according to the manual provided. Then, positive samples were used in the subsequent PCR to amplify the part of the Mlo gene and screen for the mutation presence. The amplicon was 492 bp long and included the targeted sequence in exon 2. The reaction was performed using the Q5® High-Fidelity DNA Polymerase (NEB, Ipswich, Massachusetts, USA), following the supplier’s manual. Amplicons were purified from 1% agarose gel using a gel purification kit (Macherey-Nagel GmbH & Co. KG, Dueren, Germany) and sequenced by Macrogen (Macrogen Europe, Maastricht, The Netherlands). All primer sets, PCR conditions, and sequencing primer are presented in the Supplementary Materials.
Amplicons of heterozygous samples were TOPO cloned using the pCR®4Blunt-TOPO® vector from Invitrogen (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Then, formed plasmids were transformed into STELLAR chemically competent E. coli (Takara Bio Europe SAS, Saint-Germain-en-Laye, France), following the manual. Positive colonies were picked, and plasmids were extracted using NucleoSpin Plasmid kit (Macherey-Nagel GmbH & Co. KG, Dueren, Germany) and sequenced at Macrogen using the T3 universal primer (Supplementary Materials).
Genome-edited T0 plants were selected, and their seeds were harvested at full maturity. The seeds were subsequently sown in pots (17 cm wide,14 cm deep) filled with peat moss substrate, and plants were grown in the greenhouse conditions to screen the T1 generation. As previously reported, leaf samples were collected 2 to 3 weeks after the emergence to extract gDNA. In T1, plants were not screened for the hygromycin resistance gene presence, and genotyping was performed only by amplifying and sequencing Mlo, as already described in Section 2.3. Plants with selected mlo genotypes were grown until full maturity, and their seeds were harvested (Figure 1).
Protein prediction was performed for selected mlo genotypes to assess if mutations led to premature stop codon formation or changes in amino acid sequence. The genotyping results for selected T1 plants were used to predict amino acid sequences. The amino acid analysis and sequence alignments were performed using tools available on benchling.com. With the obtained amino acid sequences, a 3D model prediction of the wild-type and mutant proteins were performed using the AlphaFold3 algorithm with default settings on https://alphafoldserver.com/ (accessed on 4 February 2026). Screenshots of the 3D model prediction are in the Supplementary Materials. Additionally, for the mutant lines without premature stop codon, differences in protein stability were assessed using the instability index (II) available at benchling.com.

4.4. Inoculation with Powdery Mildew in Generation T2

Seeds of barley cv. ‘Maythorpe’ (susceptible, Mlo) and five homozygous T1 CRISPR-made mlo mutant lines (Figure 1) were sown in pots filled with peat moss substrate (Pindstrup) and placed in a growth chamber at 20 °C/15 °C and 16 h light (150 μmol s−1 m−2)/8 h darkness cycle. About 15 seeds were sown in each pot, and each genotype had two pots. No fertilizer was applied, and watering was optimal.
For inoculation, Bh isolate C15 was used. The isolate was propagated on the susceptible barley line ‘P-02’, a near-isogenic line of cv. ‘Pallas’, placed in the above conditions. For scoring resistance to Bh, primary leaves of seven-day-old seedlings were bent over the horizontal plastic support, with adaxial side up, and secured with two rubber bands (Supplementary Materials Figure S3). Eight leaves were bent for each pot. Inoculation was performed during daylight. Pots were placed in a tray inside a settling tower. A microscope slide was placed in the tray between pots to monitor inoculum density. A few Bh-infected barley plants with freshly produced conidia were held above the settling tower, and conidiospores were blown into the tower. The spores were allowed to settle for 15–20 min, and subsequently, a spore density of four–six spores per square millimeter was estimated on microscope slide. The inoculated plants were kept in the above conditions.

4.5. Macroscopic and Microscopic Resistance Evaluation

The macroscopic resistance evaluation was performed visually 7 days post-inoculation (dpi) when the number of powdery mildew colonies were assessed.
To assess resistance microscopically, we repeated inoculation as previously described. At 4 dpi, leaf pieces (around 4 cm each) were dyed with trypan blue to score fungal penetration success and hypersensitive response (HR). The staining was performed according to Bowling, Clarke [70] with modifications. The leaves were boiled for 10 min and incubated overnight before destaining.
Four leaves per genotype were used for scoring under the light microscope, and 100 randomly selected penetration attempts were counted per leaf. Penetration was considered successful if a haustorium or fungal colony was formed, while HR was registered if cellular uptake of the stain was detected.
Average values were calculated per genotype, and data were statistically analyzed with RStudio using the agricolae 1.3.7, FSA 0.10.1, and ggplot2 4.0.1 packages. The data were visually assessed for normality, and sqrt transformation was applied if necessary. Datasets were normally distributed, so one-way ANOVA followed by Tukey’s HSD test was conducted. Significant differences were denoted using asterisks: “*” (p < 0.05), “**” (p < 0.01), and “***” (p < 0.001). Different letters indicated statistically distinct groups based on Tukey’s HSD test.

4.6. Microbiome Analysis of mlo Mutants in Generation T2

4.6.1. Experimental Setup and Rhizosphere Sampling

To assess whether mlo knockout affects the microbiome, we conducted an experiment in field-like conditions in a fine mesh tunnel at Forsøgsvej 1, Slagelse, Denmark (55.325° N, 11.391° E). Six plots were established to compare two barley genotypes: the mlo mutant and the wild type (cv. ‘Maythorpe’). Each genotype was replicated across three plots, and each plot was further divided into two rows, resulting in six replicates per genotype. We had two sampling time points, 18 and 32 days after sowing (19 September and 2 October 2023). Two plants were collected at each sampling time point in each row and pooled as one sample. Loosely attached soil was removed by shaking the roots, and the roots with the adhering rhizosphere soil were lyophilized, ground, and used for downstream analysis. In total, 24 biological samples were collected, comprising two genotypes across two time points, with six biological replicates per genotype and time point.

4.6.2. DNA Extraction

Total DNA was extracted from 0.25 g of rhizosphere soil using the PowerSoil DNA Isolation Kit (QIAGEN, Germantown, MD, USA), following the manufacturer’s protocol. The extracted DNA samples were stored at −80 °C until further use.

4.6.3. Metabarcoding

To determine the composition of the bacterial and fungal communities, we performed Illumina Miseq amplicon sequencing. First, we generated the libraries. The libraries of bacterial and fungal DNA were generated by a two-step dual-indexing PCR strategy, as previously described [71]. For bacteria, the V3/V4 region of the 16S ribosomal RNA (rRNA) gene was amplified by primers 341F (S-DBact-0341-b-S-17: 5′-CCTACGGGNGGCWGCAG-3′) and 785R (S-D-Bact-0785-a-A-21: 5′-GACTACHVGGGTATCTAATCC-3′) [72]. For fungi, the internal transcribed spacer 2 (ITS2) region of the fungal rRNA gene was amplified by primers fITS7 (5′-GTGARTCATCGAATCTTTG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [73]. PCR conditions are available in Supplementary Materials. Subsequently, a 15-cycle indexing PCR was performed with a similar PCR mixture and program as described above. A dual-indexing approach was used for index combinations [74,75], using primer sequences with internal barcodes and index previously reported by [71]. SYBR staining was used in a 1.5% agarose gel for visualization to confirm the PCR product size. The barcoded PCR products were equimolarly pooled for equal representation, followed by ethanol precipitation and resolution in 50 μL of Tris-EDTA buffer. Subsequently, the pooled DNA was separated on a 1.5% agarose gel, and the DNA band of the expected size (300 to 450 bp) was extracted using a QIAquick Gel Extraction Kit (Qiagen, USA). Finally, the amplicon sequencing was performed on a MiSeq platform by Eurofins MWG (Ebersberg, Germany).

4.6.4. Bioinformatic Analysis

The obtained sequencing data were analyzed using Quantitative Insights into Microbial Ecology 2 (QIIME2, version 2023.7). Briefly, raw paired-end reads were input, and the quality of the reads was checked by the ‘qiime demux summarize’ function. Accordingly, amplicon sequence variants (ASVs) were determined by DADA2 from raw paired-end reads using the ‘qiime dada2 denoised-paired’ function. The parameters were as follows: for bacteria, forward and reverse reads were trimmed at the 5′ end until 17 and 21 bp, respectively, to remove the primers and truncated at the 3′ end until 285 and 200 bp, respectively, to remove the low-quality base pairs; for fungi, forward and reverse reads were trimmed at the 5′ end until 19 and 20 bp, respectively, to remove the primers and truncated at the 3′ end until 300 and 220 bp, respectively, to remove the low-quality base pairs. To classify the taxonomy of bacterial ASVs, a naïve Bayes classifier was trained by the ‘feature-classifier’ function against the Silva Ref NR99 [release 132] database. The ASVs were then classified using the ‘feature-classifier classify-sklearn’ function, and non-bacteria reads such as chloroplasts and mitochondria were moved from the ASVs table. For fungi, ITS2 regions were further extracted by ITSx and clustered into Operational Taxonomic Units (OTUs) at 98.5% similarity by vsearch, as these OTUs are closer to the fungal species level. To classify the taxonomy of fungal OTUs, the ‘taxid’ of R package ‘DECIPHER’ was employed to identify the taxonomy against the UNITE database.

4.6.5. Statistical Analysis

Microbiome downstream analyses were conducted in R version 4.3.1, using ASV or OTU tables of bacterial and fungal communities, respectively. The vegan package was used for analyses of alpha and beta diversity. For alpha diversity, ASV/OTU tables were rarefied to the minimum sequencing depth across all samples using the rrarefy function. Subsequently, observed ASVs/OTUs, Pielou’s evenness index, and the Shannon diversity index were calculated to estimate community richness, evenness, and diversity. For beta diversity analysis, raw ASV/OTU counts were log2-transformed to reduce amplification bias, followed by total sum scaling (TSS) to normalize data into relative abundances. Based on the relative abundance table, Bray–Curtis dissimilarities were calculated, and principal coordinates analysis (PCoA) was performed using the cmdscale function. To test for significant differences between the mlo mutant and wild type, PERMANOVA and ANOSIM analyses were carried out using the adonis2 and anosim functions, respectively.
Finally, to identify differentially enriched bacterial and fungal ASVs/OTUs between the two treatments, DESeq2 analysis was performed on the raw count tables using the DESeq2 package (v1.47.5) in R.

5. Conclusions

In summary, we developed novel CRISPR-induced mlo alleles which confer complete resistance to powdery mildew in barley. This research also produced the first mlo barley mutant with a mutation in the extracellular region of the protein. While mlo knockout did not significantly alter overall root-associated microbiome diversity, we observed differences in the relative abundances of a few specific bacterial and fungal taxa, indicating differentially enriched microbes at various time points. Overall, our study suggests that the powdery mildew resistance acquired by the knockout of mlo did not disrupt or compromise the composition of the root microbiome.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27041846/s1.

Author Contributions

Conceptualization, J.E., M.D., T.H., C.K.M., M.V., M.N., H.T-C. and H.B-P.; validation, T.H., C.K.M., M.V., M.N., H.T.-C. and H.B.-P.; formal analysis, J.E. and M.D.; investigation, J.E., M.D., I.H. and T.P.; resources, M.V., M.N. and H.T.-C.; writing—original draft preparation, J.E. and M.D.; writing—review and editing, J.E., M.D., T.H., C.K.M., I.H., T.P., M.V., M.N., H.T.-C. and H.B.-P.; visualization, J.E. and M.D.; supervision, T.H., C.K.M., I.H., M.V., M.N., H.T.-C. and H.B.-P.; project administration, H.B.-P.; funding acquisition, H.B.-P., M.V. and M.N. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Novo Nordisk Foundation (project NovoCrops, grant number NNF19OC0056580). This article is published as part of the BarleyMicroBreed project, that has received funding from the European Union’s Horizon Europe research and innovation program under Grant Agreement No. 101060057. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Executive Agency (REA). Neither the European Union nor the granting authority can be held responsible for them.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Raw 16S rRNA and ITS2 rRNA sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1281870.

Acknowledgments

We want to thank the technical staff from the Crop Genetics and Biotechnology Section at Aarhus University for their assistance with the experimental work. Special thanks go to Rikke Jakobsen for support and help in the experimental work conducted in Flakkebjerg. Additionally, we appreciate the help from Maarit Mäenpää in statistical analysis.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Glawe, D.A. The powdery mildews: A review of the world’s most familiar (yet poorly known) plant pathogens. Annu. Rev. Phytopathol. 2008, 46, 27–51. [Google Scholar] [CrossRef] [PubMed]
  2. Kusch, S.; Panstruga, R. mlo-based resistance: An apparently universal “weapon” to defeat powdery mildew disease. Mol. Plant-Microbe Interact. 2017, 30, 179–189. [Google Scholar] [CrossRef] [PubMed]
  3. Czembor, J.H. Resistance to powdery mildew in selections from Moroccan barley landraces. Euphytica 2002, 125, 397–409. [Google Scholar] [CrossRef]
  4. Dean, R.; Van Kan, J.A.; Pretorius, Z.A.; Hammond-Kosack, K.E.; Di Pietro, A.; Spanu, P.D.; Rudd, J.J.; Dickman, M.; Kahmann, R.; Ellis, J. The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 2012, 13, 414–430. [Google Scholar] [CrossRef]
  5. Liu, M.; Braun, U.; Takamatsu, S.; Hambleton, S.; Shoukouhi, P.; Bisson, K.R.; Hubbard, K. Taxonomic revision of Blumeria based on multi-gene DNA sequences, host preferences and morphology. Mycoscience 2021, 62, 143–165. [Google Scholar] [CrossRef]
  6. Jørgensen, I.H. Discovery, characterization and exploitation of Mlo powdery mildew resistance in barley. Euphytica 1992, 63, 141–152. [Google Scholar] [CrossRef]
  7. Liao, W.; Nielsen, M.E.; Pedersen, C.; Xie, W.; Thordal-Christensen, H. Barley endosomal MONENSIN SENSITIVITY1 is a target of the powdery mildew effector CSEP0162 and plays a role in plant immunity. J. Exp. Bot. 2023, 74, 118–129. [Google Scholar] [CrossRef]
  8. Stolzenburg, M.C.; Aist, J.R.; Israel, H.W. The role of papillae in resistance to powdery mildew conditioned by the ml-o gene in barley. I Correlative evidence. Physiol. Plant Pathol. 1984, 25, 337–346. [Google Scholar] [CrossRef]
  9. Reinstädler, A.; Müller, J.; Czembor, J.H.; Piffanelli, P.; Panstruga, R. Novel induced mlo mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein. BMC Plant Biol. 2010, 10, 31. [Google Scholar] [CrossRef]
  10. Koide, H.; Hisano, H.; Yaeno, T. CRISPR/Cas9-based generation of mlo mutants for allelic complementation experiments to elucidate MLO function in barley. J. Gen. Plant Pathol. 2023, 89, 153–158. [Google Scholar] [CrossRef]
  11. Devoto, A.; Piffanelli, P.; Nilsson, I.; Wallin, E.; Panstruga, R.; von Heijne, G.; Schulze-Lefert, P. Topology, subcellular localization, and sequence diversity of the Mlo family in plants. J. Biol. Chem. 1999, 274, 34993–35004. [Google Scholar] [CrossRef]
  12. Elliott, C.; Müller, J.; Miklis, M.; Bhat, R.A.; Schulze-Lefert, P.; Panstruga, R. Conserved extracellular cysteine residues and cytoplasmic loop–loop interplay are required for functionality of the heptahelical MLO protein. Biochem. J. 2005, 385, 243–254. [Google Scholar] [CrossRef]
  13. Büschges, R.; Hollricher, K.; Panstruga, R.; Simons, G.; Wolter, M.; Frijters, A.; Van Daelen, R.; van der Lee, T.; Diergaarde, P.; Groenendijk, J. The barley Mlo gene: A novel control element of plant pathogen resistance. Cell 1997, 88, 695–705. [Google Scholar] [CrossRef]
  14. Peterhansel, C.; Freialdenhoven, A.; Kurth, J.; Kolsch, R.; Schulze-Lefert, P. Interaction analyses of genes required for resistance responses to powdery mildew in barley reveal distinct pathways leading to leaf cell death. Plant Cell 1997, 9, 1397–1409. [Google Scholar] [CrossRef] [PubMed]
  15. Piffanelli, P.; Zhou, F.; Casais, C.; Orme, J.; Jarosch, B.; Schaffrath, U.; Collins, N.C.; Panstruga, R.; Schulze-Lefert, P. The barley MLO modulator of defense and cell death is responsive to biotic and abiotic stress stimuli. Plant Physiol. 2002, 129, 1076–1085. [Google Scholar] [CrossRef] [PubMed]
  16. Jarosch, B.; Kogel, K.-H.; Schaffrath, U. The ambivalence of the barley Mlo locus: Mutations conferring resistance against powdery mildew (Blumeria graminis f. sp. hordei) enhance susceptibility to the rice blast fungus Magnaporthe grisea. Mol. Plant-Microbe Interact. 1999, 12, 508–514. [Google Scholar] [CrossRef]
  17. McGrann, G.R.; Stavrinides, A.; Russell, J.; Corbitt, M.M.; Booth, A.; Chartrain, L.; Thomas, W.T.; Brown, J.K. A trade off between mlo resistance to powdery mildew and increased susceptibility of barley to a newly important disease, Ramularia leaf spot. J. Exp. Bot. 2014, 65, 1025–1037. [Google Scholar] [CrossRef]
  18. Kumar, J.; Hückelhoven, R.; Beckhove, U.; Nagarajan, S.; Kogel, K.-H. A compromised Mlo pathway affects the response of barley to the necrotrophic fungus Bipolaris sorokiniana (teleomorph: Cochliobolus sativus) and its toxins. Phytopathology 2001, 91, 127–133. [Google Scholar] [CrossRef] [PubMed]
  19. Jacott, C.N.; Charpentier, M.; Murray, J.D.; Ridout, C.J. Mildew Locus O facilitates colonization by arbuscular mycorrhizal fungi in angiosperms. New Phytol. 2020, 227, 343–351. [Google Scholar] [CrossRef]
  20. Hilbert, M.; Novero, M.; Rovenich, H.; Mari, S.; Grimm, C.; Bonfante, P.; Zuccaro, A. MLO differentially regulates barley root colonization by beneficial endophytic and mycorrhizal fungi. Front. Plant Sci. 2020, 10, 1678. [Google Scholar] [CrossRef]
  21. Ruiz-Lozano, J.M.; Gianinazzi, S.; Gianinazzi-Pearson, V. Genes involved in resistance to powdery mildew in barley differentially modulate root colonization by the mycorrhizal fungus Glomus mosseae. Mycorrhiza 1999, 9, 237–240. [Google Scholar] [CrossRef]
  22. Ge, A.-H.; Wang, E. Exploring the plant microbiome: A pathway to climate-smart crops. Cell 2025, 188, 1469–1485. [Google Scholar] [CrossRef]
  23. Jiang, G.; Zhang, Y.; Gan, G.; Li, W.; Wan, W.; Jiang, Y.; Yang, T.; Zhang, Y.; Xu, Y.; Wang, Y. Exploring rhizo-microbiome transplants as a tool for protective plant-microbiome manipulation. ISME Commun. 2022, 2, 10. [Google Scholar] [CrossRef]
  24. Harman, G.E.; Uphoff, N. Symbiotic root-endophytic soil microbes improve crop productivity and provide environmental benefits. Scientifica 2019, 2019, 9106395. [Google Scholar] [CrossRef]
  25. Bai, B.; Liu, W.; Qiu, X.; Zhang, J.; Zhang, J.; Bai, Y. The root microbiome: Community assembly and its contributions to plant fitness. J. Integr. Plant Biol. 2022, 64, 230–243. [Google Scholar] [CrossRef] [PubMed]
  26. Acevedo-Garcia, J.; Kusch, S.; Panstruga, R. Magical mystery tour: MLO proteins in plant immunity and beyond. New Phytol. 2014, 204, 273–281. [Google Scholar] [CrossRef]
  27. UniProt. Available online: https://www.uniprot.org/uniprotkb/P93766/feature-viewer (accessed on 5 July 2025).
  28. Petersen, M.T.N.; Jonson, P.H.; Petersen, S.B. Amino acid neighbours and detailed conformational analysis of cysteines in proteins. Protein Eng. 1999, 12, 535–548. [Google Scholar] [CrossRef] [PubMed]
  29. Haag, A.F.; Kerscher, B.; Dall’Angelo, S.; Sani, M.; Longhi, R.; Baloban, M.; Wilson, H.M.; Mergaert, P.; Zanda, M.; Ferguson, G.P. Role of Cysteine Residues and Disulfide Bonds in the Activity of a Legume Root Nodule-specific, Cysteine-rich Peptide. J. Biol. Chem. 2012, 287, 10791–10798. [Google Scholar] [CrossRef] [PubMed]
  30. Ge, X.; Deng, W.; Lee, Z.Z.; Lopez-Ruiz, F.J.; Schweizer, P.; Ellwood, S.R. Tempered mlo broad-spectrum resistance to barley powdery mildew in an Ethiopian landrace. Sci. Rep. 2016, 6, 29558. [Google Scholar] [CrossRef]
  31. Ingvardsen, C.R.; Massange-Sánchez, J.A.; Borum, F.; Füchtbauer, W.S.; Bagge, M.; Knudsen, S.; Gregersen, P.L. Highly effective mlo-based powdery mildew resistance in hexaploid wheat without pleiotropic effects. Plant Sci. 2023, 335, 111785. [Google Scholar] [CrossRef]
  32. Li, S.; Lin, D.; Zhang, Y.; Deng, M.; Chen, Y.; Lv, B.; Li, B.; Lei, Y.; Wang, Y.; Zhao, L.; et al. Genome-edited powdery mildew resistance in wheat without growth penalties. Nature 2022, 602, 455–460. [Google Scholar] [CrossRef] [PubMed]
  33. Le Fevre, R.; O’Boyle, B.; Moscou, M.J.; Schornack, S. Colonization of barley by the broad-host hemibiotrophic pathogen Phytophthora palmivora uncovers a leaf development–dependent involvement of Mlo. Mol. Plant-Microbe Interact. 2016, 29, 385–395. [Google Scholar] [CrossRef] [PubMed]
  34. Pacheco-Moreno, A.; Bollmann-Giolai, A.; Chandra, G.; Brett, P.; Davies, J.; Thornton, O.; Poole, P.; Ramachandran, V.; Brown, J.K.; Nicholson, P. The genotype of barley cultivars influences multiple aspects of their associated microbiota via differential root exudate secretion. PLoS Biol. 2024, 22, e3002232. [Google Scholar] [CrossRef]
  35. Yang, C.; Yue, H.; Ma, Z.; Feng, Z.; Feng, H.; Zhao, L.; Zhang, Y.; Deakin, G.; Xu, X.; Zhu, H. Influence of plant genotype and soil on the cotton rhizosphere microbiome. Front. Microbiol. 2022, 13, 1021064. [Google Scholar] [CrossRef]
  36. Bziuk, N.; Maccario, L.; Douchkov, D.; Lueck, S.; Babin, D.; Sørensen, S.J.; Schikora, A.; Smalla, K. Tillage shapes the soil and rhizosphere microbiome of barley—But not its susceptibility towards Blumeria graminis f. sp. hordei. FEMS Microbiol. Ecol. 2021, 97, fiab018. [Google Scholar] [CrossRef] [PubMed]
  37. Chaparro, J.M.; Badri, D.V.; Vivanco, J.M. Rhizosphere microbiome assemblage is affected by plant development. ISME J. 2014, 8, 790–803. [Google Scholar] [CrossRef]
  38. Koprivova, A.; Kopriva, S. Plant secondary metabolites altering root microbiome composition and function. Curr. Opin. Plant Biol. 2022, 67, 102227. [Google Scholar] [CrossRef]
  39. Sikder, M.M.; Vestergård, M.; Kyndt, T.; Topalović, O.; Kudjordjie, E.N.; Nicolaisen, M. Genetic disruption of Arabidopsis secondary metabolite synthesis leads to microbiome-mediated modulation of nematode invasion. ISME J. 2022, 16, 2230–2241. [Google Scholar] [CrossRef]
  40. Barnes, C.J.; Bahram, M.; Nicolaisen, M.; Gilbert, M.T.P.; Vestergård, M. Microbiome selection and evolution within wild and domesticated plants. Trends Microbiol. 2024, 33, 447–458. [Google Scholar] [CrossRef]
  41. Zuccaro, A.; Langen, G. Breeding for resistance: Can we increase crop resistance to pathogens without compromising the ability to accommodate beneficial microbes? New Phytol. 2020, 227, 279–282. [Google Scholar] [CrossRef]
  42. Panstruga, R.; Kuhn, H. Mutual interplay between phytopathogenic powdery mildew fungi and other microorganisms. Mol. Plant Pathol. 2019, 20, 463–470. [Google Scholar] [CrossRef]
  43. Uwaremwe, C.; Bao, W.; Daoura, B.G.; Mishra, S.; Zhang, X.; Shen, L.; Xia, S.; Yang, X. Shift in the rhizosphere soil fungal community associated with root rot infection of Plukenetia volubilis Linneo caused by Fusarium and Rhizopus species. Int. Microbiol. 2024, 27, 1231–1247. [Google Scholar] [CrossRef]
  44. Partida-Martinez, L.P.; Hertweck, C. Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature 2005, 437, 884–888. [Google Scholar] [CrossRef]
  45. Akhtyamova, Z.; Arkhipova, T.; Sharipova, G.; Ivanov, R.; Nuzhnaya, T.; Kudoyarova, G.; Veselov, D. The Effect of plant growth-promoting bacteria Bacillus subtilis IB-22 on the hydraulic conductivity and abundance of PIP2 aquaporins in the roots of an abscisic acid-deficient barley mutant. Int. J. Mol. Sci. 2024, 25, 10706. [Google Scholar] [CrossRef] [PubMed]
  46. Menon, R.R.; Kumari, S.; Viver, T.; Rameshkumar, N. Flavobacterium pokkalii sp. nov., a novel plant growth promoting native rhizobacteria isolated from pokkali rice grown in coastal saline affected agricultural regions of southern India, Kerala. Microbiol. Res. 2020, 240, 126533. [Google Scholar] [CrossRef]
  47. Akhtyamova, Z.; Arkhipova, T.; Martynenko, E.; Nuzhnaya, T.; Kuzmina, L.; Kudoyarova, G.; Veselov, D. Growth-promoting effect of rhizobacterium (Bacillus subtilis IB22) in salt-stressed barley depends on abscisic acid accumulation in the roots. Int. J. Mol. Sci. 2021, 22, 10680. [Google Scholar] [CrossRef]
  48. Zhou, H.-Y.; Sun, P.-Y.; Yu, X.-R.; Zhou, Y.; Zhang, Z.-W.; Gao, J.-Z.; Zhao, D.-H.; Luo, Y.-L.; Hu, T.-M.; Fu, J.-J. Protective effects of Flavobacterium succinicans on perennial ryegrass under shade stress. Acta Prataculturae Sin. 2020, 29, 137–143. [Google Scholar]
  49. Belimov, A.A.; Hontzeas, N.; Safronova, V.I.; Demchinskaya, S.V.; Piluzza, G.; Bullitta, S.; Glick, B.R. Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L. Czern.). Soil Biol. Biochem. 2005, 37, 241–250. [Google Scholar] [CrossRef]
  50. Gontia-Mishra, I.; Sapre, S.; Sharma, A.; Tiwari, S. Amelioration of drought tolerance in wheat by the interaction of plant growth-promoting rhizobacteria. Plant Biol. 2016, 18, 992–1000. [Google Scholar] [CrossRef]
  51. Zanon, M.S.A.; Cavaglieri, L.R.; Palazzini, J.M.; Chulze, S.N.; Chiotta, M.L. Bacillus velezensis RC218 and emerging biocontrol agents against Fusarium graminearum and Fusarium poae in barley: In vitro, greenhouse and field conditions. Int. J. Food Microbiol. 2024, 413, 110580. [Google Scholar] [CrossRef] [PubMed]
  52. Kriuchkova, L.O. Biological control of leaf disease of barley with Bacillus strain. Biologija 2017, 63. [Google Scholar] [CrossRef]
  53. Sang, M.; Kim, K.D. The volatile-producing Flavobacterium johnsoniae strain GSE09 shows biocontrol activity against Phytophthora capsici in pepper. J. Appl. Microbiol. 2012, 113, 383–398. [Google Scholar] [CrossRef] [PubMed]
  54. Maheshwari, R.; Bhutani, N.; Bhardwaj, A.; Suneja, P. Functional diversity of cultivable endophytes from Cicer arietinum and Pisum sativum: Bioprospecting their plant growth potential. Biocatal. Agric. Biotechnol. 2019, 20, 101229. [Google Scholar] [CrossRef]
  55. Kang, S.-M.; Khan, A.L.; Hamayun, M.; Hussain, J.; Joo, G.-J.; You, Y.-H.; Kim, J.-G.; Lee, I.-J. Gibberellin-producing Promicromonospora sp. SE188 improves Solanum lycopersicum plant growth and influences endogenous plant hormones. J. Microbiol. 2012, 50, 902–909. [Google Scholar] [CrossRef]
  56. Ozimek, E.; Hanaka, A. Mortierella species as the plant growth-promoting fungi present in the agricultural soils. Agriculture 2020, 11, 7. [Google Scholar] [CrossRef]
  57. Liao, H.-L. The plant-growth-promoting fungus, Mortierella elongata: Its biology, ecological distribution, and growth-promoting activities: SS679/SL466, 3/2021. EDIS 2021, 2021. [Google Scholar] [CrossRef]
  58. Wang, Y.; Wang, L.; Suo, M.; Qiu, Z.; Wu, H.; Zhao, M.; Yang, H. Regulating root fungal community using Mortierella alpina for Fusarium oxysporum resistance in Panax ginseng. Front. Microbiol. 2022, 13, 850917. [Google Scholar] [CrossRef]
  59. Surono, S.; Zaffan, Z.R.; Soekarno, B.P.W.; Munif, A.; Nurdebyandaru, N. The first report of Pyrenochaetopsis terricola as a dark septate endophytic fungus antagonistic to Fusarium oxysporum f. sp. Lycopersici, a pathogen causing wilt disease in tomato plants. Res. Sq. 2023. [Google Scholar] [CrossRef]
  60. Bao, X.; Lu, H.; Zhao, J.; Yang, T.; Wu, L.; Zou, J.; Chen, Q.; Zhang, B. Screening and identification of two novel phosphate-solubilizing Pyrenochaetopsis tabarestanensis strains and their role in enhancing phosphorus uptake in rice. Front. Microbiol. 2025, 15, 1494859. [Google Scholar] [CrossRef]
  61. Malysheva, V.; Spirin, V. Taxonomy and phylogeny of the Auriculariales (Agaricomycetes, Basidiomycota) with stereoid basidiocarps. Fungal Biol. 2017, 121, 689–715. [Google Scholar] [CrossRef] [PubMed]
  62. Mao, Y.; Li, X.; Smyth, E.M.; Yannarell, A.C.; Mackie, R.I. Enrichment of specific bacterial and eukaryotic microbes in the rhizosphere of switchgrass (Panicum virgatum L.) through root exudates. Environ. Microbiol. Rep. 2014, 6, 293–306. [Google Scholar] [CrossRef]
  63. Bziuk, N.; Maccario, L.; Sørensen, S.J.; Schikora, A.; Smalla, K. Barley rhizosphere microbiome transplantation–A strategy to decrease susceptibility of barley grown in soils with low microbial diversity to powdery mildew. Front. Microbiol. 2022, 13, 830905. [Google Scholar] [CrossRef]
  64. Teixeira, P.J.P.; Colaianni, N.R.; Fitzpatrick, C.R.; Dangl, J.L. Beyond pathogens: Microbiota interactions with the plant immune system. Curr. Opin. Microbiol. 2019, 49, 7–17. [Google Scholar] [CrossRef] [PubMed]
  65. Holme, I.B.; Dionisio, G.; Brinch-Pedersen, H.; Wendt, T.; Madsen, C.K.; Vincze, E.; Holm, P.B. Cisgenic barley with improved phytase activity. Plant Biotechnol. J. 2012, 10, 237–247. [Google Scholar] [CrossRef] [PubMed]
  66. Mann, D.G.; LaFayette, P.R.; Abercrombie, L.L.; King, Z.R.; Mazarei, M.; Halter, M.C.; Poovaiah, C.R.; Baxter, H.; Shen, H.; Dixon, R.A. Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species. Plant Biotechnol. J. 2012, 10, 226–236. [Google Scholar] [CrossRef]
  67. Holme, I.B.; Wendt, T.; Gil-Humanes, J.; Deleuran, L.C.; Starker, C.G.; Voytas, D.F.; Brinch-Pedersen, H. Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs. Plant Mol. Biol. 2017, 95, 111–121. [Google Scholar] [CrossRef] [PubMed]
  68. Jyothishwaran, G.; Kotresha, D.; Selvaraj, T.; Srideshikan, S.; Rajvanshi, P.; Jayabaskaran, C. A modified freeze–thaw method for efficient transformation of Agrobacterium tumefaciens. Curr. Sci. 2007, 93, 770–772. [Google Scholar]
  69. Pallotta, M.; Graham, R.; Langridge, P.; Sparrow, D.; Barker, S. RFLP mapping of manganese efficiency in barley. Theor. Appl. Genet. 2000, 101, 1100–1108. [Google Scholar] [CrossRef]
  70. Bowling, S.A.; Clarke, J.D.; Liu, Y.; Klessig, D.F.; Dong, X. The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance. Plant Cell 1997, 9, 1573–1584. [Google Scholar][Green Version]
  71. Kudjordjie, E.N.; Sapkota, R.; Steffensen, S.K.; Fomsgaard, I.S.; Nicolaisen, M. Maize synthesized benzoxazinoids affect the host associated microbiome. Microbiome 2019, 7, 59. [Google Scholar] [CrossRef]
  72. Klindworth, A.; Pruesse, E.; Schweer, T.; Peplies, J.; Quast, C.; Horn, M.; Glöckner, F.O. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 2013, 41, e1. [Google Scholar] [CrossRef]
  73. Ihrmark, K.; Bödeker, I.T.; Cruz-Martinez, K.; Friberg, H.; Kubartova, A.; Schenck, J.; Strid, Y.; Stenlid, J.; Brandström-Durling, M.; Clemmensen, K.E. New primers to amplify the fungal ITS2 region–evaluation by 454-sequencing of artificial and natural communities. FEMS Microbiol. Ecol. 2012, 82, 666–677. [Google Scholar] [CrossRef] [PubMed]
  74. Siddique, A.B.; Unterseher, M. A cost-effective and efficient strategy for Illumina sequencing of fungal communities: A case study of beech endophytes identified elevation as main explanatory factor for diversity and community composition. Fungal Ecol. 2016, 20, 175–185, Corrigendum to Fungal Ecol. 2016, 22, 1.. [Google Scholar] [CrossRef]
  75. Wu, L.; Wen, C.; Qin, Y.; Yin, H.; Tu, Q.; Van Nostrand, J.D.; Yuan, T.; Yuan, M.; Deng, Y.; Zhou, J. Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis. BMC Microbiol. 2015, 15, 125. [Google Scholar] [CrossRef] [PubMed]
Ijms 27 01846 g001
Figure 1. CRISPR/Cas9 editing of Mlo in barley cv. ‘Maythorpe’. (A) Map of the construct used for transformation and genome editing. (B) Structure of the Mlo gene with the target site in exon 2 indicated in black. The target sequence is shown in bold letters, with the adjacent PAM sequence highlighted in a black box. (C) Alignment of mutant line sequences against the wild-type sequence of cv. ‘Maythorpe’. Mismatches are indicated in red.
Figure 1. CRISPR/Cas9 editing of Mlo in barley cv. ‘Maythorpe’. (A) Map of the construct used for transformation and genome editing. (B) Structure of the Mlo gene with the target site in exon 2 indicated in black. The target sequence is shown in bold letters, with the adjacent PAM sequence highlighted in a black box. (C) Alignment of mutant line sequences against the wild-type sequence of cv. ‘Maythorpe’. Mismatches are indicated in red.
Ijms 27 01846 g001
Ijms 27 01846 g002
Figure 2. Macroscopic assessment of powdery mildew resistance. Images of mutant lines ((A)—mlo-3-3, (B)—mlo-24-24, (C)—mlo-5-5, (D)—mlo-14-14, (E)—mlo-6-6) and (F)—wild-type cv. ‘Maythorpe’ samples were taken 7 dpi with Blumeria hordei. The estimated inoculation density was 4–6 spores per square millimeter.
Figure 2. Macroscopic assessment of powdery mildew resistance. Images of mutant lines ((A)—mlo-3-3, (B)—mlo-24-24, (C)—mlo-5-5, (D)—mlo-14-14, (E)—mlo-6-6) and (F)—wild-type cv. ‘Maythorpe’ samples were taken 7 dpi with Blumeria hordei. The estimated inoculation density was 4–6 spores per square millimeter.
Ijms 27 01846 g002
Ijms 27 01846 g003
Figure 3. Microscopic assessment of powdery mildew resistance. (A–C) Bar plots showing (A) the number of successfully penetrated appressoria per genotype, (B) the number of observed hypersensitive responses (HRs) per genotype, and (C) the number of Bh colonies formed per genotype. (D–F) Representative microscopic images of (D) an unsuccessful penetration attempt, (E) HR, and (F) a Bh colony. st—stoma; e—epidermal cell; sp—fungal spore; h—hyphae.
Figure 3. Microscopic assessment of powdery mildew resistance. (A–C) Bar plots showing (A) the number of successfully penetrated appressoria per genotype, (B) the number of observed hypersensitive responses (HRs) per genotype, and (C) the number of Bh colonies formed per genotype. (D–F) Representative microscopic images of (D) an unsuccessful penetration attempt, (E) HR, and (F) a Bh colony. st—stoma; e—epidermal cell; sp—fungal spore; h—hyphae.
Ijms 27 01846 g003
Ijms 27 01846 g004
Figure 4. Alpha (a,b) and beta (c,d) diversity of bacterial and fungal communities at different sampling times. Mutant data is shown in red, while wildtype data is shown in turquoise. Alpha diversity is represented by richness (the number of observed ASVs/OTUs) and evenness (the Pielou index). Beta diversity was demonstrated using principal component analysis based on the Bray–Curtis dissimilarity. Adonis and ANOSIM were used to determine the significance of the difference between the wild type and mutant. No significance is indicated by ns.
Figure 4. Alpha (a,b) and beta (c,d) diversity of bacterial and fungal communities at different sampling times. Mutant data is shown in red, while wildtype data is shown in turquoise. Alpha diversity is represented by richness (the number of observed ASVs/OTUs) and evenness (the Pielou index). Beta diversity was demonstrated using principal component analysis based on the Bray–Curtis dissimilarity. Adonis and ANOSIM were used to determine the significance of the difference between the wild type and mutant. No significance is indicated by ns.
Ijms 27 01846 g004
Ijms 27 01846 g005
Figure 5. Differentially enriched microbial taxa in the rhizosphere of mutant and wild-type plants at different sampling times. (a) Bacterial and (b) fungal taxa showing significant enrichment at different time points.
Figure 5. Differentially enriched microbial taxa in the rhizosphere of mutant and wild-type plants at different sampling times. (a) Bacterial and (b) fungal taxa showing significant enrichment at different time points.
Ijms 27 01846 g005
Table 1. Summary of mutant genotypes and their predicted alterations in protein sequence relative to the wild-type Mlo protein.
Table 1. Summary of mutant genotypes and their predicted alterations in protein sequence relative to the wild-type Mlo protein.
MutantMutation TypeEffect on ProteinPredicted Function
mlo-3-3In-frameTyr113delPartial/Modified
mlo-6-6In-frameAsp112_Tyr113delPartial/Modified
mlo-24-24In-framePro107_Cys114delPartial/Modified
mlo-5-5FrameshiftAsp112 *Null/Non-functional
mlo-14-14FrameshiftTyr110 *Null/Non-functional
Legend: Frameshift mutations with early stop codon leading to shortened peptides are indicated by an asterisk (*).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Eskildsen, J.; Dong, M.; Hanak, T.; Madsen, C.K.; Holme, I.; Plaszkó, T.; Vestergård, M.; Nicolaisen, M.; Thordal-Christensen, H.; Brinch-Pedersen, H. Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications. Int. J. Mol. Sci. 2026, 27, 1846. https://doi.org/10.3390/ijms27041846

AMA Style

Eskildsen J, Dong M, Hanak T, Madsen CK, Holme I, Plaszkó T, Vestergård M, Nicolaisen M, Thordal-Christensen H, Brinch-Pedersen H. Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications. International Journal of Molecular Sciences. 2026; 27(4):1846. https://doi.org/10.3390/ijms27041846

Chicago/Turabian Style

Eskildsen, Jovana, Menghui Dong, Tobias Hanak, Claus Krogh Madsen, Inger Holme, Tamás Plaszkó, Mette Vestergård, Mogens Nicolaisen, Hans Thordal-Christensen, and Henrik Brinch-Pedersen. 2026. "Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications" International Journal of Molecular Sciences 27, no. 4: 1846. https://doi.org/10.3390/ijms27041846

APA Style

Eskildsen, J., Dong, M., Hanak, T., Madsen, C. K., Holme, I., Plaszkó, T., Vestergård, M., Nicolaisen, M., Thordal-Christensen, H., & Brinch-Pedersen, H. (2026). Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications. International Journal of Molecular Sciences, 27(4), 1846. https://doi.org/10.3390/ijms27041846

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop