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Article

Genome-Wide Analysis of Mlo Genes and Functional Characterization of Cm-mlo38 and Cm-mlo44 in Regulating Powdery Mildew Resistance in Melon

by
Fangyi Gong
1,2,†,
Yanhong Lan
1,2,†,
Tian Zhang
1,2,
Chun Li
1,2,
Yifan Li
1,2,
Feng Xia
1,2,
Xiaojun Liu
1,2,
Duchen Liu
1,2,
Genyun Liang
1,2,
Peng Cai
1,2,* and
Chao Fang
1,2,*
1
Vegetable Germplasm Innovation and Variety Improvement Key Laboratory of Sichuan Province, Horticulture Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China
2
Sichuan Province Engineering Technology Research Center of Vegetables, Chengdu 611934, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(5), 509; https://doi.org/10.3390/horticulturae11050509
Submission received: 16 April 2025 / Revised: 6 May 2025 / Accepted: 7 May 2025 / Published: 8 May 2025
(This article belongs to the Special Issue Genomics and Genetic Diversity in Vegetable Crops)
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Abstract

:
The Mildew Resistance Locus (Mlo) gene family is reported in various species as regulators of powdery mildew (PM) resistance. However, the Mlo genes in cucurbit crops remain limited. In this study, a genome-wide investigation of Mlo genes was conducted in eight Cucurbitaceae species and in rice, maize, arabidopsis, and barley, and a total of 202 Mlo genes were identified. The phylogenetic analysis showed that 202 Mlo genes can be classified into six clades, and the Mlo genes from clades I and III are likely pivotal in regulating PM resistance in dicotyledonous and monocotyledonous plants, respectively. The Ka/Ks ratios for these homologous Mlo gene pairs ranged from 0 to 0.6, revealing that they underwent substantial purifying selection during evolution. Among 12 crops, there were the most Mlo genes (22 Cm-mlo) in melon. An expression analysis revealed that six Cm-mlo genes showed expression responses to PM infection in which Cm-mlo38 and Cm-mlo44 were phylogenetically close to Mlo genes that regulated PM resistance. Using the VIGS system for silencing, Cm-mlo38 and Cm-mlo44 enhanced resistance to PM in susceptible material. A protein interaction analysis indicated that Cm-mlo38 might regulate PM resistance through interactions with PR5 and CML proteins. These results provide a comprehensive understanding of the Mlo family in Cucurbitaceae and pave the way for future functional analysis and genetic improvement for improving PM resistance in Cucurbitaceae.

1. Introduction

The Cucurbitaceae family is an extensive group of plants comprising over 130 genera and 800 species globally [1]. It includes numerous economically significant crops that produce cucurbit fruits, such as cucumbers (Cucumis sativus L.), melons (Cucumis melo L.), watermelons (Citrullus lanatus L.), and bottle gourds (Lagenaria siceraria L.) [2]. Powdery mildew (PM) is considered one of the main biotic threats to the yield and quality of multiple species, causing serious production and economic losses in Cucurbitaceae crops [3,4]. PM is one of the most widespread and serious plant diseases of temperate climates, caused by ascomycete fungi of the order Erysiphales, and can lead to significant harvest losses in crop plants [5]. Nearly 10,000 different plants, including various cereals and horticultural crops such as melon, cucumber, pumpkin, barley, and wheat, are hosts of over 650 PM fungi in the world. Among them, Podosphaera xanthii (Px) and Golovinomyces cichoracearum (GC) are the most common pathogens responsible for PM disease [6].
The Mildew Resistance Locus (Mlo) is a gene family specific to plants and is conserved throughout the plant kingdom. In 1992, the mlo gene was first identified in barley, where loss-of-function mutations were found to confer broad-spectrum resistance to powdery mildew (PM) [7]. The mlo gene acted as a negative regulator in plant disease resistance, functioning as a susceptibility factor that promotes infection under normal conditions [8]. Resistance to PM resulting from recessive loss-of-function alleles (mlo) exhibits the following characteristics: (1) the resistance is recessive, (2) has a broad spectrum, (3) and is durable, (4) and in the absence of pathogen infection, plants with mlo mutations exhibit a spontaneous leaf cell death phenotype [7,9]. Subsequently, resistance based on mlo mutations has been successfully applied to both monocotyledonous and dicotyledonous plant species for PM control. The Mlo gene family has been identified in various plant species, including Arabidopsis thaliana [10] (15 members, AtMlo), Solanum lycopersicum [11] (17 members, SlMlo), Triticum aestivum [12] (7 members, TaMlo), Glycine max [13] (39 members, GmMlo), Oryza sativa [14] (12 members, OsMlo), C. sativus [15] (14 members, CsMlo), and C. melo [16] (14 members, CmMlo).
Mutant alleles of multiple members have been shown to function in PM resistance. For example, one natural mlo mutant (mlo-11) from an Ethiopian barley and one induced mlo mutant (mlo-9) was widely used in barley PM resistance breeding [7,17,18]. In wheat, the single mutants (aaBBDD, AAbbDD, and AABBdd) of TaMlo-A1, TaMlo-B1, or TaMlo-D1 exhibited an infection phenotype similar to the wild type (WT), while the double mutant (aabbDD, AAbbdd, and aaBBdd) and triple mutant (aabbdd) lines showed enhanced resistance compared to the WT [19]. The triple mutant of AtMlo2, AtMlo6, and AtMlo12 granted complete resistance to Arabidopsis thaliana PM [20]. In tomato, the loss of function of the Mlo SlMlo1 gene or the simultaneous silencing of SlMlo5 and SlMlo8 could enhance plant PM resistance [21,22]. In cucumber, Mlo genes of clade V are functional susceptibility genes for PM in which the loss-of-function mutations in CsaMlo8 conferred the highest resistance, while the double mutants of CsaMlo1 and CsaMlo11 exhibited middle resistance [23]. In melon, a natural mutant allele, CmMLO2_1, from the wild melon germplasm C18 confers plant broad-spectrum resistance against PM [24].
In this study, we conducted Mlo member identification on eight representative species of the Cucurbitaceae family by exploiting the newest genome sequence information. The gene structure, conserved domain, phylogenetic relationships, spatiotemporal expression pattern during plant development, and expression response to inoculation with the PM pathogen of these Mlo genes were systematically analyzed. An in-depth analysis was conducted on the Mlo gene in melon, with a focus on validating the regulatory roles of Cm-mlo38 and Cm-mlo44 in resistance to PM. The research findings establish a foundation for further elucidating the molecular mechanisms underlying the resistance of Cucurbitaceae species to PM.

2. Materials and Methods

2.1. Identification of Mlo Genes in Cucurbitaceae

The eight cucurbitaceae species (Cucurbita moschata, Cucurbita pepo, L. siceraria, Benincasa hispida, C. lanatus, Cucumis hystrix, C. sativus, and C. melo) and A. thaliana, Hordeum vulgare, O. sativa, Zea mays genome sequences, protein sequences, coding sequences, and annotation profiles were obtained from the Cucurbitaceae genomic database (CuGenDB2) version2 (http://cucurbitgenomics.org/v2/, accessed on 15 April 2025) and EnsemblPlants (https://plants.ensembl.org/info/data/ftp/index.html, accessed on 15 April 2025). The Hidden Markov Model (HMM) profile of the Mlo domain (PF03094) was downloaded from the Pfam protein family database (http://pfam.xfam.org/, accessed on 15 April 2025) and was used to identify Mlo genes from the genomes of eight species using the HMMER3.0 search tool with an E-value ≤ 10−5 [16]. The protein sequences were further checked using the National Center for Biotechnology Information (NCBI)-Conserved Domain Database (CDD) search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 15 April 2025) to identify the conserved protein domain and reject some candidate genes that were outside of the Mlo domain [25].

2.2. Sequence Analysis of Mlo Genes

The online ExPASy (https://www.expasy.org/, accessed on 15 April 2025), SMART server (http://smart.embl.de/smart/batch.pl, accessed on 15 April 2025), and ProtComp9.0 tools were used to predict the amino acid length, theoretical isoelectric point (PI), molecular weight (Mw), and subcellular localization of the Mlo proteins [16,26]. The conserved protein motifs of Mlo were predicted using the MEME Suite web server (https://meme-suite.org/meme/tools/meme, accessed on 15 April 2025), with the maximum number of motifs being specified as 15 and the optimum width of motif sets being specified as 5 to 200 amino acids. The annotation file of the Mlo genes was used to construct the exon/intron organization using the TBtools II-Gene Structure View software (v2.210) [27].

2.3. Phylogenetic and Collinearity Analyses of Mlo Genes

The amino acid sequences of all Mlo genes were aligned using the clustalW version 2.0 (https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 15 April 2025). The resulting alignments were used to construct a phylogenetic tree using the Neighbor Joining (NJ) method with 1000 bootstrap replications and the MEGA-X software and iTOL (https://itol.embl.de/upload.cgi/, accessed on 15 April 2025). The collinearity analysis of Mlo genes in O. sativa, A. thaliana, L. siceraria, C. lanatus, B. hispida, C. sativus, C. moschata, and C. melo was performed using TBtools II-One Step MCScanX and the Multiple Synteny Plot (v2.210) [27].

2.4. Expression Analysis of Mlo Genes

The expression of all Mlo genes in different tissues and the gene expression changes in all Mlo genes in plants infected with P. xanthii were obtained from the public Cucurbitaceae expression database CuGenDB2-Cucurbit Expression Atlas (http://cucurbitgenomics.org/v2/expression, accessed on 15 April 2025).

2.5. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)

Total RNA from leaf samples in wild-type melon plants and silenced plants was isolated using TRIzolTM reagent (Thermo Fisher Scientific, Tokyo, Japan). First-strand cDNA synthesis was performed using the TaKaRa PrimeScriptTM RT Reagent Kit (Takara, Dalian, China) according to the manufacturer’s instructions. Gene-specific primers were designed using Primer3 Plus software (http://www.primer3plus.com/, accessed on 15 April 2025) and Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome, accessed on 15 April 2025) (Table S1). qRT-PCR was performed on Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) in a 10 µL reaction volume containing 2 ng of cDNA, 5 µL of 1 × SYBR Premix Ex Taq (TaKaRa), and 0.5 µL (300 nM) of each primer. The 2−ΔΔCT method was used to quantify the gene expression relative to the endogenous ACTIN (EGP21177.1) control. Three biological replicates were used for each data point.

2.6. Virus-Induced Gene Silencing (VIGS)

For the VIGS experiment, the Cucurbitaceae universal vector CGMMV (derived from Cucumber Green Mottle Mosaic Virus for VIGS) was selected. Based on the cDNA sequence of the candidate gene, appropriate primers for the CGMMV vector (provided by Professor Gu Qinsheng of the Chinese Academy of Agricultural Sciences) were designed using the online SGN VIGS Tool (https://vigs.solgenomics.net/, accessed on 15 April 2025). The vector was then constructed via the In-Fusion cloning system, and the plasmid containing the target fragment was transformed into Agrobacterium tumefaciens GV3101. The positive Agrobacterium strains obtained were expanded to reach an OD600 value between 0.5 and 0.8, and then an infiltration solution was added. The germinated melon seeds were immersed in the Agrobacterium-containing solution and vacuum infiltrated at −80 kPa for 15 min, and this process was repeated once. Subsequently, the seeds were transplanted into plug trays and cultivated in an incubator until the VIGS-PDS plants exhibited the bleaching phenotype, indicative of successful gene silencing. qRT-PCR was used to detect the expression level of the target gene to determine the success of gene silencing. Thereafter, the silenced plants were inoculated with powdery mildew [28]. Silenced plants were then subjected to PM inoculation for a functional validation of the candidate gene.

2.7. Subcellular Localization

In a subcellular localization experiment, the coding sequence (CDS) of Cm-mlo38 and Cm-mlo44, with the stop codon removed, was cloned into the pCAMBIA2300-EGFP vector, which is under the control of the CaMV35S promoter. This construct was subsequently introduced into A. tumefaciens strain GV3101 and used to infiltrate Nicotiana benthamiana leaves. The N. benthamiana plants were initially kept in the dark for 24 h and then transferred to a growth chamber maintained at 20 °C with a 16 h light/8 h dark cycle for 48 h [29]. Following this, the leaves were harvested, and the fluorescence signals were observed using a laser scanning confocal microscope.

2.8. Protein Interaction Network Analysis

The protein interaction network of Mlo protein was analyzed using the online software STRING version 11.5 (https://cn.string-db.org/, accessed on 15 April 2025). The amino acid sequence of Mlo protein was mapped to C. melo protein sequences using the sequence alignment function of STRING.

2.9. Statistical Analysis

To analyze the significance of the differences in expression levels of the Cm-mlo38 and Cm-mlo44 genes between wild-type plants and silenced plants, an Analysis of Variance (ANOVA) was conducted utilizing IBM SPSS Statistics version 22, with mean comparisons conducted through Duncan’s Multiple Range Test at a significance level of 0.05.

3. Results

3.1. Identification and Sequence Analysis of Mlo Family Members

Based on the HMMER search results with an E-value ≤ 0.0001, a total of 202 genes were initially identified as potentially belonging to the Mlo gene family from 12 species, including Z. mays, O. sativa, H. vulgare, and A. thaliana, and 8 Cucurbitaceae species using the HMMER search tool with an E-value ≤ 0.0001. The most abundant Mlo gene family members were identified in C. melo (22 Cm-mlos). Totals of 14, 15, 15, 16, 16, 20, 21, 21, 13, 14, and 15 Mlo genes were found in L. siceraria (Ls-mlos), C. lanatus (Cl-mlos), B. hispida (Bh-mlos), C. sativus (Cs-mlos), C. hystrix (Ch-mlos), C. pepo (Cp-mlos), C. moschata (Cmo-mlos), Z. mays (Zm-mlos), O. sativa (Os-mlos), H. vulgare (Hv-mlos), and A. thaliana (At-mlos), respectively. Upon analysis using the NCBI-CDD tool, we confirmed that all 202 genes contained the Mlo conserved domain. A sequence analysis showed that the protein lengths of 202 Mlo genes ranged from 39 amino acids (AA) (MELO3C002216.2.1) to 1264 AAs (CmoCh01G005680.1). The theoretical pI and Mw ranged from 4.7 (Zm00001eb306720_P001) to 10.49 (Zm00001eb428720_P001) and from 4369.36 Da (MELO3C002216.2.1) to 140267.57 Da (CmoCh01G005680.1), respectively (Table S2). A total of 202 Mlo protein sequences were analyzed using the MEME tool, resulting in the identification of a total of 15 conserved motifs, with lengths varying from 12 AA (Motif 11) to 41 AA (Motif 1). Notably, a sequence analysis of these motifs indicated that Motifs 1 through 7, as well as Motifs 9, 10, 11, and 12, belonged to Mlo conserved domains (Figure S1). In the Cucurbitaceae group, eight species contain an average of 17.5 Mlo genes compared to only 15.75 in the other four species. The Mlo gene family in 12 species underwent a cumulative total of 101 gene duplication events and 204 gene loss events (Figure 1). Notably, eight Cucurbitaceae crops exhibited an amplification of 30 Mlo genes and a subsequent loss of 2 Mlo genes compared to Arabidopsis, highlighting a distinct evolutionary trajectory within the Cucurbitaceae family crops.

3.2. The Phylogenetic and Subcellular Localization Analysis of Mlo Genes

To elucidate the phylogenetic relationships within the Mlo gene family, a comprehensive phylogenetic tree was constructed using 202 Mlo genes. The analysis classified the 202 identified Mlo genes into six distinct clades (I to VI). Clade III included the most genes (50). Clade I included the least genes (six) which included two Os-mlos, two Hv-mlos and two Zm-mlos genes. Clades II, IV, V, and VI included 41, 13, 46, and 46 genes, respectively. Clade IV did not include rice, maize, and barley Mlo genes (Figure 2; Table 1). Most Mlo proteins (199 out of 202) were predicted to be located in the cell membrane (Table S2).

3.3. Intragroup and Intergroup Collinearity Analysis of Mlo Genes

To investigate the expansion and evolution mechanism of the Mlo gene family in melon, gene replication events in the melon genome were studied. We identified three pairs of homologous genes, which were MELO3C013709.2.1/MELO3C007979.2.1, MELO3C021515.2.1/MELO3C034965.2.1, and MELO3C025761.2.1/MELO3C005038.2.1 (Figure 3A). Furthermore, to gain deeper insights into the origin and function of the Cm-mlo gene family, we analyzed the synteny of the Mlo gene in melon and arabidopsis, rice, cucumber, bottlegourd, pumpkin, watermelon, and wax gourd genomes, respectively. A total of 76 pairs of homologous genes were found between melon and these species, including arabidopsis (7), rice (5), cucumber (14), bottlegourd (13), pumpkin (14), watermelon (11), and wax gourd (12) (Figure 3B). Notably, the Ka/Ks ratios for these homologous Mlo gene pairs ranged from 0 to 0.6, suggesting that they underwent substantial purifying selection during evolution. Moreover, the Ka/Ks ratios for Mlo gene pairs between melon and arabidopsis, as well as melon and rice, were notably below 0.15, significantly lower than those observed for other homologous pairs (Figure 3C). These findings hint that Mlo genes have conserved functions during evolution and are subject to negative selection, making them less susceptible to mutational changes.

3.4. Promoter Analysis of Cm-mlo Genes

We used the 2000 bp upstream sequence of ATG of 22 CmMlo genes to predict cis-elements by using PlanCARE. A total of 40 unique cis-element motifs were found, and they belong to different functional groups associated with biotic and abiotic stress responsiveness, hormone responsiveness, and fruit development. A total of 30 cis-elements related to biotic and abiotic stress responses were identified in the promoter of CmMlo genes, which suggests that CmMlo genes could mainly function in response to biotic and abiotic stresses. The hormone-responsive elements included P-box, GARE-motif, and TATC-box (gibberellin responsiveness); TCA-element and ABRE (salicylic acid responsiveness); and TGACG-motif and CGTCA-motif (MeJA responsiveness). The fruit development elements consisted of three subcategories, GCN4-motif, RY-element, and O2-site (Figure 4).

3.5. Expression Patterns in Different Tissues and Expression Response Analysis to PM

From the comprehensive RNA-seq database, we analyzed the expression patterns of these 22 Cm-mlo genes in the roots, shoots, and leaves, as well as their transcriptional responses to PM. Five Cm-mlo genes were not expressed in the roots, shoots, and leaves (FPKM < 1). Ten Cm-mlo genes were highly expressed (FPKM > 5), in which MELO3C007979.2.1 (FPKM = 117.28), MELO3C000169.2.1 (FPKM = 98.54), and MELO3C007979.2.1 (FPKM = 192.69) had the highest expression in the roots, shoots, or leaves, respectively. Each gene had different expression levels in various tissues. Some, like MELO3C034561.2.1, were only highly expressed in the roots, while others, like MELO3C007252.2.1 and MELO3C021515.2.1, were only highly expressed in the leaves. Some genes, such as MELO3C005044.2.1 and MELO3C005038.2.1, were highly expressed in both the roots and leaves. Still, others, like MELO3C012438.2.1 and MELO3C013761.2.1, were predominantly expressed in the stems and leaves. Finally, genes like MELO3C025761.2.1, MELO3C007979.2.1, MELO3C013709.2.1, MELO3C022486.2.1, and MELO3C000169.2.1 showed high expression levels in the roots, stems, and leaves (Figure 5A; Table S3).
Among these 22 genes, 9 genes were not expressed in either inoculated (PM_24h, PM_48h and PM_72h) or non-inoculated leaves (CK_24h, CK_48h and CK_72h) at 24 h, 48 h, and 72 h. Notably, the expression level of the MELO3C013709.2.1 gene showed a decreasing trend after inoculation with PM fungi. Additionally, six genes (MELO3C022486.2.1, MELO3C000169.2.1, MELO3C007979.2.1, MELO3C025761.2.1, MELO3C005044.2.1, and MELO3C012438.2.1) exhibited significantly higher expression levels in inoculated leaves compared to the control group, which was consistently observed 24 h, 48 h, and 72 h post-inoculation (Figure 5B; Table S4). Combined with the phylogenetic analysis result, MELO3C005044.2.1 and MELO3C012438.2.1 were clustered with CsaV3_1G012950.1 (CsaMLO1), CsaV3_5G036400.1 (CsaMLO11), Atmlo2 (AT1G11310), Atmlo6 (AT1G61560), and Atmlo12 (AT2G39200) (Figure 2), which have been previously identified as playing crucial roles in regulating PM resistance [20,30]. Thus, MELO3C005044.2.1 and MELO3C012438.2.1 were selected for further study and renamed as Cm-mlo44 and Cm-mlo38, respectively.

3.6. The Functional Verification of Cm-mlo38 and Cm-mlo44

To assess Cm-mlo38 and Cm-mlo44 gene functions, the VIGS system silenced these genes (VIGS-mlo38 and VIGS-mlo44) and PDS (VIGS-PDS) in a susceptible plant variety, using an empty vector-infected control (WT). The expression levels of Cm-mlo38 and Cm-mlo44 were significantly reduced in VIGS-mlo38 or VIGS-mlo44 plants compared to WT plants (Figure 6A). After exhibiting the bleaching phenotype in VIGS-PDS plants, VIGS-mlo38, VIGS-mlo44, and WT plants were inoculated with PM. Twelve days post-inoculation, the leaves of the WT were covered in PM fungi. In contrast, the leaves of VIGS-mlo38 showed white infection spots but no PM fungi, while the leaves of VIGS-mlo44 had a small amount of PM fungi (Figure 6B).

3.7. Subcellular Localization Analysis of Cm-mlo38 and Cm-mlo44 Proteins

Fluorescence microscopy was employed to evaluate the subcellular distribution of Cm-mlo38 and Cm-mlo44. This was achieved by separately transfecting Nicotiana benthamiana leaves with the CaMV35S:Cm-mlo38-EGFP or CaMV35S:Cm-mlo44-EGFP constructs. The fluorescent signal emanating from the Cm-mlo38-EGFP and Cm-mlo44-EGFP proteins was mainly aggregated into patches on the membrane (Figure 7). Further analysis of the protein sequences of Cm-mlo38 and Cm-mlo44 revealed that both contain seven transmembrane domains. These findings suggest that Cm-mlo38 and Cm-mlo44 are localized within the membrane.

3.8. Protein Interaction Network Analysis of Cm-mlo38 and Cm-mlo44 Proteins

To further investigate the functions of Cm-mlo38 and Cm-mlo44 proteins, we constructed a protein interaction network using C. melo as a reference and STRING software version 11.5. A total of 10 potential interacting proteins of Cm-mlo38 and Cm-mlo44 were identified, including ACC oxidase, ACC synthase, pathogenesis-related protein 5 (PR5), Calmodulin-like (CML), Calmodulin-7-like (CM7L), Syntaxin-121 (Syt121), Aminotransferase 1 (Amf1), Serine-glyoxylate aminotransferase-like isoform X1 (Sga X1), and two Photosystem II reaction center W proteins (PrcW) (Figure 8). Five proteins (ACC oxidase, PrcW, CML, Cm-mlo38, and Cm-mlo44) were highly enriched in the “response to stress” category (GO:0006950, FDR = 0.0468) (Figure S3). Additionally, two proteins (CML and CM7L) participated in the “plant–pathogen pathway” (Cmo04626, FDR = 0.0136) (Figure S4).

4. Discussion

The gene Mlo was initially identified as conferring broad-spectrum resistance to PM in barley when Mlo undergoes loss of function [7,9,31]. Subsequently, it was confirmed that the Mlo genes that were the homologous genes of barley Mlo in rice (OsMlo2) and wheat (TaMlo-A1, TaMlo-B1, and TaMlo-D1) conferred PM resistance to various crops in the absence of function, and PM-resistant materials were created using Mlo non-functional homologous genes [19,32]. PM was one of the main diseases affecting the yield and quality of Cucurbitaceae crops, causing serious economic losses [4,33]. We identified 202 Mlo genes with conserved structural domains across 12 crop species and analyzed their gene structures and phylogenetic relationships, revealing a range of 13–22 Mlo genes per species. Previous research on Mlo genes in Cucurbitaceae has been conducted, yet it remains incomplete. For instance, predecessors identified 14 Cs-mlo genes in the cucumber genome [15] and 14 Cm-mlo genes in the melon genome [16]. Advancements in sequencing technology have led to more refined plant genomes. This study used the latest cucumber (ChineseLong_V3) and melon (CM3.6.1) reference genomes to identify 16 and 22 Mlo gene family members, respectively. The number of gene family members reflects genetic diversity, which tends to be higher in species with more members, enhancing their environmental adaptability and survival competitiveness [34]. Among eight Cucurbitaceae crops, the most Mlo genes were found in C. melo, and the Ka/Ks ratios for Mlo gene pairs between melon and the other six crops were notably below 0.6, suggesting that the Mlo gene family expanded and was subject to negative selection during the evolution of Cucumis melo to resist PM infection. Our finding suggests that these Mlo genes may have potential application in providing new candidates for improving PM resistance.
The phylogenetic analysis revealed that clade I exclusively harbored Mlo genes from rice, maize, and barley, while clade IV contained Mlo genes solely from Cucurbitaceae and Arabidopsis. This segregation was likely due to the inherent differences between dicotyledonous plants (Cucurbitaceae and Arabidopsis) and monocotyledonous plants (rice, maize, and barley). The findings indicate that the Mlo gene pattern in clade I may be unique to monocotyledonous plants, whereas the Mlo gene pattern in clade IV appears to be specific to dicotyledonous plants. Six Mlo genes were clustered into clade I, in which HORVU.MOREX.r3.4HG0410620.1 (Mlo) was identified as conferring broad-spectrum resistance to PM in barley when it undergoes loss of function [7,9,31]. The rice Mlo gene Os03t0129100-01 (OsMlo2) was able to complement PM-resistant barley mlo mutants at the single-cell level [32]. A total of 50 Mlo genes were clustered into clade III, in which CsaV3_1G012950.1 (CsaMLO1) and CsaV3_5G036400.1 (CsaMLO11) could be negative regulators in PM resistance in cucumber [30]. The AtMLO2 mutation offers partial resistance to PM. However, full resistance is only achieved with additional mutations in AtMLO6 and AtMLO12, forming a triple mutant with less than 1% fungal entry success. Neither the AtMLO6 nor AtMLO12 mutations alone confer enhanced resistance to PM [20]. Collectively, these findings suggest that Mlo genes from clades I and III may play crucial roles in regulating PM resistance in dicotyledonous and monocotyledonous plants, respectively.
The promoter region’s cis-regulatory elements play a significant role in regulating plant control mechanisms and in genes that respond to various stimuli [35,36]. In this study, the promoter analysis of 22 Cm-mlo genes identified 40 unique cis-elements linked to stress responses, hormone signaling, and fruit development, suggesting that CmMlo may undergo diverse transcriptional regulations and possess potential functional diversity in melon. In wild melon C18, a CmMLO2 (MELO3C022486.2.1) mutant (CmMLO2_1) with an 85 bp sequence difference from cultivated species was found, and after introducing the mutant CmMLO2_1 into wild germplasm via fluorescence-mediated transformation (labeled Agrobacterium), the resulting transgenic plant was found to be susceptible to PM Race1 [24]. The expression of CmMLO3 (MELO3C005044.2.1) was significantly up-regulated in both the susceptible line (Topmark) and resistant line (PI124112) but showed no significant change in resistant line MR1 24 h and 72 h after infection. CmMLO5 (MELO3C012438.2.1) was significantly up-regulated only in Topmark, with no change in the resistant lines (MR1 and PI124112), and it showed a single base mutation (C to T) at 572 bp, leading to a T-to-I amino acid change between two PM-resistant lines (MR-1 and PI124112) and two PM-susceptible lines (X055 and Topmark) [16]. In this study, six Cm-mlo genes had expression responses to PM infection in which MELO3C012438.2.1 (Cm-mlo38) and MELO3C005044.2.1 (Cm-mlo44) were clustered with CsaMLO1, CsaMLO11, Atmlo1, Atmlo6, and Atmlo12, which had been identified as playing crucial roles in regulating resistance to PM [20,30]. Thus, using the VIGS system to silence Cm-mlo38 and Cm-mlo44 in the susceptible line (2021-1), respectively, it was found that silencing Cm-mlo38 and Cm-mlo44 enhanced resistance to PM in the susceptible line. The results suggest that Cm-mlo38 and Cm-mlo44 play a negative role in regulating PM resistance.
A protein interaction network analysis revealed that Cm-mlo38 and Cm-mlo44 potentially interacted with 10 proteins involved in stress and pathogen response, in which the CML protein belonged to an important class of Ca2+ sensors, which were crucial for the plants’ responses to environmental stresses [37,38]. PR-1 proteins, which were abundant in plants during pathogen attack, contained a C-terminal peptide (CAPE) that independently signaled defense responses against microbial pathogens [39]. Thus, we speculated that Cm-mlo38 protein could regulate PM resistance by interacting with PR5 and CML.

5. Conclusions

A total of 202 Mlo genes were identified across 12 crops. We systematically analyzed these gene structures, phylogeny, chromosomal distribution, motifs, and expression patterns. Clade I and III Mlo genes were implicated in powdery mildew (PM) resistance in dicots and monocots, respectively. In melon, Cm-mlo38 and Cm-mlo44 were found to negatively regulate PM resistance, with Cm-mlo38 potentially functioning via interactions with PR5 and CML proteins. The results of this study provide a reference for studying PM resistance in Cucurbitaceae.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11050509/s1, Table S1: List of primer sequences used in this study; Table S2: Information and physicochemical characteristics of Mlo genes; Table S3: Expression analysis of 22 Cm-mlos in roots, stems, and leaves; Table S4: Expression response analysis of 22 Cm-mlos to PM; Figure S1: The structural and compositional characteristics of the Mlo genes. A, Mlo motif of 202 genes. B, conserved motifs of 202 Mlo genes. Different motifs are represented by different colored boxes; Figure S2: The phenotypic characteristics of the PM-susceptible line after silencing of the PDS gene (VIGS-PDS); Figure S3: GO enrichment analysis of Cm-mlo38 and Cm-mlo44 interaction protein; Figure S4: KEGG enrichment analysis of Cm-mlo38 and Cm-mlo44 interaction protein.

Author Contributions

F.G., formal analysis and writing—original draft preparation. Y.L. (Yanhong Lan), investigation. T.Z., investigation. C.L., investigation. Y.L. (Yifan Li), investigation. F.X., investigation. X.L., investigation. D.L., investigation. G.L., investigation. P.C., supervision. C.F., conceptualization, project administration, funding acquisition, and manuscript revision. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the earmarked fund for the Modern Agro-industry Technology Research System of China (Grant No. CARS-25), the Sichuan Innovation Team Program of CARS (Grant No. SCCXTD-2024-22), the Talent Special Project of Sichuan Academy of Agricultural Sciences (Grant No. NKYRCZX2025006), the 1+9 Program of SAAS (Grant No. 1+9KJGG03), and the Sichuan Province Engineering Technology Research Center of Vegetables (Grant No. 2023PZSC0304).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We would like to thank the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
MloMildew resistance locus
PMPowdery mildew
PxPodosphaera xanthii
GCGolovinomyes cichoracearum
HMMHidden Markov Model
NCBINational Center for Biotechnology Information
CDDConserved Domain Database
PITheoretical isoelectric point
MwMolecular weight
NJNeighbor joining
VIGSVirus-induced gene silencing
CDSCoding sequence

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Horticulturae 11 00509 g001
Figure 1. The duplication and loss analysis of Mlo genes in 12 crops; Among these, eight crops belonging to the Cucurbitaceae family are represented in red.
Figure 1. The duplication and loss analysis of Mlo genes in 12 crops; Among these, eight crops belonging to the Cucurbitaceae family are represented in red.
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Figure 2. The phylogenetic relationships of the 202 Mlo genes (14 Ls-mlos, 15 Cl-mlos, 15 Bh-mlos, 16 Cs-mlos, 16 Ch-mlos, 20 Cp-mlos, 21 Cmo-mlos, 22 Cm-mlos, 13 Os-mlos, 14 Hv-mlos, 15 At-mlos, and 21 Zm-mlos). The clades of the Mlo genes are marked in different colors (clades I (purple), II (brown), III (blue), IV (rose red), V (green), and VI (pink)). These two genes are marked in red as they are designated for functional validation in upcoming research.
Figure 2. The phylogenetic relationships of the 202 Mlo genes (14 Ls-mlos, 15 Cl-mlos, 15 Bh-mlos, 16 Cs-mlos, 16 Ch-mlos, 20 Cp-mlos, 21 Cmo-mlos, 22 Cm-mlos, 13 Os-mlos, 14 Hv-mlos, 15 At-mlos, and 21 Zm-mlos). The clades of the Mlo genes are marked in different colors (clades I (purple), II (brown), III (blue), IV (rose red), V (green), and VI (pink)). These two genes are marked in red as they are designated for functional validation in upcoming research.
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Figure 3. Chromosome mapping and synteny analysis of melon Mlo genes. (A) Chromosomal information for Mlo genes was obtained from melon genome annotation results and mapped to corresponding chromosomes. (B) Synteny analysis of Mlo genes between genomes of C. melo and other species (O. sativa, A. thaliana, L. siceraria, C. lanatus, B. hispida, C. sativus, and C. moschata). (C) Ka/Ks ratios of Mlo genes’ collinearity pairs.
Figure 3. Chromosome mapping and synteny analysis of melon Mlo genes. (A) Chromosomal information for Mlo genes was obtained from melon genome annotation results and mapped to corresponding chromosomes. (B) Synteny analysis of Mlo genes between genomes of C. melo and other species (O. sativa, A. thaliana, L. siceraria, C. lanatus, B. hispida, C. sativus, and C. moschata). (C) Ka/Ks ratios of Mlo genes’ collinearity pairs.
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Figure 4. The cis-acting elements in the 2 kb upstream of 22 Cm-mlos. Different colors represent the quantities of cis-acting elements within the promoter, where red indicates a higher number and white denotes a lower number.
Figure 4. The cis-acting elements in the 2 kb upstream of 22 Cm-mlos. Different colors represent the quantities of cis-acting elements within the promoter, where red indicates a higher number and white denotes a lower number.
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Figure 5. Expression analysis of 22 Cm-mlos. (A) Expression analysis in roots, stems, and leaves. (B), Expression of Cm-mlo in PM_24h, PM_48h, and PM_72h (24 h, 48 h, and 72 h post-inoculation) and CK_24h, CK_48h, and CK_72h (control group).
Figure 5. Expression analysis of 22 Cm-mlos. (A) Expression analysis in roots, stems, and leaves. (B), Expression of Cm-mlo in PM_24h, PM_48h, and PM_72h (24 h, 48 h, and 72 h post-inoculation) and CK_24h, CK_48h, and CK_72h (control group).
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Figure 6. Phenotypic characteristics of PM-susceptible line after silencing of Cm-mlo38 and Cm-mlo44 and their response to PM infection in melon. (A) Expression levels of Cm-mlo38 and Cm-mlo44 in VIGS-infected and WT plants; (B) resistance of WT, VIGS-mlo38, and VIGS-mlo44 plants to PM. Error bars indicate the mean values between three replicates ± standard deviation (SD). * denotes the statistically differences at p < 0.05 (Student’s t-test).
Figure 6. Phenotypic characteristics of PM-susceptible line after silencing of Cm-mlo38 and Cm-mlo44 and their response to PM infection in melon. (A) Expression levels of Cm-mlo38 and Cm-mlo44 in VIGS-infected and WT plants; (B) resistance of WT, VIGS-mlo38, and VIGS-mlo44 plants to PM. Error bars indicate the mean values between three replicates ± standard deviation (SD). * denotes the statistically differences at p < 0.05 (Student’s t-test).
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Figure 7. Subcellular localization of Cm-mlo38 and Cm-mlo44 in Nicotiana benthamiana leaves. Green fluorescence protein: green fluorescence refers to Cm-mlo38 and Cm-mlo44 positions. Bright: bright field; Merged: indicates merging of first two.
Figure 7. Subcellular localization of Cm-mlo38 and Cm-mlo44 in Nicotiana benthamiana leaves. Green fluorescence protein: green fluorescence refers to Cm-mlo38 and Cm-mlo44 positions. Bright: bright field; Merged: indicates merging of first two.
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Figure 8. The protein interaction networks of Cm-mlo38 and Cm-mlo44.
Figure 8. The protein interaction networks of Cm-mlo38 and Cm-mlo44.
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Table 1. Phylogenetic relationships of 202 Mlo genes in clades I–VI.
Table 1. Phylogenetic relationships of 202 Mlo genes in clades I–VI.
SpeciesCommon NameNumber of Mlo GenesClade
IIIIIIIVVVI
Cucurbita moschataPumpkin21/47253
Lagenaria sicerariaGourd14/34142
Benincasa hispidaWax gourd15/36132
Citrullus lanatusWatermelon15/35142
Cucumis hystrixWild cucumber16/36142
Cucumis sativusCucumber16/45142
Cucumis meloMelon22/46354
Cucurbita pepoSquash20/47243
Arabidopsis thalianaArabidopsis15/53133
Hordeum vulgareBarley1422//28
Oryza sativaRice1322//27
Zea maysMaize21241/68
Total 20264150134646
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Gong, F.; Lan, Y.; Zhang, T.; Li, C.; Li, Y.; Xia, F.; Liu, X.; Liu, D.; Liang, G.; Cai, P.; et al. Genome-Wide Analysis of Mlo Genes and Functional Characterization of Cm-mlo38 and Cm-mlo44 in Regulating Powdery Mildew Resistance in Melon. Horticulturae 2025, 11, 509. https://doi.org/10.3390/horticulturae11050509

AMA Style

Gong F, Lan Y, Zhang T, Li C, Li Y, Xia F, Liu X, Liu D, Liang G, Cai P, et al. Genome-Wide Analysis of Mlo Genes and Functional Characterization of Cm-mlo38 and Cm-mlo44 in Regulating Powdery Mildew Resistance in Melon. Horticulturae. 2025; 11(5):509. https://doi.org/10.3390/horticulturae11050509

Chicago/Turabian Style

Gong, Fangyi, Yanhong Lan, Tian Zhang, Chun Li, Yifan Li, Feng Xia, Xiaojun Liu, Duchen Liu, Genyun Liang, Peng Cai, and et al. 2025. "Genome-Wide Analysis of Mlo Genes and Functional Characterization of Cm-mlo38 and Cm-mlo44 in Regulating Powdery Mildew Resistance in Melon" Horticulturae 11, no. 5: 509. https://doi.org/10.3390/horticulturae11050509

APA Style

Gong, F., Lan, Y., Zhang, T., Li, C., Li, Y., Xia, F., Liu, X., Liu, D., Liang, G., Cai, P., & Fang, C. (2025). Genome-Wide Analysis of Mlo Genes and Functional Characterization of Cm-mlo38 and Cm-mlo44 in Regulating Powdery Mildew Resistance in Melon. Horticulturae, 11(5), 509. https://doi.org/10.3390/horticulturae11050509

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