Screening and Functional Analysis of Genes for Resistance to Powdery Mildew in Watermelon

Screening and Functional Analysis of Genes for Resistance to Powdery Mildew in Watermelon | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Screening and Functional Analysis of Genes for Resistance to Powdery Mildew in Watermelon Xixi Zhang, Shengqi Hua, Bo Liu, Jiale Shi, Dongyang Ran, Shengcan Hou, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9080378/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Powdery mildew (PM) is a major fungal disease that causes huge economic losses by impacting thousands of plant species, including Cucurbitaceae crops, such as watermelon ( Citrullus lanatus ). Although powdery mildew is caused by a widespread watermelon pathogen, the molecular mechanism remains unclear. In this study, one watermelon powdery mildew isolate was identified as Podosphaera xanthii . According to the distinct infections of powdery mildew on DuanMan (Middle resistance to PM) and Kexi (Susceptible to PM), fifteen ClaMLO genes expression level were detected, while only ClaMLO1 and ClaMLO2 were significantly regulated at different invasion period.. Bioinformatics analysis further showed that ClaMLO gene family were very conservative and ClaMLO2 , ClaMLO7 and ClaMLO13 were clustered in clade V. Subsequent subcellular localization experiments proved that ClaMLO1 localized in the cell nucleus, and silencing ClaMLO1 in watermelon enhanced resistance to powdery mildew. Generally, our study identified one gene, named ClaMLO1 . It may be responsible for the successful invasion of watermelon powdery mildew, laying a theoretical foundation for molecular breeding of watermelon. Watermelon Powdery mildew MLO Bioinformatics analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Watermelon is one of the largest cultivated members of the Cucurbitaceae family, showing great relevance from an economic point of view, and is largely consumed around the world. Watermelon production accounts for about 7% of the world’s area dedicated to the production of vegetables, with 67% of the total quantity produced in China (Mashilo et al. 2022 ). Powdery mildew is a worldwide disease caused by obligate biotrophic ascomycete fungi belonging to the order Erysiphales. The first report of sexual stage was discovered in 1981 on cucumber in Changchun province. Powdery mildew pathogens infect host leaves, stems, and petioles but rarely fruit(Agrios et al. 2004 ). These plant parts can be infected throughout the watermelon development period, and more serious infection occurs in the middle and late stages of growth(Glawe 2008 ). Podosphaera xanthii and Golovinomyce cichoracearum are the main pathogens causing melon powdery mildew in China(Lebeda et al. 2016 ;Liang Q L et al.2010;Bakhat N et al. 2023 ). Outbreaks of the powdery mildew pathogen lead to a significant reduction in crop yield. Breeding disease-resistant varieties is an effective way to solve the harm that powdery mildew causes to melon production(Keinath et al. 2004). During the long-term co-evolution of plants and pathogens, host plants overcome pathogen infection mainly through the internal immune system, including pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI and ETI are activated by different elicitors(Jones and Dangl 2006 ). PTI is triggered by pathogen-associated molecular patterns, whereas ETI is triggered by pathogen effector proteins, which can be recognized by plant resistance proteins coded by plant resistance genes. Plant resistance genes have the advantage of being monogenic and are easier to manage in breeding programs(Fu et al. 2009 ), but the physiological races of powdery mildew vary and mutate quickly. Cultivars become susceptible more quickly under high disease pressure and rapid changes in virulence within the pathogen population(Consonni et al. 2006 ). To deal with the challenge of powdery mildew physiological races on variety resistance, breeders need to identify genetic resources with broad-spectrum resistance to maintain the continuous resistance of cultivars to powdery mildew. Disease susceptibility is the greatest concern in developing quality crops(Pavan et al. 2010 ). Exploiting plant resistance is a necessary pathway in breeding. The Mildew Locus O (MLO) protein, which is a susceptibility gene, is considered the best model for studying broad-spectrum disease resistance to powdery mildew in plants(Acevedo-Garcia et al. 2014 ;Kusch and Panstruga 2017 ). Compared with the resistance gene, the plant susceptibility gene is another resistance gene that plays a negative regulatory role in disease resistance. The first mlo mutant resistance material of barley was generated in Hordeum vulgare variety “Haisa” using X-rays (Freisleben and Lein, 1942 ). In wheat, mutations in particular Mlo genes result in broad-spectrum powdery mildew resistance(Li et al. 2022 ;Panstruga 2005 ). The MLO family has also been isolated in Arabidopsis thaliana , Triticum aestivum( Konishi et al. 2010 ), Oryza sativa (Liu and Zhu 2008 ), and other plants. In Arabidopsis, AtMlo2 , AtMlo6 , and AtMlo12 triple mutants confer full resistance because of genetic redundancy. Mutations of the BnMLO6 gene can stimulate the spontaneous accumulation of callose in leaves and activate ethylene and jasmonic acid transduction pathways, conferring resistance to powdery mildew under both field and greenhouse conditions (SHI et al. 2022 ). In pea, loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance(Pavan et al. 2011 ). The MLO gene obtained through spontaneous or artificial mutation confers broad-spectrum resistance to powdery mildew, indicating the potential application value of MLO in the breeding of plants resistant to powdery mildew. Most MLO protein commonly contain calmodulin-binding domain and transmembrane domain, but the number of transmembrane domain varies between lower and higher plants. MLO proteins with fewer than five transmembrane domains are typically found in lower plants, while MLO proteins with four to eight transmembrane domains are usually found in higher plants(Devoto et al. 2003 ). Their topological structure and subcellular localization are quite similar to G protein-coupled receptors in animals(Temple and Jones 2007 ). MLOs exist in various tissues of plants and are mainly involved in vegetative and reproductive growth and biotic and abiotic stress responses (Howlader et al. 2017 ). The expression of MLO genes increases in barley during infection by powdery mildew and mechanical damage(Piffanelli et al. 2002 ). Analysis of stress-induced expression revealed that five CmMLO genes were upregulated at 6 h after inoculation with Podosphaera xanthii Race 1 or DH487, indicating the possible role of MLO proteins in the host cell as an initial step in disease progress. Moreover, strong upregulation was observed for ClMLO12 upon powdery mildew infection, suggesting that it is a pathogen-responsive gene((Iovieno et al. 2015 ). Expression of the CaMLO2 gene in pepper is induced by abscisic acid and drought stress (Lim and Lee 2014 ). There are 16 MLO homologs in melon ( Cucumis melo ), 15 in watermelon ( Citrullus lanatus ), and 18 in zucchini ( Cucurbita pepo ) (Zhang et al. 2018 ). Among them, three melon, three watermelon, and four zucchini MLO homologs have been found to cluster together with the dicot isoforms functionally associated with powdery mildew susceptibility. However, sufficient information is not available to understand the molecular mechanism during the watermelon–powdery mildew interaction. In this study, by using the powdery mildew identified from the the experimental farm of the Henan University Genetics and Breeding Base in Kaifeng, we investigated the disease resistance of different watermelon cultivars, KeXi and DuanMan. Then, based on the available watermelon genomic database, we studied the characteristics of ClaMLO gene family. Further investigation of expression pattern of ClaMLOs help us locate at ClaMLO1 and ClaMLO2 , so we focus on exploring the function of ClaMLO1 which was localized in cell nucleus, and the genetic transformation of ClaMLO1 showed enhanced resistance to powdery mildew. 2. Materials and Methods 2.1 Watermelon cultivars and growth conditions ‘KeXi’and ‘DuanMan’ were grown at the experimental farm of the Henan University Genetics and Breeding Base in Kaifeng, China. We strictly controlled the watermelon growth conditions, with all plants cultivated under long-day conditions and examined in the greenhouse at the Henan University Genetic Breeding Base. The temperature was maintained at 25–30℃ during the day and at 15–18℃ at night. All watermelon seeds were stored in a seed storage cabinet at 4℃. 2.2 Purification and identification of watermelon powdery mildew The powdery mildew diseased leaf collected from the greenhouse of Henan University were washed with sterile water to remove surface dust, and then inoculated onto watermelon leaves by friction method and placed in incubator for propagation and preservation. For pathogen purification, referring to existing research methods, single-spore isolation technology was used to obtain pure single-spore cultures of watermelon powdery mildew.. When the conidia of watermelon powdery mildew were collected and placed on a glass slide, 1–2 drops of 3% potassium hydroxide solution were added to observe the fibrous bodies of the conidia with an optical microscope. Those with fibrous bodies are Podosphaera xanthii , and those without fibrous bodies are Golovinomyce cichoracearum. 2.3 Coomassie brilliant blue staining and microscopic observation The inoculated watermelon leaves were decolorized with decolorizing solution (alcohol: acetic acid = 3V: 1V) at 45℃ for 2 h and then soaked in 0.6% Coomassie brilliant blue dye solution (0.6 g R250/100 ml) for 2 min. The dyed leaves were first placed in clean water to remove the floating color of the dye solution. Then, the leaves were placed on a glass slide, and 1–2 drops of 50% glycerol were added and covered with the cover slide. The slides were observed under a microscope. 2.4 Identification of disease resistance with different watermelon cultivars Disease tests were performed by spraying plants with a suspension of powdery mildew conidiospores. Heavily sporulating leaves were used to release conidia and immediately placed in a 0.02% Tween 20 solution. Watermelon plants at the 2–3 true leaf stage were inoculated with an inoculum solution of 2×10 5 spores per milliliter. The spore count was calculated by the direct microscopic counting method.The leaves of infected plants were sampled randomly at 4, 8, 12, 24, 48, and 72 hpi for microscope observation and nucleic acid extraction. Symptom investigation was carried out till host get sick entirely after 10–15 days post-inoculation. The severity of the disease was divided into 6 levels. Level 0: No lesions on the plants; Level 1: White powdery lesions appear on a few of leaves, with the lesion area accounting for less than 5% of the total area, and the stems have no lesions; Level 3: A little of lesions appear on the leaves, accounting for 6% to 25% of the total area, and the stems have scattered lesions; Level 5: Moderate amounts of white powdery lesions appear on the leaves, accounting for 26% to 50% of the total area, and the stems have a little of lesions; Level 7: A number of white powdery lesions appear on the leaves, accounting for 51% to 75% of the total area, and the main stem has a little of lesions; Level 9: Many powdery mildew lesions appear on the leaves and the white powder is thick, accounting for 76% to 100% of the total area, and the stems have much more lesions. 2.5 ITS sequence and phylogenetic analysis The genomic DNA of watermelon powdery mildew was extracted from infected leaves using a MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0 (TaKaRa, Dalian, China). Fungal ribosome universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) were synthesized by Service Biotechnology Co., Ltd. Polymerase chain reactions were carried out using an S1000TM Thermal Cycler in 20 µL volume, including 1 µL of DNA template, 1 µL of forward and reverse primers, 2 µL of 10×PCR buffer (Mg 2+ ), and 15 µL of sterile deionized water. The PCR procedure was as follows: 98℃ for 5 min, 35 cycles of 94℃ for 30 s, 58℃ for 45 s, and 72℃ for 30 s, followed by a final extension at 72℃ for 10 min. PCR products were separated on 1.5% agarose and sequenced using the Beijing Genomics Institute. The MEGA 11.0 software package was used for phylogenetic analysis of the ITS sequence (Tamura et al. 2021 ). First, multiple sequence alignment (generated by CLUSTALW) was established and manually optimized. Then, a distance matrix was calculated using PROTDIST from the multiple sequence alignment and was then transformed into a tree using the neighbor-joining method. All programs were run with standard parameters. 2.6 RNA isolation and real-time quantitative reverse-transcription PCR To evaluate ClaMLO gene family transcript accumulation, total RNA was extracted from infected watermelon leaves sampled randomly at 4, 8, 12, 24, 48, and 72 hours post inoculation using a MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China), and cDNA was synthesized from 1 µg total RNA using a PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The nucleotide sequences of the primer pairs used for the amplifications are listed in Table S1. Amplification was performed using a TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Dalian, China) and Roche LightCycler®480 II System under the following program: 95℃ for 30 s, 40 cycles of 95℃ for 5 s and 60℃ for 34 s, followed by 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 15 s. The amplication efficiencies of qPCR assays ranged from 95% to 110%, and R 2 value for each calibration curves exceeded 0.98. The Ct was used to calculated the copy numbers of all above mentioned genes. For each time point, the relative expression levels were normalized with respect to the housekeeping gene β-Actin and compared to the non-inoculated control. Standard error bars refer to three biological replicates. Significant differences between the means were inferred using the Student’s t test for each time point (*P < 0.05). Relative transcript levels were calculated based on the 2 -∆∆Ct method (Livak, 2001 ). 2.7 Conserved motif and phylogenetic analysis of the MLO gene family Gene sequences of the watermelon MLO family were obtained from the cucurbit genomics official website ( http://www.cucurbitgenomics.org/ ). The conserved motifs of the watermelon MLO proteins were analyzed using Multiple Em for Motif Elicitation (MEME) online tools ( http://meme.nbcr.net/meme/cgi-bin/meme.cgi ) (Bailey et al. 2015 ). Motifs and CDS were visualized using the Gene Structure View (Advanced) of Tbtools (Chen et al. 2020 ). The parameters were set as follows: the minimum length of the conserved motif was 15; the maximum length was 40; and the maximum number of conserved motifs was 10. The other parameters were the default values. The phylogenetic tree construction of the watermelon MLO gene family and the complete Arabidopsis thaliana MLO protein family with other identified powdery mildew susceptibility genes, including SlMLO1 from Solanum lycopersicum, CaMLO2 from Capsicum annuum, PsMLO1 from Pisum sativum, LjMLO1 from Lotus japonicus, HvMLO from Hordeum vulgare, OsMLO1 from Oryza sativa, and NtMLO1 and NtMLO2 from Nicotiana tabacum, were consistent with the method given in Section 2.5 . 2.8 Subcellular localization of ClaMLO1 The subcellular localization of ClaMLO1 in onion epiderma was assessed using a plant expression vector GFP1300 containing an eGFP gene, and the full-length ClaMLO1 was fused to the N-terminus of eGFP in the SalI digestion sites. All constructs were transformed into A. tumefaciens GV3101 using the freeze–thaw method and infiltrated into the Allium cepa epidermis. The empty GFP1300 vector was used as a control. At 24 h post-infiltration, the Allium cepa epidermis was imaged using confocal laser scanning microscopy. 2.9 Watermelon transformation using Agrobacterium-mediated expression As previously described, the genetic transformation was carried out with a few minor modifications (Dong et al. 2021 ). Watermelon seeds were sown on a 0.6% agar solid medium for 2 days in the dark after being surface sterilized. Then, the cotyledons with growth points were cut as explants and infected with A. tumefaciens. Agrobacterium tumefaciens strains harboring the indicated binary vectors were co-cultivated with the cotyledon fragments in the dark for 3 days on MS solid medium containing 1 mg/L 6-BA. The cotyledon fragments were then transferred onto a selective induction medium containing 1 mg/L 6-BA, 100 mg/L Kan, and 250 mg/L Cephalosporin. The regenerated adventitious buds were excised and transferred onto an elongation medium containing 0.2 mg/L KT, 250 mg/L Cephalosporin, and 50 mg/L Kan. The elongation plants were transferred to a rooting medium containing 2 mg/L IBA and 100 mg/L Cephalosporin. Positive transgenic plants were detected using PCR. 3. Results 3.1 Morphological identification of powdery mildew on watermelon In recent years, watermelon was severely infected by powdery mildew in the greenhouse of the College of Life Sciences, Henan University. The disease primarily occurred on the leaves. At the beginning of the disease, the infected position was covered with white powder and gradually turned yellow with brown lesions. Black speckles emerged from the infected part during powdery mildew development in the later period (Fig. 1 A). The conidia of watermelon powdery mildew collected from the watermelon plant had obvious fibrous bodies (Fig. 1 B). After staining with Coomassie Brilliant Blue R250, the conidia were observed to be elliptical and clustered on the conidial stem. The number of conidia varied, ranging from 5 to 12 (Fig. 1 C). According to the morphological features, the pathogen was initially confirmed as Podosphaera xanthii (Kiss L. 2002 ). 3.2 Genetic analysis of ITS sequences for watermelon powdery mildew The ITS fragment of the pathogen was amplified using the DNA of watermelon powdery mildew as a template and the universal primers ITS1 and ITS4 of the fungal ribosomal ITS region as the primers. DNA sequencing showed that the ITS fragment length was 671 bp. Further molecular phylogeny between the ITS regions from watermelon and melon, cucumber, zucchini, adzuki bean, abelmosk, eggplant which blasted from the NCBI database was constructed. This tree was divided into three clades, but all sequences in the three clades were attributed to Podosphaera xanthii (Fig. 2 ). This analysis clearly supported the morphological observation that the powdery mildew fungus on watermelon found at Henan University belonged to Podosphaera xanthii . 3.3 Different watermelon lines display different response to powdery mildew infection In order to compare the characteristic disease symptoms of powdery mildew between KeXi and DuanMan watermelon lines, we observed the invasion procedure from conidia germination to secondary hyphal development, identified the disease resistance of KeXi and DuanMan. In the leaves of KeXi, the majority of conidia germinated producing primary germinal tubes at 12 hpi, growing new mycelium and continuously elongating between 24 and 48 hpi, and forming secondary hyphae at 72 hpi. Compared to KeXi, DuanMan showed serious retardation in conidial germination and hyphae growth and development(Fig. 3A). In addition, at 15 days post-inoculation, DuanMan still exhibited strong resistance to powdery mildew, but abundant development of conidia and severe powdery mildew pathogen symptoms were observed on KeXi(Fig. 3B). Generally, KeXi and DuanMan responded distinctly to powdery mildew infection. According to the disease index survey, DuanMan was identified as medium resistance type, KeXi was identified as susceptible type (Table 1 ). Table 1 The resistance identified result of powdery mildew of KeXi and DuanMan Cultivars Disease index Disease rating range Resistance evaluation KeXi 64.25 3–9 S DuanMan 42.86 1–9 MR 3.4 Prediction of conserved motifs and CDS with phylogenetic analysis of ClaMLO gene family MLO gene family act as susceptibility factors having an indispensable role in the occurrence of powdery mildew. To further explore the molecular mechanism of resistance to powdery mildew in different watermelon lines, KeXi and DuanMan. we utilized latest available genomic information to conduct bioinformatics analysis of the MLO gene family in watermelon. 15 predicted proteins labeled MLO like protein were matched through BLASTp analysis, and they were named from ClaMLO1 to ClaMLO15 . The number of transmembrane helices of ClaMLO gene family vary from 3—8, and ClaMLO1 , ClaMLO4 , ClaMLO5 , ClaMLO7 , ClaMLO11 , ClaMLO12 , ClaMLO13 have seven transmembrane helices (Table 2 ). The motifs of the ClaMLO gene family were predicted by MEME, and a phylogenetic tree was built with MEGA 11.0 software. We integrated CDS, motifs, and the tree using TBtools. The ClaMLO proteins had 10 conservative motifs, and all of them had motifs 1 and 5 (Fig. 4 A). Most of the families had more than eight motifs. The CDS of the gene family was distributed in the range of 0–9000 bp. These results showed that ClaMLO gene family were conservative. To study the evolution and development of ClaMLO gene family with the complete Arabidopsis thaliana MLO gene family and other identified powdery mildew susceptibility genes from different species, 23 MLO proteins were clustered in six distinct clades. Clade numbers were organized by the position of Arabidopsis AtMLO homologs and barley HvMLO (Feechan et al. 2008 ). ClaMLO2, ClaMLO7 and ClaMLO13 were clustered in clade V, which contained all MLO proteins functionally related to powdery mildew susceptibility in dicot plants. Other ClaMLOs were distributed in different clades(Fig. 4 B). Table 2 Features of the watermelon MLO gene family MLO name Locus name Chromosome Starting position(kb) Clade Introns Length(aa) Number of transmembrane helices ClaMLO1 Cla97C09G180170 9 33811195 Ⅰ 14 553 7 ClaMLO2 Cla97C05G102540 5 30786649 Ⅴ 12 508 6 ClaMLO3 Cla97C05G091880 5 9890612 Ⅰ 14 562 5 ClaMLO4 Cla97C11G224240 11 29955333 Ⅰ 14 568 7 ClaMLO5 Cla97C06G109800 6 495518 Ⅱ 12 516 7 ClaMLO6 Cla97C03G053300 3 2480883 Ⅵ 14 580 6 ClaMLO7 Cla97C02G047070 2 34811774 Ⅴ 13 600 7 ClaMLO8 Cla97C02G036300 2 17697506 Ⅳ 13 540 8 ClaMLO9 Cla97C02G026560 2 344852 Ⅱ 7 255 3 ClaMLO10 Cla97C01G020510 1 33128842 Ⅲ 14 571 8 ClaMLO11 Cla97C10G184670 10 189923 Ⅲ 14 578 7 ClaMLO12 Cla97C08G152090 8 20446868 Ⅲ 14 544 7 ClaMLO13 Cla97C03G053280 3 2425953 Ⅴ 13 582 7 ClaMLO14 Cla97C01G023190 1 34727024 Ⅳ 12 400 6 ClaMLO15 Cla97C10G185230 10 644865 Ⅰ 14 539 6 3.5 Transcription of the ClaMLO gene family in response to powdery mildew inoculation The significantly enhanced resistance of different species to powdery mildew is related to the up-regulated expression of MLO susceptibility genes. To further investigate the expression pattern of ClaMLO gene family, 15 ClaMLO genes were subjected to an assessment of their expression levels after inoculating KeXi and DuanMan with the conidia of powdery mildew. Quantification by real-time quantitative reverse-transcription PCR showed that the expression levels of ClaMLO1 and ClaMLO2 were obviously upregulated in DuanMan at 12 hpi, whereas as the inoculation time was prolonged, the expression levels of ClaMLO1 and ClaMLO2 decreased gradually. Compared with DuanMan, the expression levels of ClaMLO1 and ClaMLO2 increased continuously and reached a maximum at 48 hpi in KeXi (Fig. 5 ). These results revealed that ClaMLO1 and ClaMLO2 have different expression pattern in powdery mildew susceptible lines or resistant lines. Significantly, they may likely involved in the infection process of powdery mildew on watermelon. Unfortunately, other ClaMLO genes, except ClaMLO1 and ClaMLO2 , showed no significant difference during different invasion times, and ClaMLO7 , ClaMLO9 , and ClaMLO12 were not detected. 3.6 Subcellular localization of ClaMLO1 localized in the cell nucleus of Onion epiderma The subcellular localization of ClaMLO1 was confirmed using a transient onion expression system. ClaMLO1 was fused to the N-terminus of an enhanced green fluorescent protein gene within a GFP1300 vector and transformed into an onion expression cell by Agrobacterium tumefaciens . As visualized using fluorescence microscopy, green fluorescence accumulated in the cell nucleus of onion epiderma in the ClaMLO1-GFP1300 transformants(Fig. 6 ). In contrast, green fluorescence was uniformly distributed in the cytoplasm of the empty vector without the target gene. This suggests that ClaMLO1 is localized in the cell nucleus of onion epiderma. 3.7 Genetic transformation of ClaMLO1 in watermelon To further investigate the function of ClaMLO1 in the watermelon–powdery mildew interaction, we silenced ClaMLO1 in watermelon. In the process, we used the Agrobacterium -mediated watermelon cotyledons system, where callus tissue was differentiated from the edge of the cotyledons after two to four weeks of cultivation (Figs. 8 A-B). After 2–3 weeks of callus culture, resistant buds began to grow, and the stems elongated and thickened. The number of leaves increased, and the leaves became larger (Figs. 8 C-D). When the watermelon seedlings reached a height of 5–6 cm, the bottle cap was opened, and the seedlings were refined for 24 h. The watermelon seedlings were removed, and the agar attached to the roots was washed with water. The seedlings were transplanted into a pot filled with nutrient soil. Watermelons set fruit after pollination and matured for about 30 days (Fig. 8 E). 3.8 Silencing ClaMLO1 in watermelon enhanced resistance to powdery mildew To examined the disease resistance of ClaMLO1 silencing plants, we carried out powdery mildew inoculation assay. At the 2–3 true leaf stage, ClaMLO1 silencing plants and control plants were inoculated with powdery mildew spore suspension. Notably, at 10 days post-inoculation, the leaves of ClaMLO1 silencing plants keep green and normal. However, the leaves of control plants turns yellow and distributed with white powder (Fig. 8 A). so we also detected the expression level of ClaMLO gene family. In ClaMLO1 -silenced plants, ClaMLO1 and ClaMLO2 both have a significant increase at 12 hours post-inoculation, and they were greatly up-regulated in KeXi at 48 hours post-inoculation. These result further proved that ClaMLO1 may be a susceptible gene, having an important role in helping powdery mildew invasion. 4. Discussion Watermelon is a Cucurbitaceae crop that has sweet pulp, a cool and refreshing taste, is rich in vitamin C and various minerals, and has the effect of relieving heat and thirst. In addition to being used for food, it can also be made into a variety of drugs and processed products. According to the latest data from the Food and Agriculture Organization of the United Nations, watermelon is one of the most important fruits and vegetables in the world. Powdery mildew is a major fungal disease of thousands of plant species, including cultivated watermelon, which can limit plant photosynthesis due to whitish conidial growth on cotyledons, true leaves, leaf petioles, and stems, thus leading to a severe loss of crop quality and yield (Vielba-Fernández, 2020). At present, fungicides and resistant varieties are the main methods for controlling powdery mildew (Lebeda and Sedlakov, 2010 ). However, fungicides are expensive and harmful for the environment. Cultivars resistant to powdery mildew are fundamental to reducing the use of pesticides in agricultural practices (Zhang et al. 2011 ). The identification of powdery mildew fungi to species can be based on fungal morphology and ITS sequences (Liang et al.2013;Tang et al.2017). In powdery mildew, the internal transcribed spacer sequences (ITS) are the single available barcode for species identification (Takamatsu et al.2015). In this study, the powdery mildew of watermelon cultivars in Henan Province was evaluated. Microscope examination revealed that the fungus produced typical structures of powdery mildew conidia with fibrous bodies. This is consistent with the results of a previous taxonomic manual of Erysiphales (Branu and Cook, 2012). Since powdery mildew in tropical/subtropical environments rarely develop sexual stages with morphologically diagnostic characteristics, ITS regions of the ribosomal RNA genes obtained from the asexual stages have become an important modern tool for species identification (Yeh et al. 2021 ). To confirm the identity, the obtained 671-bp ITS sequence was blasted on the NCBI website. The subsequent phylogeny revealed that the ITS sequence was 100% identical to many records of Podosphaera xanthii . Based on both morphological and phylogenetic characteristics, the powdery mildew pathogen in watermelon was identified as Podosphaera xanthii . In addition, the Glandularia tenera powdery mildew pathogen has also been identified as Podosphaera xanthii (Pei et al, 2022 ), and mung bean planted in Taiwan can also be infected by Podosphaera xanthii , which causes severe powdery mildew disease (Sheu et al, 2021 ). Previous phylogenetic studies classified plant MLO proteins into six clades (I–VI). The available scientific literature indicates that the proteins of IV and V clades have a significant effect on powdery mildew susceptibility in monocots and dicots, respectively (Win et al,2018; Kusch et al, 2016 ). Moreover, powdery mildew pathogenesis is associated with the upregulation of MLO genes during the early stages of infection, causing the downregulation of plant defense pathways. RhMLO1 and RhMLO2 are upregulated in the rose–powdery mildew pathogen interaction (Qiu et al, 2015 ). Furthermore, specific members of the MLO gene family act as powdery mildew-susceptibility genes, as their loss-of-function mutations grant durable and broad-spectrum resistance (Pessina et al,2014;Humphry et al,2011). In watermelon, three MLO homologs, namely ClaMLO2 , ClaMLO7 and ClaMLO13 clustered together in phylogenetic clade V, indicating that they are candidates for powdery mildew susceptibility genes in watermelon. However, in our study, we also detected one upregulated gene, ClaMLO1 , after inoculation with watermelon powdery mildew, which was classified into clade I, indicating that the simple clustering in clade V is poor in recognizing a gene as a powdery mildew-susceptibility factor. A similar result has been detected in apple; a newly identified MLO gene classified in clade VII showed upregulation after inoculation with the pathogen. Furthermore, ClaMLO2 , which encodes an MLO protein in clade V, was also upregulated after powdery mildew infection, but other MLO proteins showed no significant changes in expression. Generally, we found two ClaMLO genes that may play an important role in the interaction between powdery mildew and watermelon, and they can be used as targets for functional characterization of the investigation on watermelon resistance breeding. By performing multiple alignments of watermelon MLO proteins, we found that they were very conserved in amino acid residues and motifs. This result is consistent with the occurrence of 10 conserved motifs in tomato MLO proteins and grape (Islam and Yun,2016). The existence of these conserved motifs may be related to the function of the MLO protein. At present, many MLO gene families have been found in different plants, but the function of most watermelon MLO proteins remains unknown, especially in watermelon. To solve this problem, we investigated the subcellular localization of watermelon MLO proteins. In 1999, a barley MLO protein was speculated to be localized on the cytoplasmic membrane [(Devoto et al,1999). Many studies have carried out subcellular localization of MLO proteins. For example, most MLO proteins of Rosaceae plants are distributed in the plasma membrane, followed by the endoplasmic reticulum and vacuoles, and a few are distributed in the nucleus, mitochondria, and Golgi apparatus (Xiang et al,2018). Based on the remarkable up-regulation expression levels of ClaMLO1 during powdery mildew infection process, we focused on exploring the characteristics of ClaMLO1. In this study, ClaMLO1 was primarily distributed in the cell nucleus, as shown using a subcellular localization assay. The MLO protein was detected not only around epidermal cells but also in the inner membrane and nuclear membrane in Mlo mutants without powdery mildew infection. However, after being infected with powdery mildew, it was redistributed within the plant cells, indicating the subcellular localization of the MLO protein plays an important role in the life activities of plants(Bhat et al,2005). In addition, the MLO function in pollen tube reception is dependent on MLO protein localization in Golgi (Jones et al, 2017 ). The subcelluar localization of ClaMLO1 may be greatly related to the celluar signal transduction pathway that regulate powdery mildew reponse, further disease resistance assay also prove that ClaMLO1 -silenced watermelon exhibit more resistant to powdery mildew. 5. Conclusions In this study, we identified one watermelon powdery mildew isolate belonging to Podosphaera xanthii . Microscope observation of the invasion process and disease resistance identification suggested that DuanMan displayed stronger resistance than KeXi. Further bioinformatics analysis demonstrated the molecular characteristics of ClaMLO gene family, including motif composition, numbers of transmembrane helix, phylogenetic relationships, and the transcription expression pattern. We found that ClaMLO1 localized in cell nucleus, and silencing ClaMLO1 in watermelon enhanced resistance to powdery mildew, indicating that ClaMLO1 may play an important role in the interaction between watermelon and powdery mildew. Hence, our experiments provided a direction for studying the molecular mechanism of watermelon breeding. Declarations Acknowledgements This research was funded by the National Natural Science Foundation of China (31801882) and Henan Province Key Science and Technology Projects (212102110044). Author ’s Contributions Xixi Zhang wrote original manuscript. Xixi Zhang, Shengqi Hua and Dongyang Ran performed most of the experiments. Shengqi Hua, Jiale Shi and Shengcan Hou helped data analysis. Wei Dong, Bo Liu and Junhua Li supervised spoken and written language. All authors have read and agreed to the published version of the manuscript. Conflict of interest The authors declare that they have no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9080378","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":625492559,"identity":"b4b5639f-0cc8-4cb9-82f6-f7786025159e","order_by":0,"name":"Xixi Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYDACCSjJxsB8/EOCgQ0PP38D0VrY0hgeVKTJSM44QJQWEOAxY3xw5rCNQUMCfh3ys5ufPfzaZpHYB9TyILHtPI8BwwHGDx9zcGthnHPM3FjmjIQx0GHlBoltt3nMmRuYJWduw62FWSLBTFqiQkIO6P0NEiAtlg0H2Jh58Whhk0j/Ji1hIMHDxsBgANRyjsfgQAJ+LTwSOWaSH8C2sJhJJJw5QFiLhEROmTQDxC/JBgkVyTySMw424/WL/Iz0bZI/2+oS5zcwH3z4w8DOnp+/+eCHj3i0gIOAB6z5AYzP2IBfPUjJD4JKRsEoGAWjYEQDANP7SSAlFjYKAAAAAElFTkSuQmCC","orcid":"","institution":"Henan University","correspondingAuthor":true,"prefix":"","firstName":"Xixi","middleName":"","lastName":"Zhang","suffix":""},{"id":625492560,"identity":"17a8107e-06d3-4e68-b1d4-e4fab09288c9","order_by":1,"name":"Shengqi Hua","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shengqi","middleName":"","lastName":"Hua","suffix":""},{"id":625492561,"identity":"20c30ef1-d5ee-4743-9ec1-4625d4d9af38","order_by":2,"name":"Bo Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Liu","suffix":""},{"id":625492562,"identity":"04e34ce7-432d-4fd3-a1b7-5f6c1fc54564","order_by":3,"name":"Jiale Shi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiale","middleName":"","lastName":"Shi","suffix":""},{"id":625492563,"identity":"c5133e4b-0bc4-4939-85bd-23cade2d60f0","order_by":4,"name":"Dongyang Ran","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dongyang","middleName":"","lastName":"Ran","suffix":""},{"id":625492564,"identity":"a014240b-dc5b-4cd0-a9d7-48d98afe5645","order_by":5,"name":"Shengcan Hou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shengcan","middleName":"","lastName":"Hou","suffix":""},{"id":625492565,"identity":"cb150428-ffcd-49b0-8beb-c9f9c92d7f40","order_by":6,"name":"Junhua Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Junhua","middleName":"","lastName":"Li","suffix":""},{"id":625492566,"identity":"eaa9e9a7-0962-4a6e-a388-993e4f7ac6af","order_by":7,"name":"Wei Dong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2026-03-10 07:12:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9080378/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9080378/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107913763,"identity":"911f3f88-062d-4545-85c0-6c6778a7cfc6","added_by":"auto","created_at":"2026-04-27 13:51:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":773662,"visible":true,"origin":"","legend":"\u003cp\u003eDisease symptoms and morphological characteristics of the powdery mildew fungus on watermelon. \u003cstrong\u003ea\u003c/strong\u003e Symptoms of powdery mildew on watermelon in the greenhouse of the College of Life Sciences, Henan University. \u003cstrong\u003eb\u003c/strong\u003e Powdery mildew fungus conidia with well-developed fibrosin bodies by immersion in 3% potassium hydroxide solution. \u003cstrong\u003ec\u003c/strong\u003e Microscopic micrograph of conidiophore stained with Coomassie brilliant blue R250 at 5 days post-inoculation. Scale bar = 20 μm\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9080378/v1/dbc98b912fafe04b554b16d7.png"},{"id":107913764,"identity":"ec029963-2892-4101-93bc-0258f6707f76","added_by":"auto","created_at":"2026-04-27 13:51:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":231206,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of \u003cem\u003ePodosphaera xanthii\u003c/em\u003eon watermelon. The evolutionary relationships of \u003cem\u003ePodosphaera xanthii\u003c/em\u003e on watermelon and other powdery mildew on melon (\u003cem\u003eCucumis melo\u003c/em\u003e), cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e), zucchini (\u003cem\u003eCucurbita pepo\u003c/em\u003e), adzuki bean (\u003cem\u003eVigna angularis\u003c/em\u003e), abelmosk (\u003cem\u003eAbelmoschus esculentus\u003c/em\u003e), and eggplant (\u003cem\u003eSolanum melongena\u003c/em\u003e) were determined using the maximum-likelihood algorithm. The statistical strength was assessed by bootstrap with 1000 replicates\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9080378/v1/d3d3f82590b1d49f457fb1c6.png"},{"id":107913765,"identity":"d1840e07-91b0-4088-ad0e-b643397b97d8","added_by":"auto","created_at":"2026-04-27 13:51:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":281915,"visible":true,"origin":"","legend":"\u003cp\u003eTime course of the powdery mildew infection process in KeXi and DuanMan watermelon lines. \u003cstrong\u003ea \u003c/strong\u003eCoomassie brilliant bluestained hyphae collected from watermelon lines, KeXi and DuanMan at 4, 8, 12, 24, 48, and 72 hpi with the powdery mildew fungus, scale bar = 50 μm. \u003cstrong\u003eb\u003c/strong\u003e Exemplary infection phenotypes upon powdery mildew challenge of 4–6 week old watermelon lines between DuanMan and KeXi at 15 days post-inoculation\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9080378/v1/b5f1cf249b9844ecb514f27f.png"},{"id":107913770,"identity":"2953a785-009a-4a5e-9931-cb9285fea1ee","added_by":"auto","created_at":"2026-04-27 13:51:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":170497,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatics analysis of the watermelon \u003cem\u003eClaMLO\u003c/em\u003egene family. \u003cstrong\u003ea\u003c/strong\u003e Phylogenetic and conservative motif composition of the\u003cem\u003e ClaMLO\u003c/em\u003egene family. Multiple sequence alignment of amino acids of ClaMLO proteins was used to construct the phylogenetic tree with MEGA 11.0 software. Conservative motif composition of the \u003cem\u003eClaMLO\u003c/em\u003egene family was determined using the MEME suite. \u003cstrong\u003eb\u003c/strong\u003e Phylogenetic analysis of the MLO homologs in different species. The neighbor-joining phylogenetic tree of \u003cem\u003eCitrullus lanatus\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and selected proteins that have been characterized as susceptibility genes from barley (\u003cem\u003eHordeum vulgare\u003c/em\u003e), rice (\u003cem\u003eOryza sativa\u003c/em\u003e), tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e), pepper (\u003cem\u003eCapsicum annuum\u003c/em\u003e), pea (\u003cem\u003ePisum sativum\u003c/em\u003e), and tobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e) are shown\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9080378/v1/925cfd1900ad07d84fd24e73.png"},{"id":108007497,"identity":"25f7fad6-2e01-4ec5-af3e-fba6bb70de39","added_by":"auto","created_at":"2026-04-28 13:00:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":194456,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional variation of \u003cem\u003eClaMLO\u003c/em\u003e gene family in response to powdery mildew infection. Data refer to leaves of the watermelon KeXi and DuanMan, sampled at five different time points 4, 8, 12, 24, and 48 hpi, with the powdery mildew fungus \u003cem\u003ePodosphaera xanthii\u003c/em\u003e. For each time point, the relative expression levels were normalized with respect to the housekeeping gene β-Actin and compared to the non-inoculated control. Standard error bars refer to three biological replicates. Significant differences between the means were inferred using the Student’s t test for each time point (*P \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9080378/v1/74c993f79c091d80a0291ec2.png"},{"id":107913767,"identity":"76efde44-a827-402a-804c-c219b5c20576","added_by":"auto","created_at":"2026-04-27 13:51:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":402998,"visible":true,"origin":"","legend":"\u003cp\u003eLocalization analysis of ClaMLO1 in the onion epiderma. Localization of GFP1300 and fusion protein ClaMLO1-GFP1300 in the cytoplasm and cell nucleus of onion epiderma observed under fluorescence microscopy. Scale bar= 100 μm\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9080378/v1/8e430b8fd070be6093e6e81c.png"},{"id":107913768,"identity":"2dfc82a2-33df-42b6-83ea-7e49507c7e8e","added_by":"auto","created_at":"2026-04-27 13:51:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":270988,"visible":true,"origin":"","legend":"\u003cp\u003eTransgenic watermelon seedlings constructed using the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003emethod. \u003cstrong\u003ea\u003c/strong\u003e Germination of watermelon seed; \u003cstrong\u003eb\u003c/strong\u003e Callus initiation; \u003cstrong\u003ec\u003c/strong\u003e Callus beginning to take root, differentiate, and sprout; \u003cstrong\u003ed\u003c/strong\u003e Formation of transgenic watermelon seedlings; \u003cstrong\u003ee\u003c/strong\u003e Transplantation and harvesting of transgenic watermelon seedlings\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9080378/v1/6acc33ace99ad15f77a832c9.png"},{"id":108006679,"identity":"95ea4c60-dc8d-4a12-89a0-ff5c838d915b","added_by":"auto","created_at":"2026-04-28 12:56:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":208762,"visible":true,"origin":"","legend":"\u003cp\u003eInfection assay of \u003cem\u003eClaMLO1\u003c/em\u003e-silenced plants. \u003cstrong\u003ea\u003c/strong\u003e Phenotype of \u003cem\u003eClaMLO1\u003c/em\u003e-silenced plants and control watermelon plants. Photographs were taken 10 days after inoculated with powdery mildew. \u003cstrong\u003eb \u003c/strong\u003eTranscriptional variation of\u003cem\u003e ClaMLO\u003c/em\u003e gene family in response to powdery mildew infection. Data refer to leaves of the control plants KeXi and \u003cem\u003eClaMLO1\u003c/em\u003e-silenced plants, sampled at five different time points 4, 8, 12, 24, and 48 hpi, with the powdery mildew fungus \u003cem\u003ePodosphaera xanthii\u003c/em\u003e. For each time point, the relative expression levels were normalized with respect to the housekeeping gene β-Actin and compared to the non-inoculated control. Standard error bars refer to three biological replicates. Significant differences between the means were inferred using the Student’s t test for each time point (*P \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9080378/v1/6c8f7b74ad4e12ea27a18005.png"},{"id":109318668,"identity":"998b638a-585a-4656-a89f-152578012eb2","added_by":"auto","created_at":"2026-05-15 13:05:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3241067,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9080378/v1/813f6ca7-e777-4ccc-98e6-c31a3f612b41.pdf"}],"financialInterests":"","formattedTitle":"Screening and Functional Analysis of Genes for Resistance to Powdery Mildew in Watermelon","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWatermelon is one of the largest cultivated members of the Cucurbitaceae family, showing great relevance from an economic point of view, and is largely consumed around the world. Watermelon production accounts for about 7% of the world\u0026rsquo;s area dedicated to the production of vegetables, with 67% of the total quantity produced in China (Mashilo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Powdery mildew is a worldwide disease caused by obligate biotrophic ascomycete fungi belonging to the order Erysiphales. The first report of sexual stage was discovered in 1981 on cucumber in Changchun province. Powdery mildew pathogens infect host leaves, stems, and petioles but rarely fruit(Agrios et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). These plant parts can be infected throughout the watermelon development period, and more serious infection occurs in the middle and late stages of growth(Glawe \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). \u003cem\u003ePodosphaera xanthii\u003c/em\u003e and \u003cem\u003eGolovinomyce cichoracearum\u003c/em\u003e are the main pathogens causing melon powdery mildew in China(Lebeda et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e;Liang Q L et al.2010;Bakhat N et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Outbreaks of the powdery mildew pathogen lead to a significant reduction in crop yield. Breeding disease-resistant varieties is an effective way to solve the harm that powdery mildew causes to melon production(Keinath et al. 2004).\u003c/p\u003e \u003cp\u003eDuring the long-term co-evolution of plants and pathogens, host plants overcome pathogen infection mainly through the internal immune system, including pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI and ETI are activated by different elicitors(Jones and Dangl \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). PTI is triggered by pathogen-associated molecular patterns, whereas ETI is triggered by pathogen effector proteins, which can be recognized by plant resistance proteins coded by plant resistance genes. Plant resistance genes have the advantage of being monogenic and are easier to manage in breeding programs(Fu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), but the physiological races of powdery mildew vary and mutate quickly. Cultivars become susceptible more quickly under high disease pressure and rapid changes in virulence within the pathogen population(Consonni et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). To deal with the challenge of powdery mildew physiological races on variety resistance, breeders need to identify genetic resources with broad-spectrum resistance to maintain the continuous resistance of cultivars to powdery mildew.\u003c/p\u003e \u003cp\u003eDisease susceptibility is the greatest concern in developing quality crops(Pavan et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Exploiting plant resistance is a necessary pathway in breeding. The Mildew Locus O (MLO) protein, which is a susceptibility gene, is considered the best model for studying broad-spectrum disease resistance to powdery mildew in plants(Acevedo-Garcia et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e;Kusch and Panstruga \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Compared with the resistance gene, the plant susceptibility gene is another resistance gene that plays a negative regulatory role in disease resistance. The first \u003cem\u003emlo\u003c/em\u003e mutant resistance material of barley was generated in \u003cem\u003eHordeum vulgare\u003c/em\u003e variety \u0026ldquo;Haisa\u0026rdquo; using X-rays (Freisleben and Lein, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1942\u003c/span\u003e). In wheat, mutations in particular \u003cem\u003eMlo\u003c/em\u003e genes result in broad-spectrum powdery mildew resistance(Li et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;Panstruga \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The MLO family has also been isolated in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eTriticum aestivum(\u003c/em\u003eKonishi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), \u003cem\u003eOryza sativa\u003c/em\u003e(Liu and Zhu \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and other plants. In Arabidopsis, \u003cem\u003eAtMlo2\u003c/em\u003e, \u003cem\u003eAtMlo6\u003c/em\u003e, and \u003cem\u003eAtMlo12\u003c/em\u003e triple mutants confer full resistance because of genetic redundancy. Mutations of the \u003cem\u003eBnMLO6\u003c/em\u003e gene can stimulate the spontaneous accumulation of callose in leaves and activate ethylene and jasmonic acid transduction pathways, conferring resistance to powdery mildew under both field and greenhouse conditions (SHI et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In pea, loss of \u003cem\u003ePsMLO1\u003c/em\u003e function conditions durable broad-spectrum powdery mildew resistance(Pavan et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eMLO\u003c/em\u003e gene obtained through spontaneous or artificial mutation confers broad-spectrum resistance to powdery mildew, indicating the potential application value of \u003cem\u003eMLO\u003c/em\u003e in the breeding of plants resistant to powdery mildew. Most MLO protein commonly contain calmodulin-binding domain and transmembrane domain, but the number of transmembrane domain varies between lower and higher plants. MLO proteins with fewer than five transmembrane domains are typically found in lower plants, while MLO proteins with four to eight transmembrane domains are usually found in higher plants(Devoto et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Their topological structure and subcellular localization are quite similar to G protein-coupled receptors in animals(Temple and Jones \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). MLOs exist in various tissues of plants and are mainly involved in vegetative and reproductive growth and biotic and abiotic stress responses (Howlader et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The expression of \u003cem\u003eMLO\u003c/em\u003e genes increases in barley during infection by powdery mildew and mechanical damage(Piffanelli et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Analysis of stress-induced expression revealed that five \u003cem\u003eCmMLO\u003c/em\u003e genes were upregulated at 6 h after inoculation with \u003cem\u003ePodosphaera xanthii\u003c/em\u003e Race 1 or DH487, indicating the possible role of MLO proteins in the host cell as an initial step in disease progress. Moreover, strong upregulation was observed for \u003cem\u003eClMLO12\u003c/em\u003e upon powdery mildew infection, suggesting that it is a pathogen-responsive gene((Iovieno et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Expression of the \u003cem\u003eCaMLO2\u003c/em\u003e gene in pepper is induced by abscisic acid and drought stress (Lim and Lee \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). There are 16 MLO homologs in melon (\u003cem\u003eCucumis melo\u003c/em\u003e), 15 in watermelon (\u003cem\u003eCitrullus lanatus\u003c/em\u003e), and 18 in zucchini (\u003cem\u003eCucurbita pepo\u003c/em\u003e) (Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Among them, three melon, three watermelon, and four zucchini MLO homologs have been found to cluster together with the dicot isoforms functionally associated with powdery mildew susceptibility. However, sufficient information is not available to understand the molecular mechanism during the watermelon\u0026ndash;powdery mildew interaction.\u003c/p\u003e \u003cp\u003eIn this study, by using the powdery mildew identified from the the experimental farm of the Henan University Genetics and Breeding Base in Kaifeng, we investigated the disease resistance of different watermelon cultivars, KeXi and DuanMan. Then, based on the available watermelon genomic database, we studied the characteristics of \u003cem\u003eClaMLO\u003c/em\u003e gene family. Further investigation of expression pattern of ClaMLOs help us locate at \u003cem\u003eClaMLO1\u003c/em\u003e and \u003cem\u003eClaMLO2\u003c/em\u003e, so we focus on exploring the function of ClaMLO1 which was localized in cell nucleus, and the genetic transformation of \u003cem\u003eClaMLO1\u003c/em\u003e showed enhanced resistance to powdery mildew.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Watermelon cultivars and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e\u0026lsquo;KeXi\u0026rsquo;and \u0026lsquo;DuanMan\u0026rsquo; were grown at the experimental farm of the Henan University Genetics and Breeding Base in Kaifeng, China. We strictly controlled the watermelon growth conditions, with all plants cultivated under long-day conditions and examined in the greenhouse at the Henan University Genetic Breeding Base. The temperature was maintained at 25\u0026ndash;30℃ during the day and at 15\u0026ndash;18℃ at night. All watermelon seeds were stored in a seed storage cabinet at 4℃.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Purification and identification of watermelon powdery mildew\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe powdery mildew diseased leaf collected from the greenhouse of Henan University were washed with sterile water to remove surface dust, and then inoculated onto watermelon leaves by friction method and placed in incubator for propagation and preservation. For pathogen purification, referring to existing research methods, single-spore isolation technology was used to obtain pure single-spore cultures of watermelon powdery mildew.. When the conidia of watermelon powdery mildew were collected and placed on a glass slide, 1\u0026ndash;2 drops of 3% potassium hydroxide solution were added to observe the fibrous bodies of the conidia with an optical microscope. Those with fibrous bodies are \u003cem\u003ePodosphaera xanthii\u003c/em\u003e, and those without fibrous bodies are \u003cem\u003eGolovinomyce cichoracearum.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Coomassie brilliant blue staining and microscopic observation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe inoculated watermelon leaves were decolorized with decolorizing solution (alcohol: acetic acid\u0026thinsp;=\u0026thinsp;3V: 1V) at 45℃ for 2 h and then soaked in 0.6% Coomassie brilliant blue dye solution (0.6 g R250/100 ml) for 2 min. The dyed leaves were first placed in clean water to remove the floating color of the dye solution. Then, the leaves were placed on a glass slide, and 1\u0026ndash;2 drops of 50% glycerol were added and covered with the cover slide. The slides were observed under a microscope.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Identification of disease resistance with different watermelon cultivars\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDisease tests were performed by spraying plants with a suspension of powdery mildew conidiospores. Heavily sporulating leaves were used to release conidia and immediately placed in a 0.02% Tween 20 solution. Watermelon plants at the 2\u0026ndash;3 true leaf stage were inoculated with an inoculum solution of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e spores per milliliter. The spore count was calculated by the direct microscopic counting method.The leaves of infected plants were sampled randomly at 4, 8, 12, 24, 48, and 72 hpi for microscope observation and nucleic acid extraction. Symptom investigation was carried out till host get sick entirely after 10\u0026ndash;15 days post-inoculation. The severity of the disease was divided into 6 levels. Level 0: No lesions on the plants; Level 1: White powdery lesions appear on a few of leaves, with the lesion area accounting for less than 5% of the total area, and the stems have no lesions; Level 3: A little of lesions appear on the leaves, accounting for 6% to 25% of the total area, and the stems have scattered lesions; Level 5: Moderate amounts of white powdery lesions appear on the leaves, accounting for 26% to 50% of the total area, and the stems have a little of lesions; Level 7: A number of white powdery lesions appear on the leaves, accounting for 51% to 75% of the total area, and the main stem has a little of lesions; Level 9: Many powdery mildew lesions appear on the leaves and the white powder is thick, accounting for 76% to 100% of the total area, and the stems have much more lesions.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 ITS sequence and phylogenetic analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe genomic DNA of watermelon powdery mildew was extracted from infected leaves using a MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0 (TaKaRa, Dalian, China). Fungal ribosome universal primers ITS1 (5\u0026rsquo;-TCCGTAGGTGAACCTGCGG-3\u0026rsquo;) and ITS4 (5\u0026rsquo;-TCCTCCGCTTATTGATATGC-3\u0026rsquo;) were synthesized by Service Biotechnology Co., Ltd. Polymerase chain reactions were carried out using an S1000TM Thermal Cycler in 20 \u0026micro;L volume, including 1 \u0026micro;L of DNA template, 1 \u0026micro;L of forward and reverse primers, 2 \u0026micro;L of 10\u0026times;PCR buffer (Mg\u003csup\u003e2+\u003c/sup\u003e), and 15 \u0026micro;L of sterile deionized water. The PCR procedure was as follows: 98℃ for 5 min, 35 cycles of 94℃ for 30 s, 58℃ for 45 s, and 72℃ for 30 s, followed by a final extension at 72℃ for 10 min. PCR products were separated on 1.5% agarose and sequenced using the Beijing Genomics Institute. The MEGA 11.0 software package was used for phylogenetic analysis of the ITS sequence (Tamura et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). First, multiple sequence alignment (generated by CLUSTALW) was established and manually optimized. Then, a distance matrix was calculated using PROTDIST from the multiple sequence alignment and was then transformed into a tree using the neighbor-joining method. All programs were run with standard parameters.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 RNA isolation and real-time quantitative reverse-transcription PCR\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo evaluate ClaMLO gene family transcript accumulation, total RNA was extracted from infected watermelon leaves sampled randomly at 4, 8, 12, 24, 48, and 72 hours post inoculation using a MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China), and cDNA was synthesized from 1 \u0026micro;g total RNA using a PrimeScript\u0026trade; RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The nucleotide sequences of the primer pairs used for the amplifications are listed in Table S1. Amplification was performed using a TB Green\u0026reg; Premix Ex Taq\u0026trade; II (Tli RNaseH Plus) (TaKaRa, Dalian, China) and Roche LightCycler\u0026reg;480 II System under the following program: 95℃ for 30 s, 40 cycles of 95℃ for 5 s and 60℃ for 34 s, followed by 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 15 s. The amplication efficiencies of qPCR assays ranged from 95% to 110%, and R\u003csup\u003e2\u003c/sup\u003e value for each calibration curves exceeded 0.98. The Ct was used to calculated the copy numbers of all above mentioned genes. For each time point, the relative expression levels were normalized with respect to the housekeeping gene β-Actin and compared to the non-inoculated control. Standard error bars refer to three biological replicates. Significant differences between the means were inferred using the Student\u0026rsquo;s t test for each time point (*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Relative transcript levels were calculated based on the 2\u003csup\u003e-∆∆Ct\u003c/sup\u003e method (Livak, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Conserved motif and phylogenetic analysis of the MLO gene family\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGene sequences of the watermelon MLO family were obtained from the cucurbit genomics official website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cucurbitgenomics.org/\u003c/span\u003e\u003cspan address=\"http://www.cucurbitgenomics.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The conserved motifs of the watermelon MLO proteins were analyzed using Multiple Em for Motif Elicitation (MEME) online tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme.nbcr.net/meme/cgi-bin/meme.cgi\u003c/span\u003e\u003cspan address=\"http://meme.nbcr.net/meme/cgi-bin/meme.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Bailey et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Motifs and CDS were visualized using the Gene Structure View (Advanced) of Tbtools (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The parameters were set as follows: the minimum length of the conserved motif was 15; the maximum length was 40; and the maximum number of conserved motifs was 10. The other parameters were the default values. The phylogenetic tree construction of the watermelon MLO gene family and the complete Arabidopsis thaliana MLO protein family with other identified powdery mildew susceptibility genes, including \u003cem\u003eSlMLO1\u003c/em\u003e from Solanum lycopersicum, \u003cem\u003eCaMLO2\u003c/em\u003e from Capsicum annuum, \u003cem\u003ePsMLO1\u003c/em\u003e from Pisum sativum, \u003cem\u003eLjMLO1\u003c/em\u003e from Lotus japonicus, HvMLO from \u003cem\u003eHordeum vulgare, OsMLO1\u003c/em\u003e from Oryza sativa, and \u003cem\u003eNtMLO1\u003c/em\u003e and \u003cem\u003eNtMLO2\u003c/em\u003e from Nicotiana tabacum, were consistent with the method given in Section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e2.5\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Subcellular localization of ClaMLO1\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe subcellular localization of ClaMLO1 in onion epiderma was assessed using a plant expression vector GFP1300 containing an eGFP gene, and the full-length ClaMLO1 was fused to the N-terminus of eGFP in the SalI digestion sites. All constructs were transformed into A. tumefaciens GV3101 using the freeze\u0026ndash;thaw method and infiltrated into the Allium cepa epidermis. The empty GFP1300 vector was used as a control. At 24 h post-infiltration, the Allium cepa epidermis was imaged using confocal laser scanning microscopy.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Watermelon transformation using Agrobacterium-mediated expression\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAs previously described, the genetic transformation was carried out with a few minor modifications (Dong et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Watermelon seeds were sown on a 0.6% agar solid medium for 2 days in the dark after being surface sterilized. Then, the cotyledons with growth points were cut as explants and infected with A. tumefaciens. Agrobacterium tumefaciens strains harboring the indicated binary vectors were co-cultivated with the cotyledon fragments in the dark for 3 days on MS solid medium containing 1 mg/L 6-BA. The cotyledon fragments were then transferred onto a selective induction medium containing 1 mg/L 6-BA, 100 mg/L Kan, and 250 mg/L Cephalosporin. The regenerated adventitious buds were excised and transferred onto an elongation medium containing 0.2 mg/L KT, 250 mg/L Cephalosporin, and 50 mg/L Kan. The elongation plants were transferred to a rooting medium containing 2 mg/L IBA and 100 mg/L Cephalosporin. Positive transgenic plants were detected using PCR.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Morphological identification of powdery mildew on watermelon\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eIn recent years, watermelon was severely infected by powdery mildew in the greenhouse of the College of Life Sciences, Henan University. The disease primarily occurred on the leaves. At the beginning of the disease, the infected position was covered with white powder and gradually turned yellow with brown lesions. Black speckles emerged from the infected part during powdery mildew development in the later period (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The conidia of watermelon powdery mildew collected from the watermelon plant had obvious fibrous bodies (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). After staining with Coomassie Brilliant Blue R250, the conidia were observed to be elliptical and clustered on the conidial stem. The number of conidia varied, ranging from 5 to 12 (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). According to the morphological features, the pathogen was initially confirmed as \u003cem\u003ePodosphaera xanthii\u003c/em\u003e (Kiss L. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Genetic analysis of ITS sequences for watermelon powdery mildew\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe ITS fragment of the pathogen was amplified using the DNA of watermelon powdery mildew as a template and the universal primers ITS1 and ITS4 of the fungal ribosomal ITS region as the primers. DNA sequencing showed that the ITS fragment length was 671 bp. Further molecular phylogeny between the ITS regions from watermelon and melon, cucumber, zucchini, adzuki bean, abelmosk, eggplant which blasted from the NCBI database was constructed. This tree was divided into three clades, but all sequences in the three clades were attributed to \u003cem\u003ePodosphaera xanthii\u003c/em\u003e(Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This analysis clearly supported the morphological observation that the powdery mildew fungus on watermelon found at Henan University belonged to \u003cem\u003ePodosphaera xanthii\u003c/em\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Different watermelon lines display different response to powdery mildew infection\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eIn order to compare the characteristic disease symptoms of powdery mildew between KeXi and DuanMan watermelon lines, we observed the invasion procedure from conidia germination to secondary hyphal development, identified the disease resistance of KeXi and DuanMan. In the leaves of KeXi, the majority of conidia germinated producing primary germinal tubes at 12 hpi, growing new mycelium and continuously elongating between 24 and 48 hpi, and forming secondary hyphae at 72 hpi. Compared to KeXi, DuanMan showed serious retardation in conidial germination and hyphae growth and development(Fig. 3A). In addition, at 15 days post-inoculation, DuanMan still exhibited strong resistance to powdery mildew, but abundant development of conidia and severe powdery mildew pathogen symptoms were observed on KeXi(Fig. 3B). Generally, KeXi and DuanMan responded distinctly to powdery mildew infection. According to the disease index survey, DuanMan was identified as medium resistance type, KeXi was identified as susceptible type (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e The resistance identified result of powdery mildew of KeXi and DuanMan\u003c/div\u003e\u0026nbsp;\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCultivars\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eDisease\u0026nbsp;index\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eDisease\u0026nbsp;rating\u0026nbsp;range\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eResistance\u0026nbsp;evaluation\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eKeXi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e64.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e3\u0026ndash;9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eDuanMan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e42.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1\u0026ndash;9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eMR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Prediction of conserved motifs and CDS with phylogenetic analysis of ClaMLO gene family\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eMLO gene family act as susceptibility factors having an indispensable role in the occurrence of powdery mildew. To further explore the molecular mechanism of resistance to powdery mildew in different watermelon lines, KeXi and DuanMan. we utilized latest available genomic information to conduct bioinformatics analysis of the MLO gene family in watermelon. 15 predicted proteins labeled MLO like protein were matched through BLASTp analysis, and they were named from \u003cem\u003eClaMLO1\u003c/em\u003e to \u003cem\u003eClaMLO15\u003c/em\u003e. The number of transmembrane helices of \u003cem\u003eClaMLO\u003c/em\u003e gene family vary from 3\u0026mdash;8, and \u003cem\u003eClaMLO1\u003c/em\u003e, \u003cem\u003eClaMLO4\u003c/em\u003e, \u003cem\u003eClaMLO5\u003c/em\u003e, \u003cem\u003eClaMLO7\u003c/em\u003e, \u003cem\u003eClaMLO11\u003c/em\u003e, \u003cem\u003eClaMLO12\u003c/em\u003e, \u003cem\u003eClaMLO13\u003c/em\u003e have seven transmembrane helices (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The motifs of the \u003cem\u003eClaMLO\u003c/em\u003e gene family were predicted by MEME, and a phylogenetic tree was built with MEGA 11.0 software. We integrated CDS, motifs, and the tree using TBtools. The ClaMLO proteins had 10 conservative motifs, and all of them had motifs 1 and 5 (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Most of the families had more than eight motifs. The CDS of the gene family was distributed in the range of 0\u0026ndash;9000 bp. These results showed that ClaMLO gene family were conservative.\u003c/p\u003e\n \u003cp\u003eTo study the evolution and development of ClaMLO gene family with the complete \u003cem\u003eArabidopsis thaliana\u003c/em\u003e MLO gene family and other identified powdery mildew susceptibility genes from different species, 23 MLO proteins were clustered in six distinct clades. Clade numbers were organized by the position of Arabidopsis AtMLO homologs and barley HvMLO (Feechan et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). ClaMLO2, ClaMLO7 and ClaMLO13 were clustered in clade V, which contained all MLO proteins functionally related to powdery mildew susceptibility in dicot plants. Other ClaMLOs were distributed in different clades(Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eFeatures of the watermelon MLO gene family\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eMLO name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eLocus name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eChromosome\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eStarting position(kb)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eClade\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003eIntrons\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003eLength(aa)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003eNumber of transmembrane helices\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C09G180170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e33811195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅠ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e553\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C05G102540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e30786649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅤ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e508\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C05G091880\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e9890612\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅠ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C11G224240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e29955333\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅠ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C06G109800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e495518\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅡ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e516\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C03G053300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e2480883\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅥ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e580\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C02G047070\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e34811774\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅤ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C02G036300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e17697506\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅣ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C02G026560\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e344852\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅡ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C01G020510\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e33128842\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅢ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e571\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C10G184670\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e189923\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅢ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e578\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C08G152090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e20446868\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅢ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e544\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C03G053280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e2425953\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅤ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e582\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C01G023190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e34727024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅣ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eClaMLO15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCla97C10G185230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e644865\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eⅠ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\n \u003cp\u003e539\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Transcription of the ClaMLO gene family in response to powdery mildew inoculation\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe significantly enhanced resistance of different species to powdery mildew is related to the up-regulated expression of MLO susceptibility genes. To further investigate the expression pattern of \u003cem\u003eClaMLO\u003c/em\u003e gene family, 15 \u003cem\u003eClaMLO\u003c/em\u003e genes were subjected to an assessment of their expression levels after inoculating KeXi and DuanMan with the conidia of powdery mildew. Quantification by real-time quantitative reverse-transcription PCR showed that the expression levels of \u003cem\u003eClaMLO1\u003c/em\u003e and \u003cem\u003eClaMLO2\u003c/em\u003e were obviously upregulated in DuanMan at 12 hpi, whereas as the inoculation time was prolonged, the expression levels of \u003cem\u003eClaMLO1\u003c/em\u003e and \u003cem\u003eClaMLO2\u003c/em\u003e decreased gradually. Compared with DuanMan, the expression levels of \u003cem\u003eClaMLO1\u003c/em\u003e and \u003cem\u003eClaMLO2\u003c/em\u003e increased continuously and reached a maximum at 48 hpi in KeXi (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results revealed that \u003cem\u003eClaMLO1\u003c/em\u003e and \u003cem\u003eClaMLO2\u003c/em\u003e have different expression pattern in powdery mildew susceptible lines or resistant lines. Significantly, they may likely involved in the infection process of powdery mildew on watermelon. Unfortunately, other \u003cem\u003eClaMLO\u003c/em\u003e genes, except \u003cem\u003eClaMLO1\u003c/em\u003e and \u003cem\u003eClaMLO2\u003c/em\u003e, showed no significant difference during different invasion times, and \u003cem\u003eClaMLO7\u003c/em\u003e, \u003cem\u003eClaMLO9\u003c/em\u003e, and \u003cem\u003eClaMLO12\u003c/em\u003e were not detected.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Subcellular localization of ClaMLO1 localized in the cell nucleus of Onion epiderma\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe subcellular localization of ClaMLO1 was confirmed using a transient onion expression system. ClaMLO1 was fused to the N-terminus of an enhanced green fluorescent protein gene within a GFP1300 vector and transformed into an onion expression cell by \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e. As visualized using fluorescence microscopy, green fluorescence accumulated in the cell nucleus of onion epiderma in the ClaMLO1-GFP1300 transformants(Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast, green fluorescence was uniformly distributed in the cytoplasm of the empty vector without the target gene. This suggests that ClaMLO1 is localized in the cell nucleus of onion epiderma.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Genetic transformation of ClaMLO1 in watermelon\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo further investigate the function of ClaMLO1 in the watermelon\u0026ndash;powdery mildew interaction, we silenced ClaMLO1 in watermelon. In the process, we used the \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated watermelon cotyledons system, where callus tissue was differentiated from the edge of the cotyledons after two to four weeks of cultivation (Figs. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). After 2\u0026ndash;3 weeks of callus culture, resistant buds began to grow, and the stems elongated and thickened. The number of leaves increased, and the leaves became larger (Figs. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-D). When the watermelon seedlings reached a height of 5\u0026ndash;6 cm, the bottle cap was opened, and the seedlings were refined for 24 h. The watermelon seedlings were removed, and the agar attached to the roots was washed with water. The seedlings were transplanted into a pot filled with nutrient soil. Watermelons set fruit after pollination and matured for about 30 days (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eE).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.8 Silencing ClaMLO1 in watermelon enhanced resistance to powdery mildew\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo examined the disease resistance of \u003cem\u003eClaMLO1\u003c/em\u003e silencing plants, we carried out powdery mildew inoculation assay. At the 2\u0026ndash;3 true leaf stage, \u003cem\u003eClaMLO1\u003c/em\u003e silencing plants and control plants were inoculated with powdery mildew spore suspension. Notably, at 10 days post-inoculation, the leaves of \u003cem\u003eClaMLO1\u003c/em\u003e silencing plants keep green and normal. However, the leaves of control plants turns yellow and distributed with white powder (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). so we also detected the expression level of \u003cem\u003eClaMLO\u003c/em\u003e gene family. In \u003cem\u003eClaMLO1\u003c/em\u003e-silenced plants, ClaMLO1 and ClaMLO2 both have a significant increase at 12 hours post-inoculation, and they were greatly up-regulated in KeXi at 48 hours post-inoculation. These result further proved that ClaMLO1 may be a susceptible gene, having an important role in helping powdery mildew invasion.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWatermelon is a Cucurbitaceae crop that has sweet pulp, a cool and refreshing taste, is rich in vitamin C and various minerals, and has the effect of relieving heat and thirst. In addition to being used for food, it can also be made into a variety of drugs and processed products. According to the latest data from the Food and Agriculture Organization of the United Nations, watermelon is one of the most important fruits and vegetables in the world. Powdery mildew is a major fungal disease of thousands of plant species, including cultivated watermelon, which can limit plant photosynthesis due to whitish conidial growth on cotyledons, true leaves, leaf petioles, and stems, thus leading to a severe loss of crop quality and yield (Vielba-Fern\u0026aacute;ndez, 2020). At present, fungicides and resistant varieties are the main methods for controlling powdery mildew (Lebeda and Sedlakov, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, fungicides are expensive and harmful for the environment. Cultivars resistant to powdery mildew are fundamental to reducing the use of pesticides in agricultural practices (Zhang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe identification of powdery mildew fungi to species can be based on fungal morphology and ITS sequences (Liang et al.2013;Tang et al.2017). In powdery mildew, the internal transcribed spacer sequences (ITS) are the single available barcode for species identification (Takamatsu et al.2015). In this study, the powdery mildew of watermelon cultivars in Henan Province was evaluated. Microscope examination revealed that the fungus produced typical structures of powdery mildew conidia with fibrous bodies. This is consistent with the results of a previous taxonomic manual of \u003cem\u003eErysiphales\u003c/em\u003e (Branu and Cook, 2012). Since powdery mildew in tropical/subtropical environments rarely develop sexual stages with morphologically diagnostic characteristics, ITS regions of the ribosomal RNA genes obtained from the asexual stages have become an important modern tool for species identification (Yeh et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To confirm the identity, the obtained 671-bp ITS sequence was blasted on the NCBI website. The subsequent phylogeny revealed that the ITS sequence was 100% identical to many records of \u003cem\u003ePodosphaera xanthii\u003c/em\u003e. Based on both morphological and phylogenetic characteristics, the powdery mildew pathogen in watermelon was identified as \u003cem\u003ePodosphaera xanthii\u003c/em\u003e. In addition, the Glandularia tenera powdery mildew pathogen has also been identified as \u003cem\u003ePodosphaera xanthii\u003c/em\u003e (Pei et al, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and mung bean planted in Taiwan can also be infected by \u003cem\u003ePodosphaera xanthii\u003c/em\u003e, which causes severe powdery mildew disease (Sheu et al, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious phylogenetic studies classified plant MLO proteins into six clades (I\u0026ndash;VI). The available scientific literature indicates that the proteins of IV and V clades have a significant effect on powdery mildew susceptibility in monocots and dicots, respectively (Win et al,2018; Kusch et al, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, powdery mildew pathogenesis is associated with the upregulation of \u003cem\u003eMLO\u003c/em\u003e genes during the early stages of infection, causing the downregulation of plant defense pathways. \u003cem\u003eRhMLO1\u003c/em\u003e and \u003cem\u003eRhMLO2\u003c/em\u003e are upregulated in the rose\u0026ndash;powdery mildew pathogen interaction (Qiu et al, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, specific members of the MLO gene family act as powdery mildew-susceptibility genes, as their loss-of-function mutations grant durable and broad-spectrum resistance (Pessina et al,2014;Humphry et al,2011). In watermelon, three MLO homologs, namely \u003cem\u003eClaMLO2\u003c/em\u003e, \u003cem\u003eClaMLO7\u003c/em\u003e and \u003cem\u003eClaMLO13\u003c/em\u003e clustered together in phylogenetic clade V, indicating that they are candidates for powdery mildew susceptibility genes in watermelon. However, in our study, we also detected one upregulated gene, \u003cem\u003eClaMLO1\u003c/em\u003e, after inoculation with watermelon powdery mildew, which was classified into clade I, indicating that the simple clustering in clade V is poor in recognizing a gene as a powdery mildew-susceptibility factor. A similar result has been detected in apple; a newly identified MLO gene classified in clade VII showed upregulation after inoculation with the pathogen. Furthermore, \u003cem\u003eClaMLO2\u003c/em\u003e, which encodes an MLO protein in clade V, was also upregulated after powdery mildew infection, but other MLO proteins showed no significant changes in expression. Generally, we found two \u003cem\u003eClaMLO\u003c/em\u003e genes that may play an important role in the interaction between powdery mildew and watermelon, and they can be used as targets for functional characterization of the investigation on watermelon resistance breeding.\u003c/p\u003e \u003cp\u003eBy performing multiple alignments of watermelon MLO proteins, we found that they were very conserved in amino acid residues and motifs. This result is consistent with the occurrence of 10 conserved motifs in tomato MLO proteins and grape (Islam and Yun,2016). The existence of these conserved motifs may be related to the function of the MLO protein. At present, many MLO gene families have been found in different plants, but the function of most watermelon MLO proteins remains unknown, especially in watermelon. To solve this problem, we investigated the subcellular localization of watermelon MLO proteins. In 1999, a barley MLO protein was speculated to be localized on the cytoplasmic membrane [(Devoto et al,1999). Many studies have carried out subcellular localization of MLO proteins. For example, most MLO proteins of Rosaceae plants are distributed in the plasma membrane, followed by the endoplasmic reticulum and vacuoles, and a few are distributed in the nucleus, mitochondria, and Golgi apparatus (Xiang et al,2018). Based on the remarkable up-regulation expression levels of ClaMLO1 during powdery mildew infection process, we focused on exploring the characteristics of ClaMLO1. In this study, ClaMLO1 was primarily distributed in the cell nucleus, as shown using a subcellular localization assay. The MLO protein was detected not only around epidermal cells but also in the inner membrane and nuclear membrane in \u003cem\u003eMlo\u003c/em\u003e mutants without powdery mildew infection. However, after being infected with powdery mildew, it was redistributed within the plant cells, indicating the subcellular localization of the MLO protein plays an important role in the life activities of plants(Bhat et al,2005). In addition, the MLO function in pollen tube reception is dependent on MLO protein localization in Golgi (Jones et al, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The subcelluar localization of ClaMLO1 may be greatly related to the celluar signal transduction pathway that regulate powdery mildew reponse, further disease resistance assay also prove that \u003cem\u003eClaMLO1\u003c/em\u003e-silenced watermelon exhibit more resistant to powdery mildew.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn this study, we identified one watermelon powdery mildew isolate belonging to \u003cem\u003ePodosphaera xanthii\u003c/em\u003e. Microscope observation of the invasion process and disease resistance identification suggested that DuanMan displayed stronger resistance than KeXi. Further bioinformatics analysis demonstrated the molecular characteristics of \u003cem\u003eClaMLO\u003c/em\u003e gene family, including motif composition, numbers of transmembrane helix, phylogenetic relationships, and the transcription expression pattern. We found that \u003cem\u003eClaMLO1\u003c/em\u003e localized in cell nucleus, and silencing \u003cem\u003eClaMLO1\u003c/em\u003e in watermelon enhanced resistance to powdery mildew, indicating that ClaMLO1 may play an important role in the interaction between watermelon and powdery mildew. Hence, our experiments provided a direction for studying the molecular mechanism of watermelon breeding.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis research was funded by the National Natural Science Foundation of China (31801882) and Henan Province Key Science and Technology Projects (212102110044).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;s\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eXixi Zhang wrote original manuscript. Xixi Zhang, Shengqi Hua and Dongyang Ran performed most of the experiments. Shengqi Hua, Jiale Shi and Shengcan Hou helped data analysis. Wei Dong, Bo Liu and Junhua Li supervised spoken and written language. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli dir=\"LTR\"\u003eAcevedo-Garcia, J., Kusch, S., \u0026amp; Panstruga, R. (2014). Magical mystery tour: MLO proteins in plant immunity and beyond. \u003cem\u003eNew Phytologist\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e 204, 273\u0026ndash;281. https://doi.org/10.1111/nph.12889\u003c/li\u003e\n \u003cli dir=\"LTR\"\u003eAgrios, G. N. , Hidalgo Villegas, R. , Montes Escobar, K. , Mostacedo, B. , Alarc\u0026oacute;n, A, \u0026amp; Villegas, Z. , et al. (2004). Plant pathology. \u003cem\u003eMycologia\u003c/em\u003e, 28(62), 922.https://doi.org/10.2307/3757727\u003c/li\u003e\n \u003cli dir=\"LTR\"\u003eBailey, T. L., Johnson, J., Grant, C. E., \u0026amp; Noble, W. S. (2015). 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Characterization of the \u003cem\u003eMLO\u003c/em\u003e gene family in Rosaceae and gene expression analysis in \u003cem\u003eMalus domestica\u003c/em\u003e. \u003cem\u003eBMC Genomics\u003c/em\u003e, 15, 618. https://doi.org/10.1186/1471-2164-15-618\u003c/li\u003e\n \u003cli dir=\"LTR\"\u003ePiffanelli, P., Zhou, F., Casais, C., Orme, J., Jarosch, B., Schaffrath, U., Collins, N. C., Panstruga, R., \u0026amp; Schulze-Lefert, P. (2002). The barley MLO modulator of defense and cell death is responsive to biotic and abiotic stress stimuli.\u003cem\u003e \u003c/em\u003e\u003cem\u003ePlant Physiology\u003c/em\u003e, 129, 1076\u0026ndash;1085. https://doi.org/10.1104/pp.010954\u003c/li\u003e\n \u003cli dir=\"LTR\"\u003eQiu, X., Jian, H., Wang, Q., Tang, K., \u0026amp; Bao, M. (2015). 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Genome editing of \u003cem\u003eBnMLO6\u003c/em\u003e gene by CRISPR/Cas9 for the improvement of disease resistance in \u003cem\u003eBrassica napus\u003c/em\u003e L. \u003cem\u003eActa Agronomica Sinica\u003c/em\u003e, 48, 801\u0026ndash;811. https://doi.org/10.3724/SP.J.1006.2022.14077\u003c/li\u003e\n \u003cli dir=\"LTR\"\u003eTakamatsu, S., Ito Arakawa, H., Shiroya, Y., Kiss, L., \u0026amp; Heluta, V. (2015). First comprehensive phylogenetic analysis of the genus \u003cem\u003eErysiphe\u003c/em\u003e (Erysiphales, Erysiphaceae) I. The \u003cem\u003eMicrosphaera\u003c/em\u003e lineage. \u003cem\u003eMycologia\u003c/em\u003e, 107, 475\u0026ndash;489. https://doi.org/10.3852/15-007\u003c/li\u003e\n \u003cli dir=\"LTR\"\u003eTamura, K., Stecher, G., \u0026amp; Kumar, S. (2021). 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Genome-wide identification and description of \u003cem\u003eMLO\u003c/em\u003e family genes in pumpkin (\u003cem\u003eCucurbita maxima\u003c/em\u003e Duch.). \u003cem\u003eHorticulture, Environment, and Biotechnology\u003c/em\u003e, 59, 397\u0026ndash;410. https://doi.org/10.1007/s13580-018-0036-9\u003c/li\u003e\n \u003cli dir=\"LTR\"\u003eXiang, G., Wang, K., Yan, H., Li, S., Zhou, N., Tang, K., \u0026amp; Qiu, X. (2018). Bioinformatics analysis of MLO protein family in Rosaceae plants. \u003cem\u003eGenomics and Applied Biology\u003c/em\u003e, 5, 2043\u0026ndash;2059. https://doi.org/10.13417/j.gab.037.002043\u003c/li\u003e\n \u003cli dir=\"LTR\"\u003eYeh, Y.-W., Chou, P.-Y., Hou, H.-Y., \u0026amp; Kirschner, R. (2021). First records of powdery mildew fungi (Erysiphales) on medicinal plants in Taiwan. \u003cem\u003eBotanical Studies\u003c/em\u003e, 62, 1. https://doi.org/10.1186/s40529-020-00307-0\u003c/li\u003e\n \u003cli dir=\"LTR\"\u003e Zhang, H., Guo, S., Gong, G., Ren, Y., Davis, A. R., \u0026amp; Xu, Y. (2011). Sources of resistance to race 2WF powdery mildew in U.S. watermelon plant introductions. \u003cem\u003eHortScience\u003c/em\u003e, 46, 1349\u0026ndash;1352. https://doi.org/10.21273/HORTSCI.46.10.1349\u003c/li\u003e\n \u003cli dir=\"LTR\"\u003eZhang, X. L., Zhang, J. S., Lei, B., Yu, J., \u0026amp; Zhao, D. G. (2018). Research progress of MLO proteins in plants. \u003cem\u003ePlant Physiology Journal\u003c/em\u003e, 54, 1159\u0026ndash;1171. https://doi.org/10.13592/j.cnki.ppj.2018.0160\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Watermelon, Powdery mildew, MLO, Bioinformatics analysis","lastPublishedDoi":"10.21203/rs.3.rs-9080378/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9080378/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePowdery mildew (PM) is a major fungal disease that causes huge economic losses by impacting thousands of plant species, including Cucurbitaceae crops, such as watermelon (\u003cem\u003eCitrullus lanatus\u003c/em\u003e). Although powdery mildew is caused by a widespread watermelon pathogen, the molecular mechanism remains unclear. In this study, one watermelon powdery mildew isolate was identified as \u003cem\u003ePodosphaera xanthii\u003c/em\u003e. According to the distinct infections of powdery mildew on DuanMan (Middle resistance to PM) and Kexi (Susceptible to PM), fifteen\u003cem\u003e ClaMLO\u003c/em\u003e genes expression level were detected, while only\u003cem\u003e ClaMLO1\u003c/em\u003e and \u003cem\u003eClaMLO2\u003c/em\u003ewere significantly regulated at different invasion period.. Bioinformatics analysis further showed that ClaMLO gene family were very conservative and \u003cem\u003eClaMLO2\u003c/em\u003e, \u003cem\u003eClaMLO7 \u003c/em\u003eand \u003cem\u003eClaMLO13\u003c/em\u003e were clustered in clade V. Subsequent subcellular localization experiments proved that \u003cem\u003eClaMLO1\u003c/em\u003e localized in the cell nucleus, and silencing \u003cem\u003eClaMLO1\u003c/em\u003e in watermelon enhanced resistance to powdery mildew. Generally, our study identified one gene, named \u003cem\u003eClaMLO1\u003c/em\u003e. It may be responsible for the successful invasion of watermelon powdery mildew, laying a theoretical foundation for molecular breeding of watermelon.\u003c/p\u003e","manuscriptTitle":"Screening and Functional Analysis of Genes for Resistance to Powdery Mildew in Watermelon","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 13:51:47","doi":"10.21203/rs.3.rs-9080378/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53f3cdc5-ea0b-423f-b4c8-4dfb20af7356","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Reject after review","date":"2026-05-15T09:03:31+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T13:05:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 13:51:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9080378","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9080378","identity":"rs-9080378","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}