Mildew Resistance Locus O Genes Research Articles

An Efficient Agrobacterium-Mediated Genetic Transformation System for Gene Editing in Strawberry (Fragaria × ananassa).

The octoploid-cultivated strawberry variety Benihope (Fragaria × ananassa Duch cv. Benihope) is an important commercial plant. It is highly susceptible to different diseases, which ultimately leads to a reduction in yield. Gene-editing methods, such as CRISPR/Cas9, demonstrate potential for improving disease resistance in the strawberry cv. Benihope. Establishing a plant regeneration system suitable for CRISPR/Cas9 gene editing is crucial for obtaining transgenic plants on a large scale. This research established a callus induction and plant regeneration system for Agrobacterium-mediated CRISPR/Cas9 gene editing in strawberry cv. Benihope by evaluating multiple types of explants and various plant growth regulators throughout the entire tissue culture process. The results showed that the efficiency of callus induction is strongly influenced by the type of explant and is highly sensitive to the combination of plant growth regulators. Among the different plant growth regulators employed, thidiazuron (TDZ), in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), effectively induced callus formation and plant regeneration from explants derived from nutrient tissues such as runner tips and crowns. In addition, the regeneration experiment demonstrated that the addition of polyvinylpyrrolidone (PVPP) to the shoot regeneration medium could inhibit tissue browning. The gene-edited plants in which some or all of the Fvb7-1, Fvb7-2, Fvb7-3, and Fvb7-4 genes in the MLO (Mildew resistance Locus O) gene family were knocked out by CRISPR/Cas9 system were obtained by applying the plant regeneration system developed in this study.

Analysis of Clade V MLO Gene Expressions in Hazelnut Leaves upon Exposure to Powdery Mildew

Powdery mildew affecting European hazelnut Corylus avellana L. trees in Turkey is caused by the obligate biotrophic fungus Erysiphe corylacearum. This fungal disease causes significant economic losses by reducing the yield and quality of hazelnuts. Loss-of-function mutations in the mildew resistance locus o (MLO) gene family of many plants confer high levels of broad-spectrum resistance to powdery mildew. The proteins encoded by the genes at the MLO locus are divided into approximately seven different conserved clades. Among them, the phylogenetic clade V has been shown to be involved in PM susceptibility, as inactivation of these genes leads to long-term disease resistance in dicotyledons. In this study, we examined the temporal expression pattern of three hazelnut MLO genes, previously identified as clade V, in response to powdery mildew infection in C. avellana cv. Tombul. Leaves are the main tissue affected by the powdery mildew pathogen in hazelnut plants. Analysis of MLO expression in hazelnut leaves showed that CavMLO2 and CavMLO6 were significantly upregulated after challenge with E. corylacearum, providing preliminary evidence that they may be involved in PM susceptibility. Thus, these results present a basis for the isolation and use of relevant genes in plant breeding for disease resistance. Moreover, expression profiles of the clade V MLO genes are also important to identify candidate genes that need to be silenced or edited for future molecular studies to obtain resistant hazelnut varieties.

Genome-wide identification and evolutionary analysis of MLO gene family in Rosaceae plants

Mutants lacking wild-type MLO (Mildew resistance Locus O) proteins show broad-spectrum resistance to the powdery mildew fungus, and dysregulated cell death control, with spontaneous cell death in response to developmental or abiotic stimuli. In order to understand the evolution and divergence patterns of the MLO gene family in Rosaceae plants, we analysed systematically genome-wide data from Fragaria vesca, Prunus persica, Prunus mume, Malus domestica, Pyrus bretschneideri and Rubus occidentalis based on bioinformatics methods. Using three phylogenetic methods (the neighbour-joining, maximum likelihood, and Bayesian methods), we identified 117 MLO genes from 6 Rosaceae species. The results of all three phylogenetic analysis methods supported that these genes were divided into six clades. Conserved motif analysis found that only motif 2 was present in all MLO proteins and had 3 nearly invariant amino acid residues. The findings indicated that motif 2 might be shared by the MLO gene family. The structural features of these genes showed large variations in sequence length among different species, although the lengths and the numbers of exons exhibited high degrees of similarity. Selective pressure analysis showed extremely significant differences in all 6 clades, with 2, 1, and 1 site(s) under significant positive selection detected in clades III, IV, and VI, respectively. These positive selection sites were important driving forces for the promotion of the functional differentiation of the MLO genes. Functional divergence analysis showed that the significantly divergent sites were located within the domains of the MLO genes. Functional distance analysis showed that the clade V had more conservative functions and might have retained more original functions during the evolutionary process. However, clade I may have undergone extensive altered functional constraints as a specialised functional role. Moreover, the most original function of the MLO genes in Rosaceae could be related to the evolution of their resistance to powdery mildew, which then gradually evolved into functions such as the regulation of flower development, the control of root morphology, and seed evolution due to the different evolutionary rates after gene duplication. These results provide a theoretical basis for further studies of the molecular evolutionary patterns of the plant MLO gene family.

Evolution of the MLO gene families in octoploid strawberry (Fragaria \xd7ananassa) and progenitor diploid species identified potential genes for strawberry powdery mildew resistance

Powdery mildew (PM) caused by Podosphaera aphanis is a major fungal disease of cultivated strawberry. Mildew Resistance Locus O (MLO) is a gene family described for having conserved seven-transmembrane domains. Induced loss-of-function in specific MLO genes can confer durable and broad resistance against PM pathogens. However, the genomic structure and potential role of MLO genes for PM resistance have not been characterized yet in the octoploid cultivated strawberry. In the present study, MLO gene families were characterized in four diploid progenitor species (Fragaria vesca, F. iinumae, F. viridis, and F. nipponica) and octoploid cultivated (Fragaria ×ananassa) strawberry, and potential sources of MLO-mediated susceptibility were identified. Twenty MLO sequences were identified in F. vesca and 68 identified in F. ×ananassa. Phylogenetic analysis divided diploid and octoploid strawberry MLO genes into eight different clades, in which three FveMLO (MLO10, MLO17, and MLO20) and their twelve orthologs of FaMLO were grouped together with functionally characterized MLO genes conferring PM susceptibility. Copy number variations revealed differences in MLO composition among homoeologous chromosomes, supporting the distinct origin of each subgenome during the evolution of octoploid strawberry. Dissecting genomic sequence and structural variations in candidate FaMLO genes revealed their potential role associated with genetic controls and functionality in strawberry against PM pathogen. Furthermore, the gene expression profiling and RNAi silencing of putative FaMLO genes in response to the pathogen indicate the function in PM resistance. These results are a critical first step in understanding the function of strawberry MLO genes and will facilitate further genetic studies of PM resistance in cultivated strawberry.

CmMLO17 and its partner CmKIC potentially support Alternaria alternata growth in Chrysanthemum morifolium

The Mildew Resistance Locus O (MLO) gene family has been investigated in many species. However, there are few studies on chrysanthemum MLO genes. We report in this study that CmMLO17 in Chrysanthemum morifolium was upregulated after Alternaria alternata infection. Silencing of CmMLO17 by artificial microRNA resulted in reduced susceptibility of chrysanthemum to A. alternata infection. Genes in the abscisic acid (ABA) and Ca2+ signaling pathways were upregulated in the CmMLO17-silenced line R20 compared to the wild-type plants. We speculated that CmMLO17-silenced plants had a faster and stronger defense response that was mediated by the ABA and Ca2+ signaling pathways, resulting in reduced susceptibility of chrysanthemum to A. alternata infection. In addition, a candidate gene, CmKIC, that may interact with CmMLO17 was discovered by the yeast two-hybrid assay. The interaction between CmMLO17 and CmKIC was confirmed using the yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) analysis. CmMLO17 and CmKIC were both located on the plasma membrane, and CmKIC was also located on the nucleus. CmKIC overexpression increased the susceptibility of chrysanthemum to A. alternata, whereas CmKIC silencing resulted in reduced susceptibility. Therefore, CmMLO17 and CmKIC may work together in C. morifolium to support the growth of A. alternata. The results of this study will provide insight into the potential function of MLO and improve the understanding of plant defense responses to necrotrophic pathogens.

Genome-wide identification and analysis of the MLO gene family for candidate powdery mildew susceptibility factors in Momordica charantia

Powdery mildew (PM) disease is one of the most serious threats to Momordica charantia. Specific mildew resistance locus O (MLO) genes associated with PM susceptibility have been explored in some plant species for mlo-based PM resistance. However, MLO has not been surveyed globally for candidate susceptibility factors in M. charantia. In this study, 15 MLO genes, designated McMLO1∼15, were identified in the recently reported M. charantia genome. Phylogenetic analysis clustered McMLOs into five clades. In clade V, McMLO3, McMLO4, McMLO6, and McMLO11 co-clustered with the known PM susceptibility (PMS) factors of dicotyledonous plants, making them putative PMS factors in M. charantia (McPMS). Three of the four McPMS candidates, McMLO4, McMLO6, and McMLO11, showed much higher sequence identity with PMS-related orthologues than with paralogues. Structure modelling and alignment revealed significant structural similarity between McMLO11 and McMLO4 and the functionally characterized PMS CsMLO8, CmMLO2, and CsMLO11. High structural similarity was also found between McMLO11 and McMLO6 but not between McMLO3 and any other MLO homologs. RNA-seq indicated that the four McPMS candidates were all specifically expressed at the highest level in leaves compared with other tissues. Furthermore, among the expressed McMLOs in leaves, McMLO6 was expressed at the lowest level, McMLO11, McMLO3 and McMLO4 were expressed at the first, second and fourth highest levels, respectively. During PM infection, McMLO11 was moderately induced but highly expressed at the highest level. McMLO4 and McMLO6 were significantly upregulated, but there was no significant differential expression of McMLO3. Consistent with the different expression profiles of the four McPMSs, different types and amounts of cis-acting regulatory elements were found in their promoters. Taken together, we speculate that in M. charantia McMLO4 is a minor PMS factor, and that McMLO11 is the major PMS factor and is functionally redundant with McMLO6. McMLO3 seems to be a unique PMS factor with a different functional mechanism. The results of the study provide valuable information for further study of mlo-based PM resistance improvement in M. charantia.

CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera)

Grapevine (Vitis vinifera), one of the most economically important fruit crops in the world, suffers significant yield losses from powdery mildew, a major fungal disease caused by Erysiphe necator. In addition to suppressing host immunity, phytopathogens modulate host proteins termed susceptibility (S) factors to promote their proliferation in plants. In this study, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable the targeted mutagenesis of MLO (mildew resistance Locus O) family genes that are thought to serve as S factors for powdery mildew fungi. Small deletions or insertions were induced in one or both alleles of two grapevine MLO genes, VvMLO3 and VvMLO4, in the transgenic plantlets of the powdery mildew-susceptible cultivar Thompson Seedless. The editing efficiency achieved with different CRISPR/Cas9 constructs varied from 0 to 38.5%. Among the 20 VvMLO3/4-edited lines obtained, one was homozygous for a single mutation, three harbored biallelic mutations, seven were heterozygous for the mutations, and nine were chimeric, as indicated by the presence of more than two mutated alleles in each line. Six of the 20 VvMLO3/4-edited grapevine lines showed normal growth, while the remaining lines exhibited senescence-like chlorosis and necrosis. Importantly, four VvMLO3-edited lines showed enhanced resistance to powdery mildew, which was associated with host cell death, cell wall apposition (CWA) and H2O2 accumulation. Taken together, our results demonstrate that CRISPR/Cas9 genome-editing technology can be successfully used to induce targeted mutations in genes of interest to improve traits of economic importance, such as disease resistance in grapevines.

Genome Wide Identification of the MLO Gene Family Associated with Powdery Mildew Resistance in Rubber Trees (Hevea brasiliensis)

Powdery mildew (PM) is one of the most destructive diseases affecting rubber trees (Hevea brasiliensis), leading to severe yield losses. Sulfur dusting is used to control the propagation of the disease, and no specific fungicides have been developed yet. Therefore, identification of genetic level disease resistance in the rubber trees is very crucial. In this study, we describe the whole-genome sequencing of a PM resistant rubber clone, RRIC 52, as well as a PM susceptible clone, PB 235, using massively parallel paired-end sequencing. The MLO (mildew resistance locus O) genes were identified and analyzed for their structural features, and the variations were compared in the MLO gene family between the two clones. Sequencing results showed that RRIC 52 contained 4,280,477 SNPs and 400,667 InDels, while PB 235 contained 3,651,524 SNPs and 318,899 InDels. We identified 34 MLO genes (HbMLO 1 to 34) in the rubber genome. Multiple sequence alignment identified the conserved MLO domain and its TM domain in all HbMLO proteins. Sequence analysis identified non-synonymous variations (NSVs) in 12 HbMLO proteins. Phylogenetic analysis of the HBMLO genes revealed seven different clades. Six HbMLO genes in Clade V were orthologous to Arabidopsis genes where PM interaction was previously identified. The outcomes of this study widen the understanding of the MLO gene family, which can be used in breeding disease resistant rubber varieties in the future.

Genome wide characterization revealed MnMLO2 and MnMLO6A as candidate genes involved in powdery mildew susceptibility in mulberry.

Mulberry is a fast growing economically important tree for sericulture industry and contains compounds for preventing and treating several diseases and ailments. The quality and quantity of mulberry leaf available to produce silk fibre and for medicinal purpose is greatly affected by number of foliar diseases, out of which powdery mildew is the major one. Imparting genetic resistance becomes an important approach in disease management in mulberry as spraying of fungicides has harmful effects on silkworm growth and development. Deployment of non-functional susceptible genes such as Mildew resistance Locus O (MLO) against powdery mildew in few crops stimulated to identify and characterize MLO genes in mulberry. In this study, genome wide analysis identified 16 MLO genes in Morus notabilis. Phylogenetic analysis found that MnMLO2, MnMLO6A, MnMLO6B, MnMLO12A and MnMLO12B clustered with functionally characterized MLOs associated with powdery mildew susceptibility in dicot species. Gene expression analysis indicated increased transcript abundance of MnMLO2, MnMLO6A, and MnMLO12A in response to powdery mildew infection. Further, conserved motifs exclusive to functionally characterized MLOs were identified in MnMLO1C, MnMLO2 and MnMLO6A proteins. Combined analysis of the phylogenetic relationship, conserved motif analysis and gene expression in response to infection identified MnMLO2 and MnMLO6A as potential candidate genes involved in powdery mildew susceptibility in mulberry. Identification and deployment of natural and induced mutations in the candidate genes can be useful for mulberry breeding programs to develop powdery mildew resistant varieties.

Arabidopsis mlo3 mutant plants exhibit spontaneous callose deposition and signs of early leaf senescence.

Arabidopsis thaliana mlo3 mutant plants are not affected in pathogen infection phenotypes but-reminiscent of mlo2 mutant plants-exhibit spontaneous callose deposition and signs of early leaf senescence. The family of Mildew resistance Locus O (MLO) proteins is best known for its profound effect on the outcome of powdery mildew infections: when the appropriate MLO protein is absent, the plant is fully resistant to otherwise virulent powdery mildew fungi. However, most members of the MLO protein family remain functionally unexplored. Here, we investigate Arabidopsis thaliana MLO3, the closest relative of AtMLO2, AtMLO6 and AtMLO12, which are the Arabidopsis MLO genes implicated in the powdery mildew interaction. The co-expression network of AtMLO3 suggests association of the gene with plant defense-related processes such as salicylic acid homeostasis. Our extensive analysis shows that mlo3 mutants are unaffected regarding their infection phenotype upon challenge with the powdery mildew fungi Golovinomyces orontii and Erysiphe pisi, the oomycete Hyaloperonospora arabidopsidis, and the bacterial pathogen Pseudomonas syringae (the latter both in terms of basal and systemic acquired resistance), indicating that the protein does not play a major role in the response to any of these pathogens. However, mlo3 genotypes display spontaneous callose deposition as well as signs of early senescence in 6- or 7-week-old rosette leaves in the absence of any pathogen challenge, a phenotype that is reminiscent of mlo2 mutant plants. We hypothesize that de-regulated callose deposition in mlo3 genotypes might be the result of a subtle transient aberration of salicylic acid-jasmonic acid homeostasis during development.

Genome-wide identification of mildew resistance locus O (MLO) genes in tree model poplar (Populus trichocarpa): powdery mildew management in woody plants

Poplars are economically important fast growing trees. They are exposed to broad range of fungal diseases like powdery mildew (PM). MLOs (mildew resistance locus O), as plant susceptibility genes, act as negative regulators and whose loss-of-functions confer complete resistance to PM disease. Herein, work identified the MLO gene family members in poplar, a woody model species. A total of 26 identified MLOs (annotated as PtMLO1–26) were distributed on 14 poplar chromosomes either individually or in groups of two to four. PtMLO genes encoded a polypeptide of 341–593 residues with a characteristic MLO domain structure. One tandem and eight segmental duplications were revealed in PtMLO genes. PtMLO proteins anchored at plasma membrane and had putative 5–9 TMDs with extracellular/cytosolic N- and C-terminuses. They were rich in leucine (9.1–12.9%), which is reported to play roles in defense response signaling. The C-terminal calmodulin-binding domain (CaMBD), reported to modulate the signaling mechanism in the defense response, was completely preserved in all PtMLOs, except PtMLO6. This domain was partially absent in PtMLO6 which is inferred to be a different MLO type or a pseudogene with a lost/impaired function in PM response. Besides, second and third cytoplasmic loops that are critical for PM-susceptibility were identified in PtMLOs. Particularly, PtMLO17, 18, 19, and 24 genes, inferred from Arabidopsis-poplar comparative phylogeny, were identified as potential candidates that may be involved in poplar-PM resistance. Notably, inductions of 14 PtMLO genes were detected in probes of microarray data such as GSE56865, GSE16417, and GSE23726 under different fungal infections indicating their involvements in plant defense. Overall, this work provided a basis for woody plant genomics for the effective and better management of poplar-PM disease.

Mildew Resistance Locus O Gene Cloning, Characterization, and Expression Pattern in Mulberry (Morus multicaulis) and Its Prokaryotic Expression in E. coli

MLO (mildew resistance locus O), which encodes a transmembrane protein 7TM, is considered to be a model plant gene suitable for studying broad-spectrum resistance. It is a negative regulator of powdery mildew resistance and thus has potential applications in plant breeding. In the present paper, a full cDNA sequence encoding MLO was cloned from the leaves of mulberry (Morus multicaulis) based on mulberry expressed sequence tags (EST), homologous cloning technology, and 5′-RLM-RACE using RT-PCR;the sequence was designated MMLO (GenBank accession no. KX683296). The full cDNA was 1943 bp in length with 5′-untranslated region (UTR) of 106 bp, 3′-UTR of 160 bp, and an open reading frame (ORF) of 1677 bp encoding a protein of 558 amino acids. The estimated molecular weight and isoelectric point (pI) of the putative protein were 62.48 kDa and 9.03, respectively. The MMLO protein had Mlo domain and belonged to the Mlo superfamily. Phylogenetic analysis based on the amino acid sequences encoded by the MLO gene from various species showed that mulberry was closely related to Eucalyptus grandis, Ziziphus jujube, and Juglansregia. Quantitative real-time PCR (qRT-PCR) analysis revealed that MMLO was expressed in all the tissues tested, including leaf, bud, fruit, stem, phloem, and xylem in mulberry with the highest expression in the phloem. The expression level of the mRNA increased and significantly changed under drought, cold, and salt stress treatments compared to the normal growth environment. The ORF segment of the MMLO was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. SDS-PAGE result revealed that fusion protein was successfully expressed. Overall, these results provide a better understanding of the molecular basis for the signal transduction mechanism during the stress responses in mulberry trees.

Identification and Expression Analysis of Fragaria Vesca MLO Genes Involved in Interaction with Powdery Mildew (Podosphaera Aphanis)

Strawberry powdery mildew, caused by Podosphaeraaphanis is a major fungal disease that affects strawberry yield and quality. In the model plant species Arabidopsis and the crop plants barley, tomato and pea, the Mildew resistance locus O (MLO) proteins have been found to be required for powdery mildew susceptibility. The present study, based on the sequence of a wild plum (Prunus americana) MLO protein, identified 16 MLO genes within the genome of woodland strawberry, Fragaria vesca and examined their expression pattern in response to powdery mildew infection in three diploid strawberry cultivars. Phylogenetic analysis showed that the FvMLO genes can be classified into six clades. Four FvMLO genes were grouped into clade III, which comprises MLO genes from Arabidopsis, tomato and grapevine that mediate powdery mildew susceptibility. A RNA-seq analysis of two diploid strawberry cultivars, F. vescassp. vesca accession Hawaii 4 (HW) and F. vesca f. semperflorens line “Yellow Wonder 5AF7” (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection showed the expression of 12 out of the 16 FvMLO genes. The comparison of Fragments Per Kilobase of transcript per Million mapped reads (FPKM values) detected by RNA-seq and expression values of qRT-PCR for FvMLO genes showed substantial agreement. The FvMLO3 gene, which was grouped in clade III and orthologous to the Arabidopsis,tomato and grapevine genes, was highly expressed in YW compared to other FvMLO genes across varieties. The results showed that FvMLO genes can be used as potential candidates to engineer powdery mildew resistance in strawberry based on MLO suppression or genome editing.

Arabidopsis thaliana MLO genes are expressed in discrete domains during reproductive development.

MLOs in Plant Reproduction. The MILDEW RESISTANCE LOCUS-O (MLO) protein family, comprised of 15 members, plays roles in diverse cell-cell communication processes such as powdery mildew susceptibility, root thigmomorphogenesis, and pollen tube reception. The NORTIA (NTA, AtMLO7) gene is expressed in the synergid cells of the female gametophyte where it functions in intercellular communication with the pollen tube. Discrepancies between previously published promoter::GUS and promoter::gene-GUS constructs expression patterns led us to explore the regulation of NTA expression. Here we found via NTApro::gNTA-GUS truncations that sequences within the NTA gene negatively regulate its expression in the stomata and carpel walls. This led to the hypothesis that other MLO family members may also have additional regulatory sequences within the gene. MLOpro::gMLO-GUS constructs were examined for each family member focusing specifically on flowers in order to determine whether other MLOs could play a role in reproductive cell-cell communication. Notably, several MLOs were expressed in the pollen, in the stigma, in the pollinated style, and in the synergids and central cell. These findings indicate that other MLOs in addition to NTA could play a role in reproduction. Previous studies on the MLO family showed that phylogenetically related MLOs had redundant functions in powdery mildew infection and root thigmomorphogenesis; however, MLO expression in reproductive tissues did not strictly follow phylogenetic relationships, indicating that MLOs from different evolutionary origins may have been recruited for function in sexual reproduction.

Genome-wide Identification, Classification and Expression Analysis of the Mildew Resistance Locus O ( MLO ) Gene Family in Sweet Orange ( Citrus sinensis )

ABSTRACT Mildew resistance Locus O (MLO), a gene family specific to plants, plays significant roles in the resistance to powdery mildew (PM) and response to a variety of abiotic stresses, plant growth and development. Despite their importance as barley, rice, wheat, few studies are reported in dicots except Arabidopsis; no global analysis has been performed in the burgeoning model fruit plant sweet orange (Citrus sinensis). The recent release of the genome sequences of C. sinensis provides an opportunity to conduct a comprehensive overview the evolution and features of the MLO gene family in sweet orange. In this study, amount to 14 members of the Citrus sinensis MLO gene (CisMLO) family according to their gene structures, conserved motifs, and similitude among their presumptive Arabidopsis and rice orthologs were identified in silico. Based on these analyses, all CisMLOs were grouped into six clades and expanded partly due to one tandem duplication and two segmental duplication events. Survey of their chromosomal distributions uncovered that 14 CisMLOs are localized across 6 chromosomes. Multiple-sequence alignments showed that 11 of them shared seven highly conserved transmembrane domains (TMs), while all of the sweet orange MLO proteins except CisMLO4/14 had a calmodulin-binding domain for MLO function. Expression analysis demonstrated that the MLO gene family has a diverse tissue-specific expression profiles in the sweet orange development and plays potential critical roles in stress responses. These findings will facilitate further studies of evolutionary pattern and biological functions of MLO genes in sweet orange.

A Systematic View of the MLO Family in Rice Suggests Their Novel Roles in Morphological Development, Diurnal Responses, the Light-Signaling Pathway, and Various Stress Responses.

The Mildew resistance Locus O (MLO) family is unique to plants, containing genes that were initially identified as a susceptibility factor to powdery mildew pathogens. However, little is known about the roles and functional diversity of this family in rice, a model crop plant. The rice genome has 12 potential MLO family members. To achieve systematic functional assignments, we performed a phylogenomic analysis by integrating meta-expression data obtained from public sources of microarray data and real-time expression data into a phylogenic tree. Subsequently, we identified 12 MLO genes with various tissue-preferred patterns, including leaf, root, pollen, and ubiquitous expression. This suggested their functional diversity for morphological agronomic traits. We also used these integrated transcriptome data within a phylogenetic context to estimate the functional redundancy or specificity among OsMLO family members. Here, OsMLO12 showed preferential expression in mature pollen; OsMLO4, in the root tips; OsMLO10, throughout the roots except at the tips; and OsMLO8, expression preferential to the leaves and trinucleate pollen. Of particular interest to us was the diurnal expression of OsMLO1, OsMLO3, and OsMLO8, which indicated that they are potentially significant in responses to environmental changes. In osdxr mutants that show defects in the light response, OsMLO1, OsMLO3, OsMLO8, and four calmodulin genes were down-regulated. This finding provides insight into the novel functions of MLO proteins associated with the light-responsive methylerythritol 4-phosphate pathway. In addition, abiotic stress meta-expression data and real-time expression analysis implied that four and five MLO genes in rice are associated with responses to heat and cold stress, respectively. Upregulation of OsMLO3 by Magnaporthe oryzae infection further suggested that this gene participates in the response to pathogens. Our analysis has produced fundamental information that will enhance future studies of the diverse developmental or physiological phenomena mediated by the MLO family in this model plant system.

멜론 흰가루병균 및 식물 호르몬 처리하에서 MLO 유전자군의 발현검정

Powdery mildew disease caused by Podosphaera xanthii is a major concern for Cucumis melo production worldwide. Knowledge on genetic behavior of the related genes and their modulating phytohormones often offer the most efficient approach to develop resistance against different diseases. Mildew Resistance Locus O (MLO) genes encode proteins with seven transmembrane domains that have significant function in plant resistance to powdery mildew fungus. We collected 14 MLO genes from ‘Melonomics’ database. Multiple sequence analysis of MLO proteins revealed the existence of both evolutionary conserved cysteine and proline residues. Moreover, natural genetic variation in conserved amino acids and their replacement by other amino acids are also observed. Real-time quantitative PCR expression analysis was conducted for the leaf samples of P. xanthii infected and phytohormones (methyl jasmonate and salicylic acid) treated plants in melon ‘SCNU1154’ line. Upon P. xanthii infection using 7 different races, the melon line showed variable disease reactions with respect to spread of infection symptoms and disease severity. Three out of 14 CmMLO genes were up-regulated and 7 were down-regulated in leaf samples in response to all races. The up- or down-regulation of the other 4 CmMLO genes was race-specific. The expression of 14 CmMLO genes under methyl jasmonate and salicylic acid application was also variable. Eleven CmMLO genes were up-regulated under salicylic acid treatment, and 7 were up-regulated under methyl jasmonate treatments in C. melo L. Taken together, these stress-responsive CmMLO genes might be useful resources for the development of powdery mildew disease resistant C. melo L.

Genome-wide identification of the mildew resistance locus O (MLO) gene family in novel cereal model species Brachypodium distachyon

Powdery mildew (PM) is an important plant fungal disease that adversely affects a broad range of angiosperm species, including grass families such as wheat and barley. The MLO (powdery mildew locus O) protein acts as a negative regulator in PM-resistance. Loss-of-function mutation in MLO shows complete resistance to PM disease. In this study, for the first time we reported MLO gene family members in Brachypodium distachyon, model species for grass. 11 well-conserved BdMLO genes were identified on all five chromosomes with a scattered occurrence rather than clustered. The subcellular localization and topology analyses confirmed that all BdMLO proteins anchored to plasma membrane. The seven trans-membrane (TM) and calmodulin-binding domain (CaMBD) sites were well conserved. Amino acid composition showed that BdMLO proteins were leucine-rich (9.9–13.1 %) except BdMLO5 and BdMLO8, which were alanine-rich (10.0 %) and serine-rich (8.7 %), respectively. In silico functional dissection of cis-acting elements revealed that BdMLOs were associated with mainly hormonal, stress, light response and tissue-specific signaling pathways. Phylogenetic relations of BdMLOs within distinct plant species (Arabidopsis, barley, wheat, maize, rice, tomato, pea, pepper and peach) were evaluated. Also, digital expressions of BdMLOs in drought, cold and pathogen infection conditions revealed stress-responsive MLO genes. Phylogenetic and expression analyses provided preliminary evidence that BdMLO2 could be the best susceptibility gene which may play important role in PM resistance. It was concluded that identification and characterization of MLO gene members in Brachypodium will provide essential knowledge for studying full-scale functional analysis of these genes in grass species.

Genome-wide identification, characterization, and expression analysis of the MLO gene family in Cucumis sativus

Mildew resistance locus o (MLO) is a plant-specific seven-transmembrane (TM) gene family. Several studies have revealed that certain members of the MLO gene family mediate powdery mildew susceptibility in three plant species, namely, Arabidopsis, barley, and tomato. The sequenced cucumber genome provides an opportunity to conduct a comprehensive overview of the MLO gene family. Fourteen genes (designated CsMLO01 through CsMLO14) have been identified within the Cucumis sativus genome by using an in silico cloning method with the MLO amino acid sequences of Arabidopsis thaliana and rice as probes. Sequence alignment revealed that numerous features of the gene family, such as TMs, a calmodulin-binding domain, peptide domains I and II, and 30 important amino acid residues for MLO function, are well conserved. Phylogenetic analysis of the MLO genes from cucumber and other plant species reveals seven different clades (I through VII). Three of these clades comprised MLO genes from A. thaliana, rice, maize, and cucumber, suggesting that these genes may have evolved after the divergence of monocots and dicots. In silico mapping showed that these CsMLOs were located on chromosomes 1, 2, 3, 4, 5, and 6 without any obvious clustering, except CsMLO01. To our knowledge, this paper is the first comprehensive report on MLO genes in C. sativus. These findings will facilitate the functional characterization of the MLOs related to powdery mildew susceptibility and assist in the development of disease resistance in cucumber.

The pepper MLO gene, CaMLO2, is involved in the susceptibility cell‐death response and bacterial and oomycete proliferation

Loss-of-function alleles of the mildew resistance locus O (MLO) gene provide broad-spectrum powdery mildew disease resistance. Here, we identified a pepper (Capsicum annuum) MLO gene (CaMLO2) that is transcriptionally induced by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Topology and subcellular localization analyses reveal that CaMLO2 is a plasma membrane-anchored and amphiphilic Ca²⁺-dependent calmodulin-binding protein. CaMLO2 expression is up-regulated by Xcv and salicylic acid, as well as abiotic stresses. Silencing of CaMLO2 in pepper plants confers enhanced resistance against virulent Xcv, but not against avirulent Xcv. This resistance is accompanied by a compromised susceptibility cell-death response and reduced bacterial growth, as well as an accelerated reactive oxygen species burst. Virulent Xcv infection drastically induces expression of the salicylic acid-dependent defense marker gene CaPR1 in CaMLO2-silenced leaves. CaMLO2 over-expression in Arabidopsis enhances susceptibility to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. Leaves of plants over-expressing CaMLO2 exhibit a susceptibility cell-death response and high bacterial growth during virulent Pst DC3000 infection. These are accompanied by enhanced electrolyte leakage but compromised induction of some defense response genes and the reactive oxygen species. Together, our results suggest that CaMLO2 is involved in the susceptibility cell-death response and bacterial and oomycete proliferation in pepper and Arabidopsis.