MLO Gene Family Research Articles

Computational identification, characterization, and expression analysis of MLO genes in two powdery mildew resistant and susceptible lines of sunflower

Sunflower is one of the major oilseeds producing crops in the world. However, the crop production is affected by powdery mildew in the tropics, caused by a fungal pathogen Golovinomyces latisporus. A step towards developing genetic resistance is by identifying potential genes involved. The MLO genes when silenced provide durable and broad-spectrum resistance. In this study, we identified the MLO gene family in the genome of Sunflower using computational methods. We analyzed the chromosomal distribution, gene structure conserved motifs, and phylogenetic relationship of sunflower MLO genes with MLO genes of other species. A total of 38 MLO genes were identified which are distributed unevenly on the chromosomes with varied intron/exon gene structures. These were phylogenetically distributed into six different groups. The cis elements identified in the promoter region had hormone responsive, stress responsive, development related elements along with transcription factors. We developed recombinant inbred lines by crossing H. annuus and H. praecox (F7 generation). Further, we performed RNAseq analysis of transcriptomes sequenced from powdery mildew infected leaf samples of PMR-16 (resistant) and PMS-27 (susceptible) varieties of the recombinant inbred lines. Expression analysis revealed 12 differentially expressed genes between both varieties. Our results can be used as a reference for future functional studies.

Functional and evolutionary study of MLO gene family in the regulation of Sclerotinia stem rot resistance in Brassica napus L.

BackgroundOilseed rape (Brassica napus L.) is known as one of the most important oilseed crops cultivated around the world. However, its production continuously faces a huge challenge of Sclerotinia stem rot (SSR), a destructive disease caused by the fungus Sclerotinia sclerotiorum, resulting in huge yield loss annually. The SSR resistance in B. napus is quantitative and controlled by a set of minor genes. Identification of these genes and pyramiding them into a variety are a major strategy for SSR resistance breeding in B. napus.ResultsHere, we performed a genome-wide association study (GWAS) using a natural population of B. napus consisting of 222 accessions to identify BnaA08g25340D (BnMLO2_2) as a candidate gene that regulates the SSR resistance. BnMLO2_2 was a member of seven homolog genes of Arabidopsis Mildew Locus O 2 (MLO2) and the significantly SNPs were mainly distributed in the promoter of BnMLO2_2, suggesting a role of BnMLO2_2 expression level in the regulation of SSR resistance. We expressed BnMLO2_2 in Arabidopsis and the transgenic plants displayed an enhanced SSR resistance. Transcriptome profiling of different tissues of B. napus revealed that BnMLO2_2 had the most expression level in leaf and silique tissues among all the 7 BnMLO2 members and also expressed higher in the SSR resistant accession than in the susceptible accession. In Arabidopsis, mlo2 plants displayed reduced resistance to SSR, whereas overexpression of MLO2 conferred plants an enhanced SSR resistance. Moreover, a higher expression level of MLO2 showed a stronger SSR resistance in the transgenic plants. The regulation of MLO2 in SSR resistance may be associated with the cell death. Collinearity and phylogenetic analysis revealed a large expansion of MLO family in Brassica crops.ConclusionOur study revealed an important role of BnMLO2 in the regulation of SSR resistance and provided a new gene candidate for future improvement of SSR resistance in B. napus and also new insights into understanding of MLO family evolution in Brassica crops.

Genome-wide identification, evolution, and expression analysis of MLO gene family in melon (Cucumis melo L.).

Powdery mildew (PM) is one of the main fungal diseases that appear during the cultivation of the melon fruit crop. Mildew Resistance Locus "O" (MLO) is known as a gene family and has seven conserved transmembrane domains. An induced functional loss of a specific MLO gene could mainly confer PM resistance to melons. However, the genomic structure of MLO genes and its main role in PM resistance still remain unclear in melon. In this study, bioinformatic analysis identified a total of 14 MLO gene family members in the melon genome sequence, and these genes were distributed in an uneven manner on eight chromosomes. The phylogenetic analysis divided the CmMLO genes into five different clades, and gene structural analysis showed that genes in the same clade had similar intron and exon distribution patterns. In addition, by cloning the CmMLO gene sequence in four melon lines, analyzing the CmMLO gene expression pattern after infection, and making microscopic observations of the infection pattern of PM, we concluded that the CmMLO5 (MELO3C012438) gene plays a negative role in regulating PM-resistance in the susceptible melon line (Topmark), and the critical time point for gene function was noticed at 24 and 72 hours after PM infection. The mutational analysis exhibited a single base mutation at 572 bp, which further results in loss of protein function, thus conferring PM resistance in melon. In summary, our research evidence provides a thorough understanding of the CmMLO gene family and demonstrates their potential role in disease resistance, as well as a theoretical foundation for melon disease resistance breeding.

Analysis of allelic variants of RhMLO genes in rose and functional studies on susceptibility to powdery mildew related to clade V homologs

Key messageRose has 19 MLO genes. Of these, RhMLO1 and RhMLO2 were shown to be required for powdery mildew infection, which suggests their potential as susceptibility targets towards disease resistance.Powdery mildew, caused by Podosphaera pannosa, is one of the most serious and widespread fungal diseases for roses, especially in greenhouse-grown cut roses. It has been shown that certain MLO genes are involved in powdery mildew susceptibility and that loss of function in these genes in various crops leads to broad-spectrum, long-lasting resistance against this fungal disease. For this reason, these MLO genes are called susceptibility genes. We carried out a genome-wide identification of the MLO gene family in the Rosa chinensis genome, and screened for allelic variants among 22 accessions from seven different Rosa species using re-sequencing and transcriptome data. We identified 19 MLO genes in rose, of which four are candidate genes for functional homologs in clade V, which is the clade containing all dicot MLO susceptibility genes. We detected a total of 198 different allelic variants in the set of Rosa species and accessions, corresponding to 5–15 different alleles for each of the genes. Some diploid Rosa species shared alleles with tetraploid rose cultivars, consistent with the notion that diploid species have contributed to the formation of tetraploid roses. Among the four RhMLO genes in clade V, we demonstrated using expression study, virus-induced gene silencing as well as transient RNAi silencing that two of them, RhMLO1 and RhMLO2, are required for infection by P. pannosa and suggest their potential as susceptibility targets for powdery mildew resistance breeding in rose.

Evolutionary conservation of MLO gene promoter signatures

BackgroundPowdery mildew (PM) is a widespread fungal disease of plants in temperate climates, causing significant economic losses in agricultural settings. Specific homologs of the MLO gene family are PM susceptibility factors, as their loss-of function results in durable PM resistance (mlo resistance) in several plant species. The role of MLO susceptibility genes in plant-pathogen interactions is still elusive, however it is known that they are strongly upregulated following PM infection.ResultsIn this study, we investigated the structure of 414 Putative Promoter Regions (PPRs) of MLO genes and highlighted motif and regulatory element patterns related to genomic relationships among species and phylogenetic distance among homologs. A TC box-like motif and a thymine-rich motif were found to be overrepresented in MLO genes transcriptionally upregulated upon infection with PM fungi. As proof of concept, we showed that the expression of a melon (Cucumis melo L.) gene enriched for the motifs above mentioned was strongly upregulated upon infection with the PM fungus Podosphaera xanthii.ConclusionWhile identifying a candidate MLO susceptibility gene in melon, this study provides insight on the transcriptional control of MLO genes and indicates diagnostic features useful to identify MLO susceptibility genes across species affected by the PM disease.

Genome-wide identification and description of MLO family genes in pumpkin (Cucurbita maxima Duch.)

The Mildew resistance locus o (Mlo) is a plant-specific gene family that encodes a protein with seven-transmembrane (TM) domains that play an important role in plant resistance to powdery mildew (PM). PM caused by Podosphaera xanthii is a widespread plant disease and represents the major fungal threat for many Cucurbits. The recently reported Cucurbita maxima genome sequence data provides an opportunity to identify and characterize the MLO gene family in this species. A total of twenty genes designated CmaMLO1 to CmaMLO20 have been identified using an in silico cloning method with the MLO gene sequences of Cucumis sativus, Cucumis melo, Citrullus lanatus, and Cucurbita pepo as probes. These CmaMLOs were evenly distributed on 15 of the 20 C. maxima chromosomes without any obvious clustering. Multiple sequence alignment showed that common structural features of MLO gene family, such as TM domains, a calmodulin-binding domain, and 30 important amino acid residues for MLO function, were well conserved. Phylogenetic analysis of the CmaMLO genes and other plant species revealed seven different clades (I through VII); however, clade IV is specific to monocots (rice, barley and wheat). Expression analysis showed that four of the five CmaMLO genes that belonged to clade V were up-regulated in pumpkins infected with P. xanthii. Phylogenetic and expression analysis provided preliminary evidence that these five genes could be susceptibility genes that are important for PM resistance. This study is the first comprehensive report on MLO genes in C. maxima to our knowledge. These findings will facilitate the functional analysis of MLOs related to PM susceptibility and are valuable resources for the development of disease resistance in pumpkin.

A genome-wide identification and comparative analysis of the lentil MLO genes.

Powdery mildew is a widespread fungal plant disease that can cause significant losses in many crops. Some MLO genes (Mildew resistance locus O) have proved to confer a durable resistance to powdery mildew in several species. Resistance granted by the MLO gene family members has prompted an increasing interest in characterizing these genes and implementing their use in plant breeding. Lentil (Lens culinaris Medik.) is a widely grown food legume almost exclusively consumed as dry seed with an average world production of 4.5 million tons. Powdery mildew causes severe losses on certain lentil cultivars under particular environmental conditions. Data mining of the lentil CDC Redberry draft genome allowed to identify up to 15 gene sequences with homology to known MLO genes, designated as LcMLOs. Further characterization of these gene sequences and their deduced protein sequences demonstrated conformity with key MLO protein characteristics such as the presence of transmembrane and calmodulin binding domains, as well as that of other conserved motifs. Phylogenetic and other comparative analyses revealed that LcMLO1 and LcMLO3 are the most likely gene orthologs related to powdery mildew response in other species, sharing a high similarity with other known resistance genes of dicot species, such as pea PsMLO1 and Medicago truncatula MtMLO1 and MtMLO3. Sets of primers were designed as tools to PCR amplify the genomic sequences of LcMLO1 and LcMLO3, also to screen lentil germplasm in search of resistance mutants. Primers were used to obtain the complete sequences of these two genes in all of the six wild lentil relatives. Respective to each gene, all Lens sequences shared a high similarity. Likewise, we used these primers to screen a working collection of 58 cultivated and 23 wild lentil accessions in search of length polymorphisms present in these two genes. All these data widen the insights on this gene family and can be useful for breeding programs in lentil and close related species.

Frequency of a natural truncated allele of MdMLO19 in the germplasm of Malus domestica

Podosphaera leucotricha is the causal agent of powdery mildew (PM) in apple. To reduce the amount of fungicides required to control this pathogen, the development of resistant apple cultivars should become a priority. Resistance to PM was achieved in various crops by knocking out specific members of the MLO gene family that are responsible for PM susceptibility (S-genes). In apple, the knockdown of MdMLO19 resulted in PM resistance. However, since gene silencing technologies such as RNAi are perceived unfavorably in Europe, a different approach that exploits this type of resistance is needed. This work evaluates the presence of non-functional naturally occurring alleles of MdMLO19 in apple germplasm. The screening of the re-sequencing data of 63 apple individuals led to the identification of 627 single nucleotide polymorphisms (SNPs) in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18, and MdMLO19), 127 of which were located in exons. The T-1201 insertion of a single nucleotide in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino acids, including the calmodulin-binding domain. The presence of the insertion was evaluated in 115 individuals. It was heterozygous in 64 and homozygous in 25. Twelve of the 25 individuals carrying the insertion in homozygosity were susceptible to PM. After barley, pea, cucumber, and tomato, apple would be the fifth species for which a natural non-functional mlo allele has been found.

IDENTIFICATION AND FUNCTIONAL INFERENCE ON THE MLO- FAMILY IN VIRIDIPLANTAE

Powdery mildew (PM) is a widespread plant disease of temperate climates caused by ascomycete fungi of the order Erysiphales. PM is an important agricultural issue since it can cause significant economic losses. Specific members of the MLO gene family act as susceptibility factors towards the PM disease. A step towards the stability of crop productions would be thus the characterization of MLO genes at the genomic level. We carried out a genome-wide characterization of the MLO gene family in twenty-three plant and two algal genomes providing manual curated MLO protein catalogues. In total, 180 novel proteins containing the MLO domain were identified. Evolutionary history and phylogenetic relationships were studied through maximum likelihood analysis. This highlighted eight different clades, including a new monocot-specific clade (VIII) identified for the first time. In addition, 15 and 67 putative PM susceptibility genes, clustering in clade IV and V, respectively, were identified. Results of this work may help to address further biological questions concerning MLOs involved in PM susceptibility. In follow-up studies, it could be investigated whether the silencing or loss-of-function mutations in one or more of these candidate genes may lead to PM resistance.

Mining the Cicer arietinum genome for the mildew locus O (Mlo) gene family and comparative evolutionary analysis of the Mlo genes from Medicago truncatula and some other plant species.

The mildew locus O (Mlo) gene family is ubiquitous in land plants. Some members of this gene family are involved in negative regulation of powdery mildew resistance, while others are involved in several other biological functions. Mlo proteins have characteristic seven transmembrane domains and a calmodulin-binding domain at their C-termini, and are associated with plasma membrane. The Mlo gene family has been studied in several economically important cereals, but little information is available on this gene family in the important legumes, Medicago truncatula Gaertn. and Cicer arietinum L. We carried out a comprehensive and comparative investigation of the Mlo gene family in these two species using the genome sequences available at the M. truncatula genome database (Mt v4.0) and NCBI (C. arietinum). A genome-wide homology-based search using Arabidopsis Mlo proteins as query identified 16 MtMlo (M. truncatula Mlo) and 14 CarMlo (C. arietinum Mlo) genes. The MtMlo and CarMlo genes had comparable gene structure, protein sequence and topology. Their chromosomal locations indicated the occurrence of extensive reorganization in the genomes of the two species after their divergence from the common ancestor. A multiple sequence alignment of 53 Mlo proteins from these two and several other species showed a highly conserved sequence block of seven amino acids, viz., L-ETPTW, towards their N-termini. The evolutionary phylogenetic analysis grouped the MtMlo and CarMlo members into four clusters, and most of the MtMlo and CarMlo members formed one-to-one ortholog pairs. The ka/ks analyses indicated that the MtMlo and CarMlo genes are subjected to intense purifying selection.

Comparative analysis of genome-wide Mlo gene family in Cajanus cajan and Phaseolus vulgaris.

The Mlo gene was discovered in barley because the mutant 'mlo' allele conferred broad-spectrum, non-race-specific resistance to powdery mildew caused by Blumeria graminis f. sp. hordei. The Mlo genes also play important roles in growth and development of plants, and in responses to biotic and abiotic stresses. The Mlo gene family has been characterized in several crop species, but only a single legume species, soybean (Glycine max L.), has been investigated so far. The present report describes in silico identification of 18 CcMlo and 20 PvMlo genes in the important legume crops Cajanus cajan (L.) Millsp. and Phaseolus vulgaris L., respectively. In silico analysis of gene organization, protein properties and conserved domains revealed that the C. cajan and P. vulgaris Mlo gene paralogs are more divergent from each other than from their orthologous pairs. The comparative phylogenetic analysis classified CcMlo and PvMlo genes into three major clades. A comparative analysis of CcMlo and PvMlo proteins with the G. max Mlo proteins indicated close association of one CcMlo, one PvMlo with two GmMlo genes, indicating that there was no further expansion of the Mlo gene family after the separation of these species. Thus, most of the diploid species of eudicots might be expected to contain 15-20 Mlo genes. The genes CcMlo12 and 14, and PvMlo11 and 12 are predicted to participate in powdery mildew resistance. If this prediction were verified, these genes could be targeted by TILLING or CRISPR to isolate powdery mildew resistant mutants.

TARGETED MUTAGENESIS OF MLO-HOMOLOGOUS GENES IN THE ROSE GENOME

Powdery mildew caused by Podosphaera pannosa is one of the most severe diseases of roses. Especially for greenhouse production of cut roses it depicts a significant problem. Biology and race structure of P. pannosa limit the effectiveness of race-specific, monogenic resistances making the application of fungicides indispensable. However, this application is not very desirable because of high costs, increasing legal restrictions and public concerns. An alternative to overcome this problem can be the mildew resistance locus o (mlo)-mediated resistance. The mlo-based resistance has been characterized in barley, A. thaliana, tomato and pea where the loss-of-function of specific members of the MLO gene-family leads to a recessive and durable broad-spectrum resistance. In rose four MLO homologs (RhMLO1, RhMLO2, RhMLO3 and RhMLO4) closely related to the functional ones in A. thaliana, pea and tomato have been sequenced and mapped. The aim of this work is a reverse genetics approach to verify the functionality of the four mentioned rose MLOs regarding the mediation of susceptibility towards P. pannosa. Therefore, stable transgenics of the tetraploid Rosa hybrid cultivar ‘Pariser Charme’ expressing an RNAi construct for all four genes are produced. Several shoots could be regenerated and transferred to soil and first analyses to determine the transgenic character of the plants have been performed.

Penetration resistance to Erysiphe pisi in pea mediated by er1 gene is associated with protein cross-linking but not with callose apposition or hypersensitive response

Powdery mildew caused by Erysiphe pisi DC is an important pea disease which causes high yield and quality losses. The most efficient way to control this disease is the use of resistant cultivars. Three genes of resistance are known so far in pea, namely er1, er2 and Er3. The most widely deployed gene in pea cultivars worldwide is er1. Resistance conferred by er1 is complete and considered to be durable, being recently demonstrated that er1 is in fact a member of the mlo gene family. Resistance is caused by colony abortion albeit the responsible cellular mechanisms causing this colony abortion are not known. In this study, the presence of different mechanisms related with colony abortion, such as callose apposition, protein cross-linking and hypersensitive response was quantified in a resistant pea er1 line compared to a susceptible check. Our histological studies showed that protein cross-linking, plays a determinant role in the penetration resistance conferred by er1, whereas callose apposition or hypersensitive response played little role.

Characterization of the MLO gene family in Rosaceae and gene expression analysis in Malus domestica.

BackgroundPowdery mildew (PM) is a major fungal disease of thousands of plant species, including many cultivated Rosaceae. PM pathogenesis is associated with up-regulation of MLO genes during early stages of infection, causing down-regulation of plant defense pathways. Specific members of the MLO gene family act as PM-susceptibility genes, as their loss-of-function mutations grant durable and broad-spectrum resistance.ResultsWe carried out a genome-wide characterization of the MLO gene family in apple, peach and strawberry, and we isolated apricot MLO homologs through a PCR-approach. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Homologs that are candidates for being PM susceptibility genes were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen Podosphaera leucotricha.ConclusionsGenomic tools available for Rosaceae were exploited in order to characterize the MLO gene family. Candidate MLO susceptibility genes were identified. In follow-up studies it can be investigated whether silencing or a loss-of-function mutations in one or more of these candidate genes leads to PM resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-618) contains supplementary material, which is available to authorized users.

Comparative phylogenetic analysis of genome-wide Mlo gene family members from Glycine max and Arabidopsis thaliana

Powdery mildew locus O (Mlo) gene family is one of the largest seven transmembrane protein-encoding gene families. The Mlo proteins act as negative regulators of powdery mildew resistance and a loss-of-function mutation in Mlo is known to confer broad-spectrum resistance to powdery mildew. In addition, the Mlo gene family members are known to participate in various developmental and biotic and abiotic stress response-related pathways. Therefore, a genome-wide similarity search using the characterized Mlo protein sequences of Arabidopsis thaliana was carried out to identify putative Mlo genes in soybean (Glycine max) genome. This search identified 39 Mlo domain containing protein-encoding genes that were distributed on 15 of the 20 G. max chromosomes. The putative promoter regions of these Mlo genes contained response elements for different external stimuli, including different hormones and abiotic stresses. Of the 39 GmMlo proteins, 35 were rich (8.7-13.1 %) in leucine, while five were serine-rich (9.2-11.9 %). Furthermore, all the GmMlo members were localized in the plasma membrane. Phylogenetic analysis of the GmMlo and the AtMlo proteins classified them into three main clusters, and the cluster I comprised two sub-clusters. Multiple sequence alignment visualized the location of seven transmembrane domains, and a conserved CaM-binding domain. Some of the GmMlo proteins (GmMlo10, 20, 22, 23, 32, 36, 37) contained less than seven transmembrane domains. The motif analysis yielded 27 motifs; out of these, motif 2, the only motif present in all the GmMlos, was highly conserved and three amino acid residues were essentially invariant. Five of the GmMlo members were much smaller in size; presumably they originated through deletion following a gene duplication event. The presence of a large number of GmMlo members in the G. max genome may be due to its paleopolyploid nature and the large genome size as compared to that of Arabidopsis. The findings of this study may further help in characterization and isolation of individual GmMlo members.

Loss of function in Mlo orthologs reduces susceptibility of pepper and tomato to powdery mildew disease caused by Leveillula taurica.

Powdery mildew disease caused by Leveillula taurica is a serious fungal threat to greenhouse tomato and pepper production. In contrast to most powdery mildew species which are epiphytic, L. taurica is an endophytic fungus colonizing the mesophyll tissues of the leaf. In barley, Arabidopsis, tomato and pea, the correct functioning of specific homologues of the plant Mlo gene family has been found to be required for pathogenesis of epiphytic powdery mildew fungi. The aim of this study was to investigate the involvement of the Mlo genes in susceptibility to the endophytic fungus L. taurica. In tomato (Solanum lycopersicum), a loss-of-function mutation in the SlMlo1 gene results in resistance to powdery mildew disease caused by Oidium neolycopersici. When the tomato Slmlo1 mutant was inoculated with L. taurica in this study, it proved to be less susceptible compared to the control, S. lycopersicum cv. Moneymaker. Further, overexpression of SlMlo1 in the tomato Slmlo1 mutant enhanced susceptibility to L. taurica. In pepper, the CaMlo2 gene was isolated by applying a homology-based cloning approach. Compared to the previously identified CaMlo1 gene, the CaMlo2 gene is more similar to SlMlo1 as shown by phylogenetic analysis, and the expression of CaMlo2 is up-regulated at an earlier time point upon L. taurica infection. However, results of virus-induced gene silencing suggest that both CaMlo1 and CaMlo2 may be involved in the susceptibility of pepper to L. taurica. The fact that overexpression of CaMlo2 restored the susceptibility of the tomato Slmlo1 mutant to O. neolycopersici and increased its susceptibility to L. taurica confirmed the role of CaMlo2 acting as a susceptibility factor to different powdery mildews, though the role of CaMlo1 as a co-factor for susceptibility cannot be excluded.

Genome-scale identification of MLO domain-containing genes in soybean (<i>Glycine max</i> L. Merr.)

In plants, powdery-mildew-resistance locus o (Mlo) genes encode proteins that are calmodulin-binding proteins involved in a variety of cellular processes. However, systematic characterization of this gene family in soybean (Glycine max L. Merr.) has not been yet reported. In this study, we identified MLO domain-contained members in soybean and examined their expression under phytohormone treatment and abiotic stress conditions. A total of 20 soybean Mlo genes were identified (GmMlo1-20), which are distributed on 13 chromosomes, and display diverse exon-intron structures. Phylogenetic analysis indicated that the Mlo family can be classified into four subfamilies. Sequence comparison was used to reveal the conserved calmodulin-binding domain (CaMBD) in GmMLO proteins. The expression of GmMlo genes was influenced by various phytohormone treatments and abiotic stresses, suggesting that these Mlo genes have various roles in the response of soybean to environmental stimuli. Promoter sequence analysis revealed an overabundance of stress and/or phytohormone-related cis-elements in GmMlo genes. These data provide important clues for elucidating the functions of genes of the Mlo gene family.

Pea powdery mildew er1 resistance is associated to loss-of-function mutations at a MLO homologous locus.

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F(2) individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection.

Molecular evolution of the MLO gene family in Oryza sativa and their functional divergence

The present study identified 12 MLO genes in rice that were located on chromosomes 1, 2, 3, 4, 5, 6, 10, and 11 respectively without any obvious clustering. On a genome scale we showed that the expansion of rice MLO gene family was primarily attributed to segmental duplication produced by polyploidy, rather than through tandem amplification. Gene conversion events should also play important roles in the evolution of MLO genes. The results of relative rate ratio test and maximum likelihood analysis suggested that positive selection should have occurred after gene duplication and/or speciation, prompting the formation of distinct MLO subfamilies. Functional divergence analysis provided statistical evidence for shifted evolutionary rate after gene duplication. Compared to extracellular loop 3 and Ca 2+-binding domain, much stronger functional constraints should impose on intracellular loop 2, although all of the three regions might be under purifying selection. The sliding window analysis of d N/ d S ratio values identified one sequence region where strong functional constraints must impose on, and consequently should be crucial for functionality of MLO genes.

Molecular phylogeny and evolution of the plant-specific seven-transmembrane MLO family.

Homologues of barley Mlo encode the only family of seven-transmembrane (TM) proteins in plants. Their topology, subcellular localization, and sequence diversification are reminiscent of those of G-protein coupled receptors (GPCRs) from animals and fungi. We present a computational analysis of MLO family members based on 31 full-size and 3 partial sequences, which originate from several monocot species, the dicot Arabidopsis thaliana, and the moss Ceratodon purpureus. This enabled us to date the origin of the Mlo gene family back at least to the early stages of land plant evolution. The genomic organization of the corresponding genes supports a monophyletic origin of the Mlo gene family. Phylogenetic analysis revealed five clades, of which three contain both monocot and dicot members, while two indicate class-specific diversification. Analysis of the ratio of nonsynonymous-to-synonymous changes in coding sequences provided evidence for functional constraint on the evolution of the DNA sequences and purifying selection, which appears to be reduced in the first extracellular loop of 12 closely related orthologues. The 31 full-size sequences were examined for potential domain-specific intramolecular coevolution. This revealed evidence for concerted evolution of all three cytoplasmic domains with each other and the C-terminal cytoplasmic tail, suggesting interplay of all intracellular domains for MLO function.