Mildew Resistance Locus Research Articles

An integrative AmpSeq platform for highly multiplexed marker-assisted pyramiding of grapevine powdery mildew resistance loci

Resistance breeding often requires the introgression and tracking of resistance loci from wild species into domesticated backgrounds, typically with the goal of pyramiding multiple resistance genes, to provide durable disease resistance to breeding selections and ultimately cultivars. While molecular markers are commonly used to facilitate these efforts, high genetic diversity and divergent marker technologies can complicate marker-assisted breeding strategies. Here, amplicon sequencing (AmpSeq) was used to integrate SNP markers with dominant presence/absence markers derived from genotyping-by-sequencing and other genotyping technologies, for the simultaneous tracking of five loci for resistance to grapevine powdery mildew. SNP haploblocks defined the loci for REN1, REN2 and REN3, which confer quantitative resistance phenotypes that are challenging to measure via field ratings of natural infections. Presence/absence markers for RUN1 and REN4 were validated to predict qualitative resistance phenotypes and corresponded with previous presence/absence fluorescent electrophoretic assays. Thus, 37 AmpSeq-derived markers were identified for the five loci, and markers for REN1, REN2, REN4 and RUN1 were used for multiplexed screening and selection within diverse breeding germplasm. Poor transferability of SNP markers indicated imperfect marker-trait association in some families. Together, AmpSeq SNP haploblocks and presence/absence markers provide a high-throughput, cost-effective tool to integrate divergent technologies for marker-assisted selection and genetic analysis of introgressed disease resistance loci in grapevine.

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.

MILDEW RESISTANCE LOCUS O Function in Pollen Tube Reception Is Linked to Its Oligomerization and Subcellular Distribution.

Sexual reproduction in flowering plants requires communication between synergid cells and a tip-elongating pollen tube (PT) for the successful delivery of sperm cells to the embryo sac. The reception of the PT relies on signaling within the synergid cell that ultimately leads to the degeneration of the receptive synergid and PT rupture, releasing the sperm cells for double fertilization. In Arabidopsis (Arabidopsis thaliana), NORTIA, a member of the MILDEW RESISTANCE LOCUS O (MLO) family of proteins, plays a critical role in the communication processes regulating PT reception. In this study, we determined that MLO function in PT reception is dependent on MLO protein localization into a Golgi-associated compartment before PT arrival, indicating that PT-triggered regulation of the synergid secretory system is important for synergid function during pollination. Additionally, a structure-function analysis revealed that MLO homooligomerization, mediated by the amino-terminal region of the protein, and carboxyl-terminal tail identity both contribute to MLO activity during PT reception.

Key Components of Different Plant Defense Pathways Are Dispensable for Powdery Mildew Resistance of the Arabidopsis mlo2 mlo6 mlo12 Triple Mutant.

Loss of function mutations of particular plant MILDEW RESISTANCE LOCUS O (MLO) genes confer durable and broad-spectrum penetration resistance against powdery mildew fungi. Here, we combined genetic, transcriptomic and metabolomic analyses to explore the defense mechanisms in the fully resistant Arabidopsis thaliana mlo2 mlo6 mlo12 triple mutant. We found that this genotype unexpectedly overcomes the requirement for indolic antimicrobials and defense-related secretion, which are critical for incomplete resistance of mlo2 single mutants. Comparative microarray-based transcriptome analysis of mlo2 mlo6 mlo12 mutants and wild type plants upon Golovinomyces orontii inoculation revealed an increased and accelerated accumulation of many defense-related transcripts. Despite the biotrophic nature of the interaction, this included the non-canonical activation of a jasmonic acid/ethylene-dependent transcriptional program. In contrast to a non-adapted powdery mildew pathogen, the adapted powdery mildew fungus is able to defeat the accumulation of defense-relevant indolic metabolites in a MLO protein-dependent manner. We suggest that a broad and fast activation of immune responses in mlo2 mlo6 mlo12 plants can compensate for the lack of single or few defense pathways. In addition, our results point to a role of Arabidopsis MLO2, MLO6, and MLO12 in enabling defense suppression during invasion by adapted powdery mildew fungi.

Behavior of grape breeding lines with distinct resistance alleles to downy mildew (Plasmopara viticola)

Abstract Downy mildew (Plasmopara viticola) is the main grapevine disease in humid regions. In the present investigation, marker-assisted selection (MAS) was used to develop grapevine lines homozygous in loci Rpv1 and Rpv3 for resistance against P. viticola. The experimental populations UFSC-2013-1 (n = 420) and UFSC-2013-2 (n = 237) were obtained by self-pollination of two 𝐅 𝟏 full-sib plants, originated from a cross between two distinct breeding lines containing the downy mildew resistance loci Rpv1 and Rpv3 in heterozygosity. The two experimental populations were genotyped with four microsatellite markers flanking the two downy mildew resistance loci. Among 637 genotyped plants, 300 (48.2%) were homozygous for at least one resistance locus and 10 (1.57%) were homozygous for both Rpv1 and Rpv3 alleles. These 10 plants challenged with P. viticola inoculum showed a clearly enhanced level of resistance. These plants have a great potential as resistance donors in grapevine breeding.

Mapping and validation of powdery mildew resistance loci from spring wheat cv. Naxos with SNP markers

Powdery mildew, caused by Blumeria graminis f.sp. tritici, is a major wheat disease in maritime and temperate climates. Breeding for race-non-specific or partial resistance is a cost-effective and environmentally friendly disease control strategy. The German spring wheat cultivar Naxos has proven to be a good source for partial resistance to powdery mildew. The objectives of the present study were to map the resistance loci in Naxos with use of high-density SNP markers in the Shanghai3/Catbird x Naxos inbred line population and validate the results in a different genetic background; Soru#1 x Naxos. Both populations were genotyped with the Illumina iSelect 90K wheat chip, and integrated linkage maps developed by inclusion of previously genotyped SSR and DArT markers. With the new linkage maps, we detected a total of 12 QTL for powdery mildew resistance in Shanghai3/Catbird x Naxos, of which eight were derived from Naxos. Previously reported QTL on chromosome arms 1AS and 2BL were more precisely mapped and the SNP markers enabled discovery of new QTL on 1AL, 2AL, 5AS and 5AL. In the Soru#1 x Naxos population, four QTL for powdery mildew resistance were detected, of which three had resistance from Naxos. This mapping verified the 1AS and 2AL QTL detected in Shanghai3/Catbird x Naxos, and identified a new QTL from Naxos on 2BL. In conclusion, the improved linkage maps with SNP markers enabled discovery of new resistance QTL and more precise mapping of previously known QTL. Moreover, the results were validated in an independent genetic background.

Mlo-Based Resistance: An Apparently Universal "Weapon" to Defeat Powdery Mildew Disease.

Loss-of-function mutations of one or more of the appropriate Mildew resistance locus o (Mlo) genes are an apparently reliable "weapon" to protect plants from infection by powdery mildew fungi, as they confer durable broad-spectrum resistance. Originally detected as a natural mutation in an Ethiopian barley landrace, this so-called mlo-based resistance has been successfully employed in European barley agriculture for nearly four decades. More recently, mlo-mediated resistance was discovered to be inducible in virtually every plant species of economic or scientific relevance. By now, mlo resistance has been found (as natural mutants) or generated (by induced mutagenesis, gene silencing, and targeted or nontargeted gene knock-out) in a broad range of monocotyledonous and dicotyledonous plant species. Here, we review features of mlo resistance in barley, discuss approaches to identify the appropriate Mlo gene targets to induce mlo-based resistance, and consider the issue of pleiotropic effects often associated with mlo-mediated immunity, which can harm plant yield and quality. We portray mlo-based resistance as an apparently universal and effective weapon to defeat powdery mildew disease in a multitude of plant species.

Cultivar-Based Introgression Mapping Reveals Wild Species-Derived Pm-0, the Major Powdery Mildew Resistance Locus in Squash.

Powdery mildew is a major fungal disease on squash and pumpkin (Cucurbita spp.) in the US and throughout the world. Genetic resistance to the disease is not known to occur naturally within Cucurbita pepo and only infrequently in Cucurbita moschata, but has been achieved in both species through the introgression of a major resistance gene from the wild species Cucurbita okeechobeensis subsp. martinezii. At present, this gene, Pm-0, is used extensively in breeding, and is found in nearly all powdery mildew-resistant C. pepo and C. moschata commercial cultivars. In this study, we mapped C. okeechobeensis subsp. martinezii-derived single nucleotide polymorphism (SNP) alleles in a set of taxonomically and morphologically diverse and resistant C. pepo and C. moschata cultivars bred at Cornell University that, by common possession of Pm-0, form a shared-trait introgression panel. High marker density was achieved using genotyping-by-sequencing, which yielded over 50,000 de novo SNP markers in each of the three Cucurbita species genotyped. A single 516.4 kb wild-derived introgression was present in all of the resistant cultivars and absent in a diverse set of heirlooms that predated the Pm-0 introgression. The contribution of this interval to powdery mildew resistance was confirmed by association mapping in a C. pepo cultivar panel that included the Cornell lines, heirlooms, and 68 additional C. pepo cultivars and with an independent F2 population derived from C. okeechobeensis subsp. martinezii x C. moschata. The interval was refined to a final candidate interval of 76.4 kb and CAPS markers were developed inside this interval to facilitate marker-assisted selection.

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen.

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVRa gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVRa genes and identified AVRa1 and AVRa13, encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVRa1 and AVRa13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVRA1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVRA1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVRA1 Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.

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.

Mlo‐based powdery mildew resistance in hexaploid bread wheat generated by a non‐transgenic TILLING approach

SummaryWheat is one of the most widely grown cereal crops in the world and is an important food grain source for humans. However, wheat yields can be reduced by many abiotic and biotic stress factors, including powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt). Generating resistant varieties is thus a major effort in plant breeding. Here, we took advantage of the non‐transgenic Targeting Induced Lesions IN Genomes (TILLING) technology to select partial loss‐of‐function alleles of TaMlo, the orthologue of the barley Mlo (Mildew resistance locus o) gene. Natural and induced loss‐of‐function alleles (mlo) of barley Mlo are known to confer durable broad‐spectrum powdery mildew resistance, typically at the expense of pleiotropic phenotypes such as premature leaf senescence. We identified 16 missense mutations in the three wheat TaMlo homoeologues, TaMlo‐A1, TaMlo‐B1 and TaMlo‐D1 that each lead to single amino acid exchanges. Using transient gene expression assays in barley single cells, we functionally analysed the different missense mutants and identified the most promising candidates affecting powdery mildew susceptibility. By stacking of selected mutant alleles we generated four independent lines with non‐conservative mutations in each of the three TaMlo homoeologues. Homozygous triple mutant lines and surprisingly also some of the homozygous double mutant lines showed enhanced, yet incomplete, Bgt resistance without the occurrence of discernible pleiotropic phenotypes. These lines thus represent an important step towards the production of commercial non‐transgenic, powdery mildew‐resistant bread wheat varieties.

Characterization of nine Mlo family genes and analysis of their expression against pathogen infections in Vitis flexuosa

Mildew resistance locus o (Mlo) is a specific seven-transmembrane gene family that confers resistance against several abiotic and biotic stresses in plants. In this study, we identified nine Mlo-like loci,VfMlo-like3243 (Vitis flexuosa powdery-mildew resistance locus o-like3243), VfMlo-like4098, VfMlo-like5057, VfMlo-like6833, VfMlo-like7881, VfMlo-like8120, VfMlo-like14439, VfMlo-like14557, and VfMlo-like18484, which showed differential expression upon transcriptome analysis conducted using next generation sequencing (NGS) of V. flexuosa infected with Elsinoe ampelina. These genes are 1773–2548 bp long and encode deduced full-length proteins of 482–587 amino acids. The predicted amino acid sequences of all genes show typical Mlo domains containing seven highly conserved transmembrane domains, a calmodulin-binding domain and 30 important amino acid residues for Mlo function. While 3 VfMlo-like genes were downregulated, 5 VfMlo-like genes were up-regulated in grapevines infected with Erysiphe necator, among which four (VfMlo-like6833, VfMlo-like8120, VfMlo-like14439 and VfMlo-like18484) are orthologous to the AtMlo2, AtMlo6 and AtMlo12 genes involved in powdery mildew susceptibility. All genes except VfMlo-like17881 showed upregulated expression at 48 hpi after infection of Rhizobium vitis. The results revealed that the tested genes are related in responses to pathogens of grapevines and can be utilized as useful resources for investigating their roles in disease resistant responses in grapevines.

Identification of two novel powdery mildew resistance loci, Ren6 and Ren7, from the wild Chinese grape species Vitis piasezkii.

BackgroundGrapevine powdery mildew Erysiphe necator is a major fungal disease in all grape growing countries worldwide. Breeding for resistance to this disease is crucial to avoid extensive fungicide applications that are costly, labor intensive and may have detrimental effects on the environment. In the past decade, Chinese Vitis species have attracted attention from grape breeders because of their strong resistance to powdery mildew and their lack of negative fruit quality attributes that are often present in resistant North American species. In this study, we investigated powdery mildew resistance in multiple accessions of the Chinese species Vitis piasezkii that were collected during the 1980 Sino-American botanical expedition to the western Hubei province of China.ResultsA framework genetic map was developed using simple sequence repeat markers in 277 seedlings of an F1 mapping population arising from a cross of the powdery mildew susceptible Vitis vinifera selection F2-35 and a resistant accession of V. piasezkii DVIT2027. Quantitative trait locus analyses identified two major powdery mildew resistance loci on chromosome 9 (Ren6) and chromosome 19 (Ren7) explaining 74.8 % of the cumulative phenotypic variation. The quantitative trait locus analysis for each locus, in the absence of the other, explained 95.4 % phenotypic variation for Ren6, while Ren7 accounted for 71.9 % of the phenotypic variation. Screening of an additional 259 seedlings of the F1 population and 910 seedlings from four pseudo-backcross populations with SSR markers defined regions of 22 kb and 330 kb for Ren6 and Ren7 in the V. vinifera PN40024 (12X) genome sequence, respectively.Both R loci operate post-penetration through the induction of programmed cell death, but vary significantly in the speed of response and degree of resistance; Ren6 confers complete resistance whereas Ren7 confers partial resistance to the disease with reduced colony size. A comparison of the kinetics of induction of powdery mildew resistance mediated by Ren6, Ren7 and the Run1 locus from Muscadinia rotundifolia, indicated that the speed and strength of resistance conferred by Ren6 is greater than that of Run1 which, in turn, is superior to that conferred by Ren7.ConclusionsThis is the first report of mapping powdery mildew resistance in the Chinese species V. piasezkii. Two distinct powdery mildew R loci designated Ren6 and Ren7 were found in multiple accessions of this Chinese grape species. Their location on different chromosomes to previously reported powdery mildew resistance R loci offers the potential for grape breeders to combine these R genes with existing powdery mildew R loci to produce grape germplasm with more durable resistance against this rapidly evolving fungal pathogen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0855-8) contains supplementary material, which is available to authorized users.

Lessons from a Phenotyping Center Revealed by the Genome-Guided Mapping of Powdery Mildew Resistance Loci.

The genomics era brought unprecedented opportunities for genetic analysis of host resistance, but it came with the challenge that accurate and reproducible phenotypes are needed so that genomic results appropriately reflect biology. Phenotyping host resistance by natural infection in the field can produce variable results due to the uncontrolled environment, uneven distribution and genetics of the pathogen, and developmentally regulated resistance among other factors. To address these challenges, we developed highly controlled, standardized methodologies for phenotyping powdery mildew resistance in the context of a phenotyping center, receiving samples of up to 140 grapevine progeny per F1 family. We applied these methodologies to F1 families segregating for REN1- or REN2-mediated resistance and validated that some but not all bioassays identified the REN1 or REN2 locus. A point-intercept method (hyphal transects) to quantify colony density objectively at 8 or 9 days postinoculation proved to be the phenotypic response most reproducibly predicted by these resistance loci. Quantitative trait locus (QTL) mapping with genotyping-by-sequencing maps defined the REN1 and REN2 loci at relatively high resolution. In the reference PN40024 genome under each QTL, nucleotide-binding site-leucine-rich repeat candidate resistance genes were identified-one gene for REN1 and two genes for REN2. The methods described here for centralized resistance phenotyping and high-resolution genetic mapping can inform strategies for breeding resistance to powdery mildews and other pathogens on diverse, highly heterozygous hosts.

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.

Suppression of PCD-related genes affects salt tolerance in Arabidopsis

This work aims at examining a natural exciting phenomenon suggesting that suppression of genes inducing programmed cell death (PCD) might confer tolerance against abiotic stresses in plants. PCD-related genes were induced in tobacco under oxalic acid (OA) treatment (20mM), and plant cells were characterized to confirm the incidence of PCD. The results indicated that PCD was triggered 24h after the exposure to OA. Then, RNAs were extracted from tobacco cells 0, 2, 6, 12 and 24h after treatment for deep sequencing. RNA-Seq analyses were done with a special emphasis to clusters whose PCD-related genes were upregulated after 2h of OA exposure. Accordingly, 23 tobacco PCD-related genes were knocked down via virus-induced gene silencing (VIGS), whereas our results indicated the influence of five of them on inducing or suppressing PCD. Knockout T-DNA insertion mutants of these five genes in Arabidopsis were tested under salt stress (0, 100, 150, and 200mM NaCl), and the results indicated that a mutant of an antiapoptotic gene, namely Bax Inhibitor-1 (BI-1), whose VIGS induced PCD in tobacco, was salt sensitive, while a mutant of an apoptotic gene, namely mildew resistance locus O (Mlo), whose VIGS suppressed PCD, was salt tolerant as compared to the WT (Col) control. These data support our hypothesis that retarding PCD-inducing genes can result in higher levels of salt tolerance, while retarding PCD-suppressing genes can result in lower levels of salt tolerance in plants.

Comprehensive Phylogenetic Analysis Sheds Light on the Diversity and Origin of the MLO Family of Integral Membrane Proteins.

Mildew resistance Locus O (MLO) proteins are polytopic integral membrane proteins that have long been considered as plant-specific and being primarily involved in plant–powdery mildew interactions. However, research in the past decade has revealed that MLO proteins diverged into a family with several clades whose members are associated with different physiological processes. We provide a largely increased dataset of MLO amino acid sequences, comprising nearly all major land plant lineages. Based on this comprehensive dataset, we defined seven phylogenetic clades and reconstructed the likely evolution of the MLO family in embryophytes. We further identified several MLO peptide motifs that are either conserved in all MLO proteins or confined to one or several clades, supporting the notion that clade-specific diversification of MLO functions is associated with particular sequence motifs. In baker’s yeast, some of these motifs are functionally linked to transmembrane (TM) transport of organic molecules and ions. In addition, we attempted to define the evolutionary origin of the MLO family and found that MLO-like proteins with highly diverse membrane topologies are present in green algae, but also in the distinctly related red algae (Rhodophyta), Amoebozoa, and Chromalveolata. Finally, we discovered several instances of putative fusion events between MLO proteins and different kinds of proteins. Such Rosetta stone-type hybrid proteins might be instructive for future analysis of potential MLO functions. Our findings suggest that MLO is an ancient protein that possibly evolved in unicellular photosynthetic eukaryotes, and consolidated in land plants with a conserved topology, comprising seven TM domains and an intrinsically unstructured C-terminus.

Powdery mildew resistance in the Japanese domestic tobacco cultivar Kokubu is associated with aberrant splicing of MLO orthologues

The Japanese domestic tobacco cultivar Kokubu (syn. Kou‐fan) is highly resistant to powdery mildew, and this trait is controlled by recessive alleles at two loci. To show the molecular mechanism of powdery mildew resistance of Kokubu, the transcripts and genomic sequences of tobacco Mildew resistance locus O (MLO) orthologues were compared between powdery mildew‐resistant and ‐susceptible cultivars. Two MLO orthologues (NtMLO1 and NtMLO2) corresponding to powdery mildew susceptibility were expressed in tobacco leaves. However, the transcripts of both NtMLO genes in the powdery mildew‐resistant cultivars harboured partial deletions or insertions. In a genomic DNA sequence alignment analysis, mutations in intron regions were detected in both NtMLO genes of powdery mildew‐resistant cultivars, and these mutations were linked to the powdery mildew‐resistant phenotype. Transgenic Kokubu expressing wildtype NtMLO1 or NtMLO2 exhibited severe disease symptoms, the same as those in susceptible cultivars. These results indicate that mutations of intron regions of two NtMLO genes in powdery mildew resistant cultivar Kokubu trigger the aberrant splicing of MLO transcripts and the subsequent inhibition of functional MLO protein synthesis, and that NtMLO1 and NtMLO2 are functionally redundant.

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.

Identification and mapping of PmSE5785, a new recessive powdery mildew resistance locus, in synthetic hexaploid wheat

Powdery mildew of wheat (Triticum aestivum L.) caused by Blumeria graminis f. sp. tritici, is a major fungal disease in many areas of the world. Synthetic hexaploids, created from durum wheat and Aegilops tauschii are important sources of beneficial genes for wheat improvement. Synthetic wheat ‘SE5785’ was found resistant to powdery mildew at the seedling and adult stages. Genetic analyses of the F2 population and F2:3 families developed from a cross of ‘SE5785’ with the susceptible common wheat, ‘Xiaoyan 22’, revealed that resistance in ‘SE5785’ was controlled by a single recessive gene. Bulked segregant analysis was used to map the powdery mildew resistance locus. The resulting genetic map comprised of markers Xcfd62, Xbarc59, PmSE5785, Xgwm539, and Xwmc817 spanned 16.3 cM. Markers Xbarc59 and Xgwm539 were tightly linked to the resistance locus at genetic distances of 3.6 and 4.0 cM, respectively. ‘Chinese Spring’ and its nulli-tetrasomic lines confirmed that PmSE5785 was located on chromosome 2D. Because no gene for powdery mildew resistance had ever been located on this chromosome in the genus Triticum, this must be a new locus, therefore, designated PmSE5785. N07228-1 and N07228-2 with large seeds and powdery mildew resistance were selected from the progeny of the cross ‘SE5785’/‘Xiaoyan 22’. These new lines can serve as sources of resistance to powdery mildew in wheat breeding.