mlo-based powdery mildew immunity: silver bullet or simply non-host resistance?

Durability and effectiveness against all genetic variants of a microbial species are hallmarks of so-called plant 'non-host' resistance. Highly effective immunity of monocotyledonous barley against the fungal powdery mildew pathogen, which is conferred by loss-of-function mutant alleles of the barley Mlo locus, likewise is a durable and broad-spectrum type of resistance. Although this was long considered as being a barley-specific phenomenon, recent findings indicate that mlo resistance can also occur in the distantly related dicotyledonous species Arabidopsis thaliana . Shared histological and phytopathological characteristics plus a conserved requirement for a set of genes in Arabidopsis mlo and non-host powdery mildew resistance indicate a potential common mechanism for these two seemingly distinct types of immunity.     

大麦抗白粉病基因Mlo的研究进展

野生型Mlo基因是大麦抗白粉病的负调控因子,该基因突变,赋予大麦对白粉菌的广谱抗性。综述了Mlo基因结构、功能及Mlo突变的等位基因(mlo)的抗性特点;讨论了mlo基因可能的抗病机制。为mlo抗性在麦类白粉病抗病育种中的应用提供了理论基础。     

Haplotype characterization and markers at the barley Mlo powdery mildew resistance locus as tools for marker-assisted selection.

Recessive mlo alleles of the barley Mlo gene confer resistance to almost all known isolates of the powdery mildew fungal pathogen targeting barley (Hordeum vulgare). To characterize haplotypes present in the Mlo chromosomal region of cultivated Mlo and mlo barley genotypes, we conducted a polymorphism search in 3 predicted low-copy sequence regions adjacent to the Mlo gene by examining a sample of 4 Mlo and 3 mlo cultivars. Eight single-nucleotide polymorphisms (SNPs) and 1 insertion-deletion (indel) were detected, and easy to use PCR-based markers were developed for typing the SNPs. The PCR markers were used to characterize a collection of 46 Mlo and 25 mlo barley cultivars, identifying 3 distinct mlo-11 haplotypes, 1 mlo-9 haplotype, and 4 Mlo haplotypes. We summarized the haplotype and marker information obtained here and in a previous study to help breeders identify strategies for mlo marker-assisted selection. The ability of the markers to identify mlo-resistant genotypes in segregating populations was demonstrated using 2 resistance-characterized F2 populations derived by 3-way crosses.     

Interactions between introns via exon definition in plant pre-mRNA splicing

The barley gene Mlo encodes a prototype of a novel class of plant proteins. In mlo mutants, absence of the 60 kDa wild-type Mlo protein results in broad-spectrum resistance to the powdery mildew fungus, Erysiphe graminis f. sp. hordei . To directly assess its function, Mlo was transiently expressed with a marker gene encoding a modified green fluorescent protein (GFP) in leaf epidermal cells of mlo resistant barley lines. Fungal inoculation of epidermal cells transfected with wild-type Mlo led to haustorium formation and abundant sporulation. Therefore, expression of the wild-type Mlo gene, in mlo resistant genotypes, is both necessary and sufficient to restore susceptibility to fungal attack. Complementation of mlo resistance alleles was restricted to single host cells, indicating a cell-autonomous function for the wild-type Mlo protein. We discuss our findings with respect to source–sink relationships of plants and biotrophic fungi and the potentially wide-ranging use of the transient complementation assay to analyse host compatibility and defence in response to powdery mildew attack.     

Tomato defense to Oidium neolycopersici: dominant Ol genes confer isolate-dependent resistance via a different mechanism than recessive ol-2

Tomato powdery mildew caused by Oidium neolycopersici has become a globally important disease of tomato (Lycopersicon esculentum). To study the defense responses of tomato triggered by tomato powdery mildew, we first mapped a set of resistance genes to O. neolycopersici from related Lycopersicon species. An integrated genetic map was generated showing that all the dominant resistance genes (Ol-1, Ol-3, Ol-4, Ol-5, and Ol-6) are located on tomato chromosome 6 and are organized in three genetic loci. Then, near-isogenic lines (NIL) were produced that contain the different dominant Ol genes in a L. esculentum genetic background. These NIL were used in disease tests with local isolates of O. neolycopersici in different geographic locations, demonstrating that the resistance conferred by different Ol genes was isolate-dependent and, hence, may be race-specific. In addition, the resistance mechanism was analyzed histologically. The mechanism of resistance conferred by the dominant Ol genes was associated with hypersensitive response, which varies in details depending on the Ol-gene in the NIL, while the mechanism of resistance governed by the recessive gene ol-2 on tomato chromosome 4 was associated with papillae formation.     

Acquired resistance functions in mlo barley,which is hypersusceptible to Magnaporthe grisea

Barley plants carrying a mutation in the Mlo (barley [Hordeum vulgare L.] cultivar Ingrid) locus conferring a durable resistance against powdery mildew are hypersusceptible to the rice blast fungus Magnaporthe grisea. It has been speculated that a functional Mlo gene is required for the expression of basic pathogen resistance and that the loss of Mlo function mediating powdery mildew resistance is an exception for this particular disease. Here, we report that the onset of acquired resistance (AR) after chemical as well as biological treatments is sufficient to overcome the hypersusceptible phenotype of backcross line BCIngridmlo5 (mlo) barley plants against M. grisea. Moreover, even barley plants bearing a functional Mlo gene and thus showing a moderate infection phenotype against rice blast exhibit a further enhanced resistance after induction of AR. Cytological investigations reveal that acquired resistance in mlo genotypes is manifested by the restoration of the ability to form an effective papilla at sites of attempted penetration, similarly to wild-type Mlo plants. In addition, the rate of effective papillae formation in Mlo plants was further enhanced after the onset of AR. These results demonstrate that treatments leading to the AR state in barley function independently of the Mlo/mlo phenotype and suggest that the Mlo protein is not a component of the AR signaling network. Moreover, it seems that only concomitant action of Mlo together with AR permits high level resistance in barley against blast. Higher steady state levels of PR1 and barley chemically induced mRNA correlate with higher disease severity rather than with the degree of resistance observed in this particular interaction.     

Identification of Genes Required for the Function of Non-Race-Specific mlo Resistance to Powdery Mildew in Barley

Recessive alleles (mlo) of the Mlo locus in barley mediate a broad, non-race-specific resistance reaction to the powdery mildew fungus Erysiphe graminis f sp hordei. A mutational approach was used to identify genes that are required for the function of mlo. Six susceptible M2 individuals were isolated after inoculation with the fungal isolate K1 from chemically mutagenized seed carrying the mlo-5 allele. Susceptibility in each of these individuals is due to monogenic, recessively inherited mutations in loci unlinked to mlo. The mutants identify two unlinked complementation groups, designated Ror1 and Ror2 (required for mlo-specified resistance). Both Ror genes are required for the function of different tested mlo alleles and for mlo function after challenge with different isolates of E. g. f sp hordei. A quantitative cytological time course analysis revealed that the host cell penetration efficiency in the mutants is intermediate compared with mlo-resistant and Mlo-susceptible genotypes. Ror1 and Ror2 mutants could be differentiated from each other by the same criterion. The spontaneous formation of cell wall appositions in mlo plants, a subcellular structure believed to represent part of the mlo defense, is suppressed in mlo/ror genotypes. In contrast, accumulation of major structural components in the appositions is seemingly unaltered. We conclude that there is a regulatory function for the Ror genes in mlo-specified resistance and propose a model in which the Mlo wild-type allele functions as a negative regulator and the Ror genes act as positive regulators of a non-race-specific resistance response.     

Functional conservation of wheat and rice Mlo orthologs in defense modulation to the powdery mildew fungus

Homologs of barley Mlo are found in syntenic positions in all three genomes of hexaploid bread wheat, Triticum aestivum, and in rice, Oryza sativa. Candidate wheat orthologs, designated TaMlo-A1, TaMlo-B1, and TaMlo-D1, encode three distinct but highly related proteins that are 88% identical to barley MLO and appear to originate from the three diploid ancestral genomes of wheat. TaMlo-B1 and the rice ortholog, OsMlo2, are able to complement powdery mildew-resistant barley mlo mutants at the single-cell level. Overexpression of TaMlo-B1 or barley Mlo leads to super-susceptibility to the appropriate powdery mildew formae speciales in both wild-type barley and wheat. Surprisingly, overexpression of either Mlo or TaMlo-B1 also mediates enhanced fungal development to tested inappropriate formae speciales. These results underline a regulatory role for MLO and its wheat and rice orthologs in a basal defense mechanism that can interfere with forma specialis resistance to powdery mildews.     

Linked, if not the same, Mi-1 homologues confer resistance to tomato powdery mildew and root-knot nematodes

On the short arm of tomato chromosome 6, a cluster of disease resistance (R) genes have evolved harboring the Mi-1 and Cf genes. The Mi-1 gene confers resistance to root-knot nematodes, aphids, and whiteflies. Previously, we mapped two genes, Ol-4 and Ol-6, for resistance to tomato powdery mildew in this cluster. The aim of this study was to investigate whether Ol-4 and Ol-6 are homologues of the R genes located in this cluster. We show that near-isogenic lines (NIL) harboring Ol-4 (NIL-Ol-4) and Ol-6 (NIL-Ol-6) are also resistant to nematodes and aphids. Genetically, the resistance to nematodes cosegregates with Ol-4 and Ol-6, which are further fine-mapped to the Mi-1 cluster. We provide evidence that the composition of Mi-1 homologues in NIL-Ol-4 and NIL-Ol-6 is different from other nematode-resistant tomato lines, Motelle and VFNT, harboring the Mi-1 gene. Furthermore, we demonstrate that the resistance to both nematodes and tomato powdery mildew in these two NIL is governed by linked (if not the same) Mi-1 homologues in the Mi-1 gene cluster. Finally, we discuss how Solanum crops exploit Mi-1 homologues to defend themselves against distinct pathogens.     

A small GTP-binding host protein is required for entry of powdery mildew fungus into epidermal cells of barley

Small GTP-binding proteins such as those from the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture, secondary wall formation, meristem signaling, and defense against pathogens. We isolated a RacB homolog from barley (Hordeum vulgare) to study its role in resistance to the barley powdery mildew fungus (Blumeria graminis f.sp. hordei). RacB was constitutively expressed in the barley epidermis and its expression level was not strongly influenced by inoculation with B. graminis. However, after biolistic bombardment of barley leaf segments with RacB-double-stranded RNA, sequence-specific RNA interference with RacB function inhibited fungal haustorium establishment in a cell-autonomous and genotype-specific manner. Mutants compromised in function of the Mlo wild-type gene and the Ror1 gene (genotype mlo5 ror1) that are moderately susceptible to B. graminis showed no alteration in powdery mildew resistance upon RacB-specific RNA interference. Thus, the phenotype, induced by RacB-specific RNA interference, was apparently dependent on the same processes as mlo5-mediated broad resistance, which is suppressed by ror1. We conclude that an RAC small GTP-binding protein is required for successful fungal haustorium establishment and that this function may be linked to MLO-associated functions.     

Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1

Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea.     

Novel induced <Emphasis Type="Italic">mlo</Emphasis> mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein

Background  

Recessively inherited natural and induced mutations in the barley Mlo gene confer durable broad-spectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei. Mlo codes for a member of a plant-specific family of polytopic integral membrane proteins with unknown biochemical activity. Resistant barley mlo mutant alleles identify amino acid residues that are critical for Mlo function in the context of powdery mildew susceptibility.     

Resistance genes in barley (Hordeum vulgare L.) and their identification with molecular markers

Current information on barley resistance genes available from scientific papers and on-line databases is summarised. The recent literature contains information on 107 major resistance genes (R genes) against fungal pathogens (excluding powdery mildew), pathogenic viruses and aphids identified in Hordeum vulgare accessions. The highest number of resistance genes was identified against Puccinia hordei, Rhynchosporium secalis, and the viruses BaYMV and BaMMV, with 17, 14 and 13 genes respectively. There is still a lot of confusion regarding symbols for R genes against powdery mildew. Among the 23 loci described to date, two regions Mla and Mlo comprise approximately 31 and 25 alleles. Over 50 R genes have already been localised and over 30 mapped on 7 barley chromosomes. Four barley R genes have been cloned recently: Mlo, Rpg1, Mla1 and Mla6, and their structures (sequences) are available. The paper presents a catalogue of barley resistance gene symbols, their chromosomalocation and the list of available DNA markers useful in characterising cultivars and breeding accessions.     

小麦Mlo及NBS-LRR类抗病基因同源序列的分离与鉴定

根据GenBank中公布的大麦白粉病抗性控制基因Mlo cDNA序列及一个来源于栽培一粒小麦(Triticum monococcum L.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选.结果获得两个表达基因的cDNA克隆.其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%.另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类.白粉菌诱导前后,该片段RT-PCR扩增产物存在差异,表明该片段可能与小麦抗病性相关.利用"中国春"缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上.     

Development of diagnostic PCR markers closely linked to the tomato powdery mildew resistance gene Ol-1 on chromosome 6 of tomato

Lycopersicon hirsutum G1.1560 is a wild accession of tomato that shows resistance to Oidium lycopersicum, a frequently occurring tomato powdery mildew. This resistance is largely controlled by an incompletely dominant gene Ol-1 near the Aps-1 locus in the vicinity of the resistance genes Mi and Cf-2/Cf-5. Using a new F₂ population (n=150) segregating for resistance, we mapped the Ol-1 gene more accurately to a location between the RFLP markers TG153 and TG164. Furthermore, in saturating the Ol-1 region with more molecular markers using bulked segregant analysis, we were able to identify five RAPDs associated with the resistance. These RAPDs were then sequenced and converted into SCAR markers: SCAB01 and SCAF10 were L. hirsutum-specific; SCAE16, SCAG11 and SCAK16 were L. esculentum-specific. By linkage analysis a dense integrated map comprising RFLP and SCAR markers near Ol-1 was obtained. This will facilitate a map-based cloning approach for Ol-1 and marker-assisted selection for powdery mildew resistance in tomato breeding. Received: 21 June 1999 / Accepted: 1 December 1999     

The resistance to Oidium lycopersici conferred by ol-2 gene in tomato

The powdery mildew caused by Oidium lycopersici is one of the most destructive diseases in glass-house-grown tomato and is widespreading all over the world. A high level of resistance to O. lycopersici was found in an accession of Lycopersicon esculentum var. cerasiforme at the Department of Biology and Plant Pathology, University of Bari. The genetic analysis of F1, F2 and BC plants indicated that the resistance is conferred by a single recessive gene, designed as ol-2. Studies on the infection process of O. lycopersici on susceptible and ol-2 gene resistant tomatoes were carried out at 24 °C and 90 % relative humidity. Light microscope observations on conidia germination, formation of primary appressoria, elongation of hyphae and sporulation were made on artificially inoculated basal, intermediate and apical leaves. Inoculation was made by shaking mildewed tomato leaves over each test plant. Disease development were assessed by removing the fungal structure from the leaf surface with the ceroidin film technique and by direct observations of stained inoculated leaves. The rate of conidial germination and the appressoria formation was not affected by host genotype. Mycelia growth and sporulation on leaf surface of resistant plant was strongly restricted and influenced by the leaf age. The results indicated that the resistance in ol-2 tomato is postinfectional and is not associated with a hypersensitive response. This work was supported by the MURST and CNR (Paper no. 331)     

QTLs for tomato powdery mildew resistance (Oidium lycopersici) in Lycopersicon parviflorum G1.1601 co-localize with two qualitative powdery mildew resistance genes

Tomato (Lycopersicon esculentum) is susceptible to the powdery mildew Oidium lycopersici, but several wild relatives such as Lycopersicon parviflorum G1.1601 are completely resistant. An F2 population from a cross of Lycopersicon esculentum cv. Moneymaker x Lycopersicon parviflorum G1.1601 was used to map the O. lycopersici resistance by using amplified fragment length polymorphism markers. The resistance was controlled by three quantitative trait loci (QTLs). Ol-qtl1 is on chromosome 6 in the same region as the Ol-1 locus, which is involved in a hypersensitive resistance response to O. lycopersici. Ol-qtl2 and Ol-qtl3 are located on chromosome 12, separated by 25 cM, in the vicinity of the Lv locus conferring resistance to another powdery mildew species, Leveillula taurica. The three QTLs, jointly explaining 68% of the phenotypic variation, were confirmed by testing F3 progenies. A set of polymerase chain reaction-based cleaved amplified polymorphic sequence and sequence characterized amplified region markers was generated for efficient monitoring of the target QTL genomic regions in marker assisted selection. The possible relationship between genes underlying major and partial resistance for tomato powdery mildew is discussed.