Powdery Mildew Susceptibility Research Articles

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.

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.

PAMP-triggered immune responses in barley and susceptibility to powdery mildew

ABSTRACTPathogen-associated molecular pattern-triggered immunity (PTI) builds one of the first layers of plant disease resistance. In susceptible plants, PTI is overcome by adapted pathogens. This can be achieved by suppression of PTI with the help of pathogen virulence effectors. However, effectors may also contribute to modification of host metabolism or cell architecture to ensure successful pathogenesis. Barley responds to treatment with the pathogen-associated molecular patterns flg22 or chitin with phosphorylation of mitogen-activated protein kinases and an oxidative burst. RAC/ROP GTPases can act as positive or negative modulators of these plant immune responses. The RAC/ROP GTPase RACB is a powdery mildew susceptibility factor of barley. However, RACB apparently does not negatively control early PTI responses but functions in polar cell development during invasion of the pathogen into living host epidermal cells. Here, we further discuss the incomplete picture of PTI in Triticeae.

Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine.

Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion.

Genome-Wide Study of the Tomato SlMLO Gene Family and Its Functional Characterization in Response to the Powdery Mildew Fungus Oidium neolycopersici.

The MLO (Mildew Locus O) gene family encodes plant-specific proteins containing seven transmembrane domains and likely acting in signal transduction in a calcium and calmodulin dependent manner. Some members of the MLO family are susceptibility factors toward fungi causing the powdery mildew disease. In tomato, for example, the loss-of-function of the MLO gene SlMLO1 leads to a particular form of powdery mildew resistance, called ol-2, which arrests almost completely fungal penetration. This type of penetration resistance is characterized by the apposition of papillae at the sites of plant-pathogen interaction. Other MLO homologs in Arabidopsis regulate root response to mechanical stimuli (AtMLO4 and AtMLO11) and pollen tube reception by the female gametophyte (AtMLO7). However, the role of most MLO genes remains unknown. In this work, we provide a genome-wide study of the tomato SlMLO gene family. Besides SlMLO1, other 15 SlMLO homologs were identified and characterized with respect to their structure, genomic organization, phylogenetic relationship, and expression profile. In addition, by analysis of transgenic plants, we demonstrated that simultaneous silencing of SlMLO1 and two of its closely related homologs, SlMLO5 and SlMLO8, confer higher level of resistance than the one associated with the ol-2 mutation. The outcome of this study provides evidence for functional redundancy among tomato homolog genes involved in powdery mildew susceptibility. Moreover, we developed a series of transgenic lines silenced for individual SlMLO homologs, which lay the foundation for further investigations aimed at assigning new biological functions to the MLO gene family.

Genome-wide characterization and comparative analysis of the MLO gene family in cotton

In plants, MLO (Mildew Locus O) gene encodes a plant-specific seven transmembrane (TM) domain protein involved in several cellular processes, including susceptibility to powdery mildew (PM). In this study, a genome-wide characterization of the MLO gene family in G. raimondii L., G. arboreum L. and G. hirsutum L. was performed. In total, 22, 17 and 38 homologous sequences were identified for each species, respectively. Gene organization, including chromosomal location, gene clustering and gene duplication, was investigated. Homologues related to PM susceptibility in upland cotton were inferred by phylogenetic relationships with functionally characterized MLO proteins. To conduct a comparative analysis between MLO candidate genes from G. raimondii L., G. arboreum L. and G. hirsutum L., orthologous relationships and conserved synteny blocks were constructed. The transcriptional variation of 38 GhMLO genes in response to exogenous application of salt, mannitol (Man), abscisic acid (ABA), ethylene (ETH), jasmonic acid (JA) and salicylic acid (SA) was monitored. Further studies should be conducted to elucidate the functions of MLO genes in PM susceptibility and phytohormone signalling pathways.

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.

Genomic identification, phylogeny, and expression analysis of MLO genes involved in susceptibility to powdery mildew in Fragaria vesca.

The MLO (powdery mildew locus O) gene family is important in resistance to powdery mildew (PM). In this study, all of the members of the MLO family were identified and analyzed in the strawberry (Fragaria vesca) genome. The strawberry contains at least 20 members of the MLO family, and the protein sequence contained between 171 and 1485 amino acids, with 0-34 introns. Chromosomal localization showed that the MLOs were unevenly distributed on each of the chromosomes, except for chromosome 4. The greatest number of MLOs (seven) was found on chromosome 3. A phylogenetic tree showed that the MLOs were divided into seven groups (I-VII), four of which consisted of MLOs from strawberry, Arabidopsis thaliana, rice, and maize, suggesting that these genes may have evolved after the divergence of monocots and dicots. Multiple sequence alignment showed that strawberry MLO candidates related to powdery mildew resistance possessed seven highly conserved transmembrane domains, a calmodulin-binding domain, and two conserved regions, all of which are important domains for powdery mildew resistance genes. Expressed sequence tag analysis revealed that the MLOs were induced by multiple abiotic stressors, including low and high temperature, drought, and high salinity. These findings will contribute to the functional characterization of MLOs related to PM susceptibility, and will assist in the development of disease resistance in strawberries.

Structure, evolution and functional inference on the Mildew Locus O (MLO) gene family in three cultivated Cucurbitaceae spp.

BackgroundThe powdery mildew disease affects thousands of plant species and arguably represents the major fungal threat for many Cucurbitaceae crops, including melon (Cucumis melo L.), watermelon (Citrullus lanatus L.) and zucchini (Cucurbita pepo L.). Several studies revealed that specific members of the Mildew Locus O (MLO) gene family act as powdery mildew susceptibility factors. Indeed, their inactivation, as the result of gene knock-out or knock-down, is associated with a peculiar form of resistance, referred to as mlo resistance.ResultsWe exploited recently available genomic information to provide a comprehensive overview of the MLO gene family in Cucurbitaceae. We report the identification of 16 MLO homologs in C. melo, 14 in C. lanatus and 18 in C. pepo genomes. Bioinformatic treatment of data allowed phylogenetic inference and the prediction of several ortholog pairs and groups. Comparison with functionally characterized MLO genes and, in C. lanatus, gene expression analysis, resulted in the detection of candidate powdery mildew susceptibility factors. We identified a series of conserved amino acid residues and motifs that are likely to play a major role for the function of MLO proteins. Finally, we performed a codon-based evolutionary analysis indicating a general high level of purifying selection in the three Cucurbitaceae MLO gene families, and the occurrence of regions under diversifying selection in candidate susceptibility factors.ConclusionsResults of this study may help to address further biological questions concerning the evolution and function of MLO genes. Moreover, data reported here could be conveniently used by breeding research, aiming to select powdery mildew resistant cultivars in Cucurbitaceae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2325-3) contains supplementary material, which is available to authorized users.

Monocot and dicot MLO powdery mildew susceptibility factors are functionally conserved in spite of the evolution of class-specific molecular features.

BackgroundSpecific members of the plant Mildew Locus O (MLO) protein family act as susceptibility factors towards powdery mildew (PM), a worldwide-spread fungal disease threatening many cultivated species. Previous studies indicated that monocot and dicot MLO susceptibility proteins are phylogenetically divergent.MethodsA bioinformatic approach was followed to study the type of evolution of Angiosperm MLO susceptibility proteins. Transgenic complementation tests were performed for functional analysis.ResultsOur results show that monocot and dicot MLO susceptibility proteins evolved class-specific conservation patterns. Many of them appear to be the result of negative selection and thus are likely to provide an adaptive value. We also tested whether different molecular features between monocot and dicot MLO proteins are specifically required by PM fungal species to cause pathogenesis. To this aim, we transformed a tomato mutant impaired for the endogenous SlMLO1 gene, and therefore resistant to the tomato PM species Oidium neolycopersici, with heterologous MLO susceptibility genes from the monocot barley and the dicot pea. In both cases, we observed restoration of PM symptoms. Finally, through histological observations, we demonstrate that both monocot and dicot susceptibility alleles of the MLO genes predispose to penetration of a non-adapted PM fungal species in plant epidermal cells.ConclusionsWith this study, we provide insights on the evolution and function of MLO genes involved in the interaction with PM fungi. With respect to breeding research, we show that transgenic complementation assays involving phylogenetically distant plant species can be used for the characterization of novel MLO susceptibility genes. Moreover, we provide an overview of MLO protein molecular features predicted to play a major role in PM susceptibility. These represent ideal targets for future approaches of reverse genetics, addressed to the selection of loss-of-function resistant mutants in cultivated species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0639-6) contains supplementary material, which is available to authorized users.

Expression Pattern Analysis of Four Mlo Genes from Rose

Rose (Rosa hybrida) is one of the most economically important ornamentals worldwide. Powdery mildew (Podosphaera pannosa) is a major disease in cut and potted roses. In dicots such as arabidopsis (Arabidopsis thaliana), pea (Pisum sativum), and tomato (Solanum lycopersicum), loss-of-function mutations in mildew resistance locus o (Mlo) genes confer high levels of broad-spectrum resistance to powdery mildew. Here, we present spatiotemporal expression patterns of four Mlo genes from R. hybrida based on real-time fluorescence quantitative polymerase chain reaction (qPCR). Phylogenetically closely related R. hybrida mildew resistance locus o (RhMLO) genes showed similar or overlapping tissue specificity and analogous responsiveness to external stimuli. RhMLO1 and RhMLO2 transcriptional levels were upregulated more than 2-fold by external stimuli, especially by inoculation with powdery mildew fungus P. pannosa at early time points. This phenomenon was not found for RhMLO3 or RhMLO4. The results indicated that RhMLO1 and RhMLO2 might play important roles in rose–powdery mildew pathogen interactions. Our findings may provide useful information for the study of mechanisms of powdery mildew susceptibility in rose.

Identification of candidate MLO powdery mildew susceptibility genes in cultivated Solanaceae and functional characterization of tobacco NtMLO1.

Specific homologs of the plant Mildew Locus O (MLO) gene family act as susceptibility factors towards the powdery mildew (PM) fungal disease, causing significant economic losses in agricultural settings. Thus, in order to obtain PM resistant phenotypes, a general breeding strategy has been proposed, based on the selective inactivation of MLO susceptibility genes across cultivated species. In this study, PCR-based methodologies were used in order to isolate MLO genes from cultivated solanaceous crops that are hosts for PM fungi, namely eggplant, potato and tobacco, which were named SmMLO1, StMLO1 and NtMLO1, respectively. Based on phylogenetic analysis and sequence alignment, these genes were predicted to be orthologs of tomato SlMLO1 and pepper CaMLO2, previously shown to be required for PM pathogenesis. Full-length sequence of the tobacco homolog NtMLO1 was used for a heterologous transgenic complementation assay, resulting in its characterization as a PM susceptibility gene. The same assay showed that a single nucleotide change in a mutated NtMLO1 allele leads to complete gene loss-of-function. Results here presented, also including a complete overview of the tobacco and potato MLO gene families, are valuable to study MLO gene evolution in Solanaceae and for molecular breeding approaches aimed at introducing PM resistance using strategies of reverse genetics.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-015-9878-4) contains supplementary material, which is available to authorized users.

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.

CLONING AND FUNCTIONAL CHARACTERISATION OF A PUTATIVE POWDERY MILDEW SUSCEPTIBILITY GENE IN GRAPEVINE

Grapevine is highly susceptible to a range of different fungal pathogens, including powdery mildew (Erysiphe necator), which can cause severe reductions in yield and quality. As part of multifaceted approach to improve the genetic resistance of grapevine to powdery mildew, we are investigating the grapevine pectate lyase-like (PLL) gene family because previous research in Arabidopsis has demonstrated that a specific member of the AtPLL gene family (PMR6) was required for powdery mildew susceptibility. Sixteen VvPLLs were predicted to be present in the grapevine genome. Based on sequence homology, we identified three genes, designated VvPLL1, VvPLL2 and VvPLL3, as potential orthologs of PMR6, and the coding regions were amplified from Vitis vinifera ?Cabernet Sauvignon?. All three VvPLL candidates were found to encode proteins with a C-terminal GPI-anchor, which is a characteristic of PMR6 (AtPLL13) and VvPLL-GFP fusion constructs confirmed they were targeted to the plasma membrane. Quantitative RT-PCR analysis indicated constitutive expression of VvPLL1, VvPLL2 and VvPLL3 in all tissues examined including leaves, roots, flowers and berries. In contrast, other members of the VvPLL gene family such as VvPLL9 and VvPLL10 were tissue-specific (floral). In agreement with observations for PMR6 (AtPLL13), VvPLL1, VvPLL2 and VvPLL3 were not significantly up-regulated by powdery mildew infection. Functional complementation experiments are currently underway in which the three VvPLL genes have been transformed into the Atpmr6 mutant under the control of either the CaMV 35S or Arabidopsis PMR6 promoter. Our ultimate aim is to silence these VvPLL genes in grapevine to determine if we can modify susceptibility to powdery mildew.

Evaluation of the susceptibility of apple genotypes in Queretaro, Mexico to powdery mildew (Podosphaera leucotricha)

The aim of this study was to evaluate the susceptibility to powdery mildew of apple (Malus domestica Borkh) genotypes introduced in Queretaro, Mexico. For this study, a total of 38 apple genotypes were used: 33 for the field assay and 14 for the greenhouse assay (with nine overlapping varieties). During the field assay, fungus incidence and severity were evaluated in adult plants grafted on the clonal rootstock MM 106, and this test was performed on four consecutive evaluations from May to September 2012. On the last sample, the conidial density was also determined on leaves with visible patches of powdery mildew. For the greenhouse bioassay, apple genotypes, grafted in January 2012 on MM 106, were inoculated on their adaxial side with 200μL of 7×104 conidia/mL (14,000 conidia). The incidence and severity of the fungus on the new shoots were evaluated in August and in October. In addition, the conidial densities per cm2 of leaf area and per leaf were determined for the last sample, and the correlation between the parameters evaluated was examined in each assay and between assays. ‘Rayada’ and ‘411’ showed the highest incidence (59.4 and 40.6%, respectively) and severity (120.0 and 128.3mm2, respectively) to powdery mildew in the field bioassay, in which most genotypes showed no visible damage caused by the fungus. ‘Lourdes’ and ‘436’ were the most resistant genotypes to powdery mildew in the greenhouse bioassay (no incidence, or severity), which means that they are recommended for cultivation in humid environments, and/or where little agronomic management is undertaken. The situation was different for ‘424,’ ‘428’, and ‘Royal Gala’ (with incidence rates of 32.6, 27.5, and 25.5%, and severity rates of 86.6, 229.5 and 168.8mm2, respectively), which, given their agronomic and quality advantages, could be grown under a strict phytosanitary program, or be used as an inoculum for experimental purposes. Significant correlations (P≤0.05) between incidence and severity were obtained in most cases, but no significant correlation with respect to powdery mildew susceptibility under field and greenhouse conditions was found.

CYP83A1 is required for metabolic compatibility of Arabidopsis with the adapted powdery mildew fungus Erysiphe cruciferarum

Aliphatic glucosinolates function in the chemical defense of Capparales. The cytochrome P450 83A1 monooxygenase (CYP83A1) catalyzes the initial conversion of methionine-derived aldoximes to thiohydroximates in the biosynthesis of glucosinolates, and thus cyp83a1 mutants have reduced levels of aliphatic glucosinolates. Loss of CYP83A1 function leads to dramatically reduced parasitic growth of the biotrophic powdery mildew fungus Erysiphe cruciferarum on Arabidopsis thaliana. The cyp83a1 mutants support less well the germination and appressorium formation of E.cruciferarum on the leaf surface and post-penetration conidiophore formation by the fungus. By contrast, a myb28-1 myb29-1 double mutant, which totally lacks aliphatic glucosinolates, shows a wild-type level of susceptibility to E.cruciferarum. The cyp83a1 mutants also lack very-long-chain aldehydes on their leaf surface. Such aldehydes support appressorium formation by E.cruciferarum invitro. In addition, when chemically complemented with the C26 aldehyde n-hexacosanal, cyp83a1 mutants can again support appressorium formation. The mutants further accumulate 5-methylthiopentanaldoxime, the potentially toxic substrate of CYP83A1. Loss of powdery mildew susceptibility by cyp83a1 may be explained by a reduced supply of the fungus with inductive signals from the host and an accumulation of potentially fungitoxic metabolites.

Grapevine powdery mildew resistance and susceptibility loci identified on a high-resolution SNP map

Improved efficacy and durability of powdery mildew resistance can be enhanced via knowledge of the genetics of resistance and susceptibility coupled with the development of high-resolution maps to facilitate the stacking of multiple resistance genes and other desirable traits. We studied the inheritance of powdery mildew (Erysiphe necator) resistance and susceptibility of wild Vitis rupestris B38 and cultivated V. vinifera 'Chardonnay', finding evidence for quantitative variation. Molecular markers were identified using genotyping-by-sequencing, resulting in 16,833 single nucleotide polymorphisms (SNPs) based on alignment to the V. vinifera 'PN40024' reference genome sequence. With an average density of 36 SNPs/Mbp and uniform coverage of the genome, this 17K set was used to identify 11 SNPs on chromosome 7 associated with a resistance locus from V. rupestris B38 and ten SNPs on chromosome 9 associated with a locus for susceptibility from 'Chardonnay' using single marker association and linkage disequilibrium analysis. Linkage maps for V. rupestris B38 (1,146 SNPs) and 'Chardonnay' (1,215 SNPs) were constructed and used to corroborate the 'Chardonnay' locus named Sen1 (Susceptibility to Erysiphe necator 1), providing the first insight into the genetics of susceptibility to powdery mildew from V. vinifera. The identification of markers associated with a susceptibility locus in a V. vinifera background can be used for negative selection among breeding progenies. This work improves our understanding of the nature of powdery mildew resistance in V. rupestris B38 and 'Chardonnay', while applying next-generation sequencing tools to advance grapevine genomics and breeding.

Wheat gene TaS3 contributes to powdery mildew susceptibility

Identification of TaS3 as a potential susceptibility gene encoding a protein homologous to ULP1 protease in wheat, which may regulate SUMO function facilitating powdery mildew attack. Some plant genes that are required for susceptibilities to certain pathogens are known as susceptibility genes or susceptibility factors, whose loss-of-function mutations can confer the plants resistances. To identify potential susceptibility genes to powdery mildew in wheat, differentially expressed genes in compatible and incompatible interactions between wheat and powdery mildew were examined by the cDNA chip assay. The genes exclusively expressed in the susceptible cultivar were interfered using biolistic transient transformation in wheat epidermal cells. The suppression of gene TaS3 (Triticum aestivum susceptibility) decreased the pathogen penetration by 19%, and its over-expression increased the disease susceptibility. The deduced protein from TaS3 belongs to the putative ubiquitin-like protease 1 peptidase domain family. Subcellular localization studies revealed that its protein was accumulated in the nucleus. Quantitative real-time polymerase chain reaction analysis revealed that TaS3 transcript was significantly induced in the compatible host. This suggests that TaS3 is a potential susceptible gene and its function may be related to regulate SUMO functions.

Host Cell Ploidy Underlying the Fungal Feeding Site Is a Determinant of Powdery Mildew Growth and Reproduction

Golovinomyces orontii is an obligate biotrophic powdery mildew (PM) that colonizes Arabidopsis thaliana and agronomic species. It establishes a specialized feeding structure in epidermal cells to fuel its extensive surface hyphal growth and reproduction. Previously, endoreduplication was identified in Arabidopsis mesophyll cells underlying the fungal feeding site, presumably to meet the metabolic demands imposed by the fungus. Furthermore, the cell cycle transcription factor MYB3R4 was shown to regulate this process. Herein, PM-induced endoreduplication is further characterized and three additional factors influencing host ploidy in cells underlying the fungal feeding site are identified. While mutations in PUX2 and PMR6 reduce basal ploidy, mutations in PMR5 (and MYB3R4) abrogate the PM-induced ploidy increase. Moreover, analysis of pmr5 microarray data suggests that PMR5 acts upstream of a MYB3R transcription factor such as MYB3R4 to control PM-induced ploidy. Induced endoreduplication occurs exclusively in mesophyll cells underlying the fungal feeding site at 5 days postinoculation, concomitant with PM reproduction. Gene copy number increases and chromatin remains decondensed, suggesting active, elevated gene expression. Cell ploidy underlying the fungal feeding site is highly correlated with the extent of PM growth and reproduction for these mutants, indicating that (induced) mesophyll cell ploidy is a PM susceptibility determinant.