Mildew Locus O Research Articles

Dual-Luciferase Reporter Assay for Prescreening CRISPR (d)Cas9-Mediated Epigenetic Editing on a Plant Promoter Using Human Cells.

Epigenetic editing, also known as EpiEdit, offers an exciting way to control gene expression without altering the DNA sequence. In this study, we evaluate the application of EpiEdit to plant promoters, specifically the MLO (mildew locus o) gene promoter. We use a modified CRISPR-(d)Cas9 system, in which the nuclease-deficient Cas9 (dCas9) is fused to an epigenetic modifier, to experimentally demonstrate the utility of this tool for optimizing epigenetic engineering of a plant promoter prior to in vivo plant epigenome editing. Guide RNAs are used to deliver the dCas9-epigenetic modifier fusion protein to the target gene sequence, where it induces modification of MLO gene expression. We perform preliminary experiments using a plant promoter cloned into the luciferase reporter system, which is transfected into a human system and analyzed using the dual-luciferase reporter assay. The results suggest that this approach may be useful in the early stages of plant epigenome editing, as it can aid in the selection of appropriate modifications to the plant promoter prior to conducting in vivo experiments under plant system conditions. Overall, the results demonstrate the potential of CRISPR (d)Cas9-based EpiEdit for precise and controlled regulation of gene expression.

Cloning and expression analysis of Mildew Locus O (MLO) genes related to powdery mildew resistance in Vitis pseudoreticulata, and functional characterisation of VpMLO13

ABSTRACT In many plants, Mildew Locus O (MLO) genes facilitate infection by pathogens, including Erysiphe necator, the causative agent of powdery mildew (PM) in grapevines. The function of VvMLOs in Vitis vinifera has previously been demonstrated, but the identity and function of VpMLOs in V. pseudoreticulata are not yet clear. Here, we cloned MLO genes that have been implicated in PM susceptibility from the highly PM-resistant V. pseudoreticulata accession ‘Baihe-35-1’ and the PM-susceptible V. vinifera ‘Carinena’. The open reading frames (ORFs) of MLO3, MLO6, and MLO8 differed between ‘Baihe-35-1’ and ‘Carinena’. After inoculation of the leaves with Erysiphe necator, six MLO genes exhibited two distinct expression patterns in ‘Baihe-35-1’ and ‘Carinena’. Consistent with the MLO proteins in other plants, we also found that VpMLO3, VpMLO6, VpMLO11, and VpMLO13 are all localised to the plasma membrane. To further support a role for VpMLOs in susceptibility to PM, we expressed VpMlo13 in the PM-resistant Arabidopsis thaliana MLO2/6/12 triple mutant, which showed that transgenic expression of VpMLO13 restored susceptibility to PM. In addition, a yeast two-hybrid (Y2H) assay showed that VpMLO13 interacted with calmodulin (VpCaM). These results will provide a basis for further investigating the PM resistance mechanisms in V. pseudoreticulata.

Enhancing powdery mildew resistance in soybean by targeted mutation of MLO genes using the CRISPR/Cas9 system

BackgroundPowdery mildew is a major disease that causes great losses in soybean yield and seed quality. Disease-resistant varieties, which are generated by reducing the impact of susceptibility genes through mutation in host plants, would be an effective approach to protect crops from this disease. The Mildew Locus O (MLO) genes are well-known susceptibility genes for powdery mildew in plant. In this study, we utilized the CRISPR/Cas9 system to induce targeted mutations in the soybean GmMLO genes to improve powdery mildew resistance.ResultsA dual-sgRNA CRISPR/Cas9 construct was designed and successfully transferred into the Vietnamese soybean cultivar DT26 through Agrobacterium tumefaciens-mediated transformation. Various mutant forms of the GmMLO genes including biallelic, chimeric and homozygous were found at the T0 generation. The inheritance and segregation of CRISPR/Cas9-induced mutations were confirmed and validated at the T1 and T2 generations. Out of six GmMLO genes in the soybean genome, we obtained the Gmmlo02/Gmmlo19/Gmmlo23 triple and Gmmlo02/Gmmlo19/Gmmlo20/Gmmlo23 quadruple knockout mutants at the T2 generation. When challenged with Erysiphe diffusa, a fungus that causes soybean powdery mildew, all mutant plants showed enhanced resistance to the pathogen, especially the quadruple mutant. The powdery mildew severity in the mutant soybeans was reduced by up to 36.4% compared to wild-type plants. In addition, no pleiotropic effect on soybean growth and development under net-house conditions was observed in the CRISPR/Cas9 mutants.ConclusionsOur results indicate the involvement of GmMLO02, GmMLO19, GmMLO20 and GmMLO23 genes in powdery mildew susceptibility in soybean. Further research should be conducted to investigate the roles of individual tested genes and the involvement of other GmMLO genes in this disease infection mechanism. Importantly, utilizing the CRISPR/Cas9 system successfully created the Gmmlo transgene-free homozygous mutant lines with enhanced resistance to powdery mildew, which could be potential materials for soybean breeding programs.

The First Genome-Wide Mildew Locus O Genes Characterization in the Lamiaceae Plant Family.

Powdery mildew (PM) is a widespread plant disease that causes significant economic losses in thousands crops of temperate climates, including Lamiaceae species. Multiple scientific studies describe a peculiar form of PM-resistance associated at the inactivation of specific members of the Mildew Locus O (MLO) gene family, referred to as mlo-resistance. The characterization of Lamiaceae MLO genes, at the genomic level, would be a first step toward their potential use in breeding programs. We carried out a genome-wide characterization of the MLO gene family in 11 Lamiaceae species, providing a manual curated catalog of 324 MLO proteins. Evolutionary history and phylogenetic relationships were studied through maximum likelihood analysis and motif patter reconstruction. Our approach highlighted seven different clades diversified starting from an ancestral MLO domain pattern organized in 18 highly conserved motifs. In addition, 74 Lamiaceae putative PM susceptibility genes, clustering in clade V, were identified. Finally, we performed a codon-based evolutionary analysis, revealing a general high level of purifying selection in the eleven Lamiaceae MLO gene families, and the occurrence of few regions under diversifying selection in candidate susceptibility factors. The 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.

Genome-wide characterization and expression analysis of the MLO gene family sheds light on powdery mildew resistance in Lagenaria siceraria

MLO (mildew locus O) genes play a vital role in plant disease defense system, especially powdery mildew (PM). Lagenaria siceraria is a distinct Cucurbitaceae crop, and PM is one of the most serious diseases threatening crop production and quality. Although MLOs have been exploited in many Cucurbitaceae species, genome-wide mining of MLO gene family in bottle gourd has not been surveyed yet. Here we identified 16 MLO genes in our recently assembled L. siceraria genome. A total of 343 unique MLO protein sequences from 20 species were characterized and compared to deduce a generally high level of purifying selection and the occurrence of regions related to candidate susceptibility factors in the evolutional divergence. LsMLOs were clustered in six clades containing seven conserved transmembrane domains and 10 clade-specific motifs along with deletion and variation. Three genes (LsMLO3, LsMLO6, and LsMLO13) in clade V showed high sequence identity with orthologues involved in PM susceptibility. The expression pattern of LsMLOs was tissue-specific but not cultivar-specific. Furthermore, it was indicated by qRT-PCR and RNA-seq that LsMLO3 and LsMLO13 were highly upregulated in response to PM stress. Subsequent sequence analysis revealed the structural deletion of LsMLO13 and a single nonsynonymous substitution of LsMLO3 in the PM-resistant genotype. Taken all together, it is speculated that LsMLO13 is likely a major PM susceptibility factor. The results of this study provide new insights into MLO family genes in bottle gourd and find a potential candidate S gene for PM tolerance breeding.

Insights into the Genetic Architecture and Genomic Prediction of Powdery Mildew Resistance in Flax (Linum usitatissimum L.).

Powdery mildew (PM), caused by the fungus Oidium lini in flax, can cause defoliation and reduce seed yield and quality. To date, one major dominant gene (Pm1) and three quantitative trait loci (QTL) on chromosomes 1, 7 and 9 have been reported for PM resistance. To fully dissect the genetic architecture of PM resistance and identify QTL, a diverse flax core collection of 372 accessions augmented with an additional 75 breeding lines were sequenced, and PM resistance was evaluated in the field for eight years (2010–2017) in Morden, Manitoba, Canada. Genome-wide association studies (GWAS) were performed using two single-locus and seven multi-locus statistical models with 247,160 single nucleotide polymorphisms (SNPs) and the phenotypes of the 447 individuals for each year separately as well as the means over years. A total of 349 quantitative trait nucleotides (QTNs) were identified, of which 44 large-effect QTNs (R2 = 10–30%) were highly stable over years. The total number of favourable alleles per accession was significantly correlated with PM resistance (r = 0.74), and genomic selection (GS) models using all identified QTNs generated significantly higher predictive ability (r = 0.93) than those constructed using the 247,160 genome-wide random SNP (r = 0.69), validating the overall reliability of the QTNs and showing the additivity of PM resistance in flax. The QTNs were clustered on the distal ends of all 15 chromosomes, especially on chromosome 5 (0.4–5.6 Mb and 9.4–16.9 Mb) and 13 (4.7–5.2 Mb). To identify candidate genes, a dataset of 3230 SNPs located in resistance gene analogues (RGAs) was used as input for GWAS, from which an additional 39 RGA-specific QTNs were identified. Overall, 269 QTN loci harboured 445 RGAs within the 200 Kb regions spanning the QTNs, including 45 QTNs located within the RGAs. These RGAs supported by significant QTN/SNP allele effects were mostly nucleotide binding site and leucine-rich repeat receptors (NLRs) belonging to either coiled-coil (CC) NLR (CNL) or toll interleukin-1 (TIR) NLR (TNL), receptor-like kinase (RLK), receptor-like protein kinase (RLP), transmembrane-coiled-coil (TM-CC), WRKY, and mildew locus O (MLO) genes. These results constitute an important genomic tool for resistance breeding and gene cloning for PM in flax.

Genome-Wide Characterization of the MLO Gene Family in Cannabis sativa Reveals Two Genes as Strong Candidates for Powdery Mildew Susceptibility.

Cannabis sativa is increasingly being grown around the world for medicinal, industrial, and recreational purposes. As in all cultivated plants, cannabis is exposed to a wide range of pathogens, including powdery mildew (PM). This fungal disease stresses cannabis plants and reduces flower bud quality, resulting in significant economic losses for licensed producers. The Mildew Locus O (MLO) gene family encodes plant-specific proteins distributed among conserved clades, of which clades IV and V are known to be involved in susceptibility to PM in monocots and dicots, respectively. In several studies, the inactivation of those genes resulted in durable resistance to the disease. In this study, we identified and characterized the MLO gene family members in five different cannabis genomes. Fifteen Cannabis sativa MLO (CsMLO) genes were manually curated in cannabis, with numbers varying between 14, 17, 19, 18, and 18 for CBDRx, Jamaican Lion female, Jamaican Lion male, Purple Kush, and Finola, respectively (when considering paralogs and incomplete genes). Further analysis of the CsMLO genes and their deduced protein sequences revealed that many characteristics of the gene family, such as the presence of seven transmembrane domains, the MLO functional domain, and particular amino acid positions, were present and well conserved. Phylogenetic analysis of the MLO protein sequences from all five cannabis genomes and other plant species indicated seven distinct clades (I through VII), as reported in other crops. Expression analysis revealed that the CsMLOs from clade V, CsMLO1 and CsMLO4, were significantly upregulated following Golovinomyces ambrosiae infection, providing preliminary evidence that they could be involved in PM susceptibility. Finally, the examination of variation within CsMLO1 and CsMLO4 in 32 cannabis cultivars revealed several amino acid changes, which could affect their function. Altogether, cannabis MLO genes were identified and characterized, among which candidates potentially involved in PM susceptibility were noted. The results of this study will lay the foundation for further investigations, such as the functional characterization of clade V MLOs as well as the potential impact of the amino acid changes reported. Those will be useful for breeding purposes in order to develop resistant cultivars.

Genome-wide identification and analysis of the MLO gene families in three Cucurbita species

Powdery mildew (PM) is a major fungal disease in the Cucurbita species in the world, which can cause significant yield loss. The Mildew Locus O (MLO) family genes play important roles in the PM stress response. In this paper, twenty, twenty-one, and eighteen candidate MLO genes in Cucurbita moschata, Cucurbita maxima and Cucurbita pepo, respectively, were identified and designated as CmoMLO, CmaMLO and CpeMLO, respectively. The phylogenetic analysis indicated that these MLOs were divided into five clades and the number of MLOs belonging to clade V in the Cucurbita species was more than that in other crops. Furthermore, the expression analysis in the susceptibility (S) and resistance (R) lines showed that CpeMLO1, CpeMLO2 and CpeMLO5 might be involved in the susceptibility response. CpeMLO4 and CpeMLO6 showing opposite expression patterns in the R/S lines might be involved in the resistance response. All these data would be beneficial for future functional analysis of MLOs in the Cucurbita species.

The MLO1 powdery mildew susceptibility gene in Lathyrus species: The power of high-density linkage maps in comparative mapping and synteny analysis.

Powdery mildews are major diseases for a range of crops. The loss of function of specific Mildew Locus O (MLO) genes has long been associated with pre-haustorial plant resistance to powdery mildew and has proven to be durable in several species. Erysiphe pisi is the major causal agent of powdery mildew in pea (Pisum sativum L.) and in the closely related Lathyrus sativus L. and Lathyrus cicera L. PsMLO1 has been extensively studied in pea. However, no MLO gene family members have been isolated and characterized in Lathyrus species so far. In this study, MLO1 genes were isolated and characterized in L. sativus and L. cicera genotypes with varied levels of partial resistance against powdery mildew. Phylogenetic analyses confirmed that Lathyrus MLO1 belongs to Clade V, like all dicot MLO proteins associated with powdery mildew susceptibility. A L. sativus recombinant inbred line population (RIL) was genotyped by sequencing to develop a high-density L. sativus genetic linkage map. DNA sequence polymorphisms between the analyzed genotypes allowed the location of MLO1 in the newly developed L. sativus RIL genetic linkage map. Subsequent comparative mapping between L. sativus and L. cicera genetic maps and P. sativum, Lens culinaris Medik., and Medicago truncatula Gaertn. reference genomes revealed important aspects of the conservation of the MLO1 locus position and of the overall chromosomal rearrangements occurring during legume evolution, with relevance to legume disease resistance breeding programs.

Genome-wide identification and expression analysis of the MLO gene family reveal a candidate gene associated with powdery mildew susceptibility in bitter gourd (Momordica charantia)

Powdery mildew (PM), caused by Podosphaera xanthii, is one of the most serious disease in bitter gourd (Momordica charantia). Specific members of the Mildew Locus O (MLO) gene family act as susceptibility factors by recognizing PM pathogens and the corresponding loss-of-function mutations confer broad-spectrum durable host resistance. However, the potential PM-susceptibility genes in bitter gourd have not been identified yet. Therefore, genome-wide characterization and expression analysis of the bitter gourd MLO gene family were carried out in the present study. The results suggested that 15 MLO homologs were present in the bitter gourd genome. Gene organization, cis-elements, conserved amino acid residues and motifs were also investigated and analyzed. Phylogenetic analysis revealed that these identified MLO proteins clustered in six well-defined clades. It was noteworthy that McMLO7b, a bitter gourd MLO homolog falling in clade V which clustered all the known PM susceptibility factors of dicots, was up-regulated more than 6-fold at an early infection stage (6 h) and was inferred to be a candidate gene of PM susceptibility factor. In addition, exogenous salicylic acid (SA) treatment enhanced the PM resistance of bitter gourd, accompanied by a significantly elevated level of endogenous SA. However, McMLO7b showed nearly equivalent expression in SA-treated plants compared to the mock-treated seedlings at the early PM infection stage. MLO proteins were directly targeted by PM to trigger pathogenesis, therefore, a weak effect of exogenous SA on interaction between bitter gourd and PM could be inferred. To conclude, the results will be helpful in proposing better strategies to control PM disease with either resistant cultivars or cultivation management.

Interaction Between AtCML9 and AtMLO10 Regulates Pollen Tube Development and Seed Setting.

In higher-plant reproduction, the compatibility of pollen tube germination in the pistil is essential for successful double fertilization. It has been reported that Mildew Locus O (MLO) family gene NTA (MLO7), expressing in synergid cells, can correctly guide pollen tubes. However, the molecular mechanism underlying the interacting partners to MLOs in the fertilization is still unknown. In our study, we identified the direct protein interaction between CML9 and MLO10 within a non-canonical CaMBD. In GUS reporter assays, CML9 expresses in a high level in pollens, whereas MLO10 can be specifically detected in stigma which reaches up to a peaking level before fertilization. Therefore, the spatio-temporal expression patterns of MLO10 and CML9 are required for the time-window of pollination. When we observed the pollen germination in vitro, two cml9 mutant alleles dramatically reduced germination rate by 15% compared to wild-type. Consistently, the elongation rate of pollen tubes in planta was obviously slow while manually pollinating cml9-1 pollens to mlo10-1 stigmas. Additionally, cml9-1 mlo10-1 double mutant alleles had relatively lower rate of seed setting. Taken together, protein interaction between MLO10 and CML9 is supposed to affect pollen tube elongation and further affect seed development.

Candidate gene mapping reveals VrMLO12 (MLO Clade II) is associated with powdery mildew resistance in mungbean (Vigna radiata [L.] Wilczek)

Loss/reduction of function of Mildew Locus O (MLO) genes clade V and MLO clade IV has been shown to be responsible for powdery mildew (PM) resistance in several plant species. Mungbean (Vigna radiata) genome possesses 18 MLO genes, VrMLO1 - VrMLO18. A previous study using mungbean F2 and BC1F1 populations derived from a cross between “CN60″ (susceptible) and “RUM5″ (resistance) demonstrated that QTL qPMRUM5−3 is a major QTL for PM resistance caused by Erysiphe polygoni and is the same with major QTL qPMV4718−3 that confers PM resistance in “V4718″ (resistance). In this study, bioinformatics analysis revealed VrMLO12 locates in the qPMRUM5−3 region. Fine mapping in the F2 and BC1F1 populations using newly developed DNA markers including gene-specific markers demonstrated association between VrMLO12 and the PM resistance. Sequence analyses of VrMLO12 revealed that compared to susceptible mungbeans, RUM5 and V4718 possess SNPs in exon 10 and exon 13. The SNPs caused amino acid changes of VrMLO12, A387S and A476 G, respectively. The change occurred in transmembrane 6 domain and calmodulin binding domain (CaMBD) of the VrMLO12 protein, respectively. qRT-PCR showed that transcript expression level of VrMLO12 in RUM5 challenged with and without by E. polygoni was significantly higher than that in CN60. Phylogenetic analysis revealed that in contrast to previous findings that MLO proteins associated with PM resistance belong to MLO clade V and MLO clade IV, VrMLO12 belongs to MLO clade II. The result suggested that VrMLO12 may function differently from the other MLOs that associated with PM susceptibility. Our findings provide insight into the PM resistance in mungbean and tools for mungbean breeding.

Short Communication: Identification of Mildew Locus O (MLO) genes in Durio zibethinus genome corresponding with the Powdery Mildew disease

Kemal RA, Sandjaja EBL, Santosa AP, Ivan J. 2018. Short Communication: Identification of Mildew Locus O (MLO) genes in Durio zibethinus genome corresponding with the Powdery Mildew disease. Biodiversitas 19: 2204-2212. Mildew Locus O (MLO) is a protein consisting of seven transmembrane domains and appears in the various type of plants. MLO proteins are classified into seven clades. It is known that specific clades have different roles in a plant. MLOs from Clades IV and V have been linked to plant's susceptibility to Powdery Mildew (PM) disease. This study aimed to provide an overview of MLO genes present in durian (Durio zibethinus) genome. Bioinformatic analyses were conducted to analyze the phylogeny and structure of MLO genes and proteins in durian. The result showed that there were 20 putative DzMLO genes in durian, encoding 39 putative DzMLO proteins. Durian MLOs belong to Clade I-VI with one protein belongs to Clade IV and five proteins belong to Clade V. Those six MLO proteins shared a common motif in C-terminal and second intracellular domains. Putative alternative splicing and differential expressions were observed among Clade V DzMLO genes. These findings will facilitate the functional characterization of MLO genes and proteins in durian. Functional studies, especially on C-terminal and second intracellular domains, need to be conducted to elucidate the role of MLO in PM susceptibility in durian.

Differential Expression under Podosphaera xanthii and Abiotic Stresses Reveals Candidate MLO Family Genes in Cucumis melo L

Mildew Locus O (MLO) gene family members have significant functions in plant responses to biotic and abiotic stresses. Here, we retrieved 14 MLO sequences designated as Cucumis melo MLO (CmMLO) from the melon genome database ‘Melonomics’. Phylogenetic analysis distributed the 14 predicted CmMLO proteins into five of the six distinct MLO clades. Tissue-specific reverse-transcription quantitative PCR (RT-qPCR) analysis using the C. melo ‘SCNU1154’ line revealed that 14 CmMLO genes were differentially expressed, suggesting their probable roles in specific processes of growth and development. Analysis of stress-induced expression revealed that five of the CmMLO genes were upregulated at 6 h post inoculation (dpi) with Podosphaera xanthii Race 1 or DH487, indicating a possible role for MLO proteins in the host cell as an initial step of disease progress. Seven CmMLO genes were upregulated at 10 d post inoculation (dpi) with both races, timing that corresponds to disease appearance at the later stage of infection. RT-qPCR analysis also revealed that all 14 CmMLO genes were up-regulated under drought stress, 13 were upregulated under salt stress, 10 were upregulated under heat stress, 4 were upregulated under cold, and 12 were upregulated under abscisic acid (ABA) treatment. This information regarding the stress-responsive behavior of CmMLO genes creates a window for developing stress-resistant cultivars in the Cucurbitaceae.

Functional characterization of the powdery mildew susceptibility gene SmMLO1 in eggplant (Solanum melongena L.).

Eggplant (Solanum melongena L.) is one of the most important vegetables among the Solanaceae and can be a host to fungal species causing powdery mildew (PM) disease. Specific homologs of the plant Mildew Locus O (MLO) gene family are PM susceptibility factors, as their loss of function results in a recessive form of resistance known as mlo resistance. In a previous work, we isolated the eggplant MLO homolog SmMLO1. SmMLO1 is closely related to MLO susceptibility genes characterized in other plant species. However, it displays a peculiar non-synonymous substitution that leads to a T→M amino acid change at protein position 422, in correspondence of the MLO calmodulin-binding domain. In this study, we performed the functional characterization of SmMLO1. Transgenic overexpression of SmMLO1 in a tomato mlo mutant compromised resistance to the tomato PM pathogen Oidium neolycopersici, thus indicating that SmMLO1 is a PM susceptibility factor in eggplant. PM susceptibility was also restored by the transgenic expression of a synthetic gene, named s-SmMLO1, encoding a protein identical to SmMLO1, except for the presence of T at position 422. This indicates that the T→M polymorphism does not affect the protein role as PM susceptibility factor. Overall, the results of this work are of interest for the functional characterization of MLO proteins and the introduction of PM resistance in eggplant using reverse genetics.

Candidate Susceptibility Genes for Powdery and Downy Mildew in Watermelon and Squash

Powdery mildew (PM) caused by Podosphaera xanthii and downy mildew (DM) caused by Pseudoperonospora cubensis are two of the most economically important diseases for watermelon (Citrullus lanatus) and squash (Cucurbita pepo, C. maxima and C. moschata). Traditional breeding for resistance to PM and DM is resource intensive, often requiring decades’ long phenotyping and selection processes. As an alternative, durable and broad-spectrum resistance to PM and DM can be obtained through loss-of-function of susceptibility genes in elite breeding material. Susceptibility genes for PM [Mildew-Locus-O (MLO) and Powdery Mildew Resistance (PMR)] and DM [Downy Mildew Resistance (DMR)] have been functionally proven in model plant species. Previous studies have reported candidate MLO genes for C. lanatus and C. pepo, but none for C. maxima and C. moschata. On the contrary, no PMR or DMR candidate genes have been identified for C. lanatus or any of the Cucurbita species. The current study used bioinformatics approaches based on sequence similarity, phylogenetic relationships and presence of conserved domains to predict candidate MLO genes in C. maxima and C. moschata and PMR and DMR genes in C. lanatus, C. pepo, C. maxima and C. moschata. Four MLO homologs in C. maxima and five in C. moschata clustered within Clade V, a clade containing all MLO susceptibility genes in dicots, and had highly conserved transmembrane domains and C-terminal PM interaction motif. Sixty-three candidate PMR genes were identified among the four species, 16 of which had close similarity to functionally proven PMR homologs in model species. Similarly, 37 candidate DMR genes were identified 12 among which clustered with functionally proven DMR homologs in model species. Functional analysis of the genes identified in the current study will reveal their role in pathogenesis and assess their potential for manipulation through gene editing methods to generate novel resistant plant genotypes.

The knock-down of the expression of MdMLO19 reduces susceptibility to powdery mildew (Podosphaera leucotricha) in apple (Malus domestica).

SummaryVarieties resistant to powdery mildew (PM; caused by Podosphaera leucotricha) are a major component of sustainable apple production. Resistance can be achieved by knocking‐out susceptibility S‐genes to be singled out among members of the MLO (Mildew Locus O) gene family. Candidates are MLO S‐genes of phylogenetic clade V up‐regulated upon PM inoculation, such as MdMLO11 and 19 (clade V) and MdMLO18 (clade VII). We report the knock‐down through RNA interference of MdMLO11 and 19, as well as the complementation of resistance with MdMLO18 in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock‐down of MdMLO19 reduced PM disease severity by 75%, whereas the knock‐down of MdMLO11, alone or in combination with MdMLO19, did not result in any reduction or additional reduction of susceptibility compared with MdMLO19 alone. The test in A. thaliana excluded a role for MdMLO18 in PM susceptibility. Cell wall appositions (papillae) were present in both PM‐resistant and PM‐susceptible plants, but were larger in resistant lines. No obvious negative phenotype was observed in plants with mlo genes knocked down. Apparently, MdMLO19 plays the pivotal role in apple PM susceptibility and its knock‐down induces a very significant level of resistance.

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.