ZED1-related kinase 13 is required for resistance against Pseudoidium neolycopersici in Arabidopsis accession Bla-6.

To explore specific components of resistance against the tomato-adapted powdery mildew pathogen Pseudoidium neolycopersici (On) in the model plant Arabidopsis, we performed a disease assay in 123 accessions. When testing the resistance in the F1 from crossings between resistant accessions with susceptible Col-0 or Sha, only the progeny of the cross between accession Bla-6 and Col-0 displayed a completely resistant phenotype. The resistance in Bla-6 is known to be specific for Pseudoidium neolycopersici. QTL analysis and fine-mapping through several rounds of recombinant screenings allowed us to locate a major resistance QTL in an interval on chromosome 1, containing two candidate genes and an intergenic insertion. Via CRISPR/Cas9 targeted mutagenesis, we could show that knocking out the ZED-1 RELATED KINASE 13 (ZRK13) gene compromised the On resistance in Bla-6. Several polymorphisms are observed in the ZRK13 allelic variant of Bla-6 when compared to the Col-0 protein.

Discovery and Characterization of a Novel Tomato mlo Mutant from an EMS Mutagenized Micro-Tom Population.

In tomato (Solanum lycopersicum), there are at least three SlMLO (Mildew resistance Locus O) genes acting as susceptibility genes for the powdery mildew disease caused by Oidium neolycopersici, namely SlMLO1, SlMLO5 and SlMLO8. Of the three homologs, the SlMLO1 gene plays a major role since a natural mutant allele called ol-2 can almost completely prevent fungal penetration by formation of papillae. The ol-2 allele contains a 19-bp deletion in the coding sequence of the SlMLO1 gene, resulting in a premature stop codon within the second cytoplasmic loop of the predicted protein. In this study, we have developed a new genetic resource (M200) in the tomato cv. Micro-Tom genetic background by means of ethyl methane sulfonate (EMS) mutagenesis. The mutant M200 containing a novel allele (the m200 allele) of the tomato SlMLO1 gene showed profound resistance against powdery mildew with no fungal sporulation. Compared to the coding sequence of the SlMLO1 gene, the m200 allele carries a point mutation at T65A. The SNP results in a premature stop codon L22* located in the first transmembrane domain of the complete SlMLO1 protein. The length of the predicted protein is 21 amino acids, while the SlMLO1 full-length protein is 513 amino acids. A high-resolution melting (HRM) marker was developed to distinguish the mutated m200 allele from the SlMLO1 allele in backcross populations. The mutant allele conferred recessive resistance that was associated with papillae formation at fungal penetration sites of plant epidermal cells. A comprehensive list of known mlo mutations found in natural and artificial mutants is presented, which serves as a particularly valuable resource for powdery mildew resistance breeding.

CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew.

BACKGROUND: The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene PowderyMildewResistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On). RESULTS: To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants. CONCLUSION: CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations.

Functional Characterization of a Syntaxin Involved in Tomato (Solanum lycopersicum) Resistance against Powdery Mildew.

Specific syntaxins, such as Arabidopsis AtPEN1 and its barley ortholog ROR2, play a major role in plant defense against powdery mildews. Indeed, the impairment of these genes results in increased fungal penetration in both host and non-host interactions. In this study, a genome-wide survey allowed the identification of 21 tomato syntaxins. Two of them, named SlPEN1a and SlPEN1b, are closely related to AtPEN1. RNAi-based silencing of SlPEN1a in a tomato line carrying a loss-of-function mutation of the susceptibility gene SlMLO1 led to compromised resistance toward the tomato powdery mildew fungus Oidium neolycopersici. Moreover, it resulted in a significant increase in the penetration rate of the non-adapted powdery mildew fungus Blumeria graminis f. sp. hordei. Codon-based evolutionary analysis and multiple alignments allowed the detection of amino acid residues that are under purifying selection and are specifically conserved in syntaxins involved in plant-powdery mildew interactions. Our findings provide both insights on the evolution of syntaxins and information about their function which is of interest for future studies on plant-pathogen interactions and tomato breeding.

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.

Functional characterization of cucumber (Cucumis sativus L.) Clade V MLO genes.

BACKGROUND: Powdery mildew (PM) causing fungi are well-known pathogens, infecting over 10.000 plant species, including the economically important crop cucumber (Cucumis sativus L.). Loss-of-function mutations in clade V MLO genes have previously been shown to lead to recessively inherited broad-spectrum resistance to PM in several species. In cucumber, one clade V MLO homolog (CsaMLO8) was previously identified as being a susceptibility factor to PM. Two other closely related homologs (CsaMLO1 and CsaMLO11) were found, but their function was not yet unravelled. METHODS: CsaMLO1 and CsaMLO11 were cloned from cucumber and overexpressed in a tomato mlo mutant. The transcript abundances of all three CsaMLO genes in different cucumber tissues were quantified using qRT-PCR and RNA-seq, with and without inoculation with the cucumber PM fungus Podosphaera xanthii. Allelic variation of CsaMLO1 and CsaMLO11 was screened in silico in sequenced cucumber germplasm. RESULTS: Heterologous overexpression of all three CsaMLO genes in the tomato mlo mutant restored susceptibility to PM caused by Oidium neolycopersici, albeit to a different extent: whereas overexpression of CsaMLO1 or CsaMLO8 completely restored susceptibility, overexpression of CsaMLO11 was only partially able to restore PM susceptibility. Furthermore, it was observed by qRT-PCR and RNA-seq that CsaMLO8 was significantly higher expressed in non-inoculated cucumber compared to the other two MLO genes. However, inoculation with P. xanthii led to upregulation of CsaMLO1, but not to upregulation of CsaMLO8 or CsaMLO11. CONCLUSIONS: Both CsaMLO1 and CsaMLO11 are functional susceptibility genes, although we conclude that based on the transcript abundance CsaMLO8 is probably the major clade V MLO gene in cucumber regarding providing susceptibility to PM. Potential loss-of-function mutations in CsaMLO1 and CsaMLO11 have not been identified. The generation and analysis of such mutants are interesting subjects for further investigation.

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 â†’ 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 â†’ 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.

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.

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

BACKGROUND: Specific 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. METHODS: A bioinformatic approach was followed to study the type of evolution of Angiosperm MLO susceptibility proteins. Transgenic complementation tests were performed for functional analysis. RESULTS: Our 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. CONCLUSIONS: With 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.

A transposable element insertion in the susceptibility gene CsaMLO8 results in hypocotyl resistance to powdery mildew in cucumber.

BACKGROUND: Powdery mildew (PM) is an important disease of cucumber (Cucumis sativus L.). CsaMLO8 was previously identified as a candidate susceptibility gene for PM in cucumber, for two reasons: 1) This gene clusters phylogenetically in clade V, which has previously been shown to harbour all known MLO-like susceptibility genes for PM identified in dicot species; 2) This gene co-localizes with a QTL on chromosome 5 for hypocotyl-specific resistance to PM. METHODS: CsaMLO8 alleles from susceptible and resistant cucumber were cloned and transformed to mlo-mutant tomato. Cucumber seedlings were inoculated with Podosphaera xanthii, tissues were studied for CsaMLO8 expression at several timepoints post inoculation using qRT-PCR. The occurrence of the observed loss-of-function allele of CsaMLO8 in resequenced cucumber accessions was studied in silico. RESULTS: We cloned CsaMLO8 alleles from susceptible and resistant cucumber genotypes, the latter carrying the QTL for hypocotyl resistance. We found that insertion of a non-autonomous Class LTR retrotransposable element in the resistant genotype leads to aberrant splicing of CsaMLO8 mRNA. Heterologous expression of the wild-type allele of CsaMLO8 in a tomato mlo-mutant restored PM susceptibility. However, heterologous expression of the CsaMLO8 allele cloned from the resistant cucumber genotype failed to restore PM susceptibility. Furthermore we showed that inoculation of susceptible cucumber with the PM pathogen Podosphaera xanthii induced transcriptional upregulation of CsaMLO8 in hypocotyl tissue, but not in cotyledon or leaf tissue. This coincides with the observation that the QTL at the CsaMLO8-locus causes full resistance in hypocotyl tissue, but only partial resistance in cotyledons and true leafs. We studied the occurrence of the loss-of-function allele of CsaMLO8 in cucumber germplasm by an in silico approach using resequencing data of a collection of 115 cucumber accessions, and found that this allele was present in 31 out of 115 accessions. CONCLUSIONS: CsaMLO8 was characterised as a functional susceptibility gene to PM, particularly in the hypocotyl where it was transcriptionally upregulated upon inoculation with the PM pathogen P. xanthii. A loss-of-function mutation in CsaMLO8 due to the insertion of a transposable element was found to be the cause of hypocotyl resistance to PM. This particular allele of CsaMLO8 was found to occur in 27 % of the resequenced cucumber accessions.

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.

Natural loss-of-function mutation of EDR1 conferring resistance to tomato powdery mildew in Arabidopsis thaliana accession C24.

To screen for potentially novel types of resistance to tomato powdery mildew Oidium neolycopersici, a disease assay was performed on 123 Arabidopsis thaliana accessions. Forty accessions were fully resistant, and one, C24, was analysed in detail. By quantitative trait locus (QTL) analysis of an F2 population derived from C24 × Sha (susceptible accession), two QTLs associated with resistance were identified in C24. Fine mapping of QTL-1 on chromosome 1 delimited the region to an interval of 58 kb encompassing 15 candidate genes. One of these was Enhanced Disease Resistance 1 (EDR1). Evaluation of the previously obtained edr1 mutant of Arabidopsis accession Col-0, which was identified because of its resistance to powdery mildew Golovinomyces cichoracearum, showed that it also displayed resistance to O. neolycopersici. Sequencing of EDR1 in our C24 germplasm (referred to as C24-W) revealed two missing nucleotides in the second exon of EDR1 resulting in a premature stop codon. Remarkably, C24 obtained from other laboratories does not contain the EDR1 mutation. To verify the identity of C24-W, a DNA region containing a single nucleotide polymorphism (SNP) unique to C24 was sequenced showing that C24-W contains the C24-specific nucleotide. C24-W showed enhanced resistance to O. neolycopersici compared with C24 not containing the edr1 mutation. Furthermore, C24-W displayed a dwarf phenotype, which was not associated with the mutation in EDR1 and was not caused by the differential accumulation of pathogenesis-related genes. In conclusion, we identified a natural edr1 mutant in the background of C24.

Activation tagging of ATHB13 in Arabidopsis thaliana confers broad-spectrum disease resistance.

Powdery mildew species Oidium neolycopersici (On) can cause serious yield losses in tomato production worldwide. Besides on tomato, On is able to grow and reproduce on Arabidopsis. In this study we screened a collection of activation-tagged Arabidopsis mutants and identified one mutant, 3221, which displayed resistance to On, and in addition showed a reduced stature and serrated leaves. Additional disease tests demonstrated that the 3221 mutant exhibited resistance to downy mildew (Hyaloperonospora arabidopsidis) and green peach aphid (Myzus persicae), but retained susceptibility to bacterial pathogen Pseudomonas syringae pv tomato DC3000. The resistance trait and morphological alteration were mutually linked in 3221. Identification of the activation tag insertion site and microarray analysis revealed that ATHB13, a homeodomain-leucine zipper (HD-Zip) transcription factor, was constitutively overexpressed in 3221. Silencing of ATHB13 in 3221 resulted in the loss of both the morphological alteration and resistance, whereas overexpression of the cloned ATHB13 in Col-0 and Col-eds1-2 backgrounds resulted in morphological alteration and resistance. Microarray analysis further revealed that overexpression of ATHB13 influenced the expression of a large number of genes. Previously, it was reported that ATHB13-overexpressing lines conferred tolerance to abiotic stress. Together with our results, it appears that ATHB13 is involved in the crosstalk between abiotic and biotic stress resistance pathways.

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

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

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

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