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.

Silencing of DND1 in potato and tomato impedes conidial germination, attachment and hyphal growth of Botrytis cinerea.

BACKGROUND: Botrytis cinerea, a necrotrophic pathogenic fungus, attacks many crops including potato and tomato. Major genes for complete resistance to B. cinerea are not known in plants, but a few quantitative trait loci have been described in tomato. Loss of function of particular susceptibility (S) genes appears to provide a new source of resistance to B. cinerea in Arabidopsis. RESULTS: In this study, orthologs of Arabidopsis S genes (DND1, DMR6, DMR1 and PMR4) were silenced by RNAi in potato and tomato (only for DND1). DND1 well-silenced potato and tomato plants showed significantly reduced diameters of B. cinerea lesions as compared to control plants, at all-time points analysed. Reduced lesion diameter was also observed on leaves of DMR6 silenced potato plants but only at 3 days post inoculation (dpi). The DMR1 and PMR4 silenced potato transformants were as susceptible as the control cv Desiree. Microscopic analysis was performed to observe B. cinerea infection progress in DND1 well-silenced potato and tomato leaves. A significantly lower number of B. cinerea conidia remained attached to the leaf surface of DND1 well-silenced potato and tomato plants and the hyphal growth of germlings was hampered. CONCLUSIONS: This is the first report of a cytological investigation of Botrytis development on DND1-silenced crop plants. Silencing of DND1 led to reduced susceptibility to Botrytis, which was associated with impediment of conidial germination and attachment as well as hyphal growth. Our results provide new insights regarding the use of S genes in resistance breeding.

Silencing of six susceptibility genes results in potato late blight resistance.

Phytophthora infestans, the causal agent of late blight, is a major threat to commercial potato production worldwide. Significant costs are required for crop protection to secure yield. Many dominant genes for resistance (R-genes) to potato late blight have been identified, and some of these R-genes have been applied in potato breeding. However, the P. infestans population rapidly accumulates new virulent strains that render R-genes ineffective. Here we introduce a new class of resistance which is based on the loss-of-function of a susceptibility gene (S-gene) encoding a product exploited by pathogens during infection and colonization. Impaired S-genes primarily result in recessive resistance traits in contrast to recognition-based resistance that is governed by dominant R-genes. In Arabidopsis thaliana, many S-genes have been detected in screens of mutant populations. In the present study, we selected 11 A. thaliana S-genes and silenced orthologous genes in the potato cultivar Desiree, which is highly susceptible to late blight. The silencing of five genes resulted in complete resistance to the P. infestans isolate Pic99189, and the silencing of a sixth S-gene resulted in reduced susceptibility. The application of S-genes to potato breeding for resistance to late blight is further discussed.

Down-regulation of Arabidopsis DND1 orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew.

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.

Detection of an inversion in the Ty-2 region between S. lycopersicum and S. habrochaites by a combination of de novo genome assembly and BAC cloning.

KEY MESSAGE: A chromosomal inversion associated with the tomato Ty - 2 gene for TYLCV resistance is the cause of severe suppression of recombination in a tomato Ty - 2 introgression line. Among tomato and its wild relatives inversions are often observed, which result in suppression of recombination. Such inversions hamper the transfer of important traits from a related species to the crop by introgression breeding. Suppression of recombination was reported for the TYLCV resistance gene, Ty-2, which has been introgressed in cultivated tomato (Solanum lycopersicum) from the wild relative S. habrochaites accession B6013. Ty-2 was mapped to a 300-kb region on the long arm of chromosome 11. The suppression of recombination in the Ty-2 region could be caused by chromosomal rearrangements in S. habrochaites compared with S. lycopersicum. With the aim of visualizing the genome structure of the Ty-2 region, we compared the draft de novo assembly of S. habrochaites accession LYC4 with the sequence of cultivated tomato ('Heinz'). Furthermore, using populations derived from intraspecific crosses of S. habrochaites accessions, the order of markers in the Ty-2 region was studied. Results showed the presence of an inversion of approximately 200 kb in the Ty-2 region when comparing S. lycopersicum and S. habrochaites. By sequencing a BAC clone from the Ty-2 introgression line, one inversion breakpoint was identified. Finally, the obtained results are discussed with respect to introgression breeding and the importance of a priori de novo sequencing of the species involved.

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.

Down-regulation of acetolactate synthase compromises Ol-1- mediated resistance to powdery mildew in tomato.

BACKGROUND: In a cDNA-AFLP analysis comparing transcript levels between powdery mildew (Oidium neolycopersici)-susceptible tomato cultivar Moneymaker (MM) and near isogenic lines (NILs) carrying resistance gene Ol-1 or Ol-4, a transcript-derived fragment (TDF) M11E69-195 was found to be present in NIL-Ol-1 but absent in MM and NIL-Ol-4. This TDF shows homology to acetolactate synthase (ALS). ALS is a key enzyme in the biosynthesis of branched-chain amino acids valine, leucine and isoleucine, and it is also a target of commercial herbicides. RESULTS: Three ALS homologs ALS1, ALS2, ALS3 were identified in the tomato genome sequence. ALS1 and ALS2 show high similarity, whereas ALS3 is more divergent. Transient silencing of both ALS1 and ALS2 in NIL-Ol-1 by virus-induced gene silencing (VIGS) resulted in chlorotic leaf areas that showed increased susceptibility to O. neolycopersici (On). VIGS results were confirmed by stable transformation of NIL-Ol-1 using an RNAi construct targeting both ALS1 and ALS2. In contrast, silencing of the three ALS genes individually by RNAi constructs did not compromise the resistance of NIL-Ol-1. Application of the herbicide chlorsulfuron to NIL-Ol-1 mimicked the VIGS phenotype and caused loss of its resistance to On. Susceptible MM and On-resistant line NIL-Ol-4 carrying a nucleotide binding site and leucine rich repeat (NB-LRR) resistance gene were also treated with chlorsulfuron. Neither the susceptibility of MM nor the resistance of NIL-Ol-4 was affected. CONCLUSIONS: ALS is neither involved in basal defense, nor in resistance conferred by NB-LRR type resistance genes. Instead, it is specifically involved in Ol-1-mediated resistance to tomato powdery mildew, suggesting that ALS-induced change in amino acid homeostasis is important for resistance conferred by Ol-1.

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.

Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz).

In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.

Regulatory control of high levels of carotenoid accumulation in potato tubers.

Potato (Solanum tuberosum L.) tubers contain a wide range of carotenoid contents. To decipher the key factors controlling carotenoid levels in tubers, four potato lines (Atlantic, Désirée, 91E22 and POR03) were examined by a combination of biochemical, molecular and genomics approaches. These lines contained incremental levels of carotenoids, which were found to be associated with enhanced capacity of carotenoid biosynthesis as evident from norflurazon treatment. Microarray analysis of high and low carotenoid lines (POR03 versus Atlantic) revealed 381 genes that showed significantly differential expression. The carotenoid metabolic pathway genes ß-carotene hydroxylase 2 (BCH2) and ß-carotene hydroxylase 1 (BCH1), along with zeaxanthin epoxidase (ZEP), and carotenoid cleavage dioxygenase 1A (CCD1A) were among the most highly differentially expressed genes. The transcript levels of BCH2 and BCH1 were lowest in Atlantic and highest in POR03, whereas those of ZEP and CCD1A were high in low carotenoid lines and low in high carotenoid lines. The high expression of BCH2 in POR03 line was associated with enhanced response to sugars. Our results indicate that high levels of carotenoid accumulation in potato tubers were due to an increased metabolic flux into carotenoid biosynthetic pathway, as well as the differential expression of carotenoid metabolic genes.

The Global Dimension of Tomato Yellow Leaf Curl Disease: Current Status and Breeding Perspectives.

Tomato yellow leaf curl disease (TYLCD) caused by tomato yellow leaf curl virus (TYLCV) and a group of related begomoviruses is an important disease which in recent years has caused serious economic problems in tomato (Solanum lycopersicum) production worldwide. Spreading of the vectors, whiteflies of the Bemisia tabaci complex, has been responsible for many TYLCD outbreaks. In this review, we summarize the current knowledge of TYLCV and TYLV-like begomoviruses and the driving forces of the increasing global significance through rapid evolution of begomovirus variants, mixed infection in the field, association with betasatellites and host range expansion. Breeding for host plant resistance is considered as one of the most promising and sustainable methods in controlling TYLCD. Resistance to TYLCD was found in several wild relatives of tomato from which six TYLCV resistance genes (Ty-1 to Ty-6) have been identified. Currently, Ty-1 and Ty-3 are the primary resistance genes widely used in tomato breeding programs. Ty-2 is also exploited commercially either alone or in combination with other Ty-genes (i.e., Ty-1, Ty-3 or ty-5). Additionally, screening of a large collection of wild tomato species has resulted in the identification of novel TYLCD resistance sources. In this review, we focus on genetic resources used to date in breeding for TYLCVD resistance. For future breeding strategies, we discuss several leads in order to make full use of the naturally occurring and engineered resistance to mount a broad-spectrum and sustainable begomovirus resistance.

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.

Identification of alleles of carotenoid pathway genes important for zeaxanthin accumulation in potato tubers.

We have investigated the genetics and molecular biology of orange flesh colour in potato (Solanum tuberosum L.). To this end the natural diversity in three genes of the carotenoid pathway was assessed by SNP analyses. Association analysis was performed between SNP haplotypes and flesh colour phenotypes in diploid and tetraploid potato genotypes. We observed that among eleven beta-carotene hydroxylase 2 (Chy2) alleles only one dominant allele has a major effect, changing white into yellow flesh colour. In contrast, none of the lycopene epsilon cyclase (Lcye) alleles seemed to have a large effect on flesh colour. Analysis of zeaxanthin epoxidase (Zep) alleles showed that all (diploid) genotypes with orange tuber flesh were homozygous for one specific Zep allele. This Zep allele showed a reduced level of expression. The complete genomic sequence of the recessive Zep allele, including the promoter, was determined, and compared with the sequence of other Zep alleles. The most striking difference was the presence of a non-LTR retrotransposon sequence in intron 1 of the recessive Zep allele, which was absent in all other Zep alleles investigated. We hypothesise that the presence of this large sequence in intron 1 caused the lower expression level, resulting in reduced Zep activity and accumulation of zeaxanthin. Only genotypes combining presence of the dominant Chy2 allele with homozygosity for the recessive Zep allele produced orange-fleshed tubers that accumulated large amounts of zeaxanthin.

Ty-1, a universal resistance gene against geminiviruses that is compromised by co-replication of a betasatellite.

Tomato yellow leaf curl virus (TYLCV), a begomovirus, causes large yield losses and breeding for resistance is an effective way to combat this viral disease. The resistance gene Ty-1 codes for an RNA-dependent RNA polymerase and has recently been shown to enhance transcriptional gene silencing of TYLCV. Whereas Ty-1 was earlier shown to also confer resistance to a bipartite begomovirus, here it is shown that Ty-1 is probably generic to all geminiviruses. A tomato Ty-1 introgression line, but also stable transformants of susceptible tomato cv. Moneymaker and Nicotiana benthamiana (N. benthamiana) expressing the Ty-1 gene, exhibited resistance to begomoviruses as well as to the distinct, leafhopper-transmitted beet curly top virus, a curtovirus. Stable Ty-1 transformants of N. benthamiana and tomato showed fewer symptoms and reduced viral titres on infection compared to wild-type plants. TYLCV infections in wild-type N. benthamiana plants in the additional presence of a betasatellite led to increased symptom severity and a consistent, slightly lowered virus titre relative to the high averaged levels seen in the absence of the betasatellite. On the contrary, in Ty-1 transformed N. benthamiana viral titres increased in the presence of the betasatellite. The same was observed when these Ty-1-encoding plants were challenged with TYLCV and a potato virus X construct expressing the RNA interference suppressor protein ßC1 encoded by the betasatellite. The resistance spectrum of Ty-1 and the durability of the resistance are discussed in light of antiviral RNA interference and viral counter defence strategies.

The NLR Protein Encoded by the Resistance Gene Ty-2 Is Triggered by the Replication-Associated Protein Rep/C1 of Tomato Yellow Leaf Curl Virus.

The whitefly-transmitted tomato yellow leaf curl virus (TYLCV) is one of the most destructive viral pathogens of cultivated tomato. To combat TYLCV, resistance gene Ty-2 has been introduced into cultivated tomato (Solanum lycopersicum) from wild tomato species Solanum habrochaites by interspecific crossing. Introgression lines with Ty-2 contain a large inversion compared with S. lycopersicum, which causes severe suppression of recombination and has hampered the cloning of Ty-2 so far. Here, we report the fine-mapping and cloning of Ty-2 using crosses between a Ty-2 introgression line and several susceptible S. habrochaites accessions. Ty-2 was shown to encode a nucleotide-binding leucine-rich repeat (NLR) protein. For breeding purposes, a highly specific DNA marker tightly linked to the Ty-2 gene was developed permitting marker-assisted selection. The resistance mediated by Ty-2 was effective against the Israel strain of TYLCV (TYLCV-IL) and tomato yellow leaf curl virus-[China : Shanghai2] (TYLCV-[CN : SH2]), but not against tomato yellow leaf curl Sardinia virus (TYLCSV) and leafhopper-transmitted beet curly top virus (BCTV). By co-infiltration experiments we showed that transient expression of the Rep/C1 protein of TYLCV, but not of TYLCSV triggered a hypersensitive response (HR) in Nicotiana benthamiana plants co-expressing the Ty-2 gene. Our results indicate that the Rep/C1 gene of TYLCV-IL presents the avirulence determinant of Ty-2-mediated resistance.

Resistance to Tomato Yellow Leaf Curl Virus in Tomato Germplasm.

Tomato yellow leaf curl virus (TYLCV) is a virus species causing epidemics in tomato (Solanum lycopersicum) worldwide. Many efforts have been focused on identification of resistance sources by screening wild tomato species. In many cases, the accession numbers were either not provided in publications or not provided in a consistent manner, which led to redundant screenings. In the current study, we summarized efforts on the screenings of wild tomato species for TYLCV resistance from various publications. In addition, we screened 708 accessions from 13 wild tomato species using different inoculation assays (i.e., whitefly natural infection and Agrobacterium-mediated inoculation) from which 138 accessions exhibited no tomato yellow leaf curl disease (TYLCD) symptoms. These symptomless accessions include 14 accessions from S. arcanum, 43 from S. chilense, 1 from S. chmielewskii, 28 from S. corneliomulleri, 5 from S. habrochaites, 4 from S. huaylasense, 2 from S. neorickii, 1 from S. pennellii, 39 from S. peruvianum, and 1 from S. pimpinellifolium. Most of the screened S. chilense accessions remained symptomless. Many symptomless accessions were also identified in S. arcanum, S. corneliomulleri, and S. peruvianum. A large number of S. pimpinellifolium accessions were screened. However, almost all of the tested accessions showed TYLCD symptoms. Further, we studied allelic variation of the Ty-1/Ty-3 gene in few S. chilense accessions by applying virus-induced gene silencing and allele mining, leading to identification of a number of allele-specific polymorphisms. Taken together, we present a comprehensive overview on TYLCV resistance and susceptibility in wild tomato germplasm, and demonstrate how to study allelic variants of the cloned Ty-genes in TYLCV-resistant accessions.

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.

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.

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.