A catalogue of recombination coldspots in interspecific tomato hybrids.

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties.

A chromosome scale tomato genome built from complementary PacBio and Nanopore sequences alone reveals extensive linkage drag during breeding.

The assembly and scaffolding of plant crop genomes facilitate the characterization of genetically diverse cultivated and wild germplasm. The cultivated tomato (Solanum lycopersicum) has been improved through the introgression of genetic material from related wild species, including resistance to pandemic strains of tobacco mosaic virus (TMV) from Solanum peruvianum. Here we applied PacBio HiFi and ONT Nanopore sequencing to develop independent, highly contiguous and complementary assemblies of an inbred TMV-resistant tomato variety. We show specific examples of how HiFi and ONT datasets can complement one another to improve assembly contiguity. We merged the HiFi and ONT assemblies to generate a long-read-only assembly where all 12 chromosomes were represented as 12 contiguous sequences (N50 = 68.5 Mbp). This chromosome scale assembly did not require scaffolding using an orthogonal data type. The merged assembly was validated by chromosome conformation capture data and is highly consistent with previous tomato genome assemblies that made use of genetic maps and Hi-C for scaffolding. Our long-read-only assembly reveals that a complex series of structural variants linked to the TMV resistance gene likely contributed to linkage drag of a 64.1-Mbp region of the S. peruvianum genome during tomato breeding. Through marker studies and ONT-based comprehensive haplotyping we show that this minimal introgression region is present in six cultivated tomato hybrid varieties developed in three commercial breeding programs. Our results suggest that complementary long read technologies can facilitate the rapid generation of near-complete genome sequences.

Deciphering resistance to Zymoseptoria tritici in the Tunisian durum wheat landrace accession 'Agili39'.

BACKGROUND: Septoria tritici blotch (STB), caused by Zymoseptoria tritici (Z. tritici), is an important biotic threat to durum wheat in the entire Mediterranean Basin. Although most durum wheat cultivars are susceptible to Z. tritici, research in STB resistance in durum wheat has been limited. RESULTS: In our study, we have identified resistance to a wide array of Z. tritici isolates in the Tunisian durum wheat landrace accession 'Agili39'. Subsequently, a recombinant inbred population was developed and tested under greenhouse conditions at the seedling stage with eight Z. tritici isolates and for five years under field conditions with three Z. tritici isolates. Mapping of quantitative trait loci (QTL) resulted in the identification of two major QTL on chromosome 2B designated as Qstb2B_1 and Qstb2B_2. The Qstb2B_1 QTL was mapped at the seedling and the adult plant stage (highest LOD 33.9, explained variance 61.6%), conferring an effective resistance against five Z. tritici isolates. The Qstb2B_2 conferred adult plant resistance (highest LOD 32.9, explained variance 42%) and has been effective at the field trials against two Z. tritici isolates. The physical positions of the flanking markers linked to Qstb2B_1 and Qstb2B_2 indicate that these two QTL are 5 Mb apart. In addition, we identified two minor QTL on chromosomes 1A (Qstb1A) and chromosome 7A (Qstb7A) (highest LODs 4.6 and 4.0, and explained variances of 16% and 9%, respectively) that were specific to three and one Z. tritici isolates, respectively. All identified QTL were derived from the landrace accession Agili39 that represents a valuable source for STB resistance in durum wheat. CONCLUSION: This study demonstrates that Z. tritici resistance in the 'Agili39' landrace accession is controlled by two minor and two major QTL acting in an additive mode. We also provide evidence that the broad efficacy of the resistance to STB in 'Agili 39' is due to a natural pyramiding of these QTL. A sustainable use of this Z. tritici resistance source and a positive selection of the linked markers to the identified QTL will greatly support effective breeding for Z. tritici resistance in durum wheat.

Comparative genomics reveals the in planta-secreted Verticillium dahliae Av2 effector protein recognized in tomato plants that carry the V2 resistance locus.

Plant pathogens secrete effector molecules during host invasion to promote colonization. However, some of these effectors become recognized by host receptors to mount a defence response and establish immunity. Recently, a novel resistance was identified in wild tomato, mediated by the single dominant V2 locus, to control strains of the soil-borne vascular wilt fungus Verticillium dahliae that belong to race 2. With comparative genomics of race 2 strains and resistance-breaking race 3 strains, we identified the avirulence effector that activates V2 resistance, termed Av2. We identified 277 kb of race 2-specific sequence comprising only two genes encoding predicted secreted proteins that are expressed during tomato colonization. Subsequent functional analysis based on genetic complementation into race 3 isolates and targeted deletion from the race 1 isolate JR2 and race 2 isolate TO22 confirmed that one of the two candidates encodes the avirulence effector Av2 that is recognized in V2 tomato plants. Two Av2 allelic variants were identified that encode Av2 variants that differ by a single acid. Thus far, a role in virulence could not be demonstrated for either of the two variants.

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.

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.

KEY MESSAGE: Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

Cisgenic Rvi6 scab-resistant apple lines show no differences in Rvi6 transcription when compared with conventionally bred cultivars.

MAIN CONCLUSION: The expression of the apple scab resistance gene Rvi6 in different apple cultivars and lines is not modulated by biotic or abiotic factors. All commercially important apple cultivars are susceptible to Venturia inaequalis, the causal organism of apple scab. A limited number of apple cultivars were bred to express the resistance gene Vf from the wild apple genotype Malus floribunda 821. Positional cloning of the Vf locus allowed the identification of the Rvi6 (formerly HcrVf2) scab resistance gene that was subsequently used to generate cisgenic apple lines. It is important to understand and compare how this resistance gene is transcribed and modulated during infection in conventionally bred cultivars and in cisgenic lines. The aim of this work was to study the transcription pattern of Rvi6 in three classically bred apple cultivars and six lines of 'Gala' genetically modified to express Rvi6. Rvi6 transcription was analyzed at two time points using quantitative real-time PCR (RT-qPCR) following inoculation with V. inaequalis conidia or water. Rvi6 transcription was assessed in relation to five reference genes. ß-Actin, RNAPol, and UBC were the most suited to performing RT-qPCR experiments on Malus × domestica. Inoculation with V. inaequalis conidia under conditions conducive to scab infection failed to produce any significant changes to the transcription level of Rvi6. Rvi6 expression levels were inconsistent in response to external treatments in the different apple cultivars, and transgenic, intragenic or cisgenic lines.

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

Varieties 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.

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.

Characterization of the MLO gene family in Rosaceae and gene expression analysis in Malus domestica.

BACKGROUND: Powdery 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. RESULTS: We 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. CONCLUSIONS: Genomic 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.

Dietary flavonoids from modified apple reduce inflammation markers and modulate gut microbiota in mice.

Apples are rich in polyphenols, which provide antioxidant properties, mediation of cellular processes such as inflammation, and modulation of gut microbiota. In this study we compared genetically engineered apples with increased flavonoids [myeloblastis transcription factor 10 (MYB10)] with nontransformed apples from the same genotype, "Royal Gala" (RG), and a control diet with no apple. Compared with the RG diet, the MYB10 diet contained elevated concentrations of the flavonoid subclasses anthocyanins, flavanol monomers (epicatechin) and oligomers (procyanidin B2), and flavonols (quercetin glycosides), but other plant secondary metabolites were largely unaltered. We used these apples to investigate the effects of dietary flavonoids on inflammation and gut microbiota in 2 mouse feeding trials. In trial 1, male mice were fed a control diet or diets supplemented with 20% MYB10 apple flesh and peel (MYB-FP) or RG apple flesh and peel (RG-FP) for 7 d. In trial 2, male mice were fed MYB-FP or RG-FP diets or diets supplemented with 20% MYB10 apple flesh or RG apple flesh for 7 or 21 d. In trial 1, the transcription levels of inflammation-linked genes in mice showed decreases of >2-fold for interleukin-2 receptor (Il2rb), chemokine receptor 2 (Ccr2), chemokine ligand 10 (Cxcl10), and chemokine receptor 10 (Ccr10) at 7 d for the MYB-FP diet compared with the RG-FP diet (P < 0.05). In trial 2, the inflammation marker prostaglandin E(2) (PGE(2)) in the plasma of mice fed the MYB-FP diet at 21 d was reduced by 10-fold (P < 0.01) compared with the RG-FP diet. In colonic microbiota, the number of total bacteria for mice fed the MYB-FP diet was 6% higher than for mice fed the control diet at 21 d (P = 0.01). In summary, high-flavonoid apple was associated with decreases in some inflammation markers and changes in gut microbiota when fed to healthy mice.

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.

Analysis of genetically modified red-fleshed apples reveals effects on growth and consumer attributes.

Consumers of whole foods, such as fruits, demand consistent high quality and seek varieties with enhanced health properties, convenience or novel taste. We have raised the polyphenolic content of apple by genetic engineering of the anthocyanin pathway using the apple transcription factor MYB10. These apples have very high concentrations of foliar, flower and fruit anthocyanins, especially in the fruit peel. Independent lines were examined for impacts on tree growth, photosynthesis and fruit characteristics. Fruit were analysed for changes in metabolite and transcript levels. Fruit were also used in taste trials to study the consumer perception of such a novel apple. No negative taste attributes were associated with the elevated anthocyanins. Modification with this one gene provides near isogenic material and allows us to examine the effects on an established cultivar, with a view to enhancing consumer appeal independently of other fruit qualities.

Evidence for regulation of columnar habit in apple by a putative 2OG-Fe(II) oxygenase.

Understanding the genetic mechanisms controlling columnar-type growth in the apple mutant 'Wijcik' will provide insights on how tree architecture and growth are regulated in fruit trees. In apple, columnar-type growth is controlled by a single major gene at the Columnar (Co) locus. By comparing the genomic sequence of the Co region of 'Wijcik' with its wild-type 'McIntosh', a novel non-coding DNA element of 1956 bp specific to Pyreae was found to be inserted in an intergenic region of 'Wijcik'. Expression analysis of selected genes located in the vicinity of the insertion revealed the upregulation of the MdCo31 gene encoding a putative 2OG-Fe(II) oxygenase in axillary buds of 'Wijcik'. Constitutive expression of MdCo31 in Arabidopsis thaliana resulted in compact plants with shortened floral internodes, a phenotype reminiscent of the one observed in columnar apple trees. We conclude that MdCo31 is a strong candidate gene for the control of columnar growth in 'Wijcik'.

Insights on cisgenic plants with durable disease resistance under the European Green Deal.

Significant shares of harvests are lost to pests and diseases, therefore, minimizing these losses could solve part of the supply constraints to feed the world. Cisgenesis is defined as the insertion of genetic material into a recipient organism from a donor that is sexually compatible. Here, we review (i) conventional plant breeding, (ii) cisgenesis, (iii) current pesticide-based disease management, (iv) potential economic implications of cultivating cisgenic crops with durable disease resistances, and (v) potential environmental implications of cultivating such crops; focusing mostly on potatoes, but also apples, with resistances to Phytophthora infestans and Venturia inaequalis, respectively. Adopting cisgenic varieties could provide benefits to farmers and to the environment through lower pesticide use, thus contributing to the European Green Deal target.

DNA primase large subunit is an essential plant gene for geminiviruses, putatively priming viral ss-DNA replication.

The family of Geminiviridae consists of more than 500 circular single-stranded (ss) DNA viral species that can infect numerous dicot and monocot plants. Geminiviruses replicate their genome in the nucleus of a plant cell, taking advantage of the host's DNA replication machinery. For converting their DNA into double-stranded DNA, and subsequent replication, these viruses rely on host DNA polymerases. However, the priming of the very first step of this process, i.e. the conversion of incoming circular ssDNA into a dsDNA molecule, has remained elusive for almost 30 years. In this study, sequencing of melon (Cucumis melo) accession K18 carrying the Tomato leaf curl New Delhi virus (ToLCNDV) recessive resistance quantitative trait locus (QTL) in chromosome 11, and analyses of DNA sequence data from 100 melon genomes, showed a conservation of a shared mutation in the DNA Primase Large subunit (PRiL) of all accessions that exhibited resistance upon a challenge with ToLCNDV. Silencing of (native) Nicotiana benthamiana PriL and subsequent challenging with three different geminiviruses showed a severe reduction in titers of all three viruses, altogether emphasizing an important role of PRiL in geminiviral replication. A model is presented explaining the role of PriL during initiation of geminiviral DNA replication, i.e. as a regulatory subunit of primase that generates an RNA primer at the onset of DNA replication in analogy to DNA Primase-mediated initiation of DNA replication in all living organisms.

Genetic analysis of metabolites in apple fruits indicates an mQTL hotspot for phenolic compounds on linkage group 16.

Apple (Malus×domestica Borkh) is among the main sources of phenolic compounds in the human diet. The genetic basis of the quantitative variations of these potentially beneficial phenolic compounds was investigated. A segregating F1 population was used to map metabolite quantitative trait loci (mQTLs). Untargeted metabolic profiling of peel and flesh tissues of ripe fruits was performed using liquid chromatography-mass spectrometry (LC-MS), resulting in the detection of 418 metabolites in peel and 254 in flesh. In mQTL mapping using MetaNetwork, 669 significant mQTLs were detected: 488 in the peel and 181 in the flesh. Four linkage groups (LGs), LG1, LG8, LG13, and LG16, were found to contain mQTL hotspots, mainly regulating metabolites that belong to the phenylpropanoid pathway. The genetics of annotated metabolites was studied in more detail using MapQTL®. A number of quercetin conjugates had mQTLs on LG1 or LG13. The most important mQTL hotspot with the largest number of metabolites was detected on LG16: mQTLs for 33 peel-related and 17 flesh-related phenolic compounds. Structural genes involved in the phenylpropanoid biosynthetic pathway were located, using the apple genome sequence. The structural gene leucoanthocyanidin reductase (LAR1) was in the mQTL hotspot on LG16, as were seven transcription factor genes. The authors believe that this is the first time that a QTL analysis was performed on such a high number of metabolites in an outbreeding plant species.

Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples.

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (P(MdRbc)) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar 'Gala' was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. 'Santana'.

Impairment of Tomato WAT1 Enhances Resistance to Vascular Wilt Fungi Despite Severe Growth Defects.

Verticillium dahliae is a particularly notorious vascular wilt pathogen of tomato and poses a reoccurring challenge to crop protection as limited qualitative resistance is available. Therefore, alternative approaches for crop protection are pursued. One such strategy is the impairment of disease susceptibility (S) genes, which are plant genes targeted by pathogens to promote disease development. In Arabidopsis and cotton, the Walls Are Thin 1 (WAT1) gene has shown to be a S gene for V. dahliae. In this study, we identified the tomato WAT1 homolog Solyc04g080940 (SlWAT1). Transient and stable silencing of SlWAT1, based on virus-induced gene silencing (VIGS) and RNAi, respectively, did not consistently lead to reduced V. dahliae susceptibility in tomato. However, CRISPR-Cas9 tomato mutant lines carrying targeted deletions in SlWAT1 showed significantly enhanced resistance to V. dahliae, and furthermore also to Verticillium albo-atrum and Fusarium oxysporum f. sp. lycopersici (Fol). Thus, disabling the tomato WAT1 gene resulted in broad-spectrum resistance to various vascular pathogens in tomato. Unfortunately these tomato CRISPR mutant lines suffered from severe growth defects. In order to overcome the pleiotropic effect caused by the impairment of the tomato WAT1 gene, future efforts should be devoted to identifying tomato SlWAT1 mutant alleles that do not negatively impact tomato growth and development.

The amino acid permease (AAP) genes CsAAP2A and SlAAP5A/B are required for oomycete susceptibility in cucumber and tomato.

Cucurbit downy mildew (DM), caused by the obligate biotroph Pseudoperonospora cubensis, is a destructive disease in cucumber. A valuable source of DM resistance is the Indian cucumber accession PI 197088, which harbours several quantitative trait loci (QTLs) contributing to quantitatively inherited DM resistance. With a combination of fine-mapping and transcriptomics, we identified Amino Acid Permease 2A (CsAAP2A) as a candidate gene for QTL DM4.1.3. Whole-genome and Sanger sequencing revealed the insertion of a Cucumis Mu-like element (CUMULE) transposon in the allele of the resistant near-isogenic line DM4.1.3. To confirm whether loss of CsAAP2A contributes to partial DM resistance, we performed targeting induced local lesions in genomes on a DM-susceptible cucumber genotype to identify an additional csaap2a mutant, which indeed was partially DM resistant. In view of the loss of the putative function as amino acid transporter, we measured amino acids in leaves. We found that DM-inoculated leaves of line DM4.1.3 (with the csaap2a mutation) contained significantly fewer amino acids than wild-type cucumber. The decreased flow of amino acids towards infected leaves in csaap2a plants compared to the wild type might explain the resistant phenotype of the mutant, as this would limit the available nutrients for the pathogen and thereby its fitness. To examine whether AAP genes play a conserved role as susceptibility factors in plant-oomycete interactions, we made targeted mutations in two AAP genes from tomato and studied the effect on susceptibility to Phytophthora infestans. We conclude that not only CsAAP2A but also SlAAP5A/SlAAP5B are susceptibility genes for oomycete pathogens.