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.

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.

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.

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.

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.

Analysis of QTL DM4.1 for Downy Mildew Resistance in Cucumber Reveals Multiple subQTL: A Novel RLK as Candidate Gene for the Most Important subQTL.

One of the biggest problems in cucumber cultivation is cucurbit downy mildew (DM), caused by the obligate biotroph Pseudoperonospora cubensis. Whereas DM in cucumber was previously efficiently controlled by the dm-1 gene from Indian cucumber accession PI 197087, this resistance was broken by new DM strains, prompting the search for novel sources of resistance. A promising source of resistance is the wild cucumber accession PI 197088. It was previously shown that DM resistance in this genotype inherits polygenically. In this paper, we put the focus on one of the QTL, DM4.1 that is located on chromosome 4. QTL DM4.1 was shown to consist of three subQTL: DM4.1.1 affected pathogen-induced necrosis, DM4.1.2 was shown to have an additive effect on sporulation, and DM4.1.3 had a recessive effect on chlorosis as well as an effect on sporulation. Near-isogenic lines (NILs) were produced by introgressing the subQTLs into a susceptible cucumber line (HS279) with good horticultural traits. Transcriptomic analysis revealed that many genes in general, and defense pathway genes in particular, were differentially expressed in NIL DM4.1.1/.2 compared to NIL DM4.1.3 and the susceptible parent HS279. This indicates that the resistance from subQTL DM4.1.1 and/or subQTL DM4.1.2 likely involves defense signaling pathways, whereas resistance due to subQTL DM4.1.3 is more likely to be independent of known defense pathways. Based on fine-mapping data, we identified the RLK gene CsLRK10L2 as a likely candidate for subQTL DM4.1.2, as this gene was found to have a loss-of-function mutation in the susceptible parent HS279, and was strongly upregulated by P. cubensis inoculation in NIL DM4.1.1/.2. Heterologous expression of this gene triggered necrosis, providing further evidence that this gene is indeed causal for subQTL DM4.1.2.

Dissecting the Genotypic Variation of Growth Responses to Far-Red Radiation in Tomato.

The recent development of light-emitting diodes (LEDs) and their application in modern horticulture stimulated studies demonstrating that additional far-red (FR) radiation (700-800 nm) increases plant dry mass. This effect of FR has been explained by improved photosynthesis and/or plant architecture. However, the genotypic variation in this response is largely unknown. Here, we aim to explore and explain the genotypic variation in growth responses to additional FR. We expected the genotypic variation in the responses of plant dry mass to additional FR. Further, we hypothesized that a significant improvement of both net assimilation rate (NAR) and leaf area ratio (LAR) is responsible for a strong dry mass increase under additional FR, while some genotypes respond only marginally or even negatively in NAR or LAR under FR, thus resulting in a weak FR effect on plant dry mass. To test these hypotheses, we grew 33 different tomato genotypes for 21 days with 0, 25, or 100 µmol m-2 s-1 of FR added to a common white + red LED background lighting of 150 µmol m-2 s-1. Genotypes responded similarly with respect to plant height, stem dry mass, and shoot:root ratio; i.e., they all increased with increasing FR. However, the response of total plant dry mass varied among genotypes. We categorized the genotypes into three groups (strongly, moderately, and weakly responding groups) based on their relative response in total plant dry mass to FR. Growth component analysis revealed that the strongly responding genotypes increased strongly in NAR rather than LAR. The weakly responding genotypes, however, showed a substantial increase in LAR but not NAR. The increase in LAR was due to the increase in specific leaf area. Leaf mass fraction, which is the other component of LAR, decreased with FR and did not differ between groups. In conclusion, tomato genotypes that increased strongly in NAR in response to FR were able to achieve a more substantial increase in dry mass than did other genotypes. This is the first study to explain the differences in growth responses of a large number of tomato genotypes toward FR in their light environment.

Pfcyp51 exclusively determines reduced sensitivity to 14α-demethylase inhibitor fungicides in the banana black Sigatoka pathogen Pseudocercospora fijiensis.

The haploid fungus Pseudocercospora fijiensis causes black Sigatoka in banana and is chiefly controlled by extensive fungicide applications, threatening occupational health and the environment. The 14α-Demethylase Inhibitors (DMIs) are important disease control fungicides, but they lose sensitivity in a rather gradual fashion, suggesting an underlying polygenic genetic mechanism. In spite of this, evidence found thus far suggests that P. fijiensis cyp51 gene mutations are the main responsible factor for sensitivity loss in the field. To better understand the mechanisms involved in DMI resistance, in this study we constructed a genetic map using DArTseq markers on two F1 populations generated by crossing two different DMI resistant strains with a sensitive strain. Analysis of the inheritance of DMI resistance in the F1 populations revealed two major and discrete DMI-sensitivity groups. This is an indicative of a single major responsible gene. Using the DMI-sensitivity scorings of both F1 populations and the generation of genetic linkage maps, the sensitivity causal factor was located in a single genetic region. Full agreement was found for genetic markers in either population, underlining the robustness of the approach. The two maps indicated a similar genetic region where the Pfcyp51 gene is found. Sequence analyses of the Pfcyp51 gene of the F1 populations also revealed a matching bimodal distribution with the DMI resistant. Amino acid substitutions in P. fijiensis CYP51 enzyme of the resistant progeny were previously correlated with the loss of DMI sensitivity. In addition, the resistant progeny inherited a Pfcyp51 gene promoter insertion, composed of a repeat element with a palindromic core, also previously correlated with increased gene expression. This genetic approach confirms that Pfcyp51 is the single explanatory gene for reduced sensitivity to DMI fungicides in the analysed P. fijiensis strains. Our study is the first genetic analysis to map the underlying genetic factors for reduced DMI efficacy.

Breeding Has Increased the Diversity of Cultivated Tomato in The Netherlands.

It is generally believed that domestication and breeding of plants has led to genetic erosion, including loss of nutritional value and resistances to diseases, especially in tomato. We studied the diversity dynamics of greenhouse tomato varieties in NW Europe, especially The Netherlands, over the last seven decades. According to the used SNP array, the genetic diversity was indeed very low during the 1960s, but is now eight times higher when compared to that dip. The pressure since the 1970s to apply less pesticides led to the introgression of many disease resistances from wild relatives, representing the first boost of genetic diversity. In Europe a second boost ensued, largely driven by German popular media who named poor tasting tomatoes Wasserbomben (water bombs). The subsequent collapse of Dutch tomato exports to Germany fueled breeding for fruit flavor, further increasing diversity since the 1990s. The increased diversity in composition of aroma volatiles observed starting from 1990s may reflect the efforts of breeders to improve fruit quality. Specific groups of aroma compounds showed different quantitative trend over the decades studied. Our study provides compelling evidence that breeding has increased the diversity of tomato varieties considerably since the 1970s.

Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance.

Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici-wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control.

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.

Frequency of a natural truncated allele of MdMLO19 in the germplasm of Malus domestica.

Podosphaera leucotricha is the causal agent of powdery mildew (PM) in apple. To reduce the amount of fungicides required to control this pathogen, the development of resistant apple cultivars should become a priority. Resistance to PM was achieved in various crops by knocking out specific members of the MLO gene family that are responsible for PM susceptibility (S-genes). In apple, the knockdown of MdMLO19 resulted in PM resistance. However, since gene silencing technologies such as RNAi are perceived unfavorably in Europe, a different approach that exploits this type of resistance is needed. This work evaluates the presence of non-functional naturally occurring alleles of MdMLO19 in apple germplasm. The screening of the re-sequencing data of 63 apple individuals led to the identification of 627 single nucleotide polymorphisms (SNPs) in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18, and MdMLO19), 127 of which were located in exons. The T-1201 insertion of a single nucleotide in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino acids, including the calmodulin-binding domain. The presence of the insertion was evaluated in 115 individuals. It was heterozygous in 64 and homozygous in 25. Twelve of the 25 individuals carrying the insertion in homozygosity were susceptible to PM. After barley, pea, cucumber, and tomato, apple would be the fifth species for which a natural non-functional mlo allele has been found.

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.

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.

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.

Cisgenic apple trees; development, characterization, and performance.

Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.

Effector discovery in the fungal wheat pathogen Zymoseptoria tritici.

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici-wheat pathosystem.