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The bacterial flagellar motor is a rotary machine composed of functional modular components, which can perform bidirectional rotations to control the migration behavior of the bacterial cell. It resembles a two-cogwheel gear system, which consists of small and large cogwheels with cogs at the edges to regulate rotations. Such gearset models provide elegant blueprints to design and build artificial nanomachinery with desired functionalities. In this work, we demonstrate DNA assembly of a structurally well-defined nanodevice, which can carry out programmable rotations powered by DNA fuels. Our rotary nanodevice consists of three modular components, small origami ring, large origami ring, and gold nanoparticles (AuNPs). They mimic the sun gear, ring gear, and planet gears in a planetary gearset accordingly. These modular components are self-assembled in a compact manner, such that they can work cooperatively to impart bidirectional rotations. The rotary dynamics is optically recorded using fluorescence spectroscopy in real time, given the sensitive distance-dependent interactions between the tethered fluorophores and AuNPs on the rings. The experimental results are well supported by the theoretical calculations.

Sour cherry (Prunus cerasusL.) is an economically important allotetraploid cherry species believed to have evolved in the Caspian Sea and Black Sea regions. How, when and where exactly the evolution of this species took place is unclear. It resulted from a hybridization of the tetraploid ground cherry (Prunus fruticosaPall.) and an unreduced (2n) pollen of the diploid ancestor sweet cherry (P. aviumL.). Some indications implement that the genome of sour cherry is segmental allopolyploid, but how it is structured and to what extent is unknown. To get an insight, the genome of the sour cherry cultivar ‘Schattenmorelle’ was sequenced at ~400x using Illumina NovaSeqTMshort-read and Oxford Nanopore long-read technologies (ONT R9.4.1 PromethION). Additionally, the transcriptome of ‘Schattenmorelle’ was sequenced using PacBio Sequel II SMRT cell sequencing at ~300x. The final assembly resulted in a ~629 Mbp long pseudomolecule reference genome, which could be separated into two subgenomes each split into eight chromosomes. SubgenomePceS_a which originates fromP. aviumhas a length of 269 Mbp, whereas subgenomePceS_f which originates fromP. fruticosahas a length of 299.5 Mbp. The length of unassembled contigs was 60 Mbp. The genome of the sour cherry shows a size-reduction compared to the genomes of its ancestral species. It also shows traces of homoeologous sequence exchanges throughout the genome. Comparative positional sequence and protein analyses provided evidence that the genome of sour cherry is segmental allotetraploid and that it has evolved in a very recent event in the past.

Control over multiple optical elements that can be dynamically rearranged to yield substantial three-dimensional structural transformations is of great importance to realize reconfigurable plasmonic nanoarchitectures with sensitive and distinct optical feedback. In this work, we demonstrate a transformable plasmonic helix system, in which multiple gold nanoparticles (AuNPs) can be directly transported by DNA swingarms to target positions without undergoing consecutive stepwise movements. The swingarms allow for programmable AuNP translocations in large leaps within plasmonic nanoarchitectures, giving rise to tailored circular dichroism spectra. Our work provides an instructive bottom-up solution to building complex dynamic plasmonic systems, which can exhibit prominent optical responses through cooperative rearrangements of the constituent optical elements with high fidelity and programmability.

Climate change with warmer winter and spring temperatures poses major challenges to apple fruit production. Long-term observations confirm the trend toward earlier flowering, which leads to an increased risk of frost damage. New breeding strategies are needed to generate cultivars that are able to stay largely unaffected by warmer temperatures. Recently, epigenetic variation has been proposed as a new resource for breeding purposes and seems suitable in principle for apple breeding. However, to serve as a new resource for apple breeding, it is necessary to clarify whether epigenetic variation can be induced by the environment, whether it can create phenotypic variation, and whether this variation is stable across generations. In this brief review, we summarize the impact of climate change on the timing of apple phenology, highlight how epigenetic variation can potentially support novel breeding strategies, and point out important features of epigenetic variation that are required for its application in breeding programs.

Premature leaf fall disease is caused by the hemibiotrophic ascomycete Diplocarpon mali (Y. Harada & Sawamura 1974) (anamorph Marssonina coronaria). Breeding for disease resistance and development of resistant cultivars could help to defeat the disease in the future. The first step in resistance breeding is to assess Malus genetic resources for resistance to M. coronaria. The aim was to proof a detached leaf assay as phenotyping procedure. Disease assessment was performed after 14 days post inoculation (dpi). Different symptoms obtained from the inoculated genotypes indicate a high level of susceptibility in Malus cultivars. In contrast, M. baccata accession MAL0419 was tolerant to M. coronaria. Future experiments will show whether the tolerance of M. baccata accession MAL0419 is inherited and if the genotype is a possible donor for resistance breeding toward M. coronaria.

The spotted wing drosophila (SWD) causes massive losses in red raspberry (Rubus idaeus L.) cultivation by direct oviposition leading to damages of the soft skin fruits. Knowledge of the fly's host preference could help farmers in managing the pest. We used a laboratory‐based oviposition assay for screening the germplasm of Rubus to ascertain whether the spotted wing drosophila prefers certain cultivars to others for oviposition and if preference is based on citric acid and Brix content or firmness. Correlation analyses of evaluated characters with no‐choice tests results in 3 years obtained no correlation between citric acid, Brix content and oviposition. Primocane raspberries were lower affected by SWD than floricane raspberries. The Rubus hybrid cultivar “Dorman Red” and the primocane cultivar “Pokusa” showed the lowest oviposition rate compared to the other 58 evaluated genotypes. We found that oviposition correlates to firmness of the investigated cultivars, which strongly indicates that host preference is partly connected to that character in raspberries. The results are discussed regarding the use of Rubus genetic resources in breeding and integrated pest management to control spotted wing drosophila in the field.

Pyramiding different fire blight resistance genes and QTLs in future apple cultivars is the most eco-friendly way to combat this disease. Identification of strong fire blight resistance donors, and introgression of their resistance into apple breeding material are a continuing effort of breeding programs. Thus, enormous effort is been put into breeding research to understand host – pathogen interactions and mechanisms of resistance found in Malus. The crabapple Malus fusca (accession MAL0045) is highly resistant to fire blight, and although resistance is strain-dependent, resistance of MAL0045 is not overcome by any known strain of Erwinia amylovora to date. A strong fire blight resistance locus (FB_Mfu10) was fine mapped to an interval of 0.33 Centimorgan (cM) on linkage group (LG) 10 of MAL0045 using 1888 progenies. Subsequently, a single bacterial artificial chromosome (BAC) clone (46H22), which harbours FB_Mfu10-resistance alleles, was identified in a MAL0045 BAC library and sequenced using MiSeq illumina leading to the assembly of 45 contigs. Analyses of the sequence of 46H22 led to the identification of a receptor-like kinase candidate gene. Here, we report about resequencing 46H22 using MinION Oxford Nanopore and successfully assembled the sequences into a single contig, which allowed for identifying additional candidate genes.

Apple replant disease (ARD) is a worldwide economic risk in apple production. Although several studies have shown that the wild apple accession Malus × robusta 5 (Mr5) is ARD-tolerant, the genetics of this tolerance have not yet been elucidated. A genetic mapping approach with a biparental population derived from contrasting parents involving molecular markers provides a means for marker-assisted selection of genetically complex traits and for determining candidate genes. In this study, we crossed the ARD-tolerant wild apple accession Mr5 and the ARD-susceptible rootstock ‘M9’ and analyzed the resultant progeny for ARD tolerance. Hence, a high-density genetic map using a tunable genotyping-by-sequencing (tGBS) approach was established. A total of 4804 SNPs together with 77 SSR markers were included in the parental maps comprising 17 linkage groups. The phenotypic responses to ARD were evaluated for 106 offspring and classified by an ARD-susceptibility index (ASI). A Kruskal–Wallis test identified SNP markers and one SSR marker on linkage groups (LG) 6 and 2 that correlated with ARD tolerance. We found nine candidate genes linked with these markers, which may be associated with plant response to ARD. These candidate genes provide some insight into the defense mechanisms against ARD and should be studied in more detail.

Winter dormancy – a period of low metabolic activity and no visible growth – appears as an adaptation to harsh winter conditions and can be divided into different phases. It is tightly controlled by environmental cues, with ambient temperature playing a major role. During endodormancy, a cultivar-specific amount of cold needs to be perceived, and during ecodormancy, heat hours accumulate before bud burst and anthesis in spring. Expression analysis, performed in several key fruit tree species, proved to be very useful in elucidating the molecular control of onset and release of dormancy. However, the time resolution of these experiments has been limited. Therefore, in this study, dense time-series expression analysis was conducted for 40 candidate genes involved in dormancy control, under the cool-temperate climate conditions in Dresden. Samples were taken from the cultivars ‘Pinova’ and ‘Gala,’ which differ in flowering time. The set of candidate genes included well-established dormancy genes such as DAM genes, MdFLC-like, MdICE1, MdPRE 1, and MdPIF4. Furthermore, we tested genes from dormancy-associated pathways including the brassinosteroid, gibberellic acid, abscisic acid (ABA), cytokinin response, and respiratory stress pathways. The expression patterns of well-established dormancy genes were confirmed and could be associated with specific dormancy phases. In addition, less well-known transcription factors and genes of the ABA signaling pathway showed associations with dormancy progression. The three ABA signaling genes HAB1_chr15, HAI3, and ABF2 showed a local minimum of gene expression in proximity of the endodormancy to ecodormancy transition. The number of sampling points allowed us to correlate expression values with temperature data, which revealed significant correlations of ambient temperature with the expression of the Malus domestica genes MdICE1, MdPIF4, MdFLC-like, HAB1chr15, and the type-B cytokinin response regulator BRR9. Interestingly, the slope of the linear correlation of temperature with the expression of MdPIF4 differed between cultivars. Whether the strength of inducibility of MdPIF4 expression by low temperature differs between the ‘Pinova’ and ‘Gala’ alleles needs to be tested further.

Objective The proposed candidate gene underlying the Malus fusca fire blight resistance locus on chromosome 10 was previously predicted to possess 880 amino acids and 8 exons. Eight base pair (8 bp) insertion/deletion in the first exon potentially distinguished resistant genotypes from susceptible ones. This study aimed at analyzing the candidate gene sequence in another set of original resistant and susceptible progeny, characterizing the sequence in a transgenic line transformed with the candidate gene under its own native promoter, as well as deciphering the potential genomic differences between this candidate gene and its homolog in the ‘Golden Delicious’ doubled haploid genome (GDDH13). Results Sequences of amplicons of part of the candidate gene amplified in two progenies that showed resistant and susceptible fire blight phenotypes, confirmed the 8 bp insertion that distinguishes susceptible and resistant progenies. The transgenic line was positive for the candidate gene sequence, confirming a successful transfer into the background of apple cultivar ‘Pinova’, and possessed the same genomic sequence as the progeny with a resistant phenotype. Sequence analysis showed that the homolog gene on GDDH13 possesses a significant 18 bp deletion in exon 1 leading to a difference of 15 amino acid from the protein sequence of the candidate gene.

Cherries are among the most popular fruits among consumers and are grown for industrial processing or fresh consumption. The cultivation and breeding of cherries faces new challenges in the future, not least due to climate change. Cultivation is becoming increasingly difficult due to changing climatic conditions, diseases and pests. Therefore, the market demands new varieties with high fruit quality and adaptation to locally changing conditions. Breeding for tree fruit is a long-term task, though, and tools to facilitate this process are unfortunately not yet available to a sufficient extent. Rapid progress in third-generation sequencing technologies enables breeders to explore and exploit the genomic information underlying economical important traits. We used Oxford Nanopore Technology PromethION platform and R10.3 pore type to sequence the genome of tetraploid ground cherry (Prunus fruticosa). The final sequence has been deposited at DDBJ/ENA/GenBank under the accession JAHHUK000000000. A specific description of the work can be viewed at https://biorxiv.org/cgi/content/short/2021.06.01.446499v1. The version described in this paper is version JAHHUK010000000. The data on this platform includes 17 files, supports the assembling and annotation procedure, and provides insights into this challenging work. As a result, five assemblies (MiniasmMinimap.fasta, medaka.fasta, raconpolished.fasta, purged.fasta, purged2.fasta) have been processed to generate a draft genome assembly. The final genome sequence was masked (genome.fa.masked.gz, genome.fa.out, genome.fa.tbl) and annotated using Braker 1 and Braker 2, GeMoMa pipeline and eight reference datasets from other Prunus species. The final structural annotation, coding sequences and protein predictions are provided for future studies (GeMoMa_Annotation_filtered_predictions_8ref_RNAseq_Braker.gff, GeMoMa_Annotation_filtered_predictions_8ref_RNAseq_Braker_assignment.tabular, GeMoMa_Annotation_filtered_predictions_8ref_RNAseq_Braker_cds-parts.fasta, GeMoMa_Annotation_filtered_predictions_8ref_RNAseq_Braker_proteins.fasta). The functional prediction was performed with InterProScan (Results IPS.xlsx). In addition, the sequences and annotation of the chloroplast (Pf_chloroplast_1.0.fasta, GeSeqJob-20210201-124142_utg000088l_segment0_1_GFF3.gff3) and mitochondrion (Pf_mitochondria_1.0, GeSeqJob-20210202-114557_utg001396l_trimmed_GFF3.gff3) are presented.

ObjectiveErwinia amylovora causes fire blight disease in Malus. A strong resistance QTL (Mfu10) was previously detected on linkage group 10 of Malus fusca accession MAL0045, using several strains of the bacterium. As no strain capable of breaking the resistance of MAL0045 has been found, it was hypothesized that a second resistance factor contributes to the fire blight resistance of MAL0045. However, to date, no minor locus has been detected with previously published strains of the bacterium. We detected a minor QTL only on a subset of a population following inoculation with strain Ea1038, which heterologously expresses an effector in a derivative of isolate Ea3049. Two genetic maps of MAL0045, one scarce, the other dense with markers, were used for QTL analyses.ResultsMfu10 was detected on LG10 with Ea1038, as was previously with Ea3049. Although no other QTLs of significant LOD was previously detected in other linkage groups with Ea3049, a QTL of significant LOD was detected on LG16 (Mfu16) after inoculation of a subset of 76 individuals with Ea1038, but only using the dense genetic map. Mfu16 improved the effect of Mfu10. However, when the number of individuals inoculated with Ea1038 was increased to 121, Mfu16 was no longer detected in the dense genetic map. We hypothesize some factors, which might be responsible for the instability of this QTL.

A set of 680 apple cultivars from the Fruit Gene bank in Dresden Pillnitz was evaluated for the incidence of powdery mildew and scab in two consecutive years. The incidence of both scab and powdery mildew increased significantly in the second year. Sixty and 43 cultivars with very low incidence in both years of scab and powdery mildew, respectively, were analysed with molecular markers linked to known resistance genes. Thirty-five cultivars were identified to express alleles or combinations of alleles linked to Rvi2, Rvi4, Rvi6, Rvi13, Rvi14, or Rvi17. Twenty of them, modern as well as a few traditional cultivars known before the introduction or Rvi6 from Malus floribunda 821, amplified the 159 bp fragment of marker CH_Vf1 that is linked to Rvi6. Alleles linked to Pl1, Pld, or Plm were expressed from five cultivars resistant to powdery mildew. Eleven cultivars were identified to have very low susceptibility to both powdery mildew and scab. The information on resistance/susceptibility of fruit genetic resources towards economically important diseases is important for breeding and for replanting traditional cultivars. Furthermore, our work provides a well-defined basis for the discovery of undescribed, new scab, and powdery mildew resistance.

BackgroundCherries are stone fruits and belong to the economically important plant family of Rosaceae with worldwide cultivation of different species. The ground cherry, Prunus fruticosa Pall. is one ancestor of cultivated sour cherry, an important tetraploid cherry species. Here, we present a long read chromosome-level draft genome assembly and related plastid sequences using the Oxford Nanopore Technology PromethION platform and R10.3 pore type.FindingThe final assemblies obtained from 117.3 Gb cleaned reads representing 97x coverage of expected 1.2 Gb tetraploid (2n=4x=32) and 0.3 Gb haploid (1n=8) genome sequence of P. fruticosa were calculated. The N50 contig length ranged between 0.3 and 0.5 Mb with the longest contig being ∼6 Mb. BUSCO estimated a completeness between 98.7 % for the 4n and 96.1 % for the 1n datasets.Using a homology and reference based scaffolding method, we generated a final consensus genome sequence of 366 Mb comprising eight chromosomes. The N50 scaffold was ∼44 Mb with the longest chromosome being 66.5 Mb.The repeat content was estimated to ∼190 Mb (52 %) and 58,880 protein-coding genes were annotated. The chloroplast and mitochondrial genomes were 158,217 bp and 383,281 bp long, which is in accordance with previously published plastid sequences.ConclusionThis is the first report of the genome of ground cherry (P. fruticosa) sequenced by long read technology only. The datasets obtained from this study provide a foundation for future breeding, molecular and evolutionary analysis in Prunus studies.

Malus ×arnoldiana accession MAL0004 has been found to be resistant to moderately and highly virulent strains of the fire blight causal pathogen – the Gram-negative bacterium, Erwinia amylovora. Genetic analyses with an F1 segregating population derived from crossing the highly susceptible apple cultivar ‘Idared’ and MAL0004 led to the detection and mapping of the fire blight resistance locus of M. ×arnoldiana to linkage group (LG)12 (FB_Mar12). FB_Mar12 mapped at the distal end of LG12 below the apple SSR Hi07f01 in an interval of approximately 6 cM (Centimorgan), where both the fire blight resistance loci of M. floribunda 821 and ‘Evereste’ were located. We fine mapped the region containing FB_Mar12 using 892 progenies. Mining of the region of interest (ROI) on the ‘Golden Delicious’ doubled haploid genome (GDDH13) identified the presence of 2.3 Mb (megabases) in the homologous region. Of 40 primer pairs designed within this region, 20 were polymorphic and nine were mapped, leading to the identification of 24 significant recombinant individuals whose phenotypes were informative in determining the precise position of the locus within a 0.57 cM interval. Analyses of tightly linked marker sequences on the M. baccata draft genome revealed scaffolds of interest putatively harboring the resistance loci of M. ×arnoldiana, a hybrid between M. baccata and M. floribunda. Open reading frame (ORF) analyses led to the prediction of first fire blight resistance candidate genes with serine/threonine kinase and leucine-rich repeat domains, including homologs of previously identified ‘Evereste’ candidate genes. We discuss the implications of these results on breeding for resistance to fire blight.

Mechanically interlocked molecules have marked a breakthrough in the field of topological chemistry and boosted the vigorous development of molecular machinery. As an archetypal example of the interlocked molecules, catenanes comprise macrocycles that are threaded through one another like links in a chain. Inspired by the transition metal-templated approach of catenanes synthesis, the hierarchical assembly of DNA origami catenanes templated by gold nanoparticles is demonstrated in this work. DNA origami catenanes, which contain two, three or four interlocked rings are successfully created. In particular, the origami rings within the individual catenanes can be set free with respect to one another by releasing the interconnecting gold nanoparticles. This work will set the basis for rich progress toward DNA-based molecular architectures with unique structural programmability and well-defined topology.

International audience; Apple scab, caused by Venturia inaequalis, is a major disease worldwide. To control this disease, up to 20-25 fungicide applications may be needed depending on the year and the production system. Cultivation of scab-resistant apple cultivars would reduce the chemical footprint of apple production. Breeding for durable scab resistance is an objective of most apple breeding programmes. One way to achieve durable resistance is to pyramid multiple apple scab resistance genes in a cultivar. Currently, more than 18 different apple scab resistance genes have been reported. Molecular markers are available for most of these and can be used to select seedlings carrying multiple resistance genes. Apple scab isolates are virulent to specific apple scab resistance genes. Choice of optimal resistance genes for use in a breeding program, should therefore take regional presence of these apple scab isolates into consideration. In order to learn more about the geographical distribution of apple scab isolates, the VINQUEST project (www.vinquest.ch) was initiated in 2009. Ten years after the launch of this initiative, 24 partners from 14 countries are regularly contributing data. Results obtained from the data collected during the first 10 years provide the first insights into which apple scab resistance genes may hold most promise for breeding cultivars with durable resistance.

Most of the commercial apple cultivars are highly susceptible to fire blight, which is the most devastating bacterial disease affecting pome fruits. Resistance to fire blight is described especially in wild Malus accessions such as M. × robusta 5 (Mr5), but the molecular basis of host resistance response to the pathogen Erwinia amylovora is still largely unknown. The bacterial effector protein AvrRpt2EA was found to be the key determinant of resistance response in Mr5. A wild type E. amylovora strain and the corresponding avrRpt2EA deletion mutant were used for inoculation of Mr5 to induce resistance or susceptible response, respectively. By comparison of the transcriptome of both responses, 211 differentially expressed genes (DEGs) were identified. We found that heat-shock response including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs) are activated in apple specifically in the susceptible response, independent of AvrRpt2EA. Further analysis on the expression progress of 81 DEGs by high-throughput real-time qPCR resulted in the identification of genes that were activated after inoculation with E. amylovora. Hence, a potential role of these genes in the resistance to the pathogen is postulated, including genes coding for enzymes involved in formation of flavonoids and terpenoids, ribosome-inactivating enzymes (RIPs) and a squamosa promoter binding-like (SPL) transcription factor., Scientific Reports, 11 (1), ISSN:2045-2322

Malus domestica (apple) is one of the most important fruit crops worldwide. Fire blight, caused by Erwinia amylovora, is one of the most destructive bacterial diseases that impacts apple production systems worldwide. Although it is possible to manage fire blight using antibiotics such as streptomycin, kasugamycin or oxytetracycline, the quest for sustainable and eco-friendly production makes breeding for fire blight resistance the most promising and desirable approach. Breeding for resistance is a long, resource-intensive process due to the high susceptibility of most commercial apple cultivars, and the fact that most resistance sources being characterized are from wild genetic backgrounds with unpalatable fruits, and apple’s long generation times. Nevertheless, establishment of pre-breeding materials is crucial. This review highlights the status of breeding for fire blight resistance in Malus, taking into account, 1) major and minor resistance sources and their interaction with E. amylovora, 2) progress and challenges associated with using wild species as resistance sources, 3) progress and challenges associated with using elite cultivars as resistance sources, 4) advances in biotechnology for use in enhancing the production of durable fire blight resistant cultivars.

Most of the commercial apple cultivars are highly susceptible to fire blight, which is the most devastating bacterial disease affecting pome fruits. Resistance to fire blight is described especially in wild Malus accessions such as M. × robusta 5 (Mr5), but the molecular basis of host resistance response to the pathogen Erwinia amylovora is still largely unknown. The bacterial effector protein AvrRpt2EA was found to be the key determinant of resistance response in Mr5. A wild type E. amylovora strain and the corresponding avrRpt2EA deletion mutant were used for inoculation of Mr5 to induce resistance or susceptible response, respectively. By comparison of the transcriptome of both responses, 211 differentially expressed genes (DEGs) were identified. We found that heat-shock response including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs) are activated in apple specifically in the susceptible response, independent of AvrRpt2EA. Further analysis on the expression progress of 81 DEGs by high-throughput real-time qPCR resulted in the identification of genes that were activated after inoculation with E. amylovora. Hence, a potential role of these genes in the resistance to the pathogen is postulated, including genes coding for enzymes involved in formation of flavonoids and terpenoids, ribosome-inactivating enzymes (RIPs) and a squamosa promoter binding-like (SPL) transcription factor.

Black and red raspberries (Rubus occidentalis L. and Rubus idaeus L.) are the prominent members of the genus Rubus (Rosaceae family). Breeding programs coupled with the low costs of high-throughput sequencing have led to a reservoir of data that have improved our understanding of various characteristics of Rubus and facilitated the mapping of different traits. Gene B controls the waxy bloom, a clearly visible epicuticular wax on canes. The potential effects of this trait on resistance/susceptibility to cane diseases in conjunction with other morphological factors are not fully studied. Previous studies suggested that gene H, which controls cane pubescence, is closely associated with gene B. Here, we used tunable genotyping-by-sequencing technology to identify the de novo SNPs of R. occidentalis and R. idaeus using an interspecific population that segregates for the waxy bloom phenotype. We created linkage maps of both species and mapped the identified SNPs to the seven chromosomes (Ro01&ndash, Ro07) of Rubus. Importantly, we report, for the first time, the mapping of gene B to chromosome 2 of R. occidentalis using a genetic map consisting of 443 markers spanning 479.76 cM. We observed the poor transferability of R. idaeus SSRs to R. occidentalis and discrepancies in their previously reported chromosome locations.

International audience; Apple scab, caused by Venturia inaequalis, is a major fungal disease worldwide. Cultivation of scab-resistant cultivars would reduce the chemical footprint of apple production. However, new apple cultivars carrying durable resistances should be developed to prevent or at least slow the breakdown of resistance against races of V. inaequalis. One way to achieve durable resistance is to pyramid multiple scab resistance genes in a cultivar. The choice of the resistance genes to be combined in the pyramids should take into account the frequency of resistance breakdown and the geographical distribution of apple scab isolates able to cause such breakdowns. In order to acquire this information and to make it available to apple breeders, the VINQUEST project (www.vinquest.ch) was initiated in 2009. Ten years after launching this project, 24 partners from 14 countries regularly contribute data. From 2009 to 2018 nearly 9000 data points have been collected. This information has been used to identify the most promising apple scab resistance genes for developing cultivars with durable resistance, which to date are: Rvi5, Rvi11, Rvi12, Rvi14 and Rvi15. As expected, Rvi1, together with Rvi3 and Rvi8, were often overcome, hence have little value for scab resistance breeding. Rvi10 may also belong to this group. On the other hand Rvi2, Rvi4, Rvi6, Rvi7, Rvi9, and Rvi13 are still useful for the breeding, but their use is recommended only in extended pyramids of (≥3) resistance genes.

The Gram-negative bacterium Erwinia amylovora (Burrill) Winslow. et al., causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that functions to translocate effector proteins into plant cells that collectively function to suppress host defenses and enable pathogenesis. Until now, there is only a limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. In this study, we tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent Erwinia amylovora strains, which are assumed to influence the host resistance response of fire blight-resistant Malus species. Our findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene-for-gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple ‘Evereste’ and the wild species M. floribunda 821. Our results provide new insights into the host-pathogen-interactions between Malus sp. – Erwinia amylovora. This article is protected by copyright. All rights reserved.

The apple cultivar Regia, bred in Dresden-Pillnitz, Germany, is one of the few cultivars originating from a different scab (Venturia inaequalis) resistance background than Malus floribunda 821, the source of Rvi6. Cv. Regia is a descendant of Russian apple R12740-7A and has been proved to contain the two scab resistance genes Rvi2 and Rvi4. The cultivar itself has been grown in Dresden-Pillnitz since the early 1970s, while seedling populations derived from cv. Regia have been raised in fungicide-free plots since at least 1982. In 2011, small scab lesions were found for the first time on leaves of cv. Regia trees in an experimental unsprayed orchard in Dresden-Pillnitz. Single spore isolate Regia2 (R2) was cultured from these scab lesions and propagated, and then inoculated on scions of the VINQUEST scab differential host set comprising host (0) to host (15), host (17) and cv. Regia, grafted on rootstock M9, in a greenhouse. Strong sporulation was observed on hosts (0), (1), (2), (8), (9), (10) and cv. Regia, and weak sporulation on hosts (3), (4), (13) and (17), suggesting that the new isolate possesses the respective virulences. The implications of these results are discussed in relation to the natural infection of the differential hosts in the orchard and the artificial inoculation of the grafted differential hosts with inoculum gained from in vitro culture of R2.

Although, the Pacific crabapple, Malus fusca, is a hardy and disease resistant species, studies relating to the genetics of its unique traits are very limited partly due to the lack of a genetic map of this interesting wild apple. An accession of M. fusca (MAL0045) of Julius Kühn-Institut collection in Germany is highly resistant to fire blight disease, incited by different strains of the causative pathogen—Erwinia amylovora. This is the most destructive bacterial disease of Malus of which most of the domesticated apples (Malus domestica) are susceptible. Using a scarcely dense genetic map derived from a population of 134 individuals of MAL0045 × ‘Idared’, the locus (Mfu10) controlling fire blight resistance mapped on linkage group 10 (LG10) and explained up to 66% of the phenotypic variance with different strains. Although the development of robust and tightly linked molecular markers on LG10 through chromosome walking approach led to the identification of a major candidate gene, any minor effect locus remained elusive possibly due to the lack of marker density of the entire genetic map. Therefore, we have developed a dense genetic map of M. fusca using tunable genotyping-by-sequencing (tGBS) approach. Of thousands of de novo SNPs identified, 2677 were informative in M. fusca and 90.5% of these successfully mapped. In addition, integration of SNP data and microsatellite (SSR) data resulted in a final map comprising 17 LGs with 613 loci spanning 1081.35 centi Morgan (cM). This map will serve as a template for mapping using different strains of the pathogen.

Background Although the most common path of infection for fire blight, a severe bacterial disease on apple, is via host plant flowers, quantitative trait loci (QTLs) for fire blight resistance to date have exclusively been mapped following shoot inoculation. It is not known whether the same mechanism underlies flower and shoot resistance. Results We report the detection of a fire blight resistance QTL following independent artificial inoculation of flowers and shoots on two F1 segregating populations derived from crossing resistant Malus ×robusta 5 (Mr5) with susceptible ‘Idared’ and ‘Royal Gala’ in experimental orchards in Germany and New Zealand, respectively. QTL mapping of phenotypic datasets from artificial flower inoculation of the ‘Idared’ × Mr5 population with Erwinia amylovora over several years, and of the ‘Royal Gala’ × Mr5 population in a single year, revealed a single major QTL controlling floral fire blight resistance on linkage group 3 (LG3) of Mr5. This QTL corresponds to the QTL on LG3 reported previously for the ‘Idared’ × Mr5 and an ‘M9’ × Mr5 population following shoot inoculation in the glasshouse. Interval mapping of phenotypic data from shoot inoculations of subsets from both flower resistance populations re-confirmed that the resistance QTL is in the same position on LG3 of Mr5 as that for flower inoculation. These results provide strong evidence that fire blight resistance in Mr5 is controlled by a major QTL on LG3, independently of the mode of infection, rootstock and environment. Conclusions This study demonstrates for the first time that resistance to fire blight caused by Erwinia amylovora is independent of the mode of inoculation at least in Malus ×robusta 5.

The bacterium, Erwinia amylovora, deposits effector proteins such as AvrRpt2EA into hosts through the type III secretion pathogenicity island to cause fire blight in susceptible Malus genotypes. A single nucleotide polymorphism in the AvrRpt2EA effector plays a key role in pathogen virulence on Malus hosts by exchanging one cysteine to serine in the effector protein sequence. Fire blight resistance quantitative trait loci (QTLs) were detected in a few apple cultivars and wild Malus genotypes with the resistance of wild apples generally found to be stronger than their domestic relatives. The only candidate and functionally analyzed fire blight resistance genes proposed are from wild apple genotypes. Nevertheless, the aforementioned AvrRpt2EA SNP and a couple of effector mutants of E. amylovora are responsible for the breakdown of resistance from a few Malus donors including detected QTLs and underlying R-genes. This review summarizes a key finding related to the molecular basis underpinning an aspect of virulence of E. amylovora on Malus genotypes, as well as mechanisms of host recognition and specificity, and their implications on the results of genetic mapping and phenotypic studies within the last 5-6 years. Although the knowledge gained has improved our understanding of the Malus-E. amylovora system, more research is required to fully grasp the resistance mechanisms in this genus especially as they pertain to direct interactions with pathogen effectors.

The AvrRpt2 protein of the phytopathogenic bacterium Erwinia amylovora (AvrRpt2EA) is a secreted type III effector protein, which is recognised by the FB_MR5 resistance protein of Malus × robusta 5, the only identified resistance protein from a Malus species preventing E. amylovora infection. The crystal structure of the immature catalytic domain of AvrRpt2EA, a C70 family cysteine protease and type III effector, was determined to a resolution of 1.85 A. The structure provides insights into the cyclophilin-dependent activation of AvrRpt2, and identifies a cryptic leucine of a non-canonical cyclophilin binding motif. The structure also suggests that residue Cys156, responsible for the gene induced resistance, is not involved in substrate determination, and hints that recognition by FB_MR5 is due to direct interaction.

Main conclusion Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab. The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3′-hydroxylase (F3′H). Using recombinant F3′H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3′H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents. Electronic supplementary material The online version of this article (doi:10.1007/s00425-016-2475-9) contains supplementary material, which is available to authorized users.