The interaction of roses with the leaf spot pathogen Diplocarpon rosae (the cause of black spot on roses) is an interesting pathosystem because it involves a long-lived woody perennial, with life history traits very different from most model plants, and a hemibiotrophic pathogen with moderate levels of gene flow. Here we present data on the molecular structure of the first monogenic dominant resistance gene from roses, Rdr1, directed against one isolate of D. rosae. Complete sequencing of the locus carrying the Rdr1 gene resulted in a sequence of 265,477 bp with a cluster of nine highly related TIR–NBS–LRR (TNL) candidate genes. After sequencing revealed candidate genes for Rdr1, we implemented a gene expression analysis and selected five genes out of the nine TNLs. We then silenced the whole TNL gene family using RNAi (Rdr1–RNAi) constructed from the most conserved sequence region and demonstrated a loss of resistance in the normally resistant genotype. To identify the functional TNL gene, we further screened the five TNL candidate genes with a transient leaf infiltration assay. The transient expression assay indicated a single TNL gene (muRdr1H), partially restoring resistance in the susceptible genotype. Rdr1 was found to localize within the muRdr1 gene family; the genes within this locus contain characteristic motifs of active TNL genes and belong to a young cluster of R genes. The transient leaf assay can be used to further analyze the rose black spot interaction and its evolution, extending the analyses to additional R genes and to additional pathogenic types of the pathogen.

Synchytrium endobioticum is an obligate biotrophic fungus that causes wart diseases in potato. Like other species of the class Chytridiomycetes, it does not form mycelia and its zoospores are small, approximately 3 μm in diameter, which complicates the detection of early stages of infection. Furthermore, potato wart disease is difficult to control because belowground organs are infected and resting spores of the fungus are extremely durable. Thus, S. endobioticum is classified as a quarantine organism. More than 40 S. endobioticum pathotypes have been reported, of which pathotypes 1(D1), 2(G1), 6(O1), 8(F1), and 18(T1) are the most important in Germany. No molecular methods for the differentiation of pathotypes are available to date. In this work, we sequenced both genomic DNA and cDNA of the German pathotype 18(T1) from infected potato tissue and generated 5,422 expressed sequence tags (EST) and 423 genomic contigs. Comparative sequencing of 33 genes, single-stranded confirmation polymorphism (SSCP) analysis with polymerase chain reaction fragments of 27 additional genes, as well as the analysis of 41 simple sequence repeat (SSR) loci revealed extremely low levels of variation among five German pathotypes. From these markers, one sequence-characterized amplified region marker and five SSR markers revealed polymorphisms among the German pathotypes and an extended set of 11 additional European isolates. Pathotypes 8(F1) and 18(T1) displayed discrete polymorphisms which allow their differentiation from other pathotypes. Overall, using the information of the six markers, the 16 isolates could be differentiated into three distinct genotype groups. In addition to the presented markers, the new collection of EST from genus Synchytrium might serve in the future for molecular taxonomic studies as well as for analyses of the host-pathogen interactions in this difficult pathosystem. [Formula: see text] Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Key messageWe achieved improved mapping resolution of the major wart resistance locus Xla-TNL containing also Sen1 in a dihaploid population using SNP data and developed additional markers with diagnostic value in tetraploid varieties.AbstractWe analyzed a segregating monoparental dihaploid potato population comprising 215 genotypes derived from a tetraploid variety that is highly resistant to Synchytrium endobioticum pathotypes 18 and 6. The clear bimodal segregation for both pathotypes indicated that a major dominant resistance factor in a simplex allele configuration was present in the tetraploid donor genotype. Compared to that in previous analyses of the same tetraploid donor in conventional crosses with susceptible tetraploid genotypes, a segregation pattern with a reduced genetic complexity of resistance in dihaploids was observed here. Using the 12.8 k SolCAP SNP array, we mapped a resistance locus to the Xla-TNL region containing also Sen1 on potato chromosome 11. The improved mapping resolution provided by the monoparental dihaploids allowed for the localization of the genes responsible for the resistance to both pathotypes in an interval spanning less than 800 kbp on the reference genome. Furthermore, we identified eight molecular markers segregating without recombination to pathotype 18 and pathotype 6 resistance. Also, two developed markers display improved diagnostic properties in an independent panel of tetraploid varieties. Overall, our data provide the highest resolution mapping of wart resistance genes at the Xla-TNL locus thus far.Electronic supplementary materialThe online version of this article (10.1007/s00122-018-3172-9) contains supplementary material, which is available to authorized users.

BackgroundThe resistance of plants to pathogens relies on two lines of defense: a basal defense response and a pathogen-specific system, in which resistance (R) genes induce defense reactions after detection of pathogen-associated molecular patterns (PAMPS). In the specific system, a so-called arms race has developed in which the emergence of new races of a pathogen leads to the diversification of plant resistance genes to counteract the pathogens’ effect. The mechanism of resistance gene diversification has been elucidated well for short-lived annual species, but data are mostly lacking for long-lived perennial and clonally propagated plants, such as roses. We analyzed the rose black spot resistance gene, Rdr1, in five members of the Rosaceae: Rosa multiflora, Rosa rugosa, Fragaria vesca (strawberry), Malus x domestica (apple) and Prunus persica (peach), and we present the deduced possible mechanism of R-gene diversification.ResultsWe sequenced a 340.4-kb region from R. rugosa orthologous to the Rdr1 locus in R. multiflora. Apart from some deletions and rearrangements, the two loci display a high degree of synteny. Additionally, less pronounced synteny is found with an orthologous locus in strawberry but is absent in peach and apple, where genes from the Rdr1 locus are distributed on two different chromosomes. An analysis of 20 TIR-NBS-LRR (TNL) genes obtained from R. rugosa and R. multiflora revealed illegitimate recombination, gene conversion, unequal crossing over, indels, point mutations and transposable elements as mechanisms of diversification.A phylogenetic analysis of 53 complete TNL genes from the five Rosaceae species revealed that with the exception of some genes from apple and peach, most of the genes occur in species-specific clusters, indicating that recent TNL gene diversification began prior to the split of Rosa from Fragaria in the Rosoideae and peach from apple in the Spiraeoideae and continued after the split in individual species. Sequence similarity of up to 99% is obtained between two R. multiflora TNL paralogs, indicating a very recent duplication.ConclusionsThe mechanisms by which TNL genes from perennial Rosaceae diversify are mainly similar to those from annual plant species. However, most TNL genes appear to be of recent origin, likely due to recent duplications, supporting the hypothesis that TNL genes in woody perennials are generally younger than those from annuals. This recent origin might facilitate the development of new resistance specificities, compensating for longer generation times in woody perennials.