Genome-Wide Analysis of the WRKY Transcription Factor Family in Roses and Their Putative Role in Defence Signalling in the Rose-Blackspot Interaction.

WRKY transcription factors are important players in plant regulatory networks, where they control and integrate various physiological processes and responses to biotic and abiotic stresses. Here, we analysed six rose genomes of 5 different species (Rosa chinensis, R. multiflora, R. roxburghii, R. sterilis, and R. rugosa) and extracted a set of 68 putative WRKY genes, extending a previously published set of 58 WRKY sequences based on the R. chinensis genome. Analysis of the promoter regions revealed numerous motifs related to induction by abiotic and, in some cases, biotic stressors. Transcriptomic data from leaves of two rose genotypes inoculated with the hemibiotrophic rose black spot fungus Diplocarpon rosae revealed the upregulation of 18 and downregulation of 9 of these WRKY genes after contact with the fungus. Notably, the resistant genotype exhibited the regulation of 25 of these genes (16 upregulated and 9 downregulated), while the susceptible genotype exhibited the regulation of 20 genes (15 upregulated and 5 downregulated). A detailed RT-qPCR analysis of RcWRKY37, an orthologue of AtWRKY75 and FaWRKY1, revealed induction patterns similar to those of the pathogenesis-related (PR) genes induced in salicylic acid (SA)-dependent defence pathways in black spot inoculation experiments. However, the overexpression of RcWRKY37 in rose petals did not induce the expression of any of the PR genes upon contact with black spot. However, wounding significantly induced the expression of RcWRKY37, while heat, cold, or drought did not have a significant effect. This study provides the first evidence for the role of RcWRKY37 in rose signalling cascades and highlights the differences between RcWRKY37 and AtWRKY75. These results improve our understanding of the regulatory function of WRKY transcription factors in plant responses to stress factors. Additionally, they provide foundational data for further studies.

Robust markers associated with floral traits in roses are suitable for marker-assisted selection across gene pools.

We investigated the potential of markers associated with floral traits for parental selection in a cut rose breeding program. We analysed six Kompetitive Allele Specific PCR (KASP) markers for three important floral traits, petal length, petal number and scent, derived from experiments in a garden rose population. The six markers were applied to genotype a collection of 384 parental genotypes used for commercial cut rose breeding. We phenotyped a selection of progeny derived from pairs of parents having either high or low dosages of (contrasting) marker alleles associated with these traits. Significant differences were found between the contrasting progeny groups for each of the traits, although parents with the optimal allele dosage combinations could not always be used for the crosses. This not only supports the robustness of these marker‒trait associations but also demonstrates their potential for commercial rose breeding. It also demonstrates the use of marker information generated in garden rose populations for cut rose breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01438-5.

Time course of changes in the transcriptome during russet induction in apple fruit.

BACKGROUND: Russeting is a major problem in many fruit crops. Russeting is caused by environmental factors such as wounding or moisture exposure of the fruit surface. Despite extensive research, the molecular sequence that triggers russet initiation remains unclear. Here, we present high-resolution transcriptomic data by controlled russet induction at very early stages of fruit development. During Phase I, a patch of the fruit surface is exposed to surface moisture. For Phase II, moisture exposure is terminated, and the formerly exposed surface remains dry. We targeted differentially expressed transcripts as soon as 24 h after russet induction. RESULTS: During moisture exposure (Phase I) of 'Pinova' apple, transcripts associated with the cell cycle, cell wall, and cuticle synthesis (SHN3) decrease, while those related to abiotic stress increase. NAC35 and MYB17 were the earliest induced genes during Phase I. They are therefore linked to the initial processes of cuticle microcracking. After moisture removal (Phase II), the expression of genes related to meristematic activity increased (WOX4 within 24 h, MYB84 within 48 h). Genes related to lignin synthesis (MYB52) and suberin synthesis (MYB93, WRKY56) were upregulated within 3 d after moisture removal. WOX4 and AP2B3 are the earliest differentially expressed genes induced in Phase II. They are therefore linked to early events in periderm formation. The expression profiles were consistent between two different seasons and mirrored differences in russet susceptibility in a comparison of cultivars. Furthermore, expression profiles during Phase II of moisture induction were largely identical to those following wounding. CONCLUSIONS: The combination of a unique controlled russet induction technique with high-resolution transcriptomic data allowed for the very first time to analyse the formation of cuticular microcracks and periderm in apple fruit immediately after the onset of triggering factors. This data provides valuable insights into the spatial-temporal dynamics of russeting, including the synthesis of cuticles, dedifferentiation of cells, and impregnation of cell walls with suberin and lignin.

Potato Wart Isolates from Europe and North America Form Distinct Clusters of Genetic Variation.

We have extended previously published sets of simple sequence repeat markers for Synchytrium endobioticum, selected to be polymorphic for the German-standard isolates of pathotypes P1, P2, P6, P8, and P18. These markers also complement the extensive published information on DNA polymorphisms for the mitogenomes of Synchytrium endobioticum. This extended set of 35 markers representing 73 alleles differentiated 51 isolates from Europe and North America into three large, well-separated clusters and subclusters using dendrogram analysis, principal coordinates analysis (PCoA), and population substructure analysis using STRUCTURE 2.3.4 software. This suggests a limited number of introgressions of the wart disease pathogen into current potato growing areas, followed by recombination and admixture of populations through human activities. The new markers extend the published marker sets and are useful tools for future analyses of population structure and dynamics in Synchytrium endobioticum, which are necessary to understand the biology of the interaction between the pathogen and its potato host and to develop future control strategies.

GWAS of adventitious root formation in roses identifies a putative phosphoinositide phosphatase (SAC9) for marker-assisted selection.

Rose propagation by cuttings is limited by substantial genotypic differences in adventitious root formation. To identify possible genetic factors causing these differences and to develop a marker for marker-assisted selection for high rooting ability, we phenotyped 95 cut and 95 garden rose genotypes in a hydroponic rooting system over 6 weeks. Data on rooting percentage after 3 to 6 weeks, root number, and root fresh mass were highly variable among genotypes and used in association mappings performed on genotypic information from the WagRhSNP 68 K Axiom SNP array for roses. GWAS analyses revealed only one significantly associated SNP for rooting percentage after 3 weeks. Nevertheless, prominent genomic regions/peaks were observed and further analysed for rooting percentage after 6 weeks, root number and root fresh mass. Some of the SNPs in these peak regions were associated with large effects on adventitious root formation traits. Very prominent were ten SNPs, which were all located in a putative phosphoinositide phosphatase SAC9 on chromosome 2 and showed very high effects on rooting percentage after 6 weeks of more than 40% difference between nulliplex and quadruplex genotypes. SAC9 was reported to be involved in the regulation of endocytosis and in combination with other members of the SAC gene family to regulate the translocation of auxin-efflux PIN proteins via the dephosphorylation of phosphoinositides. For one SNP within SAC9, a KASP marker was successfully derived and used to select genotypes with a homozygous allele configuration. Phenotyping these homozygous genotypes for adventitious root formation verified the SNP allele dosage effect on rooting. Hence, the presented KASP derived from a SNP located in SAC9 can be used for marker-assisted selection in breeding programs for high rooting ability in the future.

The identification of the Rosa S-locus provides new insights into the breeding and wild origins of continuous-flowering roses.

This study aims to: (i) identify the Rosa S-locus controlling self-incompatibility (SI); (ii) test the genetic linkage of the S-locus with other loci controlling important ornamental traits, such as the continuous-flowering (CF) characteristic; (iii) identify the S-alleles (SC ) of old Chinese CF cultivars (e.g, Old Blush, Slater's Crimson China) and examine the changes in the frequency of cultivars with Sc through the history of breeding; (iv) identify wild species carrying the Sc-alleles to infer wild origins of CF cultivars. We identified a new S-RNase (SC2 ) of Rosa chinensis in a contig from a genome database that has not been integrated into one of the seven chromosomes yet. Genetic mapping indicated that SC2 is allelic to the previously-identified S-RNase (SC1 ) in chromosome 3. Pollination experiments with half-compatible pairs of roses confirmed that they are the pistil-determinant of SI. The segregation analysis of an F1 -population indicated genetic linkage between the S-locus and the floral repressor gene KSN. The non-functional allele ksn is responsible for the CF characteristic. A total of five S-alleles (SC1-5 ) were identified from old CF cultivars. The frequency of cultivars with SC dramatically increased after the introgression of ksn from Chinese to European cultivars and remains high (80%) in modern cultivars, suggesting that S-genotyping is helpful for effective breeding. Wild individuals carrying SC were found in Rosa multiflora (SC1 ), Rosa chinensis var. spontanea (SC3 ), and Rosa gigantea (SC2 , SC4 ), supporting the hypothesis of hybrid origins of CF cultivars and providing a new evidence for the involvement of Rosa multiflora.

Apple fruit periderms (russeting) induced by wounding or by moisture have the same histologies, chemistries and gene expressions.

Russeting is a cosmetic defect of some fruit skins. Russeting (botanically: induction of periderm formation) can result from various environmental factors including wounding and surface moisture. The objective was to compare periderms resulting from wounding with those from exposure to moisture in developing apple fruit. Wounding or moisture exposure both resulted in cuticular microcracking. Cross-sections revealed suberized hypodermal cell walls by 4 d, and the start of periderm formation by 8 d after wounding or moisture treatment. The expression of selected target genes was similar in wound and moisture induced periderms. Transcription factors involved in the regulation of suberin (MYB93) and lignin (MYB42) synthesis, genes involved in the synthesis (CYP86B1) and the transport (ABCG20) of suberin monomers and two uncharacterized transcription factors (NAC038 and NAC058) were all upregulated in induced periderm samples. Genes involved in cutin (GPAT6, SHN3) and wax synthesis (KCS10, WSD1, CER6) and transport of cutin monomers and wax components (ABCG11) were all downregulated. Levels of typical suberin monomers (ω-hydroxy-C20, -C22 and -C24 acids) and total suberin were high in the periderms, but low in the cuticle. Periderms were induced only when wounding occurred during early fruit development (32 and 66 days after full bloom (DAFB)) but not later (93 DAFB). Wound and moisture induced periderms are very similar morphologically, histologically, compositionally and molecularly.

P Starvation in Roses Leads to Strongly Genotype-Dependent Induction of P-Transporter Genes during Black Spot Leaf Disease.

Phosphorous starvation in plants has been reported to have contrasting effects on the interaction with pathogens in different plant pathogen systems and plant species. Both increases and decreases in susceptibility have been observed in numerous reports. Here, we analysed black spot infection and the leaf expression of two plant phosphate transporters and one defence marker gene in roses after phosphorous starvation. We varied three factors: phosphate starvation versus full supply of phosphorous, black spot infection vs. mock inoculation, and different susceptible and resistant progeny of a biparental rose population. Black spot susceptibility or resistance was not significantly changed upon phosphate starvation in either compatible or incompatible interactions. The expression of phosphate transporters was strongly induced upon starvation, but in some genotypes, expression was altered by black spot interaction as well. The marker for pathogenic interactions was exclusively induced by interaction with black spot, but the expression was altered by a combination of phosphate starvation and interaction with the fungus in some genotypes. In summary, phosphate starvation has clear effects on the gene expression of phosphate transporters in rose leaves, and the interaction with a hemibiotrophic leaf pathogen is strongly genotype dependent.

First genome edited poinsettias: targeted mutagenesis of flavonoid 3'-hydroxylase using CRISPR/Cas9 results in a colour shift.

The CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3'-hydroxylase (F3'H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar 'Christmas Eve', in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3'H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3'H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3'H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3'H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11240-021-02103-5.

Analysis of allelic variants of RhMLO genes in rose and functional studies on susceptibility to powdery mildew related to clade V homologs.

KEY MESSAGE: Rose has 19 MLO genes. Of these, RhMLO1 and RhMLO2 were shown to be required for powdery mildew infection, which suggests their potential as susceptibility targets towards disease resistance. Powdery mildew, caused by Podosphaera pannosa, is one of the most serious and widespread fungal diseases for roses, especially in greenhouse-grown cut roses. It has been shown that certain MLO genes are involved in powdery mildew susceptibility and that loss of function in these genes in various crops leads to broad-spectrum, long-lasting resistance against this fungal disease. For this reason, these MLO genes are called susceptibility genes. We carried out a genome-wide identification of the MLO gene family in the Rosa chinensis genome, and screened for allelic variants among 22 accessions from seven different Rosa species using re-sequencing and transcriptome data. We identified 19 MLO genes in rose, of which four are candidate genes for functional homologs in clade V, which is the clade containing all dicot MLO susceptibility genes. We detected a total of 198 different allelic variants in the set of Rosa species and accessions, corresponding to 5-15 different alleles for each of the genes. Some diploid Rosa species shared alleles with tetraploid rose cultivars, consistent with the notion that diploid species have contributed to the formation of tetraploid roses. Among the four RhMLO genes in clade V, we demonstrated using expression study, virus-induced gene silencing as well as transient RNAi silencing that two of them, RhMLO1 and RhMLO2, are required for infection by P. pannosa and suggest their potential as susceptibility targets for powdery mildew resistance breeding in rose.

Detection of Reproducible Major Effect QTL for Petal Traits in Garden Roses.

The detection of QTL by association genetics depends on the genetic architecture of the trait under study, the size and structure of the investigated population and the availability of phenotypic and marker data of sufficient quality and quantity. In roses, we previously demonstrated that major QTL could already be detected in small association panels. In this study, we analyzed petal number, petal size and fragrance in a small panel of 95 mostly tetraploid garden rose genotypes. After genotyping the panel with the 68 K Axiom WagRhSNP chip we detected major QTL for all three traits. Each trait was significantly influenced by several genomic regions. Some of the QTL span genomic regions that comprise several candidate genes. Selected markers from some of these regions were converted into KASP markers and were validated in independent populations of up to 282 garden rose genotypes. These markers demonstrate the robustness of the detected effects independent of the set of genotypes analyzed. Furthermore, the markers can serve as tools for marker-assisted breeding in garden roses. Over an extended timeframe, they may be used as a starting point for the isolation of the genes underlying the QTL.

A highly mutable GST is essential for bract colouration in Euphorbia pulcherrima Willd. Ex Klotsch.

BACKGROUND: Mutation breeding is an extraordinary tool in plant breeding to increase the genetic variability, where mutations in anthocyanin biosynthesis are targets to generate distinctive phenotypes in ornamental species. In poinsettia, ionizing radiation is routinely applied in breeding programs to obtaining a range of colours, with nearly all pink and white varieties being obtained after γ- or X-ray mutagenesis of red varieties. In the present study we performed a thorough characterization of a potential mutagenesis target gene as the main responsible for the 'white paradox' in poinsettia. RESULTS: We identified a GST gene in poinsettia (Bract1) as an essential factor for the expression of anthocyanin-based red colouration of bracts, which presents a high phylogenetic similarity to known anthocyanin-related GSTs. Red poinsettia varieties and white mutants generated from these varieties by X-ray were analysed for polymorphisms related to the 'white paradox' in the species. A 4 bp mutation in a short repeat within the coding region of Bract1 is most likely responsible for the appearance of white phenotypes upon irradiation treatment. The polymorphism between wild-type and mutant alleles co-segregates with the phenotype in progeny from heterozygous red and white parents. Moreover, overexpression of Bract1 wild-type allele in Arabidopsis tt19 mutants restored the anthocyanin phenotype, while the Bract1 mutated allele showed to be non-functional. CONCLUSIONS: The identified repeat seems to be highly unstable, since mutated plants can be easily detected among fewer than 200 shoots derived from 10 mutated plants. Our data indicate that particular short repeat sequences, similar to microsatellite sequences or so-called dynamic mutations, might be hot spots for genetic variability. Moreover, the identification of the Bract1 mutation fills a gap on the understanding on the molecular mechanism of colour formation in poinsettia.

Russeting in Apple Is Initiated After Exposure to Moisture Ends-I. Histological Evidence.

Russeting (periderm formation) is a critical fruit-surface disorder in apple (Malus × domestica Borkh.). The first symptom of insipient russeting is cuticular microcracking. Humid and rainy weather increases russeting. The aim was to determine the ontogeny of moisture-induced russeting in 'Pinova' apple. We recorded the effects of duration of exposure to water and the stage of fruit development at exposure on microcracking, periderm formation and cuticle deposition. Early on (21 or 31 days after full bloom; DAFB) short periods (2 to 12 d) of moisture exposure induced cuticular microcracking-but not later on (66 or 93 DAFB). A periderm was not formed during moisture exposure but 4 d after exposure ended. A periderm was formed in the hypodermis beneath a microcrack. Russeting frequency and severity were low for up to 4 d of moisture exposure but increased after 6 d. Cuticle thickness was not affected by moisture for up to 8 d but decreased for longer exposures. Cuticular ridge thickness decreased around a microcrack. In general, moisture did not affect cuticular strain release. We conclude that a hypodermal periderm forms after termination of moisture exposure and after microcrack formation. Reduced cuticle deposition may cause moisture-induced microcracking and, thus, russeting.

Analysis of the Rdr1 gene family in different Rosaceae genomes reveals an origin of an R-gene cluster after the split of Rubeae within the Rosoideae subfamily.

The Rdr1 gene confers resistance to black spot in roses and belongs to a large TNL gene family, which is organized in two major clusters at the distal end of chromosome 1. We used the recently available chromosome scale assemblies for the R. chinensis 'Old Blush' genome, re-sequencing data for nine rose species and genome data for Fragaria, Rubus, Malus and Prunus to identify Rdr1 homologs from different taxa within Rosaceae. Members of the Rdr1 gene family are organized into two major clusters in R. chinensis and at a syntenic location in the Fragaria genome. Phylogenetic analysis indicates that the two clusters existed prior to the split of Rosa and Fragaria and that one cluster has a more recent origin than the other. Genes belonging to cluster 2, such as the functional Rdr1 gene muRdr1A, were subject to a faster evolution than genes from cluster 1. As no Rdr1 homologs were found in syntenic positions for Prunus persica, Malus x domestica and Rubus occidentalis, a translocation of the Rdr1 clusters to the current positions probably happened after the Rubeae split from other groups within the Rosoideae approximately 70-80 million years ago during the Cretaceous period.

Russeting in Apple is Initiated after Exposure to Moisture Ends: Molecular and Biochemical Evidence.

Exposure of the fruit surface to moisture during early development is causal in russeting of apple (Malus × domestica Borkh.). Moisture exposure results in formation of microcracks and decreased cuticle thickness. Periderm differentiation begins in the hypodermis, but only after discontinuation of moisture exposure. Expressions of selected genes involved in cutin, wax and suberin synthesis were quantified, as were the wax, cutin and suberin compositions. Experiments were conducted in two phases. In Phase I (31 days after full bloom) the fruit surface was exposed to moisture for 6 or 12 d. Phase II was after moisture exposure had been discontinued. Unexposed areas on the same fruit served as unexposed controls. During Phase I, cutin and wax synthesis genes were down-regulated only in the moisture-exposed patches. During Phase II, suberin synthesis genes were up-regulated only in the moisture-exposed patches. The expressions of cutin and wax genes in the moisture-exposed patches increased slightly during Phase II, but the levels of expression were much lower than in the control patches. Amounts and compositions of cutin, wax and suberin were consistent with the gene expressions. Thus, moisture-induced russet is a two-step process: moisture exposure reduces cutin and wax synthesis, moisture removal triggers suberin synthesis.

Hybrid de novo transcriptome assembly of poinsettia (Euphorbia pulcherrima Willd. Ex Klotsch) bracts.

BACKGROUND: Poinsettia is a popular and important ornamental crop, mostly during the Christmas season. Its bract coloration ranges from pink/red to creamy/white shades. Despite its ornamental value, there is a lack of knowledge about the genetics and molecular biology of poinsettia, especially on the mechanisms of color formation. We performed an RNA-Seq analysis in order to shed light on the transcriptome of poinsettia bracts. Moreover, we analyzed the transcriptome differences of red- and white-bracted poinsettia varieties during bract development and coloration. For the assembly of a bract transcriptome, two paired-end cDNA libraries from a red and white poinsettia pair were sequenced with the Illumina technology, and one library from a red-bracted variety was used for PacBio sequencing. Both short and long reads were assembled using a hybrid de novo strategy. Samples of red- and white-bracted poinsettias were sequenced and comparatively analyzed in three color developmental stages in order to understand the mechanisms of color formation and accumulation in the species. RESULTS: The final transcriptome contains 288,524 contigs, with 33% showing confident protein annotation against the TAIR10 database. The BUSCO pipeline, which is based on near-universal orthologous gene groups, was applied to assess the transcriptome completeness. From a total of 1440 BUSCO groups searched, 77% were categorized as complete (41% as single-copy and 36% as duplicated), 10% as fragmented and 13% as missing BUSCOs. The gene expression comparison between red and white varieties of poinsettia showed a differential regulation of the flavonoid biosynthesis pathway only at particular stages of bract development. An initial impairment of the flavonoid pathway early in the color accumulation process for the white poinsettia variety was observed, but these differences were no longer present in the subsequent stages of bract development. Nonetheless, GSTF11 and UGT79B10 showed a lower expression in the last stage of bract development for the white variety and, therefore, are potential candidates for further studies on poinsettia coloration. CONCLUSIONS: In summary, this transcriptome analysis provides a valuable foundation for further studies on poinsettia, such as plant breeding and genetics, and highlights crucial information on the molecular mechanism of color formation.

In the name of the rose: a roadmap for rose research in the genome era.

The recent completion of the rose genome sequence is not the end of a process, but rather a starting point that opens up a whole set of new and exciting activities. Next to a high-quality genome sequence other genomic tools have also become available for rose, including transcriptomics data, a high-density single-nucleotide polymorphism array and software to perform linkage and quantitative trait locus mapping in polyploids. Rose cultivars are highly heterogeneous and diverse. This vast diversity in cultivated roses can be explained through the genetic potential of the genus, introgressions from wild species into commercial tetraploid germplasm and the inimitable efforts of historical breeders. We can now investigate how this diversity can best be exploited and refined in future breeding work, given the rich molecular toolbox now available to the rose breeding community. This paper presents possible lines of research now that rose has entered the genomics era, and attempts to partially answer the question that arises after the completion of any draft genome sequence: 'Now that we have "the" genome, what's next?'. Having access to a genome sequence will allow both (fundamental) scientific and (applied) breeding-orientated questions to be addressed. We outline possible approaches for a number of these questions.

Interaction of roses with a biotrophic and a hemibiotrophic leaf pathogen leads to differences in defense transcriptome activation.

KEY MESSAGE: Transcriptomic analysis resulted in the upregulation of the genes related to common defense mechanisms for black spot and the downregulation of the genes related to photosynthesis and cell wall modification for powdery mildew. Plant pathogenic fungi successfully colonize their hosts by manipulating the host defense mechanisms, which is accompanied by major transcriptome changes in the host. To characterize compatible plant pathogen interactions at early stages of infection by the obligate biotrophic fungus Podosphaera pannosa, which causes powdery mildew, and the hemibiotrophic fungus Diplocarpon rosae, which causes black spot, we analyzed changes in the leaf transcriptome after the inoculation of detached rose leaves with each pathogen. In addition, we analyzed differences in the transcriptomic changes inflicted by both pathogens as a first step to characterize specific infection strategies. Transcriptomic changes were analyzed using next-generation sequencing based on the massive analysis of cDNA ends approach, which was validated using high-throughput qPCR. We identified a large number of differentially regulated genes. A common set of the differentially regulated genes comprised of pathogenesis-related (PR) genes, such as of PR10 homologs, chitinases and defense-related transcription factors, such as various WRKY genes, indicating a conserved but insufficient PTI [pathogen associated molecular pattern (PAMP) triggered immunity] reaction. Surprisingly, most of the differentially regulated genes were specific to the interactions with either P. pannosa or D. rosae. Specific regulation in response to D. rosae was detected for genes from the phenylpropanoid and flavonoid pathways and for individual PR genes, such as paralogs of PR1 and PR5, and other factors of the salicylic acid signaling pathway. Differently, inoculation with P. pannosa leads in addition to the general pathogen response to a downregulation of genes related to photosynthesis and cell wall modification.

Prediction of the Diplocarpon rosae secretome reveals candidate genes for effectors and virulence factors.

Rose black spot is one of the most severe diseases of field-grown roses. Though R-genes have been characterised, little information is known about the molecular details of the interaction between pathogen and host. Based on the recently published genome sequence of the black spot fungus, we analysed gene models with various bioinformatic tools utilising the expression data of infected host tissues, which led to the prediction of 827 secreted proteins. A significant proportion of the predicted secretome comprises enzymes for the degradation of cell wall components, several of which were highly expressed during the first infection stages. As the secretome comprises major factors determining the ability of the fungus to colonise its host, we focused our further analyses on predicted effector candidates. In total, 52 sequences of 251 effector candidates matched several bioinformatic criteria of effectors, contained a Y/F/WxC motif, and did not match annotated proteins from other fungi. Additional sequences were identified based on their high expression levels during the penetration/haustorium formation phase and/or by matching known effectors from other fungi. Several host genotypes that are resistant to the sequenced isolate but differ in the R-genes responsible for this resistance are available. The combination of these genotypes with functional studies of the identified candidate effectors will allow the mechanisms of the rose black spot interaction to be dissected.

The rare orange-red colored Euphorbia pulcherrima cultivar 'Harvest Orange' shows a nonsense mutation in a flavonoid 3'-hydroxylase allele expressed in the bracts.

BACKGROUND: Commercially available poinsettia (Euphorbia pulcherrima) varieties prevalently accumulate cyanidin derivatives and show intense red coloration. Orange-red bract color is less common. We investigated four cultivars displaying four different red hues with respect to selected enzymes and genes of the anthocyanin pathway, putatively determining the color hue. RESULTS: Red hues correlated with anthocyanin composition and concentration and showed common dark red coloration in cultivars 'Christmas Beauty' and 'Christmas Feeling' where cyanidin derivatives were prevalent. In contrast, orange-red bract color is based on the prevalent presence of pelargonidin derivatives that comprised 85% of the total anthocyanin content in cv. 'Premium Red' and 96% in cv. 'Harvest Orange' (synonym: 'Orange Spice'). cDNA clones of flavonoid 3'-hydroxylase (F3'H) and dihydroflavonol 4-reductase (DFR) were isolated from the four varieties, and functional activity and substrate specificity of the corresponding recombinant enzymes were studied. Kinetic studies demonstrated that poinsettia DFRs prefer dihydromyricetin and dihydroquercetin over dihydrokaempferol, and thus, favor the formation of cyanidin over pelargonidin. Whereas the F3'H cDNA clones of cultivars 'Christmas Beauty', 'Christmas Feeling', and 'Premium Red' encoded functionally active enzymes, the F3'H cDNA clone of cv. 'Harvest Orange' contained an insertion of 28 bases, which is partly a duplication of 20 bases found close to the insertion site. This causes a frameshift mutation with a premature stop codon after nucleotide 132 and, therefore, a non-functional enzyme. Heterozygosity of the F3'H was demonstrated in this cultivar, but only the mutated allele was expressed in the bracts. No correlation between F3'H-expression and the color hue could be observed in the four species. CONCLUSIONS: Rare orange-red poinsettia hues caused by pelargonidin based anthocyanins can be achieved by different mechanisms. F3'H is a critical step in the establishment of orange red poinsettia color. Although poinsettia DFR shows a low substrate specificity for dihydrokaempferol, sufficient precursor for pelargonidin formation is available in planta, in the absence of F3'H activity.