The Mechanism of Resistance of EUROPEAN Plum to Plum pox virus Mediated by Hypersensitive Response Is Linked to VIRAL NIa and Its Protease Activity.

Plum pox virus (PPV) infects Prunus trees across the globe, causing the serious Sharka disease. Breeding programs in the past 20 years have been successful, generating plum varieties hypersensitive to PPV that show resistance in the field. Recently, a single tree displaying typical PPV symptoms was detected in an orchard of resistant plums. The tree was eradicated, and infected material was propagated under controlled conditions to study the new PPV isolate. Performing overlapping PCR analysis, the viral sequence was reconstructed, cloned and tested for infectivity in different 'Jojo'-based resistant plums. The results confirmed that the isolate, named PPV-D 'Herrenberg' (PPVD-H), was able to infect all these varieties. Analyses of chimeras between PPVD-H and a PPV-D standard isolate (PPVD) revealed that the NIa region of PPD-H, carrying three amino acid changes, was enough to break the resistance of these plums. Experiments with single and double mutants showed that all changes were essential to preserve the escaping phenotype. Additionally, one of the changes at the VPg-NIapro junction suggested the involvement of controlled endopeptidase cleavage in the viral response. Transient expression experiments in Nicotiana benthamiana confirmed that NIa cleavage in PPVD-H was reduced, compared to PPVD, linking the observed behavior to an NIa cleavage modulation.

Identification of apple cultivars hypoallergenic for birch pollen-allergic individuals by a multidisciplinary in vitro and in vivo approach.

BACKGROUND: Birch pollen-related apple allergy is the most frequent IgE-mediated food allergy in Central-Northern Europe with Mal d 1 as major allergen. Its concentration in apples varies with the cultivar and storage time. Year-round appealing, hypoallergenic cultivars still are needed to satisfy the nutritional needs of affected individuals. We characterized three promising cultivars by multidisciplinary in vitro assays including long-term storage and by clinical challenges of allergic individuals before and after the birch pollen season. METHODS: Proteins were extracted from fruits of 'Santana', 'Golden Delicious' (GD), and three genuine cultivars in November 2018 and April 2019. Mal d 1-levels were analysed by mass spectrometry, SDS-PAGE, immunoblotting, competitive ELISA, and basophil activation tests. Twenty-eight allergic individuals underwent single-blinded open food challenges and skin testing with the cultivars and birch pollen in November 2018 and May 2019. Allergen-specific IgE-levels were determined. RESULTS: After storage all cultivars except 'Santana' were of appealing appearance and taste. Their Mal d 1 content had increased, also reflected by significantly amplified basophil activation and stronger reactions in clinical challenges. Besides, individuals showed boosted reactivity after pollen exposure indicated by enhanced allergen-specific IgE-levels and skin reactions to birch pollen. Still, all cultivars remained significantly less allergenic than GD and comparable to Santana in November 2018 in all assessments except for skin testing. CONCLUSIONS: Combined expertise in pomology and allergology identified promising new cultivars for allergic consumers. The evaluation of hypoallergenic apples should incorporate long-term storage and birch pollen exposure. Basophil activation tests may be suitable in the selection of promising cultivars for oral challenges.

Transcriptomic analysis of Prunus domestica undergoing hypersensitive response to plum pox virus infection.

Plum pox virus (PPV) infects Prunus trees around the globe, posing serious fruit production problems and causing severe economic losses. One variety of Prunus domestica, named 'Jojo', develops a hypersensitive response to viral infection. Here we compared infected and non-infected samples using next-generation RNA sequencing to characterize the genetic complexity of the viral population in infected samples and to identify genes involved in development of the resistance response. Analysis of viral reads from the infected samples allowed reconstruction of a PPV-D consensus sequence. De novo reconstruction showed a second viral isolate of the PPV-Rec strain. RNA-seq analysis of PPV-infected 'Jojo' trees identified 2,234 and 786 unigenes that were significantly up- or downregulated, respectively (false discovery rate; FDR≤0.01). Expression of genes associated with defense was generally enhanced, while expression of those related to photosynthesis was repressed. Of the total of 3,020 differentially expressed unigenes, 154 were characterized as potential resistance genes, 10 of which were included in the NBS-LRR type. Given their possible role in plant defense, we selected 75 additional unigenes as candidates for further study. The combination of next-generation sequencing and a Prunus variety that develops a hypersensitive response to PPV infection provided an opportunity to study the factors involved in this plant defense mechanism. Transcriptomic analysis presented an overview of the changes that occur during PPV infection as a whole, and identified candidates suitable for further functional characterization.

Identification of phenolic compounds in plum fruits (Prunus salicina L. and Prunus domestica L.) by high-performance liquid chromatography/tandem mass spectrometry and characterization of varieties by quantitative phenolic fingerprints.

Plums (Prunus domestica L. and Prunus salicina L.) are edible fruits mostly consumed in America, China, and Europe. We have used LC-MS(n) to detect and characterize the phenolic compounds in plum varieties. Forty-one phenolics were detected comprising caffeoylquinic acids, feruloylquinic acid, p-coumaroylquinic acids, methyl caffeoylquinates, methyl p-coumaroylquinate, caffeoylshikimic acids, catechin, epicatechin, rutin, esculin, quercetin, quercetin-3-O-hexosides, dimeric proanthocyanidins, trimeric proanthocyanidins, caffeoyl-glucoside, feruloyl-glucoside, p-coumaroyl-glucoside, vanillic acid-glucosides, 3,4-dihydroxybenzoyl-glucoside, quercetin-3-O-pentosides, quercetin-3-O-rhamnoside, quercetin-pentoside-rhamnosides, and 3-p-methoxycinnamoylquinic acid. This is the first time when 3-p-methoxycinnamoylquinic acid is reported in nature. Chlorogenic acids and proanthocyanidins were the major phenolics present in plums. Furthermore, HPLC with DAD and chemical reaction detection was used to generate quantitative phenolic fingerprints from the fruit flesh of 33 plum varieties. The predominant compound was 3-caffeoylquinic acid in nearly all varieties studied; generally, however, the qualitative and quantitative profiles showed high diversity even among closely related progenies.

Diversity of phenolic profiles in the fruit skin of Prunus domestica plums and related species.

The fruits of the European plum Prunus domestica exhibit a great diversity in appearance including skin colors. This study attempts to elucidate the phenylpropanoid and flavonoid profiles of 28 plum varieties belonging to P. domestica and related species as well as hybrids. A total of 49 phenolic compounds extracted from the fruit skin were quantitatively evaluated in an HPLC-DAD-based metabolomic study. The total phenolic contents of the cultivars varied among 0.4-29.9 mg/g fresh weight. The predominant anthocyanins were glycosides of cyanidin and peonidin, and rutin was the principal flavonol, whereas neochlorogenic acid and n-chlorogenic acid were the main hydroxycinnamic acids. Aside from these major phenolic classes, a group of tentatively identified flavones and several acylated flavonoids were also found. Principal component analysis revealed that anthocyanins and hydroxycinnamic acids contributed most to variety separation. The heterogeneity between the different varieties was also assessed using hierarchical cluster analysis of sample phenolics profile. A simple separation of species could not be found confirming the close relationship among them.