A fast and efficient protocol for small RNA extraction in Japanese plum and other Prunus species

Background: Small ribonucleic acids represent an important repertoire of mobile molecules that exert key roles in several cell processes including antiviral defense. Small RNA based repertoire includes both small interfering RNA (siRNA) and microRNA (miRNA) molecules. In the Prunus genus, sharka disease, caused by the Plum pox virus (PPV), first occurred on European plum ( Prunus domestica ) and then spread over among all species in this genus and thus classified as quarantine pathogen. Next-generation sequencing (NGS) was used for the study of siRNA/miRNA molecules; however, NGS relies on adequate extraction protocols. Currently, knowledge of PPV-Prunus interactions in terms of siRNA populations and miRNA species is still scarce, and siRNA/miRNA extraction protocols are limited to species such as peach, almond, and sweet cherry. Results: We describe a reliable procedure for siRNA/miRNA purification from Prunus salicina trees, in which previously used protocols did not allow adequate purification. The procedure was based on a combination of commercially available RNA purification kits and specific steps that yielded high quality purifications. The resulting molecules were adequate for library construction and NGS, leading to the development of a pipeline for analysis of both siRNAs and miRNAs in the PPV\u2013P. salicina interactions. Results showed that PPV infection led to altered siRNA profiles in Japanese plum as characterized by decreased 24-nt and increased 21- and 22-nt siRNAs. Infections showed miR164 and miR160 generation and increased miR166, miR171, miR168, miR319, miR157, and miR159. Conclusion:We propose this protocol as a reliable and reproducible small RNA isolation procedure for P. salicina and other Prunus species

Cysteamine inhibits lysosomal oxidation of low density lipoprotein in human macrophages and reduces atherosclerosis in mice

Background and aims: We have shown previously that low density lipoprotein (LDL) aggregated by vortexing is internalised by macrophages and oxidised by iron in lysosomes to form the advanced lipid/protein oxidation product ceroid. We have now used sphingomyelinase-aggregated LDL, a more pathophysiological form of aggregated LDL, to study lysosomal oxidation of LDL and its inhibition by antioxidants, including cysteamine (2-aminoethanethiol) which concentrates in lysosomes by several orders of magnitude. We have also investigated the effect of cysteamine on atherosclerosis in mice. Methods: LDL was incubated with sphingomyelinase, which increased its average particle diameter from 26 to 170 nm, and was then incubated for up to 7 days with human monocyte-derived macrophages. LDL receptor-deficient mice were fed a Western diet (19-22 per group) and some given cysteamine in their drinking water at a dose equivalent to that used in cystinosis patients. The extent of atherosclerosis in the aortic root and the rest of the aorta was measured. Results: Confocal microscopy revealed lipid accumulation in lysosomes in the cultured macrophages. Large amounts of ceroid were produced, which colocalised with the lysosomal marker LAMP2. The antioxidants cysteamine, butylated hydroxytoluene, amifostine and its active metabolite WR-1065, inhibited the production of ceroid. Cysteamine at concentrations well below those expected to be present in lysosomes inhibited the oxidation of LDL by iron ions at lysosomal pH (pH 4.5) for prolonged periods. Finally, we showed that the extent of atherosclerotic lesions in the aortic root and arch of mice was significantly reduced by cysteamine. Conclusions: These results support our hypothesis that lysosomal oxidation of LDL is important in atherosclerosis and hence antioxidant drugs that concentrate in lysosomes might provide a novel therapy for this disease

Identification of growth processes involved in QTLs for tomato fruit size and composition

Many quantitative trait loci (QTLs) for quality traits have been located on the tomato genetic map, but introgression of favourable wild alleles into large fruited species is hampered by co-localizations of QTLs with antagonist effects. The aim of this study was to assess the growth processes controlled by the main QTLs for fruit size and composition. Four nearly isogenic lines (NILs) derived from an intraspecific cross between a tasty cherry tomato (Cervil) and a normal-tasting large fruit tomato (Levovil) were studied. The lines carried one (L2, L4, and L9) or five (Lx) introgressions from Cervil on chromosomes 1, 2, 4, and 9. QTLs for fruit size could be mainly associated with cell division processes in L2 and L9, whereas cell expansion was rather homogeneous among the genotypes, except Cervil for which the low expansion rate was attributed to low cell plasticity. The link between endoreduplication and fruit size remained unclear, as cell or fruit sizes were positively correlated with the cell DNA content, but not with the endoreduplication factor. QTLs for fruit composition reflected differences in water accumulation rather than in sugar accumulation, except in L9 for which the up-regulation of sucrose unloading and hexose transport and/or starch synthesis was suggested. This may explain the increased amount of carbon allocated to cell structures in L9, which could be related to a QTL for fruit texture. In Lx, these effects were attenuated, except on fruit size and cell division. Finally, the region on top of chromosome 9 may control size and composition attributes in tomato, by a combination of QTL effects on cell division, cell wall synthesis, and carbon import and metabolism

Identification and tagging of genetic resistance factors to Sharka disease in apricot trees and Prunus davidiana

La sharka, maladie provoquée par le Plum Pox Virus (PPV), affecte gravement les arbres fruitiers à noyaux du genre Prunus. Le PPV est largement répandu en Europe de l’Est et dans les régions méditerranéennes. Il affecte, entre autre, fortement la qualité des fruits, les rendant impropres à la commercialisation. A ce jour, aucun cultivar de pêchers n’est résistant et la majorité des variétés cultivées d'abricotiers sont également sensibles. Cependant, des sources de résistance ont été identifiées chez Prunus davidiana, espèce apparentée au pêcher et chez quelques cultivars d’abricotiers. L’étude de la ségrégation de ce caractère dans des populations F1 et F2 a permis d’identifier plusieurs QTL chez chacune de ces espèces et de développer des marqueurs moléculaires utilisables en Sélection Assistée par Marqueurs. De plus, nous avons pu vérifier la robustesse de ces QTL dans différents fonds génétiques et la stabilité de la résistance selon l’inoculum et le type d’inoculations utilisés.The Plum pox virus (PPV), the causal agent of the sharka disease, is the most detrimental virus on stone-fruit trees, worldwide. Infected fruits are not marketable. To date, no stone fruit cultivar (peach, apricot, plum) is resistant. However, sources of resistance have been identified and mapped in apricot and Prunus davidiana, a wild peach-related species. Several of the mapped QTL co-localize with candidate genes previously identified. In the present study, resistance in P. davidiana was confirmed in an F2 population and two new QTL were identified. Quantitative analysis of the apricot cultivars ‘Goldrich’ and ‘SEO’ resistance was also performed. Molecular markers targeting these genes were developed as a tool for marker-assisted selection. This study made it also possible to refine boundaries of the genomic region controlling PPV resistance as well as confirming the robustness and stability of the resistance trait in both species: P. davidiana and P. armeniaca

Identification and tagging of genetic resistance factors to Sharka disease in apricot trees and Prunus davidiana

La sharka, maladie provoquée par le Plum Pox Virus (PPV), affecte gravement les arbres fruitiers à noyaux du genre Prunus. Le PPV est largement répandu en Europe de l’Est et dans les régions méditerranéennes. Il affecte, entre autre, fortement la qualité des fruits, les rendant impropres à la commercialisation. A ce jour, aucun cultivar de pêchers n’est résistant et la majorité des variétés cultivées d'abricotiers sont également sensibles. Cependant, des sources de résistance ont été identifiées chez Prunus davidiana, espèce apparentée au pêcher et chez quelques cultivars d’abricotiers. L’étude de la ségrégation de ce caractère dans des populations F1 et F2 a permis d’identifier plusieurs QTL chez chacune de ces espèces et de développer des marqueurs moléculaires utilisables en Sélection Assistée par Marqueurs. De plus, nous avons pu vérifier la robustesse de ces QTL dans différents fonds génétiques et la stabilité de la résistance selon l’inoculum et le type d’inoculations utilisés.The Plum pox virus (PPV), the causal agent of the sharka disease, is the most detrimental virus on stone-fruit trees, worldwide. Infected fruits are not marketable. To date, no stone fruit cultivar (peach, apricot, plum) is resistant. However, sources of resistance have been identified and mapped in apricot and Prunus davidiana, a wild peach-related species. Several of the mapped QTL co-localize with candidate genes previously identified. In the present study, resistance in P. davidiana was confirmed in an F2 population and two new QTL were identified. Quantitative analysis of the apricot cultivars ‘Goldrich’ and ‘SEO’ resistance was also performed. Molecular markers targeting these genes were developed as a tool for marker-assisted selection. This study made it also possible to refine boundaries of the genomic region controlling PPV resistance as well as confirming the robustness and stability of the resistance trait in both species: P. davidiana and P. armeniaca

Towards the positional cloning of the QTL Phy-P5 affecting Phytophthora blight resistance in pepper

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