Molecular Characterization of a Novel Enamovirus Infecting Raspberry.

Raspberry plants, valued for their fruits, are vulnerable to a range of viruses that adversely affect their yield and quality. Utilizing high-throughput sequencing (HTS), we identified a novel virus, tentatively named raspberry enamovirus 1 (RaEV1), in three distinct raspberry plants. This study provides a comprehensive characterization of RaEV1, focusing on its genomic structure, phylogeny, and possible transmission routes. Analysis of nearly complete genomes from 14 RaEV1 isolates highlighted regions of variance, particularly marked by indel events. The evidence from phylogenetic and sequence analyses supports the classification of RaEV1 as a distinct species within the Enamovirus genus. Among the 289 plant and 168 invertebrate samples analyzed, RaEV1 was detected in 10.4% and 0.4%, respectively. Most detections occurred in plants that were also infected with other common raspberry viruses. The virus was present in both commercial and wild raspberries, indicating the potential of wild plants to act as viral reservoirs. Experiments involving aphids as potential vectors demonstrated their ability to acquire RaEV1 but not to successfully transmit it to plants.

Clarithromycin Suppresses Apple Proliferation Phytoplasma in Explant Cultures.

Apple proliferation, caused by 'Candidatus Phytoplasma mali', is one of the most important economic threats in the field of apple production. Especially at a young age, infected trees can be affected by excessive bud proliferation and general decline. The fruit quality is also significantly reduced by this disease. To investigate treatment options, we applied a clarithromycin chemotherapy to infected in vitro cultures of 'Golden Delicious'. With increasing concentrations of clarithromycin in the media, the phytoplasma load decreased rapidly after one month of treatment, but phytotoxicity led to a pronounced mortality at 40 mg/L, which was the highest dose used in our experiment. Out of 45 initial explants, we obtained one negative mericlone and two mericlones with a concentration of phytoplasma DNA at the detection limit of PCR. The culture propagated from the mericlone that tested negative remained phytoplasma-free after 18 months of subculturing. Our results suggest the applicability of macrolide antibiotics against phytoplasma infections in vitro; however, it might be challenging to find the threshold zone where the concentration is sufficient for pathogen elimination, but not lethal for the plant material of different cultivars.

A New One-Tube Reaction Assay for the Universal Determination of Sweet Cherry (Prunus avium L.) Self-(In)Compatible MGST- and S-Alleles Using Capillary Fragment Analysis.

The sweet cherry plant (Prunus avium L.) is primarily self-incompatible, with so-called S-alleles responsible for the inability of flowers to be pollinated not only by their own pollen grains but also by pollen from other cherries having the same S-alleles. This characteristic has wide-ranging impacts on commercial growing, harvesting, and breeding. However, mutations in S-alleles as well as changes in the expression of M locus-encoded glutathione-S-transferase (MGST) can lead to complete or partial self-compatibility, simplifying orchard management and reducing possible crop losses. Knowledge of S-alleles is important for growers and breeders, but current determination methods are challenging, requiring several PCR runs. Here we present a system for the identification of multiple S-alleles and MGST promoter variants in one-tube PCR, with subsequent fragment analysis on a capillary genetic analyzer. The assay was shown to unequivocally determine three MGST alleles, 14 self-incompatible S-alleles, and all three known self-compatible S-alleles (S3', S4', S5') in 55 combinations tested, and thus it is especially suitable for routine S-allele diagnostics and molecular marker-assisted breeding for self-compatible sweet cherries. In addition, we identified a previously unknown S-allele in the 'Techlovicka´ genotype (S54) and a new variant of the MGST promoter with an 8-bp deletion in the ´Kronio´ cultivar.

High-resolution genome-wide association study of a large Czech collection of sweet cherry (Prunus avium L.) on fruit maturity and quality traits.

In sweet cherry (Prunus avium L.), quantitative trait loci have been identified for fruit maturity, colour, firmness, and size to develop markers for marker-assisted selection. However, resolution is usually too low in those analyses to directly target candidate genes, and some associations are missed. In contrast, genome-wide association studies are performed on broad collections of accessions, and assemblies of reference sequences from Tieton and Satonishiki cultivars enable identification of single nucleotide polymorphisms after whole-genome sequencing, providing high marker density. Two hundred and thirty-five sweet cherry accessions were sequenced and phenotyped for harvest time and fruit colour, firmness, and size. Genome-wide association studies were used to identify single nucleotide polymorphisms associated with each trait, which were verified in breeding material consisting of 64 additional accessions. A total of 1 767 106 single nucleotide polymorphisms were identified. At that density, significant single nucleotide polymorphisms could be linked to co-inherited haplotype blocks (median size ~10 kb). Thus, markers were tightly associated with respective phenotypes, and individual allelic combinations of particular single nucleotide polymorphisms provided links to distinct phenotypes. In addition, yellow-fruit accessions were sequenced, and a ~ 90-kb-deletion on chromosome 3 that included five MYB10 transcription factors was associated with the phenotype. Overall, the study confirmed numerous quantitative trait loci from bi-parental populations using high-diversity accession populations, identified novel associations, and genome-wide association studies reduced the size of trait-associated loci from megabases to kilobases and to a few candidate genes per locus. Thus, a framework is provided to develop molecular markers and evaluate and characterize genes underlying important agronomic traits.

Identification and Characterization of a Novel Umbra-like Virus, Strawberry Virus A, Infecting Strawberry Plants.

A novel RNA virus infecting strawberry plants was discovered using high-throughput sequencing. The analyzed plant was simultaneously infected with three different genetic variants of the virus, provisionally named strawberry virus A (StrVA). Although StrVA is phylogenetically clustered with several recently discovered, unclassified plant viruses, it has a smaller genome and several unique features in its genomic organization. A specific and sensitive qPCR system for the detection of identified StrVA genetic variants was designed. A survey conducted in the Czech Republic revealed that StrVA was present in 28.3% of strawberry samples (n = 651) from various origins (plantations, gardens, and propagation material). Sequencing of 48 randomly selected StrVA-positive strawberry samples showed that two or all three StrVA genetic variants were present in 62.5% of the samples in various proportions. StrVA was found in mixed infections with other viruses (strawberry mild yellow edge virus, strawberry crinkle virus, strawberry mottle virus, strawberry polerovirus 1, or strawberry virus 1) in 57.1% of the samples, which complicated the estimation of its biological relevance and impact on the health status of the plants.

High Incidence of Strawberry Polerovirus 1 in the Czech Republic and Its Vectors, Genetic Variability and Recombination.

In total, 332 strawberry plants from 33 different locations in the Czech Republic with or without disease symptoms were screened by RT-PCR for the presence of strawberry polerovirus 1 (SPV1) and five other viruses: strawberry mottle virus, strawberry crinkle virus, strawberry mild yellow edge virus, strawberry vein banding virus, and strawberry virus 1. SPV1 was detected in 115 tested strawberry plants (35%), including 89 mixed infections. No correlation between symptoms and the detected viruses was found. To identify potential invertebrate SPV1 vectors, strawberry-associated invertebrate species were screened by RT-PCR, and the virus was found in the aphids Aphis forbesi, A. gossypii, A. ruborum, A.sanquisorbae, Aulacorthum solani, Chaetosiphon fragaefolii, Myzus ascalonicus, and several other non-aphid invertebrate species. SPV1 was also detected in aphid honeydew. Subsequent tests of C. fragaefolii and A.gossypii virus transmission ability showed that at least 4 h of acquisition time were needed to acquire the virus. However, 1 day was sufficient for inoculation using C. fragaefolii. In conclusion, being aphid-transmitted like other tested viruses SPV1 was nevertheless the most frequently detected agent. Czech SPV1 isolates belonged to at least two phylogenetic clusters. The sequence analysis also indicated that recombination events influence evolution of SPV1 genomes.

A new one-tube reaction kit for the SSR genotyping of apple (Malus × domestica Borkh.).

Though apple genotyping is mainly used for scientific and breeding purposes, it can also be adopted by national authorities to control the authenticity of apple cultivars. To facilitate the introduction of routine apple genotyping into practice, a new apple simple sequence repeat (SSR) genotyping kit was developed (called the Ap17 in. SSR Genotyping Kit). The kit combines 17 SSR markers including those recommended by the Working Group of the European Cooperative Programme for Plant Genetic Resources (ECPGR), covering all apple linkage groups in a one-tube reaction format, using a fragment analysis method to simplify the genotyping procedure. The kit was successfully tested using 880 unique diploid apple germplasm accessions; the kit can also readily discriminate triploid and tetraploid samples. The total probability of identity for the kit and the sample collection used was calculated to be 1.73 × 10-22. Tables for converting results to enable genotype comparisons between currently-used genotyping systems and the Ap17 in. kit are provided. The kit is ideally suited for validation in laboratories genotyping a large number of apple samples, saving time, costs, and labor, while minimizing technical and human errors.

High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by "Candidatus Phytoplasma prunorum".

"Candidatus Phytoplasma prunorum" (CPp) is a highly destructive phytopathogenic agent in many stone fruit-growing regions in Europe and the surrounding countries. In this work, we focused on documenting entire bacterial community in the phloem tissues of 60 stone fruit trees. Nested PCR and two real-time PCR assays were used to select CPp-positive (group A) and CPp-negative samples (group B). Afterwards, high-throughput amplicon sequencing was performed to assess bacterial community compositions in phloem tissues. The bacterial composition in phloem tissue consisted of 118 distinct genera, represented mainly by Pseudomonas, Acinetobacter, Methylobacterium, Sphingomonas, and Rhizobium. Statistics showed that CPp influenced the bacterial composition of infected plants (group A) and that the bacterial community depended on the geographical origin of the sample. This is the first work focusing on an analysis of the influence of CPp on the bacteria coexisting in the phloem tissues of stone fruit trees.

Parental origin of deletions and duplications - about the necessity to check for cryptic inversions.

BACKGROUND: Copy number variants (CNVs) are the genetic bases for microdeletion/ microduplication syndromes (MMSs). Couples with an affected child and desire to have further children are routinely tested for a potential parental origin of a specific CNV either by molecular karyotyping or by two color fluorescence in situ hybridization (FISH), yet. In the latter case a critical region probe (CRP) is combined with a control probe for identification of the chromosome in question. However, CNVs can arise also due to other reasons, like a recombination-event based on a submicroscopic, cryptic inversion in one of the parents. RESULTS: Seventy-four patients with different MMSs and overall 81 CNVs were studied here by a novel three color FISH approach. The way how three locus-specific probes are selected (one is the CRP and two are flanking it in a distance of 5-10 Mb) enables to detect or exclude two possible parental conditions as origins of the CNV seen in the index: (i) direct parental origin of the CNV (deletion or duplication) or (ii) a parental cryptic inversion. Thus, for overall 51/81 CNVs (63%) a parental origin could be determined. 36/51 (70.5%) inherited the CNV directly from one of the parents, but 15/51 (29.5%) were due to an exclusively by three color FISH detectable parental inversion. A 2:1 ratio of maternal versus paternal inheritance was found. Also almost two times more male than female were among the index patients. CONCLUSION: The new, here suggested three color FISH approach is suited for more comprehensive parental studies of patients with MMS. The detection rate for parental origin was increased by 140% in this study. Still, for 30/81 cases (37%) no reason for the 'de novo' MMS in the affected index patient could be found by the here suggested FISH-probe set.

Parvovirus B19 induced lupus-like syndrome with nephritis.

We report a case of a 65-year-old man who developed an acute illness with fever, arthralgia and nephritic syndrome. Antinuclear antibodies were slightly positive and complement levels were low. Renal biopsy showed exudative diffuse proliferative endocapillary glomerulonephritis with diffuse immunoglobulin (IgG, IgA, IgM) and complement deposition (C3d, C4d, C1q) on immunofluorescence. The patient was first treated with corticosteroids and mycophenolate mofetil for suspected lupus with WHO class IV glomerulonephritis. The diagnosis was questioned and a diagnosis of parvovirus B19-associated nephritis was made based on elevation of serum IgM antibodies for parvovirus B19 and detection of parvovirus B19 DNA on renal biopsy. The immunosuppressive treatment was stopped and progressive spontaneous regression of clinical and laboratory abnormalities was observed. We conclude that human parvovirus B19 infection should be considered as a cause of lupus-like symptomatology and acute glomerulonephritis.

A method to identify new molecular markers for assessing minimal residual disease in acute leukemia patients.

Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include recurrent cytogenetic abnormalities and mutations in important hematological genes; unfortunately well-characterized targets are lacking in many AL patients. Here we demonstrate a technical approach for the identification and mapping of novel clone-specific chromosomal abnormalities down to the nucleotide level. We used molecular cytogenetics, chromosome microdissection, amplification of the microdissected material, and next-generation sequencing to develop PCR-based MRD assays based on unique breakpoint sequences.

Transcription factors Fli1 and EKLF in the differentiation of megakaryocytic and erythroid progenitor in 5q- syndrome and in Diamond-Blackfan anemia.

Friend leukemia virus integration 1 (Fli1) and erythroid Krüppel-like factor (EKLF) participate under experimental conditions in the differentiation of megakaryocytic and erythroid progenitor in cooperation with other transcription factors, cytokines, cytokine receptors, and microRNAs. Defective erythropoiesis with refractory anemia and effective megakaryopoiesis with normal or increased platelet count is typical for 5q- syndrome. We decided to evaluate the roles of EKLF and Fli1 in the pathogenesis of this syndrome and of another ribosomopathy, Diamond-Blackfan anemia (DBA). Fli1 and EKLF mRNA levels were examined in mononuclear blood and bone marrow cells from patients with 5q- syndrome, low-risk MDS patients with normal chromosome 5, DBA patients, and healthy controls. In 5q- syndrome, high Fli1 mRNA levels in the blood and bone marrow mononuclear cells were found. In DBA, Fli1 expression did not differ from the controls. EKLF mRNA level was significantly decreased in the blood and bone marrow of 5q- syndrome and in all DBA patients. We propose that the elevated Fli1 in 5q- syndrome protects megakaryocytic cells from ribosomal stress contrary to erythroid cells and contributes to effective though dysplastic megakaryopoiesis.

The Czech National Diamond-Blackfan Anemia Registry: clinical data and ribosomal protein mutations update.

Diamond-Blackfan anemia is a rare inherited bone marrow failure syndrome diagnosed in early infancy that is characterized by a (a) macrocytic anemia with no other significant cytopenia, (b) reticulocytopenia, and (c) normal bone marrow cellularity with a paucity of erythroid precursors. Physical anomalies are often present. Mutations in several ribosomal proteins have been associated with the disease. Here we present a detailed description of 39 patients from 34 families enrolled in the Czech National Diamond-Blackfan Anemia Registry. Erythrocyte adenosine deaminase activity and serum erythropoietin levels were measured and bone marrow analysis and clonogenic assays were carried out. Twenty-two different ribosomal proteins were sequenced. We identified mutations in five different ribosomal proteins in 28/39 patients (71.8%) from 23/34 families (67.6%). Several new mutations are described. The most interesting data relate to genotype-phenotype correlations. All patients with ribosomal protein L5 or ribosomal protein L11 mutations have a thumb defect usually with one or more other anomalies. Most of these patients were born small for gestational age and currently have short stature. We also described five patients with a ribosomal protein S26 mutation. All of the latter are transfusion-dependent and they exhibit skeletal abnormalities rather than thumb or craniofacial deformities. Patients with ribosomal protein S19 seem to bear mildest associated anomalies, usually in a craniofacial region.

Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts.

Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (e.g., RPS19) or large (e.g., RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.

Candidatus Neoehrlichia mikurensis infection identified in 2 hematooncologic patients: benefit of molecular techniques for rare pathogen detection.

Hematooncologic patients often host rare or fastidious pathogens. Using 16S rDNA sequencing and transmission electron microscopy, we have identified 2 lymphoma patients infected with Candidatus Neoehrlichia mikurensis. In both individuals, the clinical presentation suggested ehrlichiosis-like syndrome. We believe that molecular techniques open new vistas in the field of pathogen detection.

A comprehensive study of TP53 mutations in chronic lymphocytic leukemia: Analysis of 1287 diagnostic and 1148 follow-up CLL samples.

TP53 plays a pivotal role in the process of DNA repair and apoptosis. In 10-20% of patients with chronic lymphocytic leukemia (CLL), the TP53 pathway is affected. In this study, we analyzed the TP53 mutation status in 2435 consecutive CLL samples, including 1287 diagnostic samples and 1148 samples during follow-up, using FASAY (Functional Analysis of Separated Alleles in Yeast) and direct sequencing. In a cohort of 1287 diagnostic CLL samples, we identified 237 cases with TP53 variants, including mutations, temperature-sensitive variants, deletions, insertions and aberrant splicing variants (18.4%). In 1148 follow-up samples, no TP53 clonal evolution was observed.

Tyrosine 87 is vital for the activity of human protein arginine methyltransferase 3 (PRMT3).

Protein arginine methyltransferase 3 (PRMT3) is a cytosolic enzyme that catalyzes the formation of mono- and asymmetric dimethyl arginines, with ribosomal protein (RP) S2 as its main in vivo substrate. The interplay of PRMT3-RPS2 homologs in yeast is important for regulating the ribosomal subunit ratio and assembly. Prmt3-null mice display slower embryonic growth and development, although this phenotype is milder than in mouse RP gene knockouts. Defects in ribosome maturation are the hallmark of Diamond-Blackfan anemia (DBA). Sequencing of the PRMT3 gene in patients from the Czech DBA registry revealed a heterozygous mutation encoding the Tyr87Cys substitution. Although later analysis excluded this mutation as the cause of disease, we anticipated that this substitution might be important for PRMT3 function and decided to study it in detail. Tyr87 resides in a highly conserved substrate binding domain and has been predicted to be phosphorylated. To address the impact of putative Tyr87 phosphorylation on PRMT3 properties, we constructed two additional PRMT3 variants, Tyr87Phe and Tyr87Glu PRMT3, mimicking non-phosphorylated and phosphorylated Tyr87, respectively. The Tyr87Cys and Tyr87Glu-PRMT3 variants had markedly decreased affinity to RPS2 and, consequently, reduced enzymatic activity compared to the wild-type enzyme. The activity of the Tyr87Phe-PRMT3 mutant remained unaffected. No evidence of Tyr87 phosphorylation was found using mass spectrometric analysis of purified PRMT3, although phosphorylation of serines 25 and 27 was observed. In conclusion, Tyr87 is important for the interaction between PRMT3 and RPS2 and for its full enzymatic activity.

The ribosomal basis of Diamond-Blackfan Anemia: mutation and database update.

Diamond-Blackfan Anemia (DBA) is characterized by a defect of erythroid progenitors and, clinically, by anemia and malformations. DBA exhibits an autosomal dominant pattern of inheritance with incomplete penetrance. Currently nine genes, all encoding ribosomal proteins (RP), have been found mutated in approximately 50% of patients. Experimental evidence supports the hypothesis that DBA is primarily the result of defective ribosome synthesis. By means of a large collaboration among six centers, we report here a mutation update that includes nine genes and 220 distinct mutations, 56 of which are new. The DBA Mutation Database now includes data from 355 patients. Of those where inheritance has been examined, 125 patients carry a de novo mutation and 72 an inherited mutation. Mutagenesis may be ascribed to slippage in 65.5% of indels, whereas CpG dinucleotides are involved in 23% of transitions. Using bioinformatic tools we show that gene conversion mechanism is not common in RP genes mutagenesis, notwithstanding the abundance of RP pseudogenes. Genotype-phenotype analysis reveals that malformations are more frequently associated with mutations in RPL5 and RPL11 than in the other genes. All currently reported DBA mutations together with their functional and clinical data are included in the DBA Mutation Database.

Human MRCKalpha is regulated by cellular iron levels and interferes with transferrin iron uptake.

Myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha, formally known as CDC42BPA) is a serine/threonine kinase that can regulate actin/myosin assembly and activity. Recently, it has been shown that it possesses a functional iron responsive element (IRE) in the 3'-untranslated region (UTR) of its mRNA, suggesting that it may be involved in iron metabolism. Here we report that MRCKalpha protein expression is also regulated by iron levels; MRCKalpha colocalizes with transferrin (Tf)-loaded transferrin receptors (TfR), and attenuation of MRCKalpha expression by a short hairpin RNA silencing construct leads to a significant decrease in Tf-mediated iron uptake. Our results thus indicate that MRCKalpha takes part in Tf-iron uptake, probably via regulation of Tf-TfR endocytosis/endosome trafficking that is dependent on the cellular cytoskeleton. Regulation of the MRCKalpha activity by intracellular iron levels could thus represent another molecular feedback mechanism cells could use to finely tune iron uptake to actual needs.