VPg of Potato Virus Y and Potato Cap-Binding eIF4E Factors: Selective Interaction and Its Supposed Mechanism.

Potato virus Y (PVY) is one of the most common and harmful plant viruses. Translation of viral RNA starts with the interaction between the plant cap-binding translation initiation factors eIF4E and viral genome-linked protein (VPg) covalently attached to the viral RNA. Disruption of this interaction is one of the natural mechanisms of plant resistance to PVY. The multigene eIF4E family in the potato (Solanum tuberosum L.) genome contains genes for the translation initiation factors eIF4E1, eIF4E2, and eIF(iso)4E. However, which of these factors can be recruited by the PVY, as well as the mechanism of this interaction, remain obscure. Here, we showed that the most common VPg variant from the PVY strain NTN interacts with eIF4E1 and eIF4E2, but not with eIF(iso)4E. Based on the VPg, eIF4E1, and eIF4E2 models and data on the natural polymorphism of VPg amino acid sequence, we suggested that the key role in the recognition of potato cap-binding factors belongs to the R104 residue of VPg. To verify this hypothesis, we created VPg mutants with substitutions at position 104 and examined their ability to interact with potato eIF4E factors. The obtained data were used to build the theoretical model of the VPg-eIF4E2 complex that differs significantly from the earlier models of VPg complexes with eIF4E proteins, but is in a good agreement with the current biochemical data.

CRISPR/Cas9 genome editing through in planta transformation.

In this review, the application of CRISPR/Cas9 plant genome editing using alternative transformation methods is discussed. Genome editing by the CRISPR/Cas9 system is usually implemented via the generation of transgenic plants carrying Cas9 and sgRNA genes in the genome. Transgenic plants are usually developed by in vitro regeneration from single transformed cells, which requires using different in vitro culture-based methods. Despite their common application, these methods have some disadvantages and limitations. Thus, some methods of plant transformation that do not depend on in vitro regeneration have been developed. These methods are known as "in planta" transformation. The main focus of this review is the so-called floral dip in planta transformation method, although other approaches are also described. The main features of in planta transformation in the context of CRISPR/Cas9 genome editing are discussed. Furthermore, multiple ways to increase the effectiveness of this approach and to broaden its use in different plant species are considered.

Development of F1 hybrid population and the high-density linkage map for European aspen (Populus tremula L.) using RADseq technology.

BACKGROUND: Restriction-site associated DNA sequencing (RADseq) technology was recently employed to identify a large number of single nucleotide polymorphisms (SNP) for linkage mapping of a North American and Eastern Asian Populus species. However, there is also the need for high-density genetic linkage maps for the European aspen (P. tremula) as a tool for further mapping of quantitative trait loci (QTLs) and marker-assisted selection of the Populus species native to Europe. RESULTS: We established a hybrid F1 population from the cross of two aspen parental genotypes diverged in their phenological and morphological traits. We performed RADseq of 122 F1 progenies and two parents yielding 15,732 high-quality SNPs that were successfully identified using the reference genome of P. trichocarpa. 2055 SNPs were employed for the construction of maternal and paternal linkage maps. The maternal linkage map was assembled with 1000 SNPs, containing 19 linkage groups and spanning 3054.9 cM of the genome, with an average distance of 3.05 cM between adjacent markers. The paternal map consisted of 1055 SNPs and the same number of linkage groups with a total length of 3090.56 cM and average interval distance of 2.93 cM. The linkage maps were employed for QTL mapping of one-year-old seedlings height variation. The most significant QTL (LOD = 5.73) was localized to LG5 (96.94 cM) of the male linkage map, explaining 18% of the phenotypic variation. CONCLUSIONS: The set of 15,732 SNPs polymorphic in aspen and high-density genetic linkage maps constructed for the P. tremula intra-specific cross will provide a valuable source for QTL mapping and identification of candidate genes facilitating marker-assisted selection in European aspen.

α-Klotho is associated with cardiovascular and all-cause mortality in patients with stage 3b and 4 chronic kidney disease (CKD): a long-term prospective cohort study.

BACKGROUND: α-Klotho deficiency may increase cardiovascular risks and worsen survival. We evaluated the association of α-Klotho with cardiovascular and all-cause mortality in pre-dialysis chronic kidney disease (CKD) patients. METHODS: In this prospective study, 75 non-diabetic CKD stage 3b-4 patients were followed-up for a median of 8 years. Primary and secondary outcomes were all-cause and cardiovascular mortality, respectively. Human soluble α-Klotho ELISA Assay (IBL-Takara 27,998-96Well), Human Fibroblast Growth Factor-23 ELISA Assay (intact FGF23, Merck Millipore MILLENZ FGF23-32 K), and Human Sclerostin ELISA kits (Biomedica, Vienna, BI-20492) were used to measure serum α-Klotho, FGF23 and sclerostin levels in the certified laboratory at the Sechenov University according to the manufacturers' protocols. All patients underwent echocardiography to evaluate left ventricular mass index (LVMI), left ventricular ejection fraction by Simpson method, and cardiac (valve) calcification score by a semi-quantitative point scale. Lateral abdominal radiography by Kauppila method was used to estimate calcification of the abdominal aorta. Cox multivariate regression and receiver-operating characteristic curve (ROC)-analysis were used to evaluate risk factors for death and their cut-off values. RESULTS: Primary and secondary endpoints were reached in 15 (20%) and 9 (12%) patients, respectively. Median α-Klotho levels in deceased and surviving patients were 344 and 484 pg/ml, respectively (p = 0.002). In a multivariate Cox regression model, baseline α-Klotho levels (HR 0.99, 95% CI 0.98-1.00, p = 0.023), aortic calcification (HR 1.18, 95% CI 1.02-1.36, p = 0.029) and left ventricular mass index (LVMI) (HR 1.04, 95% CI 1.00-1.08, p = 0.033) were associated with the primary endpoint, whereas α-Klotho (HR 0.99, 95% CI 0.98-1.00, p = 0.029), aortic calcification (HR 1.23, 95% CI 1.07-1.42, p = 0.003) and LVMI (HR 1.04, 95% CI 1.00-1.08, p = 0.021) were associated with the secondary endpoint. α-Klotho levels had the highest area under the curve (AUC) by ROC analysis, that is, 0.766 (95% CI 0.70-0.82) for the primary endpoint and 0.842 (95% CI 0.79-0.90) for the secondary endpoint with cut-off values of 412 pg/ml (HR 3.06, 95% CI 1.36-6.89, p = 0.007) and 368 pg/ml (HR 4.84, 95% CI 1.59-14.73, p = 0.005), respectively. CONCLUSION: In pre-dialysis CKD patients, α-Klotho levels are associated with all-cause and cardiovascular mortality and may be considered an early prognostic marker.