Simultaneously induced mutations in eIF4E genes by CRISPR/Cas9 enhance PVY resistance in tobacco.

Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVYO accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVYO confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVYO in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.

Diterpene alkaloids from the roots of Aconitum moldavicum and assessment of Nav 1.2 sodium channel activity of aconitum alkaloids.

A new aconitane alkaloid, 1-O-demethylswatinine (1), was isolated from the root of Aconitum moldavicum together with the known compounds cammaconine (2), columbianine (3), swatinine (4), gigactonine (5), delcosine (6), lycoctonine (7), and ajacine (8). The structures were established by means of HRESIMS, 1D and 2D NMR spectroscopy, including 1H-1H COSY, NOESY, HSQC, and HMBC experiments, resulting in complete 1H-NMR chemical shift assignments for 1-4. The effects of the isolated compounds 4-8, together with eighteen other Aconitum diterpene and norditerpene alkaloids with different skeletal types and substitution patterns, were studied on Nav 1.2 channels by the whole-cell patch clamp technique, using the QPatch-16 automated patch clamp system. Pyroaconitine, ajacine, septentriodine, and delectinine demonstrated significant Nav 1.2 channel inhibition (57-42 %) at 10 µM concentration; several other compounds (acovulparine, acotoxicine, hetisinone, 14-benzoylaconine-8-O-palmitate, aconitine, and lycoctonine) exerted moderate inhibitory activity (30-22 %), while the rest of the tested alkaloids were considered to be inactive. On the basis of these results and by exhaustive comparison of data of previously published computerized QSAR studies on diterpene alkaloids, certain conclusions on the structure-activity relationships of Aconitum alkaloids concerning Nav 1.2 channel inhibitory activity are proposed.

Bone tuberculosis in Roman Period Pannonia (western Hungary)

The purpose of this study was to analyse a skeleton (adult female, 25-30 years) that presented evidence of tuberculous spondylitis. The skeleton, dated from the Roman Period (III-VI centuries), was excavated near the town of Győr, in western Hungary. The skeleton was examined by gross observation supplemented with mycolic acid and proteomic analyses using MALDI-TOF/TOF tandem mass spectrometry. The biomolecular analyses supported the morphological diagnosis.

Bone tuberculosis in Roman Period Pannonia (western Hungary).

The purpose of this study was to analyse a skeleton (adult female, 25-30 years) that presented evidence of tuberculous spondylitis. The skeleton, dated from the Roman Period (III-VI centuries), was excavated near the town of Gyor, in western Hungary. The skeleton was examined by gross observation supplemented with mycolic acid and proteomic analyses using MALDI-TOF/TOF tandem mass spectrometry. The biomolecular analyses supported the morphological diagnosis.