NIRS for vicine and convicine content of faba bean seed allowed GWAS to prepare for marker-assisted adjustment of seed quality of German winter faba beans

Die antinutritiven Inhaltsstoffe Vicin und Convicin (V, C) in Samen von Winterackerbohnen wurden einer GWAS unterzogen. V-, C- und V + C-Werte von 189 Inzuchtlinien (fünf Umwelten) wurden mittels NIRS ermittelt. In diesen Linien kommt das starke „vc-“-Allel (VC1-Locus) nicht vor. Labor-Resultate von 646 Proben führten zu unserer NIRS-Kalibration, die gut für V und V + C tauglich war allerdings nicht tauglich für C. Die Erblichkeit war hoch für V und V + C (0,911; 0,868) und niedriger für C (0,737). Von den 2542 kartierten SNPs waren 47 signifikant mit V und einer mit V + C assoziiert. Vier SNPs, die nahe beim VC1-Lokus kartierten, waren für V signifikant. Anscheinend trugen nicht-„vc-“-Allele an diesem Locus zur V-Variation bei. Markergestützte Züchtung in diesem Genpool kann die V + C-Gehalt auf etwa 0,44 % reduzieren, im Vergleich zur aktuell niedrigsten Linie mit 0,55 %. Weitere Forschung wird zeigen, wie diese Ergebnisse der Agronomie und Züchtung dienlich sein werden.GWAS was applied to the antinutritive compounds vicine and convicine (V, C) in winter faba bean. V, C and V + C data for 189 inbred lines (five environments) were predicted by NIRS. These lines do not carry the strong “vc-“ allele (locus VC1). Lab data for 646 samples enabled our NIRS calibration, which performed well for V and V + C yet poor for C. Heritability was high (0.911; 0.868) for V and V + C and lower for C (0.737). From the 2542 mapped SNPs, 47 were significantly associated with V and one with V + C. Four SNPs mapped near to the VC1 locus and were significant for V. Seemingly, non-“vc-“ alleles at that locus contributed to V variation. Marker-assisted breeding with this germplasm can reduce the V + C content to about 0.44%, compared to the current lowest line with 0.55%. Further research will show inasmuch this can serve agronomy and breeding

Highly Sensitive Lysine Deacetylase Assay Based on Acetylated Firefly Luciferase

Lysine deacetylases (KDACs) play important roles in many physiological processes and are implicated in many human diseases. Hence, the search for modulators of KDACs is very active, and reliable assays for monitoring their activity are key to success. Here, we describe a new KDAC assay based on Firefly luciferase harboring an acetylation on an essential active site lysine. We show that several KDACs can reverse this modification and hence activate luciferase. This new assay is extremely sensitive, reliable, and fast and can be performed in a continuous format. We used this assay to screen a small library of compounds and identified several novel effectors of SirT2 with low micromolar activity

Species-Specific Differences in the Activity of PrfA, the Key Regulator of Listerial Virulence Genes

PrfA, the master regulator of LIPI-1, is indispensable for the pathogenesis of the human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. PrfA is also present in the apathogenic species Listeria seeligeri, and in this study, we elucidate the differences between PrfA proteins from the pathogenic and apathogenic species of the genus Listeria. PrfA proteins of L. monocytogenes (PrfA(Lm) and PrfA*(Lm)), L. ivanovii (PrfA(Li)), and L. seeligeri (PrfA(Ls)) were purified, and their equilibrium constants for binding to the PrfA box of the hly promoter (Phly(Lm)) were determined by surface plasmon resonance. In addition, the capacities of these PrfA proteins to bind to the PrfA-dependent promoters Phly and PactA and to form ternary complexes together with RNA polymerase were analyzed in electrophoretic mobility shift assays, and their abilities to initiate transcription in vitro starting at these promoters were compared. The results show that PrfA(Li) resembled the constitutively active mutant PrfA*(Lm) more than the wild-type PrfA(Lm), whereas PrfA(Ls) showed a drastically reduced capacity to bind to the PrfA-dependent promoters Phly and PactA. In contrast, the efficiencies of PrfA(Lm), PrfA*(Lm), and PrfA(Li) forming ternary complexes and initiating transcription at Phly and PactA were rather similar, while those of PrfA(Ls) were also much lower. The low binding and transcriptional activation capacities of PrfA(Ls) seem to be in part due to amino acid exchanges in its C-terminal domain (compared to PrfA(Lm) and PrfA(Li)). In contrast to the significant differences in the biochemical properties of PrfA(Lm), PrfA(Li), and PrfA(Ls), the PrfA-dependent promoters of hly (Phly(Lm), Phly(L)(i), and Phly(L)(s)) and actA (PactA(Lm), PactA(L)(i), and PactA(L)(s)) of the three Listeria species did not significantly differ in their binding affinities to the various PrfA proteins and in their strengths to promote transcription in vitro. The allelic replacement of prfA(Lm) with prfA(Ls) in L. monocytogenes leads to low expression of PrfA-dependent genes and to reduced in vivo virulence of L. monocytogenes, suggesting that the altered properties of PrfA(Ls) protein are a major cause for the low virulence of L. seeligeri