Discovery and Chromosomal Location a Highly Effective Oat Crown Rust Resistance Gene Pc50-5.

Crown rust, caused by Puccinia coronata f. sp. avenae, is one of the most destructive fungal diseases of oat worldwide. Growing disease-resistant oat cultivars is the preferred method of preventing the spread of rust and potential epidemics. The object of the study was Pc50-5, a race-specific seedling crown rust resistant gene, highly effective at all growth stages, selected from the differential line Pc50 (Avena sterilis L. CW 486-1 × Pendek). A comparison of crown rust reaction as well as an allelism test showed the distinctiveness of Pc50-5, whereas the proportions of phenotypes in segregating populations derived from a cross with two crown rust-susceptible Polish oat cultivars, Kasztan × Pc50-5 and Bingo × Pc50-5, confirmed monogenic inheritance of the gene, indicating its usefulness in oat breeding programs. Effective gene introgression depends on reliable gene identification in the early stages of plant development; thus, the aim of the study was to develop molecular markers that are tightly linked to Pc50-5. Segregating populations of Kasztan × Pc50-5 were genotyped using DArTseq technology based on next-generation Illumina short-read sequencing. Markers associated with Pc50-5 were located on chromosome 6A of the current version of the oat reference genome (Avena sativa OT3098 v2, PepsiCo) in the region between 434,234,214 and 440,149,046 bp and subsequently converted to PCR-based SCAR (sequence-characterized amplified region) markers. Furthermore, 5426978_SCAR and 24031809_SCAR co-segregated with the Pc50-5 resistance allele and were mapped to the partial linkage group at 0.6 and 4.0 cM, respectively. The co-dominant 58163643_SCAR marker was the best diagnostic and it was located closest to Pc50-5 at 0.1 cM. The newly discovered, very strong monogenic crown rust resistance may be useful for oat improvement. DArTseq sequences converted into specific PCR markers will be a valuable tool for marker-assisted selection in breeding programs.

Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformations, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron variant emergence.

Reference genes expression stability in Avena sativa L. during compatible and incompatible interactions with Puccinia graminis.

A reliable qPCR experiment requires the selection of reference genes with a stable level of expression in a given experimental system. This study attempts to determine the reference genes (RGs) for the A. sativa-P. graminis experimental setup. We evaluated nine candidate reference genes in A. sativa (oat line Pg4 and the cultivar Kasztan) during compatible and incompatible interactions with different pathotypes of Puccinia graminis f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and ΔCt method). We found that the most appropriate combination of RGs for RT-qPCR data normalization were HNR (heterogeneous nuclear ribonucleoprotein 27C) + EF1A (elongation factor 1-alpha) + EIF4A (eukaryotic initiation factor 4A-3). The worst candidates for normalization in this dataset were CYP (cyclophilin) and TUA (alpha tubulin). Identified reference genes are suitable candidates for the standardization of gene expression studies in the A. sativa-P. graminis interaction system and potentially other related pathogens. To date, this is the first report of RGs selection in this pathosystem.

Validation of reference genes as an internal control for studying Avena sativa-Puccinia coronata interaction by RT-qPCR.

In this study we evaluated eleven candidate reference genes in Avena sativa during compatible and incompatible interactions with two different pathotypes of Puccinia coronata f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and ΔCt method). The results obtained confirmed that the combination of two genes would be sufficient for reliable normalization of the expression data. In general, the most stable in the tested plant-pathogen system were HNR (heterogeneous nuclear ribonucleoprotein 27C) and EF1A (elongation factor 1-alpha). ARF (ADP-ribosylation factor) and EIF4A (eukaryotic initiation factor 4A-3) could also be considered as exhibiting high expression stability. CYP (cyclophilin) was shown by all assessment methods to be the worst candidate for normalization in this dataset. To date, this is the first report of reference genes selection in A. sativa-P. coronata interaction system. Identified reference genes enable reliable and comprehensive RT-qPCR analysis of oat gene expression in response to crown rust infection. Understanding the molecular mechanisms involved in the host-pathogen interactions may expand knowledge of durable resistance strategies beneficial to modern oat breeding.

Mass spectrometry captures biased signalling and allosteric modulation of a G-protein-coupled receptor.

G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the ß1-adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor-Gs complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor-Gs complex. Further interrogation led us to propose a mechanism in which zinc ions facilitate a structural transition of the intermediate complex towards the stable state.