Destins des S-RNases et interactions moléculaires dans le tube pollinique dans le cadre de l’auto-incompatibilité gamétophytique chez Solanum chacoense

L’auto-incompatibilité (AI) est une barrière reproductive prézygotique qui permet aux pistils d’une fleur de rejeter leur propre pollen. Les systèmes d’AI peuvent prévenir l’autofertilisation et ainsi limiter l’inbreeding. Dans l’AI gamétophytique, le génotype du pollen détermine son propre phénotype d’incompatibilité, et dans ce système, les déterminants mâles et femelles de l’AI sont codés par un locus multigénique et multi-allélique désigné le locus S. Chez les Solanaceae, le déterminant femelle de l’AI est une glycoprotéine stylaire extracellulaire fortement polymorphique possédant une activité ribonucléase et désignée S-RNase. Les S-RNases montrent un patron caractéristique de deux régions hypervariables (HVa et HVb), responsables de leur détermination allélique, et cinq régions hautement conservées (C1 à C5) impliquées dans l’activité catalytique ou la stabilisation structurelle de ces protéines. Dans ce travail, nous avons investigué plusieurs caractéristiques des S-RNases et identifié un nouveau ligand potentiel aux S-RNases chez Solanum chacoense. L’objectif de notre première étude était l’élucidation du rôle de la région C4 des S-RNases. Afin de tester l’hypothèse selon laquelle la région C4 serait impliquée dans le repliement ou la stabilité des S-RNases, nous avons généré un mutant dans lequel les quatre résidus chargés présents en région C4 furent remplacés par des résidus glycine. Cette protéine mutante ne s’accumulant pas à des niveaux détectables, la région C4 semble bien avoir un rôle structurel. Afin de vérifier si C4 est impliquée dans une liaison avec une autre protéine, nous avons généré le mutant R115G, dans lequel un acide aminé chargé fût éliminé afin de réduire les affinités de liaison dans cette région. Ce mutant n’affectant pas le phénotype de rejet pollinique, il est peu probable que la région C4 soit impliquée dans la liaison des S-RNases avec un ligand ou leur pénétration à l’intérieur des tubes polliniques. Enfin, le mutant K113R, dans lequel le seul résidu lysine conservé parmi toutes les S-RNases fût remplacé par un résidu arginine, fût généré afin de vérifier si cette lysine était un site potentiel d’ubiquitination des S-RNases. Toutefois, la dégradation des S-RNases ne fût pas inhibée. Ces résultats indiquent que C4 joue probablement un rôle structurel de stabilisation des S-RNases. Dans une seconde étude, nous avons analysé le rôle de la glycosylation des S-RNases, dont un site, en région C2, est conservé parmi toutes les S-RNases. Afin d’évaluer la possibilité que les sucres conjugués constituent une cible potentielle d’ubiquitination, nous avons généré une S11-RNase dont l‘unique site de glycosylation en C2 fût éliminé. Ce mutant se comporte de manière semblable à une S11-RNase de type sauvage, démontrant que l’absence de glycosylation ne confère pas un phénotype de rejet constitutif du pollen. Afin de déterminer si l’introduction d’un sucre dans la région HVa de la S11-RNase pourrait affecter le rejet pollinique, nous avons généré un second mutant comportant un site additionnel de glycosylation dans la région HVa et une troisième construction qui comporte elle aussi ce nouveau site mais dont le site en région C2 fût éliminé. Le mutant comportant deux sites de glycosylation se comporte de manière semblable à une S11-RNase de type sauvage mais, de manière surprenante, le mutant uniquement glycosylé en région HVa peut aussi rejeter le pollen d’haplotype S13. Nous proposons que la forme non glycosylée de ce mutant constitue un allèle à double spécificité, semblable à un autre allèle à double spécificité préalablement décrit. Il est intéressant de noter que puisque ce phénotype n’est pas observé dans le mutant comportant deux sites de glycosylation, cela suggère que les S-RNases ne sont pas déglycosylées à l’intérieur du pollen. Dans la dernière étude, nous avons réalisé plusieurs expériences d’interactions protéine-protéine afin d’identifier de potentiels interactants polliniques avec les S-RNases. Nous avons démontré que eEF1A, un composant de la machinerie de traduction chez les eucaryotes, peut lier une S11-RNase immobilisée sur résine concanavaline A. Des analyses de type pull-down utilisant la protéine eEF1A de S. chacoense étiquetée avec GST confirment cette interaction. Nous avons aussi montré que la liaison, préalablement constatée, entre eEF1A et l’actine est stimulée en présence de la S11-RNase, bien que cette dernière ne puisse directement lier l’actine. Enfin, nous avons constaté que dans les tubes polliniques incompatibles, l’actine adopte une structure agrégée qui co-localise avec les S-RNases. Ces résultats suggèrent que la liaison entre eEF1A et les S-RNases pourrait constituer un potentiel lien fonctionnel entre les S-RNases et l’altération du cytosquelette d’actine observée lors des réactions d’AI. Par ailleurs, si cette liaison est en mesure de titrer les S-RNases disponibles à l’intérieur du tube pollinique, ce mécanisme pourrait expliquer pourquoi des quantités minimales ou « seuils » de S-RNases sont nécessaires au déclenchement des réactions d’AI.Self-incompatibility (SI) is a prezygotic reproductive barrier that allows the pistil of a flower to specifically reject their own (self-) pollen. SI systems can help prevent self-fertilization and avoid inbreeding. In gametophytic SI (GSI), the genotype of the pollen determines its breeding behaviour and in this system both female and male specificity determinants of SI are under the control of a multigenic and multiallelic locus called the S-locus. In Solanaceae, the female determinant of SI is a highly polymorphic stylar-expressed extracellular glycoprotein with RNase activity called the S-RNase. S-RNases show a distinct pattern of two hypervariable (HVa and HVb) regions, responsible for their allelic specificity, and five highly conserved regions (C1 to C5) thought to be involved in either the catalytic activity or the structural stabilization of the protein. In this work, we analyzed and characterized several conserved features of the S-RNases and also identified a potential novel S-RNase interactant in Solanum chacoense. The aim of our first study was to investigate the role of the C4 region of S-RNases. To test the hypothesis that the C4 region may be involved in S-RNase folding or stability, we examined a mutant in which the four charged residues in the C4 region were replaced with glycine. This mutant did not accumulate to detectable levels in styles, supporting a structural role for C4. To test the possibility that C4 might be involved in binding another protein, we prepared an R115G mutant, in which a charged amino acid was eliminated to reduce any potential binding to this region. This mutant had no effect on the pollen rejection phenotype of the protein, and thus C4 is likely not involved in either ligand binding or S-RNase entry inside pollen tubes. Finally, a K113R mutant, in which the only conserved lysine residue in all the S-RNases was replaced with arginine, was generated to test if this residue was an S-RNase ubiquitination site. However, S-RNase degradation was not disrupted in this mutant. Taken together, these results indicate that the C4 region likely plays a structural role. In a second study, we analyzed the role of S-RNase glycosylation. All S-RNases share a conserved glycosylation site in the C2 region. To test the possibility that the sugar residues might be a target for ubiquitination, a transgenic S11-RNase lacking its single glycosylation site was examined. This construct behaved similarly to a wild type S11-RNase, demonstrating that the lack of glycosylation does not confer constitutive pollen rejection. To determine if the introduction of an N-linked glycan in the HVa region would affect pollen rejection, a construct containing a second N-glycosylation site inside the HVa region of the S11-RNase and a construct containing only that N-glycosylation site inside the HVa region were prepared. The first construct rejected S11 pollen normally, but surprisingly, plants expressing the construct lacking the C2 glycosylation site rejected both S11 and S13 pollen. We propose that the non-glycosylated form is a dual specific allele, similar to a previously described dual-specific allele that also had amino acid replacements in the HV regions. Interestingly, this phenotype is not observed in the mutant containing two glycosylation sites, which suggests that the sugar residues are not removed during S-RNase entry into the pollen. In the final study, S-RNase-binding assays were performed with pollen extracts to detect potential interacting proteins. We found that concanavalin A-immobilized S11-RNase bound eEF1A, a component of the eukaryotic translational machinery. This interaction was validated by pull-down experiments using a GST-tagged S. chacoense eEF1A. We also found that a previously documented actin binding to eEF1A was markedly increased in the presence of S-RNases, although S-RNases alone do not bind actin. Lastly, we observed that actin in incompatible pollen tubes has an unusual aggregated form which also co-labels with S-RNases. This suggests that binding between S-RNases and eEF1A could provide a potential functional link between the S-RNase and the alteration of the actin cytoskeleton that occurs during the SI reaction. Furthermore, if eEF1A binding to S-RNases acted to titrate the amount of free S-RNase in the pollen tube, this binding may help explain the threshold phenomenon, where a minimum quantity of S-RNase in the style is required to trigger the SI reaction

Open data from the third observing run of LIGO, Virgo, KAGRA and GEO

The global network of gravitational-wave observatories now includes five detectors, namely LIGO Hanford, LIGO Livingston, Virgo, KAGRA, and GEO 600. These detectors collected data during their third observing run, O3, composed of three phases: O3a starting in April of 2019 and lasting six months, O3b starting in November of 2019 and lasting five months, and O3GK starting in April of 2020 and lasting 2 weeks. In this paper we describe these data and various other science products that can be freely accessed through the Gravitational Wave Open Science Center at https://gwosc.org. The main dataset, consisting of the gravitational-wave strain time series that contains the astrophysical signals, is released together with supporting data useful for their analysis and documentation, tutorials, as well as analysis software packages.Comment: 27 pages, 3 figure

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM

eEF1A Is an S-RNase Binding Factor in Self-Incompatible <i>Solanum chacoense</i>

<div><p>Self-incompatibility (SI) is a genetic mechanism that allows flowering plants to identify and block fertilization by self-pollen. In the Solanaceae, SI is controlled by a multiallelic <i>S</i>-locus encoding both S-RNases and F-box proteins as female and male determinants, respectively. S-RNase activity is essential for pollen rejection, and a minimum threshold value of S-RNases in the style is also required. Here we present biochemical evidence that eEF1A is a novel S-RNase-binding partner <i>in vitro</i>. We further show that the normal actin binding activity of eEF1A is enhanced by the presence of S-RNase. Lastly, we find that there is a co-localization of S-RNase and actin in the incompatible pollen tubes in structures reminiscent of the actin bundles formed by eEF1A. We propose that increased binding of eEF1A to actin in the presence of S-RNase could help explain the disruption of the actin cytoskeleton observed during SI reactions.</p></div

eEF1A binding to actin is increased by S₁₁-RNase.

<p>(A) A GST-eEF1A fusion protein immobilized on GST resin was mixed with known amounts of purified S₁₁-RNase and/or a commercial bovine actin. GST-eEF1A binds similar amounts of S₁₁-RNase whether actin is present or not but the amount of actin bound by eEF1A increases markedly in the presence of S₁₁-RNase. (B) A GST protein control binds neither S₁₁-RNase nor actin.</p

Regions of intense actin staining in incompatible pollen tubes also stain with S₁₁-RNase.

<p>Incompatible pollen tubes 18(A) and 24 h (B) post-pollination stained simultaneously with anti-actin (5 nm colloidal gold, black arrows) and anti-S11-RNase (20 nm colloidal gold, white arrows). Scale bars are 0.1 µm.</p

Protein purification schema and proteins identified in the final eluate.

<p>The stylar S-RNase was purified by ConA and Resource S (ion exchange) chromatography before being immobilized on ConA beads. Pollen proteins, depleted of proteins binding non-specifically to the ConA resin, were applied to the immobilized S-RNase, washed and eluted with ConA elution buffer. Only three proteins were detected in the specific eluate by LC-MS/MS.</p