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Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases, and is known to hydrolyze the bond between the ABC lactone and the D ring. Here we characterized the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using new profluorescent probes with strigolactone-like bioactivity, we found that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We demonstrated the formation of a covalent RMS3-D-ring complex, essential for bioactivity, in which the D ring was attached to histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception in which the receptor performs an irreversible enzymatic reaction to generate its own ligand.

Uncovering the basis of small molecule hormone receptors evolution is paramount to a complete understanding of how protein structure drives function. In plants, hormone receptors for strigolactones are well suited to evolutionary inquires because closely related homologs have different ligand preferences. More importantly, because of facile plant transgenic systems, receptors can be swapped and quickly assessed functionally in vivo. Here, we show only three mutations are required to switch the non-strigolactone receptor, KAI2, into a strigolactone receptor. This modified receptor still perceives KAI2 ligands and does not require receptor hydrolysis for activity. Structural and molecular dynamic modeling suggest receptor pocket flexibility is important for ligand specificity and downstream signaling partner affinity. These findings indicate a few keystone mutations link strigolactone signaling to germination, which explains how parasitic plants that devastate African agriculture evolved SL receptors to sense the presence of a host plant.

Respiratory syncytial virus (RSV) RNA synthesis occurs in cytoplasmic inclusion bodies (IBs) in which all the components of the viral RNA polymerase are concentrated. In this work, we show that RSV P protein recruits the essential RSV transcription factor M2-1 to IBs independently of the phosphorylation state of M2-1. We also show that M2-1 dephosphorylation is achieved by a complex formed between P and the cellular phosphatase PP1. We identified the PP1 binding site of P, which is an RVxF-like motif located nearby and upstream of the M2-1 binding region. NMR confirmed both P-M2-1 and P-PP1 interaction regions in P. When the P–PP1 interaction was disrupted, M2-1 remained phosphorylated and viral transcription was impaired, showing that M2-1 dephosphorylation is required, in a cyclic manner, for efficient viral transcription. IBs contain substructures called inclusion bodies associated granules (IBAGs), where M2-1 and neo-synthesized viral mRNAs concentrate. Disruption of the P–PP1 interaction was correlated with M2-1 exclusion from IBAGs, indicating that only dephosphorylated M2-1 is competent for viral mRNA binding and hence for a previously proposed post-transcriptional function.

Glutathione contributes to thiol-redox control and to extra-mitochondrial iron-sulphur cluster (ISC) maturation. To determine the physiological importance of these functions and sort out those that account for the GSH requirement for viability, we performed a comprehensive analysis of yeast cells depleted of or containing toxic levels of GSH. Both conditions triggered an intense iron starvation-like response and impaired the activity of extra-mitochondrial ISC enzymes but did not impact thiol-redox maintenance, except for high glutathione levels that altered oxidative protein folding in the endoplasmic reticulum. While iron partially rescued the ISC maturation and growth defects of GSH-depleted cells, genetic experiments indicated that unlike thioredoxin, glutathione could not support by itself the thiol-redox duties of the cell. We propose that glutathione is essential by its requirement in ISC assembly, but only serves as a thioredoxin backup in cytosolic thiol-redox maintenance. Glutathione-high physiological levels are thus meant to insulate its cytosolic function in iron metabolism from variations of its concentration during redox stresses, a model challenging the traditional view of it as prime actor in thiol-redox control.

Summary The role of histone 3 lysine 4 (H3K4) methylation is poorly understood in plant pathogenic fungi. Here, we analysed the function of CclA, a subunit of the COMPASS complex mediating H3K4 methylation, in the brassica anthracnose pathogen Colletotrichum higginsianum. We show that CclA is required for full genome‐wide H3K4 trimethylation. The deletion of cclA strongly reduced mycelial growth, asexual sporulation and spore germination but did not impair the morphogenesis of specialized infection structures (appressoria and biotrophic hyphae). Virulence of the ΔcclA mutant on plants was strongly attenuated, associated with a marked reduction in appressorial penetration ability on both plants and inert cellophane membranes. The secondary metabolite profile of the ΔcclA mutant was greatly enriched compared to that of the wild type, with three different families of terpenoid compounds being overproduced by the mutant, namely the colletochlorins, higginsianins and sclerosporide. These included five novel molecules that were produced exclusively by the ΔcclA mutant: colletorin D, colletorin D acid, higginsianin C, 13‐epi‐higginsianin C and sclerosporide. Taken together, our findings indicate that H3K4 trimethylation plays a critical role in regulating fungal growth, development, pathogenicity and secondary metabolism in C. higginsianum.

The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe241 residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries.

Adventitious root formation is essential for cutting propagation of diverse species; however, until recently little was known about its regulation. Strigolactones and ethylene have both been shown to inhibit adventitious roots and it has been suggested that ethylene interacts with strigolactones in root hair elongation. We have investigated the interaction between strigolactones and ethylene in regulating adventitious root formation in intact seedlings of Arabidopsis thaliana. We used strigolactone mutants together with 1-aminocyclopropane-1-carboxylic acid (ACC) (ethylene precursor) treatments and ethylene mutants together with GR24 (strigolactone agonist) treatments. Importantly, we conducted a detailed mapping of adventitious root initiation along the hypocotyl and measured ethylene production in strigolactone mutants. ACC treatments resulted in a slight increase in adventitious root formation at low doses and a decrease at higher doses, in both wild-type and strigolactone mutants. Furthermore, the distribution of adventitious roots dramatically changed to the top third of the hypocotyl in a dose-dependent manner with ACC treatments in both wild-type and strigolactone mutants. The ethylene mutants all responded to treatments with GR24. Wild type and max4 (strigolactone-deficient mutant) produced the same amount of ethylene, while emanation from max2 (strigolactone-insensitive mutant) was lower. We conclude that strigolactones and ethylene act largely independently in regulating adventitious root formation with ethylene controlling the distribution of root initiation sites. This role for ethylene may have implications for flood response because both ethylene and adventitious root development are crucial for flood tolerance.

Strigolactones (SLs) are intriguing phytohormones that not only regulate plant development and architecture but also interact with other organisms in the rhizosphere as root parasitic plants ( Striga, Orobanche , and Phelipanche ) and arbuscular mycorrhizal fungi. Starting with a pioneering work in 2003 for the isolation and identification of the SL receptor in parasitic weeds, fluorescence labeling of analogs has proven a major strategy to gain knowledge in SL perception and signaling. Here, we present novel chemical tools for understanding the SL perception based on the enzymatic properties of SL receptors. We designed different profluorescent SL Guillaume Clavé (GC) probes and performed structure-activity relationship studies on pea, Arabidopsis thaliana , and Physcomitrium (formerly Physcomitrella ) patens . The binding of the GC probes to PsD14/RMS3, AtD14, and OsD14 proteins was tested. We demonstrated that coumarin-based profluorescent probes were highly bioactive and well-adapted to dissect the enzymatic properties of SL receptors in pea and a resorufin profluorescent probe in moss, contrary to the commercially available fluorescein profluorescent probe, Yoshimulactone Green (YLG). These probes offer novel opportunities for the studies of SL in various plants.

Pneumoviruses include pathogenic human and animal viruses, the most known and studied being the human respiratory syncytial virus (hRSV) and the metapneumovirus (hMPV), which are the major cause of severe acute respiratory tract illness in young children worldwide, and main pathogens infecting elderly and immune-compromised people. The transcription and replication of these viruses take place in specific cytoplasmic inclusions called inclusion bodies (IBs). These activities depend on viral polymerase L, associated with its cofactor phosphoprotein P, for the recognition of the viral RNA genome encapsidated by the nucleoprotein N, forming the nucleocapsid (NC). The polymerase activities rely on diverse transient protein-protein interactions orchestrated by P playing the hub role. Among these interactions, P interacts with the NC to recruit L to the genome. The P protein also plays the role of chaperone to maintain the neosynthesized N monomeric and RNA-free (called N0) before specific encapsidation of the viral genome and antigenome. This review aims at giving an overview of recent structural information obtained for hRSV and hMPV P, N, and more specifically for P-NC and N0-P complexes that pave the way for the rational design of new antivirals against those viruses.

Secondary metabolites are essential for plant survival and reproduction. Wild undomesticated and tropical plants are expected to harbor highly diverse metabolomes. We investigated the metabolomic diversity of two morphologically similar trees of tropical Africa, Erythrophleum suaveolens and E. ivorense, known for particular secondary metabolites named the cassaine-type diterpenoids. To assess how the metabolome varies between and within species, we sampled leaves from individuals of different geographic origins but grown from seeds in a common garden in Cameroon. Metabolites were analyzed using reversed phase LC-HRMS(/MS). Data were interpreted by untargeted metabolomics and molecular networks based on MS/MS data. Multivariate analyses enabled us to cluster samples based on species but also on geographic origins. We identified the structures of 28 cassaine-type diterpenoids among which 19 were new, 10 were largely specific to E. ivorense and five to E. suaveolens. Our results showed that the metabolome allows an unequivocal distinction of morphologically-close species, suggesting the potential of metabolite fingerprinting for these species. Plant geographic origin had a significant influence on relative concentrations of metabolites with variations up to eight (suaveolens) and 30 times (ivorense) between origins of the same species. This shows that the metabolome is strongly influenced by the geographical origin of plants (i.e., genetic factors).