Cellular Electron Microscopy Imaging Reveals the Localization of the Hfq Protein Close to the Bacterial Membrane

Background: Hfq is a bacterial protein involved in several aspects of nucleic acid transactions, but one of its bestcharacterized functions is to affect the post-transcriptional regulation of mRNA by virtue of its interactions with stressrelated small regulatory (sRNA). Methodology and Principal Finding: By using cellular imaging based on the metallothionein clonable tag for electron microscopy, we demonstrate here that in addition to its localization in the cytoplasm and in the nucleoid, a significant amount of Hfq protein is located at the cell periphery. Simultaneous immunogold detection of specific markers strongly suggests that peripheral Hfq is close to the bacterial membrane. Because sRNAs regulate the synthesis of several membrane proteins, our result implies that the sRNA- and Hfq-dependent translational regulation of these proteins takes place in the cytoplasmic region underlying the membrane. Conclusions: This finding supports the proposal that RNA processing and translational machineries dedicated to membrane protein translation may often be located in close proximity to the membrane of the bacterial cell

Several wall-associated kinases participate positively and negatively in basal defense against rice blast fungus.

BACKGROUND: Receptor-like kinases are well-known to play key roles in disease resistance. Among them, the Wall-associated kinases (WAKs) have been shown to be positive regulators of fungal disease resistance in several plant species. WAK genes are often transcriptionally regulated during infection but the pathways involved in this regulation are not known. In rice, the OsWAK gene family is significantly amplified compared to Arabidopsis. The possibility that several WAKs participate in different ways to basal defense has not been addressed. Moreover, the direct requirement of rice OSWAK genes in regulating defense has not been explored. RESULTS: Here we show using rice (Oryza sativa) loss-of-function mutants of four selected OsWAK genes, that individual OsWAKs are required for quantitative resistance to the rice blast fungus, Magnaporthe oryzae. While OsWAK14, OsWAK91 and OsWAK92 positively regulate quantitative resistance, OsWAK112d is a negative regulator of blast resistance. In addition, we show that the very early transcriptional regulation of the rice OsWAK genes is triggered by chitin and is partially under the control of the chitin receptor CEBiP. Finally, we show that OsWAK91 is required for H2O2 production and sufficient to enhance defense gene expression during infection. CONCLUSIONS: We conclude that the rice OsWAK genes studied are part of basal defense response, potentially mediated by chitin from fungal cell walls. This work also shows that some OsWAKs, like OsWAK112d, may act as negative regulators of disease resistance

Culture de jeunes plants au port dressé : un support imprimé en 3D pour petits cubes de laine de roche

National audienceThe Mechanism of Plant Virus Transmission by Vector team (MOVE team) has long experience of rearing aphids, in particular the pea aphid species Acyrthosiphon pisum. These aphids are generally reared on faba beans in small pots of soil. However, the appearance of soil sciarid flies during rearing led us to opt for an inert substrate: rockwool. However, the instability of rockwool cubes made it difficult to grow upright young plants such as broad beans. We could not find any individual tutors for our rockwool cubes on the market, so we designed our own 3D printable tutors. Robust, inexpensive, and infinitely reusable, this support, which has been in use in our team for 3 years, offers a solution to anyone looking for an alternative to growing in small soil pots (3D plans attached for free access).L’équipe Mécanismes de transmission des virus de plante par vecteur (MOVE) a une longue expérience dans l’élevage de pucerons Acyrtosiphon pisum ou pucerons du pois. Ces pucerons sont généralement élevés sur fève en petit pot de terreau. L’apparition de mouches terreau au cours des élevages nous a conduits à opter pour un substrat inerte : la laine de roche. Toutefois, le manque de stabilité statique des cubes de laine de roche a rendu difficile la culture de jeunes plants au port dressé, comme les fèves. N’ayant pas trouvé de supports individuels adaptés à nos cubes de laine de roche dans le commerce, nous avons développé notre propre support imprimable en 3 D. Robuste, peu coûteux et réutilisable à souhait, ce support, utilisé dans notre équipe depuis trois ans, offre une solution à quiconque cherchant une alternative à la culture en petit pot de terreau (plans 3D joints en accès libre)

Coupling clearing and Hybridization Chain Reaction approaches to investigate gene expression in organs inside whole-mount intact insect.

Detecting RNA molecules within their natural environment inside intact arthropods has long been challenging, particularly in small organisms covered by a tanned and pigmented cuticle. Here, we have developed a methodology that enables high-resolution analysis of the spatial distribution of transcripts of interest without having to dissect tiny organs or tissues, thereby preserving their integrity. We have combined an in situ amplification approach based on Hybridization Chain Reaction, which enhances the signal-to-noise ratio, and a clearing approach that allows the visualization of inner organs beneath the cuticle. We have implemented this methodology for the first time in Hemiptera, mapping two salivary aphid ( Acyrthosiphon pisum ) transcripts, the effector c002 and the salivary sheath protein SHP. With a multiplex approach, we could simultaneously detect different mRNAs in whole-mount pea aphid head-thorax samples and show that they were distributed in distinct secretory cells of salivary glands

Coupling clearing and hybridization chain reaction approaches to investigate gene expression in organs inside intact insect heads

International audienceDetecting RNA molecules within their natural environment inside intact arthropods has long been challenging, particularly in small organisms covered by a tanned and pigmented cuticle. Here, we have developed a methodology that enables high‐resolution analysis of the spatial distribution of transcripts of interest without having to dissect tiny organs or tissues, thereby preserving their integrity. We have combined an in situ amplification approach based on hybridization chain reaction, which enhances the signal‐to‐noise ratio, and a clearing approach that allows the visualization of inner organs beneath the cuticle. We have implemented this methodology for the first time in Hemiptera, mapping two salivary aphid ( Acyrthosiphon pisum ) transcripts, the effector c002 and the salivary sheath protein SHP. With a multiplex approach, we could simultaneously detect different mRNAs in mounted pea aphid head‐thorax samples and show that they were distributed in distinct secretory cells of salivary glands. Research Highlights Combining hybridisation chain reaction and clearing allows the detection of transcripts in intact aphids heads. The transcripts of the two salivary proteins c002 and SHP are compartmentalized in distinct secretory cells of the principal glands

Synchrotron X-ray micro-tomography: an approach to unravel the biogenesis of aphid stylets

International audienceAphids are serious agricultural pests capable of making direct damages to crops and, more importantly, of transmitting hundreds of species of pathogens that cause considerable losses worldwide. Their piercing-sucking mouthparts are well-adapted structures to pierce plant tissues and uptake sap and nutrients from their host plants. They consist of four long needle-like cuticular stylets, two outer mandibles and two inner maxillae synthesized at each molt by specialized cuticular glands known as the Retort Organs (ROs). Our work has shown that proteins are not uniformly distributed in the stylets conferring local functionalization of cuticular micro-territories such as the acrostyle at the tip of maxillary stylets with stylin proteins emerging on its surface involved in virus binding. To better understand when and how the acrostyle is synthesized, we decided to explore the biogenesis of the stylets throughout the preimaginal stage of the aphid species Acyrthosiphon pisum. In this study, we used X-ray synchrotron-based micro-tomography, a non-destructive approach allowing the reconstruction of highly-resolved 3D images of internal organs. Datasets were collected at the synchrotron facility of the Paul Scherrer Institute (Villigen, CH) on synchronized aphids sampled at key time points of the 4th instar nymph and adult stages. This comparative analysis highlighted morphological and volumetric changes of the ROs during aphid development and cuticle secretion and provide an unprecedented level of detail on the complex biological process of de novo stylet synthesis

Voyage dans la tête d’un puceron. Episode 2: détecter des transcrits dans des insects transparisés.

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