Microbiota of pest insect Nezara viridula mediate detoxification and plant defense repression.

The Southern green shield bug, Nezara viridula, is an invasive piercing and sucking pest insect that feeds on crop plants and poses a threat to global food production. Given that insects are known to live in a close relationship with microorganisms, our study provides insights into the community composition and function of the N. viridula-associated microbiota and its effect on host-plant interactions. We discovered that N. viridula hosts both vertically and horizontally transmitted microbiota throughout different developmental stages and their salivary glands harbor a thriving microbial community that is transmitted to the plant while feeding. The N. viridula microbiota was shown to aid its host with the detoxification of a plant metabolite, namely 3-nitropropionic acid, and repression of host plant defenses. Our results demonstrate that the N. viridula-associated microbiota plays an important role in interactions between insects and plants and could therefore be considered a valuable target for the development of sustainable pest control strategies.

SecYEG-mediated translocation in a model synthetic cell.

Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow for transport, energy transduction, compartment growth and division. Here, we have successfully incorporated the membrane protein complex SecYEG-the key bacterial translocase that is essential for the incorporation of newly synthesized membrane proteins-in GUVs. Our method consists of fusion of small unilamellar vesicles containing reconstituted SecYEG into GUVs, thereby forming SecGUVs. These are suitable for large-scale experiments while maintaining a high protein:lipid ratio. We demonstrate that incorporation of SecYEG into GUVs does not inhibit its translocation efficiency. Robust membrane protein functionalized proteo-GUVs are promising and flexible compartments for use in the formation and growth of synthetic cells.

Conductive Polyisocyanide Hydrogels Inhibit Fibrosis and Promote Myogenesis.

Reliable in vitro models closely resembling native tissue are urgently needed for disease modeling and drug screening applications. Recently, conductive biomaterials have received increasing attention in the development of in vitro models as they permit exogenous electrical signals to guide cells toward a desired cellular response. Interestingly, they have demonstrated that they promote cellular proliferation and adhesion even without external electrical stimulation. This paper describes the development of a conductive, fully synthetic hydrogel based on hybrids of the peptide-modified polyisocyanide (PIC-RGD) and the relatively conductive poly(aniline-co-N-(4-sulfophenyl)aniline) (PASA) and its suitability as the in vitro matrix. We demonstrate that incorporating PASA enhances the PIC-RGD hydrogel's electroactive nature without significantly altering the fibrous architecture and nonlinear mechanics of the PIC-RGD network. The biocompatibility of our model was assessed through phenotyping cultured human foreskin fibroblasts (HFF) and murine C2C12 myoblasts. Immunofluorescence analysis revealed that PIC-PASA hydrogels inhibit the fibrotic behavior of HFFs while promoting myogenesis in C2C12 cells without electrical stimulation. The composite PIC-PASA hydrogel can actively change the cell fate of different cell types, providing an attractive tool to improve skin and muscle repair.

Mechanisms of extracellular electron transfer in anaerobic methanotrophic archaea.

Anaerobic methanotrophic (ANME) archaea are environmentally important, uncultivated microorganisms that oxidize the potent greenhouse gas methane. During methane oxidation, ANME archaea engage in extracellular electron transfer (EET) with other microbes, metal oxides, and electrodes through unclear mechanisms. Here, we cultivate ANME-2d archaea ('Ca. Methanoperedens') in bioelectrochemical systems and observe strong methane-dependent current (91-93% of total current) associated with high enrichment of 'Ca. Methanoperedens' on the anode (up to 82% of the community), as determined by metagenomics and transmission electron microscopy. Electrochemical and metatranscriptomic analyses suggest that the EET mechanism is similar at various electrode potentials, with the possible involvement of an uncharacterized short-range electron transport protein complex and OmcZ nanowires.

Requirement of ß1 integrin for endothelium-dependent vasodilation and collateral formation in hindlimb ischemia.

An acute increase in blood flow triggers flow-mediated dilation (FMD), which is mainly mediated by endothelial nitric oxide synthase (eNOS). A long-term increase in blood flow chronically enlarges the arterial lumen, a process called arteriogenesis. In several common human diseases, these processes are disrupted for as yet unknown reasons. Here, we asked whether ß1 integrin, a mechanosensory protein in endothelial cells, is required for FMD and arteriogenesis in the ischemic hindlimb. Permanent ligation of the femoral artery in C57BL/6 J mice enlarged pre-existing collateral arteries and increased numbers of arterioles in the thigh. In the lower leg, the numbers of capillaries increased. Notably, injection of ß1 integrin-blocking antibody or tamoxifen-induced endothelial cell-specific deletion of the gene for ß1 integrin (Itgb1) inhibited both arteriogenesis and angiogenesis. Using high frequency ultrasound, we demonstrated that ß1 integrin-blocking antibody or endothelial cell-specific depletion of ß1 integrin attenuated FMD of the femoral artery, and blocking of ß1 integrin function did not further decrease FMD in eNOS-deficient mice. Our data suggest that endothelial ß1 integrin is required for both acute and chronic widening of the arterial lumen in response to hindlimb ischemia, potentially via functional interaction with eNOS.

Kinase activity of SOBIR1 and BAK1 is required for immune signalling.

Leucine-rich repeat-receptor-like proteins (LRR-RLPs) and LRR-receptor-like kinases (LRR-RLKs) trigger immune signalling to promote plant resistance against pathogens. LRR-RLPs lack an intracellular kinase domain, and several of these receptors have been shown to constitutively interact with the LRR-RLK Suppressor of BIR1-1/EVERSHED (SOBIR1/EVR) to form signalling-competent receptor complexes. Ligand perception by LRR-RLPs initiates recruitment of the co-receptor BRI1-Associated Kinase 1/Somatic Embryogenesis Receptor Kinase 3 (BAK1/SERK3) to the LRR-RLP/SOBIR1 complex, thereby activating LRR-RLP-mediated immunity. We employed phosphorylation analysis of in planta-produced proteins, live cell imaging, gene silencing and co-immunoprecipitation to investigate the roles of SOBIR1 and BAK1 in immune signalling. We show that Arabidopsis thaliana (At) SOBIR1, which constitutively activates immune responses when overexpressed in planta, is highly phosphorylated. Moreover, in addition to the kinase activity of SOBIR1 itself, kinase-active BAK1 is essential for AtSOBIR1-induced constitutive immunity and for the phosphorylation of AtSOBIR1. Furthermore, the defence response triggered by the tomato LRR-RLP Cf-4 on perception of Avr4 from the extracellular pathogenic fungus Cladosporium fulvum is dependent on kinase-active BAK1. We argue that, in addition to the trans-autophosphorylation of SOBIR1, it is likely that SOBIR1 and BAK1 transphosphorylate, and thereby activate the receptor complex. The signalling-competent cell surface receptor complex subsequently activates downstream cytoplasmic signalling partners to initiate RLP-mediated immunity.

Avr4 promotes Cf-4 receptor-like protein association with the BAK1/SERK3 receptor-like kinase to initiate receptor endocytosis and plant immunity.

The first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the SUPPRESSOR OF BIR1 (SOBIR1) RLK contributes to RLP stability and kinase activity. As RLK activation requires transphosphorylation with a second associated RLK, it remains elusive how RLPs initiate downstream signaling. We employed live-cell imaging, gene silencing and coimmunoprecipitation to investigate the requirement of associated kinases for functioning and ligand-induced subcellular trafficking of Cf RLPs that mediate immunity of tomato against Cladosporium fulvum. Our research shows that after elicitation with matching effector ligands Avr4 and Avr9, BRI1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) associates with Cf-4 and Cf-9. BAK1/SERK3 is required for the effector-triggered hypersensitive response and resistance of tomato against C. fulvum. Furthermore, Cf-4 interacts with SOBIR1 at the plasma membrane and is recruited to late endosomes upon Avr4 trigger, also depending on BAK1/SERK3. These observations indicate that RLP-mediated resistance and endocytosis require ligand-induced recruitment of BAK1/SERK3, reminiscent of BAK1/SERK3 interaction and subcellular fate of the FLAGELLIN SENSING 2 (FLS2) RLK. This reveals that diverse classes of cell surface immune receptors share common requirements for initiation of resistance and endocytosis.

SOBIR1 requires the GxxxG dimerization motif in its transmembrane domain to form constitutive complexes with receptor-like proteins.

Receptor-like proteins (RLPs), forming an important group of transmembrane receptors in plants, play roles in development and immunity. RLPs contain extracellular leucine-rich repeats (LRRs) and, in contrast with receptor-like kinases (RLKs), lack a cytoplasmic kinase required for the initiation of downstream signalling. Recent studies have revealed that the RLK SOBIR1/EVR (SUPPRESSOR OF BIR1-1/EVERSHED) specifically interacts with RLPs. SOBIR1 stabilizes RLPs and is required for their function. However, the mechanism by which SOBIR1 associates with RLPs and regulates RLP function remains unknown. The Cf immune receptors of tomato (Solanum lycopersicum), mediating resistance to the fungus Cladosporium fulvum, are RLPs that also interact with SOBIR1. Here, we show that both the LRR and kinase domain of SOBIR1 are dispensable for association with the RLP Cf-4, whereas the highly conserved GxxxGxxxG motif present in the transmembrane domain of SOBIR1 is essential for its interaction with Cf-4 and additional RLPs. Complementation assays in Nicotiana benthamiana, in which endogenous SOBIR1 levels were knocked down by virus-induced gene silencing, showed that the LRR domain as well as the kinase activity of SOBIR1 are required for the Cf-4/Avr4-triggered hypersensitive response (HR). In contrast, the LRRs and kinase activity of SOBIR1 are not required for facilitation of Cf-4 accumulation. Together, these results suggest that, in addition to being a stabilizing scaffold for RLPs, SOBIR1 is also required for the initiation of downstream signalling through its kinase domain.

A moving view: subcellular trafficking processes in pattern recognition receptor-triggered plant immunity.

A significant challenge for plants is to induce localized defense responses at sites of pathogen attack. Therefore, host subcellular trafficking processes enable accumulation and exchange of defense compounds, which contributes to the plant on-site defenses in response to pathogen perception. This review summarizes our current understanding of the transport processes that facilitate immunity, the significance of which is highlighted by pathogens reprogramming membrane trafficking through host cell translocated effectors. Prominent immune-related cargos of plant trafficking pathways are the pattern recognition receptors (PRRs), which must be present at the plasma membrane to sense microbes in the apoplast. We focus on the dynamic localization of the FLS2 receptor and discuss the pathways that regulate receptor transport within the cell and their link to FLS2-mediated immunity. One emerging theme is that ligand-induced late endocytic trafficking is conserved across different PRR protein families as well as across different plant species.