1Cell-autonomous immunity is widespread in plant-fungus interactions and terminates fungal pathogenesis either at the cell surface or after pathogen entry. Although post-invasive resistance responses typically coincide with a self-contained cell death of plant cells undergoing attack by parasites, these cells survive pre-invasive defence. Mutational analysis in Arabidopsis identified PEN1 syntaxin as one component of two pre-invasive resistance pathways against ascomycete powdery mildew fungi 1-3 . Here we show that plasma-membrane-resident PEN1 promiscuously forms SDS-resistant soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes together with the SNAP33 adaptor and a subset of vesicle-associated membrane proteins (VAMPs). PEN1-dependent disease resistance acts in vivo mainly through two functionally redundant VAMP72 subfamily members, VAMP721 and VAMP722. Unexpectedly, the same two VAMP proteins also operate redundantly in a default secretory pathway, suggesting dual functions in separate biological processes owing to evolutionary co-option of the default pathway for plant immunity. The disease resistance function of the secretory PEN1-SNAP33-VAMP721/722 complex and the pathogen-induced subcellular dynamics of its components are mechanistically reminiscent of immunological synapse formation in vertebrates, enabling execution of immune responses through focal secretion.Arabidopsis is immune to non-adapted powdery mildew fungi such as Blumeria graminis and Erysiphe pisi, which in nature colonize grass and pea species, respectively. This non-host resistance requires both pre-and post-invasive immune responses, which are under separate genetic control 2 . The former response engages PEN1 syntaxin, peroxisomal PEN2 b-glycosyl hydrolase and the plasmamembrane-resident PEN3 ABC transporter 1-3 . PEN2 and PEN3 act in the same pathway and are implicated in the cytoplasmic synthesis and transport of small antimicrobial compounds across the plasma membrane at attempted fungal entry sites, respectively 2,3 . PEN1 syntaxin acts in a second pathway and could, by analogy to known syntaxin functions in yeast and animals, either participate in vesicle fusion processes 4 or modulate ion-channel activity through interactions with plasma-membrane-resident ion channels 5 . Genetic studies defy mechanistic interpretations but suggest direct or indirect PEN1 repressor activity in defence responses that are dependent on salicylic acid, as well as an overlapping function with the closely related syntaxin of plant 122 (SYP122) 6 . Compared with largely resistant PEN1 wild type and severely defence-compromised pen1-1 null mutants, plants containing the pen1-3 allele allow intermediate B. graminis entry rates, indicating residual PEN1-3 resistance activity ( Supplementary Fig. 1a). In the deduced PEN1-3 protein, a glycine residue is substituted by a glutamate in the SNARE domain 1 (Supplementary Fig. 1b). Because this mutation affects a hydrophobic residue that is thought to stabilize interactions with ...

β-Coronaviruses are a family of positive-strand enveloped RNA viruses that include the severe acute respiratory syndrome-CoV2 (SARS-CoV2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that β-Coronaviruses utilize lysosomal trafficking for egress, rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of β-Coronavirus results in lysosome deacidification, inactivation of lysosomal degradation enzymes and disruption of antigen presentation pathways. The β−coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

Ebola virus disease is a serious global health concern given its periodic occurrence, high lethality, and the lack of approved therapeutics. Certain drugs that alter intracellular calcium, particularly in endolysosomes, have been shown to inhibit Ebola virus infection; however, the underlying mechanism is unknown. Here, we provide evidence that Zaire ebolavirus (EBOV) infection is promoted in the presence of calcium as a result of the direct interaction of calcium with the EBOV fusion peptide (FP). We identify the glycoprotein residues D522 and E540 in the FP as functionally critical to EBOV's interaction with calcium. We show using spectroscopic and biophysical assays that interactions of the fusion peptide with Ca 2+ ions lead to lipid ordering in the host membrane during membrane fusion, and these changes are promoted at low pH and can be correlated with infectivity. We further demonstrate using circular dichroism spectroscopy that calcium interaction with the fusion peptide promotes α-helical structure of the fusion peptide, a conformational change that enhances membrane fusion, as validated using functional assays of membrane fusion. This study shows that calcium directly targets the Ebola virus fusion peptide and influences its conformation. As these residues are highly conserved across the Filoviridae, calcium's impact on fusion, and subsequently infectivity, is a key interaction that can be leveraged for developing strategies to defend against Ebola infection. This mechanistic insight provides a rationale for the use of calcium-interfering drugs already approved by the FDA as therapeutics against Ebola and enables further development of novel drugs to combat the virus.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2′ site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.