Plant diseases caused by fungi and oomycetes pose an increasing threat to food security and ecosystem health worldwide. These filamentous pathogens, while taxonomically distinct, modulate host defense responses by secreting effectors, which are typically identified based on the presence of signal peptides. Here we show that Phytophthora sojae and Verticillium dahliae secrete isochorismatases (PsIsc1 and VdIsc1, respectively) that are required for full pathogenesis. PsIsc1 and VdIsc1 can suppress salicylate-mediated innate immunity in planta and hydrolyse isochorismate in vitro. A conserved triad of catalytic residues is essential for both functions. Thus, the two proteins are isochorismatase effectors that disrupt the plant salicylate metabolism pathway by suppressing its precursor. Furthermore, these proteins lack signal peptides, but exhibit characteristics that lead to unconventional secretion. Therefore, this secretion pathway is a novel mechanism for delivering effectors and might play an important role in host–pathogen interactions.

The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryoEM structures of the yeast E complex assembled on introns, providing the first view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architecture suggests that the same spliceosome can assemble across an exon, which either remodels to span an intron for canonical linear splicing (typically on short exons) or catalyzes back-splicing generating circRNA (on long exons). The model is supported by our experiments demonstrating that E complex assembled on the yeast EFM5 or HMRA1 middle exon can be chased into circRNA when the exon is sufficiently long. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysaccharide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psl matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in improved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disassembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.

The spliceosome undergoes dramatic changes in a splicing cycle. Structures of B, Bact, C, C*, and ILS complexes revealed mechanisms of 5′ splice site (ss) recognition, branching, and intron release, but lacked information on 3′ ss recognition, exon ligation and release. Here, we report a cryoEM structure of the post-catalytic P complex at 3.3Å resolution, revealing that 3′ ss is mainly recognized through non-Watson-Crick basepairing with the 5′ ss and branch point. Furthermore, an unidentified protein becomes stably associated with the P complex, securing the 3′ exon and potentially regulating Prp22 activity. Prp22 binds nucleotides 15–21 in the 3′ exon, enabling it to pull the intron-exon or ligated exon in a 3′ to 5′ direction to achieve 3′ ss proofreading or exon release, respectively.

The intracellular infections of Mycobacterium tuberculosis, which is the causative agent of tuberculosis, are regulated by many cyclic dinucleotide signaling. Rv2837c from M. tuberculosis is a soluble, stand-alone DHH-DHHA1 domain phosphodiesterase that down-regulates c-di-AMP through catalytic degradation and plays an important role in M. tuberculosis infections. Here, we report the crystal structure of Rv2837c (2.0 Å ), and its complex with hydrolysis intermediate 5-pApA (2.35 Å ). Our structures indicate that both DHH and DHHA1 domains are essential for c-di-AMP degradation. Further structural analysis shows that Rv2837c does not distinguish adenine from guanine, which explains why Rv2837c hydrolyzes all linear dinucleotides with almost the same efficiency. We observed that Rv2837c degraded other c-di-NMPs at a lower rate than it did on c-di-AMP. Nevertheless, our data also showed that Rv2837c significantly decreases concentrations of both c-di-AMP and c-di-GMP in vivo. Our results suggest that beside its major role in c-di-AMP degradation Rv2837c could also regulate c-di-GMP signaling pathways in bacterial cell.

Increasingly, cryo-electron microscopy (cryoEM) is used to determine the structures of RNA-protein assemblies, but nearly all maps determined with this method have biologically important regions where the local resolution does not permit RNA coordinate tracing. To address these omissions, we present De novo Ribonucleoprotein modeling in Real-space through Assembly of Fragments Together with Experimental density in Rosetta (DRRAFTER). We show that DRRAFTER recovers near-native models for a diverse benchmark set of RNA-protein complexes including the spliceosome, mitochondrial ribosome, and CRISPR-Cas9-sgRNA complexes; rigorous blind tests include yeast U1 snRNP and spliceosomal P complex maps. Additionally, to aid in model interpretation, we present a method for reliable in situ estimation of DRRAFTER model accuracy. Finally, we apply DRRAFTER to recently determined maps of telomerase, the HIV-1 reverse transcriptase initiation complex, and the packaged MS2 genome, demonstrating the acceleration of accurate model building in challenging cases.

The dibenzothiophene (DBT) monooxygenase DszC, which is the key initiating enzyme in "4S" metabolic pathway, catalyzes sequential sulphoxidation reaction of DBT to DBT sulfoxide (DBTO), then DBT sulfone (DBTO2). Here, we report the crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å. Intriguingly, two distinct conformations occur in the flexible lid loops adjacent to the active site (residue 280-295, between α9 and α10). They are named "open"' and "closed" state respectively, and might show the status of the free and ligand-bound DszC. The molecular docking results suggest that the reduced FMN reacts with an oxygen molecule at C4a position of the isoalloxazine ring, producing the C4a-(hydro)peroxyflavin intermediate which is stabilized by H391 and S163. H391 may contribute to the formation of the C4a-(hydro)peroxyflavin by acting as a proton donor to the proximal peroxy oxygen, and it might also be involved in the protonation process of the C4a-(hydro)xyflavin. Site-directed mutagenesis study shows that mutations in the residues involved either in catalysis or in flavin or substrate-binding result in a complete loss of enzyme activity, suggesting that the accurate positions of flavin and substrate are crucial for the enzyme activity.

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.