Pseudomonas effector AvrB is a glycosyltransferase that rhamnosylates plant guardee protein RIN4.

The plant pathogen Pseudomonas syringae encodes a type III secretion system avirulence effector protein, AvrB, that induces a form of programmed cell death called the hypersensitive response in plants as a defense mechanism against systemic infection. Despite the well-documented catalytic activities observed in other Fido (Fic, Doc, and AvrB) proteins, the enzymatic activity and target substrates of AvrB have remained elusive. Here, we show that AvrB is an unprecedented glycosyltransferase that transfers rhamnose from UDP-rhamnose to a threonine residue of the Arabidopsis guardee protein RIN4. We report structures of various enzymatic states of the AvrB-catalyzed rhamnosylation reaction of RIN4, which reveal the structural and mechanistic basis for rhamnosylation by a Fido protein. Collectively, our results uncover an unexpected reaction performed by a prototypical member of the Fido superfamily while providing important insights into the plant hypersensitive response pathway and foreshadowing more diverse chemistry used by Fido proteins and their substrates.

Gene Replacement by a Selectable Marker in the Filamentous FungusMagnaporthe oryzae.

Magnaporthe oryzaeis a filamentous fungus responsible for the detrimental rice blast disease afflicting rice crops worldwide. For years, M. oryzae has served as an excellent model organism to study plant pathogen interactions due to its sequenced genome, its amenability to functional genetics, and its capacity to be tracked in laboratory settings. As such, techniques to genetically manipulate M. oryzae for gene deletion range from genome editing via CRISPR-Cas9 to gene replacement through homologous recombination. This protocol focuses on detailing how to perform gene replacement in the model organism, M. oryzae, through a split marker method. This technique relies on replacing the open reading frame of a gene of interest with a gene conferring resistance to a specific selectable chemical, disrupting the transcription of the gene of interest and generating a knockout mutant M. oryzae strain. Key features Comprehensive overview of primer design, PEG-mediated protoplast transformation, and fungal DNA extraction for screening.

Membrane-localized expression, production and assembly of Vibrio parahaemolyticus T3SS2 provides evidence for transertion.

It has been proposed that bacterial membrane proteins may be synthesized and inserted into the membrane by a process known as transertion, which involves membrane association of their encoding genes, followed by coupled transcription, translation and membrane insertion. Here, we provide evidence supporting that the pathogen Vibrio parahaemolyticus uses transertion to assemble its type III secretion system (T3SS2), to inject virulence factors into host cells. We propose a two-step transertion process where the membrane-bound co-component receptor (VtrA/VtrC) is first activated by bile acids, leading to membrane association and expression of its target gene, vtrB, located in the T3SS2 pathogenicity island. VtrB, the transmembrane transcriptional activator of T3SS2, then induces the localized expression and membrane assembly of the T3SS2 structural components and its effectors. We hypothesize that the proposed transertion process may be used by other enteric bacteria for efficient assembly of membrane-bound molecular complexes in response to extracellular signals.

Molecular determinants for differential activation of the bile acid receptor from the pathogen Vibrio parahaemolyticus.

Bile acids are important for digestion of food and antimicrobial activity. Pathogenic Vibrio parahaemolyticus senses bile acids and induce pathogenesis. The bile acid taurodeoxycholate (TDC) was shown to activate the master regulator, VtrB, of this system, whereas other bile acids such as chenodeoxycholate (CDC) do not. Previously, VtrA-VtrC was discovered to be the co-component signal transduction system that binds bile acids and induces pathogenesis. TDC binds to the periplasmic domain of the VtrA-VtrC complex, activating a DNA-binding domain in VtrA that then activates VtrB. Here, we find that CDC and TDC compete for binding to the VtrA-VtrC periplasmic heterodimer. Our crystal structure of the VtrA-VtrC heterodimer bound to CDC revealed CDC binds in the same hydrophobic pocket as TDC but differently. Using isothermal titration calorimetry, we observed that most mutants in the binding pocket of VtrA-VtrC caused a decrease in bile acid binding affinity. Notably, two mutants in VtrC bound bile acids with a similar affinity as the WT protein but were attenuated for TDC-induced type III secretion system 2 activation. Collectively, these studies provide a molecular explanation for the selective pathogenic signaling by V. parahaemolyticus and reveal insight into a host's susceptibility to disease.

Regulating Death and Disease: Exploring the Roles of Metacaspases in Plants and Fungi.

Identified over twenty years ago and distantly related to animal caspases are a group of cysteine proteases known as metacaspases. Throughout the years, much like caspase roles in metazoans, metacaspases have been shown to be involved in regulating cellular death in non-metazoan organisms. Yet, continued research on metacaspases describes these proteins as intricate and multifunctional, displaying striking diversity on distinct biological functions. In this review, we intend to describe the recent advances in our understanding of the divergence of metacaspase functionality in plants and fungi. We will dissect the duality of metacaspase activity in the context of plant-pathogen interactions, providing a unique lens from which to characterize metacaspases in the development, immunity, and stress responses of plants, and the development and virulence of fungi. Furthermore, we explore the evolutionary trajectory of fungal metacaspases to delineate their structure and function. Bridging the gap between metacaspase roles in immunity and pathogenicity of plant-pathogen interactions can enable more effective and targeted phytopathogen control efforts to increase production of globally important food crops. Therefore, the exploitation and manipulation of metacaspases in plants or fungi represent new potential avenues for developing mitigation strategies against plant pathogens.

Role of Two Metacaspases in Development and Pathogenicity of the Rice Blast Fungus Magnaporthe oryzae.

Rice blast disease caused by Magnaporthe oryzae is a devastating disease of cultivated rice worldwide. Infections by this fungus lead to a significant reduction in rice yields and threats to food security. To gain better insight into growth and cell death in M. oryzae during infection, we characterized two predicted M. oryzae metacaspase proteins, MoMca1 and MoMca2. These proteins appear to be functionally redundant and can complement the yeast Yca1 homologue. Biochemical analysis revealed that M. oryzae metacaspases exhibited Ca2+-dependent caspase activity in vitro Deletion of both MoMca1 and MoMca2 in M. oryzae resulted in reduced sporulation, delay in conidial germination, and attenuation of disease severity. In addition, the double ΔMomca1mca2 mutant strain showed increased radial growth in the presence of oxidative stress. Interestingly, the ΔMomca1mca2 strain showed an increased accumulation of insoluble aggregates compared to the wild-type strain during vegetative growth. Our findings suggest that MoMca1 and MoMca2 promote the clearance of insoluble aggregates in M. oryzae, demonstrating the important role these metacaspases have in fungal protein homeostasis. Furthermore, these metacaspase proteins may play additional roles, like in regulating stress responses, that would help maintain the fitness of fungal cells required for host infection.IMPORTANCEMagnaporthe oryzae causes rice blast disease that threatens global food security by resulting in the severe loss of rice production every year. A tightly regulated life cycle allows M. oryzae to disarm the host plant immune system during its biotrophic stage before triggering plant cell death in its necrotrophic stage. The ways M. oryzae navigates its complex life cycle remain unclear. This work characterizes two metacaspase proteins with peptidase activity in M. oryzae that are shown to be involved in the regulation of fungal growth and development prior to infection by potentially helping maintain fungal fitness. This study provides new insights into the role of metacaspase proteins in filamentous fungi by illustrating the delays in M. oryzae morphogenesis in the absence of these proteins. Understanding the mechanisms by which M. oryzae morphology and development promote its devastating pathogenicity may lead to the emergence of proper methods for disease control.