Structures of Phytophthora RXLR Effector Proteins: a conserved but adaptable fold underpins functional diversity

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The molecular mechanisms underlying how effectors evolve to gain new virulence functions and/or evade the plant immune system are poorly understood. Here, we report the crystal structures of the effector domains from two oomycete RXLR proteins, Phytophthora capsici AVR3a11 and Phytophthora infestans PexRD2. Despite sharin

On the evolution of decoys in plant immune systems

The Guard-Guardee model for plant immunity describes how resistance proteins (guards) in host cells monitor host target proteins (guardees) that are manipulated by pathogen effector proteins. A recently suggested extension of this model includes decoys, which are duplicated copies of guardee proteins, and which have the sole function to attract the effector and, when modified by the effector, trigger the plant immune response. Here we present a proof-of-principle model for the functioning of decoys in plant immunity, quantitatively developing this experimentally-derived concept. Our model links the basic cellular chemistry to the outcomes of pathogen infection and resulting fitness costs for the host. In particular, the model allows identification of conditions under which it is optimal for decoys to act as triggers for the plant immune response, and of conditions under which it is optimal for decoys to act as sinks that bind the pathogen effectors but do not trigger an immune response.Comment: 15 pages, 6 figure

Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein Gαo in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon Gαo binding, the determinants for GAP activity, and the structurally unique features of Gαo that likely distinguish it physiologically from other members of the larger Gαi family, affording insight to receptor, GAP and effector specificity

Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors.

Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS), which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K) channels and downregulating nicotinic acetylcholine receptors (nAChRs) in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function

The tomato Prf complex is a molecular trap for bacterial effectors based on Pto transphosphorylation

The bacteria Pseudomonas syringae is a pathogen of many crop species and one of the model pathogens for studying plant and bacterial arms race coevolution. In the current model, plants perceive bacteria pathogens via plasma membrane receptors, and recognition leads to the activation of general defenses. In turn, bacteria inject proteins called effectors into the plant cell to prevent the activation of immune responses. AvrPto and AvrPtoB are two such proteins that inhibit multiple plant kinases. The tomato plant has reacted to these effectors by the evolution of a cytoplasmic resistance complex. This complex is compromised of two proteins, Prf and Pto kinase, and is capable of recognizing the effector proteins. How the Pto kinase is able to avoid inhibition by the effector proteins is currently unknown. Our data shows how the tomato plant utilizes dimerization of resistance proteins to gain advantage over the faster evolving bacterial pathogen. Here we illustrate that oligomerisation of Prf brings into proximity two Pto kinases allowing them to avoid inhibition by the effectors by transphosphorylation and to activate immune responses

Tracking elusive cargo: Illuminating spatio-temporal type 3 effector protein dynamics using reporters

Type 3 secretion systems (T3SS) form an integral part of the arsenal of many pathogenic bacteria. These injection machines, together with their cargo of subversive effector proteins are capable of manipulating the cellular environment of the host in order to ensure persistence of the pathogen. In order to fully appreciate the functions of Type 3 effectors it is necessary to gain spatio-temporal knowledge of each effector during the process of infection. A number of genetic modifications have been exploited in order to reveal effector protein secretion, translocation and subsequent activity and localisation within host cells. In this review, we will discuss the many available approaches for tracking effector protein dynamics and discuss the challenges faced to improve the current technologies and gain a clearer picture of effector protein function

Alteration of host cell ubiquitination by the intracellular bacterial pathogen Coxiella burnetii

The intracellular bacterial agent of Q fever, Coxiella burnetii, replicates within a phagolysosomelike parasitophorous vacuole (PV) in human macrophages and delivers effector proteins to the host cytosol via a Dot/Icm type IV secretion system (T4SS). The T4SS effectors are critical for PV formation and prevention of host cell death that allows sufficient time for bacterial replication. Recruitment of ubiquitin-related components to the C. burnetii PV is also predicted to be involved in PV formation and bacterial replication and is likely controlled by effector proteins. In this study, we assessed the role of the Dot/Icm T4SS in regulating ubiquitination by comparing subcellular localization of ubiquitinated proteins between cells infected with C. burnetii and a mutant that lacks a functional T4SS. Fluorescence microscopy showed ubiquitinated proteins surrounding wild-type C. burnetii PV but not phagosomes harboring T4SS-defective organisms. Immunoblot analysis showed altered ubiquitinated protein profiles throughout infection, suggesting C. burnetii impacts post-translational modification of host cell and/or bacterial proteins. Future studies will determine how T4SS-mediated recruitment of ubiquitinated proteins impacts C. burnetii-host cell interactions and eventual development of diseas

The bacterial effector HopZ1a acetylates ZIP1 kinase to suppress Arabidopsis defence responses

During the plant-pathogen interaction, disease or resistance are determined in the plant by a series of molecular events. The plant detects Pathogen-Associated Molecular Patterns (PAMPs), such as flagellin, triggering a defence response called PTI (PAMP-Triggered Immunity). Bacterial pathogens can in turn suppress such defence response through the translocation into the plant cell cytosol of virulence proteins, called effectors, via a Type Three Secretion System (T3SS). In resistant plants, intracellular receptors known as R proteins recognize these effectors, triggering a second line of defence, more specific and intense, called ETI (Effector-Triggered Immunity), which usually leads to programmed cell death known as HR (Hypersensitive Response). Pseudomonas syringae is a phytopathogenic bacterium whose virulence depends on a T3SS and its effector repertoire. Some strains include HopZ1a, an unusual effector which is able to suppress in Arabidopsis both local (PTI and ETI), and systemic (SAR, for Systemic Acquired Resistance) defences, by means of its acetyltransferase activity. In resistant Arabidopsis plants, HopZ1a acetylates the ZED1 pseudokinase, which is proposed to function as a decoy mimicking HopZ1a target in the plant: ZED1 modification activates an R-protein (ZAR1) to trigger HopZ1a-dependent ETI. None of the Arabidopsis proteins proposed to date as HopZ1a targets is a kinase, nor fully explains the effector´s defence suppression abilities. In this work we identify an Arabidopsis kinase that functions as a positive regulator of PTI, ETI and SAR, which interacts with HopZ1a and is acetylated by this effector in lysine residues essential for its kinase activity. Further, HopZ1a can specifically suppress the defence phenotypes resulting from ZIP1 expression in Arabidopsis. We propose that ZIP1 acetylation by HopZ1a interferes with its kinase activity, and consequently with positive defence signalling.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Coxiella burnetii Blocks Intracellular Interleukin-17 Signaling in Macrophages

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting that Coxiella T4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genes Il1a, Il1b, and Tnfa, the chemokine genes Cxcl2 and Ccl5, and the antimicrobial protein gene Lcn2 We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response

Computational approach to predict species-specific type III secretion system (T3SS) effectors using single and multiple genomes

Positive (effectors), negative (non-effectors), and testing sets of the five organisms that we used in the GenSET studies. (ALL Tags denotes all proteins encoded in the genome; Positive Set denotes known effector proteins used for training; Negative Set denotes non-effector proteins used for training; Testing Set denotes all proteins in the bacteria minus the Positive and Negative Sets; Positives in Testing Set denotes known effector proteins included in the Testing Set). (XLSX 48 kb