Activity-Profiling of Vacuolar Processing Enzymes and the Proteasome during Plant-Pathogen Interactions

Programmed cell death (PCD) is an interesting natural phenomenon, common to many organisms. PCD has been extensively studied during animal apoptosis and several regulators have been identified. Cysteine proteases called caspases, and the proteasome were found to be main players in PCD in animals. In plants, PCD is regulated by vacuolar processing enzymes (VPEs) and the proteasome. Both proteolytic machineries exhibit caspase-like activities. In this work, the activity of VPEs and the proteasome were characterized using activity-based protein profiling (ABPP). ABPP involves fluorescent or biotinylated probes that react with the catalytic residue of proteases in an activity-dependent manner. Specific probes that target γVPE, the most abundant VPE in vegetative tissue, were selected from screen with legumain probes. Further characterization of γVPE activity revealed an unexpected, post-transcriptional up-regulation of γVPE activity during compatible, but not during incompatible interactions of Arabidopsis with Hyaloperonospora arabidopsidis (Hpa). Sporulation of Hpa was reduced in the absence of VPEs indicating that VPEs promote pathogen fitness. These findings introduce a new tool to study VPEs and reveal a new role of VPEs during compatible interactions. New, selective probes that target the plant proteasome are also introduced in this thesis. The proteasome is a multi-subunit proteolytic complex containing three subunits with different catalytic activities: β1, β2 and β5. ABPP was applied to further characterize the inhibition of the plant proteasome by Syringolin A (SylA), a non-ribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv. syringae. This work shows that SylA preferentially targets β2 and β5 of the plant proteasome. Structure-activity analysis revealed that dipeptide tail of SylA contributes to β2 specificity and identified a nonreactive SylA derivative. The selectivity of SylA for the catalytic subunits is discussed and the subunit selectivity is explained by crystallographic data. Importantly, it was discovered that SylA production promotes colonization of distant tissue by Pseudomonas syringae pv. syringae. SylA was found to suppress both effector-triggered immunity and salicylic acid-dependent acquired resistance. Distant colonization is a new phenomenon, common to other P. syringae strains, and undetected by classical pathogen assays

Cytokines genotypes as predictors of disease outcomes in HIV-1 infected Ukrainians

У тезах представлені дані щодо асоціації поліморфізмів генів цитокінів з опортуністичними інфекціями у українців з ВІЛ-1.В тезисах представлены данные об ассоциации полиморфизмов генов цитокинов с оппортунистическими инфекциями у украинцев с ВИЧ-1.The data of cytokines genotypes association with outcomes of the disease in Ukrainians with HIV-1 were presented

Proteases Underground: Analysis of the Maize Root Apoplast Identifies Organ Specific Papain-Like Cysteine Protease Activity

Plant proteases are key regulators of plant cell processes such as seed development, immune responses, senescence and programmed cell death (PCD). Apoplastic papain-like cysteine proteases (PL) are hubs in plant-microbe interactions and play an important role during abiotic stresses. The apoplast is a crucial interface for the interaction between plant and microbes. So far, apoplastic maize PL and their function have been mostly described for aerial parts. In this study, we focused on apoplastic PLCPs in the roots of maize plants. We have analyzed the phylogeny of maize PLCPs and investigated their protein abundance after salicylic acid (SA) treatment. Using activity-based protein profiling (ABPP) we have identified a novel root-specific PLCP belonging to the RD21-like subfamily, as well as three SA activated PLCPs. The root specific PLCP CP1C shares sequence and structural similarities to known CP1-like proteases. Biochemical analysis of recombinant CP1C revealed different substrate specificities and inhibitor affinities compared to the related proteases. This study characterized a root-specific PLCP and identifies differences between the SA-dependent activation of PLCPs in roots and leaves

Extracellular proteolytic cascade in tomato activates immune protease Rcr3

Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva) Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity

Divergent Evolution of PcF/SCR74 Effectors in Oomycetes Is Associated with Distinct Recognition Patterns in Solanaceous Plants

Plants deploy cell surface receptors known as pattern-recognition re ceptors (PRRs) that recognize non-self molecules from pathogens and microbes to defend against invaders. PRRs typically recognize microbe-associated molecular patterns (MAMPs) that are usually widely conserved, some even across kingdoms. Here, we report an oomycete-specific family of small secreted cysteine-rich (SCR) proteins that displays divergent patterns of sequence variation in the Irish potato famine pathogen Phytophthora infestans. A subclass that includes the conserved effector PcF from Phytophthora cactorum activates immunity in a wide range of plant species. In contrast, the more diverse SCR74 subclass is specific to P. infestans and tends to trigger immune responses only in a limited number of wild potato genotypes. The SCR74 response was recently mapped to a G-type lectin receptor kinase (GLecRK) locus in the wild potato Solanum microdontum subsp. gigantophyllum. The G-LecRK locus displays a high diversity in Solanum host species compared to other solanaceous plants. We propose that the diversification of the SCR74 proteins in P. infestans is driven by a fast coevolutionary arms race with cell surface immune receptors in wild potato, which contrasts the presumed slower dynamics between conserved apoplastic effectors and PRRs. Understanding the molecular determinants of plant immune responses to these divergent molecular patterns in oomycetes is expected to contribute to deploying multiple layers of disease resistance in crop plants. IMPORTANCE Immune receptors at the plant cell surface can recognize invading microbes. The perceived microbial molecules are typically widely conserved and therefore the matching surface receptors can detect a broad spectrum of pathogens. Here we describe a family of Phytophthora small extracellular proteins that consists of conserved subfamilies that are widely recognized by solanaceous plants. Remarkably, one subclass of SCR74 proteins is highly diverse, restricted to the late blight pathogen Phytophthora infestans and is specifically detected in wild potato plants. The diversification of this subfamily exhibits signatures of a coevolutionary arms race with surface receptors in potato. Insights into the molecular interaction between these potato-specific receptors and the recognized Phytophthora proteins are expected to contribute to disease resistance breeding in potato

A Role in Immunity for Arabidopsis Cysteine Protease RD21, the Ortholog of the Tomato Immune Protease C14

Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf) during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen

Subunit-selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections

The proteasome is a nuclear‐cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions, but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit‐selective inhibitors and dual‐color fluorescent activity‐based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant–microbe interactions. Our data reveal that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1‐1 [PtoDC3000(ΔhQ)] whilst the activity profile of the β1 subunit changes. Infection with wild‐type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community, and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species.Bio-organic Synthesi

An enhanced median filter for removing noise from MR images

In this paper, a novel decision based median (DBM) filter for enhancing MR images has been proposed. The method is based on eliminating impulse noise from MR images. A median-based method to remove impulse noise from digital MR images has been developed. Each pixel is leveled from black to white like gray-level. The method is adjusted in order to decide whether the median operation can be applied on a pixel. The main deficiency in conventional median filter approaches is that all pixels are filtered with no concern about healthy pixels. In this research, to suppress this deficiency, noisy pixels are initially detected, and then the filtering operation is applied on them. The proposed decision method (DM) is simple and leads to fast filtering. The results are more accurate than other conventional filters. Moreover, DM adjusts itself based on the conditions of local detections. In other words, DM operation on detecting a pixel as a noise depends on the previous decision. As a considerable advantage, some unnecessary median operations are eliminated and the number of median operations reduces drastically by using DM. Decision method leads to more acceptable results in scenarios with high noise density. Furthermore, the proposed method reduces the probability of detecting noise-free pixels as noisy pixels and vice versa

Confocal microscopy reveals in planta dynamic interactions between pathogenic, avirulent and non-pathogenic Pseudomonas syringae strains

© 2017 BSPP AND JOHN WILEY & SONS LTD Recent advances in genomics and single-cell analysis have demonstrated the extraordinary complexity reached by microbial populations within their hosts. Communities range from complex multispecies groups to homogeneous populations differentiating into lineages through genetic or non-genetic mechanisms. Diversity within bacterial populations is recognized as a key driver of the evolution of animal pathogens. In plants, however, little is known about how interactions between different pathogenic and non-pathogenic variants within the host impact on defence responses, or how the presence within a mixture may affect the development or the fate of each variant. Using confocal fluorescence microscopy, we analysed the colonization of the plant apoplast by individual virulence variants of Pseudomonas syringae within mixed populations. We found that non-pathogenic variants can proliferate and even spread beyond the inoculated area to neighbouring tissues when in close proximity to pathogenic bacteria. The high bacterial concentrations reached at natural entry points promote such interactions during the infection process. We also found that a diversity of interactions take place at a cellular level between virulent and avirulent variants, ranging from dominant negative effects on proliferation of virulent bacteria to in trans suppression of defences triggered by avirulent bacteria. Our results illustrate the spatial dynamics and complexity of the interactions found within mixed infections, and their potential impact on pathogen evolution