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

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

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

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

\u3ci\u3ePseudomonas syringae\u3c/i\u3e pv. \u3ci\u3esyringae\u3c/i\u3e Uses Proteasome Inhibitor Syringolin A to Colonize from Wound Infection Sites

Infection of plants by bacterial leaf pathogens at wound sites is common in nature. Plants defend wound sites to prevent pathogen invasion, but several pathogens can overcome spatial restriction and enter leaf tissues. The molecular mechanisms used by pathogens to suppress containment at wound infection sites are poorly understood. Here, we studied Pseudomonas syringae strains causing brown spot on bean and blossom blight on pear. These strains exist as epiphytes that can cause disease upon wounding caused by hail, sand storms and frost. We demonstrate that these strains overcome spatial restriction at wound sites by producing syringolin A (SylA), a small molecule proteasome inhibitor. Consequently, SylA-producing strains are able to escape from primary infection sites and colonize adjacent tissues along the vasculature. We found that SylA diffuses from the primary infection site and suppresses acquired resistance in adjacent tissues by blocking signaling by the stress hormone salicylic acid (SA). Thus, SylA diffusion creates a zone of SA-insensitive tissue that is prepared for subsequent colonization. In addition, SylA promotes bacterial motility and suppresses immune responses at the primary infection site. These local immune responses do not affect bacterial growth and were weak compared to effector-triggered immunity. Thus, SylA facilitates colonization from wounding sites by increasing bacterial motility and suppressing SA signaling in adjacent tissues

Activity profiling reveals changes in the diversity and activity of proteins in Arabidopsis roots in response to nematode infection

Cyst nematodes are obligate, sedentary endoparasites with a highly specialised biology and a huge economic impact in agriculture. Successful parasitism involves morphological and physiological modifications of the host cells which lead to the formation of specialised syncytial feeding structures in roots. The development of the syncytium is aided by a cocktail of nematode effectors that manipulate the host plant activities in a complex network of interactions through post-translational modifications. Traditional transcriptomic and proteomic approaches cannot display this functional proteomic information. Activity-based protein profiling (ABPP) is a powerful technology that can be used to investigate the activity of the proteome through activity-based probes. To better understand the functional proteomics of syncytium, ABPP was conducted on syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots. Our results demonstrated that the activity of several enzymes is differentially regulated in the syncytium compared to the control roots. Among those specifically activated in the syncytium are a putative S-formyl-glutathione hydrolase (SFGH), a putative methylesterase (MES) and two unidentified enzymes. In contrast, the activities of vacuolar processing enzymes (VPEs) are specifically suppressed in the syncytium. Competition labelling, quantitative gene expression and T-DNA knock-out mutants were used to further characterise the roles of the differentially regulated enzymes during plant nematode interaction. In conclusion, our study will open the door to generate a comprehensive and integrated view of the host-pathogen warfare that results in the formation of long-term feeding sites for pathogens. (C) 2015 Elsevier Masson SAS. All rights reserved

Mutant <i>rd21</i> lines are not compromised in interactions with <i>Pst</i>DC3000.

<p><b>A,</b> Compatible <i>Pst</i> interactions. (Mutant) Arabidopsis plants were spray-inoculated with <i>Pst</i>DC3000 (vir) and bacterial populations were measured at 0 and 3 dpi. Error bars represent SD of 5 independent bacterial extractions. This experiment was repeated three times with similar results. <b>B,</b> Incompatible <i>Pst</i> interactions. (Mutant) Arabidopsis plants were spray-inoculated with <i>Pst</i>DC3000 avrRpm1 (avr) and bacterial populations were measured at 0 and 3 dpi. Error bars represent SD of 5 independent bacterial extractions. This experiment was repeated three times with similar results.</p

Host apoplastic cysteine protease activity is suppressed during the mutualistic association of Lolium perenne and Epichloe festucae

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonize their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens, but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonization with their hosts. Epichloe festucae forms mutualistic associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome-wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship, and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 abundance is not altered during the mutualistic interaction and it mainly inhibits LpCP2. However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1, we propose that additional inhibitors, likely of fungal origin, are involved in the suppression of apoplastic PLCPs during E. festucae infection

Mutant <i>rd21</i> lines are unaltered in their interactions with <i>Hpa</i>.

<p><b>A,</b> Compatible <i>Hpa</i> interactions. (Mutant) Arabidopsis seedlings were infected with <i>Hpa</i> isolate Noco2, which is virulent on Col-0 but not on Ws. Spores were counted at 7 dpi in triplicate. Error bars represent standard deviation (SD) of three independent spore isolations. This experiment is repeated once with similar results. <b>B,</b> Incompatible <i>Hpa</i> interactions. (Mutant) Arabidopsis seedlings were infected with <i>Hpa</i> isolate Emwa1, which is virulent on Ws-0 but not on Col-0. Spores were counted at 7 dpi in triplicate. Error bars represent SD of three independent spore isolations.</p