An apoplastic peptide activates salicylic acid signalling in maize.

Localized control of cell death is crucial for the resistance of plants to pathogens. Papain-like cysteine proteases (PLCPs) regulate plant defence to drive cell death and protection against biotrophic pathogens. In maize (Zea mays), PLCPs are crucial in the orchestration of salicylic acid (SA)-dependent defence signalling. Despite this central role in immunity, it remains unknown how PLCPs are activated, and which downstream signals they induce to trigger plant immunity. Here, we discover an immune signalling peptide, Z. mays immune signalling peptide 1 (Zip1), which is produced after salicylic acid (SA) treatment. In vitro studies demonstrate that PLCPs are required to release bioactive Zip1 from its propeptide precursor. Conversely, Zip1 treatment strongly elicits SA accumulation in leaves. Moreover, transcriptome analyses revealed that Zip1 and SA induce highly overlapping transcriptional changes. Consequently, Zip1 promotes the infection of the necrotrophic fungus Botrytis cinerea, while it reduces virulence of the biotrophic fungus Ustilago maydis. Thus, Zip1 represents the previously missing signal that is released by PLCPs to activate SA defence signalling.

Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases.

Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes.

Dynamic hydrolase activities precede hypersensitive tissue collapse in tomato seedlings.

Hydrolases such as subtilases, vacuolar processing enzymes (VPEs) and the proteasome play important roles during plant programmed cell death (PCD). We investigated hydrolase activities during PCD using activity-based protein profiling (ABPP), which displays the active proteome using probes that react covalently with the active site of proteins. We employed tomato (Solanum lycopersicum) seedlings undergoing synchronized hypersensitive cell death by co-expressing the avirulence protein Avr4 from Cladosporium fulvum and the tomato resistance protein Cf-4. Cell death is blocked in seedlings grown at high temperature and humidity, and is synchronously induced by decreasing temperature and humidity. ABPP revealed that VPEs and the proteasome are not differentially active, but that activities of papain-like cysteine proteases and serine hydrolases, including Hsr203 and P69B, increase before hypersensitive tissue collapse, whereas the activity of a carboxypeptidase-like enzyme is reduced. Similar dynamics were observed for these enzymes in the apoplast of tomato challenged with C. fulvum. Unexpectedly, these challenged plants also displayed novel isoforms of secreted putative VPEs. In the absence of tissue collapse at high humidity, the hydrolase activity profile is already altered completely, demonstrating that changes in hydrolase activities precede hypersensitive tissue collapse.

Profiling protein kinases and other ATP binding proteins in Arabidopsis using Acyl-ATP probes.

Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)(1) probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188.

Chemical proteomics with sulfonyl fluoride probes reveals selective labeling of functional tyrosines in glutathione transferases.

Chemical probes have great potential for identifying functional residues in proteins in crude proteomes. Here we studied labeling sites of chemical probes based on sulfonyl fluorides (SFs) on plant and animal proteomes. Besides serine proteases and many other proteins, SF-based probes label Tyr residues in glutathione transferases (GSTs). The labeled GSTs represent four different GST classes that share less than 30% sequence identity. The targeted Tyr residues are located at similar positions in the promiscuous substrate binding site and are essential for GST function. The high selectivity of SF-based probes for functional Tyr residues in GSTs illustrates how these probes can be used for functional studies of GSTs and other proteins in crude proteomes.

The antimalarial natural product symplostatin 4 is a nanomolar inhibitor of the food vacuole falcipains.

The marine natural product symplostatin 4 (Sym4) has been recognized as a potent antimalarial agent. However, its mode of action and, in particular, direct targets have to date remained elusive. We report a chemical synthesis of Sym4 and show that Sym4-treatment of P. falciparum-infected red blood cells (RBCs) results in the generation of a swollen food vacuole phenotype and a reduction of parasitemia at nanomolar concentrations. We furthermore demonstrate that Sym4 is a nanomolar inhibitor of the P. falciparum falcipains in infected RBCs, suggesting inhibition of the hemoglobin degradation pathway as Sym4's mode of action. Finally, we reveal a critical influence of the unusual methyl-methoxypyrrolinone (mmp) group of Sym4 for potent inhibition, indicating that Sym4 derivatives with such a mmp moiety might represent viable lead structures for the development of antimalarial falcipain inhibitors.

Subclassification and biochemical analysis of plant papain-like cysteine proteases displays subfamily-specific characteristics.

Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific functional and structural characteristics. For example, the NPIR and KDEL localization signals are distinctive for subfamilies, and the carboxyl-terminal granulin domain occurs in two PLCP subfamilies, in which some individual members probably evolved by deletion of the granulin domains. We also discovered a conserved double cysteine in the catalytic site of SAG12-like proteases and two subfamily-specific disulfides in RD19A-like proteases. Protease activity profiling of representatives of the PLCP subfamilies using novel fluorescent probes revealed striking polymorphic labeling profiles and remarkably distinct pH dependency. Competition assays with peptide-epoxide scanning libraries revealed common and unique inhibitory fingerprints. Finally, we expand the detection of PLCPs by identifying common and organ-specific protease activities and identify previously undetected proteases upon labeling with cell-penetrating probes in vivo. This study provides the plant protease research community with tools for further functional annotation of plant PLCPs.

A para-nitrophenol phosphonate probe labels distinct serine hydrolases of Arabidopsis.

Activity-based protein profiling represents a powerful methodology to probe the activity state of enzymes under various physiological conditions. Here we present the development of a para-nitrophenol phosphonate activity-based probe with structural similarities to the potent agrochemical paraoxon. We demonstrate that this probes labels distinct serine hydrolases with the carboxylesterase CXE12 as the predominant target in Arabidopsis thaliana. The designed probe features a distinct labeling pattern and therefore represents a promising chemical tool to investigate physiological roles of selected serine hydrolases such as CXE12 in plant biology.

Mining the Active Proteome of Arabidopsis thaliana.

Assigning functions to the >30,000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied activity-based protein profiling (ABPP). ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed activities of 76 Arabidopsis proteins. These proteins represent over 10 different protein classes that contain over 250 Arabidopsis proteins, including cysteine, serine, and metalloproteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed additional protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities, e.g., of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry, and proteomics.

Correlation of [18F]-2-fluoro-deoxy-D-glucose positron emission tomography standard uptake values with the cellular composition of stage I nonsmall cell lung cancer.

BACKGROUND: The aim of the current study was to determine whether the [18F]-2-fluoro-deoxy-D-glucose (FDG) positron emission tomography (PET) standardized uptake value (SUV) in patients with a new diagnosis of stage I lung cancer correlates with a specific cellular component in the primary tumor. METHODS: This study was Health Insurance Portability and Accountability Act compliant and approved by our Institutional Review Board with a waiver of informed consent. Forty patients with stage I nonsmall cell lung cancer (NSCLC) who underwent FDG-PET imaging at the time of diagnosis followed by surgical resection were retrospectively identified. Histologic sections of the primary tumor were reviewed by a pathologist without any clinical data and scored according to the percentage of each cellular component (tumor cells, normal stroma, inflammatory cells, necrosis, fibrosis, and other). Each component was compared with maximal (SUV(max)) and mean (SUV(mean)) SUVs using Pearson correlation coefficient analysis. RESULTS: The mean SUV(max) and SUV(mean) values for 40 stage I NSCLC tumors were 8.8 and 5.4, respectively. The mean histologic composition of tumor specimens in order of frequency was as follows: tumor cells (38.9%), fibrosis (30.8%), inflammatory cells (14.8%), normal stroma (5.2%), necrosis (5.8%), and other components (4.5%); however, there was considerable variation noted among individual tumors. There was no statistically significant correlation between SUV(max) (r = .19; P = .24) or SUV(mean) (r = .017; P = .29) and the proportion of tumor cells in the tumor mass or any other cellular components. CONCLUSIONS: The cellular composition of stage I NSCLC appears to be highly variable, with no correlation noted between a specific tumor cellular component and FDG activity.

Proteasome activity profiling: a simple, robust and versatile method revealing subunit-selective inhibitors and cytoplasmic, defense-induced proteasome activities.

The proteasome plays essential roles in nearly all biological processes in plant defense and development, yet simple methods for displaying proteasome activities in extracts and living tissues are not available to plant science. Here, we introduce an easy and robust method to simultaneously display the activities of all three catalytic proteasome subunits in plant extracts or living plant tissues. The method is based on a membrane-permeable, small-molecule fluorescent probe that irreversibly reacts with the catalytic site of the proteasome catalytic subunits in an activity-dependent manner. Activities can be quantified from fluorescent protein gels and used to study proteasome activities in vitro and in vivo. We demonstrate that proteasome catalytic subunits can be selectively inhibited by aldehyde-based inhibitors, including the notorious caspase-3 inhibitor DEVD. Furthermore, we show that the proteasome activity, but not its abundance, is significantly increased in Arabidopsis upon treatment with benzothiadiazole (BTH). This upregulation of proteasome activity depends on NPR1, and occurs mostly in the cytoplasm. The simplicity, robustness and versatility of this method will make this method widely applicable in plant science.

Analysis of desiccation-induced candidate phosphoproteins from Craterostigma plantagineum isolated with a modified metal oxide affinity chromatography procedure.

Reversible protein phosphorylation/dephosphorylation is crucial for regulation of many cellular events, and increasing evidence indicates that this post-translational modification is also involved in the complex process of acquisition of desiccation tolerance. To analyze the phosphoproteome of the desiccation tolerant resurrection plant Craterostigma plantagineum, MOAC-enriched proteins from leaves at different stages of a de-/rehydration cycle were separated by 2-D PAGE and detected by phosphoprotein-specific staining. Using this strategy 20 putative phosphoproteins were identified by MALDI-TOF MS and MS/MS, which were not detected when total proteins were analyzed. The characterized desiccation-related phosphoproteins CDeT11-24 and CDeT6-19 were used as internal markers to validate the specificity of the analyses. For 16 of the identified proteins published evidence suggests that they are phosphoproteins. Comparative analysis of the 2-D gels showed that spot intensities of most identified putative phosphoproteins change during the de-/rehydration cycle. This suggests an involvement of these proteins in desiccation tolerance. Nearly all changes in the phosphoproteome of C. plantagineum, which are triggered by dehydration, are reversed within 4 days of rehydration, which is in agreement with physiological observations. Possible functions of selected proteins are discussed in the context of the de-/rehydration cycle.

Beta-lactone probes identify a papain-like peptide ligase in Arabidopsis thaliana.

New activity-based probes are essential for expanding studies on the hundreds of serine and cysteine proteases encoded by the genome of Arabidopsis thaliana. To monitor protease activities in plant extracts, we generated biotinylated peptides containing a beta-lactone reactive group. These probes cause strong labeling in leaf proteomes. Unexpectedly, labeling was detected at the N terminus of PsbP, nonproteolytic protein of photosystem II. Inhibitor studies and reverse genetics led to the discovery that this unusual modification is mediated by a single plant-specific, papain-like protease called RD21. In cellular extracts, RD21 accepts both beta-lactone probes and peptides as donor molecules and ligates them, probably through a thioester intermediate, to unmodified N termini of acceptor proteins.

Fungal effector protein AVR2 targets diversifying defense-related cys proteases of tomato.

The interaction between the fungal pathogen Cladosporium fulvum and its host tomato (Solanum lycopersicum) is an ideal model to study suppression of extracellular host defenses by pathogens. Secretion of protease inhibitor AVR2 by C. fulvum during infection suggests that tomato papain-like cysteine proteases (PLCPs) are part of the tomato defense response. We show that the tomato apoplast contains a remarkable diversity of PLCP activities with seven PLCPs that fall into four different subfamilies. Of these PLCPs, transcription of only PIP1 and RCR3 is induced by treatment with benzothiadiazole, which triggers the salicylic acid-regulated defense pathway. Sequencing of PLCP alleles of tomato relatives revealed that only PIP1 and RCR3 are under strong diversifying selection, resulting in variant residues around the substrate binding groove. The doubled number of variant residues in RCR3 suggests that RCR3 is under additional adaptive selection, probably to prevent autoimmune responses. AVR2 selectively inhibits only PIP1 and RCR3, and one of the naturally occurring variant residues in RCR3 affects AVR2 inhibition. The higher accumulation of PIP1 protein levels compared with RCR3 indicates that PIP1 might be the real virulence target of AVR2 and that RCR3 acts as a decoy for AVR2 perception in plants carrying the Cf-2 resistance gene.