Combination of transcriptomic, proteomic, and degradomic profiling reveals common and distinct patterns of pathogen-induced cell death in maize.

Regulated cell death (RCD) is crucial for plant development, as well as in decision-making in plant-microbe interactions. Previous studies revealed components of the molecular network controlling RCD, including different proteases. However, the identity, the proteolytic network as well as molecular components involved in the initiation and execution of distinct plant RCD processes, still remain largely elusive. In this study, we analyzed the transcriptome, proteome, and N-terminome of Zea mays leaves treated with the Xanthomonas effector avrRxo1, the mycotoxin Fumonisin B1 (FB1), or the phytohormone salicylic acid (SA) to dissect plant cellular processes related to cell death and plant immunity. We found highly distinct and time-dependent biological processes being activated on transcriptional and proteome levels in response to avrRxo1, FB1, and SA. Correlation analysis of the transcriptome and proteome identified general, as well as trigger-specific markers for cell death in Zea mays. We found that proteases, particularly papain-like cysteine proteases, are specifically regulated during RCD. Collectively, this study characterizes distinct RCD responses in Z. mays and provides a framework for the mechanistic exploration of components involved in the initiation and execution of cell death.

Combination of in vivo proximity labeling and co-immunoprecipitation identifies the host target network of a tumor-inducing effector in the fungal maize pathogen Ustilago maydis.

Plant pathogens secrete effectors, which target host proteins to facilitate infection. The Ustilago maydis effector UmSee1 is required for tumor formation in the leaf during infection of maize. UmSee1 interacts with maize SGT1 (suppressor of G2 allele of skp1) and blocks its phosphorylation in vivo. In the absence of UmSee1, U. maydis cannot trigger tumor formation in the bundle sheath. However, it remains unclear which host processes are manipulated by UmSee1 and the UmSee1-SGT1 interaction to cause the observed phenotype. Proximity-dependent protein labeling involving the turbo biotin ligase tag (TurboID) for proximal labeling of proteins is a powerful tool for identifying the protein interactome. We have generated transgenic U. maydis that secretes biotin ligase-fused See1 effector (UmSee1-TurboID-3HA) directly into maize cells. This approach, in combination with conventional co-immunoprecipitation, allowed the identification of additional UmSee1 interactors in maize cells. Collectively, our data identified three ubiquitin-proteasome pathway-related proteins (ZmSIP1, ZmSIP2, and ZmSIP3) that either interact with or are close to UmSee1 during host infection of maize with U. maydis. ZmSIP3 represents a cell cycle regulator whose degradation appears to be promoted in the presence of UmSee1. Our data provide a possible explanation of the requirement for UmSee1 in tumor formation during U. maydis-Zea mays interaction.

Detection of Apoplastic Protease Inhibitors Using Convolution Activity-Based Protein Profiling.

Activity-based protein profiling (ABPP) is a powerful tool in biological chemistry to monitor protein activity using chemical probes that bind covalently and irreversible to active site of enzymes such as proteases. To date, there are three different ways to experimentally use ABPP: comparative, competitive, and convolution ABPP. Here we use and describe the convolution ABPP approach, a method used to detect changes in protease inhibitor abundance in different proteomes. We have applied this method to monitor the activity of Lolium perenne apoplastic cysteine proteases during the interaction with the fungal endophyte Epichloë festucae. We describe the method to isolate apoplastic fluids from infected and uninfected L. perenne ryegrass leaves and the protocol to perform a convolution ABPP experiment. Furthermore, we report how to quantify and analyze fluorescent gels obtained from the ABPP labeling.

Host apoplastic cysteine protease activity is suppressed during the mutualistic association of Lolium perenne and Epichloë 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. Epichloë 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.

Molecular Interactions Between Smut Fungi and Their Host Plants.

Smut fungi are a large group of biotrophic plant pathogens that infect mostly monocot species, including economically relevant cereal crops. For years, Ustilago maydis has stood out as the model system to study the genetics and cell biology of smut fungi as well as the pathogenic development of biotrophic plant pathogens. The identification and functional characterization of secreted effectors and their role in virulence have particularly been driven forward using the U. maydis-maize pathosystem. Today, advancing tools for additional smut fungi such as Ustilago hordei and Sporisorium reilianum, as well as an increasing number of available genome sequences, provide excellent opportunities to investigate in parallel the effector function and evolution associated with different lifestyles and host specificities. In addition, genome analyses revealed similarities in the genomic signature between pathogenic smuts and epiphytic Pseudozyma species. This review elaborates on how knowledge about fungal lifestyles, genome biology, and functional effector biology has helped in understanding the biology of this important group of fungal pathogens. We highlight the contribution of the U. maydis model system but also discuss the differences from other smut fungi, which raises the importance of comparative genomic and genetic analyses in future research.

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.

A fungal substrate mimicking molecule suppresses plant immunity via an inter-kingdom conserved motif.

Ustilago maydis is a biotrophic fungus causing corn smut disease in maize. The secreted effector protein Pit2 is an inhibitor of papain-like cysteine proteases (PLCPs) essential for virulence. Pit2 inhibitory function relies on a conserved 14 amino acids motif (PID14). Here we show that synthetic PID14 peptides act more efficiently as PLCP inhibitors than the full-length Pit2 effector. Mass spectrometry shows processing of Pit2 by maize PLCPs, which releases an inhibitory core motif from the PID14 sequence. Mutational analysis demonstrates that two conserved residues are essential for Pit2 function. We propose that the Pit2 effector functions as a substrate mimicking molecule: Pit2 is a suitable substrate for apoplastic PLCPs and its processing releases the embedded inhibitor peptide, which in turn blocks PLCPs to modulate host immunity. Remarkably, the PID14 core motif is present in several plant associated fungi and bacteria, indicating the existence of a conserved microbial inhibitor of proteases (cMIP).

Dynamic hydrolase labelling as a marker for seed quality in Arabidopsis seeds.

Seed quality is affected by different constituents of the seed. In general, seed lots are considered to be of high quality when they exhibit fast and homogeneous germination. When seeds are stored, they undergo different degrees of damage that have detrimental effects on their quality. Therefore, accurate prediction of the seed quality and viability levels of a seed lot is of high importance in the seed-producing industry. Here, we describe the use of activity-based protein profiling of proteases to evaluate the quality of artificially and naturally aged seeds of Arabidopsis thaliana Using this approach, we have identified two protease activities with opposite behaviours in aged seeds of Arabidopsis that correlate with the quality status of the seeds. We show that vacuolar processing enzymes (VPEs) become more active during the ageing process, in both artificial and natural ageing treatments. Secondly, we demonstrate that serine hydrolases are active at the beginning of our artificial ageing treatment, but their labelling decreases along with seed viability. We present a list of candidate hydrolases active during seed germination and propose that these protease activities can be used in combination with VPEs to develop novel markers of seed quality.

Proteasome Activity Profiling Uncovers Alteration of Catalytic ß2 and ß5 Subunits of the Stress-Induced Proteasome during Salinity Stress in Tomato Roots.

The stress proteasome in the animal kingdom facilitates faster conversion of oxidized proteins during stress conditions by incorporating different catalytic ß subunits. Plants deal with similar kind of stresses and also carry multiple paralogous genes encoding for each of the three catalytic ß subunits. Here, we investigated the existence of stress proteasomes upon abiotic stress (salt stress) in tomato roots. In contrast to Arabidopsis thaliana, tomato has a simplified proteasome gene set with single genes encoding each ß subunit except for two genes encoding ß2. Using proteasome activity profiling on tomato roots during salt stress, we discovered a transient modification of the catalytic subunits of the proteasome coinciding with a loss of cell viability. This stress-induced active proteasome disappears at later time points and coincides with the need to degrade oxidized proteins during salt stress. Subunit-selective proteasome probes and MS analysis of fluorescent 2D gels demonstrated that the detected stress-induced proteasome is not caused by an altered composition of subunits in active proteasomes, but involves an increased molecular weight of both labeled ß2 and ß5 subunits, and an additional acidic pI shift for labeled ß5, whilst labeled ß1 remains mostly unchanged. Treatment with phosphatase or glycosidases did not affect the migration pattern. This stress-induced proteasome may play an important role in PCD during abiotic stress.

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 ß1 and ß5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1-1 [PtoDC3000(ΔhQ)] whilst the activity profile of the ß1 subunit changes. Infection with wild-type PtoDC3000 causes proteasome activities that range from strongly induced ß1 and ß5 activities to strongly suppressed ß5 activities, revealing that ß1 and ß5 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.

Papain-like cysteine proteases as hubs in plant immunity.

902 I. 902 II. 903 III. 903 IV. 903 V. 905 VI. 905 VII. 905 906 References 906 SUMMARY: Plants deploy a sophisticated immune system to cope with different microbial pathogens and other invaders. Recent research provides an increasing body of evidence for papain-like cysteine proteases (PLCPs) being central hubs in plant immunity. PLCPs are required for full resistance of plants to various pathogens. At the same time, PLCPs are targeted by secreted pathogen effectors to suppress immune responses. Consequently, they are subject to a co-evolutionary host-pathogen arms race. When activated, PLCPs induce a broad spectrum of defense responses including plant cell death. While the important role of PLCPs in plant immunity has become more evident, it remains largely elusive how these enzymes are activated and which signaling pathways are triggered to orchestrate different downstream responses.

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.

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination.

Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.

SNARE-RNAi results in higher terpene emission from ectopically expressed caryophyllene synthase in Nicotiana benthamiana.

Plants produce numerous terpenes and much effort has been dedicated to the identification and characterization of the terpene biosynthetic genes. However, little is known about how terpenes are transported within the cell and from the cell into the apoplast. To investigate a putative role of vesicle fusion in this process, we used Agrobacterium tumefaciens-mediated transient coexpression in Nicotiana benthamiana of an MtVAMP721e-RNAi construct (Vi) with either a caryophyllene synthase or a linalool synthase, respectively. Headspace analysis of the leaves showed that caryophyllene or linalool emission increased about five-fold when N. benthamiana VAMP72 function was blocked. RNA sequencing and protein ubiquitination analysis of the agroinfiltrated N. benthamiana leaf extracts suggested that increased terpene emissions may be attributed to proteasome malfunction based on three observations: leaves with TPS+Vi showed (1) a higher level of a DsRed marker protein, (2) a higher level of ubiquitinated proteins, and (3) coordinated induced expression of multiple proteasome genes, presumably caused by the lack of proteasome-mediated feedback regulation. However, caryophyllene or linalool did not inhibit proteasome-related protease activity in the in vitro assays. While the results are not conclusive for a role of vesicle fusion in terpene transport, they do show a strong interaction between inhibition of vesicle fusion and ectopic expression of certain terpenes. The results have potential applications in metabolic engineering.

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.

Pseudomonas syringae pv. syringae 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 of vacuolar processing enzymes reveals a role for VPE during oomycete infection.

Vacuolar processing enzymes (VPEs) are important cysteine proteases that are implicated in the maturation of seed storage proteins, and programmed cell death during plant-microbe interactions and development. Here, we introduce a specific, cell-permeable, activity-based probe for VPEs. This probe is highly specific for all four Arabidopsis VPEs, and labeling is activity-dependent, as illustrated by sensitivity for inhibitors, pH and reducing agents. We show that the probe can be used for in vivo imaging and displays multiple active isoforms of VPEs in various tissues and in both monocot and dicot plant species. Thus, VPE activity profiling is a robust, simple and powerful tool for plant research for a wide range of applications. Using VPE activity profiling, we discovered that VPE activity is increased during infection with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). The enhanced VPE activity is host-derived and EDS1-independent. Sporulation of Hpa is reduced on vpe mutant plants, demonstrating a role for VPE during compatible interactions that is presumably independent of programmed cell death. Our data indicate that, as an obligate biotroph, Hpa takes advantage of increased VPE activity in the host, e.g. to mediate protein turnover and nutrient release.

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.

Pseudomonas syringae colonizes distant tissues in Nicotiana benthamiana through xylem vessels.

The ability to move from the primary infection site and colonize distant tissue in the leaf is an important property of bacterial plant pathogens, yet this aspect has hardly been investigated for model pathogens. Here we show that GFP-expressing Pseudomonas syringae pv. syringae DC3000 that lacks the HopQ1-1 effector (PtoDC3000ΔhQ) has a strong capacity to colonize distant leaf tissue from wound-inoculated sites in N. benthamiana. Distant colonization occurs within 1 week after toothpick inoculation and is characterized by distant colonies in the apoplast along the vasculature. Distant colonization is blocked by the non-host resistance response triggered by HopQ1-1 in an SGT1-dependent manner and is associated with an explosive growth of the bacterial population, and displays robust growth differences between compatible and incompatible interactions. Scanning electron microscopy revealed that PtoDC3000ΔhQ bacteria are present in xylem vessels, indicating that they use the xylem to move through the leaf blade. Distant colonization does not require flagellin-mediated motility, and is common for P. syringae pathovars that represent different phylogroups.