902I.902II.903III.903IV.903V.905VI.905VII.905906References906 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.
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.
SUMMARYThe 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 membranepermeable, 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.
Syringolin A (SylA) is a nonribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv syringae that can inhibit the eukaryotic proteasome. The proteasome is a multisubunit proteolytic complex that resides in the nucleus and cytoplasm and contains three subunits with different catalytic activities: b1, b2, and b5. Here, we studied how SylA targets the plant proteasome in living cells using activity-based profiling and imaging. We further developed this technology by introducing new, more selective probes and establishing procedures of noninvasive imaging in living Arabidopsis (Arabidopsis thaliana) cells. These studies showed that SylA preferentially targets b2 and b5 of the plant proteasome in vitro and in vivo. Structure-activity analysis revealed that the dipeptide tail of SylA contributes to b2 specificity and identified a nonreactive SylA derivative that proved essential for imaging experiments. Interestingly, subcellular imaging with probes based on epoxomicin and SylA showed that SylA accumulates in the nucleus of the plant cell and suggests that SylA targets the nuclear proteasome. Furthermore, subcellular fractionation studies showed that SylA labels nuclear and cytoplasmic proteasomes. The selectivity of SylA for the catalytic subunits and subcellular compartments is discussed, and the subunit selectivity is explained by crystallographic data.
SUMMARYVacuolar 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.
The plant apoplast during plant-pathogen interactions is an ancient battleground that holds an intriguing range of attacking enzymes and counteracting inhibitors. Examples are pathogen xylanases and polygalacturonases that are inhibited by plant proteins like TAXI, XIP, and PGIP; and plant glucanases and proteases, which are targeted by pathogen proteins such as GIP1, EPI1, EPIC2B, and AVR2. These seven well-characterized inhibitors have different modes of action and many probably evolved from inactive enzymes themselves. Detailed studies of the structures, sequence variation, and mutated proteins uncovered molecular struggles between these enzymes and their inhibitors, resulting in positive selection for variant residues at the contact surface, where single residues determine the outcome of the interaction. Introduction Extracellular plant-pathogen interactions probably existed long before the evolution of pathogen effector translocation systems and plant resistance (R) genes. The molecular basis of these interactions is mostly undiscovered but some have been investigated in detail and reveal intriguing mechanisms. Here we will highlight major recent findings of extracellular enzyme-inhibitor interactions at the plant-pathogen interface.Although extracellular plant-pathogen interactions are complex, they can be simplified by assuming that they evolved in several stages (Figure 1). First, micro-organisms became pathogens by attacking plants using cell-wall-degrading enzymes and other hydrolases ( Figure 1A). In response to this attack, plants secrete inhibitors that suppress these hydrolases ( Figure 1B). Initially, these inhibitors were probably constitutively produced, but upon evolution of pathogen recognition systems the production and secretion of these proteins became inducible, becoming part of the arsenal of pathogenesis-related (PR) proteins. Besides suppression of pathogen attack, counter attack mechanisms also evolved in plants through the induced secretion of hydrolytic enzymes ( Figure 1C). Examples are the well-studied PR proteins including endo-b-1,3-glucanases (PR-2), chitinases (PR-3), and proteases (PR-7) [1 ]. Pathogens, in turn, responded to this counter attack by producing inhibitors that suppress these enzymes ( Figure 1D). The fifth and latest step was a sophisticated refinement of the pathogen recognition system by the evolution of R genes that recognize the manipulation of plant targets by pathogens, inducing a severe defense response that includes cell death ( Figure 1E). Aspects of this simplified model are consistent with the 'zigzag' model for the plant immune system, which explains the suppression of basal defense responses by pathogen effector proteins, followed by the evolution of efficient effector recognition by R proteins [2].Antagonistic interactions between organisms at the molecular level result in enzymes that evade inhibition, and inhibitors that adapt to these new enzymes. These 'molecular struggles' result in positive selection for variation of residues at the interactio...
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.
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.
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