Plant ESCRT protein ALIX coordinates with retromer complex in regulating receptor-mediated sorting of soluble vacuolar proteins.

Vacuolar proteins play essential roles in plant physiology and development, but the factors and the machinery regulating their vesicle trafficking through the endomembrane compartments remain largely unknown. We and others have recently identified an evolutionarily conserved plant endosomal sorting complex required for transport (ESCRT)-associated protein apoptosis-linked gene-2 interacting protein X (ALIX), which plays canonical functions in the biogenesis of the multivesicular body/prevacuolar compartment (MVB/PVC) and in the sorting of ubiquitinated membrane proteins. In this study, we elucidate the roles and underlying mechanism of ALIX in regulating vacuolar transport of soluble proteins, beyond its conventional ESCRT function in eukaryotic cells. We show that ALIX colocalizes and physically interacts with the retromer core subunits Vps26 and Vps29 in planta. Moreover, double-mutant analysis reveals the genetic interaction of ALIX with Vps26 and Vps29 for regulating trafficking of soluble vacuolar proteins. Interestingly, depletion of ALIX perturbs membrane recruitment of Vps26 and Vps29 and alters the endosomal localization of vacuolar sorting receptors (VSRs). Taken together, ALIX functions as a unique retromer core subcomplex regulator by orchestrating receptor-mediated vacuolar sorting of soluble proteins.

MTV proteins unveil ER- and microtubule-associated compartments in the plant vacuolar trafficking pathway.

The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1 Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants.

Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis.

The phytohormone cytokinin has been shown to affect many aspects of plant development ranging from the regulation of the shoot apical meristem to leaf senescence. However, some studies have reported contradictory effects of cytokinin on leaf physiology. Therefore cytokinin treatments cause both chlorosis and increased greening and both lead to decrease or increase in cell size. To elucidate this multifaceted role of cytokinin in leaf development, we have employed a system of temporal controls over the cytokinin pool and investigated the consequences of modulated cytokinin levels in the third leaf of Arabidopsis. We show that, at the cell proliferation phase, cytokinin is needed to maintain cell proliferation by blocking the transition to cell expansion and the onset of photosynthesis. Transcriptome profiling revealed regulation by cytokinin of a gene suite previously shown to affect cell proliferation and expansion and thereby a molecular mechanism by which cytokinin modulates a molecular network underlying the cellular responses. During the cell expansion phase, cytokinin stimulates cell expansion and differentiation. Consequently, a cytokinin excess at the cell expansion phase results in an increased leaf and rosette size fueled by higher cell expansion rate, yielding higher shoot biomass. Proteome profiling revealed the stimulation of primary metabolism by cytokinin, in line with an increased sugar content that is expected to increase turgor pressure, representing the driving force of cell expansion. Therefore, the developmental timing of cytokinin content fluctuations, together with a tight control of primary metabolism, is a key factor mediating transitions from cell proliferation to cell expansion in leaves.

Equilibrium dynamics of European pre-industrial populations: the evidence of carrying capacity in human agricultural societies.

Human populations tend to grow steadily, because of the ability of people to make innovations, and thus overcome and extend the limits imposed by natural resources. It is therefore questionable whether traditional concepts of population ecology, including environmental carrying capacity, can be applied to human societies. The existence of carrying capacity cannot be simply inferred from population time-series, but it can be indicated by the tendency of populations to return to a previous state after a disturbance. So far only indirect evidence at a coarse-grained scale has indicated the historical existence of human carrying capacity. We analysed unique historical population data on 88 settlements before and after the Thirty Years War (1618-1648), one the longest and most destructive conflicts in European history, which reduced the population of Central Europe by 30-50%. The recovery rate of individual settlements after the war was positively correlated with the extent of the disturbance, so that the population size of the settlements after a period of regeneration was similar to the pre-war situation, indicating an equilibrium population size (i.e. carrying capacity). The carrying capacity of individual settlements was positively determined mostly by the fertility of the soil and the area of the cadastre, and negatively by the number of other settlements in the surroundings. Pre-industrial human population sizes were thus probably controlled by negative density dependence mediated by soil fertility, which could not increase due to limited agricultural technologies.

Helping hands for budding prospects: ENTH/ANTH/VHS accessory proteins in endocytosis, vacuolar transport, and secretion.

Coated vesicles provide a major mechanism for the transport of proteins through the endomembrane system of plants. Transport between the endoplasmic reticulum and the Golgi involves vesicles with COPI and COPII coats, whereas clathrin is the predominant coat in endocytosis and post-Golgi trafficking. Sorting of cargo, coat assembly, budding, and fission are all complex and tightly regulated processes that involve many proteins. The mechanisms and responsible factors are largely conserved in eukaryotes, and increasing organismal complexity tends to be associated with a greater numbers of individual family members. Among the key factors is the class of ENTH/ANTH/VHS domain-containing proteins, which link membrane subdomains, clathrin, and other adapter proteins involved in early steps of clathrin coated vesicle formation. More than 30 Arabidopsis thaliana proteins contain this domain, but their generally low sequence conservation has made functional classification difficult. Reports from the last two years have greatly expanded our knowledge of these proteins and suggest that ENTH/ANTH/VHS domain proteins are involved in various instances of clathrin-related endomembrane trafficking in plants. This review aims to summarize these new findings and discuss the broader context of clathrin-dependent plant vesicular transport.

MTV1 and MTV4 encode plant-specific ENTH and ARF GAP proteins that mediate clathrin-dependent trafficking of vacuolar cargo from the trans-Golgi network.

Many soluble proteins transit through the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) en route to the vacuole, but our mechanistic understanding of this vectorial trafficking step in plants is limited. In particular, it is unknown whether clathrin-coated vesicles (CCVs) participate in this transport step. Through a screen for modified transport to the vacuole (mtv) mutants that secrete the vacuolar protein VAC2, we identified MTV1, which encodes an epsin N-terminal homology protein, and MTV4, which encodes the ADP ribosylation factor GTPase-activating protein nevershed/AGD5. MTV1 and NEV/AGD5 have overlapping expression patterns and interact genetically to transport vacuolar cargo and promote plant growth, but they have no apparent roles in protein secretion or endocytosis. MTV1 and NEV/AGD5 colocalize with clathrin at the TGN and are incorporated into CCVs. Importantly, mtv1 nev/agd5 double mutants show altered subcellular distribution of CCV cargo exported from the TGN. Moreover, MTV1 binds clathrin in vitro, and NEV/AGD5 associates in vivo with clathrin, directly linking these proteins to CCV formation. These results indicate that MTV1 and NEV/AGD5 are key effectors for CCV-mediated trafficking of vacuolar proteins from the TGN to the PVC in plants.

Vocal activity of lesser galagos (Galago spp.) at zoos.

Almost nothing is known about the natural vocal behavior of lesser galagos living in zoos. This is perhaps because they are usually kept in nocturnal exhibits separated from the visitors by a transparent and acoustically insulating glass barrier. The aim of the present study was therefore to fill this gap in knowledge of the vocal behavior of lesser galagos from zoos. This knowledge might be beneficial because the vocalizations of these small primates can be used for species determination. We performed a 10-day-long acoustic monitoring of vocal activity in each of seven various groups of Galago senegalensis and G. moholi living at four zoos. We quantitatively evaluated the occurrence of four loud vocalization types present in both species, including the most species-specific advertisement call. We found that qualitative as well as quantitative differences exist in the vocal behavior of the studied groups. We confirmed that the observed vocalization types can be collected from lesser galagos living at zoos, and the success can be increased by selecting larger and more diverse groups. We found two distinct patterns of diel vocal activity in the most vocally active groups. G. senegalensis groups were most vocally active at the beginning and at the end of their activity period, whereas one G. moholi group showed an opposite pattern. The latter is surprising, as it is generally accepted that lesser galagos emit advertisement calls especially at dawn and dusk, i.e., at the beginning and at the end of their diel activity.

Sortin2 enhances endocytic trafficking towards the vacuole in Saccharomyces cerevisiae.

BACKGROUND: A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae. RESULTS: A reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked. CONCLUSIONS: Overall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.

Differences in alarm calls of juvenile and adult European ground squirrels (Spermophilus citellus): Findings on permanently marked animals from a semi-natural enclosure.

The European ground squirrel (Spermophilus citellus) emits alarm calls that warn conspecifics of potential danger. Although it has been observed that inexperienced juveniles of this species emit alarm calls that sound similar to those of adults, studies focusing on juvenile alarm calls are lacking. We analyzed the acoustic structure of alarm calls emitted by six permanently marked European ground squirrels living in a semi-natural enclosure when they were juveniles and after 1 year as adults. We found that the acoustic structure of the juvenile alarm calls was significantly different from those of adults and that the alarm calls underwent nearly the same changes in all studied individuals. All juveniles emitted alarm calls consisting of one element with almost constant frequency, but their alarm calls included a second frequency-modulated element after their first hibernation as adults. Our data show that the duration of the first element is significantly shorter in adults than in juveniles. Additionally, the frequency of the first element is significantly higher in adults than in juveniles. Similar to previous findings in other Palearctic ground squirrel species, our data are inconsistent with the assumption that juvenile mammals emit vocalizations with higher fundamental frequencies than adults. However, our results do not support the previously suggested hypothesis that juvenile ground squirrels conceal information regarding their age in their alarm calls because we found significant differences in alarm calls of juveniles and adults.

Functional identification of sorting receptors involved in trafficking of soluble lytic vacuolar proteins in vegetative cells of Arabidopsis.

In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis ßFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.

A sword or a buffet: plant endomembrane system in viral infections.

The plant endomembrane system is an elaborate collection of membrane-bound compartments that perform distinct tasks in plant growth and development, and in responses to abiotic and biotic stresses. Most plant viruses are positive-strand RNA viruses that remodel the host endomembrane system to establish intricate replication compartments. Their fundamental role is to create optimal conditions for viral replication, and to protect replication complexes and the cell-to-cell movement machinery from host defenses. In addition to the intracellular antiviral defense, represented mainly by RNA interference and effector-triggered immunity, recent findings indicate that plant antiviral immunity also includes membrane-localized receptor-like kinases that detect viral molecular patterns and trigger immune responses, which are similar to those observed for bacterial and fungal pathogens. Another recently identified part of plant antiviral defenses is executed by selective autophagy that mediates a specific degradation of viral proteins, resulting in an infection arrest. In a perpetual tug-of-war, certain host autophagy components may be exploited by viral proteins to support or protect an effective viral replication. In this review, we present recent advances in the understanding of the molecular interplay between viral components and plant endomembrane-associated pathways.

Retrograde transport in plants: Circular economy in the endomembrane system.

The study of endomembrane trafficking is crucial for understanding how cells and whole organisms function. Moreover, there is a special interest in investigating endomembrane trafficking in plants, given its role in transport and accumulation of seed storage proteins and in secretion of cell wall material, arguably the two most essential commodities obtained from crops. The mechanisms of anterograde transport in the biosynthetic and endocytic pathways of plants have been thoroughly discussed in recent reviews, but, comparatively, retrograde trafficking pathways have received less attention. Retrograde trafficking is essential to recover membranes, retrieve proteins that have escaped from their intended localization, maintain homeostasis in maturing compartments, and recycle trafficking machinery for its reuse in anterograde transport reactions. Here, we review the current understanding on retrograde trafficking pathways in the endomembrane system of plants, discussing their integration with anterograde transport routes, describing conserved and plant-specific retrieval mechanisms at play, highlighting contentious issues and identifying open questions for future research.

Effect of high-speed steel screw drill geometry on cutting performance when machining austenitic stainless steel.

Drilling into the solid material is one of the basic technological operations, which creates a cylindrical hole in an appropriate time with required quality. Drilling operation demands a favourable removal of chips from the cutting area because a creation of an undesirable shape of chips can impart a lower quality of the drilled hole corresponding with the generation of excess heat due to the intense contact of the chip with drill. The solution for a proper machining is a suitable modification of the drill geometry i.e., point and clearance angles as presented in current study. The tested drills are made of M35 high-speed steel characterized by a very thin core at the point of the drill. An interesting feature of the drills is the use of cutting speed higher than 30 m min-1, with the feed of 0.2 mm per revolution. The surface roughness (Ra and Rz lower than 1 µm and 6 µm respectively), cylindricity (0.045 mm), roundness (0.025 mm), perpendicularity of the hole axis (0.025 mm), diameters and position of the individual holes were achieved for a drill with point angle 138.32°and clearance angle 6.92 respectively. The increase of the drill point angle by 6° resulted in the decrease in the feed force of more than 150 N. In addition, an increase of the clearance angle by 1° resulted with a decrease in the feed force of 70 N. The results of the experiment showed that with the correct geometry of the tool the effective machining without using internal cooling can be realised.

Influence of Aging Temperature on Mechanical Properties and Structure of M300 Maraging Steel Produced by Selective Laser Melting.

This paper deals with the study of high-strength M300 maraging steel produced using the selective laser melting method. Heat treatment consists of solution annealing and subsequent aging; the influence of the selected aging temperatures on the final mechanical properties-microhardness and compressive yield strength-and the structure of the maraging steel are described in detail. The microstructure of the samples is examined using optical and electron microscopy. The compressive test results show that the compressive yield strength increased after heat treatment up to a treatment temperature of 480 °C and then gradually decreased. The sample aged at 480 °C also exhibited the highest observed microhardness of 562 HV. The structure of this sample changed from the original melt pools to a relatively fine-grained structure with a high fraction of high-angle grain boundaries (72%).

Analysis of the Mechanical Properties of 3D-Printed Plastic Samples Subjected to Selected Degradation Effects.

The Fused Filament Fabrication (FFF) method is an additive technology that is used for the creation of prototypes within Rapid Prototyping (RP) as well as for the creation of final components in piece or small-series production. The possibility of using FFF technology in the creation of final products requires knowledge of the properties of the material and, at the same time, how these properties change due to degradation effects. In this study, the mechanical properties of the selected materials (PLA, PETG, ABS, and ASA) were tested in their non-degenerate state and after exposure of the samples to the selected degradation factors. For the analysis, which was carried out by the tensile test and the Shore D hardness test, samples of normalized shape were prepared. The effects of UV radiation, high temperature environments, high humidity environments, temperature cycles, and exposure to weather conditions were monitored. The parameters obtained from the tests (tensile strength and Shore D hardness) were statistically evaluated, and the influence of degradation factors on the properties of individual materials was assessed. The results showed that even between individual manufacturers of the same filament there are differences, both in the mechanical properties and in the behavior of the material after exposure to degradation effects.

Processing of Bimetallic Inconel 625-16Mo3 Steel Tube via Supercritical Bend: Study of the Mechanical Properties and Structure.

Incineration is currently the standard way of disposing of municipal waste. It uses components protected by high-temperature-resistant layers of materials, such as Inconel alloys. Therefore, the objective of the current paper is to study the mechanical properties and structure of a bimetallic Inconel 625-16Mo3 steel tube. The Inconel 625 layer was 3.5 mm thick and was applied to the surface of the tube with a wall thickness of 7 mm via the cold metal transfer method. The bimetallic tube was bent using a supercritical bend (d ≤ 0.7D). This paper is focused on the investigation of the material changes in the Inconel 625 layer areas influenced by the maximum tensile and compressive stresses after the bend. The change in layer thickness after the bend was evaluated and compared to the non-deformed tube. In addition, the local mechanical properties (nanohardness, Young modulus) across the indicated interfacial areas using quasistatic nanoindentation were investigated. Subsequently, a thorough microstructure observation was carried out in areas with maximum tensile and compressive stresses to determine changes in the morphology and size of dendrites related to the effect of tensile or compressive stresses induced by bending. It was found that the grain featured a stretched secondary dendrite axis in the area of tensile stress, but compressive stress imparted a prolongation of the primary dendrite axis.

Application of Carbon-Flax Hybrid Composite in High Performance Electric Personal Watercraft.

Within the herein presented research, we studied the applicability of flax fabrics for composite parts in personal watercrafts in order to enhance damping of vibrations from the engine and noise reduction (which is relatively high for contemporary carbon constructions). Since the composite parts are intended to be exposed to humid environments requiring high levels of mechanical properties, a carbon-flax composite was selected. Samples of carbon, fiberglass, flax, and hybrid carbon-flax twill and biax fabrics were subjected to tensile and three-point bending tests. The mechanical properties were also tested after exposure of the samples to a humid environment. Damping was assessed by vibration and noise measurements directly on the complete float for samples as well as real parts. The hybrid carbon-flax material exhibited lower values of tensile strength than the carbon material (760 MPa compared to 463 MPa), but, at the same time, significantly higher than the other tested materials, or flax itself (115 MPa for a twill fabric). A similar trend in the results was observed for the three-point bending tests. Vibration tests and noise measurements showed reductions in vibration amplitude and frequency when using the carbon-flax hybrid material; the frequency response function for the watercraft part assembled from the hybrid material was 50% lower than for that made of carbon. Testing of samples located in a humid environment showed the necessity of surface treatment to prevent moisture absorption (mechanical properties were reduced at minimum by 28%). The tests confirmed that the hybrid material is satisfactory in terms of strength and its contribution to noise and vibration damping.

Ecological and historical factors behind the spatial structure of the historical field patterns in the Czech Republic.

Historical field systems are an essential part of the traditional cultural landscape of societies with primarily agricultural subsistence. They embody many functions and values, as they affect the productional, ecological and hydrological functioning of the landscape, its cultural values, the way people perceive the landscape, and their impact on present-day farming. As an aspect of the historical landscape, field systems are a topic investigated in landscape archaeology, environmental studies, historical geography, landscape ecology, and related disciplines. Historical field systems can form many complex spatial structures, shapes and patterns. This paper focuses on identifying environmental and historical/cultural driving forces during the formation and the historical development of various field pattern types. We worked with 523 settlements established in the medieval to the early modern period (approx. 900-1600 AD) in the present-day Czech Republic. We have determined the proportions of different field pattern types in the examined cadastres and have statistically compared them with a variety of environmental and geographical predictors. Our results indicate a strong influence of environmental predictors (terrain undulation, cadastre size), the impact of specific historical events and associated social changes (e.g. land confiscations by the state in the seventeenth century), and a significant relationship between field pattern types and settlement layout types. Furthermore, we have observed the different adaptations of field pattern types to similar environmental conditions, as well as the impact of social and political factors on the processes of landscape formation. Our paper provides the first detailed analysis of the geographical distribution of traditional field systems on the scale of an entire modern state, and emphasizes the importance of transdisciplinary research on cultural landscapes.

Functional specialization within the vacuolar sorting receptor family: VSR1, VSR3 and VSR4 sort vacuolar storage cargo in seeds and vegetative tissues.

Two different gene families have been proposed to act as sorting receptors for vacuolar storage cargo in plants: the vacuolar sorting receptors (VSRs) and the receptor homology-transmembrane-RING H2 domain proteins (RMRs). However, functional data on these genes is scarce and the identity of the sorting receptor for storage proteins remains controversial. Through a genetic screen we have identified the mtv2 mutant, which is defective in vacuolar transport of the storage cargo VAC2 in shoot apices. Map-based cloning revealed that mtv2 is a loss of function allele of the VSR4 gene. We show that VSR1, VSR3 and VSR4, but not the remaining VSRs or RMRs, participate in vacuolar sorting of VAC2 in vegetative tissues, and 12S globulins and 2S albumins in seeds, an activity that is essential for seedling germination vigor. Finally, we demonstrate that the functional diversification in the VSR family results from divergent expression patterns and also from distinct sorting activities of the family members.

VPEgamma exhibits a caspase-like activity that contributes to defense against pathogens.

BACKGROUND: Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants. RESULTS: We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens. CONCLUSIONS: We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.