Activity-based proteomics uncovers suppressed hydrolases and a neo-functionalised antibacterial enzyme at the plant-pathogen interface.

The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonising microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in Nicotiana benthamiana upon infection with Pseudomonas syringae. Using activity-based proteomics with a cocktail of biotinylated probes, we simultaneously monitored 171 active hydrolases, including 109 serine hydrolases (SHs), 49 glycosidases (GHs) and 13 cysteine proteases (CPs). The activity of 82 of these hydrolases (mostly SHs) increases during infection, while the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active ß-galactosidase-1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor by P. syringae. One of the other suppressed hydrolases, the pathogenesis-related NbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role for NbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase, NbPR3 does not possess chitinase activity and contains an E112Q active site substitution that is essential for antibacterial activity and is present only in Nicotiana species. This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo-functionalised Nicotiana-specific antibacterial NbPR3.

Do photosynthetic cells communicate with each other during cell death? From cyanobacteria to vascular plants.

As in metazoans, life in oxygenic photosynthetic organisms relies on the accurate regulation of cell death. During development and in response to the environment, photosynthetic cells activate and execute cell death pathways that culminate in the death of a specific group of cells, a process known as regulated cell death (RCD). RCD control is instrumental, as its misregulation can lead to growth penalties and even the death of the entire organism. Intracellular molecules released during cell demise may act as 'survival' or 'death' signals and control the propagation of cell death to surrounding cells, even in unicellular organisms. This review explores different signals involved in cell-cell communication and systemic signalling in photosynthetic organisms, in particular Ca2+, reactive oxygen species, lipid derivates, nitric oxide, and eATP. We discuss their possible mode-of-action as either 'survival' or 'death' molecules and their potential role in determining cell fate in neighbouring cells. By comparing the knowledge available across the taxonomic spectrum of this coherent phylogenetic group, from cyanobacteria to vascular plants, we aim at contributing to the identification of conserved mechanisms that control cell death propagation in oxygenic photosynthetic organisms.

The sesquiterpene botrydial from Botrytis cinerea induces phosphatidic acid production in tomato cell suspensions.

MAIN CONCLUSION: The phytotoxin botrydial triggers PA production in tomato cell suspensions via PLD and PLC/DGK activation. PLC/DGK-derived PA is partially required for botrydial-induced ROS generation. Phosphatidic acid (PA) is a phospholipid second messenger involved in the induction of plant defense responses. It is generated via two distinct enzymatic pathways, either via phospholipase D (PLD) or by the sequential action of phospholipase C and diacylglycerol kinase (PLC/DGK). Botrydial is a phytotoxic sesquiterpene generated by the necrotrophic fungus Botrytis cinerea that induces diverse plant defense responses, such as the production of reactive oxygen species (ROS). Here, we analyzed PA and ROS production and their interplay upon botrydial treatments, employing tomato (Solanum lycopersicum) cell suspensions as a model system. Botrydial induces PA production within minutes via PLD and PLC/DGK. Either inhibition of PLC or DGK diminishes ROS generation triggered by botrydial. This indicates that PLC/DGK is upstream of ROS production. In tomato, PLC is encoded by a multigene family constituted by SlPLC1-SlPLC6 and the pseudogene SlPLC7. We have shown that SlPLC2-silenced plants have reduced susceptibility to B. cinerea. In this work, we studied the role of SlPLC2 on botrydial-induced PA production by silencing the expression of SlPLC2 via a specific artificial microRNA. Upon botrydial treatments, SlPLC2-silenced-cell suspensions produce PA levels similar to wild-type cells. It can be concluded that PA is a novel component of the plant responses triggered by botrydial.

From structure to function - a family portrait of plant subtilases.

Contents Summary 901 I. Introduction 901 II. Biochemistry and structure of plant SBTs 902 III. Phylogeny of plant SBTs and family organization 903 IV. Physiological roles of plant SBTs 905 V. Conclusions and outlook 911 Acknowledgements 912 References 912 SUMMARY: Subtilases (SBTs) are serine peptidases that are found in all three domains of life. As compared with homologs in other Eucarya, plant SBTs are more closely related to archaeal and bacterial SBTs, with which they share many biochemical and structural features. However, in the course of evolution, functional diversification led to the acquisition of novel, plant-specific functions, resulting in the present-day complexity of the plant SBT family. SBTs are much more numerous in plants than in any other organism, and include enzymes involved in general proteolysis as well as highly specific processing proteases. Most SBTs are targeted to the cell wall, where they contribute to the control of growth and development by regulating the properties of the cell wall and the activity of extracellular signaling molecules. Plant SBTs affect all stages of the life cycle as they contribute to embryogenesis, seed development and germination, cuticle formation and epidermal patterning, vascular development, programmed cell death, organ abscission, senescence, and plant responses to their biotic and abiotic environments. In this article we provide a comprehensive picture of SBT structure and function in plants.

Random mutagenesis of the nucleotide-binding domain of NRC1 (NB-LRR Required for Hypersensitive Response-Associated Cell Death-1), a downstream signalling nucleotide-binding, leucine-rich repeat (NB-LRR) protein, identifies gain-of-function mutations in the nucleotide-binding pocket.

Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins confer immunity to pathogens possessing the corresponding avirulence proteins. Activation of NB-LRR proteins is often associated with induction of the hypersensitive response (HR), a form of programmed cell death. NRC1 (NB-LRR Required for HR-Associated Cell Death-1) is a tomato (Solanum lycopersicum) NB-LRR protein that participates in the signalling cascade leading to resistance to the pathogens Cladosporium fulvum and Verticillium dahliae. To identify mutations in NRC1 that cause increased signalling activity, we generated a random library of NRC1 variants mutated in their nucleotide-binding domain and screened them for the ability to induce an elicitor-independent HR in Nicotiana tabacum. Screening of 1920 clones retrieved 11 gain-of-function mutants, with 10 of them caused by a single amino acid substitution. All substitutions are located in or very close to highly conserved motifs within the nucleotide-binding domain, suggesting modulation of the signalling activity of NRC1. Three-dimensional modelling of the nucleotide-binding domain of NRC1 revealed that the targeted residues are centred around the bound nucleotide. Our mutational approach has generated a wide set of novel gain-of-function mutations in NRC1 and provides insight into how the activity of this NB-LRR is regulated.

Dynamic changes of the Prf/Pto tomato resistance complex following effector recognition.

In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen recognition and activation of innate immunity. In plants, NLRs recognise pathogen-derived effector proteins and initiate effector-triggered immunity (ETI). However, the molecular mechanisms that link NLR-mediated effector recognition and downstream signalling are not fully understood. By exploiting the well-characterised tomato Prf/Pto NLR resistance complex, we identified the 14-3-3 proteins TFT1 and TFT3 as interacting partners of both the NLR complex and the protein kinase MAPKKKα. Moreover, we identified the helper NRC proteins (NLR-required for cell death) as integral components of the Prf /Pto NLR recognition complex. Notably our studies revealed that TFTs and NRCs interact with distinct modules of the NLR complex and, following effector recognition, dissociate facilitating downstream signalling. Thus, our data provide a mechanistic link between activation of immune receptors and initiation of downstream signalling cascades.

Cell Death in Cyanobacteria: Current Understanding and Recommendations for a Consensus on Its Nomenclature.

Cyanobacteria are globally widespread photosynthetic prokaryotes and are major contributors to global biogeochemical cycles. One of the most critical processes determining cyanobacterial eco-physiology is cellular death. Evidence supports the existence of controlled cellular demise in cyanobacteria, and various forms of cell death have been described as a response to biotic and abiotic stresses. However, cell death research in this phylogenetic group is a relatively young field and understanding of the underlying mechanisms and molecular machinery underpinning this fundamental process remains largely elusive. Furthermore, no systematic classification of modes of cell death has yet been established for cyanobacteria. In this work, we analyzed the state of knowledge in the field of cyanobacterial cell death. Based on that, we propose unified criterion for the definition of accidental, regulated, and programmed forms of cell death in cyanobacteria based on molecular, biochemical, and morphologic aspects following the directions of the Nomenclature Committee on Cell Death (NCCD). With this, we aim to provide a guide to standardize the nomenclature related to this topic in a precise and consistent manner, which will facilitate further ecological, evolutionary, and applied research in the field of cyanobacterial cell death.

Phosphatidic acid formation is required for extracellular ATP-mediated nitric oxide production in suspension-cultured tomato cells.

*In animals and plants, extracellular ATP exerts its effects by regulating the second messengers Ca(2+), nitric oxide (NO) and reactive oxygen species (ROS). In animals, phospholipid-derived molecules, such as diacylglycerol, phosphatidic acid (PA) and inositol phosphates, have been associated with the extracellular ATP signaling pathway. The involvement of phospholipids in extracellular ATP signaling in plants, as it is established in animals, is unknown. *In vivo phospholipid signaling upon extracellular ATP treatment was studied in (32)P(i)-labeled suspension-cultured tomato (Solanum lycopersicum) cells. *Here, we report that, in suspension-cultured tomato cells, extracellular ATP induces the formation of the signaling lipid phosphatidic acid. Exogenous ATP at doses of 0.1 and 1 mM induce the formation of phosphatidic acid within minutes. Studies on the enzymatic sources of phosphatidic acid revealed the participation of both phospholipase D and C in concerted action with diacylglycerol kinase. *Our results suggest that extracellular ATP-mediated nitric oxide production is downstream of phospholipase C/diacylglycerol kinase activation.

Plant life needs cell death, but does plant cell death need Cys proteases?

Caspases are key regulators of apoptosis in animals. This correlation has driven plant researchers for decades to look for caspases regulating programmed cell death (PCD) in plants. These studies revealed caspase-like activities, caspase-related proteases, and cysteine (Cys) proteases regulating PCD in plants, but identified no caspases and no conserved, apoptosis-like death pathway. Here, we critically review the evidence for Cys proteases implicated in PCD in plants. We discuss the role of papain-like Cys proteases, vacuolar processing enzymes, and metacaspases in PCD during the development of tracheary elements, seed coat, suspensor, and tapetum, and during the hypersensitive response. There are several convincing cases where these Cys proteases are required for PCD, but this requirement is often not conserved across different plant species. There are also cases where Cys proteases contribute to the speed, but not the timing of PCD, while other Cys proteases are nonessential for PCD, but have other roles, e.g., in the clearance of cell remains after PCD. These data illustrate the need for caution when generalizing the role of Cys proteases in regulating PCD in plants, and call for studies that further investigate plant Cys proteases and other PCD regulators.

Decoy Engineering: The Next Step in Resistance Breeding.

The recent finding that decoy engineering can expand the recognition specificity of a plant immune receptor opens a wealth of opportunities for resistance breeding. In this Spotlight we discuss which factors should be considered to successfully translate decoy engineering into crop species.