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Proteases play crucial physiological functions in all organisms by controlling the lifetime of proteins. Here, we identified an atypical protease of the subtilase family [SBT5.2(b)] that attenuates the transcriptional activation of plant defence independently of its protease activity. The SBT5.2 gene produces two distinct transcripts encoding a canonical secreted subtilase [SBT5.2(a)] and an intracellular protein [SBT5.2(b)]. Concomitant to SBT5.2(a) downregulation, SBT5.2(b) expression is induced after bacterial inoculation. SBT5.2(b) localizes to endosomes where it interacts with and retains the defence-related transcription factor MYB30. Nuclear exclusion of MYB30 results in its reduced transcriptional activation and, thus, suppressed resistance. sbt5.2 mutants, with abolished SBT5.2(a) and SBT5.2(b) expression, display enhanced defence that is suppressed in a myb30 mutant background. Moreover, overexpression of SBT5.2(b), but not SBT5.2(a), in sbt5.2 plants reverts the phenotypes displayed by sbt5.2 mutants. Overall, we uncover a regulatory mode of the transcriptional activation of defence responses previously undescribed in eukaryotes.

The recognition of pathogens by plants at the cell surface is crucial for activating plant immunity. Plants employ pattern recognition receptors (PRRs) to detect microbe-associated molecular patterns (MAMPs). However, our knowledge of the release of peptide MAMPs from their precursor proteins is very limited. Here, we explore seven protein precursors of well-known MAMP peptides and discuss the likelihood of processing being required for their recognition based on structural models and public knowledge. This analysis indicates the existence of multiple extracellular events that are likely pivotal for pathogen perception but remain to be uncovered., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Ltd.)

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 β-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., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Nicotiana benthamiana is increasingly used for transient gene expression to produce antibodies, vaccines, and other pharmaceutical proteins but transient gene expression is low in fully developed, 6-8-week old plants. This low gene expression is thought to be caused by the perception of the cold shock protein (CSP) of Agrobacterium tumefaciens. The CSP receptor is contested because both NbCSPR and NbCORE have been claimed to perceive CSP. Here, we demonstrate that CSP perception is abolished in 6-week-old plants silenced for NbCORE but not NbCSPR. Importantly, older NbCORE-silenced plants support a highly increased level of GFP fluorescence and protein upon agroinfiltration. The drastic increase in transient protein production in NbCORE-depleted plants offers new opportunities for molecular farming, where older plants with larger biomass can now be used for efficient protein expression., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Plants encode > 100 metalloproteases representing > 19 different protein families. Tools to study this large and diverse class of proteases have not yet been introduced into plant research. We describe the use of hydroxamate-based photoaffinity probes to explore plant proteomes for metalloproteases. We detected labelling of 23 metalloproteases in leaf extracts of the model plant Arabidopsis thaliana that belong to nine different metalloprotease families and localize to different subcellular compartments. The probes identified several chloroplastic FtsH proteases, vacuolar aspartyl aminopeptidase DAP1, peroxisomal metalloprotease PMX16, extracellular matrix metalloproteases and many cytosolic metalloproteases. We also identified nonproteolytic metallohydrolases involved in the release of auxin and in the urea cycle. Studies on tobacco plants (Nicotiana benthamiana) infected with the bacterial plant pathogen Pseudomonas syringae uncovered the induced labelling of PRp27, a secreted protein with implicated metalloprotease activity. PRp27 overexpression increases resistance, and PRp27 mutants lacking metal binding site are no longer labelled, but still show increased immunity. Collectively, these studies reveal the power of broad-range metalloprotease profiling in plants using hydroxamate-based probes., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Efficient plant immune responses depend on the ability to recognise an invading microbe. The 22-amino acids in the N-terminal domain and the 28-amino acids in the central region of the bacterial flagellin, called flg22 and flgII-28, respectively, are important elicitors of plant immunity. Plant immunity is activated after flg22 or flgII-28 recognition by the plant transmembrane receptors FLS2 or FLS3, respectively. There is strong selective pressure on many plant pathogenic and endophytic bacteria to overcome flagellin-triggered immunity. Here we provide an overview of recent developments in our understanding of the evasion and suppression of flagellin pattern recognition by plant-associated bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We recently developed a simple method called "agromonas" which facilitates the detection of Pseudomonas syringae growth in agroinfiltrated leaves expressing genes of interest. This protocol consists of agroinfiltration followed by inoculation of Pseudomonas syringae by either infiltration or spray inoculation. Next, bacterial growth is measured a few days later by plating leaf extracts out on P. syringae-selective medium. In this protocol, we describe all the steps required for agroinfiltration, inoculation of P. syringae by both injection and spray infection and explain how to quantify in planta live bacteria., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recognition of microbe-associated molecular patterns (MAMPs) by cell-surface receptors is pivotal in host-microbe interactions. Both pathogens and symbionts establish plant-microbe interactions using fascinating intricate extracellular strategies to avoid recognition. Here we distinguish nine different extracellular strategies to avoid recognition by the host, acting at three different levels. To avoid the accumulation of MAMP precursors (Level 1), microbes take advantage of polymorphisms in both MAMP proteins and glycans, or downregulate MAMP production. To reduce hydrolytic MAMP release (Level 2), microbes shield MAMP precursors with proteins or glycans and inhibit or degrade host-derived hydrolases. And to prevent MAMP perception directly (Level 3), microbes degrade or sequester MAMPs before they are perceived. We discuss examples of these nine strategies and envisage three additional extracellular strategies to avoid MAMP perception in plants., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth, and that transient depletion of a host immune gene and transient expression of a type-III effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure-function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers a reliable approach for further comprehensive analysis of plant immunity., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Glyco-design of proteins is a powerful tool in fundamental studies of structure-function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco-engineering facilitate customized N-glycosylation of plant-derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human-like β1,4-galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4-galactosyltransferase, many plant-derived glycoproteins still exhibit incomplete processed N-glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β-galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4- and β1,3-galactose residues on both N- and O-glycans. Transient BGAL1 down-regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β-galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N-glycans on several plant-produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N-glycans of plant-produced glycoproteins., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Plants and animals recognize conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic release before they can activate cell surface receptors. Although much of flagellin signaling is understood, little is known about the release of immunogenic fragments. We discovered that plant-secreted β-galactosidase 1 (BGAL1) of Nicotiana benthamiana promotes hydrolytic elicitor release and acts in immunity against pathogenic Pseudomonas syringae strains only when they carry a terminal modified viosamine (mVio) in the flagellin O -glycan. In counter defense, P. syringae pathovars evade host immunity by using BGAL1-resistant O -glycans or by producing a BGAL1 inhibitor. Polymorphic glycans on flagella are common to plant and animal pathogenic bacteria and represent an important determinant of host immunity to bacterial pathogens., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Proteases play crucial physiological functions in all organisms by controlling the lifetime of proteins. Here, we identified an atypical protease of the subtilase family [SBT5.2(b)] that attenuates the transcriptional activation of plant defence independently of its protease activity. The SBT5.2 gene produces two distinct transcripts encoding a canonical secreted subtilase [SBT5.2(a)] and an intracellular protein [SBT5.2(b)]. Concomitant to SBT5.2(a) downregulation, SBT5.2(b) expression is induced after bacterial inoculation. SBT5.2(b) localizes to endosomes where it interacts with and retains the defence-related transcription factor MYB30. Nuclear exclusion of MYB30 results in its reduced transcriptional activation and, thus, suppressed resistance. sbt5.2 mutants, with abolished SBT5.2(a) and SBT5.2(b) expression, display enhanced defence that is suppressed in a myb30 mutant background. Moreover, overexpression of SBT5.2(b), but not SBT5.2(a), in sbt5.2 plants reverts the phenotypes displayed by sbt5.2 mutants. Overall, we uncover a regulatory mode of the transcriptional activation of defence responses previously undescribed in eukaryotes., Competing Interests: The authors declare that no competing interests exist.

A plethora of diverse programmed cell death (PCD) processes has been described in living organisms. In animals and plants, different forms of PCD play crucial roles in development, immunity, and responses to the environment. While the molecular control of some animal PCD forms such as apoptosis is known in great detail, we still know comparatively little about the regulation of the diverse types of plant PCD. In part, this deficiency in molecular understanding is caused by the lack of reliable reporters to detect PCD processes. Here, we addressed this issue by using a combination of bioinformatics approaches to identify commonly regulated genes during diverse plant PCD processes in Arabidopsis (Arabidopsis thaliana). Our results indicate that the transcriptional signatures of developmentally controlled cell death are largely distinct from the ones associated with environmentally induced cell death. Moreover, different cases of developmental PCD share a set of cell death-associated genes. Most of these genes are evolutionary conserved within the green plant lineage, arguing for an evolutionary conserved core machinery of developmental PCD. Based on this information, we established an array of specific promoter-reporter lines for developmental PCD in Arabidopsis. These PCD indicators represent a powerful resource that can be used in addition to established morphological and biochemical methods to detect and analyze PCD processes in vivo and in planta., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Mounting of efficient plant defence responses depends on the ability to trigger a rapid defence reaction after recognition of the invading microbe. Activation of plant resistance is achieved by modulation of the activity of multiple transcriptional regulators, both DNA-binding transcription factors and their regulatory proteins, that are able to reprogram transcription in the plant cell towards the activation of defence signalling. Here we provide an overview of recent developments on the transcriptional control of plant defence responses and discuss defence-related hormone signalling, the role of WRKY transcription factors during the regulation of plant responses to pathogens, nuclear functions of plant immune receptor proteins, as well as varied ways by which microbial effectors subvert plant transcriptional reprogramming to promote disease., (Copyright © 2014 Elsevier Ltd. All rights reserved.)