The tomato P69 subtilase family is involved in resistance to bacterial wilt.

The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases-subtilases-may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post-translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D-deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum.

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.

A phloem-localized Arabidopsis metacaspase (AtMC3) improves drought tolerance.

Increasing drought phenomena pose a serious threat to agricultural productivity. Although plants have multiple ways to respond to the complexity of drought stress, the underlying mechanisms of stress sensing and signaling remain unclear. The role of the vasculature, in particular the phloem, in facilitating inter-organ communication is critical and poorly understood. Combining genetic, proteomic and physiological approaches, we investigated the role of AtMC3, a phloem-specific member of the metacaspase family, in osmotic stress responses in Arabidopsis thaliana. Analyses of the proteome in plants with altered AtMC3 levels revealed differential abundance of proteins related to osmotic stress pointing into a role of the protein in water-stress-related responses. Overexpression of AtMC3 conferred drought tolerance by enhancing the differentiation of specific vascular tissues and maintaining higher levels of vascular-mediated transportation, while plants lacking the protein showed an impaired response to drought and inability to respond effectively to the hormone abscisic acid. Overall, our data highlight the importance of AtMC3 and vascular plasticity in fine-tuning early drought responses at the whole plant level without affecting growth or yield.

The mobile SAR signal N-hydroxypipecolic acid induces NPR1-dependent transcriptional reprogramming and immune priming.

N-hydroxypipecolic acid (NHP) accumulates in the plant foliage in response to a localized microbial attack and induces systemic acquired resistance (SAR) in distant leaf tissue. Previous studies indicated that pathogen inoculation of Arabidopsis (Arabidopsis thaliana) systemically activates SAR-related transcriptional reprogramming and a primed immune status in strict dependence of FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), which mediates the endogenous biosynthesis of NHP. Here, we show that elevations of NHP by exogenous treatment are sufficient to induce a SAR-reminiscent transcriptional response that mobilizes key components of immune surveillance and signal transduction. Exogenous NHP primes Arabidopsis wild-type and NHP-deficient fmo1 plants for a boosted induction of pathogen-triggered defenses, such as the biosynthesis of the stress hormone salicylic acid (SA), accumulation of the phytoalexin camalexin and branched-chain amino acids, as well as expression of defense-related genes. NHP also sensitizes the foliage systemically for enhanced SA-inducible gene expression. NHP-triggered SAR, transcriptional reprogramming, and defense priming are fortified by SA accumulation, and require the function of the transcriptional coregulator NON-EXPRESSOR OF PR GENES1 (NPR1). Our results suggest that NPR1 transduces NHP-activated immune signaling modes with predominantly SA-dependent and minor SA-independent features. They further support the notion that NHP functions as a mobile immune regulator capable of moving independently of active SA signaling between leaves to systemically activate immune responses.

Cutting the line: manipulation of plant immunity by bacterial type III effector proteases.

Pathogens and their hosts are engaged in an evolutionary arms race. Pathogen-derived effectors promote virulence by targeting components of a host's innate immune system, while hosts have evolved proteins that sense effectors and trigger a pathogen-specific immune response. Many bacterial effectors are translocated into host cells using type III secretion systems. Type III effector proteases irreversibly modify host proteins by cleavage of peptide bonds and are prevalent among both plant and animal bacterial pathogens. In plants, the study of model effector proteases has yielded important insights into the virulence mechanisms employed by pathogens to overcome their host's immune response, as well as into the mechanisms deployed by their hosts to detect these effector proteases and counteract their effects. In recent years, the study of a larger number of effector proteases, across a wider range of pathogens, has yielded novel insights into their functions and recognition. One key limitation that remains is the lack of methods to detect protease cleavage at the proteome-wide level. We review known substrates and mechanisms of plant pathogen type III effector proteases and compare their functions with those of known type III effector proteases of mammalian pathogens. Finally, we discuss approaches to uncover their function on a system-wide level.

Mechanistic insights into CrCEP1: A dual-function cysteine protease with endo- and transpeptidase activity.

Proteases, essential regulators of plant stress responses, remain enigmatic in their precise functional roles. By employing activity-based probes for real-time monitoring, this study aimed to delve into protease activities in Chlamydomonas reinhardtii exposed to oxidative stress induced by hydrogen peroxide. However, our work revealed that the activity-based probes strongly labelled three non-proteolytic proteins-PsbO, PsbP, and PsbQ-integral components of photosystem II's oxygen-evolving complex. Subsequent biochemical assays and mass spectrometry experiments revealed the involvement of CrCEP1, a previously uncharacterized papain-like cysteine protease, as the catalyst of this labelling reaction. Further experiments with recombinant CrCEP1 and PsbO proteins replicated the reaction in vitro. Our data unveiled that endopeptidase CrCEP1 also has transpeptidase activity, ligating probes and peptides to the N-termini of Psb proteins, thereby expanding the repertoire of its enzymatic activities. The hitherto unknown transpeptidase activity of CrCEP1, working in conjunction with its proteolytic activity, unveils putative complex and versatile roles for proteases in cellular processes during stress responses.

Proteomics dataset on detached and purified Arabidopsis thaliana rosette leaf trichomes.

Trichomes are highly specialized uni- or multicellular outgrowths of epidermal cells of plant organs that, in the case of leaves, contribute to plant resistance against abiotic and biotic stress. The model plant Arabidopsis thaliana features single-celled non-glandular rosette leaf trichomes that are dispensable under laboratory conditions. Trichomes have therefore become a successful model to identify plant genes involved in cellular differentiation and cell wall development. We have recently devised an improved method for the enrichment of plant leaf trichomes that relies on the biochemical weakening of the trichome-leaf junctions and a magnetic stirrer-based mechanical stimulus for trichome release followed by density gradient purification of trichomes. Here we provide detailed information on a label-free quantitative (LFQ) shotgun proteomics dataset collected at four stages while applying this protocol to isolate trichomes from rosette leaves of A. thaliana, from (i) whole seedlings before enrichment, from (ii) trichome-depleted material after separation, from (iii) detached trichomes, and from (iv) enriched trichomes after sucrose density gradient centrifugation. Proteins were extracted, digested with trypsin and the resulting peptides identified by nanoflow-chromatography coupled to tandem mass spectrometry. This dataset informs on proteins and biochemical processes present and/or enriched in A. thaliana rosette leaf trichomes, complementing recent large-scale proteome maps. The data further enables comparative analysis with trichome proteomic data from other plant species, may be reanalyzed using different software packages or search settings, and may serve as a reference benchmark for future method refinement.

Sensitive Plant N-Terminome Profiling with HUNTER.

Protein N-termini provide unique and distinguishing information on proteolytically processed or N-terminally modified proteoforms. Also splicing, use of alternative translation initiation sites, and a variety of co- and post-translational N-terminal modifications generate distinct proteoforms that are unambiguously identified by their N-termini. However, N-terminal peptides are only a small fraction among all peptides generated in a shotgun proteome digest, are often of low stoichiometric abundance, and therefore require enrichment. Various protocols for enrichment of N-terminal peptides have been established and successfully been used for protease substrate discovery and profiling of N-terminal modification, but often require large amounts of proteome. We have recently established the High-efficiency Undecanal-based N-Termini EnRichment (HUNTER) as a fast and sensitive method to enable enrichment of protein N-termini from limited sample sources with as little as a few microgram proteome. Here we present our current HUNTER protocol for sensitive plant N-terminome profiling, including sample preparation, enrichment of N-terminal peptides, and mass spectrometry data analysis.

An advanced method for the release, enrichment and purification of high-quality Arabidopsis thaliana rosette leaf trichomes enables profound insights into the trichome proteome.

BACKGROUND: Rosette leaf trichomes of Arabidopsis thaliana have been broadly used to study cell development, cell differentiation and, more recently, cell wall biogenesis. However, trichome-specific biochemical or -omics analyses require a proper separation of trichomes from residual plant tissue. Thus, different strategies were proposed in the past for trichome isolation, which mostly rely on harsh conditions and suffer from low yield, thereby limiting the spectrum of downstream analyses. RESULTS: To take trichome-leaf separation to the next level, we revised a previously proposed method for isolating A. thaliana trichomes by optimizing the mechanical and biochemical specifications for trichome release. We additionally introduced a density gradient centrifugation step to remove residual plant debris. We found that prolonged, yet mild seedling agitation increases the overall trichome yield by more than 60% compared to the original protocol. We noticed that subsequent density gradient centrifugation further visually enhances trichome purity, which may be advantageous for downstream analyses. Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction validated a substantial enrichment upon purification of trichomes by density gradient centrifugation. Histochemical and biochemical investigation of trichome cell wall composition indicated that unlike the original protocol gentle agitation during trichome release largely preserves trichome integrity. We used enriched and density gradient-purified trichomes for proteomic analysis in comparison to trichome-depleted leaf samples and present a comprehensive reference data set of trichome-resident and -enriched proteins. Collectively we identified 223 proteins that are highly enriched in trichomes as compared to trichome-depleted leaves. We further demonstrate that the procedure can be applied to retrieve diverse glandular and non-glandular trichome types from other plant species. CONCLUSIONS: We provide an advanced method for the isolation of A. thaliana leaf trichomes that outcompetes previous procedures regarding yield and purity. Due to the large amount of high-quality trichomes our method enabled profound insights into the so far largely unexplored A. thaliana trichome proteome. We anticipate that our protocol will be of use for a variety of downstream analyses, which are expected to shed further light on the biology of leaf trichomes in A. thaliana and possibly other plant species.