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.

Subtilase-mediated biogenesis of the expanded family of SERINE RICH ENDOGENOUS PEPTIDES.

Plant signalling peptides are typically released from larger precursors by proteolytic cleavage to regulate plant growth, development and stress responses. Recent studies reported the characterization of a divergent family of Brassicaceae-specific peptides, SERINE RICH ENDOGENOUS PEPTIDES (SCOOPs), and their perception by the leucine-rich repeat receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2). Here, we reveal that the SCOOP family is highly expanded, containing at least 50 members in the Columbia-0 reference Arabidopsis thaliana genome. Notably, perception of these peptides is strictly MIK2-dependent. How bioactive SCOOP peptides are produced, and to what extent their perception is responsible for the multiple physiological roles associated with MIK2 are currently unclear. Using N-terminomics, we validate the N-terminal cleavage site of representative PROSCOOPs. The cleavage sites are determined by conserved motifs upstream of the minimal SCOOP bioactive epitope. We identified subtilases necessary and sufficient to process PROSCOOP peptides at conserved cleavage motifs. Mutation of these subtilases, or their recognition motifs, suppressed PROSCOOP cleavage and associated overexpression phenotypes. Furthermore, we show that higher-order mutants of these subtilases show phenotypes reminiscent of mik2 null mutant plants, consistent with impaired PROSCOOP biogenesis, and demonstrating biological relevance of SCOOP perception by MIK2. Together, this work provides insights into the molecular mechanisms underlying the functions of the recently identified SCOOP peptides and their receptor MIK2.

The plant nuclear lamina disassembles to regulate genome folding in stress conditions.

The nuclear lamina is a complex network of nuclear lamins and lamin-associated nuclear membrane proteins, which scaffold the nucleus to maintain structural integrity. In Arabidopsis thaliana, nuclear matrix constituent proteins (NMCPs) are essential components of the nuclear lamina and are required to maintain the structural integrity of the nucleus and specific perinuclear chromatin anchoring. At the nuclear periphery, suppressed chromatin overlapping with repetitive sequences and inactive protein-coding genes are enriched. At a chromosomal level, plant chromatin organization in interphase nuclei is flexible and responds to various developmental cues and environmental stimuli. On the basis of these observations in Arabidopsis, and given the role of NMCP genes (CRWN1 and CRWN4) in organizing chromatin positioning at the nuclear periphery, one can expect considerable changes in chromatin-nuclear lamina interactions when the global chromatin organization patterns are being altered in plants. Here we report the highly flexible nature of the plant nuclear lamina, which disassembles substantially under various stress conditions. Focusing on heat stress, we reveal that chromatin domains, initially tethered to the nuclear envelope, remain largely associated with CRWN1 and become scattered in the inner nuclear space. By investigating the three-dimensional chromatin contact network, we further reveal that CRWN1 proteins play a structural role in shaping the changes in genome folding under heat stress. Also, CRWN1 acts as a negative transcriptional coregulator to modulate the shift of the plant transcriptome profile in response to heat stress.

Sulfopeptide CLEL6 inhibits anthocyanin biosynthesis in Arabidopsis thaliana.

Posttranslationally modified peptides are now recognized as important regulators of plant stress responses. Here, we identified the small sulfated CLE-LIKE6 (CLEL6) peptide as a negative regulator of anthocyanin biosynthesis in etiolated and in light-stressed Arabidopsis (Arabidopsis thaliana) seedlings. CLEL6 function depends on proteolytic processing of the CLEL6 precursor by subtilisin-like serine proteinase 6.1 (SBT6.1) and on tyrosine sulfation by tyrosylprotein sulfotransferase (TPST). Loss-of-function mutants of either sbt6.1 or tpst showed significantly higher anthocyanin accumulation than the wild type upon light stress. The anthocyanin overaccumulation phenotype of sbt6.1 and tpst was suppressed by application of mature CLEL6. Overexpression and external application of CLEL6 inhibited the expression of anthocyanin biosynthesis genes in etiolated and light-stressed seedlings, confirming the role of CLEL6 as an inhibitor of anthocyanin biosynthesis. Small posttranslationally modified peptides are perceived by leucine-rich repeat receptor-like kinases. Using a quintuple mutant of ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors, we showed the essential function of the RGI receptor family in CLEL6 signaling. Our data indicate that overexpression or application of CLEL6 inhibits anthocyanin biosynthesis through RGI receptors. We propose that CLEL6 inhibits anthocyanin biosynthesis in etiolated seedlings, and that anthocyanin biosynthesis is derepressed when CLEL6 expression is downregulated upon light exposure. Hyperaccumulation of anthocyanins in light-stressed tpst and sbt6.1 mutant seedlings suggests that CLEL6, or related sulfopeptides, continues to act as negative regulators to limit pigment accumulation in the light.

Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates.

Proteases control plant growth and development by limited proteolysis of regulatory proteins at highly specific sites. This includes the processing of peptide hormone precursors to release the bioactive peptides as signaling molecules. The proteases involved in this process have long remained elusive. Confirmation of a candidate protease as a peptide precursor-processing enzyme requires the demonstration of protease-mediated precursor cleavage in vitro. In vitro cleavage assays rely on the availability of suitable substrates and the candidate protease with high purity. Here, we provide a protocol for the expression, purification, and characterization of tomato (Solanum lycopersicum) phytaspases as candidate proteases for the processing of the phytosulfokine precursor. We also show how synthetic oligopeptide substrates can be used to demonstrate site-specific precursor cleavage. Graphical abstract.

Analysis of Peptide Hormone Maturation and Processing Specificity Using Isotope-Labeled Peptides.

Many peptide hormones and growth factors in plants, particularly the small posttranslationally modified signaling peptides, are synthesized as larger precursor proteins. Proteolytic processing is thus required for peptide maturation, and additional posttranslational modifications may contribute to bioactivity. To what extent these posttranslational modifications impact on processing is largely unknown. Likewise, it is poorly understood how the cleavage sites within peptide precursors are selected by specific processing proteases, and whether or not posttranslational modifications contribute to cleavage site recognition. Here, we describe a mass spectrometry-based approach to address these questions. We developed a method using heavy isotope labeling to directly compare cleavage efficiency of different precursor-derived synthetic peptides by mass spectrometry. Thereby, we can analyze the effect of posttranslational modifications on processing and the specific sequence requirements of the processing proteases. As an example, we describe how this method has been used to assess the relevance of tyrosine sulfation for the processing of the Arabidopsis CIF4 precursor by the subtilase SBT5.4.

Improved Identification of Protease Cleavage Sites by In-gel Reductive Dimethylation.

A critical step in the functional characterization of proteases is the identification of physiologically relevant substrates, which often starts with a collection of candidate proteins. To test these candidates and identify specific processing sites, in vitro cleavage assays are typically used, followed by polyacrylamide gel electrophoresis (SDS-PAGE) to separate and visualize the cleavage products. For the identification of cleavage sites, the sequences at the N- or C-terminal ends of the cleavage products need to be identified, which is the most challenging step in this procedure. Here, we describe a method for the reliable identification of the N-termini of polypeptides after separation by SDS-PAGE. The procedure relies on in-gel labeling of the N-terminal-free amino group by reductive dimethylation, followed by tryptic digestion and analysis of resulting peptides by mass spectrometry. N-terminal peptides are readily identified by the 28 Da mass dimethyl tag linked to their first amino acid.

Egg cell-secreted aspartic proteases ECS1/2 promote gamete attachment to prioritize the fertilization of egg cells over central cells in Arabidopsis.

Double fertilization is an innovative phenomenon in angiosperms, in which one sperm cell first fuses with the egg cell to produce the embryo, and then the other sperm fuses with the central cell to produce the endosperm. However, the molecular mechanism of the preferential fertilization of egg cells is poorly understood. In this study, we report that two egg cell-secreted aspartic proteases, ECS1 and ECS2, play an important role in promoting preferential fertilization of egg cells in Arabidopsis. We show that simultaneous loss of ECS1 and ECS2 function resulted in an approximately 20% reduction in fertility, which can be complemented by the full-length ECS1/2 but not by corresponding active site mutants or by secretion-defective versions of ECS1/2. Detailed phenotypic analysis revealed that the egg cell-sperm cell attachment was compromised in ecs1 ecs2 siliques. Limited pollination assays with cyclin-dependent kinase a1 (cdka;1) pollen showed that preferential egg cell fertilization was impaired in the ecs1 ecs2 mutant. Taken together, these results demonstrate that egg cells secret two aspartic proteases, ECS1 and ECS2, to facilitate the attachment of sperm cells to egg cells so that preferential fertilization of egg cells is achieved. This study reveals the molecular mechanism of preferential fertilization in Arabidopsis thaliana.

Biogenesis of post-translationally modified peptide signals for plant reproductive development.

Post-translationally modified peptides (PMPs) are important regulators of plant growth and development. They are derived from larger inactive precursors by post-translational modification (PTM) and proteolytic processing to result in the bioactive peptide signals. We discuss how and why these modifications contribute to the bioactivity of inflorescence deficient in abscission (IDA), phytosulfokine (PSK), and peptides of the Casparian strip integrity factor (CIF) family, as signaling molecules during reproductive development. The emerging picture suggests that PTMs evolved to increase the specificity of interaction of PMPs with cognate receptors and of PMP precursors with processing proteases. Cleavage sites in PMP precursors are recognized by subtilases (SBTs) in a highly specific manner. SBT-mediated processing results in the activation of PMP signals regulating stress-induced flower drop, the formation of the embryonic cuticle, and pollen development.

Identification of Cognate Protease/Substrate Pairs by Use of Class-Specific Inhibitors.

Many proteins are regulated post-translationally by proteolytic processing. This includes plant signaling peptides that are proteolytically released from larger precursor proteins. The proteases involved in the biogenesis of signaling peptides and in regulation of other proteins by limited proteolysis are largely unknown. Here we describe how protease inhibitors that are specific for a certain class of proteases can be employed for the identification of proteases that are responsible for the processing of a given target protein. After having identified the protease family to which the processing enzyme belongs, candidate proteases and the GFP-tagged target protein are agro-infiltrated for transient expression in N. benthamiana leaves. Cleavage products are analyzed on immuno-blots and specificity of cleavage is confirmed by co-expression of class-specific inhibitors. For the identification of processing sites within the target protein, cleavage product(s) are purified by immunoprecipitation followed by polyacrylamide gel electrophoresis and analyzed by mass spectrometry.

Peptide Backbone Modifications for the Assessment of Cleavage Site Relevance in Precursors of Signaling Peptides.

The physiological relevance of site-specific precursor processing for the biogenesis of peptide hormones and growth factors can be demonstrated in genetic complementation experiments, in which a gain of function is observed for the cleavable wild-type precursor, but not for a non-cleavable precursor mutant. Similarly, cleavable and non-cleavable synthetic peptides can be used in bioassays to test whether processing is required for bioactivity. In genetic complementation experiments, site-directed mutagenesis has to be used to mask a processing site against proteolysis. Peptide-based bioassays have the distinctive advantage that peptides can be protected against proteolytic cleavage by backbone modifications, i.e., without changing the amino acid sequence. Peptide backbone modifications have been employed to increase the metabolic stability of peptide drugs, and in basic research, to investigate whether processing at a certain site is required for precursor maturation and formation of the bioactive peptide. For this approach, it is important to show that modification of the peptide backbone has the desired effect and does indeed protect the respective peptide bond against proteolysis. This can be accomplished with the MALDI-TOF mass spectrometry-based assay we describe here.

A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation.

The surface of pollen grains is reinforced by pollen wall components produced noncell autonomously by tapetum cells that surround developing pollen within the male floral organ, the anther. Here, we show that tapetum activity is regulated by the GASSHO (GSO) receptor-like kinase pathway, controlled by two sulfated peptides, CASPARIAN STRIP INTEGRITY FACTOR 3 (CIF3) and CIF4, the precursors of which are expressed in the tapetum itself. Coordination of tapetum activity with pollen grain development depends on the action of subtilases, including AtSBT5.4, which are produced stage specifically by developing pollen grains. Tapetum-derived CIF precursors are processed by subtilases, triggering GSO-dependent tapetum activation. We show that the GSO receptors act from the middle layer, a tissue surrounding the tapetum and developing pollen. Three concentrically organized cell types, therefore, cooperate to coordinate pollen wall deposition through a multilateral molecular dialogue.

Processing of a plant peptide hormone precursor facilitated by posttranslational tyrosine sulfation.

Most peptide hormones and growth factors are matured from larger inactive precursor proteins by proteolytic processing and further posttranslational modification. Whether or how posttranslational modifications contribute to peptide bioactivity is still largely unknown. We address this question here for TWS1 (Twisted Seed 1), a peptide regulator of embryonic cuticle formation in Arabidopsis thaliana. Using synthetic peptides encompassing the N- and C-terminal processing sites and the recombinant TWS1 precursor as substrates, we show that the precursor is cleaved by the subtilase SBT1.8 at both the N and the C termini of TWS1. Recognition and correct processing at the N-terminal site depended on sulfation of an adjacent tyrosine residue. Arginine 302 of SBT1.8 was found to be required for sulfotyrosine binding and for accurate processing of the TWS1 precursor. The data reveal a critical role for posttranslational modification, here tyrosine sulfation of a plant peptide hormone precursor, in mediating processing specificity and peptide maturation.

The Phtheirospermum japonicum isopentenyltransferase PjIPT1a regulates host cytokinin responses in Arabidopsis.

The hemiparasitic plant Phtheirospermum japonicum (Phtheirospermum) is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyze the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter-reporter, and further analyzed the subcellular localization, the enzymatic function and contribution to inducing hypertrophy by studying CRISPR-Cas9-induced Phtheirospermum mutants. PjIPT1a was expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed IPT activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a also was identified in the related weed Striga hermonthica. With PjIPT1a, we identified the IPT enzyme that induces CK responses in Phtheirospermum japonicum-infected Arabidopsis roots. We propose that PjIPT1a exemplifies how parasitism-related functions evolve through gene duplications and neofunctionalization.

Subtilase activity in intrusive cells mediates haustorium maturation in parasitic plants.

Parasitic plants that infect crops are devastating to agriculture throughout the world. These parasites develop a unique inducible organ called the haustorium that connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically toward the host vasculature. Following this, some of the intrusive cells re-differentiate to form a xylem bridge (XB) that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells remain poorly understood. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the identified intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of SBT inhibitors in intrusive cells inhibited both intrusive cell and XB development and reduced auxin response levels adjacent to the area of XB development. Therefore, we propose that subtilase activity plays an important role in haustorium development in P. japonicum.

Phytosulfokine (PSK) precursor processing by subtilase SBT3.8 and PSK signaling improve drought stress tolerance in Arabidopsis.

Increasing drought stress poses a severe threat to agricultural productivity. Plants, however, have evolved numerous mechanisms to cope with such environmental stress. Here we report that the stress-induced production of a peptide signal contributes to stress tolerance. The expression of phytosulfokine (PSK) peptide precursor genes, and transcripts of three subtilisin-like serine proteases, SBT1.4, SBT3.7, and SBT3.8, were found to be up-regulated in response to osmotic stress. Stress symptoms were more pronounced in sbt3.8 loss-of-function mutants and could be alleviated by PSK treatment. Osmotic stress tolerance was improved in plants overexpressing the PSK1 precursor (proPSK1) or SBT3.8, resulting in higher fresh weight and improved lateral root development in transgenic plants compared with wild-type plants. We further showed that SBT3.8 is involved in the biogenesis of the bioactive PSK peptide. ProPSK1 was cleaved by SBT3.8 at the C-terminus of the PSK pentapeptide. Processing by SBT3.8 depended on the aspartic acid residue directly following the cleavage site. ProPSK1 processing was impaired in the sbt3.8 mutant. The data suggest that increased expression of proPSK1 in response to osmotic stress followed by the post-translational processing of proPSK1 by SBT3.8 leads to the production of PSK as a peptide signal for stress mitigation.

Processing and Formation of Bioactive CLE40 Peptide Are Controlled by Posttranslational Proline Hydroxylation.

Small posttranslationally modified signaling peptides are proteolytically derived from larger precursor proteins and subject to several additional steps of modification, including Pro hydroxylation, Hyp glycosylation, and/or Tyr sulfation. The processing proteases and the relevance of posttranslational modifications for peptide biogenesis and activity are largely unknown. In this study these questions were addressed for the Clavata3/Endosperm Surrounding Region (CLE) peptide CLE40, a peptide regulator of stem cell differentiation in the Arabidopsis (Arabidopsis thaliana) root meristem. We identify three subtilases (SBT1.4, SBT1.7, and SBT4.13) that cleave the CLE40 precursor redundantly at two sites. C-terminal processing releases the mature peptide from its precursor and is thus required for signal biogenesis. SBT-mediated cleavage at a second site within the mature peptide attenuates the signal. The second cleavage is prevented by Pro hydroxylation, resulting in the formation of mature and bioactive CLE40 in planta. Our data reveal a role for posttranslational modification by Pro hydroxylation in the regulation of CLE40 formation and activity.

The biogenesis of CLEL peptides involves several processing events in consecutive compartments of the secretory pathway.

Post-translationally modified peptides are involved in many aspects of plant growth and development. The maturation of these peptides from their larger precursors is still poorly understood. We show here that the biogenesis of CLEL6 and CLEL9 peptides in Arabidopsis thaliana requires a series of processing events in consecutive compartments of the secretory pathway. Following cleavage of the signal peptide upon entry into the endoplasmic reticulum (ER), the peptide precursors are processed in the cis-Golgi by the subtilase SBT6.1. SBT6.1-mediated cleavage within the variable domain allows for continued passage of the partially processed precursors through the secretory pathway, and for subsequent post-translational modifications including tyrosine sulfation and proline hydroxylation within, and proteolytic maturation after exit from the Golgi. Activation by subtilases including SBT3.8 in post-Golgi compartments depends on the N-terminal aspartate of the mature peptides. Our work highlights the complexity of post-translational precursor maturation allowing for stringent control of peptide biogenesis.

Author Correction: The tomato subtilase family includes several cell death-related proteinases with caspase specificity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Systemin Signaling Cascade As Derived from Time Course Analyses of the Systemin-responsive Phosphoproteome.

Systemin is a small peptide with important functions in plant wound response signaling. Although the transcriptional responses of systemin action are well described, the signaling cascades involved in systemin perception and signal transduction at the protein level are poorly understood. Here we used a tomato cell suspension culture system to profile phosphoproteomic responses induced by systemin and its inactive Thr17Ala analog, allowing us to reconstruct a systemin-specific kinase/phosphatase signaling network. Our time-course analysis revealed early phosphorylation events at the plasma membrane, such as dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases, vesicle trafficking proteins and transcription factors. Based on a correlation analysis of systemin-induced phosphorylation profiles, we predicted substrate candidates for 44 early systemin-responsive kinases, which includes receptor kinases and downstream kinases such as MAP kinases, as well as nine phosphatases. We propose a regulatory module in which H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 mins of systemin treatment. We found the MAP kinase MPK2 to have increased phosphorylation level at its activating TEY-motif at 15 min post-treatment. The predicted interaction of MPK2 with LHA1 was confirmed by in vitro kinase assays, suggesting that the H+-ATPase LHA1 is re-activated by MPK2 later in the systemin response. Our data set provides a resource of proteomic events involved in systemin signaling that will be valuable for further in-depth functional studies in elucidation of systemin signaling cascades.