Extensive signal integration by the phytohormone protein network.

Plant hormones coordinate responses to environmental cues with developmental programs1, and are fundamental for stress resilience and agronomic yield2. The core signalling pathways underlying the effects of phytohormones have been elucidated by genetic screens and hypothesis-driven approaches, and extended by interactome studies of select pathways3. However, fundamental questions remain about how information from different pathways is integrated. Genetically, most phenotypes seem to be regulated by several hormones, but transcriptional profiling suggests that hormones trigger largely exclusive transcriptional programs4. We hypothesized that protein-protein interactions have an important role in phytohormone signal integration. Here, we experimentally generated a systems-level map of the Arabidopsis phytohormone signalling network, consisting of more than 2,000 binary protein-protein interactions. In the highly interconnected network, we identify pathway communities and hundreds of previously unknown pathway contacts that represent potential points of crosstalk. Functional validation of candidates in seven hormone pathways reveals new functions for 74% of tested proteins in 84% of candidate interactions, and indicates that a large majority of signalling proteins function pleiotropically in several pathways. Moreover, we identify several hundred largely small-molecule-dependent interactions of hormone receptors. Comparison with previous reports suggests that noncanonical and nontranscription-mediated receptor signalling is more common than hitherto appreciated.

Publisher Correction: Extensive signal integration by the phytohormone protein network.

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

A salicylic acid-associated plant-microbe interaction attracts beneficial Flavobacterium sp. to the Arabidopsis thaliana phyllosphere.

Both above- and below-ground parts of plants are constantly challenged with microbes and interact closely with them. Many plant-growth-promoting rhizobacteria, mostly interacting with the plant's root system, enhance the immunity of plants in a process described as induced systemic resistance (ISR). Here, we characterized local induced resistance (IR) triggered by the model PGPR Pseudomonas simiae WCS417r (WCS417) in Arabidopsis thaliana. Hydroponic application of WCS417 to Arabidopsis roots resulted in propagation of WCS417 in/on leaves and the establishment of local IR. WCS417-triggered local IR was dependent on salicylic acid (SA) biosynthesis and signalling and on functional biosynthesis of pipecolic acid and monoterpenes, which are classically associated with systemic acquired resistance (SAR). WCS417-triggered local IR was further associated with a priming of gene expression changes related to SA signalling and SAR. A metabarcoding approach applied to the leaf microbiome revealed a significant local IR-associated enrichment of Flavobacterium sp.. Co-inoculation experiments using WCS417 and At-LSPHERE Flavobacterium sp. Leaf82 suggest that the proliferation of these bacteria is influenced by both microbial and immunity-related, plant-derived factors. Furthermore, application of Flavobacterium Leaf82 to Arabidopsis leaves induced SAR in an NPR1-dependent manner, suggesting that recruitment of this bacterium to the phyllosphere resulted in propagation of IR. Together, the data highlight the importance of plant-microbe-microbe interactions in the phyllosphere and reveal Flavobacterium sp. Leaf82 as a new beneficial promoter of plant health.

Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea.

Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose, and pectin, and its composition can change upon stress. BETA-XYLOSIDASE4 (BXL4) belongs to a seven-member gene family in Arabidopsis (Arabidopsis thaliana), one of which encodes a protein (BXL1) involved in cell wall remodeling. We assayed the influence of BXL4 on plant immunity and investigated the subcellular localization and enzymatic activity of BXL4, making use of mutant and overexpression lines. BXL4 localized to the apoplast and was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea in a jasmonoyl isoleucine-dependent manner. The bxl4 mutants showed a reduced resistance to B. cinerea, while resistance was increased in conditional overexpression lines. Ectopic expression of BXL4 in Arabidopsis seed coat epidermal cells rescued a bxl1 mutant phenotype, suggesting that, like BXL1, BXL4 has both xylosidase and arabinosidase activity. We conclude that BXL4 is a xylosidase/arabinosidase that is secreted to the apoplast and its expression is upregulated under pathogen attack, contributing to immunity against B. cinerea, possibly by removal of arabinose and xylose side-chains of polysaccharides in the primary cell wall.

Pipecolic acid synthesis is required for systemic acquired resistance and plant-to-plant-induced immunity in barley.

Defense responses in plants are based on complex biochemical processes. Systemic acquired resistance (SAR) helps to fight infections by (hemi-)biotrophic pathogens. One important signaling molecule in SAR is pipecolic acid (Pip), accumulation of which is dependent on the aminotransferase ALD1 in Arabidopsis. While exogenous Pip primes defense responses in the monocotyledonous cereal crop barley (Hordeum vulgare), it is currently unclear if endogenous Pip plays a role in disease resistance in monocots. Here, we generated barley ald1 mutants using CRISPR/Cas9, and assessed their capacity to mount SAR. Endogenous Pip levels were reduced after infection of the ald1 mutant, and this altered systemic defense against the fungus Blumeria graminis f. sp. hordei. Furthermore, Hvald1 plants did not emit nonanal, one of the key volatile compounds that are normally emitted by barley plants after the activation of SAR. This resulted in the inability of neighboring plants to perceive and/or respond to airborne cues and prepare for an upcoming infection, although HvALD1 was not required in the receiver plants to mediate the response. Our results highlight the crucial role of endogenous HvALD1 and Pip for SAR, and associate Pip, in particular together with nonanal, with plant-to-plant defense propagation in the monocot crop barley.

Volatile terpenes - mediators of plant-to-plant communication.

Plants interact with other organisms employing volatile organic compounds (VOCs). The largest group of plant-released VOCs are terpenes, comprised of isoprene, monoterpenes, and sesquiterpenes. Mono- and sesquiterpenes are well-known communication compounds in plant-insect interactions, whereas the smallest, most commonly emitted terpene, isoprene, is rather assigned a function in combating abiotic stresses. Recently, it has become evident that different volatile terpenes also act as plant-to-plant signaling cues. Upon being perceived, specific volatile terpenes can sensitize distinct signaling pathways in receiver plant cells, which in turn trigger plant innate immune responses. This vastly extends the range of action of volatile terpenes, which not only protect plants from various biotic and abiotic stresses, but also convey information about environmental constraints within and between plants. As a result, plant-insect and plant-pathogen interactions, which are believed to influence each other through phytohormone crosstalk, are likely equally sensitive to reciprocal regulation via volatile terpene cues. Here, we review the current knowledge of terpenes as volatile semiochemicals and discuss why and how volatile terpenes make good signaling cues. We discuss how volatile terpenes may be perceived by plants, what are possible downstream signaling events in receiver plants, and how responses to different terpene cues might interact to orchestrate the net plant response to multiple stresses. Finally, we discuss how the signal can be further transmitted to the community level leading to a mutually beneficial community-scale response or distinct signaling with near kin.

Immunity-associated volatile emissions of ß-ionone and nonanal propagate defence responses in neighbouring barley plants.

Plants activate biochemical responses to combat stress. (Hemi-)biotrophic pathogens are fended off by systemic acquired resistance (SAR), a primed state allowing plants to respond faster and more strongly upon subsequent infection. Here, we show that SAR-like defences in barley (Hordeum vulgare) are propagated between neighbouring plants, which respond with enhanced resistance to the volatile cues from infected senders. The emissions of the sender plants contained 15 volatile organic compounds (VOCs) associated with infection. Two of these, ß-ionone and nonanal, elicited resistance upon plant exposure. Whole-genome transcriptomics analysis confirmed that interplant propagation of defence in barley is established as a form of priming. Although gene expression changes were more pronounced after challenge infection of the receiver plants with Blumeria graminis f. sp. hordei, differential gene expression in response to the volatile cues of the sender plants included an induction of HISTONE DEACETYLASE 2 (HvHDA2) and priming of TETRATRICOPEPTIDE REPEAT-LIKE superfamily protein (HvTPL). Because HvHDA2 and HvTPL transcript accumulation was also enhanced by exposure of barley to ß-ionone and nonanal, our data identify both genes as possible defence/priming markers in barley. Our results suggest that VOCs and plant-plant interactions are relevant for possible crop protection strategies priming defence responses in barley.

Systemic propagation of immunity in plants.

Systemic immunity triggered by local plant-microbe interactions is studied as systemic acquired resistance (SAR) or induced systemic resistance (ISR) depending on the site of induction and the lifestyle of the inducing microorganism. SAR is induced by pathogens interacting with leaves, whereas ISR is induced by beneficial microbes interacting with roots. Although salicylic acid (SA) is a central component of SAR, additional signals exclusively promote systemic and not local immunity. These signals cooperate in SAR- and possibly also ISR-associated signaling networks that regulate systemic immunity. The non-SA SAR pathway is driven by pipecolic acid or its presumed bioactive derivative N-hydroxy-pipecolic acid. This pathway further regulates inter-plant defense propagation through volatile organic compounds that are emitted by SAR-induced plants and recognized as defense cues by neighboring plants. Both SAR and ISR influence phytohormone crosstalk towards enhanced defense against pathogens, which at the same time affects the composition of the plant microbiome. This potentially leads to further changes in plant defense, plant-microbe, and plant-plant interactions. Therefore, we propose that such inter-organismic interactions could be combined in potentially highly effective plant protection strategies.

Isoprene and ß-caryophyllene confer plant resistance via different plant internal signalling pathways.

Isoprene and other terpenoids are important biogenic volatile organic compounds in terms of atmospheric chemistry. Isoprene can aid plant performance under abiotic stresses, but the fundamental biological reasons for the high emissions are not completely understood. Here, we provide evidence of a previously unrecognized ecological function for isoprene and for the sesquiterpene, ß-caryophyllene. We show that isoprene and ß-caryophyllene act as core components of plant signalling networks, inducing resistance against microbial pathogens in neighbouring plants. We challenged Arabidopsis thaliana with Pseudomonas syringae, after exposure to pure volatile terpenoids or to volatile emissions of transformed poplar or Arabidopsis plants. The data suggest that isoprene induces a defence response in receiver plants that is similar to that elicited by monoterpenes and depended on salicylic acid (SA) signalling. In contrast, the sesquiterpene, ß-caryophyllene, induced resistance via jasmonic acid (JA)-signalling. The experiments in an open environment show that natural biological emissions are enough to induce resistance in neighbouring Arabidopsis. Our results show that both isoprene and ß-caryophyllene function as allelochemical components in complex plant signalling networks. Knowledge of this system may be used to boost plant immunity against microbial pathogens in various crop management schemes.

Monoterpenes Support Systemic Acquired Resistance within and between Plants.

This study investigates the role of volatile organic compounds in systemic acquired resistance (SAR), a salicylic acid (SA)-associated, broad-spectrum immune response in systemic, healthy tissues of locally infected plants. Gas chromatography coupled to mass spectrometry analyses of SAR-related emissions of wild-type and non-SAR-signal-producing mutant plants associated SAR with monoterpene emissions. Headspace exposure of Arabidopsis thaliana to a mixture of the bicyclic monoterpenes α-pinene and ß-pinene induced defense, accumulation of reactive oxygen species, and expression of SA- and SAR-related genes, including the SAR regulatory AZELAIC ACID INDUCED1 (AZI1) gene and three of its paralogs. Pinene-induced resistance was dependent on SA biosynthesis and signaling and on AZI1 Arabidopsis geranylgeranyl reductase1 mutants with reduced monoterpene biosynthesis were SAR-defective but mounted normal local resistance and methyl salicylate-induced defense responses, suggesting that monoterpenes act in parallel with SA The volatile emissions from SAR signal-emitting plants induced defense in neighboring plants, and this was associated with the presence of α-pinene, ß-pinene, and camphene in the emissions of the "sender" plants. Our data suggest that monoterpenes, particularly pinenes, promote SAR, acting through ROS and AZI1, and likely function as infochemicals in plant-to-plant signaling, thus allowing defense signal propagation between neighboring plants.

Pipecolic Acid Is Induced in Barley upon Infection and Triggers Immune Responses Associated with Elevated Nitric Oxide Accumulation.

Pipecolic acid (Pip) is an essential component of systemic acquired resistance, priming resistance in Arabidopsis thaliana against (hemi)biotrophic pathogens. Here, we studied the potential role of Pip in bacteria-induced systemic immunity in barley. Exudates of barley leaves infected with the systemic immunity-inducing pathogen Pseudomonas syringae pv. japonica induced immune responses in A. thaliana. The same leaf exudates contained elevated Pip levels compared with those of mock-treated barley leaves. Exogenous application of Pip induced resistance in barley against the hemibiotrophic bacterial pathogen Xanthomonas translucens pv. cerealis. Furthermore, both a systemic immunity-inducing infection and exogenous application of Pip enhanced the resistance of barley against the biotrophic powdery mildew pathogen Blumeria graminis f. sp. hordei. In contrast to a systemic immunity-inducing infection, Pip application did not influence lesion formation by a systemically applied inoculum of the necrotrophic fungus Pyrenophora teres. Nitric oxide (NO) levels in barley leaves increased after Pip application. Furthermore, X. translucens pv. cerealis induced the accumulation of superoxide anion radicals and this response was stronger in Pip-pretreated compared with mock-pretreated plants. Thus, the data suggest that Pip induces barley innate immune responses by triggering NO and priming reactive oxygen species accumulation.

Comparative Proteomics Analysis of Phloem Exudates Collected during the Induction of Systemic Acquired Resistance.

Systemic acquired resistance (SAR) is a plant defense response that provides long-lasting, broad-spectrum pathogen resistance to uninfected systemic leaves following an initial localized infection. In Arabidopsis (Arabidopsis thaliana), local infection with virulent or avirulent strains of Pseudomonas syringae pv tomato generates long-distance SAR signals that travel from locally infected to distant leaves through the phloem to establish SAR In this study, a proteomics approach was used to identify proteins that accumulate in phloem exudates in response to the induction of SAR To accomplish this, phloem exudates collected from mock-inoculated or SAR-induced leaves of wild-type Columbia-0 plants were subjected to label-free quantitative liquid chromatography-tandem mass spectrometry proteomics. Comparing mock- and SAR-induced phloem exudate proteomes, 16 proteins were enriched in phloem exudates collected from SAR-induced plants, while 46 proteins were suppressed. SAR-related proteins THIOREDOXIN h3, ACYL-COENZYME A-BINDING PROTEIN6, and PATHOGENESIS-RELATED1 were enriched in phloem exudates of SAR-induced plants, demonstrating the strength of this approach and suggesting a role for these proteins in the phloem during SAR To identify novel components of SAR, transfer DNA mutants of differentially abundant phloem proteins were assayed for SAR competence. This analysis identified a number of new proteins (m-type thioredoxins, major latex protein-like protein, ULTRAVIOLET-B RESISTANCE8 photoreceptor) that contribute to the SAR response. The Arabidopsis SAR phloem proteome is a valuable resource for understanding SAR long-distance signaling and the dynamic nature of the phloem during plant-pathogen interactions.

Bacteria-triggered systemic immunity in barley is associated with WRKY and ETHYLENE RESPONSIVE FACTORs but not with salicylic acid.

Leaf-to-leaf systemic immune signaling known as systemic acquired resistance is poorly understood in monocotyledonous plants. Here, we characterize systemic immunity in barley (Hordeum vulgare) triggered after primary leaf infection with either Pseudomonas syringae pathovar japonica (Psj) or Xanthomonas translucens pathovar cerealis (Xtc). Both pathogens induced resistance in systemic, uninfected leaves against a subsequent challenge infection with Xtc. In contrast to systemic acquired resistance in Arabidopsis (Arabidopsis thaliana), systemic immunity in barley was not associated with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 or the local or systemic accumulation of salicylic acid. Instead, we documented a moderate local but not systemic induction of abscisic acid after infection of leaves with Psj. In contrast to salicylic acid or its functional analog benzothiadiazole, local applications of the jasmonic acid methyl ester or abscisic acid triggered systemic immunity to Xtc. RNA sequencing analysis of local and systemic transcript accumulation revealed unique gene expression changes in response to both Psj and Xtc and a clear separation of local from systemic responses. The systemic response appeared relatively modest, and quantitative reverse transcription-polymerase chain reaction associated systemic immunity with the local and systemic induction of two WRKY and two ETHYLENE RESPONSIVE FACTOR (ERF)-like transcription factors. Systemic immunity against Xtc was further associated with transcriptional changes after a secondary/systemic Xtc challenge infection; these changes were dependent on the primary treatment. Taken together, bacteria-induced systemic immunity in barley may be mediated in part by WRKY and ERF-like transcription factors, possibly facilitating transcriptional reprogramming to potentiate immunity.

Contrasting Roles of the Apoplastic Aspartyl Protease APOPLASTIC, ENHANCED DISEASE SUSCEPTIBILITY1-DEPENDENT1 and LEGUME LECTIN-LIKE PROTEIN1 in Arabidopsis Systemic Acquired Resistance.

Systemic acquired resistance (SAR) is an inducible immune response that depends on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). Here, we show that Arabidopsis (Arabidopsis thaliana) EDS1 is required for both SAR signal generation in primary infected leaves and SAR signal perception in systemic uninfected tissues. In contrast to SAR signal generation, local resistance remains intact in eds1 mutant plants in response to Pseudomonas syringae delivering the effector protein AvrRpm1. We utilized the SAR-specific phenotype of the eds1 mutant to identify new SAR regulatory proteins in plants conditionally expressing AvrRpm1. Comparative proteomic analysis of apoplast-enriched extracts from AvrRpm1-expressing wild-type and eds1 mutant plants led to the identification of 12 APOPLASTIC, EDS1-DEPENDENT (AED) proteins. The genes encoding AED1, a predicted aspartyl protease, and another AED, LEGUME LECTIN-LIKE PROTEIN1 (LLP1), were induced locally and systemically during SAR signaling and locally by salicylic acid (SA) or its functional analog, benzo 1,2,3-thiadiazole-7-carbothioic acid S-methyl ester. Because conditional overaccumulation of AED1-hemagglutinin inhibited SA-induced resistance and SAR but not local resistance, the data suggest that AED1 is part of a homeostatic feedback mechanism regulating systemic immunity. In llp1 mutant plants, SAR was compromised, whereas the local resistance that is normally associated with EDS1 and SA as well as responses to exogenous SA appeared largely unaffected. Together, these data indicate that LLP1 promotes systemic rather than local immunity, possibly in parallel with SA. Our analysis reveals new positive and negative components of SAR and reinforces the notion that SAR represents a distinct phase of plant immunity beyond local resistance.

ML3 is a NEDD8- and ubiquitin-modified protein.

NEDD8 (NEURAL PRECURSOR CELL-EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED PROTEIN8) is an evolutionarily conserved 8-kD protein that is closely related to ubiquitin and that can be conjugated like ubiquitin to specific lysine residues of target proteins in eukaryotes. In contrast to ubiquitin, for which a broad range of substrate proteins are known, only a very limited number of NEDD8 target proteins have been identified to date. Best understood, and also evolutionarily conserved, is the NEDD8 modification (neddylation) of cullins, core subunits of the cullin-RING-type E3 ubiquitin ligases that promote the polyubiquitylation of degradation targets in eukaryotes. Here, we show that Myeloid differentiation factor-2-related lipid-recognition domain protein ML3 is an NEDD8- as well as ubiquitin-modified protein in Arabidopsis (Arabidopsis thaliana) and examine the functional role of ML3 in the plant cell. Our analysis indicates that ML3 resides in the vacuole as well as in endoplasmic reticulum (ER) bodies. ER bodies are Brassicales-specific ER-derived organelles and, similar to other ER body proteins, ML3 orthologs can only be identified in this order of flowering plants. ML3 gene expression is promoted by wounding as well as by the phytohormone jasmonic acid and repressed by ethylene, signals that are known to induce and repress ER body formation, respectively. Furthermore, ML3 protein abundance is dependent on NAI1, a master regulator of ER body formation in Arabidopsis. The regulation of ML3 expression and the localization of ML3 in ER bodies and the vacuole is in agreement with a demonstrated importance of ML3 in the defense to herbivore attack. Here, we extend the spectrum of ML3 biological functions by demonstrating a role in the response to microbial pathogens.

Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY1 promotes systemic acquired resistance via azelaic acid and its precursor 9-oxo nonanoic acid.

Systemic acquired resistance (SAR) is a form of inducible disease resistance that depends on salicylic acid and its upstream regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). Although local Arabidopsis thaliana defence responses activated by the Pseudomonas syringae effector protein AvrRpm1 are intact in eds1 mutant plants, SAR signal generation is abolished. Here, the SAR-specific phenotype of the eds1 mutant is utilized to identify metabolites that contribute to SAR. To this end, SAR bioassay-assisted fractionation of extracts from the wild type compared with eds1 mutant plants that conditionally express AvrRpm1 was performed. Using high-performance liquid chromatography followed by mass spectrometry, systemic immunity was associated with the accumulation of 60 metabolites, including the putative SAR signal azelaic acid (AzA) and its precursors 9-hydroperoxy octadecadienoic acid (9-HPOD) and 9-oxo nonanoic acid (ONA). Exogenous ONA induced SAR in systemic untreated leaves when applied at a 4-fold lower concentration than AzA. The data suggest that in planta oxidation of ONA to AzA might be partially responsible for this response and provide further evidence that AzA mobilizes Arabidopsis immunity in a concentration-dependent manner. The AzA fragmentation product pimelic acid did not induce SAR. The results link the C9 lipid peroxidation products ONA and AzA with systemic rather than local resistance and suggest that EDS1 directly or indirectly promotes the accumulation of ONA, AzA, or one or more of their common precursors possibly by activating one or more pathways that either result in the release of these compounds from galactolipids or promote lipid peroxidation.

Analysis of Innate Immune Responses Against Pathogenic Bacteria in Arabidopsis, Tomato, and Barley.

The immune status of plants can be evaluated by monitoring the propagation of pathogens. Plants defend themselves against pathogen attack through an intricate network of phytohormone-driven innate immune responses. Of these, salicylic acid (SA)-dependent defense responses can be assessed in planta by monitoring the propagation of biotrophic and hemi-biotrophic pathogens. Here, we describe methods to monitor the propagation of the hemi-biotrophic bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana leaves. We describe protocols to (i) propagate the plants to the appropriate growth stage for infection, (ii) prepare the bacterial inoculum, (iii) inoculate plants using spray and infiltration techniques, and (iv) analyze the resulting in planta bacterial titers. The latter bacterial titers serve as a measure of plant susceptibility and negatively correlate with immunity. Based on the methods used with the A. thaliana-P. syringae model pathosystem, we include complementary methods allowing the analysis of innate immunity in the crop plants Solanum lycopersicum (tomato) in interaction with P. syringae and Hordeum vulgare (barley) in interaction with Xanthomonas translucens.

New molecules in plant defence against pathogens.

Plants host a multipart immune signalling network to ward off pathogens. Pathogen attack upon plant tissues can often lead to an amplified state of (induced) defence against subsequent infections in distal tissues; this is known as systemic acquired resistance (SAR). The interaction of plants with beneficial microbes of the rhizosphere microbiome can also lead to an induced resistance in above-ground plant tissues, known as induced systemic resistance. Second messengers such as calcium (Ca2+), reactive oxygen species (ROS), and nitric oxide (NO) are necessary for cell-to-cell signal propagation during SAR and show emergent roles in the mediation of other SAR metabolites. These include the lysine-derived signals pipecolic acid (Pip) and N-hydroxypipecolic acid (NHP), which are key signalling metabolites in SAR. Emerging evidence additionally pinpoints plant volatiles as modulators of defence signalling within and between plants. Plant volatile organic compounds (VOCs) such as monoterpenes can promote SAR by functioning through ROS. Furthermore, plant-derived and additionally also microbial VOCs can target both salicylic acid and jasmonic acid signalling pathways in plants and modulate defence against pathogens. In this review, an overview of recent findings in induced defence signalling, with a particular focus on newer signalling molecules and how they integrate into these networks is discussed.