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.

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.

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.

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.

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.

ß-D-XYLOSIDASE 4 modulates systemic immune signaling in Arabidopsis thaliana.

Pectin- and hemicellulose-associated structures of plant cell walls participate in defense responses against pathogens of different parasitic lifestyles. The resulting immune responses incorporate phytohormone signaling components associated with salicylic acid (SA) and jasmonic acid (JA). SA plays a pivotal role in systemic acquired resistance (SAR), a form of induced resistance that - after a local immune stimulus - confers long-lasting, systemic protection against a broad range of biotrophic invaders. ß-D-XYLOSIDASE 4 (BXL4) protein accumulation is enhanced in the apoplast of plants undergoing SAR. Here, two independent Arabidopsis thaliana mutants of BXL4 displayed compromised systemic defenses, while local resistance responses to Pseudomonas syringae remained largely intact. Because both phloem-mediated and airborne systemic signaling were abrogated in the mutants, the data suggest that BXL4 is a central component in SAR signaling mechanisms. Exogenous xylose, a possible product of BXL4 enzymatic activity in plant cell walls, enhanced systemic defenses. However, GC-MS analysis of SAR-activated plants revealed BXL4-associated changes in the accumulation of certain amino acids and soluble sugars, but not xylose. In contrast, the data suggest a possible role of pectin-associated fucose as well as of the polyamine putrescine as regulatory components of SAR. This is the first evidence of a central role of cell wall metabolic changes in systemic immunity. Additionally, the data reveal a so far unrecognized complexity in the regulation of SAR, which might allow the design of (crop) plant protection measures including SAR-associated cell wall components.

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.

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.

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.

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.

Systemic acquired resistance networks amplify airborne defense cues.

Salicylic acid (SA)-mediated innate immune responses are activated in plants perceiving volatile monoterpenes. Here, we show that monoterpene-associated responses are propagated in feed-forward loops involving the systemic acquired resistance (SAR) signaling components pipecolic acid, glycerol-3-phosphate, and LEGUME LECTIN-LIKE PROTEIN1 (LLP1). In this cascade, LLP1 forms a key regulatory unit in both within-plant and between-plant propagation of immunity. The data integrate molecular components of SAR into systemic signaling networks that are separate from conventional, SA-associated innate immune mechanisms. These networks are central to plant-to-plant propagation of immunity, potentially raising SAR to the population level. In this process, monoterpenes act as microbe-inducible plant volatiles, which as part of plant-derived volatile blends have the potential to promote the generation of a wave of innate immune signaling within canopies or plant stands. Hence, plant-to-plant propagation of SAR holds significant potential to fortify future durable crop protection strategies following a single volatile trigger.

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.

Bacterial medium-chain 3-hydroxy fatty acid metabolites trigger immunity in Arabidopsis plants.

In plants, cell-surface immune receptors sense molecular non-self-signatures. Lipid A of Gram-negative bacterial lipopolysaccharide is considered such a non-self-signature. The receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) mediates plant immune responses to Pseudomonas and Xanthomonas but not enterobacterial lipid A or lipopolysaccharide preparations. Here, we demonstrate that synthetic and bacterial lipopolysaccharide-copurified medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) metabolites elicit LORE-dependent immunity. The mc-3-OH-FAs are sensed in a chain length- and hydroxylation-specific manner, with free (R)-3-hydroxydecanoic acid [(R)-3-OH-C10:0] representing the strongest immune elicitor. By contrast, bacterial compounds comprising mc-3-OH-acyl building blocks but devoid of free mc-3-OH-FAs-including lipid A or lipopolysaccharide, rhamnolipids, lipopeptides, and acyl-homoserine-lactones-do not trigger LORE-dependent responses. Hence, plants sense low-complexity bacterial metabolites to trigger immune responses.

Barley susceptibility factor RACB modulates transcript levels of signalling protein genes in compatible interaction with Blumeria graminis f.sp. hordei.

RHO (rat sarcoma homologue) GTPases (guanosine triphosphatases) are regulators of downstream transcriptional responses of eukaryotes to intracellular and extracellular stimuli. For plants, little is known about the function of Rho-like GTPases [called RACs (rat sarcoma-related C botulinum substrate) or ROPs (RHO of plants)] in transcriptional reprogramming of cells. However, in plant hormone response and innate immunity, RAC/ROP proteins influence gene expression patterns. The barley RAC/ROP RACB is required for full susceptibility of barley to the powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). We compared the transcriptomes of barley plants either silenced for RACB or over-expressing constitutively activated RACB with and without inoculation with Bgh. This revealed a large overlap of the barley transcriptome during the early response to Bgh and during the over-expression of constitutively activated RACB. Global pathway analyses and stringent analyses of differentially expressed genes suggested that RACB influences, amongst others, the expression of signalling receptor kinases. Transient induced gene silencing of RACB-regulated signalling genes (a leucine-rich repeat protein, a leucine-rich repeat receptor-like kinase and an S-domain SD1-receptor-like kinase) suggested that they might be involved in RACB-modulated susceptibility to powdery mildew. We discuss the function of RACB in regulating the transcriptional responses of susceptible barley to Bgh.

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.

Systemic Responses of Barley to the 3-hydroxy-decanoyl-homoserine Lactone Producing Plant Beneficial Endophyte Acidovorax radicis N35.

Quorum sensing auto-inducers of the N-acyl homoserine lactone (AHL) type produced by Gram-negative bacteria have different effects on plants including stimulation on root growth and/or priming or acquirement of systemic resistance in plants. In this communication the influence of AHL production of the plant growth promoting endophytic rhizosphere bacterium Acidovorax radicis N35 on barley seedlings was investigated. A. radicis N35 produces 3-hydroxy-C10-homoserine lactone (3-OH-C10-HSL) as the major AHL compound. To study the influence of this QS autoinducer on the interaction with barley, the araI-biosynthesis gene was deleted. The comparison of inoculation effects of the A. radicis N35 wild type and the araI mutant resulted in remarkable differences. While the N35 wild type colonized plant roots effectively in microcolonies, the araI mutant occurred at the root surface as single cells. Furthermore, in a mixed inoculum the wild type was much more prevalent in colonization than the araI mutant documenting that the araI mutation affected root colonization. Nevertheless, a significant plant growth promoting effect could be shown after inoculation of barley with the wild type and the araI mutant in soil after 2 months cultivation. While A. radicis N35 wild type showed only a very weak induction of early defense responses in plant RNA expression analysis, the araI mutant caused increased expression of flavonoid biosynthesis genes. This was corroborated by the accumulation of several flavonoid compounds such as saponarin and lutonarin in leaves of root inoculated barley seedlings. Thus, although the exact role of the flavonoids in this plant response is not clear yet, it can be concluded, that the synthesis of AHLs by A. radicis has implications on the perception by the host plant barley and thereby contributes to the establishment and function of the bacteria-plant interaction.