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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.

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.

Little is known about plant genetic and biochemical components that coordinate immune responses with growth and environmental cues. C-TERMINALLY ENCODED PEPTIDEs (CEPs) control plant development and nitrogen demand signaling. Here, we identified CEP4 as an immune-modulatory peptide (phytocytokine) inArabidopsis thaliana. CEP4 and related CEPs are important regulators of resistance to plant pathogenic bacteria and are perceived by the tissue-specific receptor kinases CEP RECEPTOR 1 (CEPR1), CEPR2 and the phylogenetically related RECEPTOR-LIKE KINASE 7 (RLK7). CEP4 promotes flagellin-triggered responses and we provide evidence that CEPs modulate cell surface immunity upon N limitation. We propose that CEPs integrate biotic and abiotic stress-associated signals to safeguard plant health.

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. β-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.

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.

Artificial light at night (ALAN) threatens natural ecosystems globally. While ALAN research is increasing, little is known about how ALAN affects plants and interactions with other organisms. We explored the effects of ALAN on plant defence and plant–insect interactions using barley (Hordeum vulgare) and the English grain aphid (Sitobion avenae). Plants were exposed to ‘full’ or ‘part’ nights of 15–20 lux ALAN, or no ALAN ‘control’ nights, to test the effects of ALAN on plant growth and defence. Although plant growth was only minimally affected by ALAN, aphid colony growth and aphid maturation were reduced significantly by ALAN treatments. Importantly, we found strong differences between full-night and part-night ALAN treatments. Contrary to our expectations, part ALAN had stronger negative effects on aphid colony growth than full ALAN. Defence-associated gene expression was affected in some cases by ALAN, but also positively correlated with aphid colony size, suggesting that the effects of ALAN on plant defences are indirect, and regulated via direct disruption of aphid colonies, rather than via ALAN-induced upregulation of defences. Mitigating ecological side effects of ALAN is a complex problem, as reducing exposure to ALAN increased its negative impact on insect herbivores.

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.

Volatile organic compounds (VOCs) were originally identified as communication compounds between plants and insects. Today, we know that VOCs are released by organisms of all kingdoms, including bacteria and fungi, and mediate diverse intra- and interspecific interactions both above- and below-ground. Following recent trends in this research field, the majority of reviews and research papers in this special issue focus on possible biological and ecological functions and various other aspects of microbial VOCs (mVOCs). Additional reviews and research papers highlight connections between microbe-induced plant VOCs, and their possible application in future sustainable crop protection strategies.

The Arabidopsis plastid-localized ALD1 protein acts in the lysine catabolic pathway that produces infection-induced pipecolic acid (Pip), Pip derivatives, and basal non-Pip metabolite(s). ALD1 is indispensable for disease resistance associated with Pseudomonas syringae infections of naïve plants as well as those previously immunized by a local infection, a phenomenon called systemic acquired resistance (SAR). Pseudomonas syringae is known to associate with mesophyll as well as epidermal cells. To probe the importance of epidermal cells in conferring bacterial disease resistance, we studied plants in which ALD1 was only detectable in the epidermal cells of specific leaves. Local disease resistance and many features of SAR were restored when ALD1 preferentially accumulated in the epidermal plastids at immunization sites. Interestingly, SAR restoration occurred without appreciable accumulation of Pip or known Pip derivatives in secondary distal leaves. Our findings establish that ALD1 has a non-autonomous effect on pathogen growth and defense activation. We propose that ALD1 is sufficient in the epidermis of the immunized leaves to activate SAR, but basal ALD1 and possibly a non-Pip metabolite(s) are also needed at all infection sites to fully suppress bacterial growth. Thus, epidermal plastids that contain ALD1 play a key role in local and whole-plant immune signaling., The transient accumulation of ALD1 in epidermal plastids at local immunization sites activates both local disease resistance and systemic acquired resistance in the distal leaves.

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, β-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 β-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.

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.

The phytohormones salicylic acid (SA) and jasmonic acid (JA) promote two, mutually antagonistic immune pathways respectively protecting plants from biotrophic pathogens and necrotrophic pathogens or insects. This trade-off largely precludes the exploitation of SA and JA immune components for crop protection, raising the interest in immune signalling components that disrupt SA-JA antagonism. A local pathogen infection primes SA-dependent immunity in systemic tissues. This so-called systemic acquired resistance (SAR) ensures a long-lasting, broad-spectrum disease resistance that is not subject to SA-JA antagonism. Here, we show that two sequence-related LEGUME LECTIN-LIKE PROTEINs (LLPs) promote SAR through spatially separated functions with JA promoting local SAR signal generation through LLP3. In concert with LLP1, which is important for systemic recognition and propagation of SAR signals, LLP3 promotes both SA-dependent SAR and JA-mediated immunity. Thus, exploitation of LLP-associated signalling cues might allow application of plant innate immune signals to promote (crop) plant health.

Both above- and below-ground parts of plants are constantly confronted with microbes, which are main drivers for the development of plant-microbe interactions. Plant growth-promoting rhizobacteria enhance the immunity of above-ground tissues, which is known as induced systemic resistance (ISR). We show here that ISR also influences the leaf microbiome. We compared ISR triggered by the model strain Pseudomonas simiae WCS417r (WCS417) to that triggered by Bacillus thuringiensis israelensis (Bti) in Arabidopsis thaliana. In contrast to earlier findings, immunity elicited by both strains depended on salicylic acid. Both strains further relied on MYC2 for signal transduction in the plant, while WCS417-elicited ISR additionally depended on SAR-associated metabolites, including pipecolic acid. A metabarcoding approach applied to the leaf microbiome revealed a significant ISR-associated enrichment of amplicon sequence variants with predicted plant growth-promoting properties. WCS417 caused a particularly dramatic shift in the leaf microbiota with more than 50% of amplicon reads representing two bacterial species: WCS417 and 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 plant-derived factors. Together, our data connect systemic immunity with leaf microbiome dynamics and highlight the importance of plant-microbe-microbe interactions for plant health.

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 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.

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., Plants immune responses are triggered upon perception of volatile monoterpenes. Here, Wenig et al. show that a feed-forward loop featuring LEGUME LECTIN-LIKE PROTEIN1 propagates monoterpene-associated cues both within and between plants, illustrating how systemic immunity could act at a population level.

A fatty acid triggers immune responses Plants and animals respond to the microbial communities around them, whether in antagonistic or mutualistic ways. Some of these interactions are mediated by lipopolysaccharide—a large, complex, and irregular molecule on the surface of most Gram-negative bacteria. Studying the small mustard plant Arabidopsis , Kutschera et al. identified a 3-hydroxydecanoyl chain as the structural element sensed by the plant's lectin receptor kinase. Indeed, synthetic 3-hydroxydecanoic acid alone was sufficient to produce a response. A small microbial metabolite may thus suffice to trigger immune responses. Science , this issue p. 178

During Systemic Acquired Resistance (SAR), a SAR-inducing infection in one leaf initiates movement of phloem-mobile signals to uninfected distant leaves to prime plants to respond in a resistant manner to subsequent infections. Our early work with the dir1-1 (defective in induced resistance) mutant in Arabidopsis demonstrated that the DIR1 protein is required for SAR and led to the hypothesis that DIR1, a lipid transfer protein (LTP), moves to distant leaves to activate SAR. To prove this hypothesis, we monitored DIR1-GFP accumulation in phloem exudates using an estrogen-SAR assay. In this assay, estrogen treatment induces DIR1-GFP expression in one leaf of dir1-1, followed by SAR-induction in the same leaf. DIR1-GFP was detected in exudates collected from local and distant leaves of SAR-induced plants using both DIR1 and GFP antibodies. This provides compelling evidence that DIR1 moves via the phloem to distant leaves to initiate priming. Our work fills a major gap in research on SAR as no other putative SAR mobile signal has been shown to move in planta to distant leaves. To discover how DIR1 enters the phloem, we took advantage of plant lines with compromised cell-to-cell movement caused by overexpression of Plasmodesmata-Located Proteins. These lines were defective for SAR, and DIR1 was not observed in distant leaf phloem exudates, supporting the idea that cell-to-cell movement of DIR1 through plasmodesmata is important for SAR signal movement. To discover new phloem proteins that play a role during SAR, we compared phloem exudate proteomes collected from mock- and SAR-induced leaves using quantitative LC-MS/MS. Numerous proteins were enriched in SAR-induced versus mock-induced phloem exudates and T-DNA knock-out lines in some of these genes were SAR-defective, indicating they contribute to SAR. Identification of SAR-specific phloem proteins may provide clues as to the protein complement of a high molecular weight DIR1-containing complex found in phloem exudates only after SAR induction. We will take advantage of DIR1’s proteinaceous nature to identify proteins in the high molecular weight mobile signal complex, proteins associated with phloem loading of SAR signals and proteins involved in DIR1 perception in distant leaves.

Plants are exposed to numerous pathogens and fend off many of these with different phytohormone signalling pathways. Much is known about defence signalling in the dicotyledonous model plant Arabidopsisthaliana, but it is unclear to which extent knowledge from model systems can be transferred to monocotyledonous plants, including cereal crops. Here, we investigated the defence-inducing potential of Arabidopsis resistance-inducing compounds in the cereal crop barley. Salicylic acid (SA), folic acid (Fol), and azelaic acid (AzA), each inducing defence against (hemi-)biotrophic pathogens in Arabidopsis, were applied to barley leaves and the treated and systemic leaves were subsequently inoculated with Xanthomonastranslucens pv. cerealis (Xtc), Blumeria graminis f. sp. hordei (powdery mildew, Bgh), or Pyrenophora teres. Fol and SA reduced Bgh propagation locally and/or systemically, whereas Fol enhanced Xtc growth in barley. AzA reduced Bgh propagation systemically and enhanced Xtc growth locally. Neither SA, Fol, nor AzA influenced lesion sizes caused by the necrotrophic fungus P. teres, suggesting that the tested compounds exclusively affected growth of (hemi-)biotrophic pathogens in barley. In addition to SA, Fol and AzA might thus act as resistance-inducing compounds in barley against Bgh, although adverse effects on the growth of pathogenic bacteria, such as Xtc, are possible.

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

Summary Folates are essential for one-carbon transfer reactions in all organisms and contribute, for example, to de novo DNA synthesis. Here, we detected the folate precursors 7,8-dihydropteroate (DHP) and 4-amino-4-deoxychorismate (ADC) in extracts from Arabidopsis thaliana plants by Fourier transform ion cyclotron resonance-mass spectrometry. The accumulation of DHP, but not ADC, was induced after infection of plants with Pseudomonas syringae delivering the effector protein AvrRpm1. Application of folic acid or the DHP precursor 7,8-dihydroneopterin (DHN) enhanced resistance in Arabidopsis to P. syringae and elevated the transcript accumulation of the salicylic acid (SA) marker gene PATHOGENESIS-RELATED1 in both the treated and systemic untreated leaves. DHN- and folic acid-induced systemic resistance was dependent on SA biosynthesis and signalling. Similar to SA, folic acid application locally enhanced Arabidopsis susceptibility to the necrotrophic fungus Alternaria brassicicola. Together, the data associate the folic acid pathway with innate immunity in Arabidopsis, simultaneously activating local and systemic SA-dependent resistance to P. syringae and suppressing local resistance to A. brassicicola.

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 two 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.

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 β-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. A. thaliana 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, β-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 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.

• Enhanced Disease Susceptibility1 (EDS1) is an important regulator of plant basal and receptor-triggered immunity. Arabidopsis EDS1 interacts with two related proteins, Phytoalexin Deficient4 (PAD4) and Senescence Associated Gene101 (SAG101), whose combined activities are essential for defense signaling. The different sizes and intracellular distributions of EDS1-PAD4 and EDS1-SAG101 complexes in Arabidopsis leaf tissues suggest that they perform nonredundant functions. • The nature and biological relevance of EDS1 interactions with PAD4 and SAG101 were explored using yeast three-hybrid assays, in vitro analysis of recombinant proteins purified from Escherichia coli, and characterization of Arabidopsis transgenic plants expressing an eds1 mutant (eds1(L262P) ) protein which no longer binds PAD4 but retains interaction with SAG101. • EDS1 forms molecularly distinct complexes with PAD4 or SAG101 without additional plant factors. Loss of interaction with EDS1 reduces PAD4 post-transcriptional accumulation, consistent with the EDS1 physical association stabilizing PAD4. The dissociated forms of EDS1 and PAD4 are fully competent in signaling receptor-triggered localized cell death at infection foci. By contrast, an EDS1-PAD4 complex is necessary for basal resistance involving transcriptional up-regulation of PAD4 itself and mobilization of salicylic acid defenses. • Different EDS1 and PAD4 molecular configurations have distinct and separable functions in the plant innate immune response.

The plant hormone auxin (indole-3-acetic acid [IAA]) is found both free and conjugated to a variety of carbohydrates, amino acids, and peptides. We have recently shown that IAA could be converted to its methyl ester (MeIAA) by the Arabidopsis (Arabidopsis thaliana) enzyme IAA carboxyl methyltransferase 1. However, the presence and function of MeIAA in vivo remains unclear. Recently, it has been shown that the tobacco (Nicotiana tabacum) protein SABP2 (salicylic acid binding protein 2) hydrolyzes methyl salicylate to salicylic acid. There are 20 homologs of SABP2 in the genome of Arabidopsis, which we have named AtMES (for methyl esterases). We tested 15 of the proteins encoded by these genes in biochemical assays with various substrates and identified several candidate MeIAA esterases that could hydrolyze MeIAA. MeIAA, like IAA, exerts inhibitory activity on the growth of wild-type roots when applied exogenously. However, the roots of Arabidopsis plants carrying T-DNA insertions in the putative MeIAA esterase gene AtMES17 (At3g10870) displayed significantly decreased sensitivity to MeIAA compared with wild-type roots while remaining as sensitive to free IAA as wild-type roots. Incubating seedlings in the presence of [14C]MeIAA for 30 min revealed that mes17 mutants hydrolyzed only 40% of the [14C]MeIAA taken up by plants, whereas wild-type plants hydrolyzed 100% of absorbed [14C]MeIAA. Roots of Arabidopsis plants overexpressing AtMES17 showed increased sensitivity to MeIAA but not to IAA. Additionally, mes17 plants have longer hypocotyls and display increased expression of the auxin-responsive DR5:β-glucuronidase reporter gene, suggesting a perturbation in IAA homeostasis and/or transport. mes17-1/axr1-3 double mutant plants have the same phenotype as axr1-3, suggesting MES17 acts upstream of AXR1. The protein encoded by AtMES17 had a K m value of 13 μ m and a K cat value of 0.18 s−1 for MeIAA. AtMES17 was expressed at the highest levels in shoot apex, stem, and root of Arabidopsis. Our results demonstrate that MeIAA is an inactive form of IAA, and the manifestations of MeIAA in vivo activity are due to the action of free IAA that is generated from MeIAA upon hydrolysis by one or more plant esterases.

The APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily of transcription factors regulates physiological, developmental and stress responses. Most of the AP2/ERF transcription factors belong to the ERF family in both dicotyledonous and monocotyledonous plants. ERFs are implicated in the responses to both biotic and abiotic stress and occasionally impart multiple stress tolerance. Studies have revealed that ERF gene function is conserved in dicots and monocots. Moreover, successful stress tolerant phenotypes are observed on expression in heterologous systems, making ERFs promising candidates for engineering stress tolerance in plants. In this review, we summarize the role of ERFs in general stress tolerance, including responses to biotic and abiotic stress factors, and endeavor to understand the cascade of ERF regulation resulting in successful signal-to-response translation in monocotyledonous plants.

Alfalfa mosaic virus (AMV) is a positive-strand RNA virus belonging to the family Bromoviridae. It has a tripartite genome, with RNAs 1 and 2 encoding the replicase proteins P1 and P2, respectively. Of these, P1 contains regions with homology to known methyltransferase domains in its N-terminal half, as well as a superfamily I helicase-like domain in its C-terminal half (10, 33) (Fig. ​(Fig.1A).1A). P2 is the viral RNA-dependent RNA polymerase protein (RdRp) (27). AMV RNA 3 codes for the movement protein (P3) as well as the coat protein (CP), which is translated from subgenomic mRNA 4. A mixture of the genomic RNAs of AMV is infectious only when either CP or RNA 4 is added to the inoculum in a process called genome activation (7, 13). However, it was recently shown that addition of a poly(A) tail to the normally unpolyadenylated 3′-untranslated regions (UTR) of the AMV RNAs can substitute for the requirement for CP in the inoculum (25). All AMV RNAs are capped, with uncapped RNAs 1 and 2 being noninfectious. In addition, uncapped RNA 4 is unable to activate the genome. FIG. 1. (A) Schematic representation of AMV P1. Black boxes, N-terminal methyltransferase (MT)-like and C-terminal helicase (HEL)-like domain motifs. (B) Amino acid sequence alignment of the AMV methyltransferase-like domain and the methyltransferase domains ... Viruses that belong to the alphavirus-like superfamily of viruses replicate in the cytoplasm of infected cells. They use the host translation machinery for translation of their RNAs, which are capped with a 7-methyl-G(5′)ppp moiety. Since cellular mRNAs are capped in the nucleus, the host capping proteins are not accessible to alphavirus-like viruses. Consequently, these viruses encode their own capping enzymes. The capping reaction of alphavirus RNAs can be divided into four steps (3, 6): (i) dephosphorylation of the viral RNA 5′-pppN end, resulting in a 5′-ppN terminus, (ii) methylation of GTP, resulting in m7GTP, (iii) complex formation of m7GTP and the viral methyltransferase protein (MT), resulting in m7GMP-MT, and (iv) transfer of the m7GMP moiety of m7GMP-MT to the 5′-ppN terminus of the RNA, resulting in m7GpppN(pN)n. It has been shown that steps ii and iii, the methyltransferase and guanylyltransferase reactions, are supported in vitro by the proposed capping protein domains of Semliki Forest virus (SFV), tobacco mosaic virus, bamboo mosaic virus (BaMV), hepatitis E virus (HEV), and brome mosaic virus (BMV) (1, 4, 15, 20, 22, 23). In addition, the proposed capping protein of Sindbis virus (SIN) can support at least the methyltransferase reaction of the capping process in vitro (43). Recently, RNA 5′-triphosphatase activities (step i of the capping process) were assigned to the helicase-like proteins of SFV, SIN, and BaMV (21, 41). Moreover, it was shown that BaMV RNA can be capped in vitro by sequential treatment of the template with the BaMV helicase-like and methytransferase domains (21). Despite a very low sequence homology between the proposed capping proteins of alphaviruses and alphavirus-like viruses, four highly conserved sequence motifs were distinguished (33). Several conserved motifs in the proposed capping proteins of AMV, BMV, HEV, SFV, and SIN are shown in Fig. ​Fig.1B.1B. Motif I contains an invariant His residue (His-100 in AMV P1), motif II contains a conserved AspXXArg sequence (Asp-154 and Arg-157 in AMV P1), and motif IV is characterized by an invariant Tyr residue (Tyr-266 in AMV P1). In addition, two conserved Cys residues have been recognized (Cys-182 and Cys-189 in AMV P1) (33). Mutation of these conserved residues affected infectivity, methyltransferase activity, and/or guanylyltransferase activity of BMV, SFV, and SIN (1, 2, 4, 43). Previously, we showed that the replicase proteins P1 and P2 of AMV could be transiently expressed in Nicotiana benthamiana leaves by agroinfiltration (42). Expression of RNAs 1 and 2 from a transferred-DNA (T-DNA) vector resulted in formation of an RdRp complex that was active both in vivo and in vitro. Accumulation of P1 and P2 in this system was not affected by mutations that inhibited replication of RNA 1 or 2 (42). In the present study, agroinfiltration was employed to express AMV RNAs 2 and 3 in N. benthamiana together with RNA 1 sequences encoding a mutation of one of six conserved residues in the methyltransferase-like domain of P1 (Fig. ​(Fig.1B).1B). The mutations abolished infectivity of the virus and affected the accumulation of negative- and positive-strand RNA to various degrees. Several mutations that affected negative-strand RNA synthesis could be complemented in trans by coexpression of wild-type (wt) P1. Partial purification of the transiently expressed mutant replicases yielded preparations supporting transcription of positive- and negative-strand AMV template RNAs in vitro at levels ranging from 0 to 100% of those for the wt. The data indicate that, in addition to causing a putative defect in capping activity, the mutations affected other replication-associated functions.

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.

Summary Compromised systemic acquired resistance in eds1 mutant Arabidopsis plants is associated with a reduced ability of the mutant to accumulate 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.

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.

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.

Salicylic acid (SA) has been shown to regulate various aspects of growth and development; it also serves as a critical signal for activating disease resistance in Arabidopsis thaliana and other plant species. This review surveys the mechanisms involved in the biosynthesis and metabolism of this critical plant hormone. While a complete biosynthetic route has yet to be established, stressed Arabidopsis appear to synthesize SA primarily via an isochorismate-utilizing pathway in the chloroplast. A distinct pathway utilizing phenylalanine as the substrate also may contribute to SA accumulation, although to a much lesser extent. Once synthesized, free SA levels can be regulated by a variety of chemical modifications. Many of these modifications inactivate SA; however, some confer novel properties that may aid in long distance SA transport or the activation of stress responses complementary to those induced by free SA. In addition, a number of factors that directly or indirectly regulate the expression of SA biosynthetic genes or that influence the rate of SA catabolism have been identified. An integrated model, encompassing current knowledge of SA metabolism in Arabidopsis, as well as the influence other plant hormones exert on SA metabolism, is presented.

For more than 200 years, the plant hormone salicylic acid (SA) has been studied for its medicinal use in humans. However, its extensive signaling role in plants, particularly in defense against pathogens, has only become evident during the past 20 years. This review surveys how SA in plants regulates both local disease resistance mechanisms, including host cell death and defense gene expression, and systemic acquired resistance (SAR). Genetic studies reveal an increasingly complex network of proteins required for SA-mediated defense signaling, and this process is amplified by several regulatory feedback loops. The interaction between the SA signaling pathway and those regulated by other plant hormones and/or defense signals is also discussed.

We previously demonstrated that salicylic acid-binding protein 2 (SABP2) of tobacco is an integral component of systemic acquired resistance (SAR). SABP2 is a methyl salicylate (MeSA) esterase that has high affinity for SA, which feedback inhibits its esterase activity. MeSA esterase activity is required in distal, healthy tissue of pathogen-infected plants to hydrolyze MeSA, which functions as a long-distance, phloem-mobile SAR signal; this hydrolysis releases the biologically active defense hormone SA. In this study, we examined the inhibitory interaction of SA with SABP2, and identified a synthetic SA analog, 2,2,2,2′-tetra-f luoroacetophenone (tetraFA) that, like SA, competitively inhibits the activity of SABP2 and targets esterases, which utilize MeSA as a substrate. However, in contrast to SA, tetraFA does not induce downstream defense responses and, therefore, is effective in planta at blocking SAR development in tobacco mosaic virus (TMV)-infected tobacco and Pseudomonas syringae-infected Arabidopsis. These results confirm the importance of SABP2 and MeSA for SAR development in tobacco and establish similar roles for MeSA and the orthologs of SABP2 in Arabidopsis. Moreover, they demonstrate that tetraFA can be used to determine whether MeSA and its corresponding esterase(s) play a role in SAR signaling in other plant species. In planta analyses using tetraFA, in conjunction with leaf detachment assays and MeSA quantification, were used to assess the kinetics with which MeSA is generated in pathogen-infected leaves, transmitted through the phloem, and processed in the distal healthy leaves. In TMV-infected tobacco, these studies revealed that critical amounts of MeSA are generated, transmitted, and processed between 48 and 72 h post primary infection.

Systemic acquired resistance (SAR) is a form of inducible resistance that is triggered in systemic healthy tissues of locally infected plants. The nature of the mobile signal that travels through the phloem from the site of infection to establish systemic immunity has been sought after for decades. Several candidate signaling molecules have emerged in the past two years, including the methylated derivative of a well-known defense hormone (methyl salicylate), the defense hormone jasmonic acid, a yet undefined glycerolipid-derived factor, and a group of peptides that is involved in cell-to-cell basal defense signaling. Systemic SAR signal amplification increasingly appears to parallel salicylic acid-dependent defense responses, and is concomitantly fine-tuned by auxin.

Summary Salicylic acid-binding protein 2 (SABP2) is essential for the establishment of systemic acquired resistance (SAR) in tobacco; SABP2’s methyl salicylate (MeSA) esterase activity is required in healthy systemic tissues of infected plants to release the active defense phytohormone SA from MeSA, which serves as a long-distance signal for SAR. In the current study, we characterize a new gene family from Arabidopsis thaliana encoding 18 potentially active α/β fold hydrolases that share 32–57% identity with SABP2. Of 14 recombinant AtMES (MES for methyl esterase) proteins tested, five showed preference for MeSA as a substrate and displayed SA inhibition of MeSA esterase activity in vitro (AtMES1, -2, -4, -7, and -9). The two genes encoding MeSA esterases with the greatest activity, AtMES1 and -9, as well as AtMES7 were transcriptionally upregulated during infection of Arabidopsis with avirulent Pseudomonas syringae. In addition, conditional expression of AtMES1, -7, or -9 complemented SAR deficiency in SABP2-silenced tobacco, suggesting that these three members of the AtMES family are SABP2 functional homologs (orthologs). Underexpression by knockout mutation and/or RNAi-mediated silencing of multiple AtMES genes, including AtMES1, -2, -7, and -9, compromised SAR in Arabidopsis and correlated with enhanced accumulation of MeSA in the systemic tissue of SAR-induced plants. Together, the data show that several members of the AtMES gene family are functionally homologous to SABP2 and redundant for MeSA hydrolysis and probably SAR. These data suggest that MeSA is a conserved SAR signal in Arabidopsis and tobacco.