A single amino acid transporter controls the uptake of priming-inducing beta-amino acids and the associated tradeoff between induced resistance and plant growth.

Selected ß-amino acids, such as ß-aminobutyric acid (BABA) and R-ß-homoserine (RBH), can prime plants for resistance against a broad spectrum of diseases. Here, we describe a genome-wide screen of fully annotated Arabidopsis thaliana T-DNA insertion lines for impaired in RBH-induced immunity (iri) mutants against the downy mildew pathogen Hyaloperonospora arabidopsidis, yielding 104 lines that were partially affected and four lines that were completely impaired in RBH-induced resistance (IR). We confirmed the iri1-1 mutant phenotype with an independent T-DNA insertion line in the same gene, encoding the high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER 1 (LHT1). Uptake experiments with yeast cells expressing LHT1 and mass spectrometry-based quantification of RBH and BABA in leaves of lht1 mutant and LHT1 overexpression lines revealed that LHT1 acts as the main transporter for cellular uptake and systemic distribution of RBH and BABA. Subsequent characterization of lht1 mutant and LHT1 overexpression lines for IR and growth responses revealed that the levels of LHT1-mediated uptake determine the tradeoff between IR and plant growth by RBH and BABA.

Epigenetics: a catalyst of plant immunity against pathogens.

The plant immune system protects against pests and diseases. The recognition of stress-related molecular patterns triggers localised immune responses, which are often followed by longer-lasting systemic priming and/or up-regulation of defences. In some cases, this induced resistance (IR) can be transmitted to following generations. Such transgenerational IR is gradually reversed in the absence of stress at a rate that is proportional to the severity of disease experienced in previous generations. This review outlines the mechanisms by which epigenetic responses to pathogen infection shape the plant immune system across expanding time scales. We review the cis- and trans-acting mechanisms by which stress-inducible epigenetic changes at transposable elements (TEs) regulate genome-wide defence gene expression and draw particular attention to one regulatory model that is supported by recent evidence about the function of AGO1 and H2A.Z in transcriptional control of defence genes. Additionally, we explore how stress-induced mobilisation of epigenetically controlled TEs acts as a catalyst of Darwinian evolution by generating (epi)genetic diversity at environmentally responsive genes. This raises questions about the long-term evolutionary consequences of stress-induced diversification of the plant immune system in relation to the long-held dichotomy between Darwinian and Lamarckian evolution.

Transcriptomic changes during the establishment of long-term methyl jasmonate-induced resistance in Norway spruce.

Norway spruce (Picea abies) is an economically and ecologically important tree species that grows across northern and central Europe. Treating Norway spruce with jasmonate has long-lasting beneficial effects on tree resistance to damaging pests, such as the European spruce bark beetle Ips typographus and its fungal associates. The (epi)genetic mechanisms involved in such long-lasting jasmonate induced resistance (IR) have gained much recent interest but remain largely unknown. In this study, we treated 2-year-old spruce seedlings with methyl jasmonate (MeJA) and challenged them with the I. typographus vectored necrotrophic fungus Grosmannia penicillata. MeJA treatment reduced the extent of necrotic lesions in the bark 8 weeks after infection and thus elicited long-term IR against the fungus. The transcriptional response of spruce bark to MeJA treatment was analysed over a 4-week time course using mRNA-seq. This analysis provided evidence that MeJA treatment induced a transient upregulation of jasmonic acid, salicylic acid and ethylene biosynthesis genes and downstream signalling genes. Our data also suggests that defence-related genes are induced while genes related to growth are repressed by methyl jasmonate treatment. These results provide new clues about the potential underpinning mechanisms and costs associated with long-term MeJA-IR in Norway spruce.

Spodoptera frugiperda Caterpillars Suppress Herbivore-Induced Volatile Emissions in Maize.

The vast spectrum of inducible plant defenses can have direct negative effects on herbivores, or indirect effects, for instance in the form of herbivore-induced plant volatiles (HIPVs) that attract natural enemies. Various arthropods have evolved ways to suppress plant defenses. To test whether this is the case for caterpillar-induced HIPVs, we compared the volatile induction by Spodoptera frugiperda (Lepidoptera: Noctuidae), which is particularly well adapted to feed on maize (Zea mays), with the induction by three more generalist noctuid larvae. We tested the hypothesis that S. frugiperda suppresses HIPV emissions in maize, and thereby reduces attractiveness to natural enemies. HIPV emissions triggered by S. frugiperda when feeding on maize were indeed found to be significantly weaker than by Spodoptera littoralis, Spodoptera exigua, and Helicoverpa armigera. The suppression seems specific for maize, as we found no evidence for this when S. frugiperda caterpillars fed on cotton (Gossypium herbaceum). Artificially damaged maize plants treated with larval regurgitant revealed that HIPV suppression may be related to factors in the caterpillars' oral secretions. We also found evidence that differential physical damage that the caterpillars inflict on maize leaves may play a role. The suppressed induction of HIPVs had no apparent consequences for the attraction of a common parasitoid of S. frugiperda, Cotesia marginiventris (Hymenoptera: Braconidae). Nevertheless, the ability to manipulate the defenses of its main host plant may have contributed to the success of S. frugiperda as a major pest of maize, especially in Africa and Asia, which it has recently invaded.

Bacterial infection systemically suppresses stomatal density.

Many plant pathogens gain entry to their host via stomata. On sensing attack, plants close these pores to restrict pathogen entry. Here, we show that plants exhibit a second longer term stomatal response to pathogens. Following infection, the subsequent development of leaves is altered via a systemic signal. This reduces the density of stomata formed, thus providing fewer entry points for pathogens on new leaves. Arabidopsis thaliana leaves produced after infection by a bacterial pathogen that infects through the stomata (Pseudomonas syringae) developed larger epidermal pavement cells and stomata and consequently had up to 20% reductions in stomatal density. The bacterial peptide flg22 or the phytohormone salicylic acid induced similar systemic reductions in stomatal density suggesting that they might mediate this effect. In addition, flagellin receptors, salicylic acid accumulation, and the lipid transfer protein AZI1 were all required for this developmental response. Furthermore, manipulation of stomatal density affected the level of bacterial colonization, and plants with reduced stomatal density showed slower disease progression. We propose that following infection, development of new leaves is altered by a signalling pathway with some commonalities to systemic acquired resistance. This acts to reduce the potential for future infection by providing fewer stomatal openings.

Metabolite profiling of non-sterile rhizosphere soil.

Rhizosphere chemistry is the sum of root exudation chemicals, their breakdown products and the microbial products of soil-derived chemicals. To date, most studies about root exudation chemistry are based on sterile cultivation systems, which limits the discovery of microbial breakdown products that act as semiochemicals and shape microbial rhizosphere communities. Here, we present a method for untargeted metabolic profiling of non-sterile rhizosphere soil. We have developed an experimental growth system that enables the collection and analysis of rhizosphere chemicals from different plant species. High-throughput sequencing of 16SrRNA genes demonstrated that plants in the growth system support a microbial rhizosphere effect. To collect a range of (a)polar chemicals from the system, we developed extraction methods that do not cause detectable damage to root cells or soil-inhabiting microbes, thus preventing contamination with cellular metabolites. Untargeted metabolite profiling by UPLC-Q-TOF mass spectrometry, followed by uni- and multivariate statistical analyses, identified a wide range of secondary metabolites that are enriched in plant-containing soil, compared with control soil without roots. We show that the method is suitable for profiling the rhizosphere chemistry of Zea mays (maize) in agricultural soil, thereby demonstrating the applicability to different plant-soil combinations. Our study provides a robust method for the comprehensive metabolite profiling of non-sterile rhizosphere soil, which represents a technical advance towards the establishment of causal relationships between the chemistry and microbial composition of the rhizosphere.

Mechanisms of glacial-to-future atmospheric CO2 effects on plant immunity.

The impacts of rising atmospheric CO2 concentrations on plant disease have received increasing attention, but with little consensus emerging on the direct mechanisms by which CO2 shapes plant immunity. Furthermore, the impact of sub-ambient CO2 concentrations, which plants have experienced repeatedly over the past 800 000 yr, has been largely overlooked. A combination of gene expression analysis, phenotypic characterisation of mutants and mass spectrometry-based metabolic profiling was used to determine development-independent effects of sub-ambient CO2 (saCO2 ) and elevated CO2 (eCO2 ) on Arabidopsis immunity. Resistance to the necrotrophic Plectosphaerella cucumerina (Pc) was repressed at saCO2 and enhanced at eCO2 . This CO2 -dependent resistance was associated with priming of jasmonic acid (JA)-dependent gene expression and required intact JA biosynthesis and signalling. Resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) increased at both eCO2 and saCO2 . Although eCO2 primed salicylic acid (SA)-dependent gene expression, mutations affecting SA signalling only partially suppressed Hpa resistance at eCO2 , suggesting additional mechanisms are involved. Induced production of intracellular reactive oxygen species (ROS) at saCO2 corresponded to a loss of resistance in glycolate oxidase mutants and increased transcription of the peroxisomal catalase gene CAT2, unveiling a mechanism by which photorespiration-derived ROS determined Hpa resistance at saCO2 . By separating indirect developmental impacts from direct immunological effects, we uncover distinct mechanisms by which CO2 shapes plant immunity and discuss their evolutionary significance.

Chemical priming of immunity without costs to plant growth.

ß-Aminobutyric acid (BABA) induces broad-spectrum disease resistance, but also represses plant growth, which has limited its exploitation in crop protection. BABA perception relies on binding to the aspartyl-tRNA synthetase (AspRS) IBI1, which primes the enzyme for secondary defense activity. This study aimed to identify structural BABA analogues that induce resistance without stunting plant growth. Using site-directed mutagenesis, we demonstrate that the (l)-aspartic acid-binding domain of IBI1 is critical for BABA perception. Based on interaction models of this domain, we screened a small library of structural BABA analogues for growth repression and induced resistance against biotrophic Hyaloperonospora arabidopsidis (Hpa). A range of resistance-inducing compounds were identified, of which (R)-ß-homoserine (RBH) was the most effective. Surprisingly, RBH acted through different pathways than BABA. RBH-induced resistance (RBH-IR) against Hpa functioned independently of salicylic acid, partially relied on camalexin, and was associated with augmented cell wall defense. RBH-IR against necrotrophic Plectosphaerella cucumerina acted via priming of ethylene and jasmonic acid defenses. RBH-IR was also effective in tomato against Botrytis cinerea. Metabolic profiling revealed that RBH, unlike BABA, does not majorly affect plant metabolism. RBH primes distinct defense pathways against biotrophic and necrotrophic pathogens without stunting plant growth, signifying strong potential for exploitation in crop protection.

The role of DNA (de)methylation in immune responsiveness of Arabidopsis.

DNA methylation is antagonistically controlled by DNA methyltransferases and DNA demethylases. The level of DNA methylation controls plant gene expression on a global level. We have examined impacts of global changes in DNA methylation on the Arabidopsis immune system. A range of hypo-methylated mutants displayed enhanced resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis (Hpa), whereas two hyper-methylated mutants were more susceptible to this pathogen. Subsequent characterization of the hypo-methylated nrpe1 mutant, which is impaired in RNA-directed DNA methylation, and the hyper-methylated ros1 mutant, which is affected in DNA demethylation, revealed that their opposite resistance phenotypes are associated with changes in cell wall defence and salicylic acid (SA)-dependent gene expression. Against infection by the necrotrophic pathogen Plectosphaerella cucumerina, nrpe1 showed enhanced susceptibility, which was associated with repressed sensitivity of jasmonic acid (JA)-inducible gene expression. Conversely, ros1 displayed enhanced resistance to necrotrophic pathogens, which was not associated with increased responsiveness of JA-inducible gene expression. Although nrpe1 and ros1 were unaffected in systemic acquired resistance to Hpa, they failed to develop transgenerational acquired resistance against this pathogen. Global transcriptome analysis of nrpe1 and ros1 at multiple time-points after Hpa infection revealed that 49% of the pathogenesis-related transcriptome is influenced by NRPE1- and ROS1-controlled DNA methylation. Of the 166 defence-related genes displaying augmented induction in nrpe1 and repressed induction in ros1, only 25 genes were associated with a nearby transposable element and NRPE1- and/or ROS1-controlled DNA methylation. Accordingly, we propose that the majority of NRPE1- and ROS1-dependent defence genes are regulated in trans by DNA methylation.

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina.

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.

NAD Acts as an Integral Regulator of Multiple Defense Layers.

Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in Arabidopsis (Arabidopsis thaliana) that the inducible NAD-overproducing nadC lines are more resistant to an avirulent strain of Pseudomonas syringae pv tomato (Pst-AvrRpm1), which was associated with salicylic acid-dependent defense. Here, we have further characterized the NAD-dependent immune response in Arabidopsis. Quinolinate-induced stimulation of intracellular NAD in transgenic nadC plants enhanced resistance against a diverse range of (a)virulent pathogens, including Pst-AvrRpt2, Dickeya dadantii, and Botrytis cinerea Characterization of the redox status demonstrated that elevated NAD levels induce reactive oxygen species (ROS) production and the expression of redox marker genes of the cytosol and mitochondrion. Using pharmacological and reverse genetics approaches, we show that NAD-induced ROS production functions independently of NADPH oxidase activity and light metabolism but depends on mitochondrial respiration, which was increased at higher NAD. We further demonstrate that NAD primes pathogen-induced callose deposition and cell death. Mass spectrometry analysis reveals that NAD simultaneously induces different defense hormones and that the NAD-induced metabolic profiles are similar to those of defense-expressing plants after treatment with pathogen-associated molecular patterns. We thus conclude that NAD triggers metabolic profiles rather similar to that of pathogen-associated molecular patterns and discuss how signaling cross talk between defense hormones, ROS, and NAD explains the observed resistance to pathogens.

Plant perception of ß-aminobutyric acid is mediated by an aspartyl-tRNA synthetase.

Specific chemicals can prime the plant immune system for augmented defense. ß-aminobutyric acid (BABA) is a priming agent that provides broad-spectrum disease protection. However, BABA also suppresses plant growth when applied in high doses, which has hampered its application as a crop defense activator. Here we describe a mutant of Arabidopsis thaliana that is impaired in BABA-induced disease immunity (ibi1) but is hypersensitive to BABA-induced growth repression. IBI1 encodes an aspartyl-tRNA synthetase. Enantiomer-specific binding of the R enantiomer of BABA to IBI1 primed the protein for noncanonical defense signaling in the cytoplasm after pathogen attack. This priming was associated with aspartic acid accumulation and tRNA-induced phosphorylation of translation initiation factor eIF2α. However, mutation of eIF2α-phosphorylating GCN2 kinase did not affect BABA-induced immunity but relieved BABA-induced growth repression. Hence, BABA-activated IBI1 controls plant immunity and growth via separate pathways. Our results open new opportunities to separate broad-spectrum disease resistance from the associated costs on plant growth.

Optimizing Chemically Induced Resistance in Tomato Against Botrytis cinerea.

Resistance-inducing chemicals can offer broad-spectrum disease protection in crops, but can also affect plant growth and interactions with plant-beneficial microbes. We have evaluated different application methods of ß-aminobutyric acid (BABA) and jasmonic acid (JA) for long-lasting induced resistance in tomato against Botrytis cinerea. In addition, we have studied nontarget effects on plant growth and root colonization by arbuscular mycorrhizal fungi (AMF). Germinating seeds for 1 week in BABA- or JA-containing solutions promoted seed germination efficiency, did not affect plant growth, and induced resistance in 4-week-old plants. When formulating BABA and JA in carboxymethyl cellulose seed coating, only BABA was able to induce resistance in 4-week-old plants. Root treatment of 1-week-old seedlings with BABA or JA also induced resistance in 4-week-old plants. However, this seedling treatment repressed plant growth at higher concentrations of the chemicals, which was particularly pronounced in hydroponically grown plants after BABA treatment. Both seed coating with BABA, and seedling treatments with BABA or JA, did not affect AMF root colonization in soil-grown tomato. Our study has identified commercially feasible application methods of BABA and JA, which induce durable disease resistance in tomato without concurrent impacts on plant growth or colonization by plant-beneficial AMF.

Volatiles produced by soil-borne endophytic bacteria increase plant pathogen resistance and affect tritrophic interactions.

Volatile organic compounds (VOCs) released by soil microorganisms influence plant growth and pathogen resistance. Yet, very little is known about their influence on herbivores and higher trophic levels. We studied the origin and role of a major bacterial VOC, 2,3-butanediol (2,3-BD), on plant growth, pathogen and herbivore resistance, and the attraction of natural enemies in maize. One of the major contributors to 2,3-BD in the headspace of soil-grown maize seedlings was identified as Enterobacter aerogenes, an endophytic bacterium that colonizes the plants. The production of 2,3-BD by E. aerogenes rendered maize plants more resistant against the Northern corn leaf blight fungus Setosphaeria turcica. On the contrary, E. aerogenes-inoculated plants were less resistant against the caterpillar Spodoptera littoralis. The effect of 2,3-BD on the attraction of the parasitoid Cotesia marginiventris was more variable: 2,3-BD application to the headspace of the plants had no effect on the parasitoids, but application to the soil increased parasitoid attraction. Furthermore, inoculation of seeds with E. aerogenes decreased plant attractiveness, whereas inoculation of soil with a total extract of soil microbes increased parasitoid attraction, suggesting that the effect of 2,3-BD on the parasitoid is indirect and depends on the composition of the microbial community.

Role of PMR4 and PDLP1 in priming of early acting penetration defense by resistance-inducing ß-amino acids.

R-ß-homoserine (RBH) and ß-aminobutyric acid (BABA) induce resistance against the oomycete Hyaloperonospora arabidopsidis (Hpa) in Arabidopsis, which is based on priming of multiple defense layers, including early acting penetration resistance at the cell wall. Here, we have examined the molecular basis of RBH- and BABA-primed defense by cell wall papillae against Hpa. Three-dimensional reconstruction of Hpa-induced papillae by confocal microscopy revealed no structural differences between control-, RBH-, and BABA-treated plants after Hpa challenge. However, mutations affecting POWDERY MILDEW RESISTANCE 4 or PLASMODESMATA LOCATED PROTEINs (PDLPs) only impaired BABA-induced penetration resistance and not RBH-induced penetration resistance. Furthermore, PDLP1 over-expression mimicked primed penetration resistance, while the intensity of GFP-tagged PDLP1 at germinating Hpa conidiospores was increased in BABA-primed plants but not RBH-primed plants. Our study reveals new regulatory layers of immune priming by ß-amino acids and supports the notion that penetration resistance is a multifaceted defense layer that can be achieved through seperate pathways.

Mucoromycotina 'fine root endophytes': a new molecular model for plant-fungal mutualisms?

The most studied plant-fungal symbioses to date are the interactions between plants and arbuscular mycorrhizal (AM) fungi of the Glomeromycotina clade. Advancements in phylogenetics and microbial community profiling have distinguished a group of symbiosis-forming fungi that resemble AM fungi as belonging instead to the Mucoromycotina. These enigmatic fungi are now known as Mucoromycotina 'fine root endophytes' and could provide a means to understand the origins of plant-fungal symbioses. Most of our knowledge of the mechanisms of fungal symbiosis comes from investigations using AM fungi. Here, we argue that inclusion of Mucoromycotina fine root endophytes in future studies will expand our understanding of the mechanisms, evolution, and ecology of plant-fungal symbioses.

Fine tuning of reactive oxygen species homeostasis regulates primed immune responses in Arabidopsis.

Selected stimuli can prime the plant immune system for a faster and stronger defense reaction to pathogen attack. Pretreatment of Arabidopsis with the chemical agent ß-aminobutyric acid (BABA) augmented H2O2 and callose production after induction with the pathogen-associated molecular pattern (PAMP) chitosan, or inoculation with the necrotrophic fungus Plectosphaerella cucumerina. However, BABA failed to prime H2O2 and callose production after challenge with the bacterial PAMP Flg22. Analysis of Arabidopsis mutants in reactive oxygen species (ROS) production (rbohD) or ROS scavenging (pad2, vtc1, and cat2) suggested a regulatory role for ROS homeostasis in priming of chitosan- and P. cucumerina-inducible callose and ROS. Moreover, rbohD and pad2 were both impaired in BABA-induced resistance against P. cucumerina. Gene expression analysis revealed direct induction of NADPH/respiratory burst oxidase protein D (RBOHD), γ-glutamylcysteine synthetase 1 (GSH1), and vitamin C defective 1 (VTC1) genes after BABA treatment. Conversely, ascorbate peroxidase 1 (APX1) transcription was repressed by BABA after challenge with chitosan or P. cucumerina, probably to provide a more oxidized environment in the cell and facilitate augmented ROS accumulation. Measuring ratios between reduced and oxidized glutathione confirmed that augmented defense expression in primed plants is associated with a more oxidized cellular status. Together, our data indicate that an altered ROS equilibrium is required for augmented defense expression in primed plants.

Next-generation systemic acquired resistance.

Systemic acquired resistance (SAR) is a plant immune response to pathogen attack. Recent evidence suggests that plant immunity involves regulation by chromatin remodeling and DNA methylation. We investigated whether SAR can be inherited epigenetically following disease pressure by Pseudomonas syringae pv tomato DC3000 (PstDC3000). Compared to progeny from control-treated Arabidopsis (Arabidopsis thaliana; C(1)), progeny from PstDC3000-inoculated Arabidopsis (P(1)) were primed to activate salicylic acid (SA)-inducible defense genes and were more resistant to the (hemi)biotrophic pathogens Hyaloperonospora arabidopsidis and PstDC3000. This transgenerational SAR was sustained over one stress-free generation, indicating an epigenetic basis of the phenomenon. Furthermore, P(1) progeny displayed reduced responsiveness of jasmonic acid (JA)-inducible genes and enhanced susceptibility to the necrotrophic fungus Alternaria brassicicola. This shift in SA- and JA-dependent gene responsiveness was not associated with changes in corresponding hormone levels. Instead, chromatin immunoprecipitation analyses revealed that SA-inducible promoters of PATHOGENESIS-RELATED GENE1, WRKY6, and WRKY53 in P(1) plants are enriched with acetylated histone H3 at lysine 9, a chromatin mark associated with a permissive state of transcription. Conversely, the JA-inducible promoter of PLANT DEFENSIN1.2 showed increased H3 triple methylation at lysine 27, a mark related to repressed gene transcription. P(1) progeny from the defense regulatory mutant non expressor of PR1 (npr1)-1 failed to develop transgenerational defense phenotypes, demonstrating a critical role for NPR1 in expression of transgenerational SAR. Furthermore, the drm1drm2cmt3 mutant that is affected in non-CpG DNA methylation mimicked the transgenerational SAR phenotype. Since PstDC3000 induces DNA hypomethylation in Arabidopsis, our results suggest that transgenerational SAR is transmitted by hypomethylated genes that direct priming of SA-dependent defenses in the following generations.

Epigenetic processes in plant stress priming: Open questions and new approaches.

Priming reflects the capacity of plants to memorise environmental stress experience and improve their response to recurring stress. Epigenetic modifications in DNA and associated histone proteins may carry short-term and long-term memory in the same plant or mediate transgenerational effects, but the evidence is still largely circumstantial. New experimental tools now enable scientists to perform targeted manipulations that either prevent or generate a particular epigenetic modification in a particular location of the genome. Such 'reverse epigenetics' approaches allow for the interrogation of causality between individual priming-induced modifications and their role for altering gene expression and plant performance under recurring stress. Furthermore, combining site-directed epigenetic manipulation with conditional and cell-type specific promoters creates novel opportunities to test and engineer spatiotemporal patterns of priming.