Chromatin modification acts as a memory for systemic acquired resistance in the plant stress response

Priming of defence genes for amplified response to secondary stress can be induced by application of the plant hormone salicylic acid or its synthetic analogue acibenzolar S‐methyl. In this study, we show that treatment with acibenzolar S‐methyl or pathogen infection of distal leaves induce chromatin modifications on defence gene promoters that are normally found on active genes, although the genes remain inactive. This is associated with an amplified gene response on challenge exposure to stress. Mutant analyses reveal a tight correlation between histone modification patterns and gene priming. The data suggest a histone memory for information storage in the plant stress response.     

AtPME17 is a functional Arabidopsis thaliana pectin methylesterase regulated by its PRO region that triggers PME activity in the resistance to Botrytis cinerea

Pectin is synthesized in a highly methylesterified form in the Golgi cisternae and partially de-methylesterified in muro by pectin methylesterases (PMEs). Arabidopsis thaliana produces a local and strong induction of PME activity during the infection of the necrotrophic fungus Botrytis cinerea. AtPME17 is a putative A. thaliana PME highly induced in response to B. cinerea. Here, a fine tuning of AtPME17 expression by different defence hormones was identified. Our genetic evidence demonstrates that AtPME17 strongly contributes to the pathogen-induced PME activity and resistance against B. cinerea by triggering jasmonic acid–ethylene-dependent PDF1.2 expression. AtPME17 belongs to group 2 isoforms of PMEs characterized by a PME domain preceded by an N-terminal PRO region. However, the biochemical evidence for AtPME17 as a functional PME is still lacking and the role played by its PRO region is not known. Using the Pichia pastoris expression system, we demonstrate that AtPME17 is a functional PME with activity favoured by an increase in pH. AtPME17 performs a blockwise pattern of pectin de-methylesterification that favours the formation of egg-box structures between homogalacturonans. Recombinant AtPME17 expression in Escherichia coli reveals that the PRO region acts as an intramolecular inhibitor of AtPME17 activity.     

Insect eggs induce a systemic acquired resistance in Arabidopsis

Although they constitute an inert stage of the insect's life, eggs trigger plant defences that lead to egg mortality or attraction of egg parasitoids. We recently found that salicylic acid (SA) accumulates in response to oviposition by the Large White butterfly Pieris brassicae, both in local and systemic leaves, and that plants activate a response that is similar to the recognition of pathogen‐associated molecular patterns (PAMPs), which are involved in PAMP‐triggered immunity (PTI). Here we discovered that natural oviposition by P. brassicae or treatment with egg extract inhibit growth of different Pseudomonas syringae strains in Arabidopsis through the activation of a systemic acquired resistance (SAR). This egg‐induced SAR involves the metabolic SAR signal pipecolic acid, depends on ALD1 and FMO1, and is accompanied by a stronger induction of defence genes upon secondary infection. Although P. brassicae larvae showed a reduced performance when feeding on Pseudomonas syringae‐infected plants, this effect was less pronounced when infected plants had been previously oviposited. Altogether, our results indicate that egg‐induced SAR might have evolved as a strategy to prevent the detrimental effect of bacterial pathogens on feeding larvae.     

Local and systemic wound-induction of RNase and nuclease activities in Arabidopsis: RNS1 as a marker for a JA-independent systemic signaling pathway

Induction of defense-related genes is one way in which plants respond to mechanical injury. We investigated whether RNases are involved in the wound response in Arabidopsis thaliana. As in other plant systems, several activities are induced with various timings in damaged leaves, stems and seedlings in Arabidopsis, including at least three bifunctional nucleases, capable of degrading both RNA and DNA, as well as RNS1, a member of the ubiquitous RNase T(2) family of RNases. The strong induction of RNS1 is particularly interesting because it occurs both locally and systemically following wounding. The systemic induction of this RNase indicates that members of this family may be involved in defense mechanisms in addition to their previously hypothesized functions in nutrient recycling and remobilization. Additionally, the systemic induction appears to be controlled independently of jasmonic acid, and the local induction of RNS1 and the nuclease activities are independent of both JA and oligosaccharide elicitors. Consequently, a novel systemic pathway, likely involving a third signal, appears to exist in Arabidopsis.     

Plasmodesmata‐located protein overexpression negatively impacts the manifestation of systemic acquired resistance and the long‐distance movement of Defective in Induced Resistance1 in Arabidopsis

Systemic acquired resistance (SAR) is a plant defence response that provides immunity to distant uninfected leaves after an initial localised infection. The lipid transfer protein (LTP) Defective in Induced Resistance1 (DIR1) is an essential component of SAR that moves from induced to distant leaves following a SAR‐inducing local infection. To understand how DIR1 is transported to distant leaves during SAR, we analysed DIR1 movement in transgenic Arabidopsis lines with reduced cell‐to‐cell movement caused by the overexpression of Plasmodesmata‐Located Proteins PDLP1 and PDLP5. These PDLP‐overexpressing lines were defective for SAR, and DIR1 antibody signals were not observed in phloem sap‐enriched petiole exudates collected from distant leaves. Our data support the idea that cell‐to‐cell movement of DIR1 through plasmodesmata is important during long‐distance SAR signalling in Arabidopsis.     

Induction of systemic acquired resistance in cotton by BTH has a negligible effect on phytophagous insects

Whether or not chemical changes in plants in response to pests (insects and pathogens) are general or specific remains unclear. Some evidence indicates that an induced response (IR) to arthropods via the octadecanoid pathway represents a distinct mechanism from the salicylic acid-based pathway of systemic acquired resistance (SAR) to pathogens. To further test this hypothesis, young cotton seedlings were activated with benzo (1,2,3) thiadiazole-7-carbothioic acid (S) methyl ester (BTH), an elicitor of SAR. The enzymatic activities of a number of pathogenesis-related (PR) proteins in young and old leaves of control and BTH treated plants were measured. BTH applications elicited marked increases in the activity levels of chitinase, peroxidase, and -1,3-glucanase both locally and systemically. The highest levels of induction were detected systemically in young leaves. Except for some local effects on whitefly oviposition, the induction of SAR by BTH had no effect on either host preference of whiteflies Bemisia tabaci (Gennadius) or on feeding efficiency of cotton bollworms Helicoverpa armigera (Hübner). We conclude that SAR induction via the salicylic acid pathway in Acala cotton has negligible effect on the tested insect herbivores.     

Arabidopsis thaliana GLUTATHIONE‐S‐TRANSFERASE THETA 2 interacts with RSI1/FLD to activate systemic acquired resistance

A partly infected plant develops systemic acquired resistance (SAR) and shows heightened resistance during subsequent infections. The infected parts generate certain mobile signals that travel to the distal tissues and help to activate SAR. SAR is associated with epigenetic modifications of several defence‐related genes. However, the mechanisms by which mobile signals contribute to epigenetic changes are little known. Previously, we have shown that the Arabidopsis REDUCED SYSTEMIC IMMUNITY 1 (RSI1, alias FLOWERING LOCUS D; FLD), which codes for a putative histone demethylase, is required for the activation of SAR. Here, we report the identification of GLUTATHIONE‐S‐TRANSFERASE THETA 2 (GSTT2) as an interacting factor of FLD. GSTT2 expression increases in pathogen‐inoculated as well as pathogen‐free distal tissues. The loss‐of‐function mutant of GSTT2 is compromised for SAR, but activates normal local resistance. Complementation lines of GSTT2 support its role in SAR activation. The distal tissues of gstt2 mutant plants accumulate significantly less salicylic acid (SA) and express a reduced level of the SA biosynthetic gene PAL1. In agreement with the established histone modification activity of FLD, gstt2 mutant plants accumulate an enhanced level of methylated and acetylated histones in the promoters of WRKY6 and WRKY29 genes. Together, these results demonstrate that GSTT2 is an interactor of FLD, which is required for SAR and SAR‐associated epigenetic modifications.     

Micrografting device for testing systemic signaling in Arabidopsis

Grafting techniques have been applied in studies of systemic, long‐distance signaling in several model plants. Seedling grafting in Arabidopsis, known as micrografting, enables investigation of the molecular mechanisms of systemic signaling between shoots and roots. However, conventional micrografting requires a high level of skill, limiting its use. Thus, an easier user‐friendly method is needed. Here, we developed a silicone microscaled device, the micrografting chip, to obviate the need for training and to generate less stressed and more uniformly grafted seedlings. The chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro‐path with pairs of pillars for hypocotyl holding. Grafting, including seed germination, micrografting manipulation and establishment of tissue reunion, is performed on the chip. Using the micrografting chip, we evaluated the effect of temperature and the carbon source on grafting, and showed that a temperature of 27°C and a sucrose concentration of 0.5% were optimal. We also used the chip to investigate the mechanism of systemic signaling of iron status using a quadruple nicotianamine synthase (nas) mutant. The constitutive iron‐deficiency response in the nas mutant because of iron accumulation in shoots was significantly rescued by grafting of wild‐type shoots or roots, suggesting that shoot‐ and root‐ward translocation of nicotianamine–iron complexes and/or nicotianamine is essential for iron mobilization. Thus, our micrografting chip will promote studies of long‐distance signaling in plants.     

Biochemical and Physiological Responses of <i>Arabidopsis thaliana</i> Leaves to Moderate Mechanical Stimulation

Mechanical stimulation of plants can be caused by various abiotic and biotic environmental factors. Apart from the negative consequences, it can also cause positive changes, such as acclimatization of plants to stress conditions. Therefore, it is necessary to study the physiological and biochemical mechanisms underlying the response of plants to mechanical stimulation. Our aim was to evaluate the response of model plant Arabidopsis thaliana to a moderate force of 5 N (newton) for 20 s, which could be compared with the pressure caused by animal movement and weather conditions such as heavy rain. Mechanically stimulated leaves were sampled 1 h after exposure and after a recovery period of 20 h. To study a possible systemic response, unstimulated leaves of treated plants were collected 20 h after exposure alongside the stimulated leaves from the same plants. The effect of stimulation was assessed by measuring oxidative stress parameters, antioxidant enzymes activity, total phenolics, and photosynthetic performance. Stimulated leaves showed increased lipid peroxidation 1 h after treatment and increased superoxide dismutase activity and phenolic oxidation rate after a 20-h recovery period. Considering photosynthetic performance after the 20-h recovery period, the effective quantum yield of the photosystem II was lower in the stimulated leaves, whereas photochemical quenching was lower in the unstimulated leaves of the treated plants. Nonphotochemical quenching was lower in the stimulated leaves 1 h after treatment. Our study suggested that plants sensed moderate force, but it did not induce pronounced change in metabolism or photosynthetic performance. Principal component analysis distinguished three groups–leaves of untreated plants, leaves analysed 1 h after stimulation, while stimulated and unstimulated leaves of treated plants analysed 20 h after treatment formed together the third group. Observed grouping of stimulated and unstimulated leaves of treated plants could indicate signal transduction from the stimulated to distant leaves, that is, a systemic response to a local application of mechanical stimuli.     

Source verification of mis-identified Arabidopsis thaliana accessions

A major strength of Arabidopsis thaliana as a model lies in the availability of a large number of naturally occurring inbred lines. Recent studies of A. thaliana population structure, using thousands of accessions from stock center and natural collections, have revealed a robust pattern of isolation by distance at several spatial scales, such that genetically identical individuals are generally found close to each other. However, some individual accessions deviate from this pattern. While some of these may be the products of rare long‐distance dispersal events, many deviations may be the result of mis‐identification, in the sense that the data regarding location of origin data are incorrect. Here, we aim to identify such discrepancies. Of the 5965 accessions examined, we conclude that 286 deserve special attention as being potentially mis‐identified. We describe these suspicious accessions and their possible origins, and advise caution with regard to their use in experiments in which accurate information on geographic origin is important. Finally, we discuss possibilities for maintaining the integrity of stock lines.     

3-Acetonyl-3-hydroxyoxindole: a new inducer of systemic acquired resistance in plants

Systemic acquired resistance (SAR) is an inducible defence mechanism which plays a central role in protecting plants from microbial pathogen attack. Guided by bioassays, a new chemical inducer of SAR was isolated from the extracts of Strobilanthes cusia and identified to be 3-acetonyl-3-hydroxyoxindole (AHO), a derivative of isatin. Tobacco plants treated with AHO exhibited enhanced resistance to tobacco mosaic virus (TMV) and to the fungal pathogen Erysiphe cichoracearum (powdery mildew), accompanied by increased levels of pathogenesis-related gene 1 ( PR-1 ) expression, salicylic acid (SA) accumulation and phenylalanine ammonia-lyase activity. To study the mode of action of AHO, its ability to induce PR-1 expression and TMV resistance in nahG transgenic plants expressing salicylate hydroxylase, which prevents the accumulation of SA, was analysed. AHO treatment did not induce TMV resistance or PR-1 expression in nahG transgenic plants, suggesting that AHO acts upstream of SA in the SAR signalling pathway. In addition, using two-dimensional gel electrophoresis combined with mass spectrometry, five AHO-induced plant proteins were identified which were homologous to the effector proteins with which SA interacts. Our data suggest that AHO may represent a novel class of inducer that stimulates SA-mediated defence responses.     

Pathogen-associated molecular pattern recognition rather than development of tissue necrosis contributes to bacterial induction of systemic acquired resistance in Arabidopsis

Systemic acquired resistance (SAR) is usually described as a phenomenon whereby localized inoculation with a necrotizing pathogen renders a plant more resistant to subsequent pathogen infection. Here we show that Pseudomonas syringae strains for which Arabidopsis thaliana represents a non-host plant systemically elevate resistance although the underlying interactions neither trigger a hypersensitive response nor cause necrotic disease symptoms. A similar enhancement of systemic resistance was observed when elicitor-active preparations of two typical bacterial pathogen-associated molecular patterns (PAMPs), flagellin and lipopolysaccharides (LPS), were applied in a localized manner. Several lines of evidence indicate that the observed systemic resistance responses are identical to SAR. Localized applications of non-adapted bacteria, flagellin or LPS elevate levels of the SAR regulatory metabolite salicylic acid (SA) and pathogenesis-related (PR) gene expression not only in treated but also in distant leaves. All treatments also systemically increase expression of the SAR marker gene FLAVIN-DEPENDENT MONOOXYGENASE 1. Further, a whole set of SAR-deficient Arabidopsis lines, including mutants in SA biosynthesis and signalling, are impaired in establishing the systemic resistance response triggered by non-host bacteria or PAMPs. We also show that the magnitude of defence reactions such as SA accumulation, PR gene expression or camalexin accumulation induced at sites of virulent or avirulent P. syringae inoculation but not the extent of tissue necrosis during these interactions determines the extent of SAR in distant leaves. Our data indicate that PAMPs significantly contribute to SAR initiation in Arabidopsis and that tissue necroses at inoculation sites are dispensable for SAR activation.     

A novel calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings

A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A. thaliana cell suspension cultures challenged with osmotic stress. The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only. Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus. Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor. AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity. Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth. By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type. Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.     

Hrip1, a novel protein elicitor from necrotrophic fungus,Alternaria tenuissima,elicits cell death,expression of defence‐related genes and systemic acquired resistance in tobacco

Here, we report the identification, purification, characterization and gene cloning of a novel hypersensitive response inducing protein secreted by necrotrophic fungus, Alternaria tenuissima, designated as hypersensitive response inducing protein 1 (Hrip1). The protein caused the formation of necrotic lesions that mimic a typical hypersensitive response and apoptosis‐related events including DNA laddering. The protein‐encoding gene was cloned by rapid amplification of cDNA ends (RACE) method. The sequence analysis revealed that the cDNA is 495 bp in length and the open reading frame (ORF) encodes for a polypeptide of 163 amino acids with theoretical pI of 5.50 and molecular weight of 17 562.5 Da. Hrip1 induced calcium influx, medium alkalinization, activation of salicylic acid‐induced protein kinase and several defence‐related genes after infiltration in tobacco leaves. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, at a time when expression of defence‐related genes was activated. After several days, systemic acquired resistance was also induced. The tobacco plant cells that perceived the Hrip1 generated a cascade of signals acting at local, short, and long distances, and caused the coordinated expression of specific defence responses in a way similar to hypersensitivity to tobacco mosaic virus. Thus, Hrip1 represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance in necrotrophic fungi.