Elicitin‐like proteins Oli‐D1 and Oli‐D2 from Pythium oligandrum trigger hypersensitive response in Nicotiana benthamiana and induce resistance against Botrytis cinerea in tomato

The biocontrol agent Pythium oligandrum and its elicitin‐like proteins oligandrins have been shown to induce disease resistance in a range of plants. In the present study, the ability of two oligandrins, Oli‐D1 and Oli‐D2, to induce an immune response and the possible molecular mechanism regulating the defence responses in Nicotiana benthamiana and tomato were investigated. Infiltration of recombinant Oli‐D1 and Oli‐D2 proteins induced a typical immune response in N. benthamiana including the induction of a hypersensitive response (HR), accumulation of reactive oxygen species and production of autofluorescence. Agrobacterium‐mediated transient expression assays revealed that full‐length Oli‐D1 and Oli‐D2 were required for full HR‐inducing activity in N. benthamiana, and virus‐induced gene silencing‐mediated knockdown of some of the signalling regulatory genes demonstrated that NbSGT1 and NbNPR1 were required for Oli‐D1 and Oli‐D2 to induce HR in N. benthamiana. Subcellular localization analyses indicated that both Oli‐D1 and Oli‐D2 were targeted to the plasma membrane of N. benthamiana. When infiltrated or transiently expressed in leaves, Oli‐D1 and Oli‐D2 induced resistance against Botrytis cinerea in tomato and activated the expression of a set of genes involved in the jasmonic acid/ethylene (JA/ET)‐mediated signalling pathway. Our results demonstrate that Oli‐D1 and Oli‐D2 are effective elicitors capable of inducing immune responses in plants, probably through the JA/ET‐mediated signalling pathway, and that both Oli‐D1 and Oli‐D2 have potential for the development of bioactive formulae for crop disease control in practice.     

Artificial Agrobacterium tumefaciens strains exhibit diverse mechanisms to repress Xanthomonas oryzae pv. oryzae‐induced hypersensitive response and non‐host resistance in Nicotiana benthamiana

Xanthomonas oryzae pv. oryzae (Xoo) rapidly triggers a hypersensitive response (HR) and non‐host resistance in its non‐host plant Nicotiana benthamiana. Here, we report that Agrobacterium tumefaciens strain GV3101 blocks Xoo‐induced HR in N. benthamiana when pre‐infiltrated or co‐infiltrated, but not when post‐infiltrated at 4 h after Xoo inoculation. This suppression by A. tumefaciens is local and highly efficient to Xoo. The HR‐inhibiting efficiency of A. tumefaciens is strain dependent. Strain C58C1 has almost no effect on Xoo‐induced HR, whereas strains GV3101, EHA105 and LBA4404 nearly completely block HR formation. Intriguingly, these three HR‐inhibiting strains employ different strategies to repress HR. Strain GV3101 displays strong antibiotic activity and thus suppresses Xoo growth. Comparison of the genotype and Xoo antibiosis activity of wild‐type A. tumefaciens strain C58 and a set of C58‐derived strains reveals that this Xoo antibiosis activity of A. tumefaciens is negatively, but not solely, regulated by the transferred‐DNA (T‐DNA) of the Ti plasmid pTiC58. Unlike GV3101, strains LBA4404 and EHA105 exhibit no significant antibiotic effect on Xoo, but rather abolish hydrogen peroxide accumulation. In addition, expression assays indicate that strains LBA4404 and EHA105 may inhibit Xoo‐induced HR by suppression of the expression of Xoo type III secretion system (T3SS) effector genes hpa1 and hrpD6. Collectively, our results unveil the multiple levels of effects of A. tumefaciens on Xoo in N. benthamiana and provide insights into the molecular mechanisms underlying the bacterial antibiosis of A. tumefaciens and the non‐host resistance induced by Xoo.     

Induced systemic resistance (ISR) against pathogens in the context of induced plant defences

Induced systemic resistance (ISR) of plants against pathogens is a widespread phenomenon that has been intensively investigated with respect to the underlying signalling pathways as well as to its potential use in plant protection. Elicited by a local infection, plants respond with a salicylic-dependent signalling cascade that leads to the systemic expression of a broad spectrum and long-lasting disease resistance that is efficient against fungi, bacteria and viruses. Changes in cell wall composition, de novo production of pathogenesis-related-proteins such as chitinases and glucanases, and synthesis of phytoalexins are associated with resistance, although further defensive compounds are likely to exist but remain to be identified. In this Botanical Briefing we focus on interactions between ISR and induced resistance against herbivores that is mediated by jasmonic acid as a central signalling molecule. While many studies report cross-resistance, others have found trade-offs, i.e. inhibition of one resistance pathway by the other. Here we propose a framework that explains many of the thus far contradictory results. We regard elicitation separately from signalling and from production, i.e. the synthesis of defensive compounds. Interactions on all three levels can act independently from each other.     

Kinetics of the competitive reactions of isomerization and peptide bond cleavage at l‐α‐ and d‐β‐aspartyl residues in an αA‐crystallin fragment

d ‐β‐aspartyl (Asp) residue has been found in a living body such as aged lens crystallin, although l ‐α‐amino acids are constituents in natural proteins. Isomerization from l ‐α‐ to d ‐β‐Asp probably modulates structures to affect biochemical reactions. At Asp residue, isomerization and peptide bond cleavage compete with each other. To gain insight into how fast each reaction proceeds, the analysis requires the consideration of both pathways simultaneously and independently. No information has been provided, however, about these competitive processes because each reaction has been studied separately. The contribution of Asp isomers to the respective pathways has still been veiled. In this work, the two competitive reactions, isomerization and spontaneous peptide bond cleavage at Asp residue, were simultaneously observed and compared in an αA‐crystallin fragment, S⁵¹LFRTVLD⁵⁸SG⁶⁰ containing l ‐α‐ and d ‐β‐Asp58 isomers. The kinetics showed that the formation of l ‐ and d ‐succinimide (Suc) intermediate, as a first step of isomerization, was comparable at l ‐α‐ and d ‐β‐Asp. Although l ‐Suc was converted to l ‐β‐Asp, d ‐Suc was liable to return to the original d ‐β‐Asp, the reverse reaction marked enough to consider d ‐β‐Asp as apparently stable. d ‐β‐Asp was also resistant to the peptide bond cleavage. Such apparent less reactivity is probably the reason for gradual and abnormal accumulation of d ‐β‐Asp in a living body under physiological conditions. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by a Phytophthora megasperma glycoprotein elicitin

We have compared localized (LAR) and systemic (SAR) acquired resistance induced in tobacco by a hypersensitive response (HR) inducing Phytophthora megasperma glycoprotein elicitin. Three different zones were taken into account: LAR, SAR_T and SAR_S. The LAR zone was 5–10 mm wide and surrounded the HR lesion. SAR_T was the tissue of the elicitor-treated leaf immediately beyond the LAR zone. The systemic leaf was called SAR_S. Glycoprotein-treated plants showed enhanced resistance to challenge infection by tobacco mosaic virus (TMV). Disease resistance was similar in SAR_T and SAR_S, and higher in LAR. The expression pattern, in glycoprotein-treated plants, of acidic and basic PR1, PR2, PR3 and PR5 proteins and of O-methyltransferases (OMT), enzymes of the phenylpropanoid pathway, was similar to that in TMV-infected plants. OMT was stimulated in LAR but not in SAR_T and SAR_S. The four classes of acidic and basic PR proteins accumulated strongly in LAR. Reduced amounts of acidic PR1, PR2, PR3 and only minute amounts of basic PR2 and PR3 accumulated in SAR_T and SAR_S. In glycoprotein-treated plants, expression of the acidic and basic PR proteins in LAR and SAR of transgenic NahG and ETR tobacco plants and in LAR of plants treated with inhibitors of salicylic acid accumulation and of ethylene biosynthesis indicated a salicylic acid-dependent signalling pathway for acidic isoform activation and an ethylene-dependent signalling pathway for basic isoform activation.     

Stimulation and attenuation of induced resistance by elicitors and inhibitors of chemical induction in tomato (Lycopersicon esculentum) foliage

Elicitors and inhibitors of chemical induction were used to manipulate the activities of several putative defense-related proteins in leaves of the tomato, Lycopersicon esculentum Mill. The four presumptive defenses manipulated were proteinase inhibitors, polyphenol oxidase, peroxidase, and lipoxygenase. The elicitors used were jasmonic acid, methyl jasmonate, ultraviolet light, and feeding by larvae of the noctuid, Helicoverpa zea Boddie; the inhibitors used were salicylic acid and acetylsalicylic acid. These chemical manipulations were combined with short-term growth assays using larvae of the generalist noctuid, Spodoptera exigua Hubner, in order to assess the relative roles of the proteins in induced resistance to S. exigua. When activities of proteinase inhibitors and/or polyphenol oxidase in leaf tissue were high (e.g., in damaged or elicited plants), growth rates of larvae of S. exigua were low; when activities of polyphenol oxidase and proteinase inhibitors were low (e.g., in undamaged or damaged, inhibited plants), growth rates of larvae were high. In contrast, high activities of peroxidase and lipoxygenase were not associated with decreases in suitability of leaf tissue for S. exigua. The association of high levels of proteinase inhibitors and polyphenol oxidase with resistance to S. exigua – irrespective of the presence or absence of damage – strongly implicates these proteins as causal agents in induced resistance to S. exigua.     

Isolation and characterization of the plant immune-priming compounds Imprimatin B3 and -B4, potentiators of disease resistance in Arabidopsis thaliana

Plant activators are chemical crop protectants that fortify the immune system in plants. Unlike pesticides that target pathogens, plant activators provide durable effects against a broad spectrum of diseases, which have not been overcome by pathogenic microbes. Plant activators are not only useful agrochemicals, but can also help to elucidate the details of the plant immune system. Using an established high-throughput screening procedure, we previously identified 5 compounds, designated as Imprimatins, which prime plant immune response. These compounds increased disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants by inhibiting 2 salicylic acid (SA) glucosyltransferases (SAGTs), resulting in accumulation of the phytohormone SA. Here, we report the isolation of 2 additional Imprimatins, B3 and B4, which are structurally similar to Imprimatin B1 and B2. Because these compounds did not have strong inhibitory effects on SAGTs in vitro, they may exert their function after metabolic conversion in vivo.     

Salicylic acid in the machinery of hypersensitive cell death and disease resistance

Although extensive data has described the key role of salicylic acid (SA) in signaling pathogen-induced disease resistance, its function in physiological processes related to cell death is still poorly understood. Recent studies have explored the requirement of SA for mounting the hypersensitive response (HR) against an invading pathogen, where a particular cell death process is activated at the site of attempted infection causing a confined lesion. Biochemical data suggest that SA potentiates the signal pathway for HR by affecting an early phosphorylation-sensitive step preceding the generation of pro-death signals, including those derived from the oxidative burst. Accordingly, the epistatic relationship between cell death and SA accumulation, analyzed in crosses between lesion-mimic mutants (spontaneous lesion formation) and the transgenic nahG line (depleted in SA) places the SA activity in a feedback loop downstream and upstream of cell death. Exciting advances have been made in the identification of cellular protective functions and cell death suppressors that might operate in HR. Moreover, the spatio-temporal patterns of the SA accumulation (non-homogeneous distribution, biphasic kinetics) described in some HR lesions, may also reveal important clues for unraveling the complex cellular network that tightly balances pro- and anti-death functions in the hypersensitive cell death.     

Engineering d‐limonene synthase down‐regulation in orange fruit induces resistance against the fungus Phyllosticta citricarpa through enhanced accumulation of monoterpene alcohols and activation of defence

Terpene volatiles play an important role in the interactions between specialized pathogens and fruits. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with crop losses in different citrus‐growing areas worldwide. The pathogen may infect the fruit for 20–24 weeks after petal fall, but the typical hard spot symptoms appear when the fruit have almost reached maturity, caused by fungal colonization and the induction of cell lysis around essential oil cavities. d ‐Limonene represents approximately 95% of the total oil gland content in mature orange fruit. Herein, we investigated whether orange fruit with reduced d ‐limonene content in peel oil glands via an antisense (AS) approach may affect fruit interaction with P. citricarpa relative to empty vector (EV) controls. AS fruit showed enhanced resistance to the fungus relative to EV fruit. Because of the reduced d ‐limonene content, an over‐accumulation of linalool and other monoterpene alcohols was found in AS relative to EV fruit. A global gene expression analysis at 2 h and 8 days after inoculation with P. citricarpa revealed the activation of defence responses in AS fruit via the up‐regulation of different pathogenesis‐related (PR) protein genes, probably as a result of enhanced constitutive accumulation of linalool and other alcohols. When assayed in vitro and in vivo, monoterpene alcohols at the concentrations present in AS fruit showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for the development of new strategies to obtain resistance to fruit diseases.     

On‐Resin Synthesis of an Acylated and Fluorescence‐Labeled Cyclic Integrin Ligand for Modification of Poly(lactic‐co‐glycolic acid)

Cyclic Arg‐Gly‐Asp (RGD) peptides show remarkable affinity and specificity to integrin receptors and mediate important physiological effects in tumor angiogenesis. Additionally, they are one of the keyplayers in improving the biocompatibility of biomaterials. The fully biodegradable polymer poly(lactic‐co‐glycolic acid) (PLGA) is frequently used for biomedical implants and can be applied as nanoparticles for drug delivery. The aim of this work was the generation of a lipidated c[RGDfK] peptide including a second functionality for coating of hydrophobic PLGA. Therefore, we established a general and straightforward strategy for the introduction of two different modifications into the same c[RGDfK] peptide. This allowed the generation of a palmitoylated integrin‐binding lipopeptide that shows high affinity to PLGA. Additionally, we coupled 5(6)‐carboxyfluorescein to the second site for modification to enable sensitive quantification of the immobilized lipopeptide on PLGA. In conclusion, we present a synthesis protocol that enables the preparation of c[RGDfK] lipopeptides with a strong affinity to PLGA and an additional site for modifications. This will provide the opportunity to introduce a variety of effector molecules site‐specifically to the c[RGDfK] lipopeptide, which will enable the introduction of multifunctionality into c[RGDfK]‐coated PLGA devices or nanoparticles.     

Different mechanisms of Trichoderma virens‐mediated resistance in tomato against Fusarium wilt involve the jasmonic and salicylic acid pathways

In the present study, we investigated the role of Trichoderma virens (TriV_JSB100) spores or cell‐free culture filtrate in the regulation of growth and activation of the defence responses of tomato (Solanum lycopersicum) plants against Fusarium oxysporum f. sp. lycopersici by the development of a biocontrol–plant–pathogen interaction system. Two‐week‐old tomato seedlings primed with TriV_JSB100 spores cultured on barley grains (BGS) or with cell‐free culture filtrate (CF) were inoculated with Fusarium pathogen under glasshouse conditions; this resulted in significantly lower disease incidence in tomato Oogata‐Fukuju plants treated with BGS than in those treated with CF. To dissect the pathways associated with this response, jasmonic acid (JA) and salicylic acid (SA) signalling in BGS‐ and CF‐induced resistance was evaluated using JA‐ and SA‐impaired tomato lines. We observed that JA‐deficient mutant def1 plants were susceptible to Fusarium pathogen when they were treated with BGS. However, wild‐type (WT) BGS‐treated tomato plants showed a higher JA level and significantly lower disease incidence. SA‐deficient mutant NahG plants treated with CF were also found to be susceptible to Fusarium pathogen and displayed low SA levels, whereas WT CF‐treated tomato plants exhibited moderately lower disease levels and substantially higher SA levels. Expression of the JA‐responsive defensin gene PDF1 was induced in WT tomato plants treated with BGS, whereas the SA‐inducible pathogenesis‐related protein 1 acidic (PR1a) gene was up‐regulated in WT tomato plants treated with CF. These results suggest that TriV_JSB100 BGS and CF differentially induce JA and SA signalling cascades for the elicitation of Fusarium oxysporum resistance in tomato.     

The α2 adrenoceptor antagonist idazoxan alleviates l‐DOPA‐induced dyskinesia by reduction of striatal dopamine levels: an in vivo microdialysis study in 6‐hydroxydopamine‐lesioned rats

l ‐DOPA‐induced dyskinesia is characterised by debilitating involuntary movement, which limits quality of life in patients suffering from Parkinson’s disease. Here, we investigate effects of the α₂ adrenoceptor antagonist idazoxan on l ‐DOPA‐induced dyskinesia as well as on alterations of extracellular l ‐DOPA and dopamine (DA) levels in the striatum in dyskinetic rats. Male Wistar rats were unilaterally lesioned with 6‐hydroxydopamine and subsequently treated with l ‐DOPA/benserazide to induce stable dyskinetic movements. Administration of idazoxan [(9 mg/kg, intraperitoneal (i.p.)] significantly alleviated l ‐DOPA‐induced dyskinesia, whereas idazoxan (3 mg/kg, i.p.) did not affect dyskinetic behaviour. Bilateral in vivo microdialysis revealed that idazoxan 9 mg/kg reduces extracellular peak l ‐DOPA levels in the lesioned and intact striatum as well as DA levels in the lesioned striatum. In parallel, the exposure to idazoxan in the striatum was monitored. Furthermore, no idazoxan and l ‐DOPA drug–drug interaction was found in plasma, brain tissue and CSF. In conclusion, the decrease of l ‐DOPA‐derived extracellular DA levels in the lesioned striatum significantly contributes to the anti‐dyskinetic effect of idazoxan.     

Kinetics of the Accumulation of Jasmonic Acid and Its Derivatives in Systemic Leaves of Tobacco (Nicotiana tabacum cv. Xanthi nc) and Translocation of Deuterium-Labeled Jasmonic Acid from the Wounding Site to the Systemic Site

In plants, the mobile signal needed for wound-induced systemic acquired resistance (WSR) has been elusive. The signal compound involved in WSR is supposed to be JA or its derivatives. On the basis of kinetic study of the accumulation of JA or its derivatives, it was discovered that JA, JA-Ile, tuberonic acid (TA, 12-OH epi-JA), and tuberonic acid glucoside (TAG) accumulated in systemic tissues in response to mechanical wounding stress in the tobacco plant (Nicotiana tabacum). Attempts to recover deuterium-labeled JA in systemic leaves after feeding the wounded leaves with deuterium-labeled JA were successfully done. It was also found that the translocated deuterium-labeled JA was metabolized to TA in systemic leaves under feeding of deuterium-labeled JA to the wounding leaves.