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Abstract: In low nutritive environments, the uptake of N by arbuscular mycorrhizal (AM) fungi may confer competitive advantages for the host. The present study aims to understand how mycorrhizal tomato plants perceive and then prepare for an N depletion in the root environment. Plants colonized by Rhizophagus irregularis displayed improved responses to a lack of N than nonmycorrhizal (NM) plants. These responses were accomplished by a complex metabolic and transcriptional rearrangement that mostly affected the gibberellic acid and jasmonic acid pathways involving DELLA and JAZ1 genes, which were responsive to changes in the C/N imbalance of the plant. N starved mycorrhizal plants showed lower C/N equilibrium in the shoots than starved NM plants and concomitantly a downregulation of the JAZ1 repressor and the increased expression of the DELLA gene, which translated into a more active oxylipin pathway in mycorrhizal plants. In addition, the results support a priorization in AM plants of stress responses over growth. Therefore, these plants were better prepared for an expected stress. Furthermore, most metabolites that were severely reduced in NM plants following the N depletion remained unaltered in starved AM plants compared with those normally fertilized, suggesting that the symbiosis buffered the stress, improving plant development in a stressed environment. [ABSTRACT FROM AUTHOR]

The protective fungus Fusarium oxysporum Fo47 reduces the severity of wilt caused by the soilborne pathogen Verticillium dahliae in pepper. Modes of action responsible for the biocontrol activity were studied. Microscopic observations of fluorescent protein-transformed strains colonizing the root surface show that the colonization patterns of Fo47 and V. dahliae were similar. Pixel counting of the images obtained by confocal microscopy showed that Fo47 reduces colonization of the root surface by V. dahliae, suggesting a possible role of competition for nutrients at the root surface. Besides these effects on surface colonization, the hormonal pathways activated during priming of plant defence responses were identified by measuring the amount of some phytohormones and their derivatives in roots and stems of pepper. Results showed an early, slight increase of jasmonyl isoleucine, followed by a transient increase of salicylic acid during the pre-challenged phase of priming and an increase of 12-oxo-phytodienoic acid during the challenge phase of priming. The caffeic, ferulic and chlorogenic acids, known to play a role in plant defence reactions, showed a strong antimicrobial activity against V. dahliae in vitro. In pepper roots, Fo47 stimulated the biosynthesis of caffeic acid and primed that of chlorogenic acid. These results demonstrated that the effective control of V. dahliae provided by Fo47 is based on different but complementary mechanisms. [ABSTRACT FROM AUTHOR]

Plant defence peptides are paramount endogenous danger signals secreted after a challenge, intensifying the plant immune response. The peptidic hormone Systemin (Sys) was shown to participate in resistance in several plant pathosystems, although the mechanisms behind Sys-induced resistance when exogenously applied remain elusive. We performed proteomic, metabolomic, and enzymatic studies to decipher the Sys-induced changes in tomato plants in either the absence or the presence of Botrytis cinerea infection. Sys treatments triggered direct proteomic rearrangement mostly involved in carbon metabolism and photosynthesis. However, the final induction of defence proteins required concurrent challenge, triggering priming of pathogen-targeted proteins. Conversely, at the metabolomic level, Sys-treated plants showed an alternative behaviour following a general priming profile. Of the primed metabolites, the flavonoids rutin and isorhamnetin and two alkaloids correlated with the proteins 4-coumarate-CoA-ligase and chalcone-flavanone-isomerase triggered by Sys treatment. In addition, proteomic and enzymatic analyses revealed that Sys conditioned the primary metabolism towards the production of available sugars that could be fuelling the priming of callose deposition in Sys-treated plants; furthermore, PR1 appeared as a key element in Sys-induced resistance. Collectively, the direct induction of proteins and priming of specific secondary metabolites in Sys-treated plants indicated that post-translational protein regulation is an additional component of priming against necrotrophic fungi., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Mycorrhizal plants show enhanced resistance to biotic stresses, but few studies have addressed mycorrhiza-induced resistance (MIR) against biotic challenges in woody plants, particularly citrus. Here we present a comparative study of two citrus species, Citrus aurantium, which is resistant to Tetranychus urticae, and Citrus reshni, which is highly susceptible to T. urticae. Although both mycorrhizal species are protected in locally infested leaves, they show very distinct responses to MIR., Results: Previous studies have indicated that C. aurantium is insensitive to MIR in systemic tissues and MIR-triggered antixenosis. Conversely, C. reshni is highly responsive to MIR which triggers local, systemic and indirect defense, and antixenosis against the pest. Transcriptional, hormonal and inhibition assays in C. reshni indicated the regulation of jasmonic acid (JA)- and abscisic acid-dependent responses in MIR. The phytohormone jasmonic acid isoleucine (JA-Ile) and the JA biosynthesis gene LOX2 are primed at early timepoints. Evidence indicates a metabolic flux from phenylpropanoids to specific flavones that are primed at 24 h post infestation (hpi). MIR also triggers the priming of naringenin in mycorrhizal C. reshni, which shows a strong correlation with several flavones and JA-Ile that over-accumulate in mycorrhizal plants. Treatment with an inhibitor of phenylpropanoid biosynthesis C4H enzyme impaired resistance and reduced the symbiosis, demonstrating that phenylpropanoids and derivatives mediate MIR in C. reshni., Conclusion: MIR's effectiveness is inversely correlated to basal immunity in different citrus species, and provides multifaceted protection against T. urticae in susceptible C. reshni, activating rapid local and systemic defenses that are mainly regulated by the accumulation of specific flavones and priming of JA-dependent responses. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Indole-3-acetic acid (IAA) production is a pathogenicity/virulence factor in the Pseudomonas syringae complex, including Pseudomonas savastanoi . P. savastanoi pathovars (pvs.) genomes contain the iaaL gene, encoding an enzyme that catalyzes the biosynthesis of the less biologically active compound 3-indole-acetyl-ϵ-L-lysine (IAA-Lys). Previous studies have reported the identification of IAA-Lys in culture filtrates of P. savastanoi strains isolated from oleander (pv. nerii), but the conversion of IAA into a conjugate was not detectable in olive strains (pv. savastanoi). In this paper, we show the distribution of iaaL alleles in all available P. savastanoi genomes of strains isolated from woody hosts. Most strains encode two different paralogs, except for those isolated from broom (pv. retacarpa), which contain a single allele. In addition to the three previously reported iaaL alleles ( iaaL Psv , iaaL Psn and iaaL Pto ), we identified iaaL Psf , an exclusive allele of strains isolated from ash (pv. fraxini). We also found that the production of IAA-Lys in P. savastanoi pv. savastanoi and pv. nerii depends on a functional iaaL Psn allele, whereas in pv. fraxini depends on iaaL Psf . The production of IAA-Lys was detected in cultures of an olive strain heterologously expressing IaaL Psn-1 , IaaL Psf-1 and IaaL Psf-3 , but not when expressing IaaL Psv-1 . In addition, Arabidopsis seedlings treated with the strains overproducing the conjugate, and thus reducing the free IAA content, alleviated the root elongation inhibitory effect of IAA. IAA-Lys synthase activity assays with purified allozymes confirmed the functionality and specificity of lysine as a substrate of IaaL Psn-1 and IaaL Psf-3 , with IaaL Psf-3 showing the highest catalytic efficiency for both substrates. The IAA-Lys synthase activity of IaaL Psn-1 was abolished by the insertion of two additional tyrosine residues encoded in the inactive allozyme IaaL Psv-1 . These results highlight the relevance of allelic variation in a phytohormone-related gene for the modulation of auxin production in a bacterial phytopathogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pintado, Domínguez-Cerván, Pastor, Vincent, Lee, Flors and Ramos.)

The drought sensitivity of the pedunculate oak ( Quercus robur L.) poses a threat to its survival in light of climate change. Mycorrhizal fungi, which orchestrate biogeochemical cycles and particularly have an impact on the plant's defense mechanisms and metabolism of carbon, nitrogen, and phosphorus, are among the microbes that play a significant role in the mitigation of the effects of climate change on trees. The study's main objectives were to determine whether ectomycorrhizal (ECM) fungi alleviate the effects of drought stress in pedunculate oak and to investigate their priming properties. The effects of two levels of drought (mild and severe, corresponding to 60% and 30% of field capacity, respectively) on the biochemical response of pedunculate oak were examined in the presence and absence of ectomycorrhizal fungi. To examine whether the ectomycorrhizal fungi modulate the drought tolerance of pedunculate oak, levels of plant hormones and polyamines were quantified using UPLC-TQS and HPLC-FD techniques in addition to gas exchange measurements and the main osmolyte amounts (glycine betaine-GB and proline-PRO) which were determined spectrophotometrically. Droughts increased the accumulation of osmolytes, such as proline and glycine betaine, as well as higher polyamines (spermidine and spermine) levels and decreased putrescine levels in both, mycorrhized and non-mycorrhized oak seedlings. In addition to amplifying the response of oak to severe drought in terms of inducible proline and abscisic acid (ABA) levels, inoculation with ECM fungi significantly increased the constitutive levels of glycine betaine, spermine, and spermidine regardless of drought stress. This study found that compared to non-mycorrhized oak seedlings, unstressed ECM-inoculated oak seedlings had higher levels of salicylic (SA) and abscisic acid (ABA) but not jasmonic acid (JA), indicating a priming mechanism of ECM is conveyed via these plant hormones. According to a PCA analysis, the effect of drought was linked to the variability of parameters along the PC1 axe, such as osmolytes PRO, GB, polyamines, and plant hormones such as JA, JA-Ile, SAG, and SGE, whereas mycorrhization was more closely associated with the parameters gathered around the PC2 axe (SA, ODPA, ABA, and E). These findings highlight the beneficial function of the ectomycorrhizal fungi, in particular Scleroderma citrinum , in reducing the effects of drought stress in pedunculate oak.

The plant immunity system is being revisited more and more and new elements and roles are attributed to participating in the response to biotic stress. The new terminology is also applied in an attempt to identify different players in the whole scenario of immunity: Phytocytokines are one of those elements that are gaining more attention due to the characteristics of processing and perception, showing they are part of a big family of compounds that can amplify the immune response. This review aims to highlight the latest findings on the role of phytocytokines in the whole immune response to biotic stress, including basal and adaptive immunity, and expose the complexity of their action in plant perception and signaling events.

Phosphorus is an important macronutrient required for plant growth and development. It is absorbed by the roots in the form of inorganic phosphate (Pi). Under Pi limiting conditions, plants activate the Phosphate Starvation Response (PSR) system to enhance Pi acquisition. The NITROGEN LIMITATION ADAPTION (NLA) gene is a component of the Arabidopsis PSR, and its expression is post-transcriptionally regulated by miR827. We show that loss-of-function of NLA and MIR827 overexpression increases Pi level and enhances resistance to infection by the fungal pathogen Plectosphaerella cucumerina in Arabidopsis. Upon pathogen infection, high Pi plants (e.g. nla plants and wild type plants grown under high Pi supply) showed enhanced callose deposition. High Pi plants also exhibited superinduction of camalexin biosynthesis genes which is consistent with increased levels of camalexin during pathogen infection. Pathogen infection and treatment with fungal elicitors, triggered up-regulation of MIR827 and down-regulation of NLA expression. Under non-infection conditions, the nla plants showed increased levels of SA and JA compared with wild type plants, their levels further increasing upon pathogen infection. Overall, the outcomes of this study suggest that NLA plays a role in Arabidopsis immunity, while supporting convergence between Pi signaling and immune signaling in Arabidopsis., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Transcriptional regulators based on CRISPR architecture expand our ability to reprogramme endogenous gene expression in plants. One of their potential applications is the customization of plant metabolome through the activation of selected enzymes in a given metabolic pathway. Using the previously described multiplexable CRISPR activator dCasEV2.1, we assayed the selective enrichment in Nicotiana benthamiana leaves of four different flavonoids, namely, naringenin, eriodictyol, kaempferol, and quercetin. After careful selection of target genes and guide RNAs combinations, we created successful activation programmes for each of the four metabolites, each programme activating between three and seven genes, and with individual gene activation levels ranging from 4- to 1500-fold. Metabolic analysis of the flavonoid profiles of each multigene activation programme showed a sharp and selective enrichment of the intended metabolites and their glycosylated derivatives. Remarkably, principal component analysis of untargeted metabolic profiles clearly separated samples according to their activation treatment, and hierarchical clustering separated the samples into five groups, corresponding to the expected four highly enriched metabolite groups, plus an un-activated control. These results demonstrate that dCasEV2.1 is a powerful tool for re-routing metabolic fluxes towards the accumulation of metabolites of interest, opening the door for the custom-made design of metabolic contents in plants., (© 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Citrus plants are a highly mycotrophic species with high levels of fungal colonization. Citrus aurantium rootstocks typically show abundant root colonization by Rhizophagus irregularis three weeks after inoculation. Mycorrhizal symbiosis protects plants against multiple biotic stressors, however, such protection against spider mites remains controversial. We examined mycorrhiza-induced resistance (MIR) in citrus against the two-spotted spider mite Tetranychus urticae . Mycorrhized C. aurantium displayed reduced levels of damage in leaves and lower mite oviposition rates, compared to non-mycorrhized controls. Mycorrhization did not affect host choice of mites in Y-tube assays; of note, C. aurantium has innate strong antixenotic resistance against this mite. Analysis of metabolism pathways in mycorrhized citrus plants showed upregulated expression of the oxylipin-related genes LOX-2 and PR-3 early after infestation. Accordingly, jasmonic acid (JA), 12-oxo phytodienoic acid (OPDA), and JA-Ile concentrations were increased by mycorrhization. Non-targeted metabolomic analysis revealed the amino acid, oxocarboxylic acid, and phenylpropanoid metabolism as the three major pathways with more hits at 24 h post infection (hpi) in mycorrhized plants. Interestingly, there was a transition to a priming profile of these pathways at 48 hpi following infestation. Three flavonoids (i.e., malic acid, coumaric acid, and diconiferyl alcohol) were among the priming compounds. A mixture containing all these compounds provided efficient protection against the mite. Unexpectedly, systemic resistance did not improve after 72 h of primary infestation, probably due to the innate strong systemic resistance of C. aurantium . This is the first study to show that MIR is functional against T. urticae in locally infested citrus leaves, which is mediated by a complex pool of secondary metabolites and is likely coordinated by priming of JA-dependent responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Manresa-Grao, Pastor-Fernández, Sanchez-Bel, Jaques, Pastor and Flors.)

In nature, plants are concurrently exposed to a number of abiotic and biotic stresses. Our understanding of convergence points between responses to combined biotic/abiotic stress pathways remains, however, rudimentary. Here we show that MIR399 overexpression, loss-of-function of PHOSPHATE2 (PHO2), or treatment with high phosphate (Pi) levels is accompanied by an increase in Pi content and accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. High Pi plants (e.g., miR399 overexpressors, pho2 mutants, and plants grown under high Pi supply) exhibited resistance to infection by necrotrophic and hemibiotrophic fungal pathogens. In the absence of pathogen infection, the expression levels of genes in the salicylic acid (SA)- and jasmonic acid (JA)-dependent signaling pathways were higher in high Pi plants compared to wild-type plants grown under control conditions, which is consistent with increased levels of SA and JA in non-infected high Pi plants. During infection, an opposite regulation in the two branches of the JA pathway (ERF1/PDF1.2 and MYC2/VSP2) occurs in high Pi plants. Thus, while pathogen infection induces PDF1.2 expression in miR399 OE and pho2 plants, VSP2 expression is downregulated by pathogen infection in these plants. This study supports the notion that Pi accumulation promotes resistance to infection by fungal pathogens in Arabidopsis, while providing a basis to better understand interactions between Pi signaling and hormonal signaling pathways for modulation of plant immune responses., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Like in mammals, the plant immune system has evolved to perceive damage. Damaged-associated molecular patterns (DAMPs) are endogenous signals generated in wounded or infected tissue after pathogen or insect attack. Although extracellular DNA (eDNA) is a DAMP signal that induces immune responses, plant responses after eDNA perception remain largely unknown. Here, we report that signaling defenses but not direct defense responses are induced after eDNA applications enhancing broad-range plant protection. A screening of defense signaling and hormone biosynthesis marker genes revealed that OXI1, CML37 and MPK3 are relevant eDNA-Induced Resistance markers (eDNA-IR). Additionally, we observed that eDNA from several Arabidopsis ecotypes and other phylogenetically distant plants such as citrus, bean and, more surprisingly, a monocotyledonous plant such as maize upregulates eDNA-IR marker genes. Using 3,3'-Diaminobenzidine (DAB) and aniline blue staining methods, we observed that H 2 O 2 but not callose was strongly accumulated following self-eDNA treatments. Finally, eDNA resulted in effective induced resistance in Arabidopsis against the pathogens Hyaloperonospora arabidopsidis, Pseudomonas syringae, and Botrytis cinerea and against aphid infestation, reducing the number of nymphs and moving forms. Hence, the unspecificity of DNA origin and the wide range of insects to which eDNA can protect opens many questions about the mechanisms behind eDNA-IR., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hormones orchestrate the physiology of organisms. Measuring the activity of defense hormone-responsive genes can help understanding immune signaling and facilitate breeding for plant health. However, different from model species like Arabidopsis, genes that respond to defense hormones salicylic acid (SA) and jasmonic acid (JA) have not been disclosed in the soybean crop. We performed global transcriptome analyses to fill this knowledge gap. Upon exogenous application, endogenous levels of SA and JA increased in leaves. SA predominantly activated genes linked to systemic acquired resistance and defense signaling whereas JA mainly activated wound response-associated genes. In general, SA-responsive genes were activated earlier than those responding to JA. Consistent with the paradigm of biotrophic pathogens predominantly activating SA responses, free SA and here identified most robust SA marker genes GmNIMIN1, GmNIMIN1.2 and GmWRK40 were induced upon inoculation with Phakopsora pachyrhizi, whereas JA marker genes did not respond to infection with the biotrophic fungus. Spodoptera exigua larvae caused a strong accumulation of JA-Ile and JA-specific mRNA transcripts of GmBPI1, GmKTI1 and GmAAT whereas neither free SA nor SA-marker gene transcripts accumulated upon insect feeding. Our study provides molecular tools for monitoring the dynamic accumulation of SA and JA, e.g. in a given stress condition., (© 2021. The Author(s).)

To be protected from biological threats, plants have evolved an immune system comprising constitutive and inducible defenses. For example, upon perception of certain stimuli, plants can develop a conditioned state of enhanced defensive capacity against upcoming pathogens and pests, resulting in a phenotype called 'induced resistance' (IR). To tackle the confusing lexicon currently used in the IR field, we propose a widely applicable code of practice concerning the terminology and description of IR phenotypes using two main phenotypical aspects: local versus systemic resistance, and direct versus primed defense responses. Our general framework aims to improve uniformity and consistency in future scientific communication, which should help to avoid further misinterpretations and facilitate the accessibility and impact of this research field., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Plant association with arbuscular mycorrhizal fungi (AMF) can increase their ability to overcome multiple stresses, but their impact on plant interactions with herbivorous insects is controversial. Here we show higher mortality of the leaf-chewer Spodoptera exigua when fed on tomato plants colonized by the AMF Funneliformis mosseae, evidencing mycorrhiza-induced resistance. In search of the underlying mechanisms, an untargeted metabolomic analysis through ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS) was performed. The results showed that mycorrhizal symbiosis had a very limited impact on the leaf metabolome in the absence of stress, but significantly modulated the response to herbivory in the damaged area. A cluster of over accumulated metabolites was identified in those leaflets damaged by S. exigua feeding in mycorrhizal plants, while unwounded distal leaflets responded similar to those from non-mycorrhizal plants. These primed-compounds were mostly related to alkaloids, fatty acid derivatives and phenylpropanoid-polyamine conjugates. The deleterious effect on larval survival of some of these compounds, including the alkaloid physostigmine, the fatty acid derivatives 4-oxododecanedioic acid and azelaic acid, was confirmed. Thus, our results evidence the impact of AMF on metabolic reprograming upon herbivory that leads to a primed accumulation of defensive compounds., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The strawberry Fra a 1 proteins belong to the class 10 Pathogenesis-Related (PR-10) superfamily. In strawberry, a large number of members have been identified, but only a limited number is expressed in the fruits. In this organ, Fra a 1.01 and Fra a 1.02 are the most abundant Fra proteins in the green and red fruits, respectively, however, their function remains unknown. To know the function of Fra a 1.02 we have generated transgenic lines that silence this gene, and performed metabolomics, RNA-Seq, and hormonal assays. Previous studies associated Fra a 1.02 to strawberry fruit color, but the analysis of anthocyanins in the ripe fruits showed no diminution in their content in the silenced lines. Gene ontology (GO) analysis of the genes differentially expressed indicated that oxidation/reduction was the most represented biological process. Redox state was not apparently altered since no changes were found in ascorbic acid and glutathione (GSH) reduced/oxidized ratio, but GSH content was reduced in the silenced fruits. In addition, a number of glutathione-S-transferases (GST) were down-regulated as result of Fra a 1.02-silencing. Another highly represented GO category was transport which included a number of ABC and MATE transporters. Among the regulatory genes differentially expressed WRKY33.1 and WRKY33.2 were down-regulated, which had previously been assigned a role in strawberry plant defense. A reduced expression of the VQ23 gene and a diminished content of the hormones JA, SA, and IAA were also found. These data might indicate that Fra a 1.02 participates in the defense against pathogens in the ripe strawberry fruits.

Plant-microbe-arthropod (PMA) three-way interactions have important implications for plant health. However, our poor understanding of the underlying regulatory mechanisms hampers their biotechnological applications. To this end, we searched for potential common patterns in plant responses regarding taxonomic groups or lifestyles. We found that most signaling modules regulating two-way interactions also operate in three-way interactions. Furthermore, the relative contribution of signaling modules to the final plant response cannot be directly inferred from two-way interactions. Moreover, our analyses show that three-way interactions often result in the activation of additional pathways, as well as in changes in the speed or intensity of defense activation. Thus, detailed, basic knowledge of plant-microbe-arthropod regulation will be essential for the design of environmentally friendly crop management strategies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Arbuscular mycorrhizal symbiosis is restricted in roots, but it also improves shoot responses against leaf challenges, a phenomenon known as Mycorrhiza-Induced Resistance (MIR). This study focuses on mycorrhizal root signals that may orchestrate shoot defence responses. Metabolomic analysis of non-mycorrhizal and mycorrhizal plants upon Botrytis cinerea infection showed that roots rearrange their metabolome mostly in response to the symbiosis, whereas in shoots a stronger impact of the infection is observed. Specific clusters of compounds in shoots and roots display a priming profile suggesting an implication in the enhanced resistance observed in mycorrhizal plants. Among the primed pathways in roots, lignans showed the highest number of hits followed by oxocarboxylic acids, compounds of the amino acid metabolism, and phytohormones. The lignan yatein was present at higher concentrations in roots, root efflux and leaves of mycorrhizal plants This lignan displayed in vitro antimicrobial activity against B. cinerea and it was also functional protecting tomato plants. Besides, several JA defence-related genes were upregulated in mycorrhizal roots regardless of the pathogen infection, whereas PIN-II was primed in roots of mycorrhizal infected plants. These observations suggest that the enhanced resistance in shoots during MIR may be coordinated by lignans and oxylipins with the participation of roots., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)