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Several biological control agents of the hemipteran insect families Miridae, Anthocoridae and Pentatomidae, as well as mites of the family Phytoseiidae are known as zoophytophagous predators, a subset of omnivores, which are primarily predaceous but also feed on plants. It has been recently demonstrated that zoophytophagous predators are capable of inducing defenses in plants through their phytophagy. Despite the vast fundamental knowledge on plant defense mechanisms in response to herbivores, our understanding of defense induction by zoophytophagous predators and applied implications is relatively poor. In this review, we present the physiological basis of the defense mechanisms that these predators activate in plants. Current knowledge on zoophytophagous predator-induced plant defenses is summarized by groups and species for the predators of economic importance. Within each group, feeding habits and the effects of their induced-plant defenses on pests and natural enemies are detailed. Also, the ecological implications of how the induction of defenses mediated by zoophytophagous predators can interact with other plant interactors such as beneficial soil microorganisms and plant viruses are addressed. Based on the above, we propose three approaches to exploit zoophytophagous predator-induced defenses in crop protection and to guide future research. These include using predators as vaccination agents, employing biotechnological approaches, as well as applying elicitors to elicit/mimic predator-induced defenses., This work was partly supported by the Spanish Ministry of Science and Innovation through the projects RTA2017-00073-00-00 and PID2020-113234RR-I00. MLP was supported by the Onassis Foundation under the Special Grant and Support Program for Scholars’ Association Members (Grant No. R ZJ 003-2/2019–2020). GM was supported by the Dutch topsector project KV 1509-020. SB is an employee of Koppert Spain, a private company that markets benefcial arthropods. RM works for Biobest Group NV, a private company that markets benefcial arthropods.

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., This research was supported by Project RTI2018-101275-B-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” to BSS, and Plan de Promoción de la Investigación Universitat Jaume I (UJI-B2019-2) and “Servicios Centrales de Instrumentación Científica (SCIC), Universitat Jaume I to VF. MDM received the support of a fellowship from ”La Caixa” Foundation (ID 100010434), fellowship code LCF/BQ/DI20/11780039. We also acknowledge financial support from the MCIN/AEI through the “Severo Ochoa Programme for Centres of Excellence in R&D” SEV‐2015–0533 and CEX2019-000902-S, and the CERCA Programme / “Generalitat de Catalunya”. B.V-T was a recipient of a Ph. D. grant from the MINECO/AEI (BES-2016-076289)., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

BackgroundPhosphorus is an important macronutrient required for plant growth and development. It is absorbed through the roots in the form of inorganic phosphate (Pi). To cope with Pi limitation, plants have evolved an array of adaptive mechanisms to facilitate Pi acquisition and protect them from stress caused by Pi starvation. The NITROGEN LIMITATION ADAPTION (NLA) gene plays a key role in the regulation of phosphate starvation responses (PSR), its expression being regulated by the microRNA miR827. Stress caused by Pi limiting conditions might also affect the plant’s response to pathogen infection. However, cross-talk between phosphate signaling pathways and immune responses remains unclear.ResultsIn this study, we investigated whether NLA plays a role in Arabidopsis immunity. We show that loss-of-function of NLA and MIR827 overexpression causes an increase in phosphate (Pi) content which results in resistance to infection by the fungal pathogen Plectosphaerella cucumerina. The nla mutant plants accumulated callose in their leaves, a response that is also observed in wild-type plants that have been treated with high Pi. We also show that pathogen infection and treatment with fungal elicitors is accompanied by transcriptional activation of MIR827 and down-regulation of NLA. Upon pathogen challenge, nla plants exhibited higher levels of the phytoalexin camalexin compared to wild type plants. Camalexin level also increases in wild type plants treated with high Pi. Furthermore, the nla mutant plants accumulated salicylic acid (SA) and jasmonic acid (JA) in the absence of pathogen infection whose levels further increased upon pathogen.ConclusionsThis study shows that NLA acts as a negative regulator of Arabidopsis immunity. Overaccumulation of Pi in nla plants positively affects resistance to infection by fungal pathogens. This piece of information reinforces the idea of signaling convergence between Pi and immune responses for the regulation of disease resistance in Arabidopsis.

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., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., This research was supported by the Ministry of Science, Innovation and Universities/FEDER/Innovation-State Research Agency (AEI) (grant RTI2018-101275-B-I00) to BSS, the Research Promotion Plan from the Universitat Jaume I (UJI-B2019-2), and the Scientific Instrumentation Central Service from the Universitat Jaume I to VF. We acknowledge financial support from the Spanish Ministry of Science/AEI through the “Severo Ochoa Programme for Centres of Excellence in R&D” SEV-2015-0533 and CEX2019-000902-S, and the CERCA Programme/“Generalitat de Catalunya”. BVT was a recipient of a fellowship from the Ministry of Economy, Industry and Competitiveness/AEI (BES-2016-076289). MBP was a recipient of a scholarship from La Caixa Foundation.

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.

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.

The generalist predator Euseius stipulatus (Athias-Henriot) and the Tetranychidae-specialist predators Neoseiulus californicus (McGregor) and Phytoseiulus persimilis Athias-Henriot play a key role in the regulation of Tetranychus urticae Koch in Spanish citrus orchards. Previous studies have shown that sour orange (Citrus aurantium L.) and Cleopatra mandarin (Citrus reshni hort. ex Tan.) display extreme resistance and susceptibility to T. urticae, respectively. When offered a choice between these two genotypes infested by T. urticae, E. stipulatus preferred Cleopatra mandarin, whereas the specialists did not show any preference. The present study was undertaken to check whether these preferences could be related to the feeding of E. stipulatus on the host plant and/or to differences in prey feeding on the two plants. Our results demonstrate that E. stipulatus is a zoophytophagous mite, which can engage in direct plant feeding in sour orange and Cleopatra mandarin, whereas neither N. californicus nor P. persimilis do so. Whereas Cleopatra mandarin provided a higher-quality prey/feeding substrate for E. stipulatus, which may be related to its phytophagy, no differences were observed for the two specialists. As higher constitutive and faster inducible defense against T. urticae in sour orange relative to Cleopatra mandarin plants result in sour orange supporting lower T. urticae densities and plant damage, our results demonstrate that pest regulation by specialist natural enemies may be more effective when prey feed on better defended plants.

In the original publication of the article, the authors have inadvertently presented Figs.1-4 that were prepared from an incorrect data-set and were therefore inaccurate. The corrected figures and related statistical results are published herein. In addition, data supporting the corrected figures are provided as new supplementary online material (Supplementary Table 2 and Supplementary Figs. 3-6). The ecological relevance of certain responses to particular treatments were still discussed when P-values neared 0.05 (Wasserstein et al. 2019). This was the case of the preferences of Tetranychus urticae for (a) conspecific infested plants, where Cleopatra mandarin was preferred to sour orange (P ≤ 0.058), and (b) when having to choose between clean and infested Cleopatra mandarin, where infested plants were mostly preferred (P = 0.058) (Fig. 1; Supplementary Fig. 3). Similarly, the preferences of Euseius stipulatus and Neoseiulus californicus for T. urticae body odors were associated with a P-value of 0.058 (Figs. 2 and 3; Supplementary Figs. 4, 5). Finally, when having to choose between the same plant genotype, either infested or not, a P = 0.058 was obtained for N. californicus and sour orange (Fig. 3; Supplementary Fig. 5) and for Phytoseiulus persimilis and both sour orange and Cleopatra mandarin (Fig. 4; Supplementary Fig. 6). As a consequence, the conclusions discussed in the paper remain valid.

Zoophytophagous arthropods can elicit plant defense responses affecting potential prey beyond predation. Phytophagy prevails as the main trigger for these responses, as in the case of Euseius stipulatus (Athias-Henriot) (Mesostigmata: Phytoseiidae), a predator occurring in citrus. Because other triggers cannot be excluded, our aim was to examine whether other phytoseiids co-occurring with E. stipulatus but not engaged in plant feeding [Neoseiulus californicus (McGregor) and Phytoseiulus persimilis Athias-Henriot] could induce similar responses (in terms of herbivore induced plant volatiles, HIPVs, and main defensive pathways), and how these affected the behavior of conspecifics and the shared prey, Tetranychus urticae Koch (Prostigmata: Tetranychidae). N. californicus triggered plant genotype-specific defense responses, including the production of different HIPVs compared to clean plants. However, we could not observe these effects for P. persimilis. T. urticae avoided better protected plants, because of stronger direct or indirect defense. As plants with weaker direct defense levels should offer higher prey densities, and those harboring conspecific predators represent higher risk of cannibalism, predators were expected to behave similarly. However, they did not. Our results demonstrate that plant defense triggered by phytoseiids is species-specific, depend on plant genotype and can be triggered by non-feeding activities. As N. californicus is a highly efficient predator used worldwide, further studies with this species are needed. Likewise, cineol, one of the volatiles identified in the blends triggered by this phytoseiid, could be used to manipulate the prey. These studies could pave the way for a more efficient use of phytoseiids in agroecosystems.

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., Mycorrhizal symbiosis primes the accumulation of several antiherbivore compounds in response to Spodoptera exigua attack, increasing larval mortality.

Peptides are important regulators that participate in the modulation of almost every physiological event in plants, including defense. Recently, many of these peptides have been described as defense elicitors, termed phytocytokines, that are released upon pest or pathogen attack, triggering an amplification of plant defenses. However, little is known about peptides sensing and inducing resistance activities in heterologous plants. In the present study, exogenous peptides from solanaceous species, Systemins and HypSys, are sensed and induce resistance to the necrotrophic fungus Plectosphaerella cucumerina in the taxonomically distant species Arabidopsis thaliana. Surprisingly, other peptides from c1loser taxonomic clades have very little or no effect on plant protection. In vitro bioassays showed that the studied peptides do not have direct antifungal activities, suggesting that they protect the plant through the promotion of the plant immune system. Interestingly, tomato Systemin was able to induce resistance at very low concentrations (0.1 and 1 nM) and displays a maximum threshold being ineffective above at higher concentrations. Here, we show evidence of the possible involvement of the JA-signaling pathway in the Systemin-Induced Resistance (Sys-IR) in Arabidopsis. Additionally, Systemin treated plants display enhanced BAK1 and BAK1 gene expression following infection as well as increased production of ROS after PAMP treatment suggesting that Systemin sensitizes Arabidopsis perception to pathogens and PAMPs., This work was funded by the Plan de Promoción de la Investigación Universitat Jaume I UJI-B2016-43; the Spanish Ministry MICIU; RTI2018-094350-B-C33; the grant to JP-F 19I045 from the Plan Propio de Investigación Universitat Jaume I and PRIMA-INTOMED.

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 H2O2 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.

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.

Priming is an adaptive strategy that improves the defensive capacity of plants. This phenomenon is marked by an enhanced activation of induced defense mechanisms. Stimuli from pathogens, beneficial microbes, or arthropods, as well as chemicals and abiotic cues, can trigger the establishment of priming by acting as warning signals. Upon stimulus perception, changes may occur in the plant at the physiological, transcriptional, metabolic, and epigenetic levels. This phase is called the priming phase. Upon subsequent challenge, the plant effectively mounts a faster and/or stronger defense response that defines the postchallenge primed state and results in increased resistance and/or stress tolerance. Priming can be durable and maintained throughout the plant's life cycle and can even be transmitted to subsequent generations, therefore representing a type of plant immunological memory.

We prove the relevance of callose priming in mycorrhiza-induced resistance against shoot pathogens and studied the main mechanisms regulating it such as starch mobilization, sugar delivery, and vesicular trafficking., Mycorrhizal plants display enhanced resistance to several pathogens. However, the molecular mechanisms regulating mycorrhiza-induced resistance (MIR) are still elusive. We aim to study the mechanisms underlying MIR against Botrytis cinerea and the role of callose accumulation during this process. Mycorrhizal tomato plants inoculated with Rhizoglomus irregularis displayed callose priming upon B. cinerea infection. The callose inhibitor 2-deoxy-d-glucose abolished MIR, confirming the relevance of callose in the bioprotection phenomena. While studying the mechanisms underlying mycorrhiza-induced callose priming, we found that mycorrhizal plants display an enhanced starch degradation rate that is correlated with increased levels of β-amylase1 transcripts following pathogen infection. Starch mobilization in mycorrhizal plants seems coordinated with the increased transcription of sugar transporter and invertase genes. Moreover, the expression levels of genes encoding the vesicular trafficking proteins ATL31 and SYP121 and callose synthase PMR4 were higher in the mycorrhizal plants and further boosted by subsequent pathogen infection. All these proteins play a key role in the priming of callose accumulation in Arabidopsis, suggesting that callose priming is an induced resistance mechanism conserved in different plant species. This evidence highlights the importance of sugar mobilization and vesicular trafficking in the priming of callose as a defence mechanism in mycorrhiza-induced resistance.

Strigolactones (SLs) are important ex-planta signalling molecules in the rhizosphere, promoting the association with beneficial microorganisms, but also affecting plant interactions with harmful organisms. They are also plant hormones in-planta, acting as modulators of plant responses under nutrient-deficient conditions, mainly phosphate (Pi) starvation. In the present work, we investigate the potential role of SLs as regulators of early Pi starvation signalling in plants. A short-term pulse of the synthetic SL analogue 2′-epi-GR24 promoted SL accumulation and the expression of Pi starvation markers in tomato and wheat under Pi deprivation. 2′-epi-GR24 application also increased SL production and the expression of Pi starvation markers under normal Pi conditions, being its effect dependent on the endogenous SL levels. Remarkably, 2′-epi-GR24 also impacted the root metabolic profile under these conditions, promoting the levels of metabolites associated to plant responses to Pi limitation, thus partially mimicking the pattern observed under Pi deprivation. The results suggest an endogenous role for SLs as Pi starvation signals. In agreement with this idea, SL-deficient plants were less sensitive to this stress. Based on the results, we propose that SLs may act as early modulators of plant responses to P starvation., Consejo Superior de Investigaciones Científicas, Grant/Award Number: 201640I040; European Regional Development Fund, Grant/Award Numbers: AGL2017‐88‐083‐R, RTI2018‐094350‐B‐C31; Generalitat Valenciana, Grant/Award Number: CDEIGENT/2018/015; Ministerio de Ciencia, Innovación y Universidades, Grant/Award Numbers: AGL2017‐88‐083‐R, RTI2018‐094350‐B‐C31, FJCI‐2015‐23575; National Network of Carotenoids, Grant/Award Number: BIO2017‐90877‐REDT

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., This paper was inspired by scientific discussions among members of the COST Action FA1405. We thank the COST Action members that contributed to identify relevant publications. We thank Júlia Lidoy and Víctor Lidoy for drawing Figure 1. This work was financially supported by COST Action FA1405 and the Slovenian Research Agency’s research core funding No Z4-706, the Spanish Ministry of Science, Innovation and Universities (grants AGL2016-75819-C2-1-R and RTI2018-094350-B-C31), General Secreterial of Research and Technology, Greece co-financed with EU (T1- RCE-05301), and the Onassis Foundation (RZJ-003-2). A.M.M. Acknowledges funding from the program for attracting talent to Salamanca from the Fundación Salamanca Ciudad de Cultura y Saberes and Ayuntamiento de Salamanca

In nature, plants are continuously challenged by biotic stressors such as pathogenic microorganisms and herbivorous pests that often cause important yield reductions in crops. To address global food security issues but also consumer demands for healthier food production, a series of environmentally friendly crop protection strategies need to be employed in modern agriculture. In this chapter, we explore promising opportunities for controlling pests and pathogens of crops on the basis of our current knowledge on molecular, chemical and ecological parameters that determine plant defense. We present evidence about the feasibility of RNA-based molecular tools against a diversity of plant enemies. Moreover, a brief overview of current knowledge on applied aspects of plant defense priming in crop protection is provided in light of our fundamental understanding on mechanisms underlying this phenomenon. We specifically refer to beneficial organisms such as soil microbes and zoophytophagous predators and propose their use as plant vaccination agents, alongside chemicals that could be used to prime plants against future attackers. Endophytic fungi that not only mediate systemic plant resistance against pests or pathogens, but also impact entomopathogenic and plant growth promoting capabilities, are specifically addressed. Finally, we identify knowledge gaps and present future considerations about the use of the proposed biological and molecular control tools in sustainable crop production.

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., We thank the Serveis Centrals of Universitat Jaume I for the technical support. This work was financially supported by the Spanish Ministry MINECO RTL2018-094350-B-C33 and the grant to VP IJCI- 2015-24527 , by the Plan de Promoción de la Investigación-Universitat Jaume I P1.1B2015-33 and the GV/2018/115 from the program Apoyo para Grupos Emergentes, from the Generalitat Valenciana and to the Diputación de Castellón for funding the PRIMA-INTOMED consortium. We thank the grant BES-2016-077208 to NS.

Phytophthora cinnamomi (Pc) is a dangerous pathogen that causes root rot (ink disease) and threatens the production of chestnuts worldwide. Despite all the advances recently reported at molecular and physiological level, there are still gaps of knowledge that would help to unveil the defence mechanisms behind plant-Pc interactions. Bearing this in mind we quantified constitutive and Pc-induced stress-related signals (hormones and metabolites) complemented with changes in photosynthetic related parameters by exploring susceptible and resistant Castanea spp.-Pc interactions. In a greenhouse experiment, five days before and nine days after inoculation with Pc, leaves and fine roots from susceptible C. sativa and resistant C. sativa × C. crenata clonal 2-year-old plantlets were sampled (clones Cs14 and 111-1, respectively). In the resistant clone, stomatal conductance (gs) and net photosynthesis (A) decreased significantly and soluble sugars in leaves increased, while in the susceptible clone gs and A remained unchanged and proline levels in leaves increased. In the resistant clone, higher constitutive content of root SA and foliar ABA, JA and JA-Ile as compared to the susceptible clone were observed. Total phenolics and condensed tannins were highest in roots of the susceptible clone. In response to infection, a dynamic hormonal response in the resistant clone was observed, consisting of accumulation of JA, JA-Ile and ABA in roots and depletion of total phenolics in leaves. However, in the susceptible clone only JA diminished in leaves and increased in roots. Constitutive and Pc-induced levels of JA-Ile were only detectable in the resistant clone. From the hormonal profiles obtained in leaves and roots before and after infection, it is concluded that the lack of effective hormonal changes in C. sativa explains the lack of defence responses to Pc of this susceptible species. published

[EN] Sour orange, Citrus aurantium, displays higher constitutive and earlier inducible direct defenses against the two-spotted spider mite, Tetranychus urticae, than Cleopatra mandarin, Citrus reshni. Moreover, herbivore-induced plant volatiles (HIPVs) produced by sour orange upon infestation can induce resistance in Cleopatra mandarin but not vice versa. Because the role of these HIPVs in indirect resistance remains ignored, we have carried out a series of behavioral assays with three predatory mites with different levels of specialization on this herbivore, from strict entomophagy to omnivory. We have further characterized the volatile blend associated with T. urticae, which interestingly includes the HIPV methyl salicylate, as well as that produced by induced Cleopatra mandarin plants. Although a preference for less defended plants with presumably higher prey densities (i.e., C. reshni) was expected, this was not always the case. Because predators' responses changed with diet width, with omnivore predators responding to both HIPVs and prey-related odors and specialized ones mostly to prey, our results reveal that these responses depend on plant genotype, prey presence and predator diet specialization. As the different volatile blends produced by infested sour orange, induced Cleopatra mandarin and T. urticae itself are attractive to T. urticae natural enemies but not to the herbivore, they may provide clues to develop new more sustainable tools to manipulate these agriculturally relevant species., The research leading to these results was partially funded by the Spanish Ministry of Economy and Competitiveness (AGL2014-55616-C3; AGL2015-64990-2R). The authors thank M. Piquer (UJI) for technical assistance. MC received a pre-doctoral fellowship from the Spanish Ministry of Economy and Competitiveness (BES-2015-074570), and MP was the recipient of a research fellowship from INIA, Spain (subprogram DOC INIA-CCAA).

Nut production by the European sweet chestnut (Castanea sativa Mill.) in grafted orchards is under threat of increased drought stress associated with current global warming. To explore whether drought tolerance in C. sativa could be improved using drought tolerant scions and rootstocks, trees from humid (H) and xeric (X) populations in Spain were used to generate intra-family (H/H and X/X) and reciprocal inter-location (X/H and H/X) grafts. We studied the effects of the (i) scion, (ii) rootstock and (iii) the grafting itself as a wounding stress on the vegetative budbreak, secondary growth and drought tolerance of C. sativa trees. Drought tolerance was assessed by measuring leaf gas exchange, chlorophyll fluorescence, water status and leaf wilting two weeks after water deprivation. Hormone (ABA, SA, JA and JA-Ile) and proline quantification in tree leaves and fine roots were assessed, as well as tree and scion mortality. Rootstocks and scions of xeric origin induced earlier flushing and improved drought tolerance of scions and rootstocks of humid origin. Tree mortality after drought was 57 % lower in H/X than in H/H trees, and scion loss after drought was 47 % lower in H/X than in X/H trees. The ‘grafting’ (i.e., wounding) effect delayed vegetative budbreak and reduced secondary growth of trees but did not predispose trees to drought. We report for the first time differences in hormone and proline content in leaves and fine roots of chestnut trees of humid and xeric origins exposed to drought. The results suggest the use of scions and rootstocks from xeric areas to improve drought tolerance in chestnut.

Indole-3-carboxylic acid (I3CA) is an indolic compound that induces resistance in Arabidopsis adult plants against the necrotrophic fungus Plectosphaerella cucumerina through primed callose accumulation. In this study, we confirm the relevance of ATL31 and SYP121 genes involved in vesicular trafficking in I3CA priming of defenses and we discard camalexin as a mediator of I3CA-induced resistance (IR) in adult plants. In addition, we observed that an intact I3CA biosynthetic pathway is necessary for I3CA-IR functionality.

Multiple stresses are becoming common challenges in modern agriculture due to environmental changes. A large set of phytochemicals collectively known as oxylipins play a key role in responses to several stresses. Understanding the fine-tuned plant responses to multiple and simultaneous stresses could open new perspectives for developing more tolerant varieties. We carried out the molecular and biochemical profiling of genes, proteins and active compounds involved in oxylipin metabolism in response to single/combined salt and wounding stresses on Medicago truncatula. Two new members belonging to the CYP74 gene family were identified. Gene expression profiling of each of the six CYP74 members indicated a tissue- and time-specific expression pattern for each member in response to single/combined salt and wounding stresses. Notably, hormonal profiling pointed to an attenuated systemic response upon combined salt and leaf wounding stresses. Combined, these results confirm the important role of jasmonates in legume adaptation to abiotic stresses and point to the existence of a complex molecular cross-talk among signals generated by multiple stresses.

b-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)-b-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.

Grapevine (Vitis vinifera) is susceptible to many pathogens which cause significant losses to viticulture worldwide. Chemical control is available, but agro-ecological concerns have raised interest in alternative methods, especially in triggering plant immunity by elicitor treatments. The β-glucan laminarin (Lam) and its sulfated derivative (PS3) have been previously demonstrated to induce resistance in grapevine against downy mildew (Plasmopara viticola). However, if Lam elicits classical grapevine defenses such as oxidative burst, pathogenesis-related (PR)-proteins and phytoalexin production, PS3 triggered grapevine resistance via a poorly understood priming phenomenon. The aim of this study was to identify the molecular mechanisms of the PS3-induced resistance. For this purpose we studied i) the signaling events and transcriptome reprogramming triggered by PS3 treatment on uninfected grapevine, ii) grapevine immune responses primed by PS3 during P. viticola infection. Our results showed that i) PS3 was unable to elicit reactive oxygen species (ROS) production, cytosolic Ca(2+) concentration variations, mitogen-activated protein kinase (MAPK) activation but triggered a long lasting plasma membrane depolarization in grapevine cells, ii) PS3 and Lam shared a common stress-responsive transcriptome profile that partly overlapped the salicylate- (SA) and jasmonate-(JA)-dependent ones. After P. viticola inoculation, PS3 specifically primed the SA- and ROS-dependent defense pathways leading to grapevine induced resistance against this biotroph. Interestingly pharmacological approaches suggested that the plasma membrane depolarization and the downstream ROS production are key events of the PS3-induced resistance.

The protective fungus Fusarium oxysporum Fo47 reduces the severity of wilt caused by the soilborne pathogen Verticil-lium dahliae in pepper. Modes of action responsible for the biocontrol activity were studied. Microscopic observationsof fluorescent protein-transformed strains colonizing the root surface show that the colonization patterns of Fo47 andV. dahliae were similar. Pixel counting of the images obtained by confocal microscopy showed that Fo47 reduces colo-nization 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 defenceresponses were identified by measuring the amount of some phytohormones and their derivatives in roots and stems ofpepper. Results showed an early, slight increase of jasmonyl isoleucine, followed by a transient increase of salicylic acidduring the pre-challenged phase of priming and an increase of 12-oxo-phytodienoic acid during the challenge phase ofpriming. The caffeic, ferulic and chlorogenic acids, known to play a role in plant defence reactions, showed a strongantimicrobial activity against V. dahliae in vitro. In pepper roots, Fo47 stimulated the biosynthesis of caffeic acid andprimed that of chlorogenic acid. These results demonstrated that the effective control of V. dahliae provided by Fo47is based on different but complementary mechanisms. This research was funded by the Xunta de Galicia(grants INCITE07PXI103099ES, INCITE08E1R103016ES, INCITE09E2R103003ES and IN845B-2010/084).

Background: Systemin has been extensively studied since it was discovered and is described as a peptidic hormone in tomato plants and other Solanaceae. Jasmonic acid and systemin are proposed to act through a positive feed-back loop with jasmonic acid, playing synergistic roles in response to both wounding and insect attack. Despite its biological relevance, most studies regarding the function of systemin in defence have been studied via PROSYSTEMIN (PROSYS) gene expression, which encodes the propeptide prosystemin that is later cleaved to systemin (SYS). Interestingly, hardly any studies have been based on quantification of the peptide. Results: In this study, a simple and accurate method for systemin quantification was developed to understand its impact on plant metabolism. The basal levels of systemin were found to be extremely low. To study the role of endogenous systemin on plant metabolism, systemin was quantified in a transgenic line overexpressing the PROSYS gene (PS+) and in a silenced antisense line (PS-). We evaluated the relevance of systemin in plant metabolism by analysing the metabolomic profiles of both lines compared to wildtype plants through untargeted metabolomic profiling. Compounds within the lignan biosynthesis and tyrosine metabolism pathways strongly accumulated in PS+ compared to wild-type plants and to plants from the PS- line. The exogenous treatments with SYS enhanced accumulation of lignans, which confirms the role of SYS in cell wall reinforcement. Unexpectedly, PS+ plants displayed wild-type levels of jasmonic acid (JA) but elevated accumulation of 12-oxo-phytodienoic acid (OPDA), suggesting that PS+ should not be used as an over-accumulator of JA in experimental setups. Conclusions: A simple method, requiring notably little sample manipulation to quantify the peptide SYS, is described. Previous studies were based on genetic changes. In our study, SYS accumulated at extremely low levels in wild-type tomato leaves, showed slightly higher levels in the PROSYSTEMIN-overexpressing plants and was absent in the silenced lines. These small changes have a significant impact on plant metabolism. SA and OPDA, but not JA, were higher in the PROSYS-overexpressing plants., This work was financially supported by the Plan de Promoción de la Investigación de la Universitat Jaume I P1- 1B2015-33, the Spanish Ministry MINECO AGL2015-64990-C2-2-R and the Generalitat Valenciana project AICO/2016/029.

Arbuscular mycorrhizal (AM) symbioses can improve plant tolerance to multiple stresses. We compared three AM fungi (AMF) from different genera, one of them isolated from a dry and saline environment, in terms of their ability to increase tomato tolerance to moderate or severe drought or salt stress. Plant physiological parameters and metabolic profiles were compared in order to find the molecular mechanisms underlying plant protection against stress. Mycorrhizal growth response was determined, and ultrahigh-performance LC-MS was used to compare the metabolic profile of plants under the different treatments. All AMF increased plant tolerance to stress, and the positive effects of the symbiosis were correlated with the severity of the stress. The AMF isolated from the stressful environment was the most effective in improving plant tolerance to salt stress. Differentially accumulated compounds were identified and the antistress properties of some of them were confirmed. We demonstrate that AM symbioses increase plant metabolic plasticity to cope with stress. Some responses were common to all AMF tested, while others were specifically related to particular isolates. Important metabolism reprograming was evidenced upon salt stress, and we identified metabolic pathways and compounds differentially accumulated in mycorrhizas that may underlie their enhanced tolerance to stress., This research has been financed by CSIC projects (201440E046 and 201440E099) and projects AGL2015-64990-C2-1-R and CGL2015-69118-C2-2-P from the MINECO. We acknowledge exchange of ideas under COST Action FA1405, COST (European Cooperation in Science and Technology).

A fast callose accumulation has been shown to mediate defence priming in certain plant–pathogen interactions, but the events upstream of callose assembly following chemical priming are poorly understood, mainly because those steps comprise sugar transfer to the infection site. β-Amino butyric acid (BABA)-induced resistance in Arabidopsis against Plectosphaerella cucumerina is known to be mediated by callose priming. Indole-3-carboxylic acid (ICOOH, also known as I3CA) mediates BABA-induced resistance in Arabidopsis against P. cucumerina. This indolic compound is found in a common fingerprint of primed metabolites following treatments with various priming stimuli. In the present study, we show that I3CA induces resistance in Arabidopsis against P. cucumerina and primes enhancement of callose accumulation. I3CA treatment increased abscisic acid (ABA) levels before infection with P. cucumerina. An intact ABA synthesis pathway is needed to activate a starch amylase (BAM1) to trigger augmented callose deposition against P. cucumerina during I3CA-IR. To verify the relevance of the BAM1 amylase in I3CA-IR, knockdown mutants and overexpressors of the BAM1 gene were tested. The mutant bam1 was impaired to express I3CA-IR, but complemented 35S::BAM1-YFP lines in the background of bam1 restored an intact I3CA-IR and callose priming. Therefore, a more active starch metabolism is a committed step for I3CA-IR, inducing callose priming in adult plants. Additionally, I3CA treatments induced expression of the ubiquitin ligase ATL31 and syntaxin SYP131, suggesting that vesicular trafficking is relevant for callose priming. As a final element in the callose priming, an intact Powdery Mildew resistant4 (PMR4) gene is also essential to fully express I3CA-IR., The authors thank the Serveis Centrals de la Universitat Jaume I and particularly Cristian Barrera for their technical support. This work was financially supported by the Plan de Promoción de la Investigación de la Universitat Jaume I UJI‐B2016‐43, MINECO AGL2015‐64990‐C2‐2 and the Generalitat Valenciana AICO/2016/029.

BACKGROUND Zoophytophagous predators can trigger plant defenses affecting prey populations beyond predation. Euseius stipulatus is a presumed zoophytophagous phytoseiid common in citrus. The response of citrus to one of its potential prey, Tetranychus urticae, is genotype dependent, with Citrus reshni and C. aurantium exhibiting extreme susceptibility and resistance, respectively. Volatile blends produced upon infestation affected the behavior of these two mites. We wondered whether E. stipulatus could trigger similar responses. RESULTS Euseius stipulatus triggered genotype-dependent defense responses in citrus. Whereas C. aurantium upregulated the Jasmonic Acid, Salicylic Acid and flavonoids defensive pathways, C. reshni upregulated JA only. Likewise, different volatile blends were induced. These blends were exploited by E. stipulatus to select less-defended plants (i.e., those in which higher pest densities are expected) and, interestingly, did not prevent T. urticae from choosing E. stipulatus-infested plants. To the best of our knowledge, this is the first time that this type of response has been described for a zoophytophagous phytoseiid. CONCLUSION The observed responses could affect herbivore populations through plant-mediated effects. Although further research is needed to fully characterize them and include other arthropods in the system, these results open opportunities for more sustainable and effective pest control methods (i.e., combining semiochemicals and biological control). © 2018 Society of Chemical Industry.

Being sessile organisms, plants must respond to various challenges in the environment. The priming process consists of three clear stages. The first stage includes all the cellular changes in the absence of the challenge so-called pre-challenge priming stage. These changes are expected to be rather subtle, affecting the preparation of the plant to properly manage subsequent responses to pathogens with no major fitness costs. Most of the research that has been conducted at this stage has been dedicated to the study of changes in gene expression and protein phosphorylation. However, the metabolic changes that occur during the pre-challenge priming stage are poorly understood. The second stage affects the early to late stages of the defence response, which occurs after the interaction with a pathogen has been established. Most studies involving priming are dedicated to the molecular events that take place during this stage. Most studies have shown that defence priming is strongly hormonally regulated; however, there is also evidence of the involvement of phenolic derivative compounds and many other secondary metabolites, leading to stronger and faster plant responses. The third priming phase ranges from long lasting defence priming to trans-generational acquired resistance. Long-term metabolic transitions, that occur in the offspring of primed plants, remain to be elucidated. Here we review existing information in the literature that relates to the metabolic changes that occur during all three defence priming stages and highlight the metabolic transitions that are associated with the stimulation of priming and the characteristics of the pathogens whenever possible.