Intrinsically disordered Prosystemin discloses biologically active repeat motifs.

The in-depth studies over the years on the defence barriers by tomato plants have shown that the Systemin peptide controls the response to a wealth of environmental stress agents. This multifaceted stress reaction seems to be related to the intrinsic disorder of its precursor protein, Prosystemin (ProSys). Since latest findings show that ProSys has biological functions besides Systemin sequence, here we wanted to assess if this precursor includes peptide motifs able to trigger stress-related pathways. Candidate peptides were identified in silico and synthesized to test their capacity to trigger defence responses in tomato plants against different biotic stressors. Our results demonstrated that ProSys harbours several repeat motifs which triggered plant immune reactions against pathogens and pest insects. Three of these peptides were detected by mass spectrometry in plants expressing ProSys, demonstrating their effective presence in vivo. These experimental data shed light on unrecognized functions of ProSys, mediated by multiple biologically active sequences which may partly account for the capacity of ProSys to induce defense responses to different stress agents.

Corrigendum to "In silico and in vitro approaches allow the identification of the Prosystemin molecular network" [Comput Struct Biotechnol J 21 (2023) 212-223].

[This corrects the article DOI: 10.1016/j.csbj.2022.12.006.].

A soil fungus confers plant resistance against a phytophagous insect by disrupting the symbiotic role of its gut microbiota.

Plants generate energy flows through natural food webs, driven by competition for resources among organisms, which are part of a complex network of multitrophic interactions. Here, we demonstrate that the interaction between tomato plants and a phytophagous insect is driven by a hidden interplay between their respective microbiotas. Tomato plants colonized by the soil fungus Trichoderma afroharzianum, a beneficial microorganism widely used in agriculture as a biocontrol agent, negatively affects the development and survival of the lepidopteran pest Spodoptera littoralis by altering the larval gut microbiota and its nutritional support to the host. Indeed, experiments aimed to restore the functional microbial community in the gut allow a complete rescue. Our results shed light on a novel role played by a soil microorganism in the modulation of plant-insect interaction, setting the stage for a more comprehensive analysis of the impact that biocontrol agents may have on ecological sustainability of agricultural systems.

In silico and in vitro approaches allow the identification of the Prosystemin molecular network.

Tomato Prosystemin (ProSys), the precursor of Systemin, a small peptidic hormone, is produced at very low concentration in unchallenged plants, while its expression greatly increases in response to several different stressors triggering an array of defence responses. The molecular mechanisms that underpin such a wide array of defence barriers are not fully understood and are likely correlated with the intrinsically disordered (ID) structure of the protein. ID proteins interact with different protein partners forming complexes involved in the modulation of different biological mechanisms. Here we describe the ProSys-protein network that shed light on the molecular mechanisms underpinning ProSys associated defence responses. Three different approaches were used. In silico prediction resulted in 98 direct interactors, most clustering in phytohormone biosynthesis, transcription factors and signal transduction gene classes. The network shows the central role of ProSys during defence responses, that reflects its role as central hub. In vitro ProSys interactors, identified by Affinity Purification-Mass Spectrometry (AP-MS), revealed over three hundred protein partners, while Bimolecular Fluorescent Complementation (BiFC) experiments validated in vivo some interactors predicted in silico and in vitro. Our results demonstrate that ProSys interacts with several proteins and reveal new key molecular events in the ProSys-dependent defence response of tomato plant.

Not Only Systemin: Prosystemin Harbors Other Active Regions Able to Protect Tomato Plants.

Prosystemin is a 200-amino acid precursor expressed in Solanaceae plants which releases at the C-terminal part a peptidic hormone called Systemin in response to wounding and herbivore attack. We recently showed that Prosystemin is not only a mere scaffold of Systemin but, even when deprived of Systemin, is biologically active. These results, combined with recent discoveries that Prosystemin is an intrinsically disordered protein containing disordered regions within its sequence, prompted us to investigate the N-terminal portions of the precursor, which contribute to the greatest disorder within the sequence. To this aim, PS1-70 and PS1-120 were designed, produced, and structurally and functionally characterized. Both the fragments, which maintained their intrinsic disorder, were able to induce defense-related genes and to protect tomato plants against Botrytis cinerea and Spodoptera littoralis larvae. Intriguingly, the biological activity of each of the two N-terminal fragments and of Systemin is similar but not quite the same and does not show any toxicity on experimental non-targets considered. These regions account for different anti-stress activities conferred to tomato plants by their overexpression. The two N-terminal fragments identified in this study may represent new promising tools for sustainable crop protection.

Influence of Dietary Inclusion of Exhausted Bergamot By-Product in Pigs on Animal Performance, Fatty Acid Profile and Oxidative Stability of Meat and Meat Products.

An investigation was carried out on the effect in pig diet of supplementation with exhausted bergamot by-product, stemming from pharmaceutical industry after extraction of phenolic compounds, on growth performance and on the quality of meat and meat products. Twenty pigs were assigned to two dietary treatments and fed a conventional concentrate (control) or a concentrate including exhausted bergamot by-product at the level of 15% on a DM basis (EB). No significant differences between dietary treatments were found for final weight (p = 0.243), carcass weight (p = 0.679), dry matter intake (p = 0.321). In EB pork, the proportion of docosapentaenoic acid was significantly increased (p < 0.05); it tended to have a greater proportion of n-3 PUFA (p = 0.09), and the n-6/n-3 PUFA ratio was lower in EB treatment (p = 0.01). In salami from EB pigs fed, the proportion of α-linolenic acid and the total n-3 PUFA were higher than in the control group (p < 0.001). In salami, the TBARS value was lower after 5 days of storage (p < 0.001) in the EB group. Therefore, the present results suggest that the inclusion of exhausted bergamot by-product in pig diet resulted in a qualitative improvement of meat and meat products.

Tomato Prosystemin Is Much More than a Simple Systemin Precursor.

Systemin (Sys) is an octadecapeptide, which upon wounding, is released from the carboxy terminus of its precursor, Prosystemin (ProSys), to promote plant defenses. Recent findings on the disordered structure of ProSys prompted us to investigate a putative biological role of the whole precursor deprived of the Sys peptide. We produced transgenic tomato plants expressing a truncated ProSys gene in which the exon coding for Sys was removed and compared their defense response with that induced by the exogenous application of the recombinant truncated ProSys (ProSys(1-178), the Prosystemin sequence devoid of Sys region). By combining protein structure analyses, transcriptomic analysis, gene expression profiling and bioassays with different pests, we demonstrate that truncated ProSys promotes defense barriers in tomato plants through a hormone-independent defense pathway, likely associated with the production of oligogalacturonides (OGs). Both transgenic and plants treated with the recombinant protein showed the modulation of the expression of genes linked with defense responses and resulted in protection against the lepidopteran pest Spodoptera littoralis and the fungus Botrytis cinerea. Our results suggest that the overall function of the wild-type ProSys is more complex than previously shown, as it might activate at least two tomato defense pathways: the well-known Sys-dependent pathway connected with the induction of jasmonic acid biosynthesis and the successive activation of a set of defense-related genes, and the ProSys(1-178)-dependent pathway associated with OGs production leading to the OGs mediate plant immunity.

Effect of feeding pigs with bergamot by-product on fatty acid composition and oxidative stability of meat and salami.

This work investigated the effects of feeding ensiled bergamot pulp to pigs on meat and salami quality. Eighteen pigs were assigned to two experimental treatments and fed a cereal-based concentrate diet (control) or the same diet in which ensiled bergamot pulp replaced 15% dry matter of the diet fed (BP). The BP treatment increased α-linolenic acid (+250%; P < 0.05), docosapentaenoic acid (+62%; P < 0.05), docosahexaenoic acid (+43%; P < 0.05) and consequently n-3 PUFA (+15%; P < 0.01) in meat. In salami, the content of α-linolenic acid, total PUFA and n-3 PUFA increased (+320%, +25% and + 258%, respectively) by feeding the BP diet (P < 0.001). The inclusion of bergamot pulp in the diet did not alter the oxidative stability in raw and cooked meat and colour descriptors. In salami, TBARS values were lower after 5 days of storage (P < 0.001) in BP group (1.54 vs 2.96). Finally, dietary supplementation with ensiled bergamot pulp to pigs improved the nutritional value of meat and meat products.

Diversity and Relationships among Neglected Apricot (Prunus armeniaca L.) Landraces Using Morphological Traits and SSR Markers: Implications for Agro-Biodiversity Conservation.

Apricot (Prunus armeniaca L.) is an economically important tree species globally cultivated in temperate areas. Italy has an ample number of traditional varieties, but numerous landraces are abandoned and at risk of extinction because of increasing urbanization, agricultural intensification, and varietal renewal. In this work, we investigated the morphological and genetic diversity present in an ex-situ collection of 28 neglected varieties belonging to the so-called "Vesuvian apricot". Our aim was to understand the level of diversity and the possible link between the promotion of specific fruit types (e.g., by public policies) and the intraspecific variation in apricot. The combination of five continuous and seven categorical traits allowed us to phenotypically distinguish the varieties; while fruit quality-related attributes displayed high variation, both apricot size and skin colour were more uniform. The twelve fluorescent-based Simple Sequence Repeats (SSRs) markers identified cultivar-specific molecular profiles and revealed a high molecular diversity, which poorly correlated with that described by the morphological analysis. Our results highlighted the complementary information provided by the two sets of descriptors and that DNA markers are necessary to separate morphologically related apricot landraces. The observed morphological and genetic differences suggest a loss of diversity influenced by maintenance breeding of specific pomological traits (e.g., skin colour and size). Finally, our study provided evidence to recommend complementary strategies to avoid the loss of diversity in apricot. Actions should pivot on both the promotion of easily identified premium products and more inclusive biodiversity-centred on-farm strategies.

Temperature Differentially Influences the Capacity of Trichoderma Species to Induce Plant Defense Responses in Tomato Against Insect Pests.

Species of the ecological opportunistic, avirulent fungus, Trichoderma are widely used in agriculture for their ability to protect crops from the attack of pathogenic fungi and for plant growth promotion activity. Recently, it has been shown that they may also have complementary properties that enhance plant defense barriers against insects. However, the use of these fungi is somewhat undermined by their variable level of biocontrol activity, which is influenced by environmental conditions. Understanding the source of this variability is essential for its profitable and wide use in plant protection. Here, we focus on the impact of temperature on Trichoderma afroharzianum T22, Trichoderma atroviride P1, and the defense response induced in tomato by insects. The in vitro development of these two strains was differentially influenced by temperature, and the observed pattern was consistent with temperature-dependent levels of resistance induced by them in tomato plants against the aphid, Macrosiphum euphorbiae, and the noctuid moth, Spodoptera littoralis. Tomato plants treated with T. afroharzianum T22 exhibited enhanced resistance toward both insect pests at 25°C, while T. atroviride P1 proved to be more effective at 20°C. The comparison of plant transcriptomic profiles generated by the two Trichoderma species allowed the identification of specific defense genes involved in the observed response, and a selected group was used to assess, by real-time quantitative reverse transcription PCR (qRT-PCR), the differential gene expression in Trichoderma-treated tomato plants subjected to the two temperature regimens that significantly affected fungal biological performance. These results will help pave the way toward a rational selection of the most suitable Trichoderma isolates for field applications, in order to best face the challenges imposed by local environmental conditions and by extreme climatic shifts due to global warming.

The Application of Trichoderma Strains or Metabolites Alters the Olive Leaf Metabolome and the Expression of Defense-Related Genes.

Biocontrol fungal strains of the genus Trichoderma can antagonize numerous plant pathogens and promote plant growth using different mechanisms of action, including the production of secondary metabolites (SMs). In this work we analyzed the effects of repeated applications of selected Trichoderma strains or SMs on young olive trees on the stimulation of plant growth and on the development of olive leaf spot disease caused by Fusicladium oleagineum. In addition, metabolomic analyses and gene expression profiles of olive leaves were carried out by LC-MS Q-TOF and real-time RT-PCR, respectively. A total of 104 phenolic compounds were detected from olive leave extracts and 20 were putatively identified. Targeted and untargeted approaches revealed significant differences in both the number and type of phenolic compounds accumulated in olive leaves after Trichoderma applications, as compared to water-treated plants. Different secoiridoids were less abundant in treated plants than in controls, while the accumulation of flavonoids (including luteolin and apigenin derivatives) increased following the application of specific Trichoderma strain. The induction of defense-related genes, and of genes involved in the synthesis of the secoiridoid oleuropein, was also analyzed and revealed a significant variation of gene expression according to the strain or metabolite applied.

Colonization of Solanum melongena and Vitis vinifera Plants by Botrytis cinerea Is Strongly Reduced by the Exogenous Application of Tomato Systemin.

Plant defense peptides are able to control immune barriers and represent a potential novel resource for crop protection. One of the best-characterized plant peptides is tomato Systemin (Sys) an octadecapeptide synthesized as part of a larger precursor protein. Upon pest attack, Sys interacts with a leucine-rich repeat receptor kinase, systemin receptor SYR, activating a complex intracellular signaling pathway that leads to the wound response. Here, we demonstrated, for the first time, that the direct delivery of the peptide to Solanum melongena and Vitis vinifera plants protects from the agent of Grey mould (Botrytis cinerea). The observed disease tolerance is associated with the increase of total soluble phenolic content, the activation of antioxidant enzymes, and the up-regulation of defense-related genes in plants treated with the peptide. Our results suggest that in treated plants, the biotic defense system is triggered by the Sys signaling pathway as a consequence of Sys interaction with a SYR-like receptor recently found in several plant species, including those under investigation. We propose that this biotechnological use of Sys, promoting defense responses against invaders, represents a useful tool to integrate into pest management programs for the development of novel strategies of crop protection.

Zucchini Plants Alter Gene Expression and Emission of (E)-ß-Caryophyllene Following Aphis gossypii Infestation.

Zucchini (Cucurbita pepo L.) is widely cultivated in temperate regions. One of the major production challenges is the damage caused by Aphis gossypii (Homoptera: Aphididae), a polyphagous aphid, which can negatively affect its host plant, both directly by feeding and indirectly by vectoring viruses. To gain insights into the transcriptome events that occur during the zucchini-aphid interaction and to understand the early-to-late defense response through gene expression profiles, we performed RNA-sequencing (RNA-Seq) on zucchini leaves challenged by A. gossypii (24, 48, and 96 h post-infestation; hpi). Data analysis indicated a complex and dynamic pattern of gene expression and a transient transcriptional reconfiguration that involved more than 700 differentially expressed genes (DEGs), including a large number of defense-related genes. The down-regulation of key genes of plant immunity, such as leucine-rich repeat (LRR) protein kinases, transcription factors, and genes associated with direct (i.e., protease inhibitors, cysteine peptidases, etc.) and indirect (i.e., terpene synthase) defense responses, suggests the aphid ability to manipulate plant immune responses. We also investigated the emission of volatile organic compounds (VOCs) from infested plants and observed a reduced emission of (E)-ß-caryophyllene at 48 hpi, likely the result of aphid effectors, which reflects the down-regulation of two genes involved in the biosynthesis of terpenoids. We showed that (E)-ß-caryophyllene emission was modified by the duration of plant infestation and by aphid density and that this molecule highly attracts Aphidius colemani, a parasitic wasp of A. gossypii. With our results we contributed to the identification of genes involved in cucurbit plant interactions with phloem feeders. Our findings may also help pave the way toward developing tolerant zucchini varieties and to identify molecules for sustainable management of harmful insect populations.

Tomato Plants Treated with Systemin Peptide Show Enhanced Levels of Direct and Indirect Defense Associated with Increased Expression of Defense-Related Genes.

Plant defense peptides represent an important class of compounds active against pathogens and insects. These molecules controlling immune barriers can potentially be used as novel tools for plant protection, which mimic natural defense mechanisms against invaders. The constitutive expression in tomato plants of the precursor of the defense peptide systemin was previously demonstrated to increase tolerance against moth larvae and aphids and to hamper the colonization by phytopathogenic fungi, through the expression of a wealth of defense-related genes. In this work we studied the impact of the exogenous supply of systemin to tomato plants on pests to evaluate the use of the peptide as a tool for crop protection in non-transgenic approaches. By combining gene expression studies and bioassays with different pests we demonstrate that the exogenous supply of systemin to tomato plants enhances both direct and indirect defense barriers. Experimental plants, exposed to this peptide by foliar spotting or root uptake through hydroponic culture, impaired larval growth and development of the noctuid moth Spodoptera littoralis, even across generations, reduced the leaf colonization by the fungal pathogen Botrytis cinerea and were more attractive towards natural herbivore antagonists. The induction of these defense responses was found to be associated with molecular and biochemical changes under control of the systemin signalling cascade. Our results indicate that the direct delivery of systemin, likely characterized by a null effect on non-target organisms, represents an interesting tool for the sustainable protection of tomato plants.

Transcriptome and Metabolome Reprogramming in Tomato Plants by Trichoderma harzianum strain T22 Primes and Enhances Defense Responses Against Aphids.

Beneficial fungi in the genus Trichoderma are among the most widespread biocontrol agents of plant pathogens. Their role in triggering plant defenses against pathogens has been intensely investigated, while, in contrast, very limited information is available on induced barriers active against insects. The growing experimental evidence on this latter topic looks promising, and paves the way toward the development of Trichoderma strains and/or consortia active against multiple targets. However, the predictability and reproducibility of the effects that these beneficial fungi is still somewhat limited by the lack of an in-depth understanding of the molecular mechanisms underlying the specificity of their interaction with different crop varieties, and on how the environmental factors modulate this interaction. To fill this research gap, here we studied the transcriptome changes in tomato plants (cultivar "Dwarf San Marzano") induced by Trichoderma harzianum (strain T22) colonization and subsequent infestation by the aphid Macrosiphum euphorbiae. A wide transcriptome reprogramming, related to metabolic processes, regulation of gene expression and defense responses, was induced both by separate experimental treatments, which showed a synergistic interaction when concurrently applied. The most evident expression changes of defense genes were associated with the multitrophic interaction Trichoderma-tomato-aphid. Early and late genes involved in direct defense against insects were induced (i.e., peroxidase, GST, kinases and polyphenol oxidase, miraculin, chitinase), along with indirect defense genes, such as sesquiterpene synthase and geranylgeranyl phosphate synthase. Targeted and untargeted semi-polar metabolome analysis revealed a wide metabolome alteration showing an increased accumulation of isoprenoids in Trichoderma treated plants. The wide array of transcriptomic and metabolomics changes nicely fit with the higher mortality of aphids when feeding on Trichoderma treated plants, herein reported, and with the previously observed attractiveness of these latter toward the aphid parasitoid Aphidius ervi. Moreover, Trichoderma treated plants showed the over-expression of transcripts coding for several families of defense-related transcription factors (bZIP, MYB, NAC, AP2-ERF, WRKY), suggesting that the fungus contributes to the priming of plant responses against pest insects. Collectively, our data indicate that Trichoderma treatment of tomato plants induces transcriptomic and metabolomic changes, which underpin both direct and indirect defense responses.

Trichoderma atroviride P1 Colonization of Tomato Plants Enhances Both Direct and Indirect Defense Barriers Against Insects.

Numerous microbial root symbionts are known to induce different levels of enhanced plant protection against a variety of pathogens. However, more recent studies have demonstrated that beneficial microbes are able to induce plant systemic resistance that confers some degree of protection against insects. Here, we report how treatments with the fungal biocontrol agent Trichoderma atroviride strain P1 in tomato plants induce responses that affect pest insects with different feeding habits: the noctuid moth Spodoptera littoralis (Boisduval) and the aphid Macrosiphum euphorbiae (Thomas). We observed that the tomato plant-Trichoderma P1 interaction had a negative impact on the development of moth larvae and on aphid longevity. These effects were attributed to a plant response induced by Trichoderma that was associated with transcriptional changes of a wide array of defense-related genes. While the impact on aphids could be related to the up-regulation of genes involved in the oxidative burst reaction, which occur early in the defense reaction, the negative performance of moth larvae was associated with the enhanced expression of genes encoding for protective enzymes (i.e., Proteinase inhibitor I (PI), Threonine deaminase, Leucine aminopeptidase A1, Arginase 2, and Polyphenol oxidase) that are activated downstream in the defense cascade. In addition, Trichoderma P1 produced alterations in plant metabolic pathways leading to the production and release of volatile organic compounds (VOCs) that are involved in the attraction of the aphid parasitoid Aphidius ervi, thus reinforcing the indirect plant defense barriers. Our findings, along with the evidence available in the literature, indicate that the outcome of the tripartite interaction among plant, Trichoderma, and pests is highly specific and only a comprehensive approach, integrating both insect phenotypic changes and plant transcriptomic alterations, can allow a reliable prediction of its potential for plant protection.

TPS Genes Silencing Alters Constitutive Indirect and Direct Defense in Tomato.

Following herbivore attacks, plants modify a blend of volatiles organic compounds (VOCs) released, resulting in the attraction of their antagonists. However, volatiles released constitutively may affect herbivores and natural enemies' fitness too. In tomato there is still a lack of information on the genetic bases responsible for the constitutive release of VOC involved in direct and indirect defenses. Here we studied the constitutive emissions related to the two most abundant sesquiterpene synthase genes expressed in tomato and their functional role in plant defense. Using an RNA interference approach, we silenced the expression of TPS9 and TPS12 genes and assessed the effect of this transformation on herbivores and parasitoids. We found that silenced plants displayed a different constitutive volatiles emission from controls, resulting in reduced attractiveness for the aphid parasitoid Aphidius ervi and in an impaired development of Spodoptera exigua larvae. We discussed these data considering the transcriptional regulation of key-genes involved in the pathway of VOC metabolism. We provide several lines of evidence on the metabolic flux from terpenoids to phenylpropanoids. Our results shed more light on constitutive defenses mediated by plant volatiles and on the molecular mechanisms involved in their metabolic regulation.

Prosystemin, a prohormone that modulates plant defense barriers, is an intrinsically disordered protein.

Prosystemin, originally isolated from Lycopersicon esculentum, is a tomato pro-hormone of 200 aminoacid residues which releases a bioactive peptide of 18 aminoacids called Systemin. This signaling peptide is involved in the activation of defense genes in solanaceous plants in response to herbivore feeding damage. Using biochemical, biophysical and bioinformatics approaches we characterized Prosystemin, showing that it is an intrinsically disordered protein possessing a few secondary structure elements within the sequence. Plant treatment with recombinant Prosystemin promotes early and late plant defense genes, which limit the development and survival of Spodoptera littoralis larvae fed with treated plants.

Chemical Compositions, Somatic Embryogenesis, and Somaclonal Variation in Cumin.

This is the first report evaluating the relationship between the chemical compositions of cumin seeds (based on the analysis of the content of catalase, ascorbate peroxidase, proline, protein, terpenic compounds, alcohol/phenols, aldehydes, and epoxides) and the induction efficiency of somatic embryogenesis in two Iranian superior cumin landraces (Golestan and North Khorasan). Cotyledons isolated from Golestan landrace seeds cultivated on MS medium supplemented with 0.1 mg/L kinetin proved to be the best primary explant for the induction of somatic embryogenesis as well as the regeneration of the whole plantlet. Results indicated that different developmental stages of somatic embryos were simultaneously observed on a callus with embryogenic potential. The high content of catalase, ascorbate peroxidase, proline, and terpenic hydrocarbons and low content of alcoholic and phenolic compositions had a stimulatory effect on somatic embryogenesis. Band patterns of RAPD markers in regenerated plants were different from those of the mother plants. This may be related to somaclonal variations or pollination system of cumin. Generally, measurement of chemical compositions can be used as a marker for evaluating the occurrence of somatic embryogenesis in cumin. Also, somaclonal variations of regenerated plants can be applied by the plant breeders in breeding programs.

Plant-to-plant communication triggered by systemin primes anti-herbivore resistance in tomato.

Plants actively respond to herbivory by inducing various defense mechanisms in both damaged (locally) and non-damaged tissues (systemically). In addition, it is currently widely accepted that plant-to-plant communication allows specific neighbors to be warned of likely incoming stress (defense priming). Systemin is a plant peptide hormone promoting the systemic response to herbivory in tomato. This 18-aa peptide is also able to induce the release of bioactive Volatile Organic Compounds, thus also promoting the interaction between the tomato and the third trophic level (e.g. predators and parasitoids of insect pests). In this work, using a combination of gene expression (RNA-Seq and qRT-PCR), behavioral and chemical approaches, we demonstrate that systemin triggers metabolic changes of the plant that are capable of inducing a primed state in neighboring unchallenged plants. At the molecular level, the primed state is mainly associated with an elevated transcription of pattern -recognition receptors, signaling enzymes and transcription factors. Compared to naïve plants, systemin-primed plants were significantly more resistant to herbivorous pests, more attractive to parasitoids and showed an increased response to wounding. Small peptides are nowadays considered fundamental signaling molecules in many plant processes and this work extends the range of downstream effects of this class of molecules to intraspecific plant-to-plant communication.