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.

Aphidius ervi venom regulates Buchnera contribution to host nutritional suitability.

The association between the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), and the endophagous parasitoid wasp Aphidius ervi Haliday (Hymenoptera: Braconidae) offers a unique model system for studying the molecular mechanisms underlying the complex interactions between the parasitoid, its host and the associated primary symbiont. Here, we investigate in vivo the functional role of the most abundant component of A. ervi venom, Ae-γ-glutamyl transpeptidase (Ae-γ-GT), which is known to induce host castration. Microinjections of double-stranded RNA into A. ervi pupae stably knocked down Ae-γ-GT1 and Ae-γ-GT2 paralogue genes in newly emerged females. These females were used to score the phenotypic changes both in parasitized hosts and in the parasitoid's progeny, as affected by a venom blend lacking Ae-γ-GT. Ae-γ-GT gene silencing enhanced growth both of host and parasitoid, supported by a higher load of the primary bacterial symbiont Buchnera aphidicola. Emerging adults showed a reduced survival and fecundity, suggesting a trade-off with body size. This demonstrates in vivo the primary role of Ae-γ-GT in host ovary degeneration and suggests that this protein counterbalances the proliferation of Buchnera likely triggered by other venom components. Our study provides a new approach to unravelling the complexity of aphid parasitoid venom in vivo, and sheds light on a novel role for Ae-γ-GT in host regulation.

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.

Role of neuronal and non-neuronal acetylcholine signaling in Drosophila humoral immunity.

Acetylcholine (ACh) is one the major neurotransmitters in insects, whose role in mediating synaptic interactions between neurons in the central nervous system is well characterized. It also plays largely unexplored regulatory functions in non-neuronal tissues. Here we demonstrate that ACh signaling is involved in the modulation of the innate immune response of Drosophila melanogaster. Knockdown of ACh synthesis or ACh vesicular transport in neurons reduced the activation of drosomycin (drs), a gene encoding an antimicrobial peptide, in adult flies infected with a Gram-positive bacterium. drs transcription was similarly affected in Drosophila α7 nicotinic acetylcholine receptor, nAChRalpha7 (Dα7) mutants, as well as in flies expressing in the nervous system a dominant negative form (Dα7DN) of this specific receptor subunit. Interestingly, Dα7DN elicited a comparable response when it was expressed in non-neuronal tissues and even when it was specifically produced in the hemocytes. Consistently, full activation of the drs gene required Dα7 expression in these cells. Moreover, knockdown of ACh synthesis in non-neuronal cells affected drs expression. Overall, these findings uncover neural and non-neural cholinergic signals that modulate insect immune defenses and shed light on the role of hemocytes in the regulation of the humoral immune response.

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.

Secondary Metabolites, including a New 5,6-Dihydropyran-2-One, Produced by the Fungus Diplodia corticola. Aphicidal Activity of the Main Metabolite, Sphaeropsidin A.

An undescribed 5,6-dihydropyran-2-one, namely diplopyrone C, was isolated and characterized from the cultures of an isolate of the fungus Diplodia corticola recovered from Quercus suber in Algeria. The structure and relative stereostructure of (5S,6S,7Z,9S,10S)-5-hydroxy-6-(2-(3-methyloxiran-2-yl)vinyl)-5,6-dihydro-2H-pyran-2-one were assigned essentially based on NMR and MS data. Furthermore, ten known compounds were isolated and identified in the same cultures. The most abundant product, the tetracyclic pimarane diterpene sphaeropsidin A, was tested for insecticidal effects against the model sucking aphid, Acyrthosiphon pisum. Results showed a toxic dose-dependent oral activity of sphaeropsidin A, with an LC50 of 9.64 mM.

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.

Selection of Endophytic Beauveria bassiana as a Dual Biocontrol Agent of Tomato Pathogens and Pests.

Endophytic fungi (EF) can enhance both plant growth and defense barriers against pests and pathogens, contributing to the reduction of chemical pesticides and fertilizers use in agriculture. Beauveria bassiana is an entomopathogenic fungus showing endophytism in several crops, often associated with a good capacity to limit the development of pests and disease agents. However, the diversity of the protective efficacy and plant response to different strains can be remarkable and needs to be carefully assessed for the successful and predictable use of these beneficial microorganisms. This study aims to select B. bassiana strains able to colonize tomato plants as endophytes as well as to control two important disease agents, Botrytis cinerea and Alternaria alternata, and the pest aphid, Macrosiphum euphorbiae. Nine wild-type isolates and one commercial strain were screened for endophytism, then further characterized for plant-growth promotion plus inhibition of disease development and pest infestation. Four isolates proved to have a good control activity against the biotic stressors tested, but only Bb716 was also able to promote plant growth. This work provides a simple workflow for the selection of beneficial EF, paving the way towards more effective use of B. bassiana in Integrate Pest Management (IPM) of tomato.

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.

Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp.

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably influenced by the acquisition of a large number of genes by horizontal gene transfer (HGT) across different kingdoms. However, in depth-studies on the physiological traits conferred by these accidental DNA acquisitions are largely lacking. Here we elucidate the functional role of Sl gasmin, a gene of a symbiotic virus of a parasitic wasp that has been transferred to an ancestor of the moth species Spodoptera littoralis and domesticated. This gene is highly expressed in circulating immune cells (haemocytes) of larval stages, where its transcription is rapidly boosted by injection of microorganisms into the body cavity. RNAi silencing of Sl gasmin generates a phenotype characterized by a precocious suppression of phagocytic activity by haemocytes, which is rescued when these immune cells are incubated in plasma samples of control larvae, containing high levels of the encoded protein. Proteomic analysis demonstrates that the protein Sl gasmin is released by haemocytes into the haemolymph, where it opsonizes the invading bacteria to promote their phagocytosis, both in vitro and in vivo. Our results show that important physiological traits do not necessarily originate from evolution of pre-existing genes, but can be acquired by HGT events, through unique pathways of symbiotic evolution. These findings indicate that insects can paradoxically acquire selective advantages with the help of their natural enemies.

The Intestinal Microbiota of Hermetia illucens Larvae Is Affected by Diet and Shows a Diverse Composition in the Different Midgut Regions.

The larva of the black soldier fly (Hermetia illucens) has emerged as an efficient system for the bioconversion of organic waste. Although many research efforts are devoted to the optimization of rearing conditions to increase the yield of the bioconversion process, microbiological aspects related to this insect are still neglected. Here, we describe the microbiota of the midgut of H. illucens larvae, showing the effect of different diets and midgut regions in shaping microbial load and diversity. The bacterial communities residing in the three parts of the midgut, characterized by remarkable changes in luminal pH values, differed in terms of bacterial numbers and microbiota composition. The microbiota of the anterior part of the midgut showed the highest diversity, which gradually decreased along the midgut, whereas bacterial load had an opposite trend, being maximal in the posterior region. The results also showed that the influence of the microbial content of ingested food was limited to the anterior part of the midgut, and that the feeding activity of H. illucens larvae did not significantly affect the microbiota of the substrate. Moreover, a high protein content compared to other macronutrients in the feeding substrate seemed to favor midgut dysbiosis. The overall data indicate the importance of taking into account the presence of different midgut structural and functional domains, as well as the substrate microbiota, in any further study that aims at clarifying microbiological aspects concerning H. illucens larval midgut.IMPORTANCE The demand for food of animal origin is expected to increase by 2050. Since traditional protein sources for monogastric diets are failing to meet the increasing demand for additional feed production, there is an urgent need to find alternative protein sources. The larvae of Hermetia illucens emerge as efficient converters of low-quality biomass into nutritionally valuable proteins. Many studies have been performed to optimize H. illucens mass rearing on a number of organic substrates and to quantitatively and qualitatively maximize the biomass yield. On the contrary, although the insect microbiota can be fundamental for bioconversion processes and its characterization is mandatory also for safety aspects, this topic is largely overlooked. Here, we provide an in-depth study of the microbiota of H. illucens larval midgut, taking into account pivotal aspects, such as the midgut spatial and functional regionalization, as well as microbiota and nutrient composition of the feeding substrate.

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.

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.

Host regulation by the ectophagous parasitoid wasp Bracon nigricans.

The host regulation process has been widely investigated in endophagous parasitoid wasps, which in most cases finely interact with living hosts (i.e. koinobiont parasitoids). In contrast, only very limited information is available for ectophagous parasitoids that permanently paralyze and rapidly suppress their victims (i.e. idiobiont parasitoids). Here we try to fill this research gap by investigating the host regulation by Bracon nigricans, an ectophagous idiobiont wasp species. Parasitism, mainly by venom action, is able to redirect host metabolism in order to enhance its nutritional suitability for the developing parasitoid larvae and to provide the required metabolic support to host tissues. The observed alterations of the host titers of haemolymph proteins, carbohydrates and acylglycerols are associated with a parasitoid-induced mobilization of nutrients stored in the fat body. This tissue undergoes a controlled degradation mediated by a close surface interaction with haemocytes, where a cathepsin L activity is localized, as demonstrated by immunolocalization, biochemical and transcriptional data. B. nigricans parasitism does not markedly influence the survival of haemocytes, even though a persistent suppression of the immune competence is observed in parasitized hosts, which show a reduced capacity to encapsulate and melanize non-self objects. These immune alterations likely allow a more efficient food uptake and use by the ectophagous larvae. The obtained results indicate that the host regulation process in basal lineages of parasitic Hymenoptera is more complex than expected and shares functional similarities with adaptive strategies occurring in derived koinobiont species.

Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism.

Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host.

A virulence factor encoded by a polydnavirus confers tolerance to transgenic tobacco plants against lepidopteran larvae, by impairing nutrient absorption.

The biological control of insect pests is based on the use of natural enemies. However, the growing information on the molecular mechanisms underpinning the interactions between insects and their natural antagonists can be exploited to develop "bio-inspired" pest control strategies, mimicking suppression mechanisms shaped by long co-evolutionary processes. Here we focus on a virulence factor encoded by the polydnavirus associated with the braconid wasp Toxoneuron nigriceps (TnBV), an endophagous parasitoid of noctuid moth larvae. This virulence factor (TnBVANK1) is a member of the viral ankyrin (ANK) protein family, and appears to be involved both in immunosuppression and endocrine alterations of the host. Transgenic tobacco plants expressing TnBVANK1 showed insecticide activity and caused developmental delay in Spodoptera littoralis larvae feeding on them. This effect was more evident in a transgenic line showing a higher number of transcripts of the viral gene. However, this effect was not associated with evidence of translocation into the haemocoel of the entire protein, where the receptors of TnBVANK1 are putatively located. Indeed, immunolocalization experiments evidenced the accumulation of this viral protein in the midgut, where it formed a thick layer coating the brush border of epithelial cells. In vitro transport experiments demonstrated that the presence of recombinant TnBVANK1 exerted a dose-dependent negative impact on amino acid transport. These results open new perspectives for insect control and stimulate additional research efforts to pursue the development of novel bioinsecticides, encoded by parasitoid-derived genes. However, future work will have to carefully evaluate any effect that these molecules may have on beneficial insects and on non-target organisms.

Targeting the diuretic hormone receptor to control the cotton leafworm, Spodoptera littoralis.

The cotton leafworm, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae), is one of the most devastating pests of crops worldwide. Several types of treatments have been used against this pest, but many of them failed because of the rapid development of genetic resistance in the different insect populations. G protein coupled receptors have vital functions in most organisms, including insects; thus, they are appealing targets for species-specific pest control strategies. Among the insect G protein coupled receptors, the diuretic hormone receptors have several key roles in development and metabolism, but their importance in vivo and their potential role as targets of novel pest control strategies are largely unexplored. With the goal of using DHR genes as targets to control S. littoralis, we cloned a corticotropin-releasing factor-like binding receptor in this species and expressed the corresponding dsRNA in tobacco plants to knock down the receptor activity in vivo through RNA interference. We also expressed the receptor in mammalian cells to study its signaling pathways. The results indicate that this diuretic hormone receptor gene has vital roles in S. littoralis and represents an excellent molecular target to protect agriculturally-important plants from this pest.

Functional analysis of an immune gene of Spodoptera littoralis by RNAi.

Insect immune defences rely on cellular and humoral responses targeting both microbial pathogens and metazoan parasites. Accumulating evidence indicates functional cross-talk between these two branches of insect immunity, but the underlying molecular mechanisms are still largely unknown. We recently described, in the tobacco budworm Heliothis virescens, the presence of amyloid fibers associated with melanogenesis in immune capsules formed by hemocytes, and identified a protein (P102) involved in their assembly. Non-self objects coated by antibodies directed against this protein escaped hemocyte encapsulation, suggesting that P102 might coordinate humoral and cellular defence responses at the surface of foreign invaders. Here we report the identification of a cDNA coding for a protein highly similar to P102 in a related Lepidoptera species, Spodoptera littoralis. Its transcript was abundant in the hemocytes and the protein accumulated in large cytoplasmic compartments, closely resembling the localization pattern of P102 in H. virescens. RNAi-mediated gene silencing provided direct evidence for the role played by this protein in the immune response. Oral delivery of dsRNA molecules directed against the gene strongly suppressed the encapsulation and melanization response, while hemocoelic injections did not result in evident phenotypic alterations. Shortly after their administration, dsRNA molecules were found in midgut cells, en route to the hemocytes where the target gene was significantly down-regulated. Taken together, our data demonstrate that P102 is a functionally conserved protein with a key role in insect immunity. Moreover, the ability to target this gene by dsRNA oral delivery may be exploited to develop novel technologies of pest control, based on immunosuppression as a strategy for enhancing the impact of natural antagonists.