Ilaria Di Lelio | Università degli Studi di Napoli "Federico II" (original) (raw)

Papers by Ilaria Di Lelio

Research paper thumbnail of Analysis of Cellular Immune Responses in Lepidopteran Larvae

Research paper thumbnail of RESEARCH ARTICLE A Virulence Factor Encoded by a Polydnavirus Confers Tolerance to Transgenic Tobacco Plants against Lepidopteran Larvae, by Impairing Nutrient Absorption

. These authors contributed equally to this work. The biological control of insect pests is based... more . These authors contributed equally to this work. 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

Research paper thumbnail of Transgenic plants expressing immunosuppressive dsRNA improve entomopathogen efficacy against Spodoptera littoralis larvae

Journal of Pest Science, 2022

Transgenic plants that express double-stranded RNA (dsRNA) targeting vital insect genes have rece... more Transgenic plants that express double-stranded RNA (dsRNA) targeting vital insect genes have recently emerged as a valuable new tool for pest control. In this study, tobacco plants were transformed to produce dsRNA targeting Sl 102 gene that is involved in the immune response of Spodoptera littoralis larvae, a serious lepidopteran pest of several crops. Experimental larvae reared on transgenic tobacco lines showed (1) a strongly reduced level of Sl 102 transcripts, which was positively associated with food consumption; (2) a substantial impairment of the encapsulation response mediated by hemocytes; and (3) a marked increase in the susceptibility to Xentari™, a Bacillus thuringiensis-based insecticide. Importantly, this approach may allow a reduction in the doses of B. thuringiensis used for field applications and enhance its killing activity on mature larvae. The results obtained thus support the use of immunosuppressive RNAi plants to enhance the performance of microbial insectici...

Research paper thumbnail of Tomato Prosystemin is much more than a simple Systemin precursor

Systemin (Sys) is an octadecapeptide which, upon wounding, is released from the carboxy terminus ... more 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 ProSysprompted us to investigate a putative biological role of the whole precursor deprived of 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 deleted ProSys[ProSys(1-178)].By combining protein structure analyses, transcriptomic analysis, gene expression profiling and bioassays with different pests we demonstrate that the truncated ProSys, that does not induce the endogenous ProSys gene, 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...

Research paper thumbnail of Selection of Endophytic Beauveria bassiana as a Dual Biocontrol Agent of Tomato Pathogens and Pests

Pathogens

Endophytic fungi (EF) can enhance both plant growth and defense barriers against pests and pathog... more 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 p...

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

Frontiers in Plant Science

Species of the ecological opportunistic, avirulent fungus, Trichoderma are widely used in agricul... more 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...

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

Frontiers in Physiology

Numerous microbial root symbionts are known to induce different levels of enhanced plant protecti... more 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.

Research paper thumbnail of Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp

PLOS Genetics

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably in... more 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.

Research paper thumbnail of The intestinal microbiota of Hermetia illucens larvae is affected by diet and shows a diverse composition in the different midgut regions

Applied and Environmental Microbiology

The larva of the black soldier fly (Hermetia illucens) has emerged as an efficient system for the... more 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 that 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 limite...

Research paper thumbnail of Plant-to-plant communication triggered by systemin primes anti-herbivore resistance in tomato

Scientific reports, Jan 14, 2017

Plants actively respond to herbivory by inducing various defense mechanisms in both damaged (loca... more 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 -recogn...

Research paper thumbnail of Prosystemin, a prohormone that modulates plant defense barriers, is an intrinsically disordered protein

Protein science : a publication of the Protein Society, Jan 23, 2017

Prosystemin, originally isolated from Lycopersicon esculentum, is a tomato pro-hormone of 200 ami... more 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.

Research paper thumbnail of Host regulation by the ectophagous parasitoid wasp Bracon nigricans

Journal of Insect Physiology

The host regulation process has been widely investigated in endophagous parasitoid wasps, which i... more 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.

Research paper thumbnail of Uso di RNAi per il controllo degli insetti

Research paper thumbnail of Functional analysis of an immune gene of Spodoptera littoralis by RNAi

Journal of Insect Physiology, 2014

Insect immune defences rely on cellular and humoral responses targeting both microbial pathogens ... more 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.

Research paper thumbnail of Functional amyloids in insect immune response

Insect Biochemistry and Molecular Biology, 2012

The innate immune system of insects consists of humoural and cellular responses that provide prot... more The innate immune system of insects consists of humoural and cellular responses that provide protection against invading pathogens and parasites. Defence reactions against these latter include encapsulation by immune cells and targeted melanin deposition, which is usually restricted to the surface of the foreign invader, to prevent systemic damage. Here we show that a protein produced by haemocytes of Heliothis virescens (Lepidoptera, Noctuidae) larvae, belonging to XendoU family, generates amyloid fibrils, which accumulate in large cisternae of the rough endoplasmic reticulum and are released upon immune challenge, to form a layer coating non-self objects entering the haemocoel. This amyloid layer acts as a molecular scaffold that promotes localised melanin synthesis and the adhesion of immune cells around the non-self intruder during encapsulation response. Our results demonstrate a new functional role for these protein aggregates that are commonly associated with severe human diseases. We predict that insects will offer new powerful experimental systems for studying inducible amyloidogenesis, which will likely provide fresh perspectives for its prevention.

Research paper thumbnail of Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism

Proceedings of the National Academy of Sciences, 2016

Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, s... more 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. Consequ...

Research paper thumbnail of Targeting the Diuretic Hormone Receptor to Control the Cotton Leafworm, Spodoptera littoralis

Journal of Insect Science, 2014

The cotton leafworm, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae), is one of the most... more 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 agriculturallyimportant plants from this pest.

Research paper thumbnail of A Virulence Factor Encoded by a Polydnavirus Confers Tolerance to Transgenic Tobacco Plants against Lepidopteran Larvae, by Impairing Nutrient Absorption

PLoS ONE, 2014

. These authors contributed equally to this work.

Research paper thumbnail of Functional analysis of an immune gene of Spodoptera littoralis by RNAi

Journal of Insect Physiology, 2014

Insect immune defences rely on cellular and humoral responses targeting both microbial pathogens ... more 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.

Research paper thumbnail of A Virulence Factor Encoded by a Polydnavirus Confers Tolerance to Transgenic Tobacco Plants against Lepidopteran Larvae, by Impairing Nutrient Absorption

PLoS ONE, 2014

The biological control of insect pests is based on the use of natural enemies. However, the growi... more 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.

Research paper thumbnail of Analysis of Cellular Immune Responses in Lepidopteran Larvae

Research paper thumbnail of RESEARCH ARTICLE A Virulence Factor Encoded by a Polydnavirus Confers Tolerance to Transgenic Tobacco Plants against Lepidopteran Larvae, by Impairing Nutrient Absorption

. These authors contributed equally to this work. The biological control of insect pests is based... more . These authors contributed equally to this work. 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

Research paper thumbnail of Transgenic plants expressing immunosuppressive dsRNA improve entomopathogen efficacy against Spodoptera littoralis larvae

Journal of Pest Science, 2022

Transgenic plants that express double-stranded RNA (dsRNA) targeting vital insect genes have rece... more Transgenic plants that express double-stranded RNA (dsRNA) targeting vital insect genes have recently emerged as a valuable new tool for pest control. In this study, tobacco plants were transformed to produce dsRNA targeting Sl 102 gene that is involved in the immune response of Spodoptera littoralis larvae, a serious lepidopteran pest of several crops. Experimental larvae reared on transgenic tobacco lines showed (1) a strongly reduced level of Sl 102 transcripts, which was positively associated with food consumption; (2) a substantial impairment of the encapsulation response mediated by hemocytes; and (3) a marked increase in the susceptibility to Xentari™, a Bacillus thuringiensis-based insecticide. Importantly, this approach may allow a reduction in the doses of B. thuringiensis used for field applications and enhance its killing activity on mature larvae. The results obtained thus support the use of immunosuppressive RNAi plants to enhance the performance of microbial insectici...

Research paper thumbnail of Tomato Prosystemin is much more than a simple Systemin precursor

Systemin (Sys) is an octadecapeptide which, upon wounding, is released from the carboxy terminus ... more 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 ProSysprompted us to investigate a putative biological role of the whole precursor deprived of 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 deleted ProSys[ProSys(1-178)].By combining protein structure analyses, transcriptomic analysis, gene expression profiling and bioassays with different pests we demonstrate that the truncated ProSys, that does not induce the endogenous ProSys gene, 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...

Research paper thumbnail of Selection of Endophytic Beauveria bassiana as a Dual Biocontrol Agent of Tomato Pathogens and Pests

Pathogens

Endophytic fungi (EF) can enhance both plant growth and defense barriers against pests and pathog... more 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 p...

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

Frontiers in Plant Science

Species of the ecological opportunistic, avirulent fungus, Trichoderma are widely used in agricul... more 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...

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

Frontiers in Physiology

Numerous microbial root symbionts are known to induce different levels of enhanced plant protecti... more 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.

Research paper thumbnail of Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp

PLOS Genetics

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably in... more 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.

Research paper thumbnail of The intestinal microbiota of Hermetia illucens larvae is affected by diet and shows a diverse composition in the different midgut regions

Applied and Environmental Microbiology

The larva of the black soldier fly (Hermetia illucens) has emerged as an efficient system for the... more 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 that 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 limite...

Research paper thumbnail of Plant-to-plant communication triggered by systemin primes anti-herbivore resistance in tomato

Scientific reports, Jan 14, 2017

Plants actively respond to herbivory by inducing various defense mechanisms in both damaged (loca... more 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 -recogn...

Research paper thumbnail of Prosystemin, a prohormone that modulates plant defense barriers, is an intrinsically disordered protein

Protein science : a publication of the Protein Society, Jan 23, 2017

Prosystemin, originally isolated from Lycopersicon esculentum, is a tomato pro-hormone of 200 ami... more 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.

Research paper thumbnail of Host regulation by the ectophagous parasitoid wasp Bracon nigricans

Journal of Insect Physiology

The host regulation process has been widely investigated in endophagous parasitoid wasps, which i... more 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.

Research paper thumbnail of Uso di RNAi per il controllo degli insetti

Research paper thumbnail of Functional analysis of an immune gene of Spodoptera littoralis by RNAi

Journal of Insect Physiology, 2014

Insect immune defences rely on cellular and humoral responses targeting both microbial pathogens ... more 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.

Research paper thumbnail of Functional amyloids in insect immune response

Insect Biochemistry and Molecular Biology, 2012

The innate immune system of insects consists of humoural and cellular responses that provide prot... more The innate immune system of insects consists of humoural and cellular responses that provide protection against invading pathogens and parasites. Defence reactions against these latter include encapsulation by immune cells and targeted melanin deposition, which is usually restricted to the surface of the foreign invader, to prevent systemic damage. Here we show that a protein produced by haemocytes of Heliothis virescens (Lepidoptera, Noctuidae) larvae, belonging to XendoU family, generates amyloid fibrils, which accumulate in large cisternae of the rough endoplasmic reticulum and are released upon immune challenge, to form a layer coating non-self objects entering the haemocoel. This amyloid layer acts as a molecular scaffold that promotes localised melanin synthesis and the adhesion of immune cells around the non-self intruder during encapsulation response. Our results demonstrate a new functional role for these protein aggregates that are commonly associated with severe human diseases. We predict that insects will offer new powerful experimental systems for studying inducible amyloidogenesis, which will likely provide fresh perspectives for its prevention.

Research paper thumbnail of Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism

Proceedings of the National Academy of Sciences, 2016

Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, s... more 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. Consequ...

Research paper thumbnail of Targeting the Diuretic Hormone Receptor to Control the Cotton Leafworm, Spodoptera littoralis

Journal of Insect Science, 2014

The cotton leafworm, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae), is one of the most... more 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 agriculturallyimportant plants from this pest.

Research paper thumbnail of A Virulence Factor Encoded by a Polydnavirus Confers Tolerance to Transgenic Tobacco Plants against Lepidopteran Larvae, by Impairing Nutrient Absorption

PLoS ONE, 2014

. These authors contributed equally to this work.

Research paper thumbnail of Functional analysis of an immune gene of Spodoptera littoralis by RNAi

Journal of Insect Physiology, 2014

Insect immune defences rely on cellular and humoral responses targeting both microbial pathogens ... more 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.

Research paper thumbnail of A Virulence Factor Encoded by a Polydnavirus Confers Tolerance to Transgenic Tobacco Plants against Lepidopteran Larvae, by Impairing Nutrient Absorption

PLoS ONE, 2014

The biological control of insect pests is based on the use of natural enemies. However, the growi... more 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.