Can Herbivore-Induced Volatiles Protect Plants by Increasing the Herbivores' Susceptibility to Natural Pathogens?

In response to insect herbivory, plants mobilize various defenses. Defense responses include the release of herbivore-induced plant volatiles (HIPVs) that can serve as signals to alert undamaged tissues and to attract natural enemies of the herbivores. Some HIPVs can have a direct negative impact on herbivore survival, but it is not well understood by what mechanisms. Here, we tested the hypothesis that exposure to HIPVs renders insects more susceptible to natural pathogens. Exposure of the caterpillars of the noctuid Spodoptera exigua to indole and linalool, but not exposure to (Z)-3-hexenyl acetate, increased the susceptibility to Spodoptera exiguamultiple nucleopolyhedrovirus (SeMNPV). We also found that exposure to indole, but not exposure to linalool or (Z)-3-hexenyl acetate, increased the pathogenicity of Bacillus thuringiensis Additional experiments revealed significant changes in microbiota composition after forty-eight hours of larval exposure to indole. Overall, these results provide evidence that certain HIPVs can strongly enhance the susceptibility of caterpillars to pathogens, possibly through effects on the insect gut microbiota. These findings suggest a novel mechanism by which HIPVs can protect plants from herbivorous insects.IMPORTANCE Multitrophic interactions involving insect pests, their natural enemies, microorganisms, and plant hosts are increasingly being recognized as relevant factors in pest management. In response to herbivory attacks, plants activate a wide range of defenses that aim to mitigate the damage. Attacked plants release herbivore-induced plant volatiles (HIPVs), which can act as priming signals for other plants and attract natural enemies of herbivores, and which may have a direct negative impact on herbivore survival. In the present work, we show that exposure of the insects to the induced volatiles could increase the insects' susceptibility to the entomopathogens naturally occurring in the plant environment. These findings suggest a novel role for plant volatiles by influencing insect interactions with natural pathogens, probably mediated by alterations in the insect microbiota composition. In addition, this work provides evidence for selectable plant traits (production of secondary metabolites) that can have an influence on the ecology of the pests and could be relevant in the improvement of pest management strategies using natural entomopathogens.

Engineering of the baculovirus expression system for optimized protein production.

Baculoviruses are arthropod-specific large circular double-stranded DNA viruses successfully used for the control of multiple insect pests. In addition to their application in pest control, baculoviruses have become a versatile and powerful eukaryotic vector for the production of large quantities of recombinant proteins for research and biomedical purposes. Since the first recombinant protein was expressed in 1983 using the baculovirus expression system (BEVS), different strategies have been developed for the generation of recombinant viruses and to increase the stability, yield, and posttranslational modifications of recombinant proteins. In this review, we summarize the main methods and elements playing a role in the BEVS emphasizing recent progresses and future developments with respect to the main aspects involved in protein production using the BEVS.

Expression of the lef5 gene from Spodoptera exigua multiple nucleopolyhedrovirus contributes to the baculovirus stability in cell culture.

Baculoviruses are a broad group of viruses infecting insects, predominately of the order Lepidoptera. They are used worldwide as biological insecticides and as expression vectors to produce recombinant proteins. Baculoviruses replicate in their host, although several cell lines have been developed for in vitro replication. Nevertheless, replication of baculoviruses in cell culture involves the generation of defective viruses with a decrease in productivity and virulence. Transcriptional studies of the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infective process revealed differences in the expression patterns when the virus replicated under in vitro (Se301 cells) or in vivo (S. exigua larvae) conditions. The late expression factor 5 (lef5) gene was found to be highly overexpressed when the virus replicates in larvae. To test the possible role of lef5 expression in viral stability, recombinant AcMNPV expressing the lef5 gene from SeMNPV (Se-lef5) was generated and its stability was monitored during successive infection passages in Sf21 cells by evaluating the loss of several essential and non-essential genes. The gfp transgene was more stable in those viruses expressing the Se-LEF5 protein and the GFP-defective viruses were accumulated at a lower level when compared to its control viruses, confirming the positive influence of lef5 in viral stability during the multiplication process. This work describes for the first time a viral factor involved in transgene stability when baculoviruses replicate in cell culture, opening new ways to facilitate the in vitro production of recombinant proteins using baculovirus.

Shared binding sites for the Bacillus thuringiensis proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African sweet potato pest Cylas puncticollis (Brentidae).

Bacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas, which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites in Cylas puncticollis to orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-terminal sequencing of the trypsin-activated Cry7Aa fragments revealed that processing occurs at Glu(47) for the 70-kDa form or Ile(88) for the 65-kDa form. Homologous binding assays showed specific binding of the two Cry3 proteins and the 65-kDa Cry7Aa fragment to brush border membrane vesicles (BBMV) from C. puncticollis larvae. The 70-kDa fragment did not bind to BBMV. Heterologous-competition assays showed that Cry3Bb, Cry3Ca, and Cry7Aa (65-kDa fragment) competed for the same binding sites. Hence, our results suggest that pest resistance mediated by the alteration of a shared Cry receptor binding site might render all three Cry toxins ineffective.

Compatibility of mycorrhiza-induced resistance with viral and bacterial entomopathogens in the control of Spodoptera exigua in tomato.

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) are soil-borne microorganisms that establish mutualistic associations with roots of most terrestrial plants. This symbiosis results in nutritional and defensive benefits to the host plant, usually conferring protection against biotic stresses, but its indirect impact on third trophic levels is still unknown. In the present work, we explore whether the symbiosis of tomato plants with Funneliformis mosseae (and/or exposition to herbivory) influences the interaction of the generalist pest Spodoptera exigua (Lepidoptera: Noctuidae) with bacterial (Bacillus thuringiensis) and viral (baculovirus, SeMNPV) natural entomopathogens. RESULTS: Symbiosis with AMF and previous herbivory reduces the relative growth of S. exigua, increases its susceptibility to a sublethal dose of B. thuringiensis and has positive or neutral impact on the lethality of SeMNPV. Reduction of the phenoloxidase activity, a marker of the insect immune response, was associated with the larval feeding on plant material previously exposed to herbivory but not to the AMF. In addition, no changes in the insect gut microbiota could be associated with the observed changes in larval growth and susceptibility to the entomopathogens. CONCLUSION: Our findings provide the first evidence of compatibility of AMF symbiosis in tomato with the use of bacterial and viral entomopathogens, contributing to the development of novel approaches to combine the beneficial effect of AMF and entomopathogens in biological pest control. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Corrigendum: Influence of Diet, Sex, and Viral Infections on the Gut Microbiota Composition of Spodoptera exigua Caterpillars.

[This corrects the article DOI: 10.3389/fmicb.2020.00753.].

Influence of Diet, Sex, and Viral Infections on the Gut Microbiota Composition of Spodoptera exigua Caterpillars.

The gut microbiota plays essential roles in processes related with metabolism, physiology, and immunity in all organisms, including insects. In the present work, we performed a broad analysis of the Spodoptera exigua gut microbiota, a major agricultural pest. We analyzed the influence of multiple parameters such as diet, geographic location, sex, or viral infections on S. exigua caterpillar gut microbiota composition. Our study revealed a high variability in bacterial composition among individuals, and a major influence of environmental bacteria (including those acquired through diet) on the gut microbiota composition, supporting previous studies that claim resident microbiota are lacking in caterpillars. Previous studies with laboratory-reared insects showed that changes in caterpillar gut bacterial composition affect the insecticidal properties of entomopathogenic viruses and bacteria. Our study revealed different microbiota composition in field insects carrying a natural viral infection with Spodoptera exigua nucleopolyhedrovirus (SeMNPV) and/or Spodoptera exigua iflavirus 1 (SeIV1). Few taxa can be specifically associated with the infection, suggesting microbiota influence the infective process of these natural pathogens, and providing new strategies for insect pest management.

The Spodoptera exigua ABCC2 Acts as a Cry1A Receptor Independently of its Nucleotide Binding Domain II.

ABC proteins are primary-active transporters that require the binding and hydrolysis of ATP to transport substrates across the membrane. Since the first report of an ABCC2 transporter as receptor of Cry1A toxins, the number of ABC transporters known to be involved in the mode of action of Cry toxins has increased. In Spodoptera exigua, a mutation in the SeABCC2 gene is described as genetically linked to resistance to the Bt-product XentariTM. This mutation affects an intracellular domain involved in ATP binding, but not the extracellular loops. We analyzed whether this mutation affects the role of the SeABCC2 as a functional receptor to Cry1A toxins. The results show that Sf21 cells expressing the truncated form of the transporter were susceptible to Cry1A toxins. Moreover, specific Cry1Ac binding was observed in those cells expressing the truncated SeABCC2. Additionally, no differences in the irreversible Cry1Ac binding component (associated with the toxin insertion into the membrane) were observed when tested in Sf21 cells expressing either the full-length or the truncated form of the SeABCC2 transporter. Therefore, our results point out that the partial lack of the nucleotide binding domain II in the truncated transporter does not affect its functionality as a Cry1A receptor.

Highly efficient production of rabies virus glycoprotein G ectodomain in Sf9 insect cells.

In the present study, we developed a complete process to produce in insect cells a high amount of the ectodomain of rabies virus glycoprotein G (GE) as suitable antigen for detecting anti-rabies antibodies. Using the baculovirus expression vector system in Sf9 insect cells combined with a novel chimeric promoter (polh-pSeL), the expression level reached a yield of 4.1 ± 0.3 mg/L culture, which was significantly higher than that achieved with the standard polh promoter alone. The protein was recovered from the cell lysates and easily purified in only one step by metal ion affinity chromatography, with a yield of 95% and a purity of 87%. Finally, GE was successfully used in an assay to detect specific antibodies in serum samples derived from rabies-vaccinated animals. The efficient strategy developed in this work is an interesting method to produce high amounts of this glycoprotein.

Role of Bacillus thuringiensis Cry1A toxins domains in the binding to the ABCC2 receptor from Spodoptera exigua.

Cry proteins from Bacillus thuringiensis (Bt) have been used to control insect pests either as formulated sprays or as in Bt-crops. However, field-evolved resistance to Bt proteins is threatening the long-term use of Bt products. The SeABCC2 locus has been genetically linked to resistance to a Bt bioinsecticide (Xentari™) in Spodoptera exigua (a mutation producing a truncated form of the transporter lacking an ATP binding domain was found in the resistant insects). Here, we investigated the role of SeABCC2 in the mode of action of Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and two Cry1A-1Ca hybrids by expressing the receptor in Sf21 and HEK293T cell lines. Cell toxicity assays showed that Sf21 cells expressing SeABCC2 become susceptible to Cry1A proteins. HEK293T cells expressing the transporter were found susceptible to Cry1A proteins but not to Cry1Ca. The results with the Cry1A-1Ca hybrids suggest that domain II from Cry1Ab/c is crucial for the toxicity to Sf21 cells, whereas domain III from Cry1Aa/b is crucial for the toxicity to HEK293T cells. Binding assays showed that the Cry1Ac binding is of high affinity and specific to cells expressing the SeABCC2 transporter. Heterologous competition experiments support a model in which domain II of Cry1Ab/c has a common binding site in the SeABCC2 protein, whereas domain III of Cry1Aa/b binds to a different binding site in the SeABCC2 protein.

A novel baculovirus-derived promoter with high activity in the baculovirus expression system.

The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.