Bracovirus derived genes in the genome of Spodoptera exigua Hubner (Lepidoptera: Noctuidae) and their role in host susceptibility to pathogens

La asociación entre los himenópteros parasitoides, polydnavirus (PDV) y lepidópteros representa un modelo interesante para estudiar la transferencia horizontal de genes. Está bien documentado que miles de himenópteros parasitoides pertenecientes a las familias Braconidae e Ichneumonidae han domesticado virus simbióticos denominados respectivamente Bracovirus o Ichnovirus. El virus se inyecta junto con los huevos del parásito en el hemocele del lepidóptero huésped, donde se expresan proteínas específicas. Estas proteínas inhiben el sistema inmune del lepidóptero y detienen su desarrollo, lo que beneficia el desarrollo de los huevos y luego las larvas del parasitoide. En este sistema único, tres genomas están continuamente en contacto. Como resultado, este contacto puede facilitar la transferencia de genes entre los tres genomas. En este contexto, el transcriptoma del insecto plaga Spodoptera exigua reveló la presencia de un número de unigenes que codifican para proteínas altamente homólogas a proteínas de bracovirus. Estas proteínas son en su mayoría miembros de la familia de lectinas tipo C, excepto uno que codifica una proteína perteneciente a la familia BV2 de Cotesia congregata bracovirus, BV2-5. El análisis de las secuencias genómicas de BV2-5 y BLL2 (un miembro de las lectinas homólogas a lectinas de bracovirus, BLLs) confirmó el origen vírico de estos genes. Análisis adicionales de las secuencias disponibles en varias bases de datos mostró la presencia de estos genes virales en otras especies de Spodoptera. Dicha presencia sugiere eventos de integración antiguos que se produjeron en una especie ancestral de Spodoptera. Estos genes se expresan de forma constitutiva en diferentes tejidos, pero principalmente en los hemocitos, lo que sugiere que tienen un papel en la respuesta inmune del insecto. Sin embargo, uno puede preguntarse si la integración de los genes virales en la línea germinal del lepidóptero es el resultado de una estrategia del sistema virus/parasitoide para manipular el huésped o es el insecto que ha domesticado los genes virales con el fin de mejorar su inmunidad. Para responder a tal dilema y como parte de esta tesis, nos decidimos ir más allá en la investigación del papel de estos genes en la interacción insecto-patógeno. En primer lugar, hemos descifrado la función de BV2-5 (Gasmin). Para ello se construyeron varios baculovirus recombinantes expresando distintas formas de esta proteína. Observaciones mediante microscopia confocal demostraron que esta proteína es capaz de interactuar con la actina celular interrumpiendo su organización. Esta interrupción implica una drástica reducción de la multiplicación y la producción de partículas de baculovirus en condiciones de cultivo celular. Este resultado sugiere que el insecto ha domesticado este gen con el fin de hacer frente a uno de sus principales patógeno en la naturaleza: baculovirus. Por otro lado, la interrupción de la organización de la actina resultó en una disminución de la capacidad de los hemocitos para fagocitar las bacterias y, en consecuencia, resulta en un aumento en la susceptibilidad de las larvas a infecciones bacterianas. Por otra parte, se detectó una forma trucada no funcional de Gasmin en las poblaciones europeas de S. exigua. Este hecho, así como el papel divergente frente a diferentes patógenos, reflejan una posible adaptación al tipo de patógeno que es m ás abundante en cada localización geográfica. A continuación nos propusimos investigar el papel inmunológico de las lectinas homólogas a bracovirus (Se-BLLs) en la respuesta inmune del insecto. De hecho, en el transcriptoma de S. exigua se detectó la presencia de ,al menos, treinta y dos unigenes que codifican proteínas de tipo lectinas, la mayoría perteneciente a la familia de lectinas de tipo C. Debido a la gran cantidad de estas proteínas, cuatro lectinas de lepidópteros (Se-LLs), además de la Se-BLLs han sido estudiadas en detalle. El estudio detallado de los dos grupos de lectinas reveló diferencias en distintos aspectos, como son la estructura de las proteínas, su distribución en los tejidos del insecto y su respuesta transcripcional a diferentes patógenos viricos y bacterianos así como endoparasitoides. Entre las lectinas estudiadas, Se-BLL2 respondió a todos los patógenos probados incluyendo la infección por baculovirus, lo que motivó su estudio en detalle a través de su estudio funcional. Para ello se produjo de manera recombinante dicha proteína, y se comprobó que su administración conjunta con baculovirus disminuía la susceptibilidad de los insectos a baculovirus. Este resultado sugiere, nuevamente, que el insecto ha domesticado este gene de origen bracoviral con el fin de hacer frente a baculovirus. En resumen, en este estudio hemos descrito la inserción de ADN bracoviral en un genoma de lepidóptero y su posterior domesticación con el objetivo probable de proporcionar protección frente a un patógeno importante como es baculovirus.The association between parasitic hymenopteran, polydnavirus (PDVs) and lepidopteran host represent an interesting model to study the horizontal transfer of genes. It is well documented that thousands of hymenopteran parasitoids belonging to the families Braconidae and Ichneumonidae have domesticated symbiotic viruses called respectively Bracovirus or Ichnovirus. The virus is injected together with the parasitoid eggs into the hemocoel of the lepidopteran host, where it expresses specific proteins. These proteins immunocompromise the lepidopteran host and arrest its development making the development of the parasitoid eggs and then larvae successful. In this unique system, three genomes are continuously in contact which can facilitate gene transfer between them. In this context, the transcriptome of the beet armyworm Spodoptera exigua revealed the presence of a number of unigenes encoding proteins highly homologous to bracovirus proteins. All of them are members of the C-type family of lectins, except one unigene that codify for a protein belonging to the Cotesia congregata bracovirus protein family BV2, with high similarity to the viral protein BV2-5. Genomic sequence analysis of the BV2-5 and the BLL2 (a bracovirus-homolog lectin in S. exigua), confirmed the bracoviral origin of these genes. Further analysis of the available sequences showed the presence of these genes in other Spodoptera species suggesting ancient integration events in an ancestral Spodoptera species. Those genes are sustainably expressed in different tissues, but mainly in hemocytes, which suggests an immune role of them. However, one can wonder if the germ line integration of such viral genes is a strategy of the virus/parasitoid system to manipulate the host or it is the insect who has domesticated the viral genes in order to improve its immunity. In order to answer such dilemma and as part of this thesis, we decided to go further in the investigation of functional role of these genes in the insect-pathogen interaction. First, we have deciphered the function of the BV2-5 homolog, Gasmin. For that purpose, multiple recombinant baculoviruses, expressing different forms of Gasmin, where prepared and analysed. Confocal microscopy observations showed that this protein is able to interact with the cellular actin, disrupting its arrangement. This disruption leads to a drastic reduction of the multiplication and the production of baculovirus particles in cell culture experiments. This result suggests gene domestication in order to face such type of pathogen. On another hand, the disruption of actin arrangement resulted in a decrease in the capacity of host hemocytes to phagocytise bacteria, a fact that was traduced with an increasing in larval susceptibility to bacterial infection. Moreover, a non-functional truncated form of Gasmin was detected in European populations. This fact as well as the divergent role in response to different pathogens, reflect possible adaptation to the type of pathogen that is most abundant in each geographic localization. Next, we have also investigated the immune role of the S. exigua bracovirus-like lectins (Se-BLLs) on the insect immune response. Indeed, the insect transcriptome revealed the presence of at least thirty-two unigenes encoding lectin-like proteins, the majority belonging to the family of C-type lectins. Due to the large number of these proteins, four lepidopteran-like lectins (Se-LLs) in addition to the Se-BLLs have been studied in detail. Detailed study of the two groups of lectins revealed differences at different levels, such as protein structure, tissue distribution and the transcriptional response to different viral and bacterial pathogens and endoparasitoids. Among the studied lectins, Se-BLL2 responded to all the tested pathogens including baculovirus infection and its functional role was further investigated through its recombinant expression, purification and functional analysis. The administration of this protein together with the baculovirus to S. exigua larvae increased the resistance of the larvae to the baculovirus infection. Again, this result suggests an additional case of gene domestication in order to face baculovirus infection. In summary, in this study we have described the insertion of bracoviral DNA into a lepidopteran genome and its further domestication probably to provide protection against an important pathogen such as baculovirus

L-Ilf3 and L-NF90 Traffic to the Nucleolus Granular Component: Alternatively-Spliced Exon 3 Encodes a Nucleolar Localization Motif

Ilf3 and NF90, two proteins containing double-stranded RNA-binding domains, are generated by alternative splicing and involved in several functions. Their heterogeneity results from posttranscriptional and posttranslational modifications. Alternative splicing of exon 3, coding for a 13 aa N-terminal motif, generates for each protein a long and short isoforms. Subcellular fractionation and localization of recombinant proteins showed that this motif acts as a nucleolar localization signal. Deletion and substitution mutants identified four arginines, essential for nucleolar targeting, and three histidines to stabilize the proteins within the nucleolus. The short isoforms are never found in the nucleoli, whereas the long isoforms are present in the nucleoplasm and the nucleoli. For Ilf3, only the posttranslationally-unmodified long isoform is nucleolar, suggesting that this nucleolar targeting is abrogated by posttranslational modifications. Confocal microscopy and FRAP experiments have shown that the long Ilf3 isoform localizes to the granular component of the nucleolus, and that L-Ilf3 and L-NF90 exchange rapidly between nucleoli. The presence of this 13 aminoacid motif, combined with posttranslational modifications, is responsible for the differences in Ilf3 and NF90 isoforms subcellular localizations. The protein polymorphism of Ilf3/NF90 and the various subcellular localizations of their isoforms may partially explain the various functions previously reported for these proteins

Endogenous viral elements in mosquito genomes: current knowledge and outstanding questions

Integrations from non-retroviral RNA viruses (nrEVEs) have been identified across several taxa, including mosquitoes. Amongst all Culicinae species, the viral vectors Aedes aegypti and Aedes albopictus stand out for their high number of nrEVEs. In addition, Aedes nrEVEs are enriched in piRNA clusters and generate piRNAs that can silence incoming viral genomes. As such, nrEVEs represent a new form of inherited antiviral immunity. To propel this discovery into novel transmission-blocking vector control strategies, a deeper understanding of nrEVE biology and evolution is essential because differences in the landscape of nrEVEs have been identified in wild-caught mosquitoes, the piRNA profile of nrEVEs is not homogeneous and nrEVEs outside piRNA clusters exist and are expressed at the mRNA level. Here we summarise current knowledge on nrEVEs in mosquitoes and we point out the many unanswered questions and potentials of these genomic elements

Characterization of two groups of Spodoptera exigua Hübner (Lepidoptera: Noctuidae) C-type lectins and insights into their role in defense against the densovirus JcDV

Insect innate immunity relies on numerous soluble and membrane-bound receptors, named pattern recognition proteins (PRPs), which enable the insect to recognize pathogen-associated molecular patterns. C-type lectins are among the best-studied PRPs and constitute the most diverse family of animal lectins. Here we have characterized two groups of Spodoptera exigua C-type lectins that differ in their phylogeny, domain architecture, and expression pattern. One group includes C-type lectins with similar characteristics to other lepidopteran lectins, and a second group includes bracoviral-related lectins (bracovirus-like lectins, Se-BLLs) recently acquired by horizontal gene transfer. Subsequently, we have investigated the potential role of some selected lectins in the susceptibility to Junonia coenia densovirus (JcDV). For this purpose, three of the bracoviral-related lectins were expressed, purified, and their effect on the densovirus infection to two different Spodoptera species was assessed. The results showed that Se-BLL3 specifically reduce the mortality of Spodoptera frugiperda larvae caused by JcDV. In contrast, no such effect was observed with S. exigua larvae. In a previous work, we have also shown that Se-BLL2 increased the tolerance of S. exigua larvae to baculovirus infection. Taken together, these results confirm the implication of two different C-type lectins in antiviral response and reflect the biological relevance of the acquisition of bracoviral genes in Spodoptera spp

Can herbivore-induced volatiles protect plants by increasing the herbivores’ susceptibility to natural pathogens?

In response to insect herbivory, plants mobilize various defences. Defence 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. Exposing caterpillars of the noctuid Spodoptera exigua to indole and linalool, but not exposure to (Z)-3-hexenyl acetate increased the susceptibility to its 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 insects’ gut microbiota. These findings suggest a novel mechanism by which HIPVs can protect plants from herbivorous insects. Importance Multitrophic interactions involving insect pest, 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 defences that aim to mitigate the damage. Attacked plants release herbivore-induced plant volatiles (HIPVs), which can act as priming signals for other plants, attract herbivore natural enemies and 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 on the plant environment. These findings suggest a novel role for plant volatiles by influencing the insect interaction 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 entomopathogen

Subcellular localization of human eRF1 fused with short or long N-terminal sequence of Ilf3/NF90.

<p>Plasmids pCMV-heRF1-Ilf3/NF90 short N-terminal sequence (N-heRF1, mid panels) and pCMV-heRF1-Ilf3/NF90 long N-terminal sequence (NoLS-heRF1, lower panels) were transfected into HeLa cells. After 24 hours, untransfected (Control, upper panels) or transfected cells were co-stained with anti-heRF1 antibody (heRF1), anti-α-tubulin antibody (α-Tub) and DAPI. Endogenous heRF1 or heRF1 recombinant fusion proteins appear in green, α-tubulin in red and DAPI in blue. Arrows point to intranuclear foci corresponding to nucleoli.</p

Occurrence frequencies (%) of possible NoLS sequences (K/R-K/R-X-K/R) and of the twenty aminoacids at the X position in several NoLS-containing nucleolar proteins.

a<p>Among the eight possible sequence combinations (upper line), observed frequency (OF) of each aminoacid at the X position in NoLS present in 61 well-characterized nucleolar proteins and using for the analysis.</p>b<p>Mean frequency (MF) of each aminoacid observed in vertebrate proteins.</p