Densoviruses are orally infectious and lethal to insects of various orders. They are considered promising candidates, as an alternative to chemical insecticides, for the control of insect crop pests and disease vectors. Their use requires a prior in-depth understanding of viral pathogenesis and the mechanisms of their specificity. As with all orally infectious viruses, the key step that determines the initiation and success of host infection is the crossing of the intestinal barrier, although the mechanisms involved remain poorly understood. For most insects, this barrier consists of a peritrophic matrix and a monolayer epithelium, which it protects. This peritrophic matrix consists of a network of chitin fibrils associated with highly glycosylated proteins called peritrophins. The intestinal epithelium is covered by a layer of glycans, called glycocalyx, and the epithelial cells are joined by so-called "septate" junctions that ensure the impermeability of the tissue. In insect pests, the fine structure and extensive biochemical composition of the components of the intestinal barrier are not characterized, and to date, no densovirus receptors are known. The DECIDE project aims to decipher the specific molecular mechanisms used by densoviruses to cross the intestinal barrier in insects. We hypothesize that viral particles cross this barrier in two steps, first by interacting with certain glycans in the peritrophic matrix and/or intestinal epithelium, and then by accessing more specific membrane receptors that allow them to infect intestinal cells and invade the body. To test this two-step infection hypothesis, we will use as interaction models a densovirus type, the densovirus Junonia coenia (JcDV) and crop pests belonging to the genus Spodoptera (S. frugiperda and S. littoralis). Densovirus-intestinal interactions will also be evaluated in non-target species, Bombyx mori and Vanessa cardui. This project will decipher the mechanisms of interaction of the virus with the peritrophic matrix and intestinal epithelium and the consequences of these interactions on intestinal physiology. This project has four main objectives: i) To perform a complete analysis of the glycan and protein composition of the peritrophic matrix and the brush border of the intestinal epithelium; ii) To identify, among these, the glycans and/or proteins that interact with the viral particles and allow them to cross the intestinal barrier as well as a precise dissection of the capsid to identify the residues involved in these interactions; iii) Analysing the potential enzymatic activity of the capsid allowing it to cross the peritrophic matrix, as well as the molecular mechanisms leading to the increase in intestinal permeability following oral infection. Finally, iv) this project will explore a "capsid strategy" based on "natural" or synthetic pseudo-particles (VLPs) as biocontrol tools. This project will address virology, insect gut physiology, cellular biology and glycobiology issues in lepidopteran pest models, combining a wide range of methods including mainly glycomic, proteomic, high resolution imaging and synthetic biology approaches. This original project provides a conceptual and experimental framework to study the impact of the use of entomopathogenic agents in biological control of insect pests or vectors. It could lead to the development of new strategies, based on the use of densoviruses, that specifically target the gut of insects.

Relevance of the project to the specific call: The ongoing process of global warming is causing dramatic changes in environmental conditions worldwide. Predictions show that many regions will become more arid, which will impact on local crop cultivation. In many Mediterranean Countries grapevine is one of the most economically and culturally important crop. As a woody perennial plant, grapevine is particularly sensitive to environmental stresses. Novel and sustainable strategies are urgently needed to maintain grapevine productivity in future climatic conditions. Objective of the project. In the last decade, microbiomes have surged as important players in the physiology of many biological systems. In this project, we aim at exploiting the natural endophytes biodiversity existing in grapevines cultivated in regions of the Mediterranean characterized by arid conditions, to investigate possible application to ameliorate grapevine resilience to drought stress. Methodology. Microbial endophyte consortia conferring the highest resistance to drought stress in traditional grapevine cultivars will be selected under controlled conditions and applied to sanitized grapevine plants, either by grafting or direct inoculation of culturable fraction Plant response will be characterized at physiological and molecular levels by a multidisciplinary approach to determine the mechanisms underlying the modulation of the grapevine response to drought due to microbiomes. Transcriptome, methylome and metabolome analysis will be correlated to the physiological characterization of plants, including optogenetical measurements of the stress hormone Abscisic Acid, Ca2+ homeostasis, photosynthesis, and stomatal conductance. Beneficial microbial associations will be identified by means of metagenomic approaches. By the end of the project, we will be able to offer new technological procedures (double grafting or direct inoculation) to produce commercial material enriched in beneficial endophytes that can be safely used in the field. The project outcome will be the identification of beneficial microbial consortia to be used in environmentally friendly agricultural practices thus increasing the sustainability of the system as a major challenge to face and overcome the problem of increasing water scarcity.