The project aims to provide an entomopathogenic nematode (EPN) with increased efficacy against different insect pests by promoting the overexpression of self-virulence factors. EPN are insect pathogens widely used in biological control of agricultural pests. Despite they are safe, the generalized use of these organisms face still a few challenges, particularly related to the virulence that is variable from an insect to another. This limitation is normally solved by searching and assaying new strains with desired efficacy in the target insect. However, the screen for new nematodes is laborious and time consuming. Recent works based in genomic and transcriptomic analysis of high and low virulent strains toward different insects point to a few genes that are associated to specific virulence. These genes must be the key targets to improve beneficial traits. Association analysis based in a library of transcripts of high and low virulent nematode strains in different insects, allowed to the identification of a set of putative genes related to virulence. Our proposal aims to identify the most determinant putative genes in the virulence and to demonstrate at what extend the engineering of these genes should allow for a more effective nematode. We propose to develop this investigation based in functional genomics approach that has been frequently suggested but not yet applied in H. bacteriophora. Therefore, we will develop protocols for the application of dCas9 system to the set of candidate genes. We will focus particularly in three key points of the interaction nematode-insect: i) the resistance of the nematode to the immune responses of the insect; ii) the effect of nematode effectors in the response of the insects and iii) in the induction of apoptosis and histolysis caused by nematode effectors. Validation of gene induction/repression will be done by RT-PCR. The functional effect in the parasitic process will be tested using Drosophila has model insect and in vitro assays. The functional analysis will allow the identification of a functional map in which genes really targeting virulence will be identified. Genes which functionality will be proved to be more hazardous to Drosophila will be evaluated as bioinsecticide against agricultural pests by performing a nematode with constitutive overexpression of the selected gene based in CRISPR-Cas9 system and testing its efficacy against lepidopteran, coleopteran and dipteran pests. The project is expected to get for a protocol for the improvement of nematodes in which useful genes and procedures to improve nematodes will be protected. Dissemination of the results will be done near end users of insecticides (agricultures and farmers) but also near entrepreneurs in science and technology parks and students of biotechnologies, motivating people to produce the new bioinsecticide.
FCT - Fundação para a Ciencia e Tecnologia