Project PTDC/BAA-AGR/30609/2017

We will focus particularly on three key points of the nematode-insect interaction:

(i) nematode resistance to insect immune responses; (ii) the effect of the effectors of the nematode parasitic process on the insect response, and

(iii) in the induction of apoptosis and histolysis caused by nematode effector molecules. The validation of the gene induction will be done by RT-PCR and by the analysis of the production of the virulence factors in question

The project aims to improve the effectiveness of an entomopathogenic nematode (EPN) against different insect pests by inducing the over-expression of virulence factors. EPN are insect pathogens widely used in the biological control of agricultural pests. Despite being safe, the widespread use of these organisms still faces some challenges, particularly related to virulence, which varies from one insect to another. This limitation is usually resolved by screening and testing new strains with the desired efficacy on the target insect. However, the search for new nematodes is laborious and time-consuming. Recent research based on comparative genomics and transcriptomics analysis of high and low virulent isolates for different insects points to some genes that are associated with virulence. These genes should be key targets to improve beneficial traits of these organisms. Our proposal aims to demonstrate that the overexpression of these genes will make it possible to obtain a more efficient nematode. We propose to develop this investigation based on functional genomics which, despite being frequently suggested, has not yet been applied to H. bacteriophora. Therefore, we will develop protocols for the application of the dCas9 system to the candidate gene pool.

The functional effect of the modification on the expression of each gene will be tested using Drosophila as model insects and in vitro assays. The transiently transformed nematode isolates will be tested against agricultural pests to identify the modification that confers an increase in efficacy that will be selected. The selected modification will be used to make a new nematode strain with constitutive overexpression of the selected gene based on the CRISPR-Cas9 system. The efficacy and safety against non-target plants and organisms of this new strain will be tested. The project is expected to obtain a protocol for the improvement of entomopathogenic nematodes based on the overexpression of known genes whose intellectual property is protected. The results will be disseminated to users of insecticides, farmers, for example, but also to entrepreneurs in science and technology parks and biotechnology students, motivating them to produce the new bioinsecticide.