Resumo

The entomopathogenic filamentous fungus Metarhizium anisopliae infects and kills its hosts with great efficiency having relevance for its use in biological control. The fungus also has been used as a model for pathogen-host interactions and the main focus of research is the infection cycle. The infection cycle has well-delineated steps: conidia adhesion to the exoskeleton host, conidia germination, germ-tube differentiation into apressoria, cuticle penetration, differentiation into blastospores – hyphal bodies, host colonization, extrusion to the host cadaver surface and conidiophores and conidia production on the host cadaver. One of the most important steps in the infection cycle is the cuticle penetration since it will define the establishment or not of a productive infection. The cuticle is composed mainly by chitin fibrils in a lipo-proteic matrix and the successful penetration depends on mechanical pressure, generated by appresoria turgor pressure, and hydrolitic enzymes secretion, to hydrolyze cuticle components. One class of such enzymes is the chitinases, involved not only in the penetration mechanism but also in fungus morphogenesis and nutrition. A genome survey revealed that M. anisopliae strain E6 has 23 putative chitinases. In order to study the function of these enzymes four chitinase genes ChiMaA1, ChiMaA7, ChiMaB5 e ChiMaB7 have been selected to generate null mutants based on previous transcriptional profiles. To generate this cassettes, fusion PCR was employed, where 5’ and 3’ flanking regions from the specific targets were fuse to the gene BAR, which confer resistance to glufosinate, and the final amplicon generated the functional selective marker BAR flanked by the 5’ and 3’ regions of each target gene. The final disrupted constructions were cloned into pCR2.1 - TOPO vector. The constructions will be used on microprojectile bombardment experiments in order to generate mutant strains for these genes in M. anisopliae. As perspective, phenotypic analysis, bioassays as well as transcriptional analysis will be performed to elucidate the function of these genes. Financial Support: CNPq, CAPES and FAPERGS.