Resumo

RNA interference (RNAi) is a post-transcriptional silencing mechanism in which small RNA molecules interfere with gene expression through targeted degradation of mRNA or through the suppression of translation. For this reason this mechanism has been used as genomic functional analysis tool. Our purpose is to test RNAi mechanism in Metarhizium anisopliae by silencing subgroup C (sgC) chitinase genes. The entomopathogenic fungus M. anisopliae is a model organism to examine host-pathogen interactions and the chitinases have been shown to be involved in chitin hydrolysis, the most important component of arthropod exoskeleton. Furthermore, the subgroup C chitinases are unique to filamentous fungi. First, we performed a bioinformatic analysis and RNAi proteins were searched in the genome of M. anisopliae strain E6 using M. acridum, M.robertsii, Neurospora crassa, Cordyceps militaris and Beauveria bassiana amino acid sequences as query. It was possible to detect orthologs for all proteins involved in this biological process on M.anisopliae genome. To investigate the functionality of RNA interference in our model, we constructed a vector with dual promoter in opposite direction (pgpd and ptrpc from A. nidulans) based on pPZPbar which contained a selectable marker, the bar gene (phosphinothricin acetyltransferase gene), generating the pPZPBDP. To analyze the function of sgC chitinases chiMaC1, chiMaC2, chiMaC3 and chiMaC4, the genes regions were amplified by PCR, generating four fragments of 267 bp, 203 bp, 202 bp and 201 bp, respectively. The vector pPZPBDP were digested with EcoRV and the four overlapping fragments, were fusioned using In-Fusion^® EcoDry^TM Cloning Kit (Clontech) and transformed in E. coli strain OmniMaxTM (Invitrogen) chemically competent cells. The vector generated pPZPBDPC was transformed into Agrobacterium tumefaciens strain EHA105, and Agrobacterium-mediated transformation is being performed. As a control experiment, an RNAi vector to silence ade2 gene is under development, in which a 250 bp fragment of the gene was amplified and cloned into the vector pPZPBDP. Disruption of ade2 gene is non-lethal and the colony color selection method is effective during screening of adenine auxotrophic mutants. The efficiency of the RNAi method will be performed by reverse transcription quantitative PCR analysis from the obtained mutants. It will also be carried out growth assays, cell differentiation and virulence.