Silenciamento do gene SGE1 por meio de recombinação homóloga e RNA de interferência em Fusarium oxysporum f. sp. cubense

Abstract

Fusarium oxysporum f. sp. cubense (Foc) is a filamentous fungus causal agent of Panama disease. The four pathogenic races of banana (R1, R2, TR4 and STR4) are capable of generating large socioeconomic losses to banana plantations sector in climates Tropical and Subtropical. In Brazil and abroad the banana crop shows economic growth and the spread of the disease contributes to the considerable reduction in the productivity of most banana cultivars. Information about which genes or components of metabolic pathways which act effectively in pathogenicity contribute to the development of various disease control strategies, such as breeding for resistance of the host, of inducing resistance to fungi by means of strains subjected to muting or silencing of gene-specific only reaches the pathogen, without prejudice to other beneficial microorganisms plant. Thus, this study aimed to obtain transformants through homologous recombination (knockout) and RNA interference (knockdown), and analyze them as the knockout and / or silencing of the gene encoding the transcription factor Sge1 in F. oxysporum f. sp. cubense. For this purpose, cassettes of different constructions were used for homologous recombination, one of the tape only had a checkmark concerning the hph gene and the other had the hph gene and the reporter gene GFP. For RNA interference, we used a tape with sense and antisense sequences of the SGE1 gene. Obtaining the transformants for analysis was possible in both methodologies "knockout and knockdown". But in the first part of the work was to analyze the occurrence of knockout did not identify any strain with homologous recombination in the target region (SGE1) because all transformants analyzed showed ectopic integration and transformants with cassette integration hph/gfp perfectly issued the green fluorescence. Taking into consideration the difficulties for the occurrence of homologous recombination, it is necessary observations in some ways. In the second part of this work we were isolated 120 transformants integration of the cassette for silencing via RNA interfência, in which only 13 were selected for analysis, and presented different morphological characteristics like radial size of the colonies, pigmentation and sporulation capacity, and Different relative expression levels observed before and after the mitotic stability test, of these, three (T2-30%; T4-27% e T7-47%) showed significant reduction in levels of relative expression of transcripts of the SGE1 gene indicating gene silencing. In the analysis of silencing by the pathogenicity test the selected transformants (T4 and T11), have achieved the rhizome and pseudostem of banana plants inoculated, but when compared to the wild type F. oxysporum cubense they managed to induce the symptoms of the disease in banana more slowly. Given these results, we can conclude that the RNAi mechanism is present and active in F. oxysporum cubense, proving to be an effective technique, opening new avenues for the development of mechanisms that block or reduce the pathogen action as well as functionally explore genomic data.

Keywords: gene silencing, F. oxysporum f. sp. cubense, SGE1.