Análise do processo da penetração à colonização de Fusarium decemcellulare em guaranazeiro e obtenção de mutantes para genes relacionados à patogenicidade por meio de CRISPR/Cas9

Abstract

Given the need to understand the mechanisms of interaction between Fusarium decemcellulare and the guarana plant, as well as to analyze genes that may play an important role in pathogenicity and/or virulence, this work aimed to analyze the pathogen-host interaction process through: 1- the establishment of efficient conditions for genetic transformation of F. decemcellulare (Fdc) and obtaining transformed strains expressing Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP) for use in the analysis of penetration and colonization; 2- identification of the form of colonization of Fdc in guarana plants to assist in the establishment of disease control methods; and 3- obtaining Fdc strains with knockouts for genes related to auxin production by the pathogen. In the results, it was shown that the genetic transformation method mediated by Agrobacterium tumefaciens (ATMT) did not allow the obtaining of Fdc transformants, while transformation via polyethylene glycol (PEG) had an average efficiency of three transformants per µg of DNA expressing fluorescent proteins GFP or RFP. These transformed strains were then used in studies on pathogen colonization in the plant, analyzed through confocal and scanning microscopy. The analyses showed that Fdc presents systemic colonization, both in tomato and guaraná plants, being isolated upstream and downstream of the inoculation point with 30 to 120 days in both plants, but requires injuries to succeed in the penetration process. It also has a high association with intact and ruptured trichomes in guaraná plants, with no preference for invasion through natural openings (such as stomata), for example. In this work, in addition to reports on the oversprouting complex, we are reporting for the first time in guarana plants symptoms that resemble those of the Dieback disease caused by F. decemcellulare. The established transformation method was also useful as a first step in the genome editing process using the CRISPR/Cas9 system in Fdc, which was evaluated for obtaining single or triple knockouts. 23 transformants were obtained, none of which showed double or triple knockouts, and for deletion of a single gene, eight transformants were obtained, three of which had a knockout for the aldh gene located on chromosome 9, four knockouts for the aldh gene on chromosome 2, and one for the yucca gene.