Abstracto
- Two Fol strains (named Fol59 and Fol60) out of 119 fungal isolates obtained from wilted tomato plants collected in the Colombian Andean Region, were identi ed as Fol race 2 by PCR ampli cation of SIX1, SIX3 and SIX4 e ector genes and sequencing analysis of SIX3 (Figure 1). In this study, two transformation constructs were generated in the pPK2HPH backbone vector (provided by Professor Martijn Rep, University of Amsterdam), in order to knock out SIX1 and SIX3 e ectors by homologous recombination with a hyg cassette (Figure 2). Furthermore, two vectors containing 3 cassettes consisting of single guide RNAs of either SIX7 or SIX12, driven by the U6 promoter, the hyg cassette from the pPK2HPH vector and the fungal codon-optimized Cas9 endonuclease driven by the gpd promoter were transformed into Agrobacterium tumefaciens LBA4404 to initiate e ector gene editing studies (Figure 3). An additional vector containing a GFP cassette with the SIX1 promoter and terminator regions, were used to transform Fol59 (Figure 4), for further analysis of the disease progress in the xylem. Pathogenicity tests plants using monosporic cultures from six Fol59 mutant strains, carrying the GFP construct were performed on susceptible tomato plants, in order to test whether the mutation compromised the virulence of Fol59 wild type (Figure 5).