Development of a system in Fusarium oxysporum for the search of new antifungals


M. Carmen Limon 1 José Carlos Castilla-Alcantara 1 Belen Muñoz-Mayoral 1 Cees A.M.M. van den Hondel 2
1Genetics, University of Seville, Seville, Spain
2Molecular Microbiology & Biotechnology, Institute of Biology, Leiden, Netherlands

Fusarium species are well-known plant pathogens that produce a big array of secondary metabolites including mycotoxins that produce diseases in animals fed with contaminated cereal grains. Superficial human infections such as keratitis and onychomycosis are commonly provoked by Fusarium. Fusarium invasive infections are mainly found in immunocompromised patients. A molecular strategy originally designed to screen cell wall mutants of Aspergillus niger has been used to detect induction of cell wall integrity (CWI) pathway in Fusarium oxysporum. The method consists in the detection of expression of a reporter gene under control of a CWI pathway-responding promoter. F. oxysporum was transformed with a mluc reporter gene under control of Aspergillus agsA promoter (PagsA::mluc). The promoter was previously engineered with 3 boxes for the transcription factor RlmA and luminescence was measured in transformant SX76 with luciferin after addition of calcofluor white (CFW). Although results showed that it is functional in F. oxysporum, the system should be optimized. Promoter sequences were analyzed to find canonical Rlm1 boxes that could be used for an improved cassette. Promoters were selected based on data of upregulated genes detected in RNA-seq of wild-type A. fumigatus treated with CFW or detected in microarray of samples treated with Congo red in comparison to non-treated Aspergillus (Rocha et al, 2008). The up-regulated candidate genes should also be uninduced in deltaRlmA mutants. Expression of three selected genes was studied by RT-PCR in F. oxysporum treated with CFW at different times and compared to non-treated mycelia. Our current data are very promising for optimization of this reporter system in Fusarium.

Reference

Rocha CM et al (2016). Aspergillus fumigatus MADS-Box transcription factor rlmA is required for regulation of the cell wall integrity and virulence. G3: Genes, Genomes, Genetics 6: 2983-3002.