Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker

The fungus Fusarium oxysporum f. sp. radicis-lycopersici is the causal agent of tomato foot and root rot disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Transformation of F oxysporum f. sp. radicis-lycopersici was very efficient and gfp expression was stable for at least nine subcultures. Microscopic analysis of the transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the chlamydospores and conidia. To our knowledge, this is the first report in which this is shown. The transformation did not affect the pathogenicity. Using confocal laser scanning microscopy, colonization, infection, and disease development on tomato roots were visualized in detail and several new aspects of these processes were observed, such as (i) the complete colonization pattern of the tomato root system; (ii) the very first steps of contact between the fungus and the host, which takes place at the root hair zone by mingling and by the attachment of hyphae to the root hairs; (iii) the preferential colonization sites on the root surface, which are the grooves along the junctions of the epidermal cells; and (iv) the absence of specific infection sites, such as sites of emergence of secondary roots, root tips, or wounded tissue, and the absence of specific infection structures, such as appressoria. The results of this work prove that the use of GFP as a marker for F. oxysporum f. sp. radicis-lycopersici is a convenient, fast, and effective approach for studying plant-fungus interactions.