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In order to monitor Rosellinia necatrix infection of avocado roots, we generated a plasmid vector (pCPXHY1eGFP) constitutively expressing EGFP and developed a protoplast transformation protocol. Using this protocol, four R. necatrix isolates were efficiently transformed and were shown to stably express EGFP homogeneously while not having any observable effect on pathogenicity. Confocal laser scanning microscopy (CLSM) images of avocado roots infected with the highly virulent isolate CH53-GFP demonstrated that fungal penetration of avocado roots occurs simultaneously at several random sites, but it occurs preferentially in the crown region as well as throughout the lenticels and in the junctions between epidermal cells. Not only were R. necatrix hyphae observed invading the epidermal and cortical root cells, but they were also able to penetrate the primary and secondary xylem. Scanning electron …