The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs. To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens-mediated transformation. We generated PcBMM-GFP and Pc2127-GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T-DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM-GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers. By comparing the colonization of Arabidopsis wild-type plants and hypersusceptible (agb1-1 and cyp79B2cyp79B3) and resistant (irx1-6) mutants by PcBMM-GFP or Pc2127-GFP, we found that the plant immune response was already mounted at 12–18 h post-inoculation, and that Arabidopsis resistance to these fungi correlated with the time course of spore germination and hyphal growth on the leaf surface. The virulence of a subset of the PcBMM T-DNA insertional transformants was determined in Arabidopsis wild-type plants and agb1-1 mutant, and several transformants were identified that showed altered virulence in these genotypes in comparison with that of untransformed PcBMM. The T-DNA flanking regions in these fungal mutants were successfully sequenced, further supporting the utility of these functional genomics tools in the molecular characterization of the pathogenicity of necrotrophic fungi.