The soil borne fungus, Fusarium virguliforme, causes sudden death syndrome (SDS) in soybean, which is a serious foliar and root rot disease. The pathogen has never been isolated from the diseased foliar tissues; phytotoxins produced by the pathogen are believed to cause foliar SDS symptoms. One of these toxins, a 13.5-kDa acidic protein named FvTox1, has been hypothesized to interfere with photosynthesis in infected soybean plants and cause foliar SDS. The objective of this study is to determine if FvTox1 is involved in foliar SDS development. We created and studied five independent knockout fvtox1 mutants to study the function of FvTox1. We conducted Agrobacterium tumefaciens-mediated transformation to accomplish homologous recombination of FvTox1 with a hygromycin B resistance gene, hph, to generate the fvtox1 mutants. Approximately 40 hygromycin-resistant transformants were obtained from 10(6) conidial spores of the F. virguliforme Mont-1 isolate when the spores were co-cultivated with the A. tumefaciens EHA105 but not with LBA4044 strain carrying a recombinant binary plasmid, in which the hph gene encoding hygromycin resistance was flanked by 5'- and 3'-end FvTox1 sequences. We observed homologous recombination-mediated integration of hph into the FvTox1 locus among five independent fvtox1 mutants. In stem-cutting assays using cut soybean seedlings fed with cell-free F. virguliforme culture filtrates, the knockout fvtox1 mutants caused chlorophyll losses and foliar SDS symptoms, which were over twofold less than those caused by the virulent F. virguliforme Mont-1 isolate. Similarly, in root inoculation assays, more than a twofold reduction in foliar SDS development and chlorophyll losses was observed among the seedlings infected with the fvtox1 mutants as compared to the seedlings infected with the wild-type Mont-1 isolate. These results suggest that FvTox1 is a major virulence factor involved in foliar SDS development in soybean. It is expected that interference of the function of this toxin in transgenic soybean plants will lead to generation of SDS-resistant soybean cultivars.