Investigation of the Fusarium virguliforme fvtox1 mutants revealed that the FvTox1 toxin is involved in foliar sudden death syndrome development in soybean
- 638 Downloads
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 106 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.
KeywordsFusariumvirguliforme FvTox1 Agrobacteriumtumefaciens ATMT-mediated homologous recombination pRF-HU2 Knockout mutants
We thank Dr. Rasmus John Normand Frandsen from Center for Microbial Biotechnology (CMB), Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark for kindly providing the pRF-HU2 plasmid for this study and Dr. Catherine Brooke for reviewing this manuscript. We are thankful to Iowa Soybean Association for financial support in the form of a grant.
- Adachi K, Nelson GH, Peoples KA, Frank SA, Montenegro-Chamorro MV, DeZwaan TM, Ramamurthy L, Shuster JR, Hamer L, Tanzer MM (2002) Efficient gene identification and targeted gene disruption in the wheat blotch fungus Mycosphaerella graminicola using TAGKO. Curr Genet 42:123–127PubMedCrossRefGoogle Scholar
- Lightfoot DA, Meksem K, Gibson PT (2007) Method of determining soybean sudden death syndrome resistance in a soybean plant: greenhouse assays. United States PatentGoogle Scholar
- Sambrook J, Russell DW, Irwin N, Janssen KA (2001) Molecular cloning. A laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar