Current Genetics

, Volume 59, Issue 3, pp 107–117 | Cite as

Investigation of the Fusarium virguliforme fvtox1 mutants revealed that the FvTox1 toxin is involved in foliar sudden death syndrome development in soybean

  • Ramesh N. Pudake
  • Sivakumar Swaminathan
  • Binod B. Sahu
  • Leonor F. Leandro
  • Madan K. BhattacharyyaEmail author
Research Article


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.


Fusariumvirguliforme 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.

Supplementary material

294_2013_392_MOESM1_ESM.pptx (54 kb)
Supplemental figure 1 Representation of the vector constructs used in this investigation. a Schematic representation of the strategy used in targeted knockout construct of the FvTox1 gene. Locations of primers for PCR amplification of the promoter and terminator of the FvTox1 gene are shown with triangles. PCR amplified promoter and terminator fragments were ligated to the linearized pRF-HU2 vector carrying the marker hph gene. The recombinant plasmid was transformed into E. coli and then into A. tumefaciens. A. tumefaciens containing recombinant pRF-HU2 plasmid was used to infect conidial spores of the F. virguliforme Mont-1 isolate. b Diagramatic representation of the locations of primers and restriction endonucleases sites used for analyses of the fvtox1 mutants. The FvTox1 locus and proposed structure of this locus after integration of hph through homologous recombination are shown. The boxed arrows show the FvTox1, and hph genes. The primers used for PCR amplification of promoters and terminators and molecular characterization of mutants are shown in the figure with arrows and details of primers are presented in Table 1. The sizes of different restriction fragments are shown on the map. Probes used in Southern blot analyses are shown with arrows. X; XbaI site, E; EcoRI site, hph; hygromycin B resistance gene. RB and LB: T-DNA left and right border repeat sequences (PPTX 53 kb)
294_2013_392_MOESM2_ESM.pptx (5.4 mb)
Supplemental figure 2 PCR analysis of F. virguliforme genes. Genomic DNA was extracted from the fvtox1 mutants and the F. virguliforme Mont-1 isolate infested sorghum meals and PCR was conducted for the 38 F. virguliforme genes using primers presented in Supplemental table 1. Lane 1 to 6 represent Mont-1, fvtox1-1, fvtox1-2, fvtox1-3, fvtox1-4 and fvtox1-5, respectively. Information about the genes can be found in the website: (PPTX 5481 kb)
294_2013_392_MOESM3_ESM.pptx (83 kb)
Supplemental figure 3 Southern blot analysis of fvtox1 mutants with the left T-DNA border-specific probe. a The blot carrying the pRF-HU2::FvTox1 construct and the pRF-HU2 was hybridized to left T-DNA border-specific probe amplified by LBF and LBR primers (Table 1). b The blot of the XbaI digested genomic DNA from both Mont-1 and fvtox1 mutants was hybridized with the left T-DNA border-specific probe used in (a) (PPTX 82 kb)
294_2013_392_MOESM4_ESM.pptx (65 kb)
Supplemental figure 4 Foliar SDS symptoms development 15 days following feeding of cut soybean seedlings with cell-free F. virguliforme culture filtrates. Soybean seedlings fed with the cell-free fvtox1 culture filtrates showed over 2 fold less foliar disease incidence as compared to that in soybean seedlings fed with cell-free Mont-1 culture filtrates. Control plants fed with only water did not show any symptoms. 7D, soybean seedlings 7 days post feeding with culture filtrates; 9D, soybean seedlings 9 days post feeding with culture filtrates; 11D, soybean seedlings 11 days post feeding with culture filtrates; 13D, soybean seedlings 13 days post feeding with culture filtrates; 15D, soybean seedlings 15 days post feeding with culture filtrates (PPTX 64 kb)
294_2013_392_MOESM5_ESM.pptx (54 kb)
Supplemental figure 5 Foliar SDS symptoms development 27 days after planting of soybean seeds in soil mixed with F. virguliforme inocula. Soybean seedlings grown in soil infested with Mont-1 showed severe disease symptoms compared to seedlings grown in soil infested with fvtox1 inocula. Control plants grown in soil with sorghum meal alone did not show any trace of symptoms. 15D, 15 days post inoculation; 18D, 18 days post inoculation; 21D, 21 days post inoculation; 24D, 24 days post inoculation; 27D, 27 days post inoculation (PPTX 53 kb)
294_2013_392_MOESM6_ESM.docx (16 kb)
Supplemental table 1 (DOCX 54 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Ramesh N. Pudake
    • 1
    • 3
  • Sivakumar Swaminathan
    • 1
  • Binod B. Sahu
    • 1
  • Leonor F. Leandro
    • 2
  • Madan K. Bhattacharyya
    • 1
    Email author
  1. 1.Department of AgronomyIowa State UniversityAmesUSA
  2. 2.Department of Plant Pathology and MicrobiologyIowa State UniversityAmesUSA
  3. 3.School of Biotechnology and BiosciencesLovely Professional UniversityPhagwaraIndia

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