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Hirsutella rhossiliensis  and  H. minnesotensis  are endoparasitic fungi of the second-stage juvenile (J2) of the soybean cyst nematode ( Heterodera glycines ) in nature. They also parasitize both  H. glycines  J2 and  Caenorhabditis elegans  on agar plates.  Agrobacterium tumefaciens -mediated transformation conditions were established for these  Hirsutella  spp. The resulting transformants were similar to the corresponding wild-type strains. The infection processes of  H. glycines  J2 and  C. elegans  second larval stage (L2) by  H. minnesotensis  expressing ZsGreen were microscopically analyzed. Conidia of  H. minnesotensis  adhered to passing nematodes within 8 h post-inoculation (hpi), formed an infection peg between 8 and 12 hpi, and penetrated the nematode cuticle between 12 and 24 hpi for  C. elegans  L2 and between 12 and 32 hpi for  H. glycines  J2. Hyphal proliferation inside of the nematode coelom was observed at approximately 32 hpi for  C. elegans  L2 and at approximately 40 hpi for  H. glycines  J2. The fungus consumed the whole body and grew out to produce conidia at approximately 156 and 204 hpi for  C. elegans  L2 and  H. glycines  J2, respectively. The efficient transformation protocol and a better understanding of infection process provide a solid foundation for studying the molecular and cellular mechanisms underlying fungal parasitism of nematodes.

Development of a transformation system for Hirsutella spp. and visualization of the mode of nematode infection by GFP-labeled H. minnesotensis