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

Sun J, Park SY, Kang S, Liu X, Qiu J, Xiang M - Sci Rep (2015)

Bottom Line: The resulting transformants were similar to the corresponding wild-type strains.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 3 Park 1, West Beichen Road, Chaoyang District, Beijing 100101, China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Detection of the hph gene in twelve randomly selected transformants using PCR.The presence of the hph gene in transformants of H. minnesotensis AS3.9869 and H. rhossiliensis AS6.0004 was detected using PCR. This PCR product was absent in AS3.9869 and AS6.0004.
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f1: Detection of the hph gene in twelve randomly selected transformants using PCR.The presence of the hph gene in transformants of H. minnesotensis AS3.9869 and H. rhossiliensis AS6.0004 was detected using PCR. This PCR product was absent in AS3.9869 and AS6.0004.

Mentions: The sensitivities of Hirsutella spp. to hygromycin B and geneticin were determined. The minimum concentrations of hygromycin B and geneticin resulting in complete growth inhibition were 150 μg/ml and 400 μg/ml, respectively, for H. minnesotensis AS3.9869 and 100 μg/ml and 400 μg/ml, respectively, for H. rhossiliensis AS6.0004. Growth responses of these isolates to hygromycin B on potato dextrose agar (PDA) and cornmeal agar (CMA) were indistinguishable. Based on their higher sensitivity to hygromycin B than geneticin, we chose the hph gene, which confers resistance to hygromycin B, to select transformants. Binary vectors containing hph and the AsRed, AsCyan or ZsGreen gene were used for transformation. PCR analysis of 12 randomly selected transformants revealed that the hph gene in the inserted T-DNA was detected in all selected transformants but not in the wild-type (wt) AS3.9869 or AS6.0004 strains (Fig. 1). Microscopic observations of slide cultures showed that all three fluorescent proteins were expressed strongly in hyphae and conidia of AS3.9869 and AS6.0004 transformants (Fig. 2).


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

Sun J, Park SY, Kang S, Liu X, Qiu J, Xiang M - Sci Rep (2015)

Detection of the hph gene in twelve randomly selected transformants using PCR.The presence of the hph gene in transformants of H. minnesotensis AS3.9869 and H. rhossiliensis AS6.0004 was detected using PCR. This PCR product was absent in AS3.9869 and AS6.0004.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507137&req=5

f1: Detection of the hph gene in twelve randomly selected transformants using PCR.The presence of the hph gene in transformants of H. minnesotensis AS3.9869 and H. rhossiliensis AS6.0004 was detected using PCR. This PCR product was absent in AS3.9869 and AS6.0004.
Mentions: The sensitivities of Hirsutella spp. to hygromycin B and geneticin were determined. The minimum concentrations of hygromycin B and geneticin resulting in complete growth inhibition were 150 μg/ml and 400 μg/ml, respectively, for H. minnesotensis AS3.9869 and 100 μg/ml and 400 μg/ml, respectively, for H. rhossiliensis AS6.0004. Growth responses of these isolates to hygromycin B on potato dextrose agar (PDA) and cornmeal agar (CMA) were indistinguishable. Based on their higher sensitivity to hygromycin B than geneticin, we chose the hph gene, which confers resistance to hygromycin B, to select transformants. Binary vectors containing hph and the AsRed, AsCyan or ZsGreen gene were used for transformation. PCR analysis of 12 randomly selected transformants revealed that the hph gene in the inserted T-DNA was detected in all selected transformants but not in the wild-type (wt) AS3.9869 or AS6.0004 strains (Fig. 1). Microscopic observations of slide cultures showed that all three fluorescent proteins were expressed strongly in hyphae and conidia of AS3.9869 and AS6.0004 transformants (Fig. 2).

Bottom Line: The resulting transformants were similar to the corresponding wild-type strains.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 3 Park 1, West Beichen Road, Chaoyang District, Beijing 100101, China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus