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A split luciferase complementation assay for studying in vivo protein–protein interactions in filamentous ascomycetes

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Abstract

Protein–protein interactions play important roles in controlling many cellular events. To date, several techniques have been developed for detection of protein–protein interactions in living cells, among which split luciferase complementation has been applied in animal and plant cells. Here, we examined whether the split luciferase assay could be used in filamentous ascomycetes, such as Gibberella zeae and Cochliobolus heterostrophus. The coding sequences of two strongly interacting proteins (the F-box protein, FBP1, and its partner SKP1) in G. zeae, under the control of the cryparin promoter from Cryphonectria parasitica, were translationally fused to the C- and N-terminal fragments of firefly luciferase (luc), respectively. Each fusion product inserted into a fungal transforming vector carrying the gene for resistance to either geneticin or hygromycin B, was transformed into both fungi. We detected complementation of split luciferase proteins driven by interaction of the two fungal proteins with a high luminescence intensity-to-background ratio only in the fungal transformants expressing both N-luc and C-luc fusion constructs. Using this system, we also confirmed a novel protein interaction between transcription factors, GzMCM1 and FST12 in G. zeae, which could hardly be proven by the yeast two-hybrid method. This is the first study demonstrating that monitoring of split luciferase complementation is a sensitive and efficient method of studying in vivo protein–protein interactions in filamentous ascomycetes.

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Acknowledgments

This research was supported by the Agricultural Research Center program of the Ministry for Food, Agriculture, Forestry and Fisheries, Korea, and by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (2009-0075256). We thank Ae Ran Park, Seoul National University for technical assistance in the western blot analysis.

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Authors and Affiliations

  1. Department of Medical Biotechnology, Soonchunhyang University, Asan, 336-745, South Korea

    Hee-Kyoung Kim, Eun Ji Cho, Seong mi Jo, Bo Reum Sung, Seunghoon Lee & Sung-Hwan Yun

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  1. Hee-Kyoung Kim
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  2. Eun Ji Cho
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  3. Seong mi Jo
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  4. Bo Reum Sung
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  5. Seunghoon Lee
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Correspondence to Sung-Hwan Yun.

Additional information

Communicated by U. Kueck.

H.-K. Kim and E. J. Cho equally contributed.

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Kim, HK., Cho, E.J., Jo, S. et al. A split luciferase complementation assay for studying in vivo protein–protein interactions in filamentous ascomycetes. Curr Genet 58, 179–189 (2012). https://doi.org/10.1007/s00294-012-0375-5

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