In this study, we aimed to generate a series of versatile tagging plasmids that can be used in diverse molecular biological studies of the fungal pathogen Cryptococcus neoformans. We constructed 12 plasmids that can be used to tag a protein of interest with a GFP, mCherry, 4×FLAG, or 6×HA, along with nourseothricin-, neomycin-, or hygromycin-resistant selection markers. Using this tagging plasmid set, we explored the adenylyl cyclase complex (ACC), consisting of adenylyl cyclase (Cac1) and its associated protein Aca1, in the cAMP-signaling pathway, which is critical for the pathogenicity of C. neoformans. We found that Cac1-mCherry and Aca1-GFP were mainly colocalized as punctate forms in the cell membrane and non-nuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by co-immunoprecipitation, using Cac1-6×HA and Aca1-4×FLAG tagging strains. Bimolecular fluorescence complementation further confirmed the in vivo interaction of Cac1 and Aca1 in live cells. Finally, protein pull-down experiments using aca1∆::ACA1-GFP and aca1∆::ACA1-GFP cac1∆ strains and comparative mass spectrometry analysis identified Cac1 and a number of other novel ACC-interacting proteins. Thus, this versatile tagging plasmid system will facilitate diverse mechanistic studies in C. neoformans and further our understanding of its biology.
Bibliographical noteFunding Information:
We thank Shinae Maeng for her technical assistance in constructing the tagging plasmid set. This work was supported by a National Research Foundation of Korea grant (2015R1A2A1A15055687) from MEST and by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agricultural Microbiome R&D Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (916006-2).
© 2017 by The Korean Society for Microbiology and Biotechnology.
All Science Journal Classification (ASJC) codes
- Applied Microbiology and Biotechnology