Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes

Abstract

The green fluorescent protein (GFP) has been established as the premier in vivo reporter for investigations of gene expression, protein localization, and cell and organism dynamics. The fungal transformation vector pCT74, with sGFP under the control of the ToxA promoter from Pyrenophora tritici-repentis, effectively expresses GFP in a diverse group of filamentous ascomycetes. Due to the versatility of ToxA promoter-driven expression of GFP, we constructed an additional set of fluorescent protein expression vectors to expand the color palette of fluorescent markers for use in filamentous fungi. EYFP, ECFP and mRFP1 were successfully expressed from the ToxA promoter in its fungus of origin, P. tritici-repentis, and a distant relative, Verticillium dahliae. Additionally the ToxB promoter from P. tritici-repentis drove expression of sGFP in V. dahliae, suggesting a similar potential to the ToxA promoter for heterologous expression in ascomycetes. The suite of fungal transformation vectors presented here promise to be useful for a variety of fungal research applications.

• fluorescent proteins
• fungal promoter
• heterologous expression
• Pyrenophora tritici-repentis
• ToxA
• ToxB

We would like to express our gratitude to those who contributed isolates and constructs, including: Dr. Gary Buchenau, South Dakota State University, Brookings, for the P. tritici-repentis isolate SD-19; Dr. Leonard Francl and Dr. Shaukat Ali, North Dakota State University, Fargo, for the P. tritici-repentis isolate DW7; Dr. Mary Powelson, Oregon State University, Corvallis, for the V. dahliae isolate; Dr. Andreas Nebenführ, University of Tennessee, Knoxville, for pAN94 and pAN95; and Dr. Valerian Dolja, Oregon State University, Corvallis, for pCB302Hsp70h-mRFP. We would also like to thank Viola Manning for thoughtful discussion and review, as well as Dr. Jeffery Stone and Dr. Thomas Wolpert for review. This work was financially supported in part by the National Research Initiative of the USDA Cooperative State Research Education and Extension Service, grant number 2003-35319-13476, and by the Agricultural Research Foundation at Oregon State University, grant number ARF 4420. The authors wish to acknowledge the Confocal Microscopy Facility (made possible in part by grant number 1S10RR107903-01 from the National Institutes of Health) of the Center for Genome Research and Biocomputing and the Environmental and Health Sciences Center at Oregon State University.