Vectors for fluorescent protein tagging in Phytophthora: tools for functional genomics and cell biology Audrey M. V. AH-FONG, Howard S. JUDELSON* Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA article info Article history: Received 26 April 2011 Received in revised form 28 June 2011 Accepted 5 July 2011 Available online 18 July 2011 Corresponding Editor: Meritxell Riquelme Keywords: Cell biology Fluorescent tagging Oomycete Phytophthora Subcellular Transformation abstract Fluorescent tagging has become the strategy of choice for examining the subcellular local- isation of proteins. To develop a versatile community resource for this method in oomy- cetes, plasmids were constructed that allow the expression of either of four spectrally distinct proteins [cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry], alone or fused at their N- or C-termini, to se- quences of interest. Equivalent sets of plasmids were made using neomycin or hygromycin phosphotransferases (nptII, hpt) as selectable markers, to facilitate double-labelling and aid work in diverse species. The fluorescent proteins and drug-resistance markers were fused to transcriptional regulatory sequences from the oomycete Bremia lactucae, which are known to function in diverse oomycetes, although the promoter in the fluorescence cas- sette (ham34) can be replaced easily by a promoter of interest. The function of each plasmid was confirmed in Phytophthora infestans. Moreover, fusion proteins were generated using targeting sequences for the endoplasmic reticulum, Golgi, mitochondria, nuclei, and perox- isomes. Studies of the distribution of the fusions in mycelia and sporangia provided insight into cellular organisation at different stages of development. This toolbox of vectors should advance studies of gene function and cell biology in Phytophthora and other oomycetes. ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Advances in genome sequencing have accelerated the discov- ery of the gene content of many organisms, and accordingly have catalysed the advancement of tools for functional geno- mics. Genomes are now analysed for several oomycetes, a group of fungus-like microbes that include major plant and animal pathogens as well as saprophytes (Lamour et al. 2006). One of the most important oomycetes is Phytophthora infestans, which causes the late blight diseases of potato and tomato (Fry 2008). Other oomycetes having economic impact include more than 100 additional species of Phytophthora that cause disease on diverse plants, Pythium, which contains plant and animal pathogens, Saprolegnia, which causes significant losses in aqua- culture, and several groups of downy mildews. Determining the subcellular localisation of a protein is valuable for understanding its function, in addition to helping reveal details of cellular organisation. Fluorescence tagging has emerged as one of the most powerful methods for this, complementing cell fractionation, immunohistochemistry, and bioinformatics studies (Lunn 2007). Tags with distinct spectroscopic properties have now been developed, following the initial work using green fluorescent protein (GFP) from Aequorea victoria (Lippincott-Schwartz & Patterson 2003). In addition to localising single proteins, it is now possible to use multiple tags to test two or more proteins for colocalisa- tion or interaction. Sets of vectors for expressing proteins fused to either of several fluorescent tags have been developed for several taxa including bacteria, yeast, and plants (Sheff & Thorn 2004; Nelson et al. 2007; Norris et al. 2010). * Corresponding author. Tel.: þ1 951 827 4199; fax: þ1 951 827 4294. E-mail address: howard.judelson@ucr.edu journal homepage: www.elsevier.com/locate/funbio fungal biology 115 (2011) 882 e890 1878-6146/$ e see front matter ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2011.07.001