A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potato Anna O. Avrova, 1 ** Petra C. Boevink, 1 Vanessa Young, 1 Laura J. Grenville-Briggs, 2 Pieter van West, 2 Paul R. J. Birch 3 and Stephen C. Whisson 1 * 1 Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK. 2 Aberdeen Oomycete Group, College of Life Sciences and Medicine, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB24 2ZD, UK. 3 Division of Plant Sciences, College of Life Sciences, University of Dundee at SCRI, Invergowrie, Dundee DD2 5DA, UK. Summary Phytophthora infestans causes late-blight, a devastat- ing and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans- membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appresso- rium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that pro- vides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic struc- tures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to suc- cessful host colonization by P. infestans. Introduction Filamentous eukaryotic plant pathogenic microorganisms such as fungi and oomycetes frequently form highly spe- cialized structures during invasion and colonization of their hosts. For many plant pathogens, host invasion com- mences with the differentiation of a domed or swollen structure, called an appressorium, at the terminus of a germination tube or hypha from a spore (Talbot, 2003; Grenville-Briggs and van West, 2005). The appressorium secretes adhesive compounds to maintain contact between host and pathogen during infection (Tucker and Talbot, 2001; Grenville-Briggs and van West, 2005), and can generate mechanical force (Howard et al., 1991; Bechinger et al., 1999) and/or hydrolytic enzymes to pene- trate host cell walls (Francis et al., 1996; Pryce-Jones et al., 1999). After invading a compatible host, fungal and oomycete hyphae grow either intercellularly or intracellularly, with or without biotrophic structures called haustoria (Mendgen and Hahn, 2002). For example, Cladosporium fulvum produces entirely intercellular hyphae without haustoria, Magnaporthe grisea grows in planta as intra- cellular hyphae without haustoria (Kankanala et al., 2007), while obligate biotrophs, such as the rust fungi, produce intercellular hyphae with intracellular haustoria. Haustoria are formed by invagination of the host cell membrane and are thus in intimate contact with the host cell. The haustorial plasma membrane of fungal plant pathogens is differentiated from the intercellular hyphal plasma membrane and is distinct in its possession of proteins for the transport of nutrients such as sugars and amino acids (Hahn et al., 1997; Struck et al., 1998; Voegele et al., 2001). Haustoria are also the site of secretion for fungal and oomycete effector proteins, some of which may act inside the host cell to modulate Received 29 April, 2008; revised 30 June, 2008; accepted 7 July, 2008. For correspondence. *E-mail Steve.Whisson@scri.ac.uk; Tel. (+44) 1382 562731; Fax (+44) 1382 562426; **E-mail Anna.Avrova@ scri.ac.uk; Tel. (+44) 1382 562731; Fax (+44) 1382 562426. Cellular Microbiology (2008) doi:10.1111/j.1462-5822.2008.01206.x © 2008 Scottish Crop Research Institute Journal compilation © 2008 Blackwell Publishing Ltd