Fungal endophyte N-acetylglucosaminidase expression in the infected host grass Huaijun Michael LI, Jo Anne CROUCH and Faith C. BELANGER* Department of Plant Biology and Pathology and The Biotechnology Center for Agriculture & the Environment, Cook College, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08903, USA. E-mail : belanger@aesop.rutgers.edu Received 15 June 2004; accepted 15 October 2004. Fungal endophytes of the genera Neotyphodium and Epichloe¨ are important mutualistic symbionts and pathogens of many cool-season grass species. Here we report the characterization of a secreted N-acetylglucosaminidase from the Neotyphodium sp. endophyte that infects the grass Poa ampla. The enzyme was expressed at low levels within the host, and activity could be detected in the apoplastic protein fraction. Low-level expression could also be detected in endophyte-infected perennial ryegrass (Lolium perenne), Chewings fescue (Festuca rubra subsp. fallax), and tall fescue (L. arundinaceum). The enzyme may function in the recycling of chitin oligomers generated from turnover of the fungal cell wall. This is the first report of a secreted N-acetylglucosaminidase expressed by an endophytic fungus in the infected host plant. INTRODUCTION Fungal endophytes of the genera Neotyphodium and Epichloe¨ are important mutualistic symbionts of many grass species (Clay & Schardl 2002, Schardl 2001, Scott 2001). The fungi exist exclusively in the intercellular spaces of the leaf sheaths and blades of the host grasses, never invading the plant cells. Endophyte-infected plants are protected from insect and animal herbivory due to toxic alkaloids synthesized by the fungi (Breen 1994, Bush, Wilkinson & Schardl 1997). For some en- dophyte/grass combinations, however, the endophytes can be considered pathogens since they result in choke disease in which the fungus forms a stroma that inter- feres with the flowering of the host plant resulting in reduction or elimination of seed-set (Kirby 1961). The fungal endophytes are not found free-living in nature, apparently requiring association with their plant hosts for their nutrition and protection. How the fungal endophytes are obtaining the required nutritional compounds from the nutrient-poor apoplastic spaces of their hosts is not understood. In general, fungi satisfy their nutritional needs by secreting hydrolytic enzymes that act on polymeric substances in the en- vironment releasing monomers that can be transported into the fungal cells. We are interested in endophyte-secreted proteins since they may be important in the nutrition and growth of the fungus within the infected host. Endophyte secreted proteins may also have roles in the interspecies communication. We are using the grass Poa ampla infected with a Neotyphodium sp. endophyte as a model system for the host grass/fungal endophyte interaction. This is a good system for such investi- gations since, because of apomixis, we have identical plant genotypes that are infected and uninfected with the fungal endophyte. We previously characterized a fungal invertase, serine proteinase, beta-1,6-glucanase, and chitinase that are expressed in endophyte-infected plants (Lam et al. 1995, Reddy, Lam & Belanger 1996, Moy et al. 2002, Li et al. 2004). These secreted hydro- lytic enzymes may be involved in the nutrition and/or growth of the fungal hyphae through the apoplast. During the purification of the endophyte chitinase it became apparent that the Neotyphodium sp. endophyte was also producing another secreted chitinolytic enzyme, N-acetyl-b-glucosaminidase (Li et al. 2004). N-acetylglucosaminidases catalyze the release of N-acetylglucosamine (GlcNAc) monomers from chitin oligomers generated by endo- and exochitinases (Cohen-Kupiec & Chet 1998). Here we report the characterization of this enzyme and its presence in the apoplast of infected plants. * Corresponding author. Mycol. Res. 109 (3): 363–373 (March 2005). f The British Mycological Society 363 doi:10.1017/S0953756204002205 Printed in the United Kingdom.