1 Physical and Genetic Interaction Between Ammonium Transporters and the Signaling 1

ABSTRACT 20 Dimorphic transitions between yeast-like and filamentous forms occur in many fungi and are 21 often associated with pathogenesis. One of the cues for such a dimorphic switch is the 22 availability of nutrients. Under conditions of nitrogen limitation, fungal cells (like 23 Saccharomyces cerevisiae and Ustilago maydis) switch from budding to pseudohyphal or 24 filamentous growth. Ammonium transporters (AMTs) are responsible for uptake and, in some 25 cases, for sensing availability of ammonium, a preferred nitrogen source. Homo- and/or 26 heterodimer formation may be required for regulating the activity of the AMTs. To investigate 27 the potential interactions of Ump1 and Ump2, the AMTs of the maize pathogen, U. maydis, we 28 first used the split ubiquitin system followed by a modified split YFP system to validate the 29 interactions in vivo. Such analysis showed formation of homo- and hetero-oligomers by Ump1 30 and Ump2. We also demonstrated interaction of the high affinity ammonium transporter, Ump2, 31 with the protein, Rho1 GTPase, a central character in signaling, with roles in controlling 32 polarized growth. This is the first demonstration in eukaryotes of physical interaction in vivo of 33 an ammonium transporter with the signaling protein, Rho1. Moreover, interaction of the Ump 3