Physical and Genetic Interaction between Ammonium Transporters and the Signaling Protein Rho1 in the Plant Pathogen Ustilago maydis

Abstract

Dimorphic transitions between yeast-like and filamentous forms occur in many fungi and are often associated with pathogenesis. One of the cues for such a dimorphic switch is the availability of nutrients. Under conditions of nitrogen limitation, fungal cells (such as those of Saccharomyces cerevisiae and Ustilago maydis) switch from budding to pseudohyphal or filamentous growth. Ammonium transporters (AMTs) are responsible for uptake and, in some cases, for sensing the availability of ammonium, a preferred nitrogen source. Homodimer and/or heterodimer formation may be required for regulating the activity of the AMTs. To investigate the potential interactions of Ump1 and Ump2, the AMTs of the maize pathogen U. maydis, we first used the split-ubiquitin system, followed by a modified split-YFP (yellow fluorescent protein) system, to validate the interactions in vivo. This analysis showed the formation of homo- and hetero-oligomers by Ump1 and Ump2. We also demonstrated the interaction of the high-affinity ammonium transporter, Ump2, with the Rho1 GTPase, a central protein in signaling, with roles in controlling polarized growth. This is the first demonstration in eukaryotes of the physical interaction in vivo of an ammonium transporter with the signaling protein Rho1. Moreover, the Ump proteins interact with Rho1 during the growth of cells in low ammonium concentrations, a condition required for the expression of the Umps. Based on these results and the genetic evidence for the interaction of Ump2 with both Rho1 and Rac1, another small GTPase, we propose a model for the role of these interactions in controlling filamentation, a fundamental aspect of development and pathogenesis in U. maydis.

Regulation of haplid phenotypes in Ustilago maydis by ammonium transporters and components of the b mating locus.

“…This protein appeared to localize to the cell membrane, as has been previously reported (Paul et al, 2014). Cells containing the ump2-YFP construct and the overxpression construct were also visualized in this manner, and ump2 localization showed a similar pattern (Figure 34c).…”

“…The YFP-tagged ump2 construct (Paul et al, 2014) was used to transform FB1 wild-type cells, using hygromyocin resistance as a selectable marker.…”

“…Earlier investigations hypothesized that the connection of ump2 to the PKA signaling pathway occurs at multiple levels. More recently, Ump2 has been found to interact physically with Rho1 (Paul, Barati, Cooper, & Perlin, 2014), a small G protein required for viability in haploid U.…”

Exploring the role of PTN1 in ustilago maydis.

“…In U. maydis, low ammonium plays an indispensable role in regulating the dimorphic 1/2 2/9 1/2 Δaga1 2/9 Δaga1 1/2 2/9 1/2 Δaga1 2/9 Δaga1 switch in response to nutrient limitation, specifically nitrogen in the form of ammonium (Paul, Barati, Cooper, & Perlin, 2014). This indicates that aga1 may have a role in the growth of the fungus under low ammonium conditions and play a role in the filamentous response pathway in U. maydis.…”

Atmospheric nitrogen assimilation in Ustilago maydis.

“…The role of these ammonium transporters in the dimorphic transition was further elucidated by confirmation that the signaling protein Rho1 directly interacts with both Ump1 and Ump2 (Paul et al, 2014). Rho1, a small GTPase, has been shown to localize at the growing tip and septum of U. maydis cells during cytokinesis as well as modulating b-induced filament formation (Pham et al, 2009).…”