The Non-Phototrophic Hypocotyl3 (NPH3)-domain protein NRL5 is a trafficking-associated GTPase essential for drought resistance.

The mechanisms of plant resistance to low water potential ({psi}w) during drought are unclear but may involve signaling and trafficking at the plasma membrane as well as metabolic reprogramming, including proline accumulation. Forward genetic screening using a Proline Dehydrogenase 1 (ProDH1) promoter:reporter line identified a mutant with extreme low {psi}w hypersensitivity due to a single amino acid substitution (P335L) in the Non-Phototrophic Hypocotyl3 (NPH3) domain of NPH3/RPT2-Like5 (NRL5)/Naked Pins in Yucca8 (NPY8). Further experiments found that NRL5, and other NPH3-domain proteins, are GTPases. NRL5 interacted with RAB small GTPases and the SNARE proteins VAMP721/722 and had polar localization. NRL5P335L had greatly reduced GTPase activity, impaired RAB and VAMP721/722 interaction and disrupted polar localization. These data demonstrate that NRL5-mediated restraint of proline catabolism is required for drought resistance and also more broadly define unexpected functions of the NPH3 domain such that the role of NPH3-domain proteins in signaling, trafficking, and cellular polarity can be critically re-evaluated. One-Sentence SummaryA protein containing the plant-specific NPH3-domain has GTPase activity, trafficking interaction and drought resistance function.