Dissecting the functional domains of the Arabidopsis thaliana nonhost resistance 2B (AtNHR2B) protein.

The Arabidopsis thaliana nonhost resistant 2B (AtNHR2B) is involved in plant defense responses by mediating the deposition of the ß-1,3-glucan polymer callose to the cell wall in response to bacterial pathogens. Despite having a critical role in plant immunity, the exact mechanism of how this protein functions is not known and its protein sequence does not have any similarity to known proteins characterized to date. Using in silico analysis we identified three transmembrane domains and two nuclear localization signals (NLS). To validate these predictions, we generated truncated versions of the protein fused to the green fluorescent protein (GFP) to analyze their subcellular localization by laser scanning confocal microscopy. We found that the in silico predictions matched the subcellular localization of the truncated versions. Specifically, the presence of at least one of the transmembrane domain was required for membrane-bound subcellular compartments. Intriguingly, the localization of the transmembrane domains and the nuclear localization signals correspond to overlapping regions of the protein at the C-terminus and found one truncation that enabled protein localization to the nucleus. These results highlight that AtNHR2B is a unique protein composed of various domains that enable the protein to localize to diverse subcellular compartments and, by virtue of these multiple localizations, likely functions in multiple biological processes.