Establishment of a high-efficiency transient expression system for subcellular localization studies in plants based...

Agro-infiltration-mediated transient expression in Nicotiana benthamiana leaves is a widely used method to analyzing the subcellular localization of fluorescence protein (FP)-tagged fusion proteins. However, this technique is largely restricted to leaf tissues and a limited number of plant species. Here, we established a vacuum infiltration-based transient expression system and successfully expressed proteins with various subcellular localizations utilizing the roots and hypocotyls of 5-day-old seedlings as explants from different plant species. This approach extends the applicability of vacuum infiltration beyond cotyledons and Arabidopsis, which efficiently bypasses the tissue specificity of conventional agro-infiltration, shortens the experimental cycle, and is readily adaptable to multiple plant species. Collectively, these advantages make this method a rapid, versatile, and broadly applicable system for investigating protein subcellular localizations across diverse plant models.