Development of a rapid transformation and amplification system in Byblis guehoi unveils an atypical protein secretion pathway in sessile trichome cells.

The leaves of the carnivorous plant Byblis guehoi are densely covered with structurally simple sessile trichomes, each composed of eight flattened cells arranged in a ring. These trichomes exhibit robust protein exocytosis and endocytosis, making them a suitable system for studying these processes. To track protein secretion, we established an Agrobacterium-mediated transformation protocol using root explants. Calli were induced on Byblis callus induction medium, and transgenic shoots were regenerated within two months on hygromycin-containing Byblis selection and regeneration medium. In addition, a temporary immersion system was implemented to rapidly propagate transgenic lines, enhancing growth and facilitating acclimation. Four 35S promoter-driven constructs expressing β-glucuronidase, betalain reporter (RUBY), modified green fluorescent protein (mGFP), or CLAVATA3 signal peptide fusion modified green fluorescent protein (CLV3sp-mGFP5) validated the protocol, yielding transformation efficiencies of 24.9%-59.4% and regeneration efficiencies of 22.3%-73.7%. Confocal microscopy revealed predominantly cytoplasmic signals for mGFP5 and plasma membrane-localized signals for CLV3sp-mGFP5 in epidermal cells. Unexpectedly, both constructs exhibited GFP signals at the plasma membrane and in the extracellular space of sessile trichome cells, regardless of the presence of a signal peptide. Western blot analysis further confirmed that mGFP5 was secreted into the mucilage independently of signal peptide presence. These findings reveal signal peptide-independent extracellular protein accumulation in glandular trichomes, deviating from the classical endoplasmic reticulum-Golgi pathway. Given that sessile trichomes of B. guehoi possess a specialized cuticular pore lacking a rigid cell wall, this structural feature may provide a plausible interface for nonclassical protein export. This study establishes a rapid transformation-propagation pipeline for B. guehoi, highlighting its potential as an experimental platform for investigating protein secretion in plant glandular cells.