Direct protein delivery into intact plant cells using polyhistidine peptides

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Polyhistidine peptides (PHPs) are short sequences of histidine residues.
• PHPs function as effective cell-penetrating peptides (CPPs) for plant cells.
• Protein delivery into plant cells is crucial for various biotechnological applications.

Purpose of the Study:

• To evaluate the efficacy of PHP-fusion proteins for delivering proteins into cultured plant cells.
• To investigate the influence of PHP length on protein cellular uptake.
• To assess the functionality of delivered proteins using adenylate cyclase (CyaA) as a reporter.

Main Methods:

• Constructed fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and varying lengths of PHPs (His8-His20).
• Co-cultivated fusion proteins with cultured plant cells from *Nicotiana tabacum*, *Oryza sativa*, and *Cryptomeria japonica*.
• Utilized CyaA as a reporter to measure protein delivery and functional activity by assessing intracellular fluorescence and cyclic adenosine monophosphate (cAMP) production.

Main Results:

• All tested PHP-fusion proteins demonstrated cellular uptake in all plant cell lines.
• The fusion protein with the longest PHP (MBP-RFP-His20) exhibited maximum intracellular fluorescence.
• The delivered CyaA reporter protein successfully increased intracellular fluorescence and cAMP production in all tested cell lines.

Conclusions:

• Polyhistidine peptides are highly effective carriers for delivering proteins into the intracellular space of diverse cultured plant cells.
• PHP-based protein delivery systems show significant potential for advancing plant biotechnology and research.
• The length of the polyhistidine tag influences the efficiency of protein delivery into plant cells.