Abstract
Plant cells contain groups of biomolecules that participate together in a particular biological process. Exogenous codelivery of multiple biomolecules is an essential step for elucidation of the biological significance of these molecules and enables various biotechnological applications in plants. However, the currently existing biomolecule delivery methods face difficulties in delivering multiple components into plant cells, mediating transgene expression, and maintaining the stability of the numerous components and lead to delays in biomolecular function. Cell-penetrating peptides (CPPs) have demonstrated remarkable abilities to introduce diverse biomolecules into various plant species. Here, we employed the engineered CPP KH9-BP100 as a carrier to deliver multiple biomolecules into plant cells and performed a bimolecular fluorescence complementation assay to assess the simultaneous introduction of multiple biomolecules. We demonstrate that multiple biomolecule/CPP cargos can be simultaneously internalized by a particular plant cell, albeit with different efficiencies. We present a cutting-edge technique for codelivery of multiple biomolecules into plant cells that can be used for elucidation of functional correlations and for metabolic engineering.
Highlights
Plant cells create functional proteins that synergistically interact with each other to function in particular metabolic processes and in cellular mechanism
We further detected the colocalization of complemented sfGN155-methylxanthine methyltransferase 1 (MxMT)/sfGC155-MxMT homodimers with FM4-64-stained vesicles in the cytoplasm (Fig. 5f and Fig. S14b†), and the results indicated that the two protein/Cell-penetrating peptides (CPPs) complexes colocalized with vesicles when the colocalization coefficient (R) was higher than 0.75, associated with vesicles when 0.25 ≤ R ≤ 0.75, or were distinctly released when R was lower than 0.25 (Fig. S14b†)
Our findings showed that the transcript and protein abundances of the two nonfluorescent superfolder GFP (sfGFP) fragment-fused MxMT components in plant leaves transfected with pDNA and singlestranded RNA (ssRNA)/CPP cargos were comparable
Summary
Plant cells create functional proteins that synergistically interact with each other to function in particular metabolic processes and in cellular mechanism How these interacting biomolecules participate in specific biological reactions is intriguing, because protein interactions are highly complex and dynamic. Rational fine-tuning of multiple transgene expression approaches in plants is critical but is one of the drawbacks of such biomolecule delivery methods This fine-tuning is necessary because the regulation of gene expression is strictly controlled at both the transcription and translation steps.[8] It is unclear whether different gene expression cassettes produce similar levels of mRNA molecules in the same plant tissue.[9] Individual mRNA molecules can correspond to numerous proteins that interact each other and might not behave in a single plant cell.[10,11,12] multiple proteins participating in the same event may have distinct vulnerabilities to protein degradation over the duration of an experiment.[13] exogenous expression of multiple genes through conventional biomolecule introduction methods constrains the measurable functional parameters to only the average biomolecule synthesis rate.
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