The production of recombinant cationic α‐helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum

Abstract

Summary Synthetic linear antimicrobial peptides with cationic α‐helical structures, such as BP100, are valuable as novel therapeutics and preservatives. However, they tend to be toxic when expressed at high levels as recombinant peptides in plants, and they can be difficult to detect and isolate from complex plant tissues because they are strongly cationic and display low extinction coefficient and extremely limited immunogenicity. We therefore expressed BP100 with a C‐terminal tag which preserved its antimicrobial activity and demonstrated significant accumulation in plant cells. We used a fluorescent tag to trace BP100 following transiently expression in Nicotiana benthamiana leaves and showed that it accumulated in large vesicles derived from the endoplasmic reticulum (ER) along with typical ER luminal proteins. Interestingly, the formation of these vesicles was induced by BP100. Similar vesicles formed in stably transformed Arabidopsis thaliana seedlings, but the recombinant peptide was toxic to the host during latter developmental stages. This was avoided by selecting active BP100 derivatives based on their low haemolytic activity even though the selected peptides remained toxic to plant cells when applied exogenously at high doses. Using this strategy, we generated transgenic rice lines producing active BP100 derivatives with a yield of up to 0.5% total soluble protein.

Production of Phytotoxic Cationic α-Helical Antimicrobial Peptides in Plant Cells Using Inducible Promoters

“…The sequences encoding BP100.2 [7], BP100-DsRed-tag54 and DsRed-tag54 [8] were subcloned into these plasmids using the Spe I and BamH I restriction sites.…”

“…Plant expression of recombinant cationic α-helical peptides such as BP100 is preferable for industrial and phytosanitary applications. We have recently showed that active BP100-derived peptides can be expressed as recombinant peptides in plants [7], [8], demonstrated by the increased resistance of GM plants to some rice pathogens [7] and by in vitro growth inhibition assays [8]. These recombinant BP100-derived peptides include endoplasmic reticulum (ER) retention motifs to minimize toxicity to the host plant.…”

“…This did not affect the antimicrobial activity of the products in vitro , demonstrated using bacterial growth inhibition tests. Following transient expression in Nicotiana benthamiana and stable expression in Arabidopsis thaliana seedlings, the peptides accumulated in large ER-derived vesicles, along with typical ER luminal proteins [8]. However, the authors found that the recombinant peptides were often toxic to the host during later developmental stages.…”

“…However, the authors found that the recombinant peptides were often toxic to the host during later developmental stages. Similarly, most transgenic Oryza sativa plants constitutively expressing recombinant ER-targeted BP100-derived peptides failed to achieve maturity, with only a few peptides coupling potent antimicrobial and low hemolytic activity accumulating in transgenic rice lines, with a yield of up to 0.5% total soluble protein (TSP) [8]. Not many cationic α-helical peptides can be expressed in transgenic plants following this strategy, although they have potent activities against other types of pathogenic cells making them valuable as novel therapeutics and preservatives.…”

Concatemerization increases the inhibitory activity of short, cell-penetrating, cationic and tryptophan-rich antifungal peptides

“…Specific challenges for the biotechnological production of short AMPs in cell factories are their small length, susceptibility to degradation, and potential toxicity to the producer host. Fusion to other protein motifs has been used to stabilize AMP in the producer host (Company et al 2014;Viana et al 2013;Wang et al 2014) or to target AMP to cell compartments such as the endoplasmic reticulum (ER) or protein bodies (Bundó et al 2014;Coca et al 2006;Company et al 2014) where they can accumulate in high amounts.…”