The production of recombinant cationic α-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum
The production of recombinant cationic α-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum
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DOI：
10.1111/pbi.12119
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发表时间：
2014-01-01
影响因子：
13.8
通讯作者：
Pla, Maria
中科院分区：
文献类型：
--
作者：
Company, Nuri;Nadal, Anna;Pla, Maria
具有阳离子螺旋结构的合成线性抗微生物肽，如BP 100，作为新的治疗剂和防腐剂是有价值的。然而，当在植物中以高水平表达为重组肽时，它们往往是有毒的，并且它们可能难以从复杂的植物组织中检测和分离，因为它们是强阳离子的并且显示出低消光系数和极其有限的免疫原性。因此，我们表达了具有C-末端标签的BP 100，其保留了其抗微生物活性，并在植物细胞中表现出显著的积累。我们使用荧光标记来追踪BP 100在本氏烟草叶中瞬时表达后，并显示它与典型的内质网腔蛋白沿着积累在来自内质网（ER）的大囊泡中。有趣的是，这些囊泡的形成是由BP 100诱导的。在稳定转化的拟南芥幼苗中形成类似的囊泡，但重组肽在后期发育阶段对宿主有毒。这通过基于其低溶血活性选择活性BP 100衍生物来避免，即使当以高剂量外源施用时，所选肽对植物细胞保持毒性。使用这种策略，我们产生了转基因水稻品系生产活性BP 100衍生物的产量高达0.5%的总可溶性蛋白。
Synthetic linear antimicrobial peptides with cationic -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.