Protein body formation in leaves of Nicotiana benthamiana: A concentration-dependent mechanism influenced by the presence of fusion tags

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles originally found in seeds whose function is to accumulate seed storage proteins. It has been shown that PB formation is not limited to seeds and green fluorescent protein (GFP) fused to either elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags induce the formation of PBs in leaves of N. benthamiana. In this study, we compared the ELP- and HFBI-induced PBs and showed that ELP-induced PBs are larger than HFBI-induced PBs. The size of ELP- and HFBI-induced PBs increased over time along with the accumulation levels of their fused protein. Our results show that PB formation is a concentration-dependent mechanism in which proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. Our results show that the presence of fusion tags is not necessary for the formation of PBs, but affects the distribution pattern and size of PBs. This was confirmed by PBs induced by fluorescent proteins as well as fungal xylanases. We noticed that in the process of PB formation, secretory and ER-resident molecules are passively sequestered into the lumen of PBs. We propose to use this property of PBs as a tool to increase the accumulation levels of erythropoietin and human interleukin-10 by co-expression with PB-inducing proteins.