Influenza viruses are a major public health threat that causes repetitive outbreaks. In recent years, genotype 4 (G4) reassortant Eurasian avian-like (EA) H1N1 (G4 EA H1N1) has garnered attention as a potential novel pandemic strain. The necessity of developing vaccines against G4 EA H1N1 is growing because of the increasing cases of human infection and the low cross-reactivity of the strain with current immunity. In this study, we produced a G4 EA H1N1-derived neuraminidase (G4NA) as a vaccine candidate in Nicotiana benthamiana. The expressed G4NA was designed to be accumulated in the endoplasmic reticulum (ER). The M-domain of the human receptor-type tyrosine-protein phosphatase C was incorporated into the expression cassette to enhance the translation of G4NA. In addition, the family 3 cellulose-binding module and Brachypodium distachyon small ubiquitin-like modifier sequences were used to enable the cost-effective purification and removal of unnecessary domains after purification, respectively. The G4NA produced in plants displayed high solubility and assembled as a tetramer, which is required for the efficacy of an NA-based vaccine. In a mouse immunization model, the G4NA produced in plants could induce significant humoral immune responses. The plant-produced G4NA also stimulated antigen-specific CD4 T cell activation. These G4NA vaccine-induced immune responses were intensified by the administration of the antigen with a vaccine adjuvant. These results suggest that G4NA produced in plants has great potential as a vaccine candidate against G4 EA H1N1.

Subcellular organelles in eukaryotes are surrounded by lipid membranes. In an endomembrane system, vesicle trafficking is the primary mechanism for the delivery of organellar proteins to specific organelles. However, organellar proteins for chloroplasts, mitochondria, the nucleus, and peroxisomes that are translated in the cytosol are directly imported into their target organelles. Chloroplasts are a plant‐specific organelle with outer and inner envelope membranes, a dual‐membrane structure that is similar to mitochondria. Interior chloroplast proteins translated by cytosolic ribosomes are thus translocated through TOC and TIC complexes (translocons in the outer and inner envelope of chloroplasts, respectively), with stromal ATPase motor proteins playing a critical role in pulling pre‐proteins through these import channels. Over the last three decades, the identity and function of TOC/TIC components and stromal motor proteins have been actively investigated, which has shed light on the action mechanisms at a molecular level. However, there remains some disagreement over the exact composition of TIC complexes and genuine stromal motor proteins. In this review, we discuss recent findings on the mechanisms by which proteins are translocated through TOC/TIC complexes and discuss future prospects for this field of research.