Golden Gate vectors for apoplastic protein expression in Nicotiana benthamiana

作者：Nick Moritz Eilmann, Tolga O. Bozkurt, Jiorgos Kourelis · 发表于：Zenodo (CERN European Organization for Nuclear Research) · 年份：2026 · DOI：10.5281/zenodo.21458824 · 研究领域：Transgenic Plants and Applications、Plant-Microbe Interactions and Immunity、Plant Virus Research Studies

Many pathogen effectors are secreted into the apoplast, where plant cell-surface immune receptors perceive them. Testing their function by transient expression in Nicotiana benthamiana requires a signal peptide to direct the expressed protein to the apoplast, together with a strong promoter and terminator for high accumulation. We describe three Golden Gate compatible plasmids for apoplastic expression in N. benthamiana. pNEC-0019 and pNE-C-0021 are binary transfer DNA (T-DNA) vectors carrying the long cauliflower mosaic virus (CaMV) 35S promoter, the tobacco NtPR1a signal peptide for apoplastic secretion, and the Arabidopsis thaliana heat-shock protein 18.2 (HSP18.2) terminator. A Golden Gate cloning site lies between the signal peptide and the terminator. A coding sequence (CDS) cloned into pNE-C-0019 is expressed as an untagged secreted protein. pNE-C-0021 has a GCTT fusion site at the 3′ cloning arm and gives an untagged or a C-terminally tagged protein, depending on whether a tag module is included in the assembly. pNE-CDS-0061 contains the C-terminal signalling region of the tomato receptor-like protein (RLP) Cf-4 as a module for building chimeric receptors.