Systemic and Phloem-Specific Protein Targeting by High Affinity Nanobodies Expressed From a Plant RNA Virus Vector

The emergence of nanobodies (Nbs) has kindled an avid interest for their use in genetic engineering and plant biotechnology. In planta expression of Nbs has relied on either stable or transient transformation approaches that are lengthy and cannot support systemic expression, respectively. In addition, there is no precedence for studies on tissue-specific expression of Nbs. To address these issues, viral vectors could be used as an alternative, but this has not been shown. Here, this proof-of-concept study establishes a platform to demonstrate the phloem-specific targeting of proteins by Nbs expressed from a citrus tristeza virus-based vector. The vector facilitates anti-green fluorescent protein (GFP) Nb production within the phloem of transgenic Nicotiana benthamiana plants expressing a GFP-fused endoplasmic reticulum-targeting peptide and that of a microtubule marker line expressing GFP-fused α-tubulin 6. The interaction between anti-GFP Nb and the GFP-tagged peptide/protein is corroborated by both pull-down assays and fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) measurements. This proof-of-concept platform-including validation of Nb-antigen interaction in the phloem by FRET-FLIM analysis, which has not been described in the literature-is novel for exploring Nb-mediated functions applicable to targeting or identifying phloem proteins and those co-opted into the virus infection process.