Chen Angel Y S, Spigolon Giada, Scipioni Lorenzo, Ng James C K
Department of Microbiology and Plant Pathology, University of California, Riverside, California, USA.
Beckman Institute, Caltech, Pasadena, California, USA.
Mol Plant Pathol. 2025 Jun;26(6):e70105. doi: 10.1111/mpp.70105.
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.
纳米抗体（Nbs）的出现引发了人们对其在基因工程和植物生物技术中应用的浓厚兴趣。在植物中表达纳米抗体分别依赖于稳定或瞬时转化方法，而这两种方法耗时较长且无法支持系统性表达。此外，关于纳米抗体组织特异性表达的研究尚无先例。为了解决这些问题，可以使用病毒载体作为替代方案，但尚未得到证实。在此，这项概念验证研究建立了一个平台，以证明基于柑橘衰退病毒载体表达的纳米抗体对蛋白质的韧皮部特异性靶向作用。该载体促进了抗绿色荧光蛋白（GFP）纳米抗体在表达GFP融合内质网靶向肽的转基因本氏烟草植物的韧皮部以及表达GFP融合α-微管蛋白6的微管标记系的韧皮部中的产生。通过下拉试验和荧光共振能量转移-荧光寿命成像显微镜（FRET-FLIM）测量，证实了抗GFP纳米抗体与GFP标记的肽/蛋白之间的相互作用。这个概念验证平台——包括通过FRET-FLIM分析在韧皮部中验证纳米抗体-抗原相互作用，这在文献中尚未描述——对于探索适用于靶向或鉴定韧皮部蛋白以及那些参与病毒感染过程的蛋白的纳米抗体介导的功能来说是新颖的。
